Abstract:
:NSC 613862 (S)-(-) and NSC 613863 (R)-(+) are the two chiral isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3, 4-b]pyrazin-7-yl carbamate. Both compounds bind to tubulin in a region that overlaps the colchicine site. They induce formation of abnormal polymers from purified GTP-Mg-tubulin, the active assembly form of tubulin, in glycerol-free buffer with magnesium [De Ines, C., Leynadier, D., Barasoain, I., Peyrot, V., Garcia, P., Briand, C., Rener, G. A., and Temple, C., Jr. (1994) Cancer Res. 54, 75-84]. In this study, we observed that the S-isomer can promote polymerization of GDP-tubulin, the inactive assembly-incompetent form of tubulin, into nonmicrotubular structures at a critical protein concentration of 1 mg/mL (12 mM MgCl2). Neither the R-isomer nor colchicine have this ability. By electron microscopy, these tubulin polymers showed the same poorly defined filamentous structure when GDP-tubulin or GTP-Mg-tubulin were used. By HPLC measurements, we demonstrated that a dissociated GTP hydrolysis and exchange of nucleotide occurred during the isomer-induced abnormal assembly. Both isomers inhibited the Mg2+-induced tubulin self-association leading to 42 S double ring formation from GTP-Mg-tubulin or GDP-tubulin. Measurement of their binding under nonassociation conditions revealed a 3-fold decrease in the apparent equilibrium binding constant of the R-isomer to GDP-tubulin relative to GTP-Mg-tubulin. For the S-isomer, the decrease in the binding constant was less pronounced. Binding data, analyzed in terms of a system of linked conformational and association equilibria, provide evidence that the active ("straight") rather than the inactive ("curved") conformation of tubulin differentially recognizes these ligands. Whereas binding of colchicine to tubulin is well-known to induce GTP hydrolysis, this is the first case in which the interaction of a ligand with the colchicine site is shown to be sensitive to the presence of GDP or GTP at the distant nucleotide binding site.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Barbier P,Peyrot V,Leynadier D,Andreu JMdoi
10.1021/bi970568tsubject
Has Abstractpub_date
1998-01-13 00:00:00pages
758-68issue
2eissn
0006-2960issn
1520-4995pii
bi970568tjournal_volume
37pub_type
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