1. Competition between species Communication Defense against predators Defense against pathogens Why so many biologically active compounds from invertebrates?

2. Sponges!! Cnidarians (e.g. Corals) Microorganisms Tunicates Echinoderms Mollusca Red Algae Brown Algae Green Algae Drugs from the Sea: Invertebrates

3. Overview Introduction to Sponges (Porifera) Okadaic Acid: Protein Phosphatase Inhibitor Discodermolide: Potential Anticancer Drug?

4. Drugs from the Sea: Sponges Phylum Porifera > 10,000 species known Oldest multicellular animal In Out Sessile

5. Hungry Fish 150,000 bites/m 2 /day

6. Chemical Defenses of Sponges Percentage (%) Eaten SpongeControl*Treated Acanthella acuta100.06.3 Aplysina aerophoba89.88.2 Ianthella basta94.06.0 Axinella sp.100.093.8 Crambe crambe94.42.8 Stylissa massa100.02.8 Dysidea avara97.727.9 Ircinia fasciculata100.068.9 Petrosia ficiformis97.517.5 Extract Mix with artificial food Present to fish * Control = No extract added. Yuck! No, thank you! Reject Mmmm! Spongey. Accept Paul and Puglisi (2004), Nat. Prod. Rep., 21:189-209; Paul et al. (2006) Nat. Prod. Rep., 23:153-80.

7. Okadaic Acid Halichondria okadai Halichondrin B Bioactive Compounds from Sponges: Okadaic Acid

8. Halichondria okadai 1.MeOH (3x)/Acetone Extraction 2. Remove organic solvent (70% aq.) 3.Hexane Wash (“de-fatting”) 4. EtOAc Extraction Polystyrene Gel, MeOH LH-20, MeOH Si Gel, n-Hexane/Acetone (5:1) Crystallization (from MeOH) Re-Crystallization (from CH 2 Cl 2 /Hex.) Colorless Crystalline Solid (0.0001% wet wt.) Mouse (i.p.) LC 50 = 192 µg/kg KB Cytotoxicity 30% Inhibition (2.5 ng/mL) 80% Inhibition (5 ng/mL) Isolation of Okadaic Acid #1 (Tachibana and Scheuer, Univ. of Hawaii; Van Engen and Clardy, Cornell University) Tachibana et al. (1981) J. Am. Chem. Soc., 103: 2469-71

9. H. melanodocia Isolation of Okadaic Acid #2 (Gopichand and Schmitz, Univ. of Oklahoma) 1. 2-Propanol Extraction/H 2 O dilution 2. CH 2 Cl 2 Extraction 3. 10% MeOH Suspension 4. 10-30% MeOH/Water Suspension 5. Hexane and CCl 4 Wash/CHCl 3 Ext. LH-20 (MeOH/CHCl 3, 1:1) Silica Gel (CHCl 3 to CHCl 3 /5% MeOH) Crystallization (from benzene) Crystallization (from benzene/CHCl 3 ) White Crystalline Solid (0.0001% wet wt.) Mouse (i.p.) >120 µg/kg Cytotoxicity P388 - ED 50 = 1.7 x 10 3 L1210 - ED 50 - 1.7 x 10 2 Tumor Inhibition None (≤subtoxic dose) Tachibana et al. (1981) J. Am. Chem. Soc., 103: 2469-71

10. Okadaic Acid: Structure Elucidation Tachibana et al. (1981) J. Am. Chem. Soc., 103: 2469-71 UV, IR: Uninformative EI-MS: m/z 786 (C 44 H 66 O 12 ) 1 H and 14 C NMR MW 804.47 C 44 H 8 O 13 Okadaic Acid Diazomethane Treatment: Methyl Okadaate -> 1H-NMR Acetylation (AcO, pyridine, 20 h, r.t.): Tetraacetate (i.e. 4 hydroxyls) Comparison to Acanthifolicin: Absolute Stereochemistry

11. Okadaic Acid: Structure Elucidation Triethyl-Ammonium Okadaate + o-Bromobenzyl Bromide (in acetone), 36 h (reflux) o-Bromobenzyl Okadaate Si Gel Chromatography Crystallization (2x), CH 2 Cl 2 /Hexane X-Ray Diffraction

12. CO 2 + Okadaic Acid is a “Linear Polyether-Type” Polyketide

13. Okadaic Acid: Type 1/2A Phosphatase Inhibitor

14. Kinase Phosphatase Protein Kinases/Phosphatases: Biochemical “On/Off Switches” Serine Threonine Tyrosine ATPADP

15. Ser/Thr Protein Phosphatases (PP) PP1 PP2APP4PP5PP2B (Calcineurin) PP2C

16. PP1PP2A Catalytic SubunitPP1 c (37 Kda)PP2A c (36 Kda) Ser/Thr Protein Phosphatases 1 and 2A (PP1/2A) DistributionMyosin, Glycogen,Widely Chromatin, S.R. Endogenous I-1/DARPP-32, I-2, I-1 PP2A, I-2 PP2A InhibitorsDopamine, NIPP-1

17. PhosphataseSubstrate ID 50 (nM) PP1PMLC 315 Phosphorylase a 272 PP2A c PMLC 1.2 Phosphorylase a 1.6 PCMPMLC 205 Phosphorylase a 72 PP2BPMLC4530 p-Nitrophenyl Phosphate3600 PP2CPMLC>10,000 Phosphorylase a>10,000 Tyr Phosphatase-->10,000 Inositol-1,4,5-triPP-->10,000 Acid Phosphatase-->10,000 Alkaline -->10,000 Phosphatase Okadaic Acid is a PP1/2A-Specific Inhibitor Bialojan and Takai (1988) Biochem. J., 256: 283-90

18. The “Okadaic Acid Class of Inhibitors” Peptides Terpenoids Other Polyketides Microcystins (“Blue-Green Algae”, e.g. Microcystis) (+ Nodularins) Thyrsiferyl-23-Acetate (L. obtusa, a “Red Alga”) Cantharidin (Insects) Dinophysisotoxin (Dinoflagellate) (+)-Calyculin (Sponge) Tautomycin (Streptomyces)

19. Lucaya Discodermia dissoluta Depth: 33 m Discodermolide: Discovery

20. Discodermolide: Isolation Frozen/Thawed 434 g Extracted: MeOH/Toluene (3:1) Partitioned: EtOAc/Water EtOAcWater Column Chromatography (Silica Gel, CH 2 Cl 2 /MeOH) Reverse-Phase Chromatography (C18, H 2 O/MeOH) RP-HPLC (C18, 5µm, 250 x10 mm): 48% H 2 O/MeOH 7 mg (0.002%) Gunasekara et al. (1990) J. Org. Chem., 55: 4912-4915

21. Discodermolide: Structure White crystalline solid, mp = 115-6° C UV (MeOH): max 235 nm - conjugated dienes IR (CHCl 3 ) : 3600-3500, 1725 cm -1 - hydroxyl and carbonyl Low Resolution FAB-MS: 550 Daltons (M+1) + - CONH 2 NMR: 1H, 13C, COSY, HMQC, HMBC NOT Stable at room temperature! Gunasekara et al. (1990) J. Org. Chem., 55: 4912-4915

22. Discodermolide: Structure 5.0 mg (in 1 mL pyridine) 0.5 mL acetic anhydride (overnight) RP-HPLC (C18, 20% H 2 O/CH 3 CN) 4.5 mg Acetylation Gunasekara et al. (1990) J. Org. Chem., 55: 4912-4915

23. Discodermolide: Structure X-Ray Crystallography

24. Discodermolide: Synthesis/Structure ( + )-Discodermolide ( - )-Discodermolide Nerenberg et al. (1993) J. Am. Chem. Soc., 115:12621-2 (and subsequent work by Schreiber Group)

25. Discodermolide: Synthesis Novartis® Synthesis Scheme

26. Discodermolide Inhibits Proliferation of Cells Estrogen-Receptor Positive/Negative Breast Carcinoma (MCF-7/MDA-MB231): IC 50 = 2.4 nM (48 h) Ter Haar et al. (1996) Biochemistry, 35:243-50 Purified Murine (i.e. “mouse”) T-Cell: IC 50 = 9 nM Longley et al. (1991) Transplantation, 52: 650-656 Various Human and Murine Cell-Lines: IC 50 = 3-80 nM Hung et al. (1994) Chem. Biol., 1:67-71 NIH3T3 Cells: IC 50 Stage (+)-Discodermolide7 nM(G2/M) (-)-Discodermolide135 nM(S) Hung et al. (1996) J. Am. Chem. Soc., 118:11054-80

27. G1G1 G2G2 S M Mitosis-Promoting Factor (MPF) Cyclin A/B Cdk1 (a.k.a. cdc2) G0G0 G0G0 Cyclin D Cdk4/6 Cyclin E Cdk2 “Restriction Point” Cyclin A Cdk2 A, T, G, C + DNA Polymerase Prophase Metaphase Anaphase Telophase

28. + -   Microtubules Comprised of Polymers of the Dimer Tubulin

29. Tubulin Polymerization Dependent on GTP/GDP Hydrolysis GTP + + GDP GTP GDP

30. Tubulin-GTP Tubulin-GDP Dynamic Instability of Microtubules

31. Tubulin-GTP Tubulin-GDP Dynamic Instability of Microtubules

32. Tubulin-GTP Tubulin-GDP Dynamic Instability of Microtubules “GTP Cap”

33. Tubulin-GTP Tubulin-GDP Dynamic Instability of Microtubules

34. Tubulin-GTP Tubulin-GDP Dynamic Instability of Microtubules

35. Tubulin-GTP Tubulin-GDP Dynamic Instability of Microtubules

36. Dynein +- + - Kinesin Tubulin-Polymerization Tubulin-Depolymerization Tubulin Polymerization and Depolymerization Aligns Chromosomes During Metaphase

37. Dynein +- + - Kinesin Polymerized Tubulin

38. Tubulin Polymerization and Depolymerization Separates Chromosomes During Anaphase Dynein +-+ - Tubulin Depolymerizes

39. Tubulin Polymerization and Depolymerization Separates Chromosomes During Anaphase Dynein +-+ -

40. (+)-Discodermolide Prevents Depolymerization of Tubulin Dynein +-+ - Tubulin Depolymerizes

41. Control+ Discodermolide (+)-Discodermolide Stabilizes Microtubules (i.e. Inhibits Depolymerization)

42. G1G1 G2G2 S M Mitosis-Promoting Factor (MPF) Cyclin A/B Cdk1 (a.k.a. cdc2) Prophase Metaphase Anaphase Telophase (+)-Discodermolide inhibits depolymerization of tubulin (+)-Discodermolide prevents breakdown of Cyclin B

43. Taxol™ (Paclitaxel) * From bark of “Pacific Yew” (Taxus brevifolia)

44. EC 50 (+)-Discodermolide3.2 µM Taxol™ (Paclitaxel)23 µM Discodermolide Stabilizes Microtubules More Than Taxol™ + 10 µM Taxol, or 10 µM (+)-Discodermolide

45. Multi-Drug Resistant Cancer Cells Less Resistant to Discodermolide “Level of Resistance”* ColonOvarianCarcinoma (+)-Discodermolide25-fold89-fold Taxol900-fold2800-fold *Compared to parent line

46. (+)-Discodermolide Binds to Same (or Overlapping Site) as Taxol

47. Drug Approval: An Overview Discovery Pre-Clinical Toxicity/Pharmacology in vitro and in vivo (animal models, e.g. rodents) How much of the drug is absorbed in the blood? How is the drug broken down in the body? What is the toxicity of the drug and its breakdown products? How quickly does the body excrete the drug and its by-products? Synthesis and/or Purification Clinical Trials Phase 1: 20-80 patients; safety, safe dose, side-effects Phase 2: 40-100 patients; effectiveness, further safety Phase 3: 200+ patients; effectiveness, comparison, further safety Phase 4: After drug marketed; safety in particular groups, long-term effects FDA