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Discovery and Identification of Antibacterial Agents for Dental Use

Discovery and Identification of Antibacterial Agents for Dental Use. Adam Kirkpatrick HHMI: Summer 2005 Dr. Bill Gerwick. Tooth Decay. Est. 90% of U.S. have at least one cavity 2 nd only to common cold $68 billion annually. Potential Outcomes. Useful molecule

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Discovery and Identification of Antibacterial Agents for Dental Use

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  1. Discovery and Identification of Antibacterial Agents for Dental Use Adam Kirkpatrick HHMI: Summer 2005 Dr. Bill Gerwick

  2. Tooth Decay • Est. 90% of U.S. have at least one cavity • 2nd only to common cold • $68 billion annually

  3. Potential Outcomes • Useful molecule • Could help defeat dental disease • Dental caries (“cavities”)

  4. Organism Crude Extract Prefractionated Extract Pure Compound Molecular Structure Gerwick Lab Research

  5. Test Antimicrobial Pure Compound? Pure Compound My Research

  6. Photograph from http://www.mrcophth.com/finalmrcophthmcqss/16parasitology/1strep-mutans.jpg2 Procedure • Obtain bacteria • The Mutans Streptococci • Broad term • 7 species of Streptococcus • Cause dental caries (“cavities”) • S. mutans • ATCC # UAB 308 • Choose compounds • Culture the bacteria • Freezer stock • Working stock

  7. Photograph from http://www.austin.cc.tx.us/microbugz/48antibio.html Antibiotic Susceptibility Testing • Bacteria “lawn” • Impregnate disc • Plates • Controls • Incubate • Zones of inhibition

  8. Results • 21 pure compounds tested • 1 antimicrobial pure compound • Allolaurinterol

  9. Photograph from http://www.biol.tsukuba.ac.jp/~inouye/ino/r/Martensia.GIF Allolaurinterol Properties • Structure • Name of Source Organism • Martensia • Laurencia • Found • Curacao • 1991 • Other known activity • Anti-cancer • Antibiotic resistant bacteria

  10. Current Project • Other strains? • Toxic? • Mouthwash?

  11. Test Antimicrobial Crude and Prefractionated Extracts? Crude and Prefractionated Extracts Purify Pure Compound Test Antimicrobial Pure Compound? Structure My Research – 2nd Facet

  12. Fractionation Technique • Vacuum Liquid Chromatography (VLC)

  13. Pre-fractionation of Crude Extracts to Produce Reduced Complexity, Higher Titer Screening Materials A C E G I B F D H

  14. Reverse Phase HPLC Traces from Pre-fractions A-I Most Polar Least Polar

  15. Purification Scheme A B C D F G H I TLC E VLC Solid Phase Extraction Chromatography (SPEC) HPLC

  16. Characterization of Purified Compounds • Mass Spectrometry • IR Spectroscopy • 1H, 13C and 2-Dimensional NMR Spectroscopy Photograph from http://www.biology.arizona.edu/biochemistry/problem_sets/aa/Graphics/MolStruct/L-leucine.jpg3

  17. Results • 30 organisms tested • 9 fractions, 1 crude extract • 300 total • 89 antimicrobial crude extracts and fractions • No isolated compounds • Newly discovered molecule?

  18. Results

  19. Photograph from http://www.answers.com/main/content/wp/en/thumb/c/cb/400px-CellMembraneDrawing.jpg Other Potential Research • Other bacterial strains • Human toxicity • Antimicrobial interaction

  20. Dr. Kevin Ahern Howard Hughes Medical Institute Dr. Taifo Mahmud Dr. Bill Gerwick Acknowledgements

  21. Dr. Roger Linington Carla Sorrels Eric Andrianosolo Jennifer Kepler Harry Gross Aishu Ramakrishnan Mirjam Musafija-Girt Acknowledgements

  22. You!

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