1. Parazoa Porifera Radiata e.g. Cnidaria Acoelomates Pseudocoelomates Protostomes (Schizocoelomates) Deuterostomes (Enterocoelomates) Mollusca Annelida Arthropoda

2. Cnidaria Produce Nematocyst Venoms Nematocysts Nematocysts (peptide toxins/venoms)

3. Parazoa Porifera Radiata e.g. Cnidaria Acoelomates Pseudocoelomates Protostomes (Schizocoelomates) Deuterostomes (Enterocoelomates) Mollusca Annelida Arthropoda

4. Acoelomates/ Pseudocoelomates Protostomes Endoprocta Ectoprocta Brachiopoda Phoronida Echinodermata Urochordata (“tunicates”) Vertebrata “Bryozoa” ? “Lophophores”

5. Acoelomates/ Pseudocoelomates Protostomes Endoprocta Ectoprocta Brachiopoda Phoronida Echinodermata Urochordata (“tunicates”) Vertebrata “Bryozoa” ? “Lophophores”

6. Phylum Bryozoa (Ectoprocta)

7. Approximately 5,000 species Mostly marine (~ 50 freshwater species) Colonial (except one species, Monobryozoan ambulans)

8. Phylum Bryozoa (Ectoprocta) “zooid” Zooecium Larva Ancestrula Colony

9. 500 kg (“wet animal”) CH 2 Cl 2 Extraction Solvent Partitioning: 9:1 to 4:1 MeOH/water with ligroin to CCl 4 “CCl 4 Fraction” (214 g) LH-20 Sephadex Si Gel Column Chromatography Recrystallize: CH 2 Cl 2 /MeOH Bryostatin-1 from Bugula neritina Bugula neritina Pettit et al. (1982) J. Am. Chem. Soc., 104:6846-8. “parallelepiped crystals”

10. Bryostatin 1 Pettit et al. (1982) J. Am. Chem. Soc., 104:6846-8. (1) m.p. 230-235°C; (2) EI-MS m/z 886 (M + - H 2 O, C 47 H 66 O 16 ; (3) FAB-MS m/z 904 (M + ); (4) Exact Mass 886.4375 amu (for C 47 H 66 O 16 ); (5) UV (MeOH) max 233 and 263 nm; (6) IR (KBr); (7) 400 MHz NMR; (8) X-Ray Crystallography.

11. 1 R=OAc, R’=OCO(CH) 4 n-Pr 2 R=OH, R’=OCO(CH) 4 n-Pr 4 R=OCOC(CH 3 ) 3, R’=OCOn-Pr 5 R=OCOC(CH 3 ) 3, R’=OAc 6 R=OCOn-Pr, R’=OAc 7 R=OAc, R’=OAc 8 R=OCOn-Pr. R’=OCOn-Pr 9 R=OAc, R’=OCOn-Pr 10 R=OCOC(CH 3 ) 3, R’=H 11 R=OAc, R’=H 12 R=OCO(CH) 3 n-Pr, R’=OCOn-Pr 13 R=OCOn-Pr, R’=H 14 R=OCOC(CH 3 ) 3, R’=OH 15 R=OAc, R’=OCO(CH) 4 CH(OH)Et 16 X=H, Y=COOCH 3 17 X=COOCH 3, Y=H 3 18 7 20

12. Bryostatins Activate Protein Kinase C (PKC)

13. Phosphatidylinositol- 4,5-Bisphosphate (PIP 2 ) Phospholipase C (PLC) R Protein Kinase C INACTIVE    G-Protein GDP

14. Phospholipase C (PLC)    RL Phosphatidylinositol- 4,5-Bisphosphate (PIP 2 ) Protein Kinase C INACTIVE G-Protein GTP

15. Diacylglycerol (DAG) 1, 4, 5-Phosphoinositol (IP 3 ) Phospholipase C (PLC)   RL Protein Kinase C INACTIVE  G-Protein GTP

16. Endoplasmic Reticulum Phospholipase C (PLC)   RL Protein Kinase C INACTIVE  G-Protein GTP

17. Endoplasmic Reticulum Ca 2+ Phospholipase C (PLC)   RL Protein Kinase C INACTIVE  G-Protein GTP

18. Phospholipase C (PLC)   RL Protein Kinase C Ca 2+  G-Protein GTP

19. Phospholipase C (PLC)   RL Protein Kinase C ACTIVE Ca 2+  G-Protein GTP Protein OPO 3 - ATP ADP

20. Phospholipase C (PLC)   RL Protein Kinase C ACTIVE Ca 2+  G-Protein GTP OPO 3 - ATP ADP

21. How does bryostatin inhibit cell proliferation? 1.Binds PKC Regulatory Domain (at same site as DAG) 2.Activates PKC via Self-Phosphorylation

22. Phospholipase C (PLC)   RL Protein Kinase C ACTIVE Ca 2+  G-Protein GTP OPO 3 - C1C1 C 26 C 19

23. Bind RequireDAG/ Ca2+? Bryostatin? Conventional YesYes ( ,  1,  2,  Novel NoYes (  Atypical NoNo (  Sub-Types of PKC

24. Kinase (Catalytic) Domain Cys-Rich Domain 2 (Binds Ca 2+ ) Cys-Rich Domain 1a/b (Binds DAG) Pseudo- Substrate Domain Kinase (Catalytic) Domain CRD2-Like Domain Cys-Rich Domain 1a/b (Binds DAG) Pseudo- Substrate Domain Kinase (Catalytic) Domain CRD1-Like Domain Pseudo- Substrate Domain Regulatory Catalytic Conventional NovelAtypical

25. Problem: Bryostatins and Tumor-Promoting Phorbol Esters Bind Same Site! Binds PKC CRD1 Promotes Cell- Proliferation (Tumor Promoter) Inhibits Cell- Proliferation (Tumor Inhibitor)

26. Phospholipase C (PLC)   RL PKC  ACTIVE Ca 2+  G-Protein GTP OPO 3 -

27. Phospholipase C (PLC)   RL Protein Kinase C ACTIVE Ca 2+  G-Protein GTP OPO 3 - C1C1 C 26 C 19

28. Phospholipase C (PLC)   RL Protein Kinase C ACTIVE Ca 2+  G-Protein GTP OPO 3 - C1C1 C 26 C 19

29. Phospholipase C (PLC)   RL  G-Protein GTP Ca 2+

30. Phospholipase C (PLC)   RL PKC  ACTIVE Ca 2+  G-Protein GTP OPO 3 - C1C1 C 26 C 19

31. Bryostatin Inhibits Down-Regulation of PKC  Phorbol Ester (e.g. TPA) Down- Regulates PKC 

32. Bryostatin Inhibits Down-Regulation of PKC  Bryostatin-1 Inhibits Down- Regulation of PKC 

33. Bryostatin-1 Down-Regulates PKC  in HOP-92 Cells (PMA Does NOT!) Inhibition of PKCd Down-Regulation is Cell- and Dose-Specific Choi et al. (2006) Cancer Res., 66: 7261-9 Bryostatin-1 Down- Regulation is Dose- Dependent

34. How does bryostatin inhibit cell proliferation? 1.Binds PKC Regulatory Domain (at same site as DAG) 2.Activates PKC via Self-Phosphorylation 3. Reduces BAX:Bcl-2 Leading to Apoptosis

35. Apoptosis is Programmed Cell-Death

36. Ratio of BAX:Bcl-2 is Determinant of Apoptosis BAX (Pro-Apoptotic) Bcl-2 (Anti-Apoptotic)

37. Bryostatin Reduces BAX:Bcl-2 1.Bryostatin-1 Increases Proteolytic Degradation of Bcl-2 2.Bryostatin-1 Decreases Bcl-2 mRNA (Wall et al. (2000) Int. J. Mol. Med., 5: 165-71) 3. Bryostatin Prevents Degradation of BAX (Wall et al. (1999) Leuk. Res., 23: 881-8)

38. Bryostatin Enhances Other Anticancer Drugs Cell-LineCytotoxic Cmpd.Effect HL-60Ara-CDoubled apoptotic cells U937TaxolDoubled apoptotic cells P388Tamoxifen200x growth inhibition RehAuristatin PE,Enhanced number of Vincristine apoptotic cells WSU-CLL2-CdAIncreased delayed of tumor growth (37 to 76 days) Cf. Mutter and Wills (2000) Bioorg. Med. Chem., 8: 1841-60

39. Bryostatin Pre-Clinical and Clinical Trials Pre-Clinical 1.[C26-3H]-Labelled Bryostatin-1 in CD1/F2 Mice - Half-Life (I.v.) = 22.97 h; Unmodified. - Urinary excretion (24 h), Fecal Excretion (>72 h) - Wide Distribution; Highest in Liver, Lungs and Bone Marrow (Zhang et al., 1996, Cancer Res., 56: 802) 2. Dose-Dependent Inhibition of Rabbit Papillomas (Bodily et al., 1999, Cancer Lett., 136: 67) 3. Inhibited Growth in WSU-CLL-Bearing SCID Mice with Co-Administration of 2-CdA (Mohammad et al., 1998, Clin. Cancer Res., 4: 445) 4. Cured (5/5) in WSU-CLL-Bearing SCID Mice with Co- Administration of Auristatin PE (Mohammad et al., 1998, Clin. Cancer Res., 4: 1337)

40. Bryostatin Pre-Clinical and Clinical Trials Clinical 1.43 Separate Phase I and II Clinical Trials 2.Side-Effects (Dose-Limiting Toxicity [LDT]): Myalgia 3.Maximum Dose 120 µg/m 2 per course (72-h infusion); Phase II Dose = 25-35 µg/m 2 Weekly 1 h, or 24 h 3x Every 4 weeks. 4.No “Cures” Alone; “Stable Disease State” for 19 Months 5.Co-Administration with Cisplatin or Vincristine: Partial Responses in 1 and 2 Patients, Respectively.

41. Bryostatin Present in Very Small Quantities in Bryozoans 1000 kg B. neritina 3.1 to 306 mg (3.1 x 10 -7 % to 3.0 x 10 -5 %) 1.5 to 8.6 g (1.5 to 8.6 x 10 -4 %) Highest ever reported: 15 mg from 1.5 kg (10 -3 %)

42. Cost - $500-600 / 50 µg Strategies Cultivation - CalBioChem Biosynthesis Synthetic studies Bryostatin Supply Issues

43. Biosynthesis of Bryostatins Cell-Free Extract from B. neritina Radiolabeled Precursor Acetate Glycerol S-Adenosylmethionine Propionate N-Butyrate Isobutyrate Succinate Cell-Free Extract from B. neritina Bryostatins Kerr et al. (1996) Tetrahedron Lett., 37: 8305.

44. Cost - $500-600 / 50 µg Strategies Cultivation - CalBioChem Biosynthesis Synthetic studies Bryostatin Supply Issues

45. Evan’s Total Synthesis of Bryostatin 2 Evans et al. (1999) J. Am. Chem. Soc., 121: 7540

46. Ecological role of bryostatins? Bryostatins Larva Adult