Why so many biologically active compounds from invertebrates?

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Why so many biologically active compounds from
invertebrates?
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Drugs from the Sea Invertebrates
Microorganisms
Green Algae
Sponges!!
Brown Algae
Red Algae
Tunicates
Echinoderms
Mollusca
Cnidarians (e.g. Corals)
3
Overview
Introduction to Sponges (Porifera) Okadaic
Acid Protein Phosphatase Inhibitor
Discodermolide Potential Anticancer Drug?
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Drugs from the Sea Sponges
Out
Phylum Porifera
gt 10,000 species known
In
Oldest multicellular animal
Sessile
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Hungry Fish
150,000 bites/m2/day
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Chemical Defenses of Sponges
Mix with artificial food
Present to fish
Extract
Percentage () Eaten Sponge Control Treat
ed Acanthella acuta 100.0 6.3 Aplysina
aerophoba 89.8 8.2 Ianthella basta 94.0 6.0 A
xinella sp. 100.0 93.8 Crambe
crambe 94.4 2.8 Stylissa massa 100.0 2.8 Dys
idea avara 97.7 27.9 Ircinia
fasciculata 100.0 68.9 Petrosia
ficiformis 97.5 17.5
Control No extract added.
Paul and Puglisi (2004), Nat. Prod. Rep.,
21189-209 Paul et al. (2006) Nat. Prod. Rep.,
23153-80.
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Bioactive Compounds from Sponges Okadaic Acid
Halichondrin B
Okadaic Acid
Halichondria okadai
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Isolation of Okadaic Acid 1 (Tachibana and
Scheuer, Univ. of Hawaii Van Engen and Clardy,
Cornell University)
Halichondria okadai
1.MeOH (3x)/Acetone Extraction 2. Remove organic
solvent (70 aq.) 3.Hexane Wash (de-fatting) 4.
EtOAc Extraction
Mouse (i.p.) LC50 192 µg/kg KB Cytotoxicity
30 Inhibition (2.5 ng/mL) 80 Inhibition (5
ng/mL)
Polystyrene Gel, MeOH
LH-20, MeOH
Si Gel, n-Hexane/Acetone (51)
Crystallization (from MeOH)
Re-Crystallization (from CH2Cl2/Hex.)
Colorless Crystalline Solid (0.0001 wet wt.)
Tachibana et al. (1981) J. Am. Chem. Soc., 103
2469-71
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Isolation of Okadaic Acid 2 (Gopichand and
Schmitz, Univ. of Oklahoma)
H. melanodocia
1. 2-Propanol Extraction/H2O dilution 2. CH2Cl2
Extraction 3. 10 MeOH Suspension 4. 10-30
MeOH/Water Suspension 5. Hexane and CCl4
Wash/CHCl3 Ext.
Mouse (i.p.) gt120 µg/kg Cytotoxicity P388 -
ED50 1.7 x 103 L1210 - ED50 - 1.7 x 102 Tumor
Inhibition None (subtoxic dose)
LH-20 (MeOH/CHCl3, 11)
Silica Gel (CHCl3 to CHCl3 /5 MeOH)
Crystallization (from benzene)
Crystallization (from benzene/CHCl3)
White Crystalline Solid (0.0001 wet wt.)
Tachibana et al. (1981) J. Am. Chem. Soc., 103
2469-71
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Okadaic Acid Structure Elucidation
Okadaic Acid
MW 804.47 C44H8O13
UV, IR Uninformative
EI-MS m/z 786 (C44H66O12)
1H and 14C NMR
Acetylation (AcO, pyridine, 20 h, r.t.)
Tetraacetate (i.e. 4 hydroxyls)
Diazomethane Treatment Methyl Okadaate -gt 1H-NMR
Comparison to Acanthifolicin Absolute
Stereochemistry
Tachibana et al. (1981) J. Am. Chem. Soc., 103
2469-71
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Okadaic Acid Structure Elucidation
Triethyl-Ammonium Okadaate
X-Ray Diffraction
o-Bromobenzyl Bromide (in acetone), 36 h
(reflux)
Si Gel Chromatography
Crystallization (2x), CH2Cl2/Hexane
o-Bromobenzyl Okadaate
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Okadaic Acid is a Linear Polyether-Type
Polyketide
CO2

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Okadaic Acid Type 1/2A Phosphatase Inhibitor
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Protein Kinases/Phosphatases Biochemical
On/Off Switches
ATP
ADP
Kinase
Serine
Threonine
Phosphatase
Tyrosine
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Ser/Thr Protein Phosphatases (PP)
PP1
PP2A
PP4
PP5
PP2B (Calcineurin)
PP2C
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Ser/Thr Protein Phosphatases 1 and 2A (PP1/2A)
PP1 PP2A Catalytic Subunit PP1c (37
Kda) PP2Ac (36 Kda)
Distribution Myosin, Glycogen, Widely Chromat
in, S.R.
Endogenous I-1/DARPP-32, I-2, I-1PP2A,
I-2PP2A Inhibitors Dopamine, NIPP-1
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Okadaic Acid is a PP1/2A-Specific Inhibitor
Phosphatase Substrate ID50
(nM) PP1 PMLC 315 Phosphorylase a
272 PP2Ac PMLC 1.2 Phosphorylase a
1.6 PCM PMLC 205 Phosphorylase a
72 PP2B PMLC 4530 p-Nitrophenyl
Phosphate 3600 PP2C PMLC gt10,000 Phosphoryl
ase a gt10,000 Tyr Phosphatase -- gt10,000 Inosi
tol-1,4,5-triPP -- gt10,000 Acid
Phosphatase -- gt10,000 Alkaline
-- gt10,000 Phosphatase
Bialojan and Takai (1988) Biochem. J., 256 283-90
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The Okadaic Acid Class of Inhibitors
Peptides
( Nodularins)
Microcystins (Blue-Green Algae, e.g.
Microcystis)
Terpenoids
Cantharidin (Insects)
Thyrsiferyl-23-Acetate (L. obtusa, a Red Alga)
Other Polyketides
Dinophysisotoxin (Dinoflagellate) ()-Calyculin
(Sponge) Tautomycin (Streptomyces)
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Discodermolide Discovery
Depth 33 m
Lucaya
Discodermia dissoluta
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Discodermolide Isolation
Frozen/Thawed 434 g
Extracted MeOH/Toluene (31)
Partitioned EtOAc/Water
EtOAc
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
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Discodermolide Structure
White crystalline solid, mp 115-6 C UV
(MeOH) lmax 235 nm - conjugated dienes IR
(CHCl3) 3600-3500, 1725 cm-1 - hydroxyl and
carbonyl Low Resolution FAB-MS 550 Daltons
(M1) - CONH2 NMR 1H, 13C, COSY, HMQC,
HMBC NOT Stable at room temperature!
Gunasekara et al. (1990) J. Org. Chem., 55
4912-4915
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Discodermolide Structure
5.0 mg (in 1 mL pyridine)
0.5 mL acetic anhydride (overnight)
Acetylation
RP-HPLC (C18, 20 H2O/CH3CN)
4.5 mg
Gunasekara et al. (1990) J. Org. Chem., 55
4912-4915
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Discodermolide Structure
X-Ray Crystallography
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Discodermolide Synthesis/Structure
?
()-Discodermolide
(-)-Discodermolide
Nerenberg et al. (1993) J. Am. Chem. Soc.,
11512621-2 (and subsequent work by Schreiber
Group)
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Discodermolide Synthesis
Novartis Synthesis Scheme
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Discodermolide Inhibits Proliferation of Cells
Purified Murine (i.e. mouse) T-Cell IC50 9
nM Longley et al. (1991) Transplantation, 52
650-656
Various Human and Murine Cell-Lines IC50 3-80
nM Hung et al. (1994) Chem. Biol., 167-71
Estrogen-Receptor Positive/Negative Breast
Carcinoma (MCF-7/MDA-MB231) IC50 2.4 nM (48
h) Ter Haar et al. (1996) Biochemistry, 35243-50
NIH3T3 Cells IC50 Stage ()-Discodermolide
7 nM (G2/M) (-)-Discodermolide 135
nM (S) Hung et al. (1996) J. Am. Chem. Soc.,
11811054-80
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S
G2
Prophase
Metaphase
G1
Anaphase
M
Telophase
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Microtubules Comprised of Polymers of the Dimer
Tubulin
b

a
-
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Tubulin Polymerization Dependent on GTP/GDP
Hydrolysis
GDP
GTP
GTP
GTP
GTP
GDP


GTP
GTP
GTP
GTP
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Dynamic Instability of Microtubules
Tubulin-GTP
Tubulin-GDP
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Dynamic Instability of Microtubules
Tubulin-GTP
Tubulin-GDP
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Dynamic Instability of Microtubules
Tubulin-GTP
Tubulin-GDP
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Dynamic Instability of Microtubules
Tubulin-GTP
Tubulin-GDP
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Dynamic Instability of Microtubules
Tubulin-GTP
Tubulin-GDP
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Dynamic Instability of Microtubules
Tubulin-GTP
Tubulin-GDP
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Tubulin Polymerization and Depolymerization
Aligns Chromosomes During Metaphase
Tubulin-Polymerization
Dynein
-


-
Kinesin
Tubulin-Depolymerization
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Tubulin Polymerization and Depolymerization
Aligns Chromosomes During Metaphase
Polymerized Tubulin
Dynein
-


-
Kinesin
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Tubulin Polymerization and Depolymerization
Separates Chromosomes During Anaphase
Dynein
-

-

Tubulin Depolymerizes
Tubulin Depolymerizes
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Tubulin Polymerization and Depolymerization
Separates Chromosomes During Anaphase
Dynein
-

-

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()-Discodermolide Prevents Depolymerization of
Tubulin
Dynein
-

-

Tubulin Depolymerizes
Tubulin Depolymerizes
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()-Discodermolide Stabilizes Microtubules (i.e.
Inhibits Depolymerization)
Control
Discodermolide
42
S
G2
Mitosis-Promoting Factor (MPF)
Cyclin A/B
Cdk1 (a.k.a. cdc2)
Prophase
Metaphase
G1
Anaphase
M
Telophase
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Taxol (Paclitaxel)
From bark of Pacific Yew (Taxus brevifolia)
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Discodermolide Stabilizes Microtubules More Than
Taxol
10 µM Taxol, or 10 µM ()-Discodermolide
EC50 ()-Discodermolide 3.2 µM Taxol
(Paclitaxel) 23 µM
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Multi-Drug Resistant Cancer Cells Less Resistant
to Discodermolide
Level of Resistance Colon Ovarian
Carcinoma Carcinoma ()-Discodermolide 25-fold
89-fold Taxol 900-fold 2800-fold Compar
ed to parent line
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()-Discodermolide Binds to Same (or Overlapping
Site) as Taxol
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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 Trials Phase 1
20-80 patients safety, safe dose,
side-effects Phase 2 40-100 patients
effectiveness, further safety Phase 3 200
patients effectiveness, comparison, further
safety Phase 4 After drug marketed safety in
particular groups, long-term effects