Abstract
The inward rectifier potassium (Kir) channel Kir4.1 (KCNJ10) carries out important physiologic 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. Although 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-isopropylphenoxy)-N-(2,2,6,6-tetramethylpiperidin-4-yl)acetamide (VU0134992). In whole-cell patch-clamp electrophysiology experiments, VU0134992 inhibits Kir4.1 with an IC50 value 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 unbound fraction (fu) 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.
Footnotes
- Received March 1, 2018.
- Accepted May 30, 2018.
Work in the Denton laboratory was funded by National Institutes of Health (NIH) Grants [5R21-NS-073097-01S1 and R01-DK-082884]. E.F. was supported by NIH Training Grant [5T32- GM-007628]. Work in the Meiler laboratory is supported by NIH Grants [R01- GM-080403, R01-HL-122010] and National Science Foundation Grant [CHE 1305874]. Work in the Satlin laboratory is supported through NIH Grants [DK-038470 and P30-DK-079307, The Pittsburgh Center for Kidney Research]. The WaveFront Biosciences Panoptic kinetic imaging plate reader is housed and managed within the Vanderbilt High-Throughput Screening Core Facility, an institutionally supported core, and was funded by NIH Shared Instrumentation Grant [1S10-OD-021734]. C.D.W. is an owner of WaveFront Biosciences, manufacturer of the Panoptic plate reader, receives royalties from the sales of Thallos via a licensing agreement with Vanderbilt University. No other potential conflicts of interest relevant to this article are reported.
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- Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics
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