Why so many biologically active
compounds from invertebrates?
Communication
Competition between species
Defense against pathogens
Defense
against
predators
Drugs from the Sea: Invertebrates
Microorganisms
15%
Green Algae
Brown Algae
Red Algae
Tunicates
1%
Sponges!!
2%
38%
6%
7%
Echinoderms 3%
Mollusca
7%
21%
Cnidarians
(e.g. Corals)
Overview
• Introduction to Sponges (Porifera)
• Okadaic Acid: Protein Phosphatase Inhibitor
• Discodermolide: Potential Anticancer Drug?
Drugs from the Sea: Sponges
Out
In
Phylum Porifera
> 10,000 species known
Oldest multicellular animal
Sessile
Hungry Fish
150,000 bites/m2/day
Chemical Defenses of Sponges
Mix with
artificial
food
Present
to fish
Extract
Sponge
Acanthella acuta
Aplysina aerophoba
Ianthella basta
Axinella sp.
Crambe crambe
Stylissa massa
Dysidea avara
Ircinia fasciculata
Petrosia ficiformis
Percentage (%) Eaten
Control*
Treated
100.0
6.3
89.8
8.2
94.0
6.0
100.0
93.8
94.4
2.8
100.0
2.8
97.7
27.9
100.0
68.9
97.5
17.5
Yuck! No,
thank you!
Reject
Mmmm!
Spongey.
Accept
* Control = No extract added.
Paul and Puglisi (2004), Nat. Prod. Rep., 21:189-209; Paul et al. (2006) Nat. Prod. Rep., 23:153-80.
Bioactive Compounds from Sponges:
Okadaic Acid
Halichondrin B
Okadaic Acid
Halichondria okadai
H
O
H
O
O
O
O
O
H
CH3
O
O
OH
O
H
H
O
O
O
H
O
H
O
H3C
O
H
HO
H3C
OH
O
OH
O
O
CH3
O
HO
O
O
H
H
OH
CH3
H
HO
HO
H
O
O
O
Isolation of Okadaic Acid #1
(Tachibana and Scheuer, Univ. of Hawaii; Van Engen and Clardy, Cornell University)
Halichondria okadai
Mouse (i.p.)
LC50 = 192 µg/kg
KB Cytotoxicity
30% Inhibition (2.5 ng/mL)
80% Inhibition (5 ng/mL)
1.MeOH (3x)/Acetone Extraction
2. Remove organic solvent (70% aq.)
3.Hexane Wash (“de-fatting”)
4. EtOAc Extraction
Polystyrene Gel, MeOH
LH-20, MeOH
Si Gel, n-Hexane/Acetone (5:1)
Crystallization (from MeOH)
Re-Crystallization (from CH2Cl2/Hex.)
Tachibana et al. (1981) J. Am.
Chem. Soc., 103: 2469-71
Colorless Crystalline Solid
(0.0001% wet wt.)
Isolation of Okadaic Acid #2
(Gopichand and Schmitz, Univ. of Oklahoma)
H. melanodocia
Mouse (i.p.)
>120 µg/kg
Cytotoxicity
P388 - ED50 = 1.7 x 103
L1210 - ED50 - 1.7 x 102
Tumor Inhibition
None (≤subtoxic dose)
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.
LH-20 (MeOH/CHCl3, 1:1)
Silica Gel (CHCl3 to CHCl3 /5% MeOH)
Crystallization (from benzene)
Crystallization (from benzene/CHCl3)
Tachibana et al. (1981) J. Am.
Chem. Soc., 103: 2469-71
White Crystalline Solid
(0.0001% wet wt.)
Okadaic Acid: Structure Elucidation
Okadaic Acid
O
MW 804.47
C44H8O13
OH
O
O
HO
OH
O
OH
O
O
O
UV, IR: Uninformative
EI-MS: m/z 786 (C44H66O12)
1H and 14C NMR
O
OH
Acetylation (AcO, pyridine, 20 h, r.t.):
Tetraacetate (i.e. 4 hydroxyls)
Diazomethane Treatment: Methyl Okadaate -> 1H-NMR
Comparison to Acanthifolicin: Absolute Stereochemistry
Tachibana et al. (1981) J. Am. Chem. Soc., 103: 2469-71
Okadaic Acid: Structure Elucidation
O
OH
O
O
HO
OH
O
OH
O
O
O
O
OH
Triethyl-Ammonium Okadaate
X-Ray Diffraction
+ o-Bromobenzyl Bromide (in acetone),
36 h (reflux)
Si Gel Chromatography
Crystallization (2x), CH2Cl2/Hexane
o-Bromobenzyl Okadaate
O
O
HOOC
OH
OH
O
Okadaic Acid is a
“Linear Polyether-Type”
Polyketide
O
HOOC
S
CoA
S
CoA
O
O
OH
O
O
O
HO
OH
HOOC
S
O
O
O
OH
Enz
S
O
O
ACP
OH
CO2
O
O
S
O
ACP
O
O
O
+
S
Enz
HOOC
S
ACP
O
O
O
Okadaic Acid: Type 1/2A
Phosphatase Inhibitor
Protein Kinases/Phosphatases:
Biochemical “On/Off Switches”
ATP
ADP
O
Kinase
OH
O
O
O
N
H
OH
O-
N
H
O
O
Serine
OH
O
O
O
N
H
Threonine
P
O
P
OH
O-
N
H
O
O
O
OH
O
P
O-
O
O
N
H
Tyrosine
N
H
O
O
Phosphatase
OH
Ser/Thr Protein Phosphatases (PP)
PP1
PP2A
PP4
PP5
PP2B
(Calcineurin)
PP2C
Ser/Thr Protein Phosphatases 1 and 2A
(PP1/2A)
PP1
PP2A
Catalytic Subunit PP1c (37 Kda)
PP2Ac (36 Kda)
Distribution
Myosin, Glycogen,
Chromatin, S.R.
Widely
Endogenous
Inhibitors
I-1/DARPP-32, I-2,
Dopamine, NIPP-1
I-1PP2A, I-2PP2A
Okadaic Acid is a PP1/2A-Specific Inhibitor
Phosphatase
PP1
PP2Ac
PCM
PP2B
PP2C
Tyr Phosphatase
Inositol-1,4,5-triPP
Acid Phosphatase
Alkaline
Phosphatase
Substrate
PMLC
Phosphorylase a
PMLC
Phosphorylase a
PMLC
Phosphorylase a
PMLC
p-Nitrophenyl Phosphate
PMLC
Phosphorylase a
-----
ID50 (nM)
315
272
1.2
1.6
205
72
4530
3600
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
Bialojan and Takai (1988) Biochem. J., 256: 283-90
The “Okadaic Acid Class of Inhibitors”
CO2H
CH3
O
N
HN
NH
H3C
O
OCH 3
Peptides
CH2
H3C
O
O
NH
CH3
CH3
H
N
CH3
H
N
(+ Nodularins)
CH 3
HN
NH2
O
OH
CO2 H
O
Microcystins
(“Blue-Green Algae”, e.g. Microcystis)
HN
Terpenoids
CH 3
O
O
H
HN
OAc
O
O
O
O
OH
O
H3C
O
H
Br
H
Thyrsiferyl-23-Acetate
(L. obtusa, a “Red Alga”)
Other Polyketides
Dinophysisotoxin (Dinoflagellate)
(+)-Calyculin (Sponge)
Tautomycin (Streptomyces)
O
H3C
Cantharidin
(Insects)
Discodermolide: Discovery
Depth: 33 m
Lucaya
Discodermia dissoluta
Discodermolide: Isolation
Frozen/Thawed
434 g
Extracted: MeOH/Toluene (3:1)
EtOAc
Partitioned: EtOAc/Water
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: Structure
O
O
OH
OH
O
O
OH
OH
NH2
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 (M+1)+ - CONH2
NMR: 1H, 13C, COSY, HMQC, HMBC
NOT Stable at room temperature!
Gunasekara et al. (1990) J. Org. Chem., 55: 4912-4915
Discodermolide: Structure
O
O
OH
OH
O
O
OH
OH
NH2
5.0 mg (in 1 mL pyridine)
0.5 mL acetic anhydride (overnight)
Acetylation
RP-HPLC (C18, 20% H2O/CH3CN)
O
O
O
O
O
O
O
O
NH2
4.5 mg
Gunasekara et al. (1990) J. Org. Chem., 55: 4912-4915
Discodermolide: Structure
X-Ray Crystallography
Discodermolide:
Synthesis/Structure
HO
O
OH
OH
O

O
O
OH
NH 2
(+)-Discodermolide
HO
O
OH
OH
O
O
O
OH
NH 2
(-)-Discodermolide
Nerenberg et al. (1993) J. Am. Chem. Soc., 115:12621-2 (and subsequent work by Schreiber Group)
Discodermolide: Synthesis
Novartis® Synthesis Scheme
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., 1:67-71
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
NIH3T3 Cells:
(+)-Discodermolide
(-)-Discodermolide
Hung et al. (1996) J. Am. Chem. Soc., 118:11054-80
IC50
7 nM
135 nM
Stage
(G2/M)
(S)
A, T, G, C
+ DNA Polymerase
S
Cyclin A
G2
Cdk2
Mitosis-Promoting
Factor (MPF)
Cyclin A/B
Cdk1 (a.k.a. cdc2)
“Restriction
Point”
Prophase
Cyclin E
Metaphase
Cdk2
G1
G0
Cyclin D
Cdk4/6
G0
Anaphase
Telophase
M
Microtubules Comprised of
Polymers of the Dimer Tubulin
b
+
a
-
Tubulin Polymerization
Dependent on GTP/GDP
O
O
N
HN
O
-O
P
O-
O
O
P
O-
O
O
H2N
P
O
N
N
Hydrolysis
O
HO
CH2
P
O
O-
O-
OH
GTP
H2N
P
O
N
CH2
OH
GTP GTP
GDP
GTP
+
GTP
N
O-
OH
+
GTP
O
GDP
GTP
GTP
N
HN
GTP GTP
GTP
GTP
OH
Dynamic
Instability of
Microtubules
Tubulin-GTP
Tubulin-GDP
Dynamic
Instability of
Microtubules
Tubulin-GTP
Tubulin-GDP
Dynamic
Instability of
Microtubules
Tubulin-GTP
Tubulin-GDP
“GTP Cap”
Dynamic
Instability of
Microtubules
Tubulin-GTP
Tubulin-GDP
Dynamic
Instability of
Microtubules
Tubulin-GTP
Tubulin-GDP
Dynamic
Instability of
Microtubules
Tubulin-GTP
Tubulin-GDP
Tubulin Polymerization and
Depolymerization Aligns
Chromosomes During Metaphase
Tubulin-Polymerization
Dynein
-
+
+
-
Kinesin
Tubulin-Depolymerization
Tubulin Polymerization and
Depolymerization Aligns
Chromosomes During Metaphase
Polymerized Tubulin
Dynein
-
+
+
Kinesin
-
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
(+)-Discodermolide Stabilizes
Microtubules (i.e. Inhibits
Depolymerization)
Control
+ Discodermolide
S
G2
Cyclin A/B
(+)-Discodermolide
inhibits
depolymerization
of tubulin
Cdk1 (a.k.a. cdc2)
Prophase
Metaphase
G1
Anaphase
Telophase
Mitosis-Promoting
Factor (MPF)
M
(+)-Discodermolide
prevents breakdown
of Cyclin B
O
CH3
O
O
H3C
O
O
CH3
N
H
OH
O
OH
HO
O
OH C
3
O
O
O
Taxol™ (Paclitaxel)
* From bark of “Pacific Yew” (Taxus brevifolia)
Discodermolide Stabilizes
Microtubules More Than Taxol™
+ 10 µM Taxol, or 10 µM
(+)-Discodermolide
(+)-Discodermolide
EC50
3.2 µM
Taxol™ (Paclitaxel)
23 µM
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
*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
FDA
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