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

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

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

5. My Research Pure Compound Antimicrobial Pure Compound? Test

6. Procedure Obtain bacteria Obtain bacteria The Mutans Streptococci The Mutans Streptococci Broad term Broad term 7 species of Streptococcus 7 species of Streptococcus Cause dental caries (“cavities”) Cause dental caries (“cavities”) S. mutans S. mutans ATCC # UAB 308 ATCC # UAB 308 Choose compounds Choose compounds Culture the bacteria Culture the bacteria Freezer stock Freezer stock Working stock Working stock Photograph from http://www.mrcophth.com/finalmrcophthmcqss/16parasitolo gy/1strep-mutans.jpg 2 http://www.mrcophth.com/finalmrcophthmcqss/16parasitolo gy/1strep-mutans.jpg

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

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

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

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

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

12. Fractionation Technique Vacuum Liquid Chromatography (VLC) Vacuum Liquid Chromatography (VLC)

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

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

15. Purification Scheme Solid Phase Extraction Chromatography (SPEC) VLC HPLC TLC ABCD E FGHI

16. Characterization of Purified Compounds Mass Spectrometry Mass Spectrometry IR Spectroscopy IR Spectroscopy 1 H, 13 C and 2-Dimensional NMR Spectroscopy 1 H, 13 C and 2-Dimensional NMR Spectroscopy Photograph from http://www.biology.arizona.edu/biochemistry/problem_sets/aa/Gra phics/MolStruct/L-leucine.jpg 3 http://www.biology.arizona.edu/biochemistry/problem_sets/aa/Gra phics/MolStruct/L-leucine.jpg

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

18. Results Fraction Number Organism Name Collected From ZOI Diameter (mm) 1303 E Red Alga Papua New Guinea 13.5 0988 D Laminaria Hokkaido, Japan 13 1512 A Brown Tufts Madagascar14.5 1500 A Symploca Papua New Guinea 12 1460 H Red Brown Fuzzy Papua New Guinea 11, very clear

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

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

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

22. You!