Combinatorial Combinatorial Chemistry and Chemistry and

Drug Discovery LabDrug Discovery Lab

Jasmine Erfe Miramar College/Lab Technician

Ericka Senegar-Mitchell Science in the City/ Director; Junipero Serra High School/Science Educator

Sandra Slivka Southern CA Biotechnology Center @ Miramar College/Director

BackgrounBackgrounddThis lab protocol wasThis lab protocol was

adapted from the originaladapted from the originalwork of Scott Wolkenbergwork of Scott Wolkenbergand Andrew Su of Theand Andrew Su of TheScripps Research InstituteScripps Research Institutein La Jolla, California. Thein La Jolla, California. Theexperiment was originallyexperiment was originallypublished in the June 2001published in the June 2001issue of the Journal ofissue of the Journal ofChemical Education andChemical Education andimplemented in the Sanimplemented in the SanDiego area from May 2002Diego area from May 2002thru 2005.thru 2005.CITATION:Wolkenberg, Scott E.; Su, Andrew I. J Chem.Educ.2001 78 784

OverviewOverviewOverviewOverview• In this lab students will identify a drug In this lab students will identify a drug

that kills bacteria by producing libraries that kills bacteria by producing libraries of compounds based on the A-B model.of compounds based on the A-B model.

• They will test the mixtures for antibiotic They will test the mixtures for antibiotic activity and then isolate the individual activity and then isolate the individual compound(s) which possess antibiotic compound(s) which possess antibiotic properties. properties.

• Students will screen the mixtures by Students will screen the mixtures by utilizing techniques used to conduct utilizing techniques used to conduct Kirby-Bauer and Ouchterlony tests. Kirby-Bauer and Ouchterlony tests.

California California Science Science Content Content

StandardsStandards

California California Science Science Content Content

StandardsStandardsSubject Area Content Standard

Chemistry

2.a. Chemical Bonds – formation of ionic and covalent (peptide) bonds3.g. Stoichiometry – redox reactions, dehydration synthesis (condensation)10.b. Organic Chemistry – bonding characteristics of carbon10.e. Functional Groups – formation of a hydrazone from an aldehyde and a hydrazine, identification and analysis of amine groups

Biology1.a. Membrane Regulation – membrane structure and function1.c. Prokaryotic and Eukaryotic Cells – structure and function1.h. Macromolecules – structure and function

Genetics (Molecular Biology)4.c. Mutations – antibiotic expression4.e. Proteins – structure4.f. Proteins – function/chemical properties

Genetics (Biotechnology)5.c. Biotechnology – production of novel biomedical and agricultural products

Evolution7.a. Natural Selection – phenotype vs. genotype7.d. Genetic Variation – influence of environmental factors on the natural selection of adaptive traits8.a. Natural Selection – selective fitness; differential survival of groups

Physiology10.b. Immune Response – antibody/antigen response10.d. Bacterial Infections – use of antibiotics in treating bacterial infections; use of antibacterial agents to control the growth of bacteria

National National Science Science

Education Education StandardsStandardsCategory Content Standard

Unifying Concepts and ProcessesChange, constancy and measurement – process of generating chemical libraries and screening for effective compounds, combinatorial dataEvidence, models, and explanation – Kirby-Bauer & Ouchterlony TestsForm and function – cells, macromolecules, prokaryotes (E.coli )

Science as InquiryAbilities necessary to do scientific inquiry – production of mixtures (libraries) of compounds using the A-B model; deconvolution (separation) of the mixtures to identify the compound(s) with antibiotic properties

Life ScienceThe Cell – types of cells, cell membrane structure and functionBiological evolution – development of antibiotic resistanceBehavior of organisms – growing bacterial cultures (lag vs. log phase), interpretation of bacterial plates (deconvolution)

Science and TechnologyUnderstandings about science and technology – process and design in combinatorial chemistry; bringing a new pharmaceutical to market

Science in Personal and Social Perspectives

Personal and community health – drug discovery, pharmacology, diagnosis of human disease and course of treatmentNatural and human-induced hazards – aseptic (sterile) technique, safety protocols including Material Safety Data Sheets (MSDS)

History and Nature of ScienceScience as a human endeavor – biomedical research, clinical trials, bioethics of the biotechnology industry

ChemicalsChemicals

ChemicalsChemicalsCHEMICAL VENDOR CATALOG # PRICE*

A1: 2-nitrobenzaldehyde Sigma-Aldrich 772780-50g $116.50

A2: 5-nitro-2-furaldehyde Sigma-Aldrich 170968-25g $102.50

A3: 3-nitrobenzaldehyde Sigma-AldrichN10845-100g

  $17.80

B1: 4-bromophenylhydrazine hydrochloride

Sigma-Aldrich 143219-10g $101.50

B2: 4-cyanophenylhydrazine hydrochloride

Sigma-Aldrich 453471-5g  $42.20

B3: aminoguanidine bicarbonate

Sigma-Aldrich109266-100g

 $15.00

Total: Total: $395.50$395.50

Suggested Suggested MaterialsMaterials

Preparing Overnight Culture of E. coli1.Transfer 10ml of sterile LB broth in a culture tube. 2.To obtain a sample of E.coli, take the inoculating loop and dip into the frozen E. coli stock. Be certain that some of the stock has adhered to the loop.3.Dip the inoculating loop into the culture tube containing the 10ml of LB broth. Stir the broth using the loop to ensure that the E. coli is thoroughly mixed in the broth.4.Cap the tube and incubate the E. coli culture overnight at 37°C with agitation.

Presenter Presenter NotesNotes

Growing E. Coli into Log Phase1.Transfer 25ml of sterile LB broth in a 50ml Erlenmeyer flask.2.Take 1ml of the prepared overnight E. coli culture and transfer the sample into the Erlenmeyer flask containing 25ml of LB broth.3.Cap the Erlenmeyer flask and incubate at 37°C with agitation.

Presenter Presenter NotesNotes

Spectrophotometer 1.Turn on the spectrophotometer and set to O.D. 550.2.Take a 3ml sample of the E. coli culture and transfer into a test tube. Wipe and handle the test tube with Kimwipes and place into the spectrophotometer.3.Note the reading. The culture will reach log phase once the O.D.550 reaches 0.3-0.4.4.If the reading has not reached log phase, repeat steps 2 and 3 using a clean test tube and waiting 30 minutes between each new reading.5.Once the culture has reached log phase, note the total volume of culture that remains in the flask. According to the volume, add glycerol in the amount of 15% of the total volume to the flask and immediately transfer 1 ml aliquots of the log phase culture into cryogenic tubes and freeze at -80ºC.

Presenter NotesPresenter Notes

Prepare stock Prepare stock solutionssolutions

Instructor Instructor NotesNotes

You have been provided 6 uniquely labeled conical tubes, each containing a specific chemical. Add 12 mL of deionized water to each tube and shake vigorously for approximately ten seconds.

If mixtures are not completely If mixtures are not completely dissolving using a hot water bath dissolving using a hot water bath may be effective. may be effective.

Instructor Instructor NotesNotes

Student Student ProtocolProtocol

Combinatorial Combinatorial Chemistry and Chemistry and Drug Discovery Drug Discovery

LabLab

Lab SafetyLab SafetyTips for handling E.coli:

1.Wipe down the lab bench or station with a 10% bleach solution or 70% isopropanol solution at the beginning and end of each laboratory session. 2.When creating mixtures and transferring chemical solutions or liquid bacterial cultures, keep nose and mouth away from the opening of the tube to avoid inhaling any aerosols that may be created.3.All spills should be reported to your instructor and cleaned up immediately according to the Material Safety Data Sheets (MSDS) for each chemical used in the experiment. Be sure to wear proper footwear (closed toe) to prevent injury.4.Dispose of any materials that have come in contact with bacterial cultures (i.e. tubes, pipettes) in special waste containers as provided by your instructor.5.Wash hands with soap and water before leaving the lab.

• This lab protocol was adapted This lab protocol was adapted from the original work of from the original work of Scott Wolkenberg and Scott Wolkenberg and Andrew Su of The Scripps Andrew Su of The Scripps Research Institute in La Jolla, Research Institute in La Jolla, CA. The experiment was CA. The experiment was originally published in the originally published in the June 2001 issue of the Journal June 2001 issue of the Journal of Chemical Education.of Chemical Education.

• Combinatorial ChemistryCombinatorial Chemistry is a is a technique used to synthesize technique used to synthesize a library of compounds and a library of compounds and screen for a desired property. screen for a desired property. Instead of screening one Instead of screening one compound at a time, the compound at a time, the compounds are screened compounds are screened more efficiently in mixtures.more efficiently in mixtures.

CITATION:Wolkenberg, Scott E.; Su, Andrew I. J Chem. Educ. 2001 78

784

OvervieOvervieww

When microorganisms are introduced into fresh culture medium usually no immediate increase in cell number occurs therefore this is referred to as the LAG PHASE.

During the EXPONENTIAL or LOG PHASE bacteria are growing and dividing at the maximal rate given their genetic potential, nature of the medium and conditions under which they are growing.

In the STATIONARY PHASE, the total number of viable bacteria remains constant. This may result from a balance between cell division and cell death or cells may cease to divide while remaining metabolically active.

What limiting factors would cause a microbial population to enter the

stationary phase?

Detrimental environmental conditions such as lack of nutrients and waste buildup lead to the decline, usually logarithmic, in the number of viable cells. This is characteristic of the DEATH PHASE.

Kirby-Bauer Kirby-Bauer TestTest•Disk-diffusion method used

for routine testing in a clinical laboratory in which an isolated microbe is tested for susceptibility to numerous antibiotics.

•The isolated organism is uniformly placed on an agar plate with paper disk of fixed concentrations of antibiotics.

•Growth of the organism and diffusion of the antibiotic occur simultaneously resulting in a circular zone of inhibition if the antibiotic has antibacterial properties.

Measuring Antibiotic Measuring Antibiotic ResistanceResistance

Ouchterlony Ouchterlony TestTest•A double diffusion technique developed by Organ

Ouchterlony more than 40 years ago.

•A technique in which reaction partners, antigen and antibody, are allowed to diffuse to each other in an agar gel in a precipitation reaction. •Classical procedure used to detect the presence of antibodies and determine their specificity by visualization of "lines of identity" or precipitin lines.

Student/Group Lab Set-Student/Group Lab Set-upup

 3 Luria Broth (LB) agar plates

6, 15mL conical tubes containing stock solutions:•A1: 2-nitrobenzaldehyde•A2: 5-nitro-2-furaldehyde•A3: 3-nitrobenzaldehyde•B1: 4-bromophenylhydrazine hydrochloride•B2: 4-cyanophenylhydrazine hydrochloride•B3: aminoguanidine bicarbonate

1 cryotube (orange cap) containing 1.0 mL E. coli 1 cell spreader 15 disposable transfer pipettes or P-1000 micropipette 9 eppendorf tubes  1 plastic straw, wrapped Conical tube rack Sharpie Marker

1 sterile wrapped transfer pipette

Label transfer Label transfer pipettespipettes1. Label 6 of the transfer pipettes A1, A2, A3, B1, B2, B3. These will be used to

prepare your compounds.2. Label 9 transfer pipettes M1, M2, M3, M4, M5, M6, A#B1, A#B2, A#B3. These

will be used to transfer your compounds onto the plates. Each student or group will be assigned mixtures to test for confirmation of antibiotic activity (i.e. # = 1, 2, or 3 for A1B1, A1B2, A1B3; A2B2… ).

Label epitubesLabel epitubes

Create wells in Create wells in plates plates

Label Petri Label Petri dishesdishes

M1 M2

M3

M4 M5

M6

A#B1 A#B2

A#B3

M1 M2

M3

M4 M5

M6

A#B1 A#B2

A#B3

Each student or group will be assigned mixtures to test for confirmation of antibiotic activity. (i.e. # = 1, 2, or 3 for A1B1, A1B2, A1B3; A2B2...).

Carefully invert the tube containing Carefully invert the tube containing 1.0 ml of thawed E. coli several times 1.0 ml of thawed E. coli several times before opening. Spread the E. coli before opening. Spread the E. coli culture evenly on the surface of each culture evenly on the surface of each agar plate.agar plate.

Prepare Prepare mixtures mixtures (libraries)(libraries)

Tube # Add 5 drops Then 5 dropsThen 5 drops

Then 15 drops

M1 B1 B2 B3 A1

M2 B1 B2 B3 A2

M3 B1 B2 B3 A3

M4 A1 A2 A3 B1

M5 A1 A2 A3 B2

M6 A1 A2 A3 B3

1. Make sure that theproper transfer pipette

isused for each solution

toavoid contamination.

2. Add solutions in theorder indicated in the

table.Record your

observations.

NOTE: 15 drops = ~ 750µL 5 drops = ~ 250µL 1 drop = .05 mL = 50 µL

Add compounds to wells and Add compounds to wells and wait approximately 15-20 wait approximately 15-20 minutes for liquid to absorb.minutes for liquid to absorb.

Incubate at 37ºC or store at Incubate at 37ºC or store at room temperature room temperature

overnight.overnight. (48 hours is optimal for room temperature (48 hours is optimal for room temperature

option)option)

1. If no incubator is available: Plates canbe stored overnight atroom temp. with theagar side down.

2. If incubator is available:

Allow compounds toabsorb into agar thenincubate agar side

up.

Chemical Chemical StructuresStructures

A carbonyl group at the end of the

carbon skeleton

indicates that the compound

is an ALDEHYDE

The amino group (-NH2) consists of a

nitrogen atom bonded to two

hydrogen atoms; the

compound is a HYDRAZINE

Wolkenberg, Scott E.; Su, Andrew I. J Chem. Educ. 2001 78 784

Chemical Structures of Chemical Structures of Synthesized Compounds Synthesized Compounds A special type of

covalent bond called a peptide bond forms between the carbon and the nitrogen creating a newly synthesized compound called a HYDRAZONE.

The oxygen from the carbonyl group of the aldehyde leaves to bond with the hydrogen from the amino group of the hydrazine to form water. This represents a condensation reaction or dehydration synthesis.

H 2

O

Wolkenberg, Scott E.; Su, Andrew I. J Chem. Educ. 2001 78 784

A-B Model

Data Analysis/Data Analysis/ResultsResults

Combinatorial Combinatorial Chemistry and Chemistry and Drug Discovery Drug Discovery

LabLab

Data AnalysisData Analysis

Mixture Contents Result

M1 A1, B1, B2, B3

M2 A2, B1, B2, B3

M3 A3, B1, B2, B3

M4 B1, A1, A2, A3

M5 B2, A1, A2, A3

M6 B3, A1, A2, A3

--++------++

- - = no inhibition of growth = no inhibition of growth ++ = zone of inhibition of growth = zone of inhibition of growth

Data AnalysisData Analysis

Mixtures(Libraries) M1 M2 M3

M4

M5

M6

A1-B1

A1-B2 A2-B2

A1-B3 A3-B3

A3-B2

A3-B1A2-B1

A2-B3

Using this table, we can determine which compound in our active mixtures has antibiotic properties. First, shade in the column to indicate the mixture that shows antibiotic activity for compounds M1 thru M3. Next, shade in the row that corresponds to the mixture M4 thru M6 that shows antibiotic activity. The position in the table where the shaded column and shaded row intersect is the active compound.

Extension: Kirby–Bauer Extension: Kirby–Bauer Test Test

Determining Class Average Zone Size1. After 24 hours in the incubator or 48 hours at room temperature check for the presence of antibiotic activity. This is done by looking for a clear area, called a zone of inhibition, surrounding a well. Remember to never open the Petri dish for a better view. Use the agar side to observe and measure any zone of inhibition.

2.Using a ruler, measure the diameter (in millimeters) of any zone of inhibition and record your individual group data on the data table provided.

3.Now gather class data to determine the average zones of inhibition, if present, for each of the different compounds M1 thru M6 and record the averages.

4.After recording the average class data for the diameters of the zones of inhibition, decide whether your groups sample of E.coli bacteria is susceptible (sensitive) displaying a CLEAR zone of inhibition, unaffected (resistant) showing no observable zone of inhibition or intermediate (somewhere in between) for each of the compounds M1 thru M6. Record your conclusion in the final column of the data table.

Extension: Kirby–Bauer Extension: Kirby–Bauer Test Test

Determining Class Average Zone Size

Using a ruler, measure the diameter (in millimeters) of any zone of inhibition

and record your individual group data on the data table provided.

Extension: Zone Size Interpretive Extension: Zone Size Interpretive Chart Chart ANTIMICROBIAL

AGENT  DISC CODE R = mm    or less

 I = mm    range

  MS =  S = mm    or more 

 amoxicillin (Staph)

AMC 19     20

 amoxicillin (other bacteria)

AMC 13 14-17   18

 ampicillin (Staph)

AM 28     29

 ampicillin (other bacteria)

AM 11 12-13   14

 carbenicillin (Pseudomonas)

CB 13 14-16   17

 carbenicillin (other bacteria)

CB 17 18-22   23

 cefoxatime CTX 14   15-22 23 cephalothin CF 14 15-17   18 chloramphenicol

C 12 13-17   18

 erythromycin E 13 14-22   23

 gentamycin GM 12 13-14   15 methicillin (used for Staph only)

M (or DP) 9 10-13   14

 penicillin P 28     29

 streptomycin S 11 12-14   15

 sulfamethoxazole-trimethoprim

SXT-TMP 10 11-15   16

 tetracycline TE 14 15-18   19

Concept ReviewSuggest a reason why groups may have reported different diameters for the zones of inhibition created in response to the six compounds tested. Why do you suppose most antibiotics are allowed a range of diameters for their zones of inhibition on the SIR table?

Expected Results at 37ºCExpected Results at 37ºC

Results at Room Results at Room TemperatureTemperature

Chemical Structure of the Chemical Structure of the Active CompoundActive Compound

H 2

O

One of the resulting compounds created in our library of mixtures is “discovered” to have antibacterial properties. The active compound is a known antibiotic called guanofuracin which was discovered in the 1950’s and developed for both human and veterinary use.

5-nitro-2-furaldehyde

aminoguanidine bicarbonate

Post-lab Post-lab DiscussionDiscussion

Combinatorial Combinatorial Chemistry and Chemistry and Drug Discovery Drug Discovery

LabLab

Autoclaving is the most effective and most efficient means of sterilization. All autoclaves operate on a time/ temperature relationship. The usual standard temperature/ pressure employed is 121ºC/15 psi for 15 minutes.

AntibacteriaAntibacterial Agentsl Agents

Three Major Classes of Three Major Classes of Antibacterial AgentsAntibacterial Agents

Controlling the growth of microorganims usually involves the use of physical or chemical agents which either kill or prevent the growth of microorganisms.

1) Bactericidal – agents that kill bacteria are called cidal agents; also referred to as bactericides.

2)Bacteriostatic – agents which inhibit the growth of cells (without killing them) are referred to as static agents.

3) Bacteriolytic – agents that have the ability to lyse or break apart, dissolve, and destroy bacteria by the use of an enzyme or other agent.

Bacteria have the ability to develop resistance following repeated or subclinical (insufficient) doses, so more advanced antibiotics and synthetic antimicrobials are continually required to overcome them.

Gram – Gram – positivepositivebacteriabacteria

Gram – Gram – negativenegativebacteriabacteria

Variations in bacterial cell wall structures not only cause differences in staining but the anatomy of each cell wall also leads to differences in the susceptibility of bacteria to antibiotics. Some antibiotics easily penetrate Gram-positive cell walls while others are more capable of penetrating Gram-negative cell walls.

Antibacterial Sites of Antibacterial Sites of ActionAction

ConceptConcept ReviewReview

• Analyze the graph by describing the phase of growth being represented by lines A thru E: LAG, LOG, STATIONARY, or DEATH.

• If the graph shown is the result of antibiotic resistance testing, what is the effect of the antibiotics on the growth of the bacteria at A thru E: NORMAL, BACTERIOSTATIC, RESISTANT or BACTERICIDAL?

Great Job Jasmine Great Job Jasmine Erfe!Erfe!