Discovery and Identification of Antibacterial Agents for Dental Use Adam Kirkpatrick HHMI: Summer...
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Transcript of Discovery and Identification of Antibacterial Agents for Dental Use Adam Kirkpatrick HHMI: Summer...
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
2
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
3
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!