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Marine Natural Products Drug Discovery Amy Wright Ph.D. Center for Marine Biomedical and Biotechnology Research HARBOR BRANCH Oceanographic Institute at Florida Atlantic University Definition 1: Primary metabolites A primary metabolite is a chemical substance which is required for cells to survive and replicate and is therefore required for survival of the organism Examples are: – – – – Proteins Carbohydrates Lipids Nucleic acids Definition 2: Secondary Metabolites (aka Natural products) • A secondary metabolite is a chemical compound produced by an organism which is not required for survival of the organism but presumably confers an evolutionary advantage O H HH H H OH H N H O O O H HO O HO O H O H O NH OH O O O OH O O OH O H H N O O O O H H H O H OH Why does nature make natural products? Defense Reproduction Why can marine natural products be used as medicines? • Barnacles use a protein with homology to a histamine/adrenergic receptor to analyze surfaces prior to settlement • In humans adrenergic receptors regulate vasodilation, muscle contraction, etc. • Active sites of proteins are often conserved between species, but can have very different functions. Nature As a Source of Medicines • Over 62% of small molecule agents approved for use as drugs can be traced back to natural products • Some examples: • aspirin (willow/birch ) • morphine (Poppy) • penicillin (fungus) • Lovastatin (fungus) • Adriamycin/dauxorubicin (bacterium) • Taxol™ (yew tree) A Historical Perspective: Florida as a Source of Important Natural Products Florida Historical Perspective O O HN HO HN N O CH3 HO O N O O HO HO OH OH spongouridine • 1940’s and 1950’s • spongouridine and spongothymidine reported from Cryptotethia crypta collected near Elliott Key • Bergmann JOC 1951 15: 981 • Bergmann JOC 1955 20:1502 spongothymidine NH2 N HO N O O HO OH Nucleoside antiviral and Anticancer agents Ara-C AZT etc. Florida Historical Perspective O • 1969 Siegel and co-workers at the University of Miami • Discover potent antitumor and immunomodulatory properties of the mangrove tunicate Ecteinascidia turbinata • Approved for use against soft tissue sarcoma in Europe 2007 Siegel et. al. Food Drugs from the Sea 1969 Wright et. al. JOC 1990 Rinehart et. al. JOC 1990 O HO O H HH S O O O N N H OH O O NH HO Ecteinascidin 743 The first marine derived drug approved by the FDA is: Prialt™ • Prialt (SNX-111) is a synthetic form of the omega-conotoxin MVIIA • It is 100-1000x more potent than morphine • It has been approved for use by the FDA for chronic pain The Process of Marine Drug Discovery • • • • • Collection Biological Screening Natural Products Chemistry Secondary Testing/Pharmacology Production of material for clinical evaluation • Clinical investigation Marine Natural Product Drug Discovery Requires a Multidisciplinary Team • Biologists – Marine Biologists – Cell Biologists; Immunologists, Virologists, Microbiologists – Pharmacologists/Biochemists – Molecular Biologists • Chemists – Natural Products Chemists – Spectroscopists – Synthetic/Medicinal Chemists • Business Professionals – Marketing/Technology Transfer/ Patent Attorneys Step 1: Collections Before you collect-Get the right permits • International Convention on Biological Diversity – http://www.biodiv.org/convention/articles.asp • You must have informed consent of host country – Permits may be issued at a local or country level – Work with US State Department for Foreign Countries – Work with State Governments for State waters; National marine Fisheries for Federal – Special Permits for Marine Sanctuaries • Often a Memorandum of Understanding is negotiated prior to collection – – – – Participation by Host Nation in Project Return of Income/Benefit to Host Country Reporting requirements Sharing of specimens and information on samples • John E. Fogarty Center (part of NIH) regulates this – http://www.fic.nih.gov/programs/oecdub.html Collection Methods • • • • Wading Scuba Trawling Submersibles – AUV (mapping/documentation) – ROV (mapping/documentation/collections) – HOV (mapping/documentation/collections) Harbor Branch Operates the Johnson-Sea-Link class subs Depth capability: 3000 ft An Excellent View A Variety of Manipulator Tools Unique Work Platforms How do we choose dive sites? Multi-Beam Echo Sounding produces a 3-D image of the seafloor with a width of about 4 times water depth 600–800m Resolution 20-50m From: Kongsberg Simrad Reconnaissance Mapping Tool : EM 1002 Depth range 585-750m 1 km Slide Courtesy of Grasmueck et al. CSL U. Miami 2006 Highres Mapping Tool : AUV C-Surveyor II AUV: Autonomous Underwater Vehicle Free swimming- No tether- no cables Pre-programmed to perform set tasks Sensors 200 kHz Multibeam Echo Sounder 120 kHz Side Scan Sonar 2-8 kHz Sub-Bottom Profiler Acoustic Doppler Current Profiler Temperature, Salinity, Methane 40m Resolution 1-3 m Picture courtesy of Reson Inc. and Bluefin Robotics Inc. Highres Mapping Tool : AUV C-Surveyor II Depth range 585-750m Grasmueck et al. CSL U. Miami 2006 1 km Reconnaissance Mapping Tool: EM 1002 High resolution Mapping Tool: AUV C-Surveyor II Grasmueck et al. CSL U. Miami 2006 Depth range 585-750m 40 m Ground-truthing GBB5z 900 m 0.5 km Correa et al., 2006 CSL Meeting Collection Strategies Biological Diversity = Chemical Diversity Habitat Diversity = Chemical Diversity Myrmekioderma metabolites are depth dependent OH H OH O O < 33 m, Anti-tumor Activity H O 33 m < X < 66 m Anti-tumor Activity > 66 m Antiviral Activity (HSV-1) Sennett et. al. J. Nat. Prod 1992, 55,1421 Kashman et. al. Tet. Lett. 1987, 28, 546. Invertebrate Diversity=Microbial Diversity • Sponges can have up to 40% of their biomass made from associated microbes • The evidence is mounting that many “sponge derived” compounds are synthesized by microbial associates Thin section of the sponge Discodermia There is notable structural similarity between certain marine natural products and those produced by microorganisms H H NH NH H H O H H H N O O H H H OH HO H HO NH H H HO H H H NH O H H O OH H N O O Alteramide Alteramide Ikarugamycin Discodermide O Ikarugamycin O O OH Discodermide Alteromonas associated with Halichondria Terrestrial Streptomycete Discodermia dissoluta H Biosynthetic Gene clusters localized to bacterial symbionts O O N S N H Patellamide A produced by the tunicate symbiont Prochloron didemni N O HN NH O N S N H N Gene cluster expressed in E. coli Schmidt et. al. PNAS 2005 102: 7315 O O O N H Onnamide from sponge Theonella swinhoeii Gene cluster sequenced- associated with bacterium Piel et. Al. PNAS 2004, 101: 16222 COOH HO O O OH O H2N O H N O O NH NH O The challenge remains to culture these microbes! Step 2: Screening/Biological Assays Screening Approach Forward Chemical Genetics Approach Reverse Chemical Genetics Approach CELL Purified Protein Treat with small molecule Treat with small molecule Detect desired Cell Phenotype Detect small molecules which bind to target Determine HOW small molecule causes phenotype Define WHAT EFFECTS the binding has on the Phenotype Current Research Focus Finding Compounds to treat: • Cancer – Pancreatic Cancer – Multidrug resistant cancers – Cancer “Specific” Agents (NCDDG) • Infectious Disease – Drug resistant Staphylococcus aureus – Anti-malarial- collaborative UCF, WRAIR • Neurodegenerative Disease- collaborative – Alzheimer’s & Parkinson’s Diseases – Neuroprotection (Stroke) • Inflammation –collaborative Types of Assays Run at HBOITumor Cell Lines Tumor Cell Line Panel/MTT assay • A549 (lung) • PANC-1 (pancreatic) • ASPC-1 (pancreatic) • Mia PaCa2 (pancreatic) • BxPC-3 (pancreatic) • MCF-7 (breast) • DLD-1 (colon) • NCI-ADR-Res (ovarian) • P388 (murine leukemia) Cartoon of a Cell-based Reporter Assay LIGHT LIGHT The Cytoblot Assay Horse radish peroxidase LIGHT LIGHT LIGHT O3PO Secondary Antibody Primary Antibody Protein of Interest • The target protein increases or decreases in level after treatment • The protein target is detected with sequential treatment of antibodies, one of which is labeled with a horseradish peroxidase • Differences in light emitted from control values indicate if something is active Stockwell et. al. Chemistry & Biology 1999, 6:71-83. Target directed assays • MAP Kinase Signaling – p-ERK, p-MEK – cell survival, invasion, resistance to apoptosis • Inhibition of GSK-3 – regulates NF-B in pancreatic and breast cancer- controls cell proliferation • Apoptosis – Restoring sensitivity to TRAIL- induced apoptosis in resistant tumor cells • Cancer and Inflammation – Regulation of NF-B, STAT3. mast cell migration • HEDGEHOG signaling – targeting cancer stem cells through regulation of Gli proteins Step 3: Natural Products Chemistry Purify and Determine the structures of the Active Natural Products Extracts are Complex Mixtures of Natural Products Natural Products Chemistry The Traditional Way Chromatography Bioassay Chromatography Bioassay Pure Active Compound Extracts are Complex Mixtures of Natural Products • Mixtures are too complex for modern high throughput screening operations • Solution: make a “Peak Library” HBOI Peak Library Generation Prepare Extract Solvent partition Chromatograph using COMBIFLASH Assay/Analyze Fractions Deep-water Verongid sponge Enriched fraction Enriched fraction Enriched fraction Enriched fraction Pure Compound Spectroscopy is used to define the Structures O HO HO HO O Cl O O O O Cl N O HO HO OH NH2 O OCH3 600 MHz Nuclear Magnetic Resonance Spectrometer Step 4: Determine the Mechanism of Action For example: How does the compound kill cancer cells? Confocal Microscopy Flow Cytometry Western Analysis for measuring responses at the Protein level Immuno-blots Affinity Chromatography MW Phospho-Akt (Thr 473) OH H O H H N H N H N O OH H H H O O O HO H H 2 3 4 5 6 So, we find a potent drug candidate… It comes from a Deep Water Sponge… How do we get enough drug to treat anybody? (Without destroying the environment) We have a number of projects focused on Sustainable Use of Marine Resources Chemical Synthesis O O OH OH OH OH O O NH2 We have a number of projects focused on Sustainable Use of Marine Resources Invertebrate Cell Culture We have a number of projects focused on Sustainable Use of Marine Resources Aquaculture Polymastia in Rope Culture We have a number of projects focused on Sustainable Use of Marine Resources Recombinant Production Case Studies: Aphrocallistin Hexactinellida- “glass sponges” The Sea Urchin Embryo Assay 1.5 hours post fertilization Vehicle control Treated with antimitotic agent Embryos at 4-8 cell stage Cell Division arrested Rapid assay that detects compounds which block cell division Hexactinellida- Glass sponges Extraction with Ethanol n-butanol Partition C-18 Vacuum Column Chromatography Reverse phase MPLC with Combiflash Companion Crude extract Active Compound PANC-1 cells % Inhibition= 70% at 5 g/ml M+H+ m/z observed 539.04279 Sea urchin embryo division 100% Calculated for C20H25O2N6Br2 +2.2 mmu Inhibition at 100 g/ml 1H NMR Spectrum Molecular Formula: C20H24Br2O2N6 Structure of Major Compound N H H2N H2N N N H3C Br O OH O N Br CH2 N NH2 CH3 molokaiamine Modest activity against cancer cell lines PANC-1 IC50= 12 g/ml NCI-ADR-Res IC50= 15 g/ml CH3 Cell Cycle Analysis—Aphrocallistin Panc-1 cells; 24 hour treatment nontreated methanol 12.5 μg/ml aphrocallistin Aphrocallistin induces a G1 block in the cell cycle Flow cytometry on PANC-1 pancreatic carcinoma cells: Chk1 untreated Methanol control Aphrocallistin 12.5 µg/ml Example of Flow cytometry on PANC-1 cells: Chk2 untreated Methanol control Aphrocallistin 12.5 µg/ml Clear increase in levels of P-Chk2 (thr68) Aphrocallistin acts via ATM/ATR in PANC-1 cells Growth factors DNA damage Stress Chk1 P-Chk1 Chk2 P-Chk2 P-cyclinD G1 G0 Cyclin D CDK4/6 ATM/ATR CDC25A M p16INK4 Chk1/2 p19 p53 p27 CDK7 Cyclin A/B CDC2 p21 CDK2 Cyclin A/E Abl Rb DP1 E2F S DP1 G2 E2F Total Synthesis of Aphrocallistin Burnham Institute for Medical Research Aphrocallistin-Like Library Synthesis Stepwise Process: 1. Assemble key Building block: 1 2. Install Linker: 4 3. Add Heterocycle: 4 x 4 = 16 4. Deprotect then Add Acyl Groups: 16 x 12 = 192 Example 2: Leiodermatolide: A Potent Antimitotic Agent The cytoblot assay 24 hrs 1. Plate cells and allow to adhere 48 hrs 2. Treat with HBOI compounds and known inhibitor 3. Fix, Permeabilize, and block cells ECL LIGHT 4. Treat with targetspecific antibody 5. Treat with HRPconjugated secondary antibody Adapted from a protocol by Stockwell et al Chemistry and Biology 1999 6:71-83 The Phosphonucleolin Cytoblot TAXOL “ACTIVE” EXTRACT CYTOTOXIC Nucleolin is phosphorylated prior to mitosis Cells which are blocked at mitosis are detected using an antibody to phosphonucleolin and a secondary antibody tagged with luciferase Assay adapted from Stockwell et. al. Chemistry & Biology 1999, 6:71-83. Leiodermatium sp. • Extracts shows strong response in PN cytoblot assay • Cytotoxic against tumor cell lines: – PANC-1 – A549 – NCI/ADR-Res Purification Extraction with Ethanol-Ethyl acetate Solvent Partitioning Ethyl Acetate-Water Silica gel Vacuum Column Chromatography C-18 Reverse Phase HPLC Pure Compound Leiodermatolide H3C H H CH3 OH CH3 H H2N CH3 O O O H3C H HO O O O H H3C H CH3 Cell Line A549 (human lung) IC50 nM 3.3 nM PANC-1 (pancreatic) DLD-1 (colon) NCI-ADR-Res P388 (murine leukemia) 5.0 nM 8.3 nM 233 nM 3.3 nM Leiodermatolide blocks cell cycle progression at the G2/M checkpoint Methanol 10nM Leiodermatolide 100nM paclitaxel 1nM Leiodermatolide A549 Stained for Tubulin and with Propidium Iodide - Adherent Nontreated 1nM Leiodermatolide Methanol 10nM Leiodermatolide 100nM Taxol 100nM Leiodermatolide Leiodermatolide 10 nM Leiodermatolide does not directly bind tubulin Work is on-going to define its molecular target Microbial Production? H3C H H CH3 OH CH3 H H2N CH3 O O O H3C H HO O O O H H3C H CH3 Has O-carbamate Has signature C-1 alkylation Probably produced by Type I PKS This gives us key genes to search for •Ketosynthase •O-carbamoyl transferase •HMG-CO-A synthase Conclusions • The marine environment holds a wealth of natural products • If the compounds are coupled to the wealth of molecular targets defined by studies on the human genome and proteome… • and biotechnology is harnessed to produce them….then • the Oceans can play a major role in improving Human Health Many thanks to: • All my colleagues in the CMBBR • Mark Grasmuek and Gregor Eberli, RSMAS • Greg Roth, Daniela Divlianska, Jennifer Hoffman BIMR Lake Nona • Jill Roberts- Leiodermatolide; Diana Pechter –Aphrocallistin • Esther Guzmán, Tara Pitts, Pat Linley: Cell Biology • Our Funding Sources – National Institutes of Health, NCI – Center of Excellence in Biomedical and Marine Biotechnology – State of Florida, FWRI – National Science Foundation – Florida Sea Grant College Program – NOAA Office of Ocean Exploration – Gertrude E. Skelly Charitable Foundation – The Atlantic Foundation