Discovery and Identification Discovery and Identification of Antibacterial Agents for of Antibacterial Agents for

Dental UseDental Use

Adam KirkpatrickAdam Kirkpatrick

HHMI: Summer 2005HHMI: Summer 2005

Dr. Bill GerwickDr. Bill Gerwick

Tooth DecayTooth Decay

Est. 90% of U.S. Est. 90% of U.S. have at least one have at least one cavitycavity

22ndnd only to common only to common coldcold

$68 billion annually$68 billion annually

Potential OutcomesPotential Outcomes

Useful moleculeUseful molecule

Could help defeat dental diseaseCould help defeat dental disease Dental caries (“cavities”)Dental caries (“cavities”)

Gerwick Lab ResearchGerwick Lab Research

Crude Extract

Prefractionated Extract

Pure Compound

Molecular Structure

Organism

My ResearchMy Research

Pure Compound

Antimicrobial Pure Compound?

Test

ProcedureProcedure

Obtain bacteriaObtain bacteria The Mutans StreptococciThe Mutans Streptococci

Broad termBroad term 7 species of 7 species of StreptococcusStreptococcus Cause dental caries (“cavities”)Cause dental caries (“cavities”)

S. mutansS. mutans ATCC # UAB 308ATCC # UAB 308

Choose compoundsChoose compounds Culture the bacteriaCulture the bacteria

Freezer stockFreezer stock Working stockWorking stock

Photograph from http://www.mrcophth.com/finalmrcophthmcqss/16parasitology/1strep-mutans.jpg

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Antibiotic Susceptibility TestingAntibiotic Susceptibility Testing

Bacteria “lawn”Bacteria “lawn” Impregnate discImpregnate disc PlatesPlates ControlsControls IncubateIncubate Zones of inhibitionZones of inhibition

Photograph from http://www.austin.cc.tx.us/microbugz/48antibio.html

ResultsResults

21 pure compounds tested21 pure compounds tested 1 antimicrobial pure compound1 antimicrobial pure compound

AllolaurinterolAllolaurinterol

Photograph from http://www.biol.tsukuba.ac.jp/~inouye/ino/r/Martensia.GIF

Allolaurinterol PropertiesAllolaurinterol Properties

StructureStructure Name of Source OrganismName of Source Organism

MartensiaMartensia LaurenciaLaurencia

FoundFound CuracaoCuracao 19911991

Other known activityOther known activity Anti-cancerAnti-cancer Antibiotic resistant bacteriaAntibiotic resistant bacteria

OH

Br

Current ProjectCurrent Project

Other strains?Other strains?

Toxic?Toxic?

Mouthwash?Mouthwash?

My Research – 2My Research – 2ndnd Facet FacetCrude and Prefractionated

Extracts

Antimicrobial Crude and Prefractionated Extracts?

Pure Compound

Antimicrobial Pure Compound?

Test

Purify

Structure

Test

Fractionation TechniqueFractionation Technique

Vacuum Liquid Chromatography Vacuum Liquid Chromatography (VLC)(VLC)

Pre-fractionation of Crude Extracts to Produce Reduced Complexity, Higher Titer Screening Materials

Crude Extract (preferred 2.0 gms)

50 mg for parallel crude extract testing

100% H

10% E/H

20% E/H 100% M100% E

Size of VLC (for 2.0 gm, use 350 mL VLC funnel)

Silica gel cm wide x cm high

Volume of solvent = 300 mL of each

Extract in the middle of column

Silica GelTLC grade

25% M/E

Use Course PorosityVacuum Filter Funnel

Enter # mgper fraction

40% E/H 80% E/H

60% E/H

A

B

C

D

E

F

G

H

I

Reverse Phase HPLC Traces from Pre-fractions A-I

Most Polar Least Polar

Purification SchemePurification Scheme

Solid Phase Extraction Chromatography (SPEC)

VLC

HPLC

TLC

A B C D

E

F G H I

Characterization of Purified Characterization of Purified CompoundsCompounds

Mass SpectrometryMass Spectrometry IR SpectroscopyIR Spectroscopy 11H, H, 1313C and 2-Dimensional NMR C and 2-Dimensional NMR

SpectroscopySpectroscopy

Photograph from http://www.biology.arizona.edu/biochemistry/problem_sets/aa/Graphics/MolStruct/L-leucine.jpg

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ResultsResults

30 organisms tested30 organisms tested 9 fractions, 1 crude extract9 fractions, 1 crude extract 300 total300 total

89 antimicrobial crude extracts and 89 antimicrobial crude extracts and fractionsfractions No isolated compoundsNo isolated compounds Newly discovered molecule?Newly discovered molecule?

ResultsResultsFraction Fraction NumberNumber

Organism Organism NameName

Collected Collected FromFrom

ZOI Diameter ZOI Diameter (mm)(mm)

1303 E1303 E Red AlgaRed Alga Papua New Papua New GuineaGuinea

13.513.5

0988 D0988 D LaminariaLaminaria Hokkaido, Hokkaido, JapanJapan

1313

1512 A1512 A Brown TuftsBrown Tufts MadagascarMadagascar 14.514.5

1500 A1500 A SymplocaSymploca Papua New Papua New GuineaGuinea

1212

1460 H1460 H Red Brown Red Brown FuzzyFuzzy

Papua New Papua New GuineaGuinea

11, very clear11, very clear

Other Potential ResearchOther Potential Research

Other bacterial strainsOther bacterial strains

Human toxicityHuman toxicity

Antimicrobial interactionAntimicrobial interaction

Photograph from http://www.answers.com/main/content/wp/en/thumb/c/cb/400px-CellMembraneDrawing.jpg

AcknowledgementsAcknowledgements

Dr. Bill Gerwick

Howard Hughes Medical Institute Dr. Kevin Ahern

Dr. Taifo Mahmud

AcknowledgementsAcknowledgements

Mirjam Musafija-Girt

Carla Sorrels Jennifer Kepler

Aishu Ramakrishnan

Dr. Roger Linington

Harry GrossEric Andrianosolo

You!You!