Targeting the “Undruggable” STAT3 DBD: Free-Energy Perturbation-Guided Design of Covalent Micheliolide Derivatives Targeting Cysteine 367

These derivatives retain comparable antiproliferative potency but exhibit markedly improved physicochemical properties, including enhanced kinetic aqueous solubility and optimized lipophilicity. Microscale thermophoresis confirms C367-dependent binding, while microsecond-scale molecular dynamics simulations and pair-interaction energy analyses highlight stable complexes in which electrostatic interactions predominate, with major contributions from key residues (Lys365, Val366, and Ile368).

This study establishes C367 as a druggable covalent site in the historically challenging STAT3 DBD, validates FEP-guided optimization of covalent inhibitors, and identifies compound <b>16</b> as a promising next-generation lead with a superior drug-like profile for targeting STAT3-dependent cancers. All in all, these findings provide a foundation for advancing selective STAT3 DBD inhibitors toward clinical translation.