Mechanism of action cryptophycin. Interaction with the Vinca alkaloid domain of tubulin.
Cryptophycin is a potent antitumor agent that depletes microtubules in intact cells, including cells with the multidrug resistance phenotype. To determine the mechanism of action of cryptophycin, its effects on tubulin function in vitro were analyzed. Cryptophycin reduced the in vitro polymerization of bovine brain microtubules by 50% at a drug:tubulin ratio of 0.1. Cryptophycin did not alter the critical concentration of tubulin required for polymerization, but instead caused substoichiometric reductions in the amount of tubulin that was competent for assembly. Consistent with its persistent effects on intact cells, cryptophycin-treated microtubule protein remained polymerization-defective even after cryptophycin was reduced to sub-inhibitory concentrations. The effects of cryptophycin were not due to denaturation of tubulin and were associated with the accumulation of rings of microtubule protein. The site of cryptophycin interaction with tubulin was examined using functional and competitive binding assays. Cryptophycin blocked the formation of vinblastine-tubulin paracrystals in intact cells and suppressed vinblastine-induced tubulin aggregation in vitro. Cryptophycin inhibited the binding of [3H]vinblastine and the hydrolysis of [gamma32P]GTP by isolated tubulin, but did not block the binding of colchicine. These results indicate that cryptophycin disrupts the Vinca alkaloid site of tubulin; however, the molecular details of this interaction are distinct from those of other antimitotic drugs.
Smith
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Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Zhang
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D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D002417:Cattle; D003078:Colchicine / Q000378:metabolism / Q000494:pharmacology; D047630:Depsipeptides; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D010456:Peptides, Cyclic / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011108:Polymers / Q000737:chemistry / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D014747:Vinblastine / Q000378:metabolism / Q000494:pharmacology
D000970:Antineoplastic Agents; D047630:Depsipeptides; D010456:Peptides, Cyclic; D011108:Polymers; D014404:Tubulin; C088419:cryptophycin; D014747:Vinblastine; D006160:Guanosine Triphosphate; D003078:Colchicine