Abstract
Cyclostreptin (1), a natural product from Streptomyces sp. 9885, irreversibly stabilizes cellular microtubules, causes cell cycle arrest, evades drug resistance mediated by P-glycoprotein in a tumor cell line and potently inhibits paclitaxel binding to microtubules, yet it only weakly induces tubulin assembly. In trying to understand this paradox, we observed irreversible binding of synthetic cyclostreptin to tubulin. This results from formation of covalent crosslinks to β-tubulin in cellular microtubules and microtubules formed from purified tubulin in a 1:1 total stoichiometry distributed between Thr220 (at the outer surface of a pore in the microtubule wall) and Asn228 (at the lumenal paclitaxel site). Unpolymerized tubulin was only labeled at Thr220. Thus, the pore region of β-tubulin is an undescribed binding site that (i) elucidates the mechanism by which taxoid-site compounds reach the kinetically unfavorable lumenal site and (ii) explains how taxoid-site drugs induce microtubule formation from dimeric and oligomeric tubulin.
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Acknowledgements
The authors thank J. Vilarrasa for helpful discussions, P. Lastres for his help with flow cytometry, and Matadero Madrid Norte S.A. and José Luis Gancedo S.L. for providing calf brains for tubulin purification. This work was supported in part by grant BFU2004-00358 from Ministerio de Educación y Ciencia and grant 200520M061 from Comunidad Autonoma de Madrid to J.F.D. R.M.B. was supported by a Beca de Formación de Profesorado Universitario del Ministerio de Educación y Ciencia fellowship.
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R.M.B. performed research; E.C. performed research; I.B. designed and performed research; O.P. performed research; M.C.E. performed research; R.M. performed research; G.C. performed research; C.D.V. contributed materials and performed research; B.W.D. contributed materials and edited the manuscript; E.J.S. contributed materials; J.A.L. interpreted data; J.M.A. designed research, interpreted data and edited the manuscript; E.H. interpreted data and wrote the paper; J.F.D. designed and performed research, interpreted data and wrote the paper.
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Supplementary information
Supplementary Fig. 1
Characterization of peptide chemical modifications by triple quadrupole MS. (PDF 154 kb)
Supplementary Fig. 2
Analysis of tubulin tryptic peptides by nanoliquid chromatography coupled to 3D ion-trap MS. (PDF 131 kb)
Supplementary Fig. 3
Analysis of tubulin isolated from cyclostreptin-treated cells. (PDF 101 kb)
Supplementary Fig. 4
Model of cyclostreptin binding. (PDF 240 kb)
Supplementary Fig. 5
Characterization of the cyclostreptin molecule by MS. (PDF 130 kb)
Supplementary Table 1
Cell cycle distribution of PtK2 cells treated for 7 h with cyclostreptin or paclitaxel with further incubation without drug and cell cycle distribution of PtK2, A549, A2780 and A2780/AD cells treated with cyclostreptin and paclitaxel. (PDF 23 kb)
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Buey, R., Calvo, E., Barasoain, I. et al. Cyclostreptin binds covalently to microtubule pores and lumenal taxoid binding sites. Nat Chem Biol 3, 117–125 (2007). https://doi.org/10.1038/nchembio853
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DOI: https://doi.org/10.1038/nchembio853