Bringing Back Nature to Drug Discovery Natural Molecules in an Antibacterial Program

2. Bringing Back Nature to Drug Discovery Natural Molecules in an Antibacterial Program Mark O’Neil-Johnson

4. 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 Strengths 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 Collaborator 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 delivers compounds to Collaborator • Sequoia elucidates • the chemical structure • of an Active Compound Collaborator rescreens purified compounds

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

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

10. CapNMR Probe The Ultimate NMR Probe for Samples of Limited Mass

11. High-throughput Natural Products Chemistry CapNMRTM Probe 2 mg Quinidine in MeOH,d4 5mm TXI probe @ 900 MHz 2 mg in 550 mL MeOH,d4 CapNMR probe @ 600 MHz 2 mg in 8 mL MeOH,d4; Infuse 5 mL

12. 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.

13. 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

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. HPLC / ELSD of Hit 620 of Fraction 8, tube 34

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

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

18. SF_4356 SF_4351 SF_4335 SF_4331 SF_4329

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

20. Plant Family: Orchidaceae Species: Phragmipediumcalurum

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

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

23. High-throughput Natural Products Chemistry Sequoia Sciences, Inc. 64 Scans 512 Scans Data base peak search

24. Bacterial Biofilms Nasty Stuff!

25. High-throughput Natural Products Chemistry Bacteria…..Some Nasty Stuff! Test Summary: Pseudomonas Disinfection and Regrowth in Pipes • R. Anderson (Center for Disease Control) filled plastic pipes with water • contaminated with two strains of Pseudomonas. • Incubate for 8 weeks • Empty and douse pipes with germ-killing biocides, include chlorine, for • 7 days • Refill with sterile water and periodically sample “clean water” • Both strains of Pseudomonas survived and reestablished colonies!

26. 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

27. 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

28. 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

29. 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

30. High-throughput Natural Products Chemistry Screening Challenge • Minimum Inhibitory Concentrations (MIC) against planktonic bacteria not relevant to biofilms • New biofilm susceptibility methods need to be developed: biofilm inhibition conc. (BIC) or minimum biofilm eradication conc. (MBEC) • Variability in BIC and MBEC values suggest characteristics of biofilms is related to model used to grow them in • 96 Well plate format • Rotating Disk Reactor (RDR)

31. 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

32. High-throughput Natural Products Chemistry Rotating Disk Reactor (RDR) Figure 1 Rotating Disk Reactor showing test coupon holder with growth coupons inserted Figure 2 Test coupons: disk on left is polycarbonate, Disk on right is stainless steel

33. High-throughput Natural Products Chemistry Airway Infections in CF Patients • Pseudomonas aeruginosa airway infections: leading cause of death in Cystic Fibrosis patients • Chronic infection that is resistant to antibiotic therapies • Standard treatment: tobramycin and azithromycin • RDR, a physiological valid screening method? • Test Sequoia compounds in RDR with tobramycin and azithromycin or ciprofloxacin combination

35. High-throughput Natural Products Chemistry Rotating Disk Reactor (RDR) 255+toby 255+water toby control

36. High-throughput Natural Products Chemistry Rotating Disk Reactor (RDR)

37. High-throughput Natural Products Chemistry Rotating Disk Reactor (RDR) C = ciprofloxacin T = tobramycin

38. High-throughput Natural Products Chemistry Screening for Biofilms • Summary • Asiatic Acid and Corosolic Acid in a physiological relevant biofilm screen • are not true biofilm inhibitors but biofilm modulators • Synergistic effect on P. aeruginosa biofilm by Sequoia compounds indicate • that they do affect the tolerance of antibiotic to PA • Sequoia compounds not true biofilm inhibitors, suggesting a different MOA • by modification of biofilm • P. aeruginosa grown in presence of 10 mg/ml Asiatic Acid makes biofilm sensitive to • 10 mg/ml ciprofloxacin • Sequoia Compounds are excellent biofilm potentiator candidates

39. Jin-Feng Hu Eliane Garo Gary Eldridge James Norcross Tim Peck Dean Olson High-throughput Natural Products Chemistry Sequoia Sciences, Inc. Special Thanks to: Bill Costerton Garth James