Pironetin Binds Covalently to αCys316 and Perturbs a Major Loop and Helix of α-Tubulin to Inhibit Microtubule Formation

Abstract

Microtubule-targeting agents are among the most powerful drugs used in chemotherapy to treat cancer patients. Pironetin is a natural product that displays promising anticancer properties by binding to and potently inhibiting tubulin assembly into microtubules; however, its molecular mechanism of action remained obscure. Here, we solved the crystal structure of the tubulin-pironetin complex and found that the compound covalently binds to Cys316 of α-tubulin. The structure further revealed that pironetin perturbs the T7 loop and helix H8 of α-tubulin. Since both these elements are essential for establishing longitudinal tubulin contacts in microtubules, this result explains how pironetin inhibits the formation of microtubules. Together, our data define the molecular details of the pironetin binding site on α-tubulin and thus offer a promising basis for the rational design of pironetin variants with improved activity profiles. They further extend our knowledge on strategies evolved by natural products to target and perturb the microtubule cytoskeleton.

Mechanism of microtubule stabilization by taccalonolide AJ

“…12) and pironetin to αC316 (refs 13, 14). AJ has also been suggested to react covalently with tubulin and the binding region has been narrowed down by mass spectrometry to the β212–230 peptide11.…”

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

“…Therefore, we investigated whether molecular modeling could be used as a tool for the design of future analogues. Analogues 6 , 19 and 32 – 34 were docked into the pironetin binding site in α‐tubulin . Because pironetin is a covalent inhibitor, docking scores were calculated using the CovDock module in the Schrödinger Maestro software package .…”

“…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.…”

“…Osada and co‐workers had originally proposed that pironetin forms a covalent bond with lysine 352 of α‐tubulin via conjugate addition into the α,β‐unsaturated lactone . However, the X‐ray crystal structure of pironetin‐bound α‐tubulin showed a covalent adduct being formed between cysteine 316 instead of lysine 352 . Although pironetin has potent antiproliferative activity in vitro against various cancer cell lines including cell lines which overexpress P‐glycoprotein while maintaining inactivity against normal lung fibroblasts, the natural product has not been developed as a chemotherapeutic agent.…”

Structure-activity relationships, biological evaluation and structural studies of novel pyrrolonaphthoxazepines as antitumor agents

“…The most well-characterized sites include: i) the well-known taxane site, targeted by compounds such as taxol (1), located on β-tubulin in a profound hydrophobic cleft; ii) the site targeted by laulimalide (2) that binds to β-tubulin in a different pocket from the taxane site; iii) the vinca site located at the plus-end edge near the GTP binding site of β-tubulin, targeted by compounds such as vinblastine (3); and iv) the colchicine site situated at the tubulin intradimer interface between the αand β-tubulin subunit and targeted by MTAs such as colchicine (4) and nocodazole ( Figure 1) [8]. Additional very well characterized sites are the maytansine site [9] and the pironetin site [10]. The colchicine site is one of the most important binding pockets for MTAs and compounds acting on this site (nocodazole, 5) are well-known microtubuledestabilizing agents (MDAs).…”