competition between species communication defense against predators defense against pathogens why so...
TRANSCRIPT
Competition between species
Communication
Defense against predators
Defense against pathogens
Why so many biologically active Why so many biologically active compounds from invertebrates?compounds from invertebrates?
Sponges!!
Cnidarians (e.g. Corals)
Microorganisms
Tunicates
Echinoderms
Mollusca
Red Algae
Brown Algae
Green Algae
38%
21%
7%
3%
7%
6%
2%
1%
15%
Drugs from the Sea: Drugs from the Sea: InvertebratesInvertebrates
Overview
• Introduction to Sponges (Porifera)
• Okadaic Acid: Protein Phosphatase Inhibitor
• Discodermolide: Potential Anticancer Drug?
Drugs from the Sea: Drugs from the Sea: SpongesSponges
Phylum Porifera> 10,000 species known
Oldest multicellular animalIn
Out
Sessile
Hungry FishHungry Fish
150,000 bites/m2/day
Chemical Defenses of SpongesChemical Defenses of Sponges
Percentage (%) EatenSponge Control* TreatedAcanthella acuta 100.0 6.3Aplysina aerophoba 89.8 8.2Ianthella basta 94.0 6.0Axinella sp. 100.0 93.8Crambe crambe 94.4 2.8Stylissa massa 100.0 2.8Dysidea avara 97.7 27.9Ircinia fasciculata 100.0 68.9Petrosia ficiformis 97.5 17.5
Extract
Mix with artificial
foodPresent 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.
Okadaic Acid Halichondria okadai
HO
O
O O
O
O O
O
O
CH3
CH3H
CH3
H
HOH
OH
H
OH
H3C OH
H3C
HO
O
HO
HO
OO
O
O
O
O
O
OO
O
O
O
OOO
H
H
H
H
H
H
H
H
Halichondrin B
Bioactive Compounds from Sponges: Bioactive Compounds from Sponges: Okadaic AcidOkadaic Acid
Halichondria okadai
1.MeOH (3x)/Acetone Extraction2. Remove organic solvent (70% aq.)3.Hexane Wash (“de-fatting”)4. EtOAc Extraction
Polystyrene Gel, MeOH
LH-20, MeOHSi Gel, n-Hexane/Acetone (5:1)
Crystallization (from MeOH)
Re-Crystallization (from CH2Cl2/Hex.)
Colorless Crystalline Solid (0.0001% wet wt.)
Mouse (i.p.) LC50 = 192 µg/kgKB Cytotoxicity 30% Inhibition (2.5 ng/mL) 80% Inhibition (5 ng/mL)
Isolation of Okadaic Acid #1Isolation of Okadaic Acid #1(Tachibana and Scheuer, Univ. of Hawaii; Van Engen and Clardy, Cornell University)(Tachibana and Scheuer, Univ. of Hawaii; Van Engen and Clardy, Cornell University)
Tachibana et al. (1981) J. Am. Chem. Soc., 103: 2469-71
H. melanodocia
Isolation of Okadaic Acid #2Isolation of Okadaic Acid #2(Gopichand and Schmitz, Univ. of Oklahoma)(Gopichand and Schmitz, Univ. of Oklahoma)
1. 2-Propanol Extraction/H2O dilution2. CH2Cl2 Extraction3. 10% MeOH Suspension4. 10-30% MeOH/Water Suspension5. Hexane and CCl4 Wash/CHCl3 Ext.
LH-20 (MeOH/CHCl3, 1:1)
Silica Gel (CHCl3 to CHCl3 /5% MeOH)
Crystallization (from benzene)
Crystallization (from benzene/CHCl3)
White Crystalline Solid (0.0001% wet wt.)
Mouse (i.p.) >120 µg/kgCytotoxicity P388 - ED50 = 1.7 x 103
L1210 - ED50 - 1.7 x 102
Tumor Inhibition None (≤subtoxic dose)
Tachibana et al. (1981) J. Am. Chem. Soc., 103: 2469-71
HO
O
O O
O O
OO
OH
OH
OH
O
OH
Okadaic Acid: Structure ElucidationOkadaic Acid: Structure Elucidation
Tachibana et al. (1981) J. Am. Chem. Soc., 103: 2469-71
UV, IR: UninformativeEI-MS: m/z 786 (C44H66O12)1H and 14C NMR
MW 804.47C44H8O13
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
Okadaic Acid: Structure ElucidationOkadaic Acid: Structure Elucidation
HO
O
O O
O O
OO
OH
OH
OH
O
OH
Triethyl-Ammonium Okadaate
+ o-Bromobenzyl Bromide (in acetone), 36 h (reflux)
o-Bromobenzyl Okadaate
Si Gel Chromatography
Crystallization (2x), CH2Cl2/Hexane
X-Ray Diffraction
OH
O
OH
O
HOOC
S
O
CoA S
O
HOOC
CoA
S
O
Enz S
O
HOOC
ACP
S
O
ACP
O
S
O
Enz
O OOO O
CO2
S
O
HOOC
ACP
+
HO
O
O O
O O
OO
OH
OH
OH
O
OH
Okadaic Acid is a “Linear Polyether-Type”
Polyketide
Okadaic Acid: Type 1/2A Okadaic Acid: Type 1/2A Phosphatase InhibitorPhosphatase Inhibitor
NH
O
O
OH
NH
O
O
OP
O
O-
OH
NH
O
O
OH
NH
O
O
OP
O
O-
OH
NH
O
O
OH
NH
O
O
OP
O
OH
O-
Kinase
Phosphatase
Protein Kinases/Phosphatases: Protein Kinases/Phosphatases: Biochemical “On/Off Switches”Biochemical “On/Off Switches”
Serine
Threonine
Tyrosine
ATP ADP
Ser/Thr Protein Phosphatases (PP)Ser/Thr Protein Phosphatases (PP)
PP1 PP2A PP4 PP5 PP2B(Calcineurin)
PP2C
PP1 PP2A
Catalytic Subunit PP1c (37 Kda) PP2Ac (36 Kda)
Ser/Thr Protein Phosphatases 1 and 2A Ser/Thr Protein Phosphatases 1 and 2A (PP1/2A)(PP1/2A)
Distribution Myosin, Glycogen, WidelyChromatin, S.R.
Endogenous I-1/DARPP-32, I-2, I-1PP2A, I-2PP2A
Inhibitors Dopamine, NIPP-1
Phosphatase Substrate ID50 (nM)PP1 PMLC 315
Phosphorylase a 272PP2Ac PMLC 1.2
Phosphorylase a 1.6PCM PMLC 205
Phosphorylase a 72PP2B PMLC 4530
p-Nitrophenyl Phosphate 3600PP2C PMLC >10,000
Phosphorylase a >10,000Tyr Phosphatase -- >10,000Inositol-1,4,5-triPP -- >10,000Acid Phosphatase -- >10,000Alkaline -- >10,000 Phosphatase
Okadaic Acid is a Okadaic Acid is a PP1/2A-SpecificPP1/2A-Specific Inhibitor Inhibitor
Bialojan and Takai (1988) Biochem. J., 256: 283-90
The “Okadaic Acid Class of Inhibitors”
OCH3
CH3 CH3
HN
N
NH
HNNH
HN
O
HN
CH3
CH3
O
O
HN
HN NH2
O
O CH2
O
CO2H
CH3
CO2H
H3C
CH3
H3C
O
O
O
O
OH
OOAc
OH
H H
H
Br
O
OO
O
H3C
H3C
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)
Lucaya
Discodermia dissoluta
Depth: 33 m
Discodermolide: DiscoveryDiscodermolide: Discovery
Discodermolide: IsolationDiscodermolide: Isolation
Frozen/Thawed434 g
Extracted: MeOH/Toluene (3:1)
Partitioned: EtOAc/WaterEtOAc Water
Column Chromatography (Silica Gel, CH2Cl2/MeOH)
Reverse-Phase Chromatography (C18, H2O/MeOH)
RP-HPLC (C18, 5µm, 250 x10 mm):48% H2O/MeOH
7 mg(0.002%)
Gunasekara et al. (1990) J. Org. Chem., 55: 4912-4915
Discodermolide: StructureDiscodermolide: Structure
White crystalline solid, mp = 115-6° C
UV (MeOH): max 235 nm - conjugated dienesIR (CHCl3) : 3600-3500, 1725 cm-1 - hydroxyl and carbonyl
Low Resolution FAB-MS: 550 Daltons (M+1)+ - CONH2
NMR: 1H, 13C, COSY, HMQC, HMBC
NOT Stable at room temperature!
OO
OH
OH
OH O
O
NH2OH
Gunasekara et al. (1990) J. Org. Chem., 55: 4912-4915
Discodermolide: StructureDiscodermolide: Structure
OO
OH
OH
OH O
O
NH2OH
5.0 mg (in 1 mL pyridine)
0.5 mL acetic anhydride (overnight)
RP-HPLC (C18, 20% H2O/CH3CN)
OO
O
O
O O
O
NH2O
4.5 mg
Acetylation
Gunasekara et al. (1990) J. Org. Chem., 55: 4912-4915
Discodermolide: StructureDiscodermolide: Structure
X-Ray Crystallography
Discodermolide: Discodermolide: Synthesis/StructureSynthesis/Structure
O OH
OH
OH O
O
NH2
HO
O
O OH
OH
OH O
O
NH2
HO
O
(+)-Discodermolide
(-)-Discodermolide
Nerenberg et al. (1993) J. Am. Chem. Soc., 115:12621-2 (and subsequent work by Schreiber Group)
Discodermolide: SynthesisDiscodermolide: Synthesis
Novartis® Synthesis Scheme
Discodermolide Inhibits Proliferation of Cells
Estrogen-Receptor Positive/Negative Breast Carcinoma (MCF-7/MDA-MB231): IC50 = 2.4 nM (48 h)Ter Haar et al. (1996) Biochemistry, 35:243-50
Purified Murine (i.e. “mouse”) T-Cell: IC50 = 9 nMLongley et al. (1991) Transplantation, 52: 650-656
Various Human and Murine Cell-Lines: IC50 = 3-80 nMHung et al. (1994) Chem. Biol., 1:67-71
NIH3T3 Cells:IC50 Stage
(+)-Discodermolide 7 nM (G2/M)(-)-Discodermolide 135 nM (S)
Hung et al. (1996) J. Am. Chem. Soc., 118:11054-80
G1
G2S
M
Mitosis-Promoting Factor (MPF)
Cyclin A/B
Cdk1 (a.k.a. cdc2)
G0
G0
Cyclin D
Cdk4/6
Cyclin E
Cdk2
“Restriction Point”
Cyclin A
Cdk2
A, T, G, C + DNA Polymerase
Prophase
Metaphase
AnaphaseTelophase
+
-
Microtubules Comprised of Polymers of the Dimer Tubulin
Tubulin Polymerization Dependent on GTP/GDP
HN
N
N
N
O
OHOH
CH2OP
O
O-
OP
O
O-
OP
O
O-
-O
H2N
HN
N
N
N
O
OHOH
CH2OP
O
O-
OP
O
O-
HO
H2NHydrolysis
GTP
GTP+
GTP
GTP
GTP
GTP
GTP
GTP
GTP
GTP+
GTP
GDP
GTP GDP
Tubulin-GTP
Tubulin-GDP
Dynamic Instability of Microtubules
Tubulin-GTP
Tubulin-GDP
Dynamic Instability of Microtubules
Tubulin-GTP
Tubulin-GDP
Dynamic Instability of Microtubules
“GTP Cap”
Tubulin-GTP
Tubulin-GDP
Dynamic Instability of Microtubules
Tubulin-GTP
Tubulin-GDP
Dynamic Instability of Microtubules
Tubulin-GTP
Tubulin-GDP
Dynamic Instability of Microtubules
Dynein
+ -+-
Kinesin
Tubulin-Polymerization
Tubulin-Depolymerization
Tubulin Polymerization and Depolymerization Aligns
Chromosomes During Metaphase
Tubulin Polymerization and Depolymerization Aligns
Chromosomes During Metaphase
Dynein
+ -+-
Kinesin
Polymerized Tubulin
Tubulin Polymerization and Depolymerization Separates
Chromosomes During Anaphase
Dynein
+ -+-
Tubulin Depolymerizes Tubulin Depolymerizes
Tubulin Polymerization and Depolymerization Separates
Chromosomes During Anaphase
Dynein
+ -+-
(+)-Discodermolide Prevents Depolymerization of Tubulin
Dynein
+ -+-
Tubulin Depolymerizes Tubulin Depolymerizes
Control + Discodermolide
(+)-Discodermolide Stabilizes Microtubules (i.e. Inhibits
Depolymerization)
G1
G2S
M
Mitosis-Promoting Factor (MPF)
Cyclin A/B
Cdk1 (a.k.a. cdc2)
Prophase
Metaphase
AnaphaseTelophase
(+)-Discodermolide inhibits depolymerization of tubulin
(+)-Discodermolide prevents breakdown of Cyclin B
O
NH
O
O
OH
H3CO
O
O
CH3
O
O
CH3 OH
OO
H3C O
HO
Taxol™ (Paclitaxel)
* From bark of “Pacific Yew” (Taxus brevifolia)
EC50
(+)-Discodermolide 3.2 µM
Taxol™ (Paclitaxel) 23 µM
Discodermolide Stabilizes Microtubules More Than Taxol™
+ 10 µM Taxol, or 10 µM (+)-Discodermolide
Multi-Drug Resistant Cancer Cells Less Resistant to Discodermolide
“Level of Resistance”*
Colon OvarianCarcinoma Carcinoma
(+)-Discodermolide 25-fold 89-fold
Taxol 900-fold 2800-fold
*Compared to parent line
(+)-Discodermolide Binds to Same (or Overlapping Site) as
Taxol
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 TrialsPhase 1: 20-80 patients; safety, safe dose, side-effectsPhase 2: 40-100 patients; effectiveness, further safetyPhase 3: 200+ patients; effectiveness, comparison, further safetyPhase 4: After drug marketed; safety in particular groups, long-term effects
FDA