parazoa porifera
DESCRIPTION
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 PresentationTRANSCRIPT
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