PT - JOURNAL ARTICLE AU - Sujay V. Kharade AU - Haruto Kurata AU - Aaron Bender AU - Anna L. Blobaum AU - Eric E. Figueroa AU - Amanda M. Duran AU - Meghan Kramer AU - Emily Days AU - Paige Vinson AU - Daniel Flores AU - Lisa M. Satlin AU - Jens Meiler AU - C. David Weaver AU - Craig W. Lindsley AU - Corey R. Hopkins AU - Jerod S. Denton TI - Discovery, characterization, and effects on renal fluid and electrolyte excretion of the Kir4.1 potassium channel pore blocker, VU0134992 AID - 10.1124/mol.118.112359 DP - 2018 Jan 01 TA - Molecular Pharmacology PG - mol.118.112359 4099 - http://molpharm.aspetjournals.org/content/early/2018/06/13/mol.118.112359.short 4100 - http://molpharm.aspetjournals.org/content/early/2018/06/13/mol.118.112359.full AB - The inward rectifier potassium (Kir) channel Kir4.1 (KCNJ10) carries out important physiological roles in epithelial cells of the kidney, astrocytes in the central nervous system, and stria vascularis of the inner ear. Loss-of-function mutations in KCNJ10 lead to EAST/SeSAME syndrome, which is characterized by epilepsy, ataxia, renal salt wasting, and sensorineural deafness. While genetic approaches have been indispensable for establishing the importance of Kir4.1 in the normal function of these tissues, the availability of pharmacological tools for acutely manipulating the activity of Kir4.1 in genetically normal animals has been lacking. We therefore carried out a high-throughput screen of 76,575 compounds from the Vanderbilt Institute of Chemical Biology library for small-molecule modulators of Kir4.1. The most potent inhibitor identified was 2-(2-Bromo-4-iso-propylphenoxy)-N-(2,2,6,6-tetramethylpiperidin-4-yl)acetamide (VU0134992). In whole-cell patch clamp electrophysiology experiments, VU0134992 inhibits Kir4.1 with an IC50 of 0.97 μM and is 9-fold selective for homomeric Kir4.1 over Kir4.1/5.1 concatemeric channels (IC50=9 μM) at -120 mV. In thallium (Tl+) flux assays, VU0134992 is greater than 30-fold selective for Kir4.1 over Kir1.1, Kir2.1, and Kir2.2, is weakly active toward Kir2.3, Kir6.2/SUR1, and Kir7.1, and is equally active toward Kir3.1/3.2, Kir3.1/3.4, and Kir4.2. This potency and selectivity profile is superior to Kir4.1 inhibitors amitriptyline, nortriptyline, and fluoxetine. Medicinal chemistry identified components of VU0134992 that are critical for inhibiting Kir4.1. Patch clamp electrophysiology, molecular modeling, and site-directed mutagenesis identified pore-lining glutamate 158 and isoleucine 159 as critical residues for block of the channel. VU0134992 displayed a large free fraction in rat plasma (fu = 0.213). Consistent with the known role of Kir4.1 in renal function, oral dosing of VU0134992 led to a dose-dependent diuresis, natriuresis, and kaliuresis in rats. Thus, VU0134992 represents the first in vivo-active tool compound for probing the therapeutic potential of Kir4.1 as a novel diuretic target for the treatment of hypertension.