Discovery and characterization of allosteric WNK kinase inhibitors.

Protein kinases are known for their highly conserved adenosine triphosphate (ATP)-binding site, rendering the discovery of selective inhibitors a major challenge. In theory, allosteric inhibitors can achieve high selectivity by targeting less conserved regions of the kinases, often with an added benefit of retaining efficacy under high physiological ATP concentration. Although often overlooked in favor of ATP-site directed approaches, performing a screen at high ATP concentration and/or stringent hit triaging with high ATP concentration offer conceptually simple methods of identifying allosteric inhibitors. Here we applied the latter approach to the With-No-Lysine (K) (WNK) kinases to discover lead molecules for a next-generation anti-hypertensive that requires a stringent safety profile. This strategy yielded several ATP non-competitive WNK1-4 kinase inhibitors, whose optimization enabled co-crystallization with WNK1, revealing an allosteric binding mode consistent with the observed exquisite specificity for WNK1-4 kinases. The optimized compound inhibited rubidium uptake by sodium chloride co-transporter 1 (NKCC1) in HT29 cells, consistent with the known WNK biology.