Abstract 2789: Interaction of (+)-discodermolide-Taxol hybrids with microtubules

Abstract

Abstract Taxol and discodermolide are both microtubule stabilizing agents that bind to the β-tubulin subunit of microtubules. Based on our results that the hydrophobic binding pocket of β-tubulin is occupied by the Taxol side chain but not by (+)-discodermolide, and that the two drugs act synergistically in cancer cell lines, a small library of (+)-discodermolide-Taxol hybrids was synthesized (J. Med. Chem. 54:6319, 2011). Cytotoxicity assays demonstrated a 2-9-fold increase in antiproliferative activity by the hybrids compared to discodermolide in the human cancer cell lines, A549 and MCF-7. Hybrids containing a tether of 3 carbons that connect discodermolide with the Taxol side chain, exhibited the best activity. Tubulin assembly assays were performed with the two most potent hybrid molecules. Like discodermolide, these two hybrids increased tubulin assembly rapidly without a lag period. Tubulin polymerization studies using purified bovine brain tubulin demonstrated that they also had the greatest effect on the formation of polymerized microtubules. These results also were observed in intact A549 cells and in 100,000 x g supernatants prepared from these cells. We have previously shown that [3H]2-(m-azidobenzoyl)Taxol photolabels a peptide containing amino acid residues 217-231 of β-tubulin. A 5-fold molar excess of unlabeled compound inhibited the photolabeling of purified bovine brain tubulin by 94%, demonstrating the specificity of this photolabeling. Microtubule stabilizing agents (MSAs), such as Taxol, epothilone B, discodermolide and ixabepilone, each at a 5-fold molar excess, inhibited the photolabeling by 24%, 92%, 100% and 41%, respectively, indicating that discodermolide is the most potent inhibitor of the photolabeling. In contrast, two other MSAs, laulimalide and peloruside that are known to bind to a different site in β-tubulin, exhibited stimulatory effects on the photolabeling. Both drugs, at a 5-fold molar excess, increased the labeling by 30-40%. [3H]2-(m-azidobenzoyl)Taxol (0.5 - 20 µM) was used to study the kinetics of the inhibitory effects of the hybrid molecules on photoaffinity labeling of tubulin. Discodermolide-Taxol hybrids inhibited the photolabeling of bovine brain tubulin in a dose dependent manner. The concentrations that inhibited by 50% were lowest for the two most potent hybrid molecules. Therefore, the tubulin polymerization activity and the binding affinity of the hybrids to β-tubulin correlated with their antiproliferative activity. Other biological properties of the discodermolide-Taxol hybrids including senescence and antitumor activity are being evaluated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2789. doi:1538-7445.AM2012-2789