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

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

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

Collaborator rescreens Sequoia Sciences, Inc. Biological Activity to Chemical Structure 1 Collaborator identifies an Active Sample 4 Structure elucidation of an Active Compound Bruker Avance test 600 MHz Magnet Flow Probe Flow Probe 5 5 m m L L Flow cell Flow cell 1.5 1.5 m m L L Active volume Active volume Coil Coil Sequoia acquires NMR spectra on 5 to 100 ug of an Active Compound 2 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

What can I do with only micrograms of a sample?

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

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

CapNMRTM Probe 2 mg Quinidine in MeOH,d4 5mm TXI probe @ 900 MHz 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

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

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

CapNMRTM Probe Amount isolated: 4 mg. Dilute with 7mL MeOD Infuse 5 mL Center in flow cell with 7 mL 4 mg.

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

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

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

SF_4356 SF_4351 SF_4335 SF_4331 SF_4329

High-throughput Natural Products Chemistry Sequoia Sciences, Inc.

Plant Family: Orchidaceae Species: Phragmipedium calurum

High-throughput Natural Products Chemistry Sequoia Sciences, Inc.

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

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

Bacterial Biofilms Nasty Stuff!

Bacteria…..Some Nasty Stuff! 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!

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

Type of Biofilms Industrial Biofilms Medical Biofilms 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

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

Screening for Biofilms 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

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)

Screening Challenge Bacterial strain: Pseudomonas aeruginosa (PA01) 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

Rotating Disk Reactor (RDR) 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

Airway Infections in CF Patients 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

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

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

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

Screening for Biofilms 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

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