Discovery of Novel HPK1 Inhibitors via High‐Throughput Virtual Screening, Biological Evaluation, and Molecular Dynamics Simulation

ABSTRACT Hematopoietic progenitor kinase 1 (HPK1) acts as a negative regulator of T‐cell receptor signaling, making it a high‐priority target for cancer immunotherapy. Addressing the scarcity of structurally novel HPK1 inhibitors, we constructed and validated a comprehensive high‐throughput virtual screening (HTVS) workflow to screen the ChemDiv database. This hierarchical protocol, characterized by a hybrid docking strategy and MM/GBSA rescoring, identified 10 candidates for biological testing. Experimental evaluation confirmed compound 8 as a potent hit, exhibiting an IC 50 of 1585 nM and 91.65% inhibition at 25 μM. Subsequent 500 ns molecular dynamics simulations validated the stability of the compound 8 –HPK1 complex ( ΔG binding = −41.59 kcal/mol), driven by critical interactions with residues GLU92 and CYS94. These findings establish compound 8 as a promising scaffold for structural optimization and demonstrate the robustness of our computational framework for HPK1 inhibitor discovery.