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

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

Plants to Compound Library Sequoia Sciences, Inc. Analysis performed on each sample 3 Normalized sample weights 4 zz Utilize ELSD data to deliver normalized weights of each sample ELSD Chromatogram Screening concentrations between 1 to 20 µM Sequoia’s Platform 1 Parallel Purification Technologies State-of-the-art NMR Probe Technology Taxonomically Diverse Plant Collection 2 Five step purification process to remove non-drug-like compounds 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 8-channel MUX LC/MS with 8 ELSD’s Mass Spectrum TIC Chromatogram m/z ~ 110 samples / plant 1.1 to 5 compounds per sample 2.Samples of known quantities 3.Drug-like compounds a.Average MW < 400 daltons b.Log P from 0 to 5

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

Ground Plant 8765 Extract A EFG C Extreme ly lipophili c fraction is discarde d Preparative HPLC 4 x mL chromatographic fractions D A - 50:50ethyl acetate : ethanol B - 100methanol C - 75:25hexane : ethyl acetate D - 25:75hexane : ethyl acetate E - 100ethyl acetate F - 70:30ethyl acetate : methanol G - 50:50ethyl acetate : methanol H – 50:50methanol:water 1% acetic acid C18 SPE to remove lipophilics LC/MS analysis, and removal of tannins with polyamide SPE Flash Chromatography Extract B H 9

High-throughput Natural Products Chemistry 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

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

SF_4329 SF_4331 SF_4335 SF_4351 SF_4356

High-throughput Natural Products Chemistry Sequoia Sciences, Inc.

CapNMR TM  Probe 4  g. Amount isolated: 4 mg. Dilute with 7mL MeOD Infuse 5 mL Center in flow cell with 7 mL

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

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

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

Plant Families Underrepresented in a Drug Discovery Program

Courtesy of Michelle Goering

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

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

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

What can I do with only micrograms of a sample?

High-throughput Natural Products Chemistry Move to Miniaturize 100 mL1 L10 mL1 mL 100  L10  L

CapNMR TM Probe High-throughput Natural Products Chemistry 1.5  lit. (~1 mm) 5  lit. 5 mm std. NMR tube 270  lit. (~17 mm) 5 mm Rf Coil

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

Courtesy of Missouri Botanical Garden Orchidaceae

Plant Family: Orchidaceae Species: Phragmipedium calurum

High-throughput Natural Products Chemistry Sequoia Sciences, Inc.

Stilbenes isolated from Phragmipedium sp.

High-throughput Natural Products Chemistry Nepenthes

High-throughput Natural Products Chemistry Sequoia Sciences, Inc. Combine tubes C7, D7, E7 and F7 SepFraction  g per collection

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

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

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

Bacterial Biofilms

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

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

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

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

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