A Photoaffinity Analogue of Discodermolide Specifically Labels a Peptide in β-Tubulin

Abstract

Discodermolide is a potentially important antitumor agent that stabilizes microtubules and blocks cells at the G2/M phase of the cell cycle in a manner similar to that of Taxol. Discodermolide also has unique properties that distinguish it from Taxol. In the present study, photoaffinity-labeled discodermolide analogues are used to investigate their binding site in tubulin. Three photoaffinity-labeled discodermolide analogues were synthesized, all of which promoted microtubule polymerization in the absence of GTP. The analogue, C19-[4-(4-³H-benzoyl-phenyl)-carbamate]-discodermolide (C19-[³H]BPC-discodermolide), was selected for photolabeling studies because it had the highest extent of photoincorporation, ∼1%, of the three radiolabeled discodermolide analogues explored. Although compared to discodermolide, C19-BPC-discodermolide revealed no hypernucleation effect in the in vitro microtubule polymerization assay, it was more cytotoxic than discodermolide, and, like discodermolide, demonstrated synergism with Taxol. These results suggest that the hypernucleation effect of discodermolide is not involved in its cytotoxic activity. Similar to discodermolide, C19-BPC-discodermolide can effectively displace [³H]Taxol from microtubules, but Taxol cannot effectively displace C19-[³H]BPC-discodermolide binding. Discodermolide can effectively displace C19-[³H]BPC-discodermolide binding. Formic acid hydrolysis, immunoprecipitation experiments, and subtilisin digestion indicate that C19-BPC-discodermolide labels amino acid residues 305−433 in β-tubulin. Further digestion with Asp-N and Arg-C enzymes suggested that C19-BPC-discodermolide binds to amino acid residues, 355−359, in β-tubulin, which is in close proximity to the Taxol binding site. Molecular modeling guided by the above evidence led to a putative binding model for C19-BPC-discodermolide in tubulin.