Identification of Potent Reverse Indazole Inhibitors for HPK1

Abstract

Hematopoietic progenitor kinase (HPK1), a negative regulator of TCR-mediated T-cell activation, has been recognized as a novel antitumor immunotherapy target. Structural optimization of kinase inhibitor 4 through a systematic two-dimensional diversity screen of pyrazolopyridines led to the identification of potent and selective compounds. Crystallographic studies with HPK1 revealed a favorable water-mediated interaction with Asp155 and a salt bridge to Asp101 with optimized heterocyclic solvent fronts that were critical for enhanced potency and selectivity. Computational studies of model systems revealed differences in torsional profiles that allowed for these beneficial protein-ligand interactions. Further optimization of molecular properties led to identification of potent and selective reverse indazole inhibitor 36 that inhibited phosphorylation of adaptor protein SLP76 in human PBMC and exhibited low clearance with notable bioavailability in in vivo rat studies.

Figure 1

Co-crystal of HPK1 enzyme with different RI ligands show two-point hinge contacts with Cys94 and Glu92. (a) Co-crystal of HPK1 with 11 (PDB: 7L24, resolution 2.7 Å). (b) Co-crystal of HPK1 with 18. Aza of core generates water network with Asp155 (PDB: 7L25, resolution 1.9 Å). (c) Co-crystal of HPK1 with 38. Displacement of unfavorable water molecule and Asp155 moves closer to ligand (PDB: 7L26, resolution 2.3 Å).

Figure 2

(a) Lowest energy conformer of phenyl analogue of 16. Dihedral angles of RI core to phenyl group calculated to be 35°. (b) Lowest energy conformer of 2-pyridyl analogue 18. Dihedral angles of RI core to 2-pyridyl group calculated to be 0°. (c) Lowest energy conformer of 4-pyridyl analogue of 19. Dihedral angles of RI core to 4-pyridyl group calculated to be 29°. Calculated with M06-2X/6-31G**.