Parazoa Porifera Radiata e.g. Cnidaria Acoelomates Pseudocoelomates Protostomes (Schizocoelomates) Deuterostomes (Enterocoelomates) Mollusca Annelida Arthropoda
Parazoa Porifera
Feb 05, 2016
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. - PowerPoint PPT Presentation
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ParazoaPorifera
Radiatae.g. Cnidaria
Acoelomates
Pseudocoelomates
Protostomes(Schizocoelomates)
Deuterostomes(Enterocoelomates)
Mollusca
Annelida
Arthropoda
Cnidaria Produce Nematocyst Venoms
Nematocysts
Nematocysts (peptide toxins/venoms)
ParazoaPorifera
Radiatae.g. Cnidaria
Acoelomates
Pseudocoelomates
Protostomes(Schizocoelomates)
Deuterostomes(Enterocoelomates)
Mollusca
Annelida
Arthropoda
Acoelomates/Pseudocoelomates
Protostomes
Endoprocta
Ectoprocta
Brachiopoda
Phoronida
Echinodermata
Urochordata (“tunicates”)
Vertebrata
“Bryozoa”?
“Lophophores”
Acoelomates/Pseudocoelomates
Protostomes
Endoprocta
Ectoprocta
Brachiopoda
Phoronida
Echinodermata
Urochordata (“tunicates”)
Vertebrata
“Bryozoa”
?
“Lophophores”
Phylum Bryozoa (Ectoprocta)
Phylum Bryozoa (Ectoprocta)
Approximately 5,000 species
Mostly marine (~ 50 freshwater species)
Colonial (except one species, Monobryozoan ambulans)
Phylum Bryozoa (Ectoprocta)
“zooid”
Zooecium
Larva Ancestrula Colony
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
Bryostatin-1 from Bugula neritina
Bugula neritina
Pettit et al. (1982) J. Am. Chem. Soc., 104:6846-8.
“parallelepiped crystals”
O O
O O
CH3
OH H
H3C
O
H3C
H3C
OH
OH
O
H3COHO
OAc
O
O
OCH3
OH3C
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+- H2O, C47H66O16; (3) FAB-MS m/z 904 (M+); (4) Exact Mass 886.4375 amu (for C47H66O16); (5) UV (MeOH) max 233 and 263 nm; (6) IR (KBr); (7) 400 MHz NMR; (8) X-Ray Crystallography.
O O
O O
CH3
R
OH H
H3C
O
H3C
H3C
R' OH
OH
O
H3COHO
O
OCH3
O O
O O
CH3
OAc
OH H
H3C
O
H3C
H3C
O OH
OH
O
H3COHO
On-Pr
O
O
O O
O O
CH3
OH H
H3C
O
H3C
H3C
OH
OH
O
H3COHO
O
O
O
H3CO
O O
O O
CH3
OH H
H3C
O
H3C
H3C
OH
OH
O
H3COHO
O
O
X Y
1 R=OAc, R’=OCO(CH)4n-Pr2 R=OH, R’=OCO(CH) 4n-Pr4 R=OCOC(CH3)3, R’=OCOn-Pr5 R=OCOC(CH3)3, R’=OAc6 R=OCOn-Pr, R’=OAc7 R=OAc, R’=OAc8 R=OCOn-Pr. R’=OCOn-Pr9 R=OAc, R’=OCOn-Pr10 R=OCOC(CH3)3, R’=H11 R=OAc, R’=H12 R=OCO(CH)3n-Pr, R’=OCOn-Pr13 R=OCOn-Pr, R’=H14 R=OCOC(CH3)3, R’=OH15 R=OAc, R’=OCO(CH)4CH(OH)Et
16 X=H, Y=COOCH3
17 X=COOCH3, Y=H3
18
7
20
Bryostatins Activate Protein Kinase C (PKC)
O
O
O
P
O
O
H
O-O
O
HO
OPO32-
2 -O3PO
OH
HO
Phosphatidylinositol- 4,5-Bisphosphate
(PIP2)
Phospholipase C (PLC)
R
Protein Kinase CINACTIVE
G-Protein
GDP
Phospholipase C (PLC)
R L
O
O
O
P
O
O
H
O-O
O
HO
OPO32-
2 -O3PO
OH
HO
Phosphatidylinositol- 4,5-Bisphosphate
(PIP2)
Protein Kinase CINACTIVE
G-Protein
GTP
O
O
OH
O
O
HP
O-O
O
HO
OPO32-
2 -O3PO
OH
HO
O-
Diacylglycerol(DAG)
1, 4, 5-Phosphoinositol(IP3)
Phospholipase C (PLC)
R L
Protein Kinase CINACTIVE
G-Protein
GTP
O
O
OH
O
O
HP
O-O
O
HO
OPO32-
2 -O3PO
OH
HO
O-
Endoplasmic Reticulum
Phospholipase C (PLC)
R L
Protein Kinase CINACTIVE
G-Protein
GTP
O
O
OH
O
O
H
Endoplasmic Reticulum
Ca2+
Phospholipase C (PLC)
R L
Protein Kinase CINACTIVE
G-Protein
GTP
O
O
OH
O
O
H
Phospholipase C (PLC)
R L
Protein Kinase CCa2+
G-Protein
GTP
O
O
OH
O
O
H
Phospholipase C (PLC)
R L
Protein Kinase CACTIVE
Ca2+
G-Protein
GTP
Protein
OPO3-
ATP
ADP
O
O
OH
O
O
H
Phospholipase C (PLC)
R L
Protein Kinase CACTIVE
Ca2+
G-Protein
GTP
OPO3-
ATP
ADP
How does bryostatin inhibit cell proliferation?
1. Binds PKC Regulatory Domain (at same site as DAG)
2. Activates PKC via Self-Phosphorylation
O
O
OH
O
O
H
Phospholipase C (PLC)
R L
Protein Kinase CACTIVE
Ca2+
G-Protein
GTP
OPO3-
O O
O O
CH3
OH H
H3 C
O
H3C
H3C
OH
OH
O
H3COHO
OAc
O
O
OCH3
OH3C
C1
C26
C19
BindRequire DAG/ Ca2+? Bryostatin?
Conventional Yes Yes(, 1, 2,
Novel No Yes(
Atypical No No(
Sub-Types of PKC
Kinase (Catalytic)Domain
Cys-RichDomain 2
(Binds Ca2+)
Cys-RichDomain 1a/b(Binds DAG)
Pseudo-SubstrateDomain
Kinase (Catalytic)Domain
CRD2-Like Domain
Cys-RichDomain 1a/b(Binds DAG)
Pseudo-SubstrateDomain
Kinase (Catalytic)Domain
CRD1-LikeDomain
Pseudo-SubstrateDomain
Regulatory
Catalytic
Conventional Novel Atypical
Problem: Bryostatins and Tumor-Promoting
Phorbol Esters Bind Same Site!
O
H
HO
O
C13H27
O
CH3
OCH3
CH3
O
H3C
OH
HO
H
H
H3C
O O
O O
CH3
OH H
H3C
O
H3C
H3C
OH
OH
O
H3COHO
OAc
O
O
OCH3
OH3C
Binds PKC
CRD1
Binds PKC
CRD1
Promotes Cell-Proliferation
(Tumor Promoter)
Inhibits Cell-Proliferation
(Tumor Inhibitor)
O
O
OH
O
O
H
Phospholipase C (PLC)
R L
PKCACTIVE
Ca2+
G-Protein
GTP
OPO3-
O
H
HO
O
C13H27
O
CH3
OCH3
CH3
O
H3C
OH
HO
H
H
H3C
O O
O O
CH3
OH H
H3C
O
H3C
H3C
OH
OH
O
H3COHO
OAc
O
O
OCH3
OH3C
O
O
OH
O
O
H
Phospholipase C (PLC)
R L
Protein Kinase CACTIVE
Ca2+
G-Protein
GTP
OPO3-
O O
O O
CH3
OH H
H3 C
O
H3C
H3C
OH
OH
O
H3COHO
OAc
O
O
OCH3
OH3C
C1
C26
C19
O
O
OH
O
O
H
Phospholipase C (PLC)
R L
Protein Kinase CACTIVE
Ca2+
G-Protein
GTP
OPO3-
O O
O O
CH3
OH H
H3 C
O
H3C
H3C
OH
OH
O
H3COHO
OAc
O
O
OCH3
OH3C
C1
C26
C19
O
O
OH
O
O
H
Phospholipase C (PLC)
R L
G-Protein
GTP
Ca2+
O O
O O
CH3
OH H
H3 C
O
H3C
H3C
OH
OH
O
H3COHO
OAc
O
O
OCH3
OH3C
O
O
OH
O
O
H
Phospholipase C (PLC)
R L
PKCACTIVE
Ca2+
G-Protein
GTP
OPO3-
O O
O O
CH3
OH H
H3 C
O
H3C
H3C
OH
OH
O
H3COHO
OAc
O
O
OCH3
OH3C
C1
C26
C19
Bryostatin Inhibits Down-Regulation of PKC
Phorbol Ester (e.g. TPA) Down-Regulates PKC
Bryostatin Inhibits Down-Regulation of PKC
Bryostatin-1 Inhibits Down-Regulation of
PKC
Bryostatin-1 Down-Regulates PKCin 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
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
Apoptosis is Programmed Cell-Death
Ratio of BAX:Bcl-2 is Determinant of Apoptosis
BAX (Pro-Apoptotic)
Bcl-2 (Anti-Apoptotic)
Bryostatin Reduces BAX:Bcl-2
1. Bryostatin-1 Increases Proteolytic Degradation of Bcl-22. 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)
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 ofVincristine apoptotic cells
WSU-CLL 2-CdA Increased delayed oftumor growth (37 to 76 days)
Cf. Mutter and Wills (2000) Bioorg. Med. Chem., 8: 1841-60
Bryostatin Pre-Clinical and Clinical Trials
Pre-Clinical1. [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)
Bryostatin Pre-Clinical and Clinical Trials
Clinical1. 43 Separate Phase I and II Clinical Trials
2. Side-Effects (Dose-Limiting Toxicity [LDT]): Myalgia
3. 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.
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.
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 %)
Cost - $500-600 / 50 µg
Strategies
Cultivation - CalBioChem
Biosynthesis
Synthetic studies
Bryostatin Supply Issues
Biosynthesis of Bryostatins
Cell-Free Extract from B. neritina
Radiolabeled Precursor
AcetateGlycerol
S-Adenosylmethionine
PropionateN-ButyrateIsobutyrateSuccinate
Cell-Free Extract from B. neritina
Bryostatins
Bryostatins
Kerr et al. (1996) Tetrahedron Lett., 37: 8305.
Cost - $500-600 / 50 µg
Strategies
Cultivation - CalBioChem
Biosynthesis
Synthetic studies
Bryostatin Supply Issues
Evan’s Total Synthesis of Bryostatin 2
Evans et al. (1999) J. Am. Chem. Soc., 121: 7540
Ecological role of bryostatins?Ecological role of bryostatins?
0
20
40
60
80
100
120
Control LarvalExtract
AdultExtract
O
OO
O
OO
OH
OH
OH
O
O
O
OH
O O
Bryostatins
Larva Adult
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