Zampanolide, a potent new microtubule-stabilizing agent, covalently reacts with the taxane luminal site in tubulin α,β-heterodimers and microtubules

Zampanolide 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.JRS was supported by a fellowship from Programa de Cooperacion Cientıfica entre el Ministerio de Ciencia, Tecnologıas y Medio Ambiente de la Republica de Cuba (CITMA) y el CSIC. J.F. received a short-term fellowship from EMBO and a Professional Development Grant from the Genesis Oncology Trust. This work was supported in part by grants BIO2010-16351 and CTQ2009-08536 from Ministerio de Economia y Competitividad to J.F.D. and J.J.B., respectively, and grant S2010/BMD-2457 BIPEDD2 from Comunidad Autonoma de Madrid to J.F.D., the Cancer Society of New Zealand, and the Wellington Medical Research Foundation (J.M.). The CNIC is supported by the Ministerio de Economıa y Competitividad and the Fundacion Pro CNIC.S