Discovery and Characterization of Allosteric WNK Kinase Inhibitors

Abstract

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 or stringent hit triaging with high ATP concentration offers conceptually simple methods of identifying inhibitors that bind outside the ATP pocket. Here, we applied the latter approach to the With-No-Lysine (K) (WNK) kinases to discover lead molecules for a next-generation antihypertensive that requires a stringent safety profile. This strategy yielded several ATP noncompetitive WNK1-4 kinase inhibitors, the optimization of which enabled cocrystallization 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 cotransporter 1 (NKCC1) in HT29 cells, consistent with the reported physiology of WNK kinases in renal electrolyte handling.

Cheminformatics Analysis of Dynamic WNK‐Inhibitor Interactions

“…Dataset compilation and curation . For this study, we assembled a dataset of WNK inhibitors extracted from the recent literature, and the CHEMBL22 database . Overall, a set of 210 compounds with their respective experimental activity towards one (or several) WNK isoforms was retrieved.…”

“…Due to the high sequence and structural similarity of the ATP binding sites of WNK proteins (and more generally kinases), it is extremely challenging to develop WNK‐selective inhibitors. Therefore, the recent emergence of allosteric WNK inhibitors is sought to boost the drug discovery process based on the WNK kinase family. A high‐throughput screening campaign using a single‐point screen and 1.2 million in‐house compound library enabled Yamada et al.…”

“…Therefore, the recent emergence of allosteric WNK inhibitors is sought to boost the drug discovery process based on the WNK kinase family. A high‐throughput screening campaign using a single‐point screen and 1.2 million in‐house compound library enabled Yamada et al. at Novartis to discover an ATP noncompetitive, allosteric WNK1 kinase inhibitor called WNK476 (IC 50 =0.18 μM).…”

Docking simulation of fragment library compounds to find new leads for specific WNK kinase inhibitors

“…Among several crystal structures (PDB code: 3FPQ [6], 4PWN [7], 4Q2A [7], 5DRB [4], 5TF9 [5]) of WNK1 reported so far, we focused on 3FPQ-B ( Figure S1b), which possesses a deep back pocket and contains glycerols in the hinge region and at the bottom of the back pocket. Docking simulation targeting a glycerol at the bottom of the back pocket was performed with the MOE-Dock program in the Molecular Operating Environment (MOE) [8].…”

Competitive fragment assay for the selective inhibitor of WNKs kinase

“…Accordingly, we hypothesized that compounds interacting with the back pocket and Lys233 in WNK1, but not with the hinge region, would be promising candidates for specific inhibitors. Among reported WNK1 and WNK4 kinase inhibitors, only one is so far known to interact with the back pocket [3,[5][6].…”