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Communication. Competition between species. Defense against pathogens. Defense against predators. Why so many biologically active compounds from invertebrates?. Drugs from the Sea: Invertebrates. Microorganisms. Green Algae. Sponges!!. Brown Algae. Red Algae. Tunicates. Echinoderms.

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slide1

Communication

Competition between species

Defense against pathogens

Defense against predators

Why so many biologically active compounds from invertebrates?

slide2

Drugs from the Sea: Invertebrates

Microorganisms

Green Algae

Sponges!!

Brown Algae

Red Algae

Tunicates

Echinoderms

Mollusca

Cnidarians (e.g. Corals)

slide3

Overview

•Introduction to Sponges (Porifera)

•Okadaic Acid: Protein Phosphatase Inhibitor

•Discodermolide: Potential Anticancer Drug?

slide4

Drugs from the Sea: Sponges

Out

Phylum Porifera

> 10,000 species known

In

Oldest multicellular animal

Sessile

slide5

Hungry Fish

150,000 bites/m2/day

slide6

Yuck! No,

thank you!

Reject

Mmmm!

Spongey.

Accept

Chemical Defenses of Sponges

Mix with artificial food

Present to fish

Extract

Percentage (%) Eaten

SpongeControl*Treated

Acanthella acuta 100.0 6.3

Aplysina aerophoba 89.8 8.2

Ianthella basta 94.0 6.0

Axinella sp. 100.0 93.8

Crambe crambe 94.4 2.8

Stylissa massa 100.0 2.8

Dysidea 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., 21:189-209; Paul et al. (2006) Nat. Prod. Rep., 23:153-80.

slide7

Bioactive Compounds from Sponges: Okadaic Acid

Halichondrin B

Okadaic Acid

Halichondria okadai

slide8

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 (5:1)

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

slide9

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.)

>120 µg/kg

Cytotoxicity

P388 - ED50 = 1.7 x 103

L1210 - ED50 - 1.7 x 102

Tumor Inhibition

None (≤subtoxic dose)

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.)

Tachibana et al. (1981) J. Am. Chem. Soc., 103: 2469-71

slide10

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 -> 1H-NMR

Comparison to Acanthifolicin: Absolute Stereochemistry

Tachibana et al. (1981) J. Am. Chem. Soc., 103: 2469-71

slide11

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

slide14

Protein Kinases/Phosphatases:

Biochemical “On/Off Switches”

ATP

ADP

Kinase

Serine

Threonine

Tyrosine

Phosphatase

slide15

Ser/Thr Protein Phosphatases (PP)

PP1

PP2A

PP4

PP5

PP2B

(Calcineurin)

PP2C

slide16

Ser/Thr Protein Phosphatases 1 and 2A (PP1/2A)

PP1 PP2A

Catalytic Subunit PP1c(37 Kda) PP2Ac(36 Kda)

Distribution Myosin, Glycogen, Widely

Chromatin, S.R.

Endogenous I-1/DARPP-32, I-2, I-1PP2A, I-2PP2A

Inhibitors Dopamine, NIPP-1

slide17

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 >10,000

Phosphorylase a >10,000

Tyr Phosphatase -- >10,000

Inositol-1,4,5-triPP -- >10,000

Acid Phosphatase -- >10,000

Alkaline -- >10,000

Phosphatase

Bialojan and Takai (1988) Biochem. J., 256: 283-90

slide18

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)

slide19

Discodermolide: Discovery

Depth: 33 m

Lucaya

Discodermia dissoluta

slide20

Discodermolide: Isolation

Frozen/Thawed

434 g

Extracted: MeOH/Toluene (3:1)

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

slide21

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 (M+1)+ - CONH2

NMR: 1H, 13C, COSY, HMQC, HMBC

NOT Stable at room temperature!

Gunasekara et al. (1990) J. Org. Chem., 55: 4912-4915

slide22

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

slide23

Discodermolide: Structure

X-Ray Crystallography

slide24

Discodermolide: Synthesis/Structure

(+)-Discodermolide

(-)-Discodermolide

Nerenberg et al. (1993) J. Am. Chem. Soc., 115:12621-2 (and subsequent work by Schreiber Group)

slide25

Discodermolide: Synthesis

Novartis® Synthesis Scheme

slide26

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:

IC50 Stage

(+)-Discodermolide 7 nM (G2/M)

(-)-Discodermolide 135 nM (S)

Hung et al. (1996) J. Am. Chem. Soc., 118:11054-80

slide27

A, T, G, C

+ DNA Polymerase

Mitosis-Promoting Factor (MPF)

Cyclin A

Cdk2

Cyclin A/B

Cdk1 (a.k.a. cdc2)

“Restriction Point”

Cyclin E

Cdk2

G0

Cyclin D

Cdk4/6

G0

S

G2

Prophase

Metaphase

G1

Anaphase

M

Telophase

slide29

GTP

GTP

GTP

GTP

Tubulin Polymerization Dependent on GTP/GDP

Hydrolysis

GDP

GTP

GTP

GTP

GDP

GTP

+

+

GTP

GTP

GTP

GTP

slide32

“GTP Cap”

Dynamic Instability of Microtubules

Tubulin-GTP

Tubulin-GDP

slide36

Tubulin Polymerization and Depolymerization Aligns Chromosomes During Metaphase

Tubulin-Polymerization

Dynein

-

+

+

-

Kinesin

Tubulin-Depolymerization

slide37

Tubulin Polymerization and Depolymerization Aligns Chromosomes During Metaphase

Polymerized Tubulin

Dynein

-

+

+

-

Kinesin

slide38

Tubulin Polymerization and Depolymerization Separates Chromosomes During Anaphase

Dynein

-

+

+

-

Tubulin Depolymerizes

Tubulin Depolymerizes

slide39

Tubulin Polymerization and Depolymerization Separates Chromosomes During Anaphase

Dynein

-

+

+

-

slide40

(+)-Discodermolide Prevents Depolymerization of Tubulin

Dynein

-

+

+

-

Tubulin Depolymerizes

Tubulin Depolymerizes

slide41

(+)-Discodermolide Stabilizes Microtubules (i.e. Inhibits Depolymerization)

Control

+ Discodermolide

slide42

(+)-Discodermolide inhibits depolymerization of tubulin

(+)-Discodermolide prevents breakdown of Cyclin B

S

G2

Mitosis-Promoting Factor (MPF)

Cyclin A/B

Cdk1 (a.k.a. cdc2)

Prophase

Metaphase

G1

Anaphase

M

Telophase

slide43

Taxol™ (Paclitaxel)

* From bark of “Pacific Yew” (Taxus brevifolia)

slide44

Discodermolide Stabilizes Microtubules More Than Taxol™

+ 10 µM Taxol, or 10 µM (+)-Discodermolide

EC50

(+)-Discodermolide 3.2 µM

Taxol™ (Paclitaxel) 23 µM

slide45

Multi-Drug Resistant Cancer Cells Less Resistant to Discodermolide

“Level of Resistance”*

Colon Ovarian

CarcinomaCarcinoma

(+)-Discodermolide 25-fold 89-fold

Taxol 900-fold 2800-fold

*Compared to parent line

slide47

FDA

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