Localization of the discodermolide binding site in beta-tubulin

Abstract

Discodermolide is one of the microtubule stabilizing drugs. It has similarities and differences from Taxol, which is the first drug identified to stabilize microtubules. Discodermolide has very strong hypernucleation effects in the in vitro microtubule polymerization assay. Cell lines resistant to Taxol and epothilone B demonstrate little cross resistance to discodermolide. Discodermolide also induces accelerated senescence in cancer cell lines, which is not a significant characteristic of Taxol. Taxol and discodermolide are a synergistic drug combination that inhibits cell growth in culture and in an ovarian xenograft tumor model. These observations have encouraged us to investigate the nature of the discodermolide binding site in tubulin.;In the present study, photoaffinity labeled discodermolide analogs are used to investigate their binding site in tubulin. Three photoaffinity-labeled discodermolide analogs were synthesized, all of which promoted microtubule polymerization in the absence of GTP. The analog, C19-[4-(4-3H-Benzoyl-phenyl)-carbamate]-discodermolide (C19-[3H]BPC-discodermolide), was selected for photolabeling studies because it had the highest photoincorporation, ∼ 1%, of the three radiolabeled discodermolide analogs. Although, compared to discodermolide, C19-BPC-discodermolide revealed no hypernucleation effect in the in vitro microtubule polymerization assay, it demonstrated synergism with Taxol, but not with discodermolide in cells. These suggest that the hypernucleation effect of discodermolide is not involved in its cytotoxic activity. C19-BPC-Discodermolide binds to tubulin with a stoichiometry approaching 1 per alphabeta-tubulin dimer. Similar to discodermolide, C19-BPC-discodermolide could effectively displace [3H]Taxol from microtubules, but Taxol could not effectively displace C19-[3H]BPC-discodermolide binding. C19-BPC-discodermolide could also competitively inhibit [3H]Taxol binding to microtubules. Formic acid hydrolysis, immunoprecipitation experiments, and subtilisin digestion indicated that C19-BPC-discodermolide labels amino acid residues 305--433 in beta-tubulin. Further digestion with Asp-N and Arg-C enzymes suggested that C19-BPC-discodermolide binds to amino acid residues, 355--359, in beta-tubulin, which is in close proximity to the Taxol binding site. Docking of C19-BPC-discodermolide into the binding pocket by molecular modeling in beta-tubulin revealed a conformational change at the S9--S10 loop. When discodermolide was docked into this site, no conformation change occurred in the S9--S10 loop. A comparison of the binding conformation of C19-BPC-discodermolide and discodermolide with Taxol demonstrated similarities, but also differences in the binding pocket.