Cyclostreptin binds covalently to microtubule pores and lumenal taxoid binding sites

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 beta-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 beta-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.

Microtubule Active Agents: Beyond the Taxane Frontier

“…Cyclostreptin was originally recovered from the fermentation broth of a bacterium from the Streptomyces species (54). Although it is less cytotoxic than paclitaxel in vitro, it is active in taxane-resistant cell lines possibly because of a novel mechanism of action involving covalently cross-linking with h-tubulin (54).…”

15-Deoxi-Δ12,14-prostaglandin J2 is a tubulin-binding agent that destabilizes microtubules and induces mitotic arrest

“…To this end, we first analyzed the incorporation of biotinylated 15d-PGJ 2 into -and -tubulin by A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 15 Neutravidin-gel pull down followed by Western blot with anti--tubulin or anti--tubulin antibodies. triple-quadrupole mass spectrometry to find the 15d-PGJ 2 -binding site(s) [28].…”

Synthesis and Biological Evaluation As Microtubule-Active Agents of Several Tetrahydrofuran and Spiroacetal Derivatives

“…Human A549 non small lung carcinoma cells were cultured in RPMI 1640 supplemented with 10% FCS, glutamine, and antibiotics as previously described [38]. Human ovarian carcinomas A2780 and A2780AD (MDR overexpressing P-glycoprotein) were cultured as above with the addition of 0.25 units/mL of bovine insulin.…”