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Cyclostreptin binds covalently to microtubule pores and lumenal taxoid binding sites

Nature Chemical Biology volume 3, pages 117–125 (2007)Cite this article

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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Figure 1: Biochemistry of the cyclostreptin-microtubule interaction.
Figure 2: Inhibition of binding of Flutax-2 to PtK2 cytoskeletons by preincubation with cyclostreptin and irreversibility of microtubule effects on PtK2 cells grown in cyclostreptin.
Figure 3: MS analyses of cyclostreptin binding to 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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Author notes

  1. Rubén M Buey and Enrique Calvo: These authors contributed equally to this work.

Authors and Affiliations

  1. Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, Madrid, 28040, Spain

    Rubén M Buey, Isabel Barasoain, Ruth Matesanz, José Manuel Andreu & J Fernando Díaz

  2. Unidad de Proteómica, Centro Nacional de Investigaciones Cardiovasculares, Melchor Fernández Almagro 3, Madrid, 28029, Spain

    Enrique Calvo & Juan Antonio López

  3. Departament de Química Orgànica, Facultat de Química, Universitat de Barcelona, Av. Diagonal 647, Barcelona, 08028, Spain

    Oriol Pineda

  4. Division of Cancer Treatment and Diagnosis, Toxicology and Pharmacology Branch, Developmental Therapeutics Program, National Cancer Institute at Frederick, National Institutes of Health, Frederick, 21702, Maryland, USA

    Michael C Edler & Ernest Hamel

  5. PharmaMar, S.A. Avda. de los Reyes 1, Colmenar Viejo, 28770, Spain

    Gemma Cerezo

  6. Department of Chemistry, University of California, Irvine, 92697, California, USA

    Christopher D Vanderwal

  7. Departments of Pharmaceutical Sciences and Chemistry, University of Pittsburgh, Pittsburgh, 15261, Pennsylvania, USA

    Billy W Day

  8. Department of Chemistry, Princeton University, Princeton, 08544, New Jersey, USA

    Erik J Sorensen

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  1. Rubén M Buey
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Contributions

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.

Corresponding author

Correspondence to J Fernando Díaz.

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The authors declare no competing financial interests.

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)

Supplementary Methods (PDF 87 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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