Structure-based discovery of novel BCL-X_L inhibitors through molecular docking and key interaction-guided virtual screening

BCL-X_L, an anti-apoptotic member of the BCL-2 protein family, is an attractive therapeutic target for overcoming apoptosis resistance in cancer. However, the large, shallow, and predominantly hydrophobic nature of its BH3-binding groove has complicated the discovery of potent, drug-like small-molecule inhibitors. Here, we employed structure-based virtual screening with key interaction constraints to identify novel BCL-X_L inhibitor scaffolds with relatively low molecular weights (MWs). Approximately 312,000 drug-like compounds were docked into the BH3-binding groove, and the top-ranked molecules were filtered for simultaneous hydrogen-bond interactions with Ser106 and Arg139, yielding 26 candidates for experimental evaluation. Biochemical competitive binding assays identified two previously unreported inhibitors, 1 and 2, with IC₅₀ values of 0.34 and 2.51 μM, respectively. Both compounds possess relatively low MWs and moderate lipophilicity, providing favorable starting points for further medicinal chemistry optimization. Docking analyses indicated that both inhibitors occupy the canonical BH3-binding groove and are stabilized by the targeted hydrogen bonds with Ser106 and Arg139, together with extensive hydrophobic contacts. Notably, 1 forms an additional hydrogen bond with Arg139 through its benzo[c]isoxazole moiety, which may contribute to its higher potency. These findings establish 1 and 2 as novel BCL-X_L inhibitor scaffolds and support the use of key interaction constraints to improve docking-based virtual screening against challenging protein-protein interaction targets. Future studies will focus on structure-guided optimization of these scaffolds to strengthen their interactions within the BH3-binding groove, improve inhibitory potency toward the nanomolar range, and evaluate their cellular activity and drug-like properties.