Structure-guided discovery of a novel BTK inhibitor inducing apoptosis and G1 phase arrest in tumor cells.

Bruton's tyrosine kinase (BTK) is a pivotal component of multiple signaling pathways in hematopoietic cells and serves as a critical pharmacological target in B-cell malignancies. Despite the availability of clinically approved BTK inhibitors, therapeutic resistance and limited efficacy in certain patient populations necessitate the discovery of novel candidates. In this study, virtual high-throughput screening of the ZINC database was employed to identify potential BTK inhibitors. Compounds were prioritized based on molecular docking scores, binding patterns, and free energy calculations. ZINC000045971961 (ZINC1961) emerged as a promising lead compound, forming stable hydrogen bonds with Glu475 and Met477 key residues also targeted by the reference inhibitor Ibrutinib. Molecular dynamics simulations and MM/GBSA free energy analysis further confirmed the stability and favorable binding affinity of ZINC1961. Biological evaluation in primary tumor cells demonstrated potent cytotoxicity, with an IC₅₀ of 80 ± 0.5 µM, and pronounced apoptosis confirmed by AO/EB/DAPI triple staining, Annexin-V/PI assay, and scanning electron microscopy. Additionally, ZINC1961 induced G1 phase cell cycle arrest, contributing to its antiproliferative effects. Collectively, these findings not only highlight ZINC1961 as a novel BTK inhibitor but also underscore the power of integrative in silico and in vitro approaches in accelerating early-stage cancer drug discovery.