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Parazoa Porifera

Parazoa Porifera. Radiata e.g. Cnidaria. Acoelomates. Pseudocoelomates. Mollusca. Protostomes (Schizocoelomates). Annelida. Arthropoda. Deuterostomes (Enterocoelomates). Cnidaria Produce Nematocyst Venoms. Nematocysts. Nematocysts (peptide toxins/venoms). Parazoa Porifera.

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Parazoa Porifera

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

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

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

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

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

  6. Phylum Bryozoa (Ectoprocta)

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

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

  9. Bryostatin-1 from Bugula neritina Bugula neritina 500 kg (“wet animal”) CH2Cl2 Extraction Solvent Partitioning: 9:1 to 4:1 MeOH/water with ligroin to CCl4 “CCl4 Fraction” (214 g) LH-20 Sephadex Si Gel Column Chromatography Recrystallize: CH2Cl2 /MeOH “parallelepiped crystals” Pettit et al. (1982) J. Am. Chem. Soc., 104:6846-8.

  10. Bryostatin 1 (1) m.p. 230-235°C; (2) EI-MSm/z 886 (M+- H2O, C47H66O16; (3) FAB-MSm/z 904 (M+); (4) Exact Mass 886.4375 amu (for C47H66O16); (5) UV (MeOH) lmax 233 and 263 nm; (6) IR (KBr); (7) 400 MHz NMR; (8) X-Ray Crystallography. Pettit et al. (1982) J. Am. Chem. Soc., 104:6846-8.

  11. 1 R=OAc, R’=OCO(CH)4n-Pr 2 R=OH, R’=OCO(CH) 4n-Pr 4 R=OCOC(CH3)3, R’=OCOn-Pr 5 R=OCOC(CH3)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(CH3)3, R’=H 11 R=OAc, R’=H 12 R=OCO(CH)3n-Pr, R’=OCOn-Pr 13 R=OCOn-Pr, R’=H 14 R=OCOC(CH3)3, R’=OH 15 R=OAc, R’=OCO(CH)4CH(OH)Et 7 20 18 16 X=H, Y=COOCH3 17 X=COOCH3, Y=H 3

  12. Bryostatins Activate Protein Kinase C (PKC)

  13. G-Protein b R a g Phospholipase C (PLC) Phosphatidylinositol- 4,5-Bisphosphate (PIP2) GDP Protein Kinase C INACTIVE

  14. Phospholipase C (PLC) G-Protein b R L a g Phosphatidylinositol- 4,5-Bisphosphate (PIP2) GTP Protein Kinase C INACTIVE

  15. Phospholipase C (PLC) 1, 4, 5-Phosphoinositol (IP3) Diacylglycerol (DAG) G-Protein b R L a g GTP Protein Kinase C INACTIVE

  16. Endoplasmic Reticulum Phospholipase C (PLC) G-Protein b R L a g GTP Protein Kinase C INACTIVE

  17. Phospholipase C (PLC) G-Protein b Endoplasmic Reticulum R L a g GTP Ca2+ Protein Kinase C INACTIVE

  18. Phospholipase C (PLC) G-Protein b R L a g GTP Ca2+ Protein Kinase C

  19. Phospholipase C (PLC) ATP OPO3- ADP Protein G-Protein b R L a g GTP Ca2+ Protein Kinase C ACTIVE

  20. Phospholipase C (PLC) G-Protein b R L a g GTP ADP OPO3- Ca2+ ATP Protein Kinase C ACTIVE

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

  22. Phospholipase C (PLC) OPO3- G-Protein b R L a g GTP C1 C19 Ca2+ C26 Protein Kinase C ACTIVE

  23. Sub-Types of PKC Bind Require DAG/ Ca2+? Bryostatin? Conventional Yes Yes (a, b1, b2, g) Novel No Yes (d, e, h, q, m) Atypical No No (z, l)

  24. Conventional Novel Atypical Pseudo- Substrate Domain Pseudo- Substrate Domain CRD2-Like Domain CRD1-Like Domain Cys-Rich Domain 1a/b (Binds DAG) Pseudo- Substrate Domain Regulatory Kinase (Catalytic) Domain Cys-Rich Domain 2 (Binds Ca2+) Cys-Rich Domain 1a/b (Binds DAG) Kinase (Catalytic) Domain Kinase (Catalytic) Domain Catalytic

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

  26. Phospholipase C (PLC) OPO3- G-Protein b R L a g GTP Ca2+ PKCd ACTIVE

  27. Phospholipase C (PLC) OPO3- G-Protein b R L a g GTP C1 C19 Ca2+ C26 Protein Kinase C ACTIVE

  28. Phospholipase C (PLC) OPO3- G-Protein b R L a g GTP C1 C19 Ca2+ C26 Protein Kinase C ACTIVE

  29. Phospholipase C (PLC) G-Protein b R L a g GTP Ca2+

  30. Phospholipase C (PLC) OPO3- G-Protein b R L a g GTP C1 C19 Ca2+ C26 PKCd ACTIVE

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

  32. Bryostatin Inhibits Down-Regulation of PKCd Bryostatin-1 Inhibits Down-Regulation of PKCd

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

  34. How does bryostatin inhibit cell proliferation? Binds PKC Regulatory Domain (at same site as DAG) 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 Bryostatin-1 Increases Proteolytic Degradation of Bcl-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-Line Cytotoxic Cmpd. Effect HL-60 Ara-C Doubled apoptotic cells U937 Taxol Doubled apoptotic cells P388 Tamoxifen 200x growth inhibition Reh Auristatin PE, Enhanced number of Vincristine apoptotic cells WSU-CLL 2-CdA Increased 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 [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 43 Separate Phase I and II Clinical Trials Side-Effects (Dose-Limiting Toxicity [LDT]): Myalgia Maximum Dose 120 µg/m2 per course (72-h infusion); Phase II Dose = 25-35 µg/m2 Weekly 1 h, or 24 h 3x Every 4 weeks. No “Cures” Alone; “Stable Disease State” for 19 Months 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. Bryostatin Supply Issues Cost - $500-600 / 50 µg Strategies Cultivation - CalBioChem Biosynthesis Synthetic studies

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

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

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

  46. Bryostatins Ecological role of bryostatins? Adult Larva

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