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 in silico,
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 8b. Guided by
four series of structure–activity relationship studies, our
efforts afforded the optimized compound 53 (HPK1 IC50 = 1.7 nM). Compound 53 suppressed SLP76 phosphorylation,
enhanced IL-2 release in the cell, and displayed low CYP/hERG risk.
Moreover, compound 53 demonstrated potent in
vivo antitumor efficacy in both mouse models, which highlighted
its potential as a preclinical immunotherapy candidate.