Studies of (-)-Pironetin Binding to α-Tubulin: Conformation, Docking, and Molecular Dynamics

A comprehensive conformational analysis for the anticancer agent pironetin (1) was achieved by molecular modeling using density functional theory calculations at the B3PW91/DGTZVP level in combination with calculated and experimental H-1-H-1 coupling constants comparison. Two solvent-dependent conformational families (L and M) were revealed for the optimum conformations. Docking studies of the pironetin-tubulin complex determined a quantitative model for the hydrogen-bond interactions of pironetin through the alpha Asn249, alpha Asn258, and alpha Lys352 amino groups in alpha-tubulin, which supported the formation of a covalent adduct between the alpha Lys352 and the beta carbon atom of the alpha,beta-unsaturated lactone. Saturation-transfer difference NMR spectroscopy confirmed that pironetin binds to tubulin, while molecular dynamics exposed a distortion of the tubulin secondary structure at the H8 and HIO10 alpha-helices as well as at the S9 beta-sheet, where alpha Lys352 is located. A large structural perturbation in the M-loop geometry between the alpha Ile274 and alpha Leu285 residues, an essential region for molecular recognition between alpha-alpha and beta-beta units of protofilaments, was also identified and provided a rationale for the pironetin inhibitory activity.