Antibiotics are compounds of natural, semi-synthetic, or synthetic origin which inhibit growth of microorganisms without significant toxicity to the

human or animal host.

The key concept of antibiotic therapy is selectivity. The independent evolutionary history of bacterial (prokaryotic) and host

(eukaryotic) cells led to a significant difference in cell organization, biochemical pathways and structures of proteins and RNA.

These differences form the basis for drug selectivity.

Definition of antibiotics

1. The target of an antibiotic can be present only in bacteria but not in the eukaryotic host.

2. The target in bacteria is different from the homologous target in the eukaryotic host.

Bases of antibiotic selectivity

Modern genomics provide a great tool for identifying targets of new selective antibiotics

Selectivity of antibiotics is not ‘natural’

Natural antibiotics are weapons that bacteria or fungi use to compete with other microorganisms.

Selectivity is not a ‘natural’ feature of antibiotics.

Most of clinically-useful antibiotics are fortuitously selective antibacterials.

Many antibiotics are omni-potent and inhibit growth of a wide variety of organisms. Such antibiotics can be developed into selective drugs

through modification of their chemical structures.

Antibiotics with a bactericidal mode of action are preferred, especially for treatment of immunocompromised patients. The mode (static vs. cidal) of

antibiotic action may differ for different pathogens and may depend on the drug concentration.

The basis of bactericidal versus bacteriostatic effects is poorly understood but maybe related to the accumulation of reactive oxygen radicals in the bacterial

cells upon treatment with bactericidal drugs.

Antibiotics are classified as bacteriostatic or bactericidal.

Bacteriostatic drugs make bacteria dormant, but do not kill them.

Most bacterial cells resume growth after removal of the antibiotic

(e.g. chloramphenicol)

Bactericidal drugs kill bacteria (e.g. ciprofloxacin)

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2000

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After the golden era of the 1940s-1950s, the progress in antibiotic discovery has significantly slowed down until the year 2000

Golden era in antibiotic discovery No principally new antibiotics

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There are two major mechanisms by which bacteria can become resistant to antibiotics:

1. Mutation of a “normal” bacterial gene resulting in antibiotic resistance.2. Acquisition of a resistance gene from the environment.

Sensitive bacteriaSensitive bacteria

Resistant bacteriaResistant bacteria Resistant bacteriaResistant bacteria

spontaneous mutationin the bacterial gene

acquisition of a resistance gene(often brought by a genetic vector)

vertical transfer(to the descendants)

horizontal transfer (gene exchange)

vertical transfer(to the descendants)

The appearance and spread of antibiotic resistance calls for new antibiotics. Resistance to the available

antibiotics is a result of Darwinian selection

Modification of the drug (aminoglycoside modifying enzymes)

Modification of the drug target (Erm methylases, target site mutations)

Reducing the drug’s intracellular concentration: drug-specific transporters (mefA), multi-drug transporters (bmr), reduced drug uptake (mutations in porins)

There are three major types of resistance mechanisms:

The number of targets of clinically-useful antibiotics is very limited.

Cell Wall

Biosynthesis

Beta-lactamsGlycopeptides

Bacitracin

Protein synthesis

AminoglycosidesOxazolidinonesTetracyclinesMacrolides

ChloramphenicolLincosamides

Streptogramins

DNA Gyrase Fluoroquinolones

RNA Polymerase Rifampicin

Folate Biosynthesis

Sulfonamides

Membrane

Integrity

Cationic peptidesLipopeptides

Many resistant strains are cross-resistant to different drugs acting upon the same target.

Originally, screening for new antibiotics was based on testing the bacterial or fungal extracts.

A number of drugs developed in the middle of the 20th century still remain among the best.

These include aminoglycosides, cephalosporins, tetracyclines, macrolides, etc.

There is now a renewed interest in screening natural sources (especially, non-traditional, e.g. marine microorganisms)

New antibiotics

New antibiotics: genome-based approaches

Genomics holds a good potential for identifying new drug targets.

New antibiotics: Functional genomics approach

gene

target

lead

lead optimization

drug candidate

preclinical

clinical

HTS enzyme inhibitors

On average: only one lead candidate is developed from 14 high throughput screening experiments!

“easy”

hard

In the functional genomics approach, the enzymatic target is first defined and then inhibitors are searched for in high-throughput screening assays

(HTS).

New antibiotics: Reverse genomics approach

In the reverse genomics approach the lead is first identified in the whole cell assay and then the target is searched for.

lead

lead optimization

drug candidate

preclinical

clinical

target mode of action

HTS growth inhibitors

Most of the bacteria have a rigid cell wall which protects the cell from changes in osmotic pressure. Presence of the cell wall is critical for the survival of

bacterial cell.

The structure and composition of bacterial cell wall is dramatically different from the cell envelope of the

eukaryotic cell. Therefore, enzymes of cell wall biosynthesis are unique to bacteria and presents an

excellent target for antibiotics.

According to the structure of their cell wall, all bacteria are divided into Gram-positive and Gram-

negative according to a staining procedure developed by Christian Gram in 1884.

Cell wall as antibiotic target

Outside of the cytoplasmic membrane of Gram-positive bacteria lies a thick layer of peptidoglycan which determines the rigidity of the cell wall. In Gram-positive bacteria, peptidoglycan accounts for 50% of the cell weight and up to 90% of the weight of the cell wall. Peptidoglycan layer is 20-80 nm thick and

rather unstructured.

Cell wall of Gram-positive bacteria is thick and unstructured

The cell wall of Gram-negative bacteria consists of the cytoplasmic membrane, a thin layer of peptidoglycan, and an outer membrane. The area between the

cytoplasmic membrane and peptidoglycan layer is called the periplasmic space.

Periplasmic space

Cell wall of Gram-negative bacteria is thin and neatly organized

glycan

tetrapeptide

NAG

NAM

pentaglycine bridge

Peptidoglycan consists of polysaccharide chains cross-linked by

peptide ‘bridges’. 35% to 50% of peptides attached to

polysaccharide chains are crosslinked.

Peptidoglycan composition

Peptide tails protrude from the polysaccharide

helically and thus crosslink to different polysaccharide chains. This accounts for the rigidity of the cell wall.

D-AlaL-Ala

racemaseD-Ala-D-Ala synthetase

UDPMurNAc

UDPL-Ala

UDPD-GluL-Lys

UDP

(1)(2)(3)

UDP

pp

UDPGlcNAc

pp

cytoplasm

membrane

periplasmic space

p p

p

(4)

(8)

(9)

(10)transpeptidase

(5)

(6)

phospholipid

pp

(7)

pp

N-acetyl muramic acidN-acetyl glucosamin

pentaglycin

pentapeptide

The precursor monomers of the

peptidoglycan polymer, disaccharide-

pentapeptides, are synthesized in the cell cytoplasm, transported

across cytoplasmic membrane, and then

attached to the growing peptidoglycan polymer.

Peptidoglycan biosynthesis

TPase

TPase

TPase

Crosslinking between glycan strands is catalyzed by the enzyme called transpeptidase (TPase). In the course of the reaction, TPase hydrolyzes peptide bond between two terminal D-Ala residues of the precursor and forms a transient covalent link with the

precursor peptide. The intermediate is then resolved with the formation of a new peptide bond.

The reaction of formation of the covalent intermediate is targeted by -lactam antibiotics.

Transpeptidase

D-Ala D-AlaD-Ala

Development of methods for growing Penicillium notatum and purifying penicillin by Florey and Chain made it into a drug. The deep

fermentation method, the use of corn steep liquor and the discovery of P. chrysogenum by Mary Hunt made the commercial production of

penicillin possible.

Penicillin was discovered by A. Fleming in 1929

Alexander Fleming

Howard Florey

Ernst ChainPhoto: L. Segovia

-lactam antibiotics

N

SRCONH

CH3

CH3

O CO2H

penicillins

N

SNHCO

OCO

2H

CH3

R

cephalosporins

NO CO

2H

SNH

2

H

CH3

H HOH

carbapenems

NO

N

SO3H

R

O

monobactams

The most important class of antibiotics affecting cell wall biosynthesis are -lactams. -lactam group (a four-atom cyclic amide) is the pharmacophore of all

-lactam antibiotics. -lactam rings were unknown before the discovery of penicillin and it took big effort to determine the structure of the drug.

The most important classes of -lactam antibiotics are penicillins, cephalosporins, carbapenems and monobactams.

The mechanism of action of -lactam antibiotics is based on the similarity of structures of the C - N bond in the -lactam ring and the

peptide bond connecting two D-alanine residues of the peptidoglycan precursor. TPase recognizes the -lactam as its substrate and forms a covalent bond with the antibiotic molecule. This adduct is very stable

and because of that TPase is irreversibly inactivated.

NH

C

CH3

C COOH

CH3

NH

C

O

NH C C

H S

R C

O

H

CO N

C

CH

COOH

CH

3

O

Ser

TPase

CH3

D-Ala-D-Ala penicillin

Covalent complex of penicillin with TPase

Mechanism of -lactam action

N

SRCONH

CH3

CH3

O CO2H

Penicillin binding proteins (PBPs)

Several other enzymes of cell wall biosynthesis with a mechanism of action comparable to TPase are also targeted by -lactams.

These proteins can be detected on a gel by their ability to bind penicillin. These proteins are called ‘penicillin binding proteins’ or PBPs.

Penicillins : penicillin G

In penicillins, the -lactam ring is fused to thiazolidine ring. Originally, penicillin was produced in the form of Penicillin G

(benzylpenicillin) by fermenting Penicillium mold in the presence of phenyl acetic acid

N

S

H

N

C

H

3

C

H

3

O

C

O

2

H

O

Good activity, but only against Gram-positive bacteria

Acid- and alkali-labile

Sensitive to the action of inactivating penicillinases

N

SH2N

O

6-APA

Presently, many penicillins are produced semisynthetically starting from 6-aminopenicillanic acid (6-APA) as a precursor.

6-APA can be generated from penicillin G by cleaving off the benzyl moiety of penicillin G.

Various new side chains can be then attached to the penicillin molecule through the amino group of 6-APA

N

SRCONHCH3

OCO2H

New penicillins

Various penicillins differ mainly by the nature of the N6 side chain R

6-APA

N

S

H

N

C

H

3

C

H

3

O

C

O

2

H

O

Benzyl-penicillin

CH3CH3

CH3

CO2H

Penicillin improvements: better acid stability

The amide bond in the β-lactam ring is highly strained and relatively unstable in acidic solutions. The rate of acid hydrolysis depends on the

chemical nature of the side chain. Electron-withdrawing side chains decrease the rate of acid hydrolysis. Because of that, amoxicillin or

cloxacillin are more acid-stable: they can withstand the acidic pH of the stomach and can be used orally.

amide bond

N

SC O NH

C H3

C H3

O C O2H

CH

N H2

OH

Amoxicillin

N

SCONH

CH3

CH3

O CO2H

CH3OH

N

Cl

Cloxacillin

Penicillin improvements: broader spectrum

Penicillins enter the periplasmic space of Gram-negative bacteria through the ‘holes’ in the outer membrane (porins). Hydrophobic side chains (e.g. benzyl

group in penicillin G) interfere with passage through porins. More polar groups, such as -NH2 or -COOH facilitate crossing the outer membrane and increase access of β-lactmas to the periplasmic space of

Gram-negative bacteria.

N

S

CH3

CH3

O CO2H

CH

NH2

ampicillinCONH

N

SCONH

CH3

CH3

O CO2H

CH

NH2

OH

amoxicillin

N

S

CH3

CH3

O CO2H

CH

COOH

carbenicillinCONH

Ampicillin has a very broad spectrum of activity. It can be used orally or parenterally. But it has low bioavailability.

A more lipophilic pro-drug, pivampicillin, has a better oral bioavailability. Pivampicillin is an ester of ampicillin; the ester bond is slowly

hydrolyzed in the blood resulting in the release of the active ampicillin.

N

S

CH3

CH3

O CO2H

CH

NH2

ampicillin

N

SCONHCH3

CH3

O CO2CH2OCOC(CH3)3

CH

NH2

pivampicillin

The pro-drug approach can be used to increase bioavailability of some penicillins

CONH

Penicillin improvements: resistance to -lactamases

The main mechanism of resistance to penicillin is based on the secretion by bacteria of enzymes -lactamases that can hydrolyze amide bond in -lactam ring. The presence of a bulky side chain in the drug may hinder

access of a -lactamase to the amide bond. Therefore, penicillin derivatives containing bulky side chains are fairly resistant to the -lactamase action.

N

SCONH

CH3

CH3

O CO2H

OCH3

OCH3

methicillin

N

SCONH

CH3

CH3

O CO2H

CH3OH

N

Cl

cloxacillin

-lactamase inhibitors

Clavulanic acid

N

O

OCOOH

CH2OH

Sulbactam

N

S

OCOOH

O O

A useful way to overcome -lactamase-based resistance is to administer a -lactam drug in combination with -lactamase inhibitors. Such inhibitors (clavulanic acid,

sulbactam, tazobactam) possess a -lactam ring and generally resemble -lactam antibiotics. They function by binding to the -lactamase enzymes and inactivating the

enzyme without being degraded. Such inhibitors, which look like -lactam antibiotics, have only weak antimicrobial

activity.

Popular combinations are amoxicillin with clavulanate (augmentin) or ampicillin with sulbactam (unasyn).

Other mechanisms of resistance to -lactams

An important mechanism of resistance to -lactams involves mutations in transpeptidases and other penicillin-binding proteins (PBPs) involved in

bacterial cell wall biosynthesis.

Resistance mechanism found in methicillin-resistant Staphylococcus aureus (MRSA) is based on acquisition of a mecA gene which encodes a resistant mutant protein, PBP2’. PBP2’ has a very low affinity for -lactam

antibiotics and can support cell wall biosynthesis even when all other PBPs are covalently inactivated by the drug.

Genetic analysis show that mecA gene has been independently transferred to S. aureus at least 5 times resulting in 5 independent lineages of MRSA

Cephalosporins have been first obtained from a fungus Cephalosporium acremonium. Similar to penicillins, many cephalosporins are produced semi-synthetically either starting from 7-aminocephalosporanic acid (7-

ACA) or by converting relevant penicillins into cephalosporins.

N

SH2N

OCO2H

O CH3

O

7-aminocephalosporanic acid (7-ACA)

2

3

7

The activity of cephalosporins is modulated not only by the nature of substitutions R2 at C7 (as in penicillins) but also by the side chain R1 at C3.

Cephalosporins

Active against Gram-positive cocci and streptococci.

cephalothin cafazolin cephalexin

Cephalosporins are classified by generations

N

SNHC O

O

CO2H

O C H3

O

S S

N N

N

SNHCO

OCO

2H

S CH3

NN

NN

N

SNHCO

OCO

2H

CH3

NH2

I

II

cephaclorcefamandole nofate

N

S

O

CO2H

Cl

NHCO

NH2

N

SNHCO

OCO

2H

CH2S

OCHO

N N

NN

CH3

cefotaxime cefixime

N

SNHC O

O

C O2H

CH2OC OC H

3

N OC H3

N

SNH

2N

SNHCO

O

CO2H

CH2

NOCH2CO

2H

N

SNH

2

Improved activity against some Gram-negatives, for example, H. influenzae.

Better activity for Gram-negatives though, somewhat reduced activity against Gram-positive pathogens.

III

COOH

OH

N

N N

NN

SNHCO

OCO2H

OCH3

S

CH37

Similar to penicillins, cephalosporins can be inactivated by -lactamases.Resistance to -lactamases increases in drugs such as moxalactam which have bulky side chains. The presence of 7--methoxy group

increases moxalactam resistance to -lactamase hydrolysis even further.

Moxalactam

Carbapenems combine chemical features of penicillins and cephalosporins. Prototype carbapenem thienamycin, has been isolated from Streptomyces

cattleva. It exhibits excellent activity against a broad spectrum of Gram-positive and Gram-negative organisms.

thienamycin

Thienamycin penetrates very easily through the outer membrane of Gram-negative bacteria (through porin "holes") . It is resistant to the action of extended spectrum -lactamases (ESBL) which can inactivate penicillins and cephalosporins. In contrast to penicillins and cephalosporins which target only PBPs, carbapenems can target

another enzyme of the cell wall biosynthesis, Ld transferase (LdT) which sometimes can help cell to bypass the need for TPase. Therefore, carbapenems shows

excellent activity against some Gram-positive strains which developed resistance to penicillins and cephalosporins.

Carbapenems

NO CO2H

SNH2

H

H3C

H HOH

In concentrated solutions, the side amino group of thienamycin can react with the amide bond in the -lactam ring of another thienamycin molecule making the drug unstable in concentrated solutions. This problem has been solved in

the thienamycin derivative, imipenem by modifying the side chain. Imipenem was the first parenteral carbapenem.

Imipenem

thienamycin

NO CO2H

SNH2

H

H3C

H HOHimipenem

NH

NHN

O CO2H

SH

H3C

H HOH

doripenemertapenem meropenem

The newer drugs of this class are meropenem, ertapenem and doripenemThe drawback of carbapenems is that they are acid-labile and therefore used only intravenously. In

addition, they are very expensive.

Monobactams

Aztreonam (Azactam)

Monobactams were developed as narrow-spectrum antibiotics specifically targeting aerobic Gram-negative bacteria. Monobactams are particularly

useful for the treatment of individuals allergic to penicillin. Such patients can still be treated with the monobactams, which are sufficiently structurally

different to not induce allergic reaction.

S

O

3

H

N

O

C

H

3

C

H

3

C

O

O

H

N

H

2

N

O

C

H

3

N

O

S

N

Cell wall inhibitors of non--lactam type: Bacitracin

A number of peptide antibiotics affect cell wall biosynthesis.Bacitracin, a polypeptide antibiotic produced by licheniformis group of

Bacillus subtilis, inhibits cell wall synthesis by interfering with dephosphorylation of the lipid carrier that moves the peptidoglycan

precursors across the cytoplasmic membrane. Blocking regeneration of the lipid carrier aborts cell wall synthesis.

Side effect of bacitracin is based on its interference with sterol biosynthesis in mammalian cells which accounts for its certain toxicity in humans.

Therefore, it is used exclusively in topical formulations.

An important class of antibiotics that interfere with synthesis of cell wall are glycopeptides. The most important of them is vancomycin.

Vancomycin is isolated from a bacterium Nocardia orientalis. Vancomycin is a tricyclic glycopeptide with a large molecular weight of 1449 D.

It is active against most Gram-positive bacteria. It is especially important in the treatment of infections due to methicillin- and

cephalosporin-resistant organisms. Vancomycin is bactericidal against most of the susceptible bacteria.

Cell wall inhibitors of non--lactam type: Vancomycin

Vancomycin binds very tightly to the D-Ala - D-Ala residues at the ‘end’ of the peptidoglycan precursor peptide. Because of that, the peptide bond between

two D-Ala residues becomes inaccessible to TPase so that peptidoglycan strands cannot be crosslinked.

Vancomycin

D-Ala-D-Ala vancomycin

Resistance to vancomycin

Vancomycin was kept as a ‘reserve’ antibiotic and was usually prescribed only when other drugs proved to be inactive. However, even in spite of its relatively infrequent usage, resistant strains eventually appeared.

Vancomycin resistant enterococci (VRE) account now for up to 25% hospital strains of enterococci.

First vancomycin resistance appeared in staphylococci in ‘vancomycin intermediate susceptible S. aureus’ - VISA.VISA cells have abnormal peptidoglycan: the cell wall is thicker and less crosslinked. Therefore more D-Ala - D-Ala remain in the cell wall and are exposed. They work as vancomycin trap (sponge). However, because of the abnormal cell wall, VISA strains are sick (high fitness cost) and do not spread very rapidly.

In 2002 the first Vancomycin-resistant S. aureus (VRSA) strain was reported.The Van gene they have acquired replaces D-Ala - D-Ala in the precursor for D-Ala - D-lactate. Since Van genes are active only when cells are exposed to vancomycin, VRSA strains are more fit than VISA.

New glycopeptides

Newer versions of glycopeptide antibiotics include teicoplanin and dalbavancin (ZevenTM). These drugs are similar to vancomycin but have

additional hydrophobic side chains. Teicoplanin and dalbavancin are active against all vancomycin-sensitive

strains, plus against a number of resistant strains and do not induce expression of VanB resistance gene.

Both teicoplanin and dalbavancin are rapidly cidal.Dalbavancin is very tightly bound to serum proteins and is therefore very

stable. Because of that, it can be administered once a week.

dalbavancin