Discovery, characterization, and preclinical development of a Kir4.1 (KCNJ10) inhibitor for the treatment of hypertension

Abstract

Kir4.1 (encoded by KCNJ10) inward rectifier potassium channels are emerging as key regulators of sodium reabsorption in the distal nephron of the kidney. Kir4.1 is expressed in the basolateral membrane of the thick ascending limb of Henle, distal convoluted tubule, and collecting duct of nephron. Kir4.1 can form homotetrameric channels or heterotetrameric channels with Kir5.1, although the most common channel subtype in the kidney is the heteromeric Kir4.1/5.1 form. Genetic ablation of Kir4.1 in mice or humans causes severe salt and water wasting resembling clinical phenotypes associated with diuretic use. These human validation data and recent animal studies suggest that Kir4.1 might represent a novel diuretic target for the management of hypertension through inhibition of distal nephron sodium reabsorption. The aim of this study was to develop a selective small‐molecule inhibitor for evaluating the therapeutic potential of renal Kir4.1 channels. From a high‐throughput screen of 76,575 compounds from the Vanderbilt Institute of Chemical Biology library, we identified an inhibitor, termed VU992, which inhibits homotetrameric Kir4.1 with an IC50 of ~0.9 μM and heterotetrameric Kir4.1/5.1 with an IC50 of ~ 9 μM. Lead optimization failed to identify analogs with improved potency toward Kir4.1. The selectivity screening against other Kir channels revealed that VU992 does not block Kir1.1, Kir2.x, Kir6.1/SUR1 and Kir7.1 channels. The voltage dependent unblock suggested that VU992 probably binds in the pore of the Kir4.1 channel. Computational docking analysis using a Kir4.1 homology model revealed several possible interacting residues in the pore. Experimental binding site analysis using mutagenesis and patch clamp electrophysiology suggested that the pore lining glutamic acid residue at 158 (E158) is crucial for the binding of VU992 to the Kir4.1. Mutating E158 to corresponding asparagine residue in Kir1.1 (E158N) abolished the VU992 block completely. Pharmacokinetic studies in rats showed that VU992 does not cross the blood brain barrier and therefore should not inhibit Kir4.1 channels expressed in the CNS. Finally, oral administration of VU992 dose‐dependently increased urine output and Na+ and K+ loss in volume‐loaded, freely behaving rats. This study shows proof‐of‐concept that homotetrameric Kir4.1 might represent a molecular target for a novel class of diuretic for the treatment of hypertension. Its basolateral localization might offer advantages over other diuretic targets that are located on the luminal membrane of the nephron.Support or Funding InformationNIH 5R21 NS073097‐01S1This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.