Molecular mechanisms of antimicrotubule agents, paclitaxel and colchicine

Molecular interactions of paclitaxel and colchicine with tubulin have been investigated in detail. The contribution of 2'-hydroxyl group and the role of the N-benzoyl group in paclitaxel-microtubule association haa been determined by studying the effect of paclitaxel deletion analogs on tubulin assembly. The affinities of these taxanes for microtubules and their cytotoxicities were also measured. It was found that the 2'-hydroxyl group alone contributes 25% to the standard free energy of binding. This contribution also accounts for about 80% of the binding free energy attributed to the entire C-13 side chain. Molecular modeling analysis suggests that 2'-OH group forms a hydrogen bond with D26 of beta-tubulin. The N-benzoyl group therefore may serve as an anchor for appropriate conformation of C-13 side chain, facilitating the formation of the hydrogen binding interaction between the 2'-hydroxyl group and protein. These findings help define structural requirements for high affinity microtubule binding and may consequently be significant toward the development of improved paclitaxel analogs.;The structure of colchicine binding site on tubulin isotype beta VI has been established by computational analysis of colchicine binding site as well as the investigation of the tubulin-binding and microtubule assembly-inhibitory properties of colchicine site agents. The studies performed simultaneously on bovine brain tubulin and tubulin isotype betaVI have revealed that colchicine binding site on tubulin isotype betaVI is relatively more hydrophobic, has a shrunken A ring binding pocket and prefers polarizable substituents on the C ring of colchicine. Since tubulin in erythropoetic cells is 95% betaVI isotype, these studies are important for the evaluation of hematological toxicity of potential new colchicine site drugs.;In vitro tubulin assembly assays have been developed for quantitative evaluation of activity of paclitaxel analogs that are poorly soluble under normal assembly conditions. In these new assays, GDP-tubulin replaces GTP-tubulin allowing the cosolvent (DMSO) concentration up to 15%. These conditions improved the solubility of the ligand and normal microtubule assembly was observed. Since many paclitaxel site drugs are very hydrophobic, the results are important because they provide conditions by which activities of such molecules can be quantitatively assessed.;Paclitaxel analogs of varying conformations have been evaluated for tubulin bioactivity in order to test the proposed T-Taxol conformation model for microtubule-bound paclitaxel. Analogs derived from inactive taxanes and restricted to "T-Conformation" were found to be as active as paclitaxel. Analogs that can not adopt this conformation were found to be inactive. Simplified paclitaxel analogs with approximate T-Taxol conformation displayed weak activity. The results provide evidence that T-Taxol is the bioactive conformation of paclitaxel.;In addition, taxane conjugated cyanonilutamides have been studied for the tubulin assembly, microtubule binding and cytotoxicity for prostrate cancer cells. The C-10 conjugates among these agents have been found to be significantly active, indicating their potential effectiveness against castrate-resistant prostrate cancer.