Mark ONeil-JohnsonAdvanced Analytical Technologies as Applied to Natural Products for Drug Discovery:Segment Deux

Parallel Purification TechnologiesState-of-the-art NMR Probe Technology Taxonomically Diverse PlantCollectionHigh-throughput Natural Products ChemistrySequoia Sciences, Inc.

Plants to Compound LibrarySequoia Sciences, Inc.Analysis performed on each sampleNormalized sample weightszzSequoias PlatformParallel Purification TechnologiesState-of-the-art NMR Probe TechnologyTaxonomically Diverse PlantCollectionFive step purification process to removenon-drug-like compoundsApproximately 90% of plant extracts are composed of non-drug-like compoundsSequoias process removes these non-drug-like compoundsGenerates approximately 110 preparative HPLC fractions (sample) per plant8-channel MUX LC/MS with 8 ELSDsMass SpectrumTIC Chromatogram506.2447.2244.9336.0

Sheet1

Highly Lipophilic Compounds> Log P of 5

Highly Hydrophilic Compounds< Log P of 0

High Molecular Weight Compounds> 1000 Daltons

Miscellaneous Compounds

i.e. Carbohydrates, Tannins

Sheet2

Sheet3

Flow ProbeCoil5 mLFlow cell1.5 mLActive volumeBruker AvanceFlow ProbeCoil5 mLFlow cell1.5 mLActive volume Biology rescreens purified compounds Biological Activity to Chemical StructureSequoia Sciences, Inc.Biology identifies anActive SampleSequoia acquires NMR spectra on 5 to 100 ug of an Active Compound Structure elucidation of an Active CompoundSequoia sends pure compounds to Biology Sequoia isolates compounds from an Active SampleSequoia elucidates the chemical structure of an Active Compound600 MHz Magnet34

Ground Plant8765ExtractAEFGCExtremelylipophilicfraction is discardedPreparative HPLC4 x 40 20 mL chromatographic fractionsDA - 50:50ethyl acetate : ethanolB - 100methanolC - 75:25hexane : ethyl acetateD - 25:75hexane : ethyl acetateE - 100ethyl acetateF - 70:30ethyl acetate : methanolG - 50:50ethyl acetate : methanolH 50:50methanol:water 1% acetic acid

C18 SPE to remove lipophilicsLC/MS analysis, and removal of tannins with polyamide SPEFlash ChromatographyExtractBH9

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

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

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

SF_4329SF_4331SF_4335SF_4351SF_4356

CapNMRTM Probe4 mg. Amount isolated: 4 mg. Dilute with 7mL MeOD Infuse 5 mL Center in flow cell with 7 mL

High-throughput Natural Products ChemistrySequoia Sciences, Inc.C7D7E7F7Combine tubes C7, D7, E7 and F7SepFraction 15827 9 mg total (2 HPLC Collections)

High-throughput Natural Products ChemistrySequoia Sciences, Inc.Compound 553

High-throughput Natural Products ChemistrySequoia Sciences, Inc.Compound 553

From Hit 1212IC50: 0.38 mM

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 collectedNumber of GeneraNumber of Genera SampledRef: Biologically active natural products: pharmaceuticals, 2000, CRC pressCourtesy of Jim Miller

Conclusion of NCI collection surveyCollection 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 Miniaturize100 mL1 L10 mL1 mL1 mL100 mL10 mL

CapNMRTM ProbeHigh-throughput Natural Products Chemistry1.5 mlit.(~1 mm)5 mlit. 5 mm std. NMR tube270 mlit.(~17 mm)5 mm Rf Coil

Sequoia Sciences, Inc.High-Throughput Natural Products ChemistrySample Submission Hardware, Solvent and Waste Recoverymini-tube10 ml gas tight syringe96-well plateWaste recoveryvial

Courtesy of Missouri Botanical Garden Orchidaceae

Plant Family: OrchidaceaeSpecies: Phragmipedium calurum

Stilbenes isolated from Phragmipedium sp.

High-throughput Natural Products ChemistryNepenthes

High-throughput Natural Products ChemistrySequoia Sciences, Inc.Combine tubes C7, D7, E7 and F7SepFraction 1668115 mg per collection

Secoiridoids isolated from Sarracenia alataHigh-throughput Natural Products ChemistrySequoia Sciences, Inc.Secoiridoids isolated from Sarracenia alata

Combine tubes F10, G10 and H10SepFraction 155096 mg total (2 HPLC runs)To NMRPlant Family: AsteraceaeSpecies: Layia platyglossa

512 Scans64 ScansData base peak search

Bacterial Biofilms

Bacteria attach on surface, begin to secrete polysaccharide matrix

Cells grow into a community, complete with nutrient channels

Community thrives, protected by polysaccharide matrix

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

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

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

96 Well plate format for initial screen (initial screen at 10 mg/ml)

Asiatic Acid Corosolic Acid Ursolic Acid

High-throughput Natural Products ChemistrySequoia Sciences, Inc.Very Special Thanks to: Brent Lefebvre and Ryan Sasaki of ACDPeter RavenJames Miller

*******************We do collarborate with MBG. They provide us with the plant collection.*For most drug discovery program, taxonomy is a reliable indicator of the distribution of chemical diversity in nature. Therefore the goal of most of these program is to gather a set of species that are representative of the botanical diversity that exist in that region.Jim Miller did an interesting survey on the taxonomic composition of a plant collection. To have representative results, they took the NCI collection in Madagascar over a period of 10 years and looked at the biological diversity of plant collection.*Collections are not generating botanical diversity that exists in an area..Woody plants that are major element of tropical forest have been sampled at much greater rate than small, herbaceous taxa. Considering the amount of material required for a drug discovery program (> 300g) it is not surprising that collection is biased toward taxa that are present in large populations, such as Rubiaceae, Euphorbiaceae and Fabaceae.Herbaceae plant families that are small or occur in sparse populations will be underrepresented, which is the case of Poaceae and Orchidaceae.*If the botanist has to collect 300-400 g dry weight, it is clear that some families will never be sampledIt might be these species, that are excluded from almost every screening program because of their small size, that are most likely to yield previously unknown compounds simply because they remain completely unstudied.*****Orchidaceae: 19,000 species, 750 generaIn order to preserve the botanical diversity of Orchidaceae, the botanist has to decide whether the collection of the orchid will have a negative impact on the species, and see if there would be enough plant material to satisfy quantity requirement of conventional drug discovery programs*****************