SEA URCHIN EMBRYO - A PROMISING MODEL IN SCREENING FOR TUBULIN DESTABILIZING ACTIVITY

MICROTUBULES  HeLa pf

FUNCTIONS OF TUBULIN/MICROTUBULE MT-ROLES INTRACELLULAR TRANSPORT CELL SHAPE AND MIGRATION MITOSIS CILIA & FLAGELLA

TUBULIN - TARGET  MICROTUBULE HETERODIMER DESTABILIZING AGENTS: INHIBIT POLYMERIZATION DISASSEMBLE MICROTUBULES STABILIZING AGENTS: ALTER MICROTUBULE DYNAMICS MITOTIC ARREST, ANTI-PROLIFERATIVE ACTIVITY SHUTDOWN OF TUMOR BLOOD SUPPLY

DESTAB/STRUCTURES   COLCHICINE PODOFYLLOTOXIN COMBRETASTATIN D NOCODAZOLE DOLASTATIN VINBLASTINE NOSTODIONE 2-METHOXY- ESTRADIOL

STABILIZERS +    + Diaz et al., J Biol Chem, 2000 TAXOL DISCODERMOLIDE EPOTHILONES

NEW ANTI-TUB. DRUGS

0.5 M KCl SPAWNING & FERT. Paracentrotus lividus FERTILIZATION ~ EGGS/FEMALE

STAGES PRISMEARLY PLUTEUS ACTIVE FEEDING CLEAVAGEMESENCHYME BLASTULA MID-PLUTEUS 2-3 DAYS  LATE GASTRULA ~ 140  ~ 115  FERTILIZED EGG HATCHING 9-16 h BLASTULA

SU_EMBR. TUBULIN Gong & Brandhorst, Dev Biol, 1988 (Lytechinus pictus) I II III IV  HATCHING

TUBULIN SU & VERT HIGHLY CONSERVATIVE POLYPEPTIDE BACKBONE ~ 450 АMINO ACIDS 2-17% VARIABILITY VARIABLE DOMAIN ~ АA COOH NH 2  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 Control 6-11 h 1-30 h TAXOL

EFFECT/ANTIPROLIF ER. TARGET - DNA 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. SEE TUBULIN DESTABILIZERS DATABASE ON PUBCHEM >>>

SCREENING METHOD  a FERTILIZED EGG CLEAVAGE ARREST SWIMMING BLASTULA SPINNING CLEAVAGE

TREATMENT PROCEDURE EXPOSED STAGES HATCHED BLASTULA 50  M Control 10  M 0.05  M0.2  M1  M 5 ml SUSPENSION IN SEA WATER, EMBRYOS/ML ONE FEMALE EMBRYOS  ~ 30 PLATES 9 h (20-23 о ) 16 h (14-17 о ) FERTILIZED EGG 5 мM5 мM 100  M 10  M STOCK SOLUTIONS (IN DMSO, ETHANOL)

OGT-TEST 2005 LATE BLASTULA CONTROL COLCHICINE, NOCODAZOLE, D NEW COMPOUNDS

EXPENSES $ IN VITRO TUBULIN POLYMERIZATION $ IN VIVO TEST ~ 200 $ SEA URCHIN EMBRYO TEST 750 $ CELL CULTURE TEST IC50, FACS, D10