1. Marine Biodiversity What Is It Good For ? David J. Newman, D.Phil. Chief, Natural Products Branch Developmental Therapeutics Program Division of Cancer Treatment & Diagnosis NCI-Frederick, Frederick, MD, 21702, USA +1.301.846.5397 Voice +1.301.846.6178 Facsimile Email dn22a@nih.govdn22a@nih.gov

2. Why Look There ? A question that is very frequently asked is the title of this short talk, and what I intend to try to do in the next 15 or so minutes is to show you what is going on from the perspective of drug discovery in utilization of the marine resources, meaning coral reefs, marine muds and sessile invertebrates, but the emphasis as will become apparent is on the organisms we cannot see. Because of my background, the focus is on cancer, but any of the materials may (will ?) have utility in other diseases.

3. -Complex polyketide natural product -Not very effective by itself in Phase II trials -Was in Phase I and Phase II trials with other cytotoxic drug therapy (e.g. Vincristine) to melanoma, kidney cancer and lymphoma Biosynthetic Source? Pettit,GR Fortschritte der Chemie organischer Naturstoffe. 1991, 57, 153-195. Bryostatin 1 Modulates Protein Kinase C

4. Bryozoan-Bacterial Symbiosis Produces the Anticancer Bryostatins (Haygood/Sherman) Not yet cultivated Found in almost all tested populations, adults and larvae (by PCR) Symbiont is a novel gamma proteobacterium (by 16S rRNA) –named “Candidatus Endobugula sertula” Pathway cloned with Sherman Lab Many novel biosynthetic aspects In situ hybridization with symbiont specific probe in larva Haygood et al, Chem Biol 2005, 12, 397 Sherman et al, J. Nat Prod 2007, 70, 67

5. Microbes and Tubulin Interactive Agents Tubulin as a target has a relatively long history, though not with agents from microbes (though just wait until a little later). With the discovery of the mechanism of action of Taxol ® by Susan Horwitz in 1979, a new target came into play for antitumor agents. The marine environment in particular has yielded some extremely interesting molecules that interact with tubulin in a variety of ways and that probably also involve microbes in their biosynthesis.

6. Potentially Microbial

7. Some Microbial Involvement ? May well be a new binding site

8. Other Marine-Derived Agents Tubulin, Proteasome, VDA and VoATPase

9. Kishi Synthesis of Halichondrin B, 1992 “Acyclic Stereochemistry Control” –Synthetic access to highly complex natural products New Ni/Cr-mediated coupling reaction to form C-C bonds –Nozaki-Hiyama-Kishi reaction Why halichondrin B? –Showcase Ni/Cr-mediated coupling –Highly potent anticancer activity (Hirata & Uemura, 1986) was “added value” Halichondrin B total synthesis: –Aicher TD, Buszek KR, Fang FG, Forsyth CJ, Jung SH, Kishi Y, Matelich MC, Scola PM, Spero DM, Yoon SK (1992) JACS 114:3162-3164 Professor Yoshito Kishi Department of Chemistry Harvard University 1,2,3-butane triol Asymmetric carbons: 2 Possible stereoisomers: 2 n = 2 2 = 4 Halichondrin B Asymmetric carbons: 32 Possible stereoisomers: 2 n = 2 32 = 4.3 x 10 9 Total synthesis created an opportunity to develop halichondrin B-based drugs from renewable resources

10. E7389: Synthetic Macrocyclic Ketone Analog of Halichondrin B’s “Right Half” ~200 analogues MW = 1110 32 stereocenters 0.2 nM (MDA-MB-435) MW = 730 19 stereocenters 0.1 nM (MDA-MB-435) Paclitaxel MW = 854 11 stereocenters 2.5 nM (MDA-MB-435) Eribulin, E7389 = NSC-707389 previously ER-086526, B1939

11. “Piece de Resistance”; “Seabed to Sickbed” The first “Direct from the Sea” drug to be approved for Cancer Treatment. EMEA 20SEP07 Just about every technique used; large-scale harvesting, aquaculture and semisynthesis from Cyanosafracin B

12. “You are what you eat” Dolabella auricularia Dolastatins come from a Symploca species that they graze on

13. Dolastatin 10 Dolastatin 10 and a Synthetic Analogue Auristatin PE Phase I (II)

14. Marine Sediments: Nereus Pharmaceuticals Marine Microbe Culture Collection Over 15,000 Strains ~50 % Actinomycetes; 10 New genera discovered ~50 % Fungi Fenical et al., Angew. Chem. Int. Ed., 42, 355-357 (2003) Sediment sampler

15. DISCOVERY AND DEVELOPMENT OF NPI-0052, A NOVEL PROTEASOME INHIBITOR FOR THE TREATEMENT OF CANCER Feb 2003: NPI-0052 (Salinosporamide A) structure, cytotoxicity and proteasome inhibitory activity established (Feling et al) 2Q 2007: Phase I Multiple Myeloma 2Q 2006: Phase I Solid Tumors and Lymphoma Mar 2006: X-Ray Crystal Structure in complex with 20S proteasome Dec 2005: NPI-0052 efficacy in mouse multiple myeloma xenograft models (Chauhan et al) Nov 2006: NPI-0052 efficacy in mouse colon cancer xenograft models (Cusack et al) Dec 2005: NPI-0052 IND Filed Preclinical Development (30 months) Preclinical models (in vitro/in vivo) API manufacturing (saline fermentation) Formulation development Drug product manufacturing Toxicology May 2003: FDA approves Velcade™ for treatment of multiple myeloma, validating the proteasome as a target for cancer treatment May 2004: Total synthesis (EJ Corey) June 2005: Total synthesis (Danishefsky) June 2007: Total synthesis (Ling et al) Oct 2002: Novel Marine actinomycete Salinispora discovered (Mincer et al) Salinosporamide Development Time Line Ray Lam, Nereus Pharma

16. Imperial Purple and The Cell Cycle Hexaplex trunculus (A) was extracted for Tyrian purple (B), various brominated indirubins (1–4) (C), and indigos. Oxime derivatives (5–8) and N1-methylated analogs (9–11) of these indirubins were synthesized, as well as the methoxime and acetoxime of 6-bromoindirubin (12, 13). 6-bromoindirubin-3-oxime (BIO) (7) and its control analog 1-methyl-6-bromoindirubin-3-oxime (MeBIO) (11) were used in the biological models.

17. Isolation of Indirubin-Binding Proteins Potential Anti-Alzheimer’s Treatment

18. Cell Cycle and Natural Products Modified from Meijer, 2003