Optimization of Allosteric With-No-Lysine (WNK) Kinase Inhibitors and Efficacy in Rodent Hypertension Models.

The observed structure-activity relationship of three distinct ATP noncompetitive With-No-Lysine (WNK) kinase inhibitor series, together with a crystal structure of a previously disclosed allosteric inhibitor bound to WNK1, led to an overlay hypothesis defining core and side-chain relationships across the different series. This in turn enabled an efficient optimization through scaffold morphing, resulting in compounds with a good balance of selectivity, cellular potency, and pharmacokinetic profile, which were suitable for in vivo proof-of-concept studies. When dosed orally, the optimized compound reduced blood pressure in mice overexpressing human WNK1, and induced diuresis, natriuresis and kaliuresis in spontaneously hypertensive rats (SHR), confirming that this mechanism of inhibition of WNK kinase activity is effective at regulating cardiovascular homeostasis.

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 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.

Kinetic mechanism and inhibitor characterization of WNK1 kinase.

Pseudohypoaldosteronism type II (PHAII) is caused by the mutation of two members of the WNK (with-no-K[Lys] kinase) kinase family. We describe here the development of an in vitro WNK1 microfluidic mobility shift assay for kinetic mechanism studies. Assays using capillary electrophoresis on a microfluidic chip are suitable for both compound selection and mechanistic studies, because of the robustness of this method, as well as its high-throughput feature and insensitivity to the ATP concentration. Double-reciprocal plots of the initial rates versus the concentration of the substrate revealed that the random sequential activity of WNK catalyzed OXSR1 (oxidative stress response kinase-1) phosphorylation. WNK1 inhibitors were then found from among 86 kinases in a commercially available library. Interestingly, the Hck, Lck, and Src inhibitors, PP1 and PP2, exhibited positive inhibition against WNK1. The inhibition mode of PP1 was analyzed to be pure ATP competition with a K(i) value of 12.7 microM, showing noncompetitive inhibition against the OXSR1 peptide. From the structure-based comparison, we found that, since the WNK1 enzymes are categorized as STEs (homologues of yeast Sterile 7, Sterile 11, and Sterile 20 kinases) and Hck belongs to the TK (tyrosine kinase) family on the basis of the results of the Human Kinome Project, the residues at the catalytic site of the WNK1 that interact with PP1 were well-conserved in Hck. We concluded that the compound-based structural alignment enabled us to find interesting relationships among the kinases. This information helps us to screen specific WNK1 therapeutic reagents with no inhibition of the Src, Hck, and Lck kinases for the treatment of hypertension.