In vitro effect of cryptophycin 52 on microtubule assembly and tubulin: molecular modeling of the mechanism of action of a new antimitotic drug.
Cryptophycin 52 (C52) is a new synthetic compound of the cryptophycin family of antitumor agents that is currently undergoing clinical evaluation for cancer chemotherapy. The cryptophycin class of compounds acts on microtubules. This report details the mechanism by which C52 substoichiometrically inhibits tubulin self-assembly into microtubules. The inhibition data were analyzed through a model described by Perez-Ramirez [Perez-Ramirez, B., Andreu, J. M., Gorbunoff, M. J., and Timasheff, S. N. (1996) Biochemistry 35, 3277-3285]. We thereby determined the values of the apparent binding constant of the tubulin-C52 complex to the end of a growing microtubule (K(i)) and the apparent binding constant of C52 to tubulin (K(b)). The binding of C52 depended on tubulin concentration, and binding induced changes in the sedimentation pattern of tubulin, which indicates that C52 induces the self-association of tubulin and tubulin aggregates other than microtubules. Using analytical ultracentrifugation and electron microscopy, we show that C52 induces tubulin to form ring-shaped oligomers (single rings). We also show that C52 inhibits the formation of double rings from either GTP- or GDP-tubulin. In addition, the advances made by electron crystallography in understanding the structure of the tubulin and the microtubule allowed us to visualize the putative binding site of C52 and to reconstruct C52-induced ring oligomers by molecular modeling.
Barbier
P
P
UMR-CNRS 6032, Faculté de Pharmacie, and Laboratoire de Biophysique 1, Faculté de Pharmacie, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France.
Gregoire
C
C
Devred
F
F
Sarrazin
M
M
Peyrot
V
V
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D047630:Depsipeptides; D007769:Lactams / Q000494:pharmacology*; D007783:Lactones / Q000494:pharmacology*; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D008958:Models, Molecular; D013552:Swine; D014404:Tubulin / Q000187:drug effects / Q000378:metabolism*
D000903:Antibiotics, Antineoplastic; D000970:Antineoplastic Agents; D047630:Depsipeptides; D007769:Lactams; D007783:Lactones; D014404:Tubulin; C114033:cryptophycin 52