Abstact

The phosphatidylinositol 5-phosphate 4-kinases (PIP4Ks) are an evolutionarily conserved family of lipid kinases that phosphorylate phosphatidylinositol 5-phosphate to generate phosphatidylinositol 4,5-bisphosphate. In mammals, the catalytically active alpha and beta isoforms, encoded by PIP4K2A and PIP4K2B, respectively, localize to distinct cellular compartments and have been implicated in metabolism, immune regulation and tumorigenesis, prompting interest in their pharmacological inhibition. Notably, most reported small-molecule inhibitors display substantially higher potency towards the alpha isoform than the beta isoform, suggesting intrinsic structural features that limit the effective targeting of PIP4K2B. Here, we report the crystal structure of PIP4K2A in complex with 422A, a potent dual alpha/beta inhibitor with improved metabolic stability. The structure reveals an unexpected, water-mediated interaction in which a pyridyl nitrogen of the inhibitor engages a conserved structured water molecule in the roof of the specificity pocket, constraining the orientation of the pyridylmethyl side chain and stabilizing a rigid, high-affinity binding mode. Comparative structural analysis with the PIP4K2A-selective inhibitor BAY-091 shows that deeper penetration into the specificity pocket enhances PIP4K2A binding but is accompanied by local steric constraints that are likely to be less well tolerated in PIP4K2B. Together, these findings define structural determinants of isoform-dependent inhibitor binding within the PIP4K family and provide a framework for structure-guided optimization of lipid kinase inhibitors with improved isoform balance.