Transcript 10 Mb

SEA URCHIN EMBRYO - A PROMISING MODEL
IN SCREENING FOR TUBULIN DESTABILIZING
ACTIVITY
MICROTUBULES
HeLa
10-15 pf
FUNCTIONS OF
MT-ROLES
TUBULIN/MICROTUBULE
INTRACELLULAR
TRANSPORT
CELL SHAPE
AND MIGRATION
MITOSIS
CILIA & FLAGELLA
TUBULIN - TARGET
a
b
HETERODIMER
STABILIZING AGENTS:
ALTER MICROTUBULE
DYNAMICS
DESTABILIZING AGENTS:
INHIBIT POLYMERIZATION
DISASSEMBLE MICROTUBULES
MICROTUBULE
• MITOTIC ARREST, ANTI-PROLIFERATIVE ACTIVITY
• SHUTDOWN OF TUMOR BLOOD SUPPLY
OH
O
H3 CO
O
O
DESTAB/STRUCTURES
O
CH3
NH
H 3 CO
H3 CO
O
OCH 3
NH
OH
HO
COMBRETASTATIN
2-METHOXYESTRADIOL
O
O
N
b
D-24851
NH
N
a
Cl
O
O
S
H
H 3 CO
COLCHICINE
PODOFYLLOTOXIN
O
H
H 3 CO
OCH 3
OCH 3
O
H
H 3 CO
H 3 CO
H3 CO
O
H 3 CO
OH
CH3
NOSTODIONE
N
OH
OH
OCH3
NH
N
N
H
NH
NOCODAZOLE
H3 CO
N
O
H3 CO
O
NH
N
O
DOLASTATIN
N
H
NH
H3 CO
O
OCH 3
O
S
OH
N
O
H
O
O
OCH 3
NH
N
VINBLASTINE
NH
STABILIZERS
AcO
O OH
O
O
O
H
OH
OH
O
OH
HO
O
O
H
AcO
O
OH
OH
O
NH2
O
DISCODERMOLIDE
TAXOL
+
S
N
O
+
O
OH
O
O
OH
b
a
b
EPOTHILONES
Diaz et al., J Biol Chem, 2000
NEW ANTI-TUB. DRUGS
COMPANY NAME
MOLECULE
BINDING SITE
CLINICAL TRIALS
TUBULIN DESTABILIZERS (7)
Asta Medica AG
D-24851
New
Phase II
Astra Zeneca
ZD-6186
Colchicine
Phase II
Aventis
AVE-8062
Colchicine
Phase II
OxiGene
Combretastatine 4P
Colchicine
Phase II
Abbott
ABT-751
Colchicine
Phase I
Wyeth
HTI-286
Colchicine
Phase I
EntrMed
2-Methoxyestradiol
Colchicine
Phase I/II
TUBULIN STABILIZERS (8)
Aventis
LIT-976, taxoid
Taxol
Phase I/II
Bayer
Taxane Bay-59-8862
Taxol
Phase I/II
BMS
Taxanes BMS247550
Taxol
Phase I/II
Wyeth
Taxane Mac 321
Taxol
Phase I/II
BMS
Epothilones
Taxol
Phase I/II
Kosan/Roche
Epothilone KOS862
Taxol
Phase I/II
Shering/Metagen
Epothilones
Taxol
Phase I/II
Novartis
Epothilone EPO-906
Taxol
Phase I/II
SPAWNING & FERT.
0.5 M KCl
~ 1 000 000 EGGS/FEMALE
Paracentrotus lividus
FERTILIZATION
STAGES
HATCHING
9-16 h
FERTILIZED EGG
CLEAVAGE
BLASTULA
~ 115 m
MESENCHYME
BLASTULA
LATE GASTRULA
450 - 500 m
~ 140 m
ACTIVE
FEEDING
PRISM
EARLY PLUTEUS
MID-PLUTEUS
2-3 DAYS
9
V, relative units
a
8
b

SU_EMBR. TUBULIN
HATCHING
7

6

IV
5
III
I
4

II
3
2




1 


0
0
10
20
30
40
50
60
Age, h
Gong & Brandhorst, Dev Biol, 1988 (Lytechinus pictus)
70
TUBULIN SU & VERT
HIGHLY CONSERVATIVE POLYPEPTIDE BACKBONE
~ 450 АMINO ACIDS
2-17% VARIABILITY
VARIABLE
DOMAIN
~ 15-17 АA
COOH
NH2
ab
CROSS-PRECIPITATION WITH ANTIBODIES TO VERTEBRATE BRAIN TUBULIN
(CHICKEN, RAT, PIG)
FITC - LABELED
PIG BRAIN TUBULIN
INCLUDES INTO
MITOTIC SPINDLE
Y.Hamagushi et al., J Cell Biol, 1985 (Clypeaster japonicus)
EFFECT/TUB.
DESTAB.
COLCHICINE
NOCODAZOLE
VINBLASTINE
DOLASTATIN 15
D-24851
TAXOL
1-30 h
6-11 h
Control
TARGET - DNA
EFFECT/ANTIPROLIF
ER.
AZIDOTHYMIDINE
АPIGENIN
HYDROXYUREA
MITOXANTRONE
MITOMYCIN С
RUBOXYL
4-PHENYLBUTYRIC ACID
5-FLUOROURACYL
APHIDICOLIN
5-AZACYTIDINE
DOXORUBICIN
CHLORAMBUCIL
TARGET - CYTOSKELETON
ACRYLAMIDE
CYTOCHALASIN D
ABNORMAL
CLEAVAGE
NO SPINNING
EFFECTIVE CONC.
EC minimum, mM
IC 50, mM (LITERATURE DATA)
SEA URCHIN
EMBRYOS CLEAVAGE
ALTERATION
TUMOR CELL
CULTURES
CYTOTOXICITY
VINBLASTINE
0.2
0.23
D-24851
0.01
0.036 - 5.4
0.3
DOLASTATIN 15
0.005
0.003 - 0.005
23
COLCHICINE
50
0.005 - 0.8
2-8
2-METOXYESTRADIOL
0.2
0.08 - 1.8
1.9 - 4.7
NOCODAZOLE
0.005
0.05 - 0.3
0.01
REAGENT
PURIFIED
TUBULIN
POLYMERIZATIO
N
1.5
SEE TUBULIN DESTABILIZERS DATABASE ON PUBCHEM >>>
SCREENING METHOD
CLEAVAGE ARREST
CLEAVAGE
FERTILIZED EGG
b
a
O
NH
O
N
N
Cl
SWIMMING
BLASTULA
SPINNING
TREATMENT
PROCEDURE
EXPOSED STAGES
5 мM
100 mM
STOCK SOLUTIONS
10 mM
(IN DMSO, ETHANOL)
FERTILIZED EGG
HATCHED BLASTULA
Control
50 mM
10 mM
1 mM
0.2 mM
0.05 mM
9 h (20-23о)
16 h (14-17о)
5 ml SUSPENSION IN SEA WATER, 700-3000 EMBRYOS/ML
ONE FEMALE EMBRYOS  ~ 30 PLATES
CONTROL
COLCHICINE, NOCODAZOLE, D-24851
3 NEW COMPOUNDS
OGT-TEST 2005
LATE BLASTULA
EXPENSES
IN VIVO TEST
2500 - 10 000 $
CELL CULTURE TEST
IC50, FACS, D10
750 $
IN VITRO TUBULIN POLYMERIZATION
SEA URCHIN EMBRYO TEST
350 - 400 $
~ 200 $