WNK (With No Lysine [K]) kinases are serine/threonine kinases implicated in familial hyperkalemic hypertension (FHHt). Beyond blood pressure regulation, WNKs are emerging therapeutic targets for stroke and certain cancers, including triple-negative breast cancer and glioblastoma. In this study, we aimed to identify and characterize novel inhibitors of WNK kinases. A high-throughput screening of approximately 210,000 compounds was performed, followed by compound re-acquisition, secondary assays, commercial selectivity profiling, and crystallographic analysis to discover inhibitors with isoform-specific activity against WNK kinases. Five distinct classes of compounds were found to inhibit WNK1 kinase activity: quinoline derivatives, halo-sulfones, cyclopropane-containing thiazoles, piperazine-containing compounds, and nitrophenol-based molecules. These compounds broadly inhibit all four WNK isoforms, demonstrating strong pan-WNK selectivity. Notably, a subset of quinoline derivatives displayed isoform-selective potency, with greater inhibition of WNK3 compared to WNK1. Structural analysis of the quinoline compound SW120619 bound to WNK3’s kinase domain revealed active site engagement, and comparison with WNK1 provided insights into the molecular basis of isoform specificity. The identified compound classes provide promising scaffolds for the development of pharmacological tools and potential therapeutic agents targeting hypertension and cancer.
