Mark O’Neil-Johnson

2. Advanced Analytical Technologies as Applied to Natural Products for Drug Discovery: Segment Deux Mark O’Neil-Johnson

3. High-throughput Natural Products Chemistry Sequoia Sciences, Inc. State-of-the-art NMR Probe Technology Taxonomically Diverse Plant Collection Parallel Purification Technologies

5. 1 4 2 ELSD Chromatogram • ~ 110 samples / plant • 1 to 5 compounds per sample • Samples of known quantities • Drug-like compounds • Average MW < 400 daltons • Log P from 0 to 5 Utilize ELSD data to deliver normalized weights of each sample 200 300 400 500m/z Screening concentrations between 1 to 20 µM 3 Sequoia Sciences, Inc. Plants to Compound Library Analysis performed on each sample Sequoia’s Platform 8-channel MUX LC/MS with 8 ELSD’s State-of-the-art NMR Probe Technology Taxonomically Diverse Plant Collection Parallel Purification Technologies Mass Spectrum TIC Chromatogram 506.2 447.2 244.9 336.0 Five step purification process to remove non-drug-like compounds Normalized sample weights zz • Approximately 90% of plant extracts are composed of “non-drug-like” compounds • Sequoia’s process removes these “non-drug-like” compounds • Generates approximately 110 preparative HPLC fractions (“sample”) per plant

6. 1 test 2 Sequoia Sciences, Inc. Biological Activity to Chemical Structure Biology identifies an Active Sample 4 Structure elucidation of an Active Compound Bruker Avance 600 MHz Magnet Flow Probe Flow Probe m m 5 5 L L Flow cell Flow cell m m 1.5 1.5 L L Active volume Active volume Coil Coil • Sequoia acquires NMR • spectra on 5 to 100 ug • of an Active Compound Sequoia isolates compounds from an Active Sample 3 Sequoia sends pure compounds to Biology • Sequoia elucidates • the chemical structure • of an Active Compound Biology rescreens purified compounds

7. A - 50:50 ethyl acetate : ethanol B - 100 methanol C - 75:25 hexane : ethyl acetate D - 25:75 hexane : ethyl acetate E - 100 ethyl acetate F - 70:30 ethyl acetate : methanol G - 50:50 ethyl acetate : methanol H – 50:50 methanol:water 1% acetic acid A B Ground Plant Extract Extract D E F G H C Flash Chromatography Extremely lipophilic fraction is discarded 5 6 7 8 9 C18 SPE to remove lipophilics LC/MS analysis, and removal of tannins with polyamide SPE Preparative HPLC 4 x 40 20 mL chromatographic fractions

9. High-throughput Natural Products Chemistry Sequoia Sciences, Inc. HPLC / ELSD of Hit 620 of Fraction 8, tube 34

10. High-throughput Natural Products Chemistry Sequoia Sciences, Inc. HPLC / ELSD of Hit 620 of Fraction 8, tube 34

11. High-throughput Natural Products Chemistry Sequoia Sciences, Inc. Active Fractions From Hit 620 Active Fractions for Rescreen and NMR

12. SF_4356 SF_4351 SF_4335 SF_4331 SF_4329

13. High-throughput Natural Products Chemistry Sequoia Sciences, Inc.

14. CapNMRTMProbe • Amount isolated: 4 mg. • Dilute with 7mL MeOD • Infuse 5 mL • Center in flow cell with 7 mL 4 mg.

15. High-throughput Natural Products Chemistry Sequoia Sciences, Inc. Combine tubes C7, D7, E7 and F7 C7 D7 SepFraction 15827 9 mg total (2 HPLC Collections) E7 F7

16. High-throughput Natural Products Chemistry Sequoia Sciences, Inc. Compound 553

17. High-throughput Natural Products Chemistry Sequoia Sciences, Inc. Compound 553 From Hit 1212 IC50: 0.38 mM

18. Plant Families Underrepresented in a Drug Discovery Program

19. Courtesy of Michelle Goering

20. Botanical/Chemical diversity of plant collection? Courtesy of Missouri Botanical Garden

21. Study of NCI collection in Madagascar: Representative families genera in flora versus collected Number of Genera Sampled Number of Genera Courtesy of Jim Miller Ref: Biologically active natural products: pharmaceuticals, 2000, CRC press

22. Conclusion of NCI collection survey Collection could be improved by greater attention to rare plants with provisions to allow for smaller samples to be collected for certain taxa Advanced technology that allow the analysis of small samples Plants grown in greenhouses

23. What can I do with only micrograms of a sample?

24. High-throughput Natural Products Chemistry Move to Miniaturize 1 mL 100 mL 10 mL 1 L 100 mL 10 mL 1 mL

25. High-throughput Natural Products Chemistry CapNMRTM Probe 5 mm std. NMR tube 5 mm Rf Coil 270 mlit. (~17 mm) 1.5 mlit. (~1 mm) 5 mlit.

26. High-Throughput Natural Products Chemistry Sequoia Sciences, Inc. Sample Submission Hardware, Solvent and Waste Recovery 96-well plate Waste recovery vial 10 ml gas tight syringe mini-tube

27. Orchidaceae Courtesy of Missouri Botanical Garden

28. Plant Family: Orchidaceae Species: Phragmipediumcalurum

29. High-throughput Natural Products Chemistry Sequoia Sciences, Inc.

30. Stilbenes isolated from Phragmipedium sp.

31. High-throughput Natural Products Chemistry Nepenthes

32. High-throughput Natural Products Chemistry Sequoia Sciences, Inc. Combine tubes C7, D7, E7 and F7 SepFraction 16681 15 mg per collection

33. High-throughput Natural Products Chemistry Secoiridoids isolated from Sarracenia alata Secoiridoids isolated from Sarracenia alata Sequoia Sciences, Inc.

34. Plant Family: Asteraceae Species: Layiaplatyglossa Combine tubes F10, G10 and H10 To NMR SepFraction 15509 6 mg total (2 HPLC runs)

35. High-throughput Natural Products Chemistry ACD Software 64 Scans 512 Scans Data base peak search

36. Bacterial Biofilms

37. High-throughput Natural Products Chemistry What is a Biofilm? • Bacteria attach on surface, begin to secrete polysaccharide matrix • Cells grow into a community, complete with nutrient channels • Community thrives, protected by polysaccharide matrix

38. High-throughput Natural Products Chemistry Type of Biofilms • Industrial Biofilms • Ship bottoms and pipes • Cooling towers • Air ventilation systems • Drinking water filtration and distribution systems • Air filtration units • Dental water lines • Hemodialysis units • Food processing systems • Medical Biofilms • Urinary tract infections • Lung infections • Sinus infections • Ear infections • Chronic wounds • Dental caries • Medical device implants • Acne

39. High-throughput Natural Products Chemistry Biofilms • Resistant to antibiotics at concentrations from 1000 to 1500 times higher than conventional use • Involved in two-thirds of human bacterial infections • No specific biofilm inhibitor is commercially available

40. High-throughput Natural Products Chemistry Screening Challenge • Bacterial strain: Pseudomonas aeruginosa (PA01) • Ciprofloxacin active against planktonic bacteria at 1 mg/ml • Ciprofloxacin active against a bacterial biofilm at 50 mg/ml • Develop a valid physiological screening assay

41. High-throughput Natural Products Chemistry Screening for Biofilms • 96 Well plate format for initial screen • (initial screen at 10 mg/ml) • Asiatic Acid • Corosolic Acid • Ursolic Acid

42. Eliane Garo Jin-Feng Hu Gary Eldridge James Norcross Tim Peck Dean Olson High-throughput Natural Products Chemistry Sequoia Sciences, Inc. Peter Raven James Miller Very Special Thanks to: Brent Lefebvre and Ryan Sasaki of ACD