Discovery of Novel HPK1 Inhibitors via Chemistry-Based Direct-to-Bioassay Screening and SAR Optimization

Currently, efficient exploration of biologically relevant chemical space remains a significant challenge in lead discovery. Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T cell activation and a high-priority target for cancer immunotherapy. Herein, we report an integrated combinatorial chemistry-biological assay approach to accelerate lead identification for HPK1. First, an in-house HPK1 inhibitor library was fragmented and recombined <i>in silico</i>, followed by molecular docking, yielding preferred fragments. Then, selected fragments were assembled in a microplate, and the resulting compounds were used directly to perform biological assays, leading to the identification of lead compound <b>8b</b>. Guided by four series of structure-activity relationship studies, our efforts afforded the optimized compound <b>53</b> (HPK1 IC₅₀ = 1.7 nM). Compound <b>53</b> suppressed SLP76 phosphorylation, enhanced IL-2 release in the cell, and displayed low CYP/hERG risk. Moreover, compound <b>53</b> demonstrated potent <i>in vivo</i> antitumor efficacy in both mouse models, which highlighted its potential as a preclinical immunotherapy candidate.