23396069 Lecture3 Screening for Microbes and Strain Improvement
Transcript of 23396069 Lecture3 Screening for Microbes and Strain Improvement
copy 2009 Maulik P Suthar
Screening for microbes and strain improvement
Maulik P Suthar
copy 2009 Maulik P Suthar
Microbial products
bull Primary metabolites Made during the organismrsquos growth phase Essential to an organismrsquos metabolism and can be intermediate metabolites or end
bull Secondary metabolites Not essential to cell function or growth and are usually made late in the organismrsquos growth cycle Usually derived from primary metabolites or intermediates of primary metabolites Most likely give the organism an advantage over competitors
Primary Secondary
copy 2009 Maulik P Suthar
Methods for screening
bull Microbial bull Genotypicbull Phenotypic bull Chemical
copy 2009 Maulik P Suthar
Strain improvement
bull Stable production strain with a high titre and ideal production profile which makes a simple purification process possible To obtain such strains we use a combination of traditional methods such as random mutagenisation and genetic engineering
bull Mutagenisation and selection very effectively increase the titres of desired compounds Gene technology is used to obtain an ideal production profile eg to inactivate genes leading to side products and to improve the self-resistance of the production strain which increases both stability and titre
copy 2009 Maulik P Suthar
What are Antibiotics
bull Antibiotics = ldquoagainst liferdquobull Antibiotics = molecules that stop microbes both bacteria and fungi
from growing or kill them outrightbull Antibiotics can be either natural products or man-made synthetic
chemicalsbull Most of the antibiotics identified over the past 60 years have been
natural products produced by one micro-organism within a particular environment to affect neighbouring microbes
bull Can cause microbe death or regulate the growth of the neighbouring microbes
bull These antibiotic agents are produced by both bacteria and fungi
copy 2009 Maulik P Suthar
Antibiotic Production
bull Antibiotics are produced industrially by a process of fermentation where the source microorganism is grown in large amounts
bull As the antibiotics are produced by the microorganisms they have to be removed to ensure they do not effect the bacterial population
bull Certain bacterial species are selectively mutated to produce the maximum amount of the antibiotic agent
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
Mutations and Genetic Instability
bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts
copy 2009 Maulik P Suthar
Induced Mutations
UV radiation
Chemical Mutations
1 4-Nitroquinoline-1-oxide
2 Hydroxylamine
3 Methyl Methanesulfonate
4 Ethyl Methanesulfonate
5 N-methyl-N-nitro-N-nytrosoguanidine
copy 2009 Maulik P Suthar
Mutagenesis and Strain Development
Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques
Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today
1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity
copy 2009 Maulik P Suthar
Plate Based Techniques
bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic
diffused into the surrounding agar
copy 2009 Maulik P Suthar
Antibiotic Production
bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value
Why screening bull In order to identify the useful antibiotics a process of screening is
often employed bull Using this method isolates of a large number of micro-organisms
are cultured and then tested for production of diffusible products which inhibit the growth of test organisms
copy 2009 Maulik P Suthar
Production with microbes
Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and
maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage
copy 2009 Maulik P Suthar
Steps in Industrial fermentation
Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)
(raw material)
copy 2009 Maulik P Suthar
Industrial organism
copy 2009 Maulik P Suthar
AN INDUSTRIAL MICROORGANISM MUST
1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the
substratendash COMMODITY chemicals are inexpensive chemicals produced in
bulk including ethanol citric acid and many others - DEE antibiotics)
3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable
4 Be non-pathogenic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Microbial products
bull Primary metabolites Made during the organismrsquos growth phase Essential to an organismrsquos metabolism and can be intermediate metabolites or end
bull Secondary metabolites Not essential to cell function or growth and are usually made late in the organismrsquos growth cycle Usually derived from primary metabolites or intermediates of primary metabolites Most likely give the organism an advantage over competitors
Primary Secondary
copy 2009 Maulik P Suthar
Methods for screening
bull Microbial bull Genotypicbull Phenotypic bull Chemical
copy 2009 Maulik P Suthar
Strain improvement
bull Stable production strain with a high titre and ideal production profile which makes a simple purification process possible To obtain such strains we use a combination of traditional methods such as random mutagenisation and genetic engineering
bull Mutagenisation and selection very effectively increase the titres of desired compounds Gene technology is used to obtain an ideal production profile eg to inactivate genes leading to side products and to improve the self-resistance of the production strain which increases both stability and titre
copy 2009 Maulik P Suthar
What are Antibiotics
bull Antibiotics = ldquoagainst liferdquobull Antibiotics = molecules that stop microbes both bacteria and fungi
from growing or kill them outrightbull Antibiotics can be either natural products or man-made synthetic
chemicalsbull Most of the antibiotics identified over the past 60 years have been
natural products produced by one micro-organism within a particular environment to affect neighbouring microbes
bull Can cause microbe death or regulate the growth of the neighbouring microbes
bull These antibiotic agents are produced by both bacteria and fungi
copy 2009 Maulik P Suthar
Antibiotic Production
bull Antibiotics are produced industrially by a process of fermentation where the source microorganism is grown in large amounts
bull As the antibiotics are produced by the microorganisms they have to be removed to ensure they do not effect the bacterial population
bull Certain bacterial species are selectively mutated to produce the maximum amount of the antibiotic agent
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
Mutations and Genetic Instability
bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts
copy 2009 Maulik P Suthar
Induced Mutations
UV radiation
Chemical Mutations
1 4-Nitroquinoline-1-oxide
2 Hydroxylamine
3 Methyl Methanesulfonate
4 Ethyl Methanesulfonate
5 N-methyl-N-nitro-N-nytrosoguanidine
copy 2009 Maulik P Suthar
Mutagenesis and Strain Development
Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques
Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today
1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity
copy 2009 Maulik P Suthar
Plate Based Techniques
bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic
diffused into the surrounding agar
copy 2009 Maulik P Suthar
Antibiotic Production
bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value
Why screening bull In order to identify the useful antibiotics a process of screening is
often employed bull Using this method isolates of a large number of micro-organisms
are cultured and then tested for production of diffusible products which inhibit the growth of test organisms
copy 2009 Maulik P Suthar
Production with microbes
Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and
maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage
copy 2009 Maulik P Suthar
Steps in Industrial fermentation
Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)
(raw material)
copy 2009 Maulik P Suthar
Industrial organism
copy 2009 Maulik P Suthar
AN INDUSTRIAL MICROORGANISM MUST
1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the
substratendash COMMODITY chemicals are inexpensive chemicals produced in
bulk including ethanol citric acid and many others - DEE antibiotics)
3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable
4 Be non-pathogenic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Methods for screening
bull Microbial bull Genotypicbull Phenotypic bull Chemical
copy 2009 Maulik P Suthar
Strain improvement
bull Stable production strain with a high titre and ideal production profile which makes a simple purification process possible To obtain such strains we use a combination of traditional methods such as random mutagenisation and genetic engineering
bull Mutagenisation and selection very effectively increase the titres of desired compounds Gene technology is used to obtain an ideal production profile eg to inactivate genes leading to side products and to improve the self-resistance of the production strain which increases both stability and titre
copy 2009 Maulik P Suthar
What are Antibiotics
bull Antibiotics = ldquoagainst liferdquobull Antibiotics = molecules that stop microbes both bacteria and fungi
from growing or kill them outrightbull Antibiotics can be either natural products or man-made synthetic
chemicalsbull Most of the antibiotics identified over the past 60 years have been
natural products produced by one micro-organism within a particular environment to affect neighbouring microbes
bull Can cause microbe death or regulate the growth of the neighbouring microbes
bull These antibiotic agents are produced by both bacteria and fungi
copy 2009 Maulik P Suthar
Antibiotic Production
bull Antibiotics are produced industrially by a process of fermentation where the source microorganism is grown in large amounts
bull As the antibiotics are produced by the microorganisms they have to be removed to ensure they do not effect the bacterial population
bull Certain bacterial species are selectively mutated to produce the maximum amount of the antibiotic agent
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
Mutations and Genetic Instability
bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts
copy 2009 Maulik P Suthar
Induced Mutations
UV radiation
Chemical Mutations
1 4-Nitroquinoline-1-oxide
2 Hydroxylamine
3 Methyl Methanesulfonate
4 Ethyl Methanesulfonate
5 N-methyl-N-nitro-N-nytrosoguanidine
copy 2009 Maulik P Suthar
Mutagenesis and Strain Development
Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques
Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today
1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity
copy 2009 Maulik P Suthar
Plate Based Techniques
bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic
diffused into the surrounding agar
copy 2009 Maulik P Suthar
Antibiotic Production
bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value
Why screening bull In order to identify the useful antibiotics a process of screening is
often employed bull Using this method isolates of a large number of micro-organisms
are cultured and then tested for production of diffusible products which inhibit the growth of test organisms
copy 2009 Maulik P Suthar
Production with microbes
Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and
maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage
copy 2009 Maulik P Suthar
Steps in Industrial fermentation
Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)
(raw material)
copy 2009 Maulik P Suthar
Industrial organism
copy 2009 Maulik P Suthar
AN INDUSTRIAL MICROORGANISM MUST
1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the
substratendash COMMODITY chemicals are inexpensive chemicals produced in
bulk including ethanol citric acid and many others - DEE antibiotics)
3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable
4 Be non-pathogenic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Strain improvement
bull Stable production strain with a high titre and ideal production profile which makes a simple purification process possible To obtain such strains we use a combination of traditional methods such as random mutagenisation and genetic engineering
bull Mutagenisation and selection very effectively increase the titres of desired compounds Gene technology is used to obtain an ideal production profile eg to inactivate genes leading to side products and to improve the self-resistance of the production strain which increases both stability and titre
copy 2009 Maulik P Suthar
What are Antibiotics
bull Antibiotics = ldquoagainst liferdquobull Antibiotics = molecules that stop microbes both bacteria and fungi
from growing or kill them outrightbull Antibiotics can be either natural products or man-made synthetic
chemicalsbull Most of the antibiotics identified over the past 60 years have been
natural products produced by one micro-organism within a particular environment to affect neighbouring microbes
bull Can cause microbe death or regulate the growth of the neighbouring microbes
bull These antibiotic agents are produced by both bacteria and fungi
copy 2009 Maulik P Suthar
Antibiotic Production
bull Antibiotics are produced industrially by a process of fermentation where the source microorganism is grown in large amounts
bull As the antibiotics are produced by the microorganisms they have to be removed to ensure they do not effect the bacterial population
bull Certain bacterial species are selectively mutated to produce the maximum amount of the antibiotic agent
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
Mutations and Genetic Instability
bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts
copy 2009 Maulik P Suthar
Induced Mutations
UV radiation
Chemical Mutations
1 4-Nitroquinoline-1-oxide
2 Hydroxylamine
3 Methyl Methanesulfonate
4 Ethyl Methanesulfonate
5 N-methyl-N-nitro-N-nytrosoguanidine
copy 2009 Maulik P Suthar
Mutagenesis and Strain Development
Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques
Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today
1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity
copy 2009 Maulik P Suthar
Plate Based Techniques
bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic
diffused into the surrounding agar
copy 2009 Maulik P Suthar
Antibiotic Production
bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value
Why screening bull In order to identify the useful antibiotics a process of screening is
often employed bull Using this method isolates of a large number of micro-organisms
are cultured and then tested for production of diffusible products which inhibit the growth of test organisms
copy 2009 Maulik P Suthar
Production with microbes
Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and
maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage
copy 2009 Maulik P Suthar
Steps in Industrial fermentation
Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)
(raw material)
copy 2009 Maulik P Suthar
Industrial organism
copy 2009 Maulik P Suthar
AN INDUSTRIAL MICROORGANISM MUST
1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the
substratendash COMMODITY chemicals are inexpensive chemicals produced in
bulk including ethanol citric acid and many others - DEE antibiotics)
3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable
4 Be non-pathogenic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
What are Antibiotics
bull Antibiotics = ldquoagainst liferdquobull Antibiotics = molecules that stop microbes both bacteria and fungi
from growing or kill them outrightbull Antibiotics can be either natural products or man-made synthetic
chemicalsbull Most of the antibiotics identified over the past 60 years have been
natural products produced by one micro-organism within a particular environment to affect neighbouring microbes
bull Can cause microbe death or regulate the growth of the neighbouring microbes
bull These antibiotic agents are produced by both bacteria and fungi
copy 2009 Maulik P Suthar
Antibiotic Production
bull Antibiotics are produced industrially by a process of fermentation where the source microorganism is grown in large amounts
bull As the antibiotics are produced by the microorganisms they have to be removed to ensure they do not effect the bacterial population
bull Certain bacterial species are selectively mutated to produce the maximum amount of the antibiotic agent
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
Mutations and Genetic Instability
bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts
copy 2009 Maulik P Suthar
Induced Mutations
UV radiation
Chemical Mutations
1 4-Nitroquinoline-1-oxide
2 Hydroxylamine
3 Methyl Methanesulfonate
4 Ethyl Methanesulfonate
5 N-methyl-N-nitro-N-nytrosoguanidine
copy 2009 Maulik P Suthar
Mutagenesis and Strain Development
Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques
Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today
1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity
copy 2009 Maulik P Suthar
Plate Based Techniques
bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic
diffused into the surrounding agar
copy 2009 Maulik P Suthar
Antibiotic Production
bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value
Why screening bull In order to identify the useful antibiotics a process of screening is
often employed bull Using this method isolates of a large number of micro-organisms
are cultured and then tested for production of diffusible products which inhibit the growth of test organisms
copy 2009 Maulik P Suthar
Production with microbes
Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and
maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage
copy 2009 Maulik P Suthar
Steps in Industrial fermentation
Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)
(raw material)
copy 2009 Maulik P Suthar
Industrial organism
copy 2009 Maulik P Suthar
AN INDUSTRIAL MICROORGANISM MUST
1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the
substratendash COMMODITY chemicals are inexpensive chemicals produced in
bulk including ethanol citric acid and many others - DEE antibiotics)
3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable
4 Be non-pathogenic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Antibiotic Production
bull Antibiotics are produced industrially by a process of fermentation where the source microorganism is grown in large amounts
bull As the antibiotics are produced by the microorganisms they have to be removed to ensure they do not effect the bacterial population
bull Certain bacterial species are selectively mutated to produce the maximum amount of the antibiotic agent
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
Mutations and Genetic Instability
bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts
copy 2009 Maulik P Suthar
Induced Mutations
UV radiation
Chemical Mutations
1 4-Nitroquinoline-1-oxide
2 Hydroxylamine
3 Methyl Methanesulfonate
4 Ethyl Methanesulfonate
5 N-methyl-N-nitro-N-nytrosoguanidine
copy 2009 Maulik P Suthar
Mutagenesis and Strain Development
Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques
Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today
1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity
copy 2009 Maulik P Suthar
Plate Based Techniques
bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic
diffused into the surrounding agar
copy 2009 Maulik P Suthar
Antibiotic Production
bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value
Why screening bull In order to identify the useful antibiotics a process of screening is
often employed bull Using this method isolates of a large number of micro-organisms
are cultured and then tested for production of diffusible products which inhibit the growth of test organisms
copy 2009 Maulik P Suthar
Production with microbes
Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and
maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage
copy 2009 Maulik P Suthar
Steps in Industrial fermentation
Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)
(raw material)
copy 2009 Maulik P Suthar
Industrial organism
copy 2009 Maulik P Suthar
AN INDUSTRIAL MICROORGANISM MUST
1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the
substratendash COMMODITY chemicals are inexpensive chemicals produced in
bulk including ethanol citric acid and many others - DEE antibiotics)
3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable
4 Be non-pathogenic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
Mutations and Genetic Instability
bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts
copy 2009 Maulik P Suthar
Induced Mutations
UV radiation
Chemical Mutations
1 4-Nitroquinoline-1-oxide
2 Hydroxylamine
3 Methyl Methanesulfonate
4 Ethyl Methanesulfonate
5 N-methyl-N-nitro-N-nytrosoguanidine
copy 2009 Maulik P Suthar
Mutagenesis and Strain Development
Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques
Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today
1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity
copy 2009 Maulik P Suthar
Plate Based Techniques
bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic
diffused into the surrounding agar
copy 2009 Maulik P Suthar
Antibiotic Production
bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value
Why screening bull In order to identify the useful antibiotics a process of screening is
often employed bull Using this method isolates of a large number of micro-organisms
are cultured and then tested for production of diffusible products which inhibit the growth of test organisms
copy 2009 Maulik P Suthar
Production with microbes
Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and
maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage
copy 2009 Maulik P Suthar
Steps in Industrial fermentation
Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)
(raw material)
copy 2009 Maulik P Suthar
Industrial organism
copy 2009 Maulik P Suthar
AN INDUSTRIAL MICROORGANISM MUST
1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the
substratendash COMMODITY chemicals are inexpensive chemicals produced in
bulk including ethanol citric acid and many others - DEE antibiotics)
3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable
4 Be non-pathogenic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
Mutations and Genetic Instability
bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts
copy 2009 Maulik P Suthar
Induced Mutations
UV radiation
Chemical Mutations
1 4-Nitroquinoline-1-oxide
2 Hydroxylamine
3 Methyl Methanesulfonate
4 Ethyl Methanesulfonate
5 N-methyl-N-nitro-N-nytrosoguanidine
copy 2009 Maulik P Suthar
Mutagenesis and Strain Development
Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques
Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today
1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity
copy 2009 Maulik P Suthar
Plate Based Techniques
bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic
diffused into the surrounding agar
copy 2009 Maulik P Suthar
Antibiotic Production
bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value
Why screening bull In order to identify the useful antibiotics a process of screening is
often employed bull Using this method isolates of a large number of micro-organisms
are cultured and then tested for production of diffusible products which inhibit the growth of test organisms
copy 2009 Maulik P Suthar
Production with microbes
Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and
maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage
copy 2009 Maulik P Suthar
Steps in Industrial fermentation
Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)
(raw material)
copy 2009 Maulik P Suthar
Industrial organism
copy 2009 Maulik P Suthar
AN INDUSTRIAL MICROORGANISM MUST
1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the
substratendash COMMODITY chemicals are inexpensive chemicals produced in
bulk including ethanol citric acid and many others - DEE antibiotics)
3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable
4 Be non-pathogenic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
copy 2009 Maulik P Suthar
Mutations and Genetic Instability
bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts
copy 2009 Maulik P Suthar
Induced Mutations
UV radiation
Chemical Mutations
1 4-Nitroquinoline-1-oxide
2 Hydroxylamine
3 Methyl Methanesulfonate
4 Ethyl Methanesulfonate
5 N-methyl-N-nitro-N-nytrosoguanidine
copy 2009 Maulik P Suthar
Mutagenesis and Strain Development
Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques
Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today
1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity
copy 2009 Maulik P Suthar
Plate Based Techniques
bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic
diffused into the surrounding agar
copy 2009 Maulik P Suthar
Antibiotic Production
bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value
Why screening bull In order to identify the useful antibiotics a process of screening is
often employed bull Using this method isolates of a large number of micro-organisms
are cultured and then tested for production of diffusible products which inhibit the growth of test organisms
copy 2009 Maulik P Suthar
Production with microbes
Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and
maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage
copy 2009 Maulik P Suthar
Steps in Industrial fermentation
Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)
(raw material)
copy 2009 Maulik P Suthar
Industrial organism
copy 2009 Maulik P Suthar
AN INDUSTRIAL MICROORGANISM MUST
1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the
substratendash COMMODITY chemicals are inexpensive chemicals produced in
bulk including ethanol citric acid and many others - DEE antibiotics)
3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable
4 Be non-pathogenic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Mutations and Genetic Instability
bull Spontaneous mutations include a variety of molecular insults including deletions duplications transpositions insertions base pair substitutions and reading frame shifts
copy 2009 Maulik P Suthar
Induced Mutations
UV radiation
Chemical Mutations
1 4-Nitroquinoline-1-oxide
2 Hydroxylamine
3 Methyl Methanesulfonate
4 Ethyl Methanesulfonate
5 N-methyl-N-nitro-N-nytrosoguanidine
copy 2009 Maulik P Suthar
Mutagenesis and Strain Development
Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques
Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today
1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity
copy 2009 Maulik P Suthar
Plate Based Techniques
bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic
diffused into the surrounding agar
copy 2009 Maulik P Suthar
Antibiotic Production
bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value
Why screening bull In order to identify the useful antibiotics a process of screening is
often employed bull Using this method isolates of a large number of micro-organisms
are cultured and then tested for production of diffusible products which inhibit the growth of test organisms
copy 2009 Maulik P Suthar
Production with microbes
Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and
maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage
copy 2009 Maulik P Suthar
Steps in Industrial fermentation
Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)
(raw material)
copy 2009 Maulik P Suthar
Industrial organism
copy 2009 Maulik P Suthar
AN INDUSTRIAL MICROORGANISM MUST
1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the
substratendash COMMODITY chemicals are inexpensive chemicals produced in
bulk including ethanol citric acid and many others - DEE antibiotics)
3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable
4 Be non-pathogenic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Induced Mutations
UV radiation
Chemical Mutations
1 4-Nitroquinoline-1-oxide
2 Hydroxylamine
3 Methyl Methanesulfonate
4 Ethyl Methanesulfonate
5 N-methyl-N-nitro-N-nytrosoguanidine
copy 2009 Maulik P Suthar
Mutagenesis and Strain Development
Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques
Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today
1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity
copy 2009 Maulik P Suthar
Plate Based Techniques
bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic
diffused into the surrounding agar
copy 2009 Maulik P Suthar
Antibiotic Production
bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value
Why screening bull In order to identify the useful antibiotics a process of screening is
often employed bull Using this method isolates of a large number of micro-organisms
are cultured and then tested for production of diffusible products which inhibit the growth of test organisms
copy 2009 Maulik P Suthar
Production with microbes
Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and
maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage
copy 2009 Maulik P Suthar
Steps in Industrial fermentation
Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)
(raw material)
copy 2009 Maulik P Suthar
Industrial organism
copy 2009 Maulik P Suthar
AN INDUSTRIAL MICROORGANISM MUST
1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the
substratendash COMMODITY chemicals are inexpensive chemicals produced in
bulk including ethanol citric acid and many others - DEE antibiotics)
3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable
4 Be non-pathogenic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Mutagenesis and Strain Development
Mutation induction in Streptomyces is a complex process Not all mutagens are capable of inducing a high level of mutation in all streptomycetes1 Treat spore suspension with chemical or UV mutagen2 Isolate improved mutants using screening techniques
Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today
1 Treat with mutagen2 Plate out on growth medium at low density3 Use agar plugs to isolate single colonies4 Innoculate liquid cultures with test colony5 Screen for increased productivity
copy 2009 Maulik P Suthar
Plate Based Techniques
bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic
diffused into the surrounding agar
copy 2009 Maulik P Suthar
Antibiotic Production
bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value
Why screening bull In order to identify the useful antibiotics a process of screening is
often employed bull Using this method isolates of a large number of micro-organisms
are cultured and then tested for production of diffusible products which inhibit the growth of test organisms
copy 2009 Maulik P Suthar
Production with microbes
Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and
maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage
copy 2009 Maulik P Suthar
Steps in Industrial fermentation
Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)
(raw material)
copy 2009 Maulik P Suthar
Industrial organism
copy 2009 Maulik P Suthar
AN INDUSTRIAL MICROORGANISM MUST
1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the
substratendash COMMODITY chemicals are inexpensive chemicals produced in
bulk including ethanol citric acid and many others - DEE antibiotics)
3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable
4 Be non-pathogenic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Plate Based Techniques
bull As before treat spore suspension and plate at low densitybull Culture the spores into colonies on the platebull Overlay the colonies with membrane innoculated with test organismbull Zones of exclusion are measured using image analysisbull Largest zone of exclusion highest concentration of antibiotic
diffused into the surrounding agar
copy 2009 Maulik P Suthar
Antibiotic Production
bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value
Why screening bull In order to identify the useful antibiotics a process of screening is
often employed bull Using this method isolates of a large number of micro-organisms
are cultured and then tested for production of diffusible products which inhibit the growth of test organisms
copy 2009 Maulik P Suthar
Production with microbes
Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and
maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage
copy 2009 Maulik P Suthar
Steps in Industrial fermentation
Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)
(raw material)
copy 2009 Maulik P Suthar
Industrial organism
copy 2009 Maulik P Suthar
AN INDUSTRIAL MICROORGANISM MUST
1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the
substratendash COMMODITY chemicals are inexpensive chemicals produced in
bulk including ethanol citric acid and many others - DEE antibiotics)
3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable
4 Be non-pathogenic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Antibiotic Production
bull Despite the wide variety of known antibiotics less than 1 of antimicrobial agents have any medical or commercial value
Why screening bull In order to identify the useful antibiotics a process of screening is
often employed bull Using this method isolates of a large number of micro-organisms
are cultured and then tested for production of diffusible products which inhibit the growth of test organisms
copy 2009 Maulik P Suthar
Production with microbes
Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and
maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage
copy 2009 Maulik P Suthar
Steps in Industrial fermentation
Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)
(raw material)
copy 2009 Maulik P Suthar
Industrial organism
copy 2009 Maulik P Suthar
AN INDUSTRIAL MICROORGANISM MUST
1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the
substratendash COMMODITY chemicals are inexpensive chemicals produced in
bulk including ethanol citric acid and many others - DEE antibiotics)
3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable
4 Be non-pathogenic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Production with microbes
Fermentation bull Ability of microbes to produce lsquousefulrsquo productbull Usually a naturally produce substances for growth and
maintenancebull Metabolitesbull Primary metabolite ndash produce during growthbull Secondary metabolite ndash produce after growth stage
copy 2009 Maulik P Suthar
Steps in Industrial fermentation
Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)
(raw material)
copy 2009 Maulik P Suthar
Industrial organism
copy 2009 Maulik P Suthar
AN INDUSTRIAL MICROORGANISM MUST
1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the
substratendash COMMODITY chemicals are inexpensive chemicals produced in
bulk including ethanol citric acid and many others - DEE antibiotics)
3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable
4 Be non-pathogenic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Steps in Industrial fermentation
Isolation of microbes that produce product of interest bull Screen for the best producing strain naturally or mutationbull Optimize production condition (growth ndash basically) in labbull Scale up from lab scale (up to 10 L) to industrial scale (gt10000 L)
(raw material)
copy 2009 Maulik P Suthar
Industrial organism
copy 2009 Maulik P Suthar
AN INDUSTRIAL MICROORGANISM MUST
1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the
substratendash COMMODITY chemicals are inexpensive chemicals produced in
bulk including ethanol citric acid and many others - DEE antibiotics)
3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable
4 Be non-pathogenic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Industrial organism
copy 2009 Maulik P Suthar
AN INDUSTRIAL MICROORGANISM MUST
1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the
substratendash COMMODITY chemicals are inexpensive chemicals produced in
bulk including ethanol citric acid and many others - DEE antibiotics)
3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable
4 Be non-pathogenic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
AN INDUSTRIAL MICROORGANISM MUST
1 Produce the product of interest in high yield2 Grow rapidly on inexpensive culture media available in bulk quantity
(corn steep liquor whey) [ALL NON-animal cf Mad Cow Disease ndash BSE]ndash A major production cost of commodity chemicals is the
substratendash COMMODITY chemicals are inexpensive chemicals produced in
bulk including ethanol citric acid and many others - DEE antibiotics)
3 Be amenable to genetic manipulation ndash mutation genetically engineered (NYT Feb) - yet stable
4 Be non-pathogenic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Industrial micro-organisms
bull Often fungi or streptomycetes (bacteria)bull Ideally produce spores or some other reproductive cell form that can
be easily inoculated into large fermentersbull Capable of growth amp product formation in large scale culturebull Grow rapidly amp produce product in relatively short periodbull Organisms can be manipulated in large-scale culture to produce one
or more products in high concentrationbull Organisms are often genetically altered by mutation or recombinationbull Industrial strains can be very different from ldquowild typerdquo of the organism
found in naturebull Able to grow in inexpensive liquid culture media ndash waste from other
industries- egndash Corn steep liquor from corn milling (rich in N amp growth factors)ndash Whey from cheese production (rich in lactose amp minerals)
bull Should not be pathogenic (humans animals plants)bull Amenable to genetic manipulation
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Antibiotic producing organisms
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Level of antimicrobial activity
bull Effectiveness expressed in two ways
1 Minimal Inhibitory Concentration (MIC) lowest concentration of drug that inhibits growth of pathogen
- Dilution of series of antimicrobial compounds made
- Lowest conc That inhibits is MIC
2 Minimal Lethal Concentration (MLC) lowest concentration of drug that kills pathogen
- Subculturing into fresh medium can reveal MLC
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Antimicrobial tests
bull Dilution Susceptibility Tests involves inoculating media containing different concentrations of drug
bull Disk Diffusion Tests disks impregnated with specific drugs are placed on agar plates inoculated with test microbe 1048708 drug diffuses from disk into agar establishing concentration gradient observe clear zones (no growth) around disksndash Standardized bacterial culture on spread on agar ndash Filter paper disk impregnated with antimicrobial compound is placed on
agarndash Compound diffuses into agar creating a conc Gradient ndash Diameter of zone of Inhibition reflects the sensitivity of organism to
compound
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
The Etest- strip test
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
How do the microbes produce theseproducts
bull Genetically modified microorganismsbull Clone genes that encode protein of interest into vectors and express
this genes in microbes used in productionbull Able to produce high amount of compounds than in original
organisms with ease of manipulationbull Microbes able to utilize waste from other process as a raw material
for fermentation
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Search for new antibiotics
Two approaches
1 Bioprospecting
2 Use of molecular microbiology computational chemistry
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Bioprospecting
Investigating naturally occurring compounds for antimicrobial activity
ndash Soil organisms
ndash streptomycetes actinomycetes fungi
ndash Marine actinomycetes
ndash Plants
ndash Corals and other marine organisms
ndash Products from insects and animals eg frog skin secretions
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Bioprospecting - Step 1
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Bioprospecting - step 2
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Molecular approaches
bull Molecular approaches ndash enables target identification and expression rarr facilitates in-vitro screening amp identification of lead molecules
bull Limited success with antimicrobials ndash many leads unable to cross the bacterial cell wallmembrane eg t-RNA synthetase inhibitors FabH carbapenamase FabH
bull More successful examples ndash benzimidazole amp indazole gyrase inhibitors
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
DNA microarrays
bull High-density DNA microarrays allow researchers to monitor the relative expression levels of thousands of genes (an entire microbial genome)
bull Enables studies of coordinated gene expression that occurs under various microbial growth conditions
bull Expose organism to various sub-lethal concentrations of antibioticsbull Enables identification of genes that are more active or less active in
the presence of antibioticsbull Could provide targets for further study ndasheg mechanism of action
(MOA) or identify targets with unknown MOA
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
The Design Bicycle
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Novel approaches
bull Virtual screeningndash Enzyme purification crystallization and 3-D structural
determination of a drug target (X-ray crystallography NMR)ndash Once target site identified ndash molecules with potential to target can
be identified using computer modellingbull Approach only works for targets amenable to 3-D structure
determination ndash many targets are too complexndash Various known or unknown structures docked to target site and
those with best fit are selected for ldquowet screeningrdquondash Rational approach has been used for lead optimization for a
number of targets eg gyrase inhibitors HIV protease ndash not many significant leads for antibacterial targets
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Novel approaches
2 Genome-wide antibacterial targets - sequencing of bacterial genomesndash Genomes of around 80 bacteria now completely sequenced - gt 100 others underway Eg S
aureusbull Using microarray methods - estimated that genome contained 265-359 essential
genesbull 60 broadly conserved in clinically relevant bacteriabull Currently marketed drugs already target about 15 of the essential gene productsbull Identification of targets
ndash Targets whose genes are essential for in-vitro growthbull All currently marketed antibiotics inhibit or kill bacteria grown in vitro
ndash Targets that only expressed in vivo or are essential for growth and survival in the infected host eg P pilus gene in UTI E coli
bull Biofilm production by Pseudomonas aeruginosabull Bioinformatic analysis of genomic data can assistndash Identify genes conserved in multiple bacterial speciesbull Determining if a gene is essential
ndash Random mutagenesisndash Targeted mutagenesis
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Identification of targets
bull Target gene then cloned and sequencedbull Corresponding protein expressed in a standard expression system
(eg Pichia pastoris Baculovirus or E coli)bull Target protein is purifiedbull Biochemical assay developed for screening large number of diverse
low molecular wt compounds to identify target inhibitors (ldquohitsrdquo)bull ldquoHitsrdquo characterized in terms of potency mechanism of inhibition
and enzyme spectrum and selectivity
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Genomics and target basedidentification of new antimicrobials
bull Once whole cell activity is established need to check if the antimicrobial activity is linked to the intended target or via an unintended mechanismndash Use defined mechanism of action (MOA) assaysndash These are decisive in guiding medicinal chemistry efforts with
respect to optimizing potency spectrum and selectivity
bull As lead compound series are identified and optimized DMPK (drug metabolism and pharmacokinetic) studies are undertaken and animal models of infection are applied
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 1 ndash primary screen
Elementary tests to determine activity in-vitro against small number of key organisms
bull Stage 2 - secondary screen
Selected compounds tested more extensively ndash aim to determine if compound could be useful in clinical medicine
bull Stage 3 ndash toxicity testing
Compounds thoroughly tested in animals
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Search for new antibiotics
bull Stage 4 ndash human pharmacokinetics
Measurement of distribution of the compound in the human body
Potential dosage regimes and routes of administrationbull Stage 5 clinical trials
Effectiveness amp acceptability of the compound tested in patients
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Demonstrable activity against target pathogen(s)bull Breadth of antibacterial spectrum amp degree of activity of the
compoundbull Bacteriostatic or bactericidalbull Any cross-resistance with existing antimicrobialsbull How readily resistance emerges in important pathogensbull How resistant it is to destruction by bacterial enzymes
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Parameters which determine theusefulness of an antibiotic
bull Stability to degradation by animal tissuesbull Extent to which it is bound to serum proteinsbull Pharmacokinetics in experimental animals following administration
by various routesbull Ability to protect animals from experimental infectionsbull Freedom from toxic effects in animalsbull Human pharmacokinetics in volunteers ndash after administration via
various routesbull Side effects in volunteers ndash pain on injection nausea headache or
other symptoms not predicted by toxicity tests in animals
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Antibiotic Targets
bull The major classes of antibiotics affect 1 of 3 targets in bacteria cells(1) Cell wall biosynthesis
ndash penicillinsndash cephalosporinsndash vancomycin(non-ribosomal peptide)
(2) Protein synthesisndash erythromycin (macrolidepolyketides)ndash tetracycline (aromatic polyketides)ndash streptomycin kanamycin(aminoglycosides)
(3) DNA replicationndash quinolones(Cipro)
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Antibiotic Targets
Antibiotics work by exploiting biochemical differences between our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis-block synthesis of peptidoglycan the covalently cross-linked
peptideglycannetwork which imparts osmotic resistanceto cell(2) Protein synthesis
-target 23S rRNA+ associated proteins in peptidyltransferase center of bacterial ribosome
-stop elongation of growing peptide chains(3) DNA replication
-inhibit gyrase essential enzyme that uncoilsintertwined circles of DNA after replication of the circular bacterial chromosome
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic
copy 2009 Maulik P Suthar
Questions
bull Importance of industrial microorganismsbull Properties of useful industrial organismsbull Differentiate primary and secondary metabolitesbull Importance of control of microbial growth processesbull Outline the key steps in the production of penicillin and
semisynthetic penicillinsbull Outline the steps in the production of tetracyclinesbull Describe the key approaches in the search for new antibioticsbull Describe the steps involved in isolation of new antibiotics from soil
organismsbull Describe the importance of molecular advances in the search for
new antibioticsbull Outline the steps in the development of a new antibioticbull Describe the parameters that determine the usefulness of a new
antibiotic