Presentation on theme: "Bringing Back Nature to Drug Discovery Natural Molecules in an Antibacterial Program Mark O’Neil-Johnson."— Presentation transcript:
Bringing Back Nature to Drug Discovery Natural Molecules in an Antibacterial Program Mark O’Neil-Johnson
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 Strengths 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 Collaborator rescreens purified compounds Biological Activity to Chemical Structure Sequoia Sciences, Inc. Collaborator identifies an Active Sample Sequoia acquires NMR spectra on 5 to 100 ug of an Active Compound Structure elucidation of an Active Compound Sequoia delivers compounds to Collaborator Sequoia isolates compounds from an Active Sample Sequoia elucidates the chemical structure of an Active Compound 600 MHz Magnet test
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 Probe The Ultimate NMR Probe for Samples of Limited Mass
CapNMR TM Probe High-throughput Natural Products Chemistry 2 g Quinidine in MeOH,d 4 2 g in 550 L MeOH,d4 2 g in 8 L MeOH,d4; Infuse 5 L 5mm TXI 900 MHz CapNMR 600 MHz
High-throughput Natural Products Chemistry Sequoia Sciences, Inc.
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 Sequoia Sciences, Inc. 512 Scans 64 Scans Data base peak search
Bacterial Biofilms 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
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 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 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)
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
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 High-throughput Natural Products Chemistry Rotating Disk Reactor (RDR)
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
High-throughput Natural Products Chemistry Rotating Disk Reactor (RDR) 255+toby 255+water toby control
High-throughput Natural Products Chemistry Rotating Disk Reactor (RDR)
High-throughput Natural Products Chemistry Rotating Disk Reactor (RDR) C = ciprofloxacin T = tobramycin
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 g/ml Asiatic Acid makes biofilm sensitive to 10 g/ml ciprofloxacin Sequoia Compounds are excellent biofilm potentiator candidates
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