Discovery of highly selective and potent HPK1 inhibitors for cancer immunotherapy

Hematopoietic progenitor kinase 1 (HPK1) is an intracellular negative regulator of T cell receptor (TCR) signaling having been considered as an important target for cancer immunotherapy. Pharmacological studies in preclinical models have shown that the inhibition or depletion of HPK1 achieved the reinvigoration of exhausted T cells and enhanced anti-tumor immunity. Although HPK1 inhibitors in clinical trials have shown preliminary clinical benefits, lager scale verification is still needed. High selectivity, especially within the MAP4K family, is critical for the development of HPK1 inhibitors in clinic. Herein, guided by structural pharmacology, we developed a novel HPK1 inhibitor, compound 34, with excellent selectivity and potency. Compound 34 robustly inhibited HPK1 kinase activity with an IC₅₀ value lower than 5 nM and displayed 1257-fold selectivity over the MAP4K kinase family member GLK. Cocrystal structures combining with docking analysis unveiled that the stable U-shaped non-planar conformation of these serials of compounds renders high selectivity over GLK. Treatment with compound 34 inhibited TCR-induced phosphorylation of SLP76 at Ser376 in a dose-dependent manner. Pharmacokinetic evaluation revealed high oral bioavailability of compound 34. Furthermore, oral dosing of compound 34 combined with anti-PD-1 achieved significant anti-tumor effect (TGI = 62.90 %) in the CT-26 syngeneic model. These findings demonstrated compound 34 as a promising preclinical candidate worthy of further investigation.