Pironetin reacts covalently with cysteine-316 of α-tubulin to destabilize microtubule

Abstract

Molecules that alter the normal dynamics of microtubule assembly and disassembly include many anticancer drugs in clinical use. So far all such therapeutics target β-tubulin, and structural biology has explained the basis of their action and permitted design of new drugs. However, by shifting the profile of β-tubulin isoforms, cancer cells become resistant to treatment. Compounds that bind to α-tubulin are less well characterized and unexploited. The natural product pironetin is known to bind to α-tubulin and is a potent inhibitor of microtubule polymerization. Previous reports had identified that pironetin reacts with lysine-352 residue however analogues designed on this model had much lower potency, which was difficult to explain, hindering further development. We report crystallographic and mass spectrometric data that reveal that pironetin forms a covalent bond to cysteine-316 in α-tubulin via a Michael addition reaction. These data provide a basis for the rational design of α-tubulin targeting chemotherapeutics.

SKLB060 Reversibly Binds to Colchicine Site of Tubulin and Possesses Efficacy in Multidrug-Resistant Cell Lines

“…The preparation of the crystals of the T2R-TTL complex (T2: αβ-tubulin heterodimer, R: the stathmin-like protein RB3, TTL: tubulin tyrosine ligase) is described in our previous study [14]. To soak the compounds into the crystals, 0.1 µL of the compound solution (10 mM in DMSO) was added to the 2-µL crystal-containing drops for 24 h at 20°C.…”

“…Beamlines BL17U1 and BL19U1 at the Shanghai Synchrotron Radiation Facility were used to obtain X-ray diffraction data. Determination of the structure and the refinement protocols were the same as those in our previous study [14]. PYMOL and Discovery Studio 4.5 Client were used to generate the Fig., …”

“…Apart from the well-known taxane or vinca alkaloid binding sites, there are other binding sites on tubulin, such as the colchicine [11], laulimalide [12], maytansine [13] and pironetin [14] (binding to α-tubulin) binding sites. Among them, the colchicine binding site may be a promising target because most tubulin inhibitors that bind to this site are not substrates of MDR proteins [15].…”

Synthesis and Cytotoxicity Evaluation of C4‐ and C5‐Modified Analogues of the α,β‐Unsaturated Lactone of Pironetin

“…Analogues 6, 19 and 32-34 were docked into the pironetin binding site in a-tubulin. [24,25] Because pironetin is ac ovalent inhibitor,d ocking scoresw ere calculated using the CovDock module in the Schrçdinger Maestro software package. [51] While we were able to dock our analoguesi nto the binding site, ac orrelation was unfortunately not observed between the CovDock scores and the observed antiproliferativea ctivity.…”

“…[23] However,t he X-ray crystal structure of pironetin-bound a-tubulins howed ac ovalent adduct being formed between cysteine 316 insteado fl ysine 352. [24,25] Although pironetin has potent antiproliferative activity in vitro against variousc ancerc ell lines including cell lines which overexpress P-glycoprotein [21] while maintaining inactivity against normall ung fibroblasts, [22] the natural product has not been developed as achemotherapeutic agent.…”