discovery and identification of antibacterial agents for dental use adam kirkpatrick hhmi: summer...
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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
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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
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Potential OutcomesPotential Outcomes
Useful moleculeUseful molecule
Could help defeat dental diseaseCould help defeat dental disease Dental caries (“cavities”)Dental caries (“cavities”)
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Gerwick Lab ResearchGerwick Lab Research
Crude Extract
Prefractionated Extract
Pure Compound
Molecular Structure
Organism
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My ResearchMy Research
Pure Compound
Antimicrobial Pure Compound?
Test
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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
2
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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
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ResultsResults
21 pure compounds tested21 pure compounds tested 1 antimicrobial pure compound1 antimicrobial pure compound
AllolaurinterolAllolaurinterol
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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
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Current ProjectCurrent Project
Other strains?Other strains?
Toxic?Toxic?
Mouthwash?Mouthwash?
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My Research – 2My Research – 2ndnd Facet FacetCrude and Prefractionated
Extracts
Antimicrobial Crude and Prefractionated Extracts?
Pure Compound
Antimicrobial Pure Compound?
Test
Purify
Structure
Test
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Fractionation TechniqueFractionation Technique
Vacuum Liquid Chromatography Vacuum Liquid Chromatography (VLC)(VLC)
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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
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Reverse Phase HPLC Traces from Pre-fractions A-I
Most Polar Least Polar
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Purification SchemePurification Scheme
Solid Phase Extraction Chromatography (SPEC)
VLC
HPLC
TLC
A B C D
E
F G H I
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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
3
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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?
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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
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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
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AcknowledgementsAcknowledgements
Dr. Bill Gerwick
Howard Hughes Medical Institute Dr. Kevin Ahern
Dr. Taifo Mahmud
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AcknowledgementsAcknowledgements
Mirjam Musafija-Girt
Carla Sorrels Jennifer Kepler
Aishu Ramakrishnan
Dr. Roger Linington
Harry GrossEric Andrianosolo
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You!You!