Zampanolide, a Potent New Microtubule-Stabilizing Agent, Covalently Reacts with the Taxane Luminal Site in Tubulin α,β-Heterodimers and Microtubules

SummaryZampanolide and its less active analog dactylolide compete with paclitaxel for binding to microtubules and represent a new class of microtubule-stabilizing agent (MSA). Mass spectrometry demonstrated that the mechanism of action of both compounds involved covalent binding to β-tubulin at residues N228 and H229 in the taxane site of the microtubule. Alkylation of N228 and H229 was also detected in α,β-tubulin dimers. However, unlike cyclostreptin, the other known MSA that alkylates β-tubulin, zampanolide was a strong MSA. Modeling the structure of the adducts, using the NMR-derived dactylolide conformation, indicated that the stabilizing activity of zampanolide is likely due to interactions with the M-loop. Our results strongly support the existence of the luminal taxane site of microtubules in tubulin dimers and suggest that microtubule nucleation induction by MSAs may proceed through an allosteric mechanism.

Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (287 K)Download as PowerPoint slideHighlights► Zampanolide covalently binds to the luminal paclitaxel site at β-tubulin N228 and H229 ► Labeling occurs in both tubulin dimers and microtubules ► Bound zampanolide models indicate interaction with the M-loop at the paclitaxel site ► Microtubule nucleation may proceed through both matchmaking and allosteric mechanisms