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

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

2. a b MICROTUBULES MICROTUBULES HeLa 10-15 pf

3. INTRACELLULAR TRANSPORT MITOSIS CELL SHAPE AND MIGRATION CILIA & FLAGELLA THE ROLES OF MICROTUBULES IN CELL FUNCTIONS OF TUBULIN/MICROTUBULE MT-ROLES

4. DESTABILIZING AGENTS: INHIBIT POLYMERIZATION DISASSEMBLE MICROTUBULES a b STABILIZING AGENTS: ALTER MICROTUBULE DYNAMICS TUBULIN: TUBULIN - TARGET A TARGET FOR ANTI-TUMOR DRUGS HETERODIMER MICROTUBULE • MITOTIC ARREST, ANTI-PROLIFERATIVE ACTIVITY • SHUTDOWN OF TUMOR BLOOD SUPPLY

5. MICROTUBULE DESTABILIZING DRUGS DESTAB/STRUCTURES 2-METHOXY-ESTRADIOL COLCHICINE PODOFYLLOTOXIN COMBRETASTATIN b D-24851 a NOSTODIONE NOCODAZOLE VINBLASTINE DOLASTATIN

6. + + b a b Diaz et al., J Biol Chem, 2000 MICROTUBULE STABILIZERS STABILIZERS DISCODERMOLIDE TAXOL EPOTHILONES

7. NEW ANTI-TUMOR TUBULIN DESTABILIZING DRUGS NEW ANTI-TUB. DRUGS

8. ARTIFICIAL SPAWNING SPAWNING & FERT. AND FERTILIZATION 0.5 M KCl ~ 1 000 000 EGGS/FEMALE Paracentrotus lividus FERTILIZATION

9. BLASTULA ACTIVE FEEDING SEA URCHIN EMBRYONIC DEVELOPMENT STAGES HATCHING 9-16 h CLEAVAGE MESENCHYME BLASTULA LATE GASTRULA FERTILIZED EGG ~ 115 m 450 - 500 m ~ 140 m PRISM EARLY PLUTEUS MID-PLUTEUS 2-3 DAYS

10. a b TUBULIN SYNTHESIS DURING SEA URCHIN EMBRYO DEVELOPMENT SU_EMBR. TUBULIN HATCHING IV III I II Gong & Brandhorst, Dev Biol, 1988 (Lytechinus pictus)

11. NH CROSS-PRECIPITATION WITH ANTIBODIES TO VERTEBRATE BRAIN TUBULIN (CHICKEN, RAT, PIG) HIGHLY CONSERVATIVE POLYPEPTIDE BACKBONE ~ 450 АMINO ACIDS2-17% VARIABILITY VARIABLE DOMAIN ~ 15-17 АA 2 a b COOH SIMILARITY OF TUBULINS FROM SEA URCHIN EMBRYOS AND VERTEBRATES TUBULIN SU & VERT I II III FITC - LABELED PIG BRAIN TUBULIN INCLUDES INTO MITOTIC SPINDLE Y.Hamagushi et al., J Cell Biol, 1985 (Clypeaster japonicus)

12. TAXOL EFFECT OF STANDARD TUBULIN DESTABILIZERS EFFECT/TUB. DESTAB. COLCHICINE NOCODAZOLE VINBLASTINE DOLASTATIN 15 D-24851 1-30 h 6-11 h Control

13. EFFECT OF ANTI-PROLIFERATIVE AGENTS EFFECT/ANTIPROLIFER. TARGET - DNA AZIDOTHYMIDINE АPIGENIN HYDROXYUREA MITOXANTRONE MITOMYCIN С RUBOXYL 4-PHENYLBUTYRIC ACID 5-FLUOROURACYL APHIDICOLIN 5-AZACYTIDINE DOXORUBICIN CHLORAMBUCIL TARGET - CYTOSKELETON ACRYLAMIDE CYTOCHALASIND ABNORMAL CLEAVAGE NO SPINNING

14. EFFECTIVE CONCENTRATIONS EFFECTIVE CONC. OF TUBULIN DESTABILIZING DRUGS SEE TUBULIN DESTABILIZERS DATABASE ON PUBCHEM >>>

15. b a SCREENING PROCEDURE FOR TUBULIN DESTABILIZERS SCREENING METHOD CLEAVAGE ARREST A CLEAVAGE FERTILIZED EGG Б SWIMMING BLASTULA SPINNING

16. 100mM 10 mM 5мM FERTILIZED EGG 0.05mM 0.2mM 50 mM 10 mM 1 mM Control 5 ml SUSPENSION IN SEA WATER, 700-3000 EMBRYOS/ML ONE FEMALE EMBRYOS  ~ 30 PLATES TREATMENT PROCEDURE TREATMENT PROCEDURE EXPOSED STAGES STOCK SOLUTIONS(IN DMSO, ETHANOL) HATCHED BLASTULA 9 h (20-23о) 16 h (14-17о)

17. LATE BLASTULA OREGON GREEN TAXOL MICROTUBULE VISUALIZATION OGT-TEST 2005 CONTROL COLCHICINE, NOCODAZOLE, D-24851 3 NEW COMPOUNDS

18. IN VIVO TEST 2500 - 10 000 $ CELL CULTURE TEST IC50, FACS, D10 750 $ SEA URCHIN EMBRYO TEST IN VITRO TUBULIN POLYMERIZATION ~ 200 $ 350 - 400 $ PRICES OF ONE COMPOUND TEST (USA) EXPENSES