Discovery, Characterization, and Effects on Renal Fluid and Electrolyte Excretion of the Kir4.1 Potassium Channel Pore Blocker, VU0134992

Abstract

The inward rectifier potassium (Kir) channel Kir4.1 () 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 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)--(2,2,6,6-tetramethylpiperidin-4-yl)acetamide (VU0134992). In whole-cell patch-clamp electrophysiology experiments, VU0134992 inhibits Kir4.1 with an IC value of 0.97 M and is 9-fold selective for homomeric Kir4.1 over Kir4.1/5.1 concatemeric channels (IC = 9 M) 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 () in rat plasma ( = 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.

Regulation of the Renal NaCl Cotransporter and Its Role in Potassium Homeostasis

“…Moreover, it is conceivable that drugs that inhibit the RAAS or block ENaC may exert their beneficial effects in part by increasing plasma K ϩ and, consequently, decreasing NCC phosphorylation. Clinically, the pharmacological treatment of hypertension may also benefit from the novel insights into the regulation of NCC, as several novel diuretics targeting the DCT are developed, including inhibitors of Kir4.1, ClC-K, WNK, and SPAK kinases (51,136,150,152,175). Genome-wide association studies (GWAS) suggest that polymorphisms in the genes encoding NCC, SPAK, or WNK kinases may explain differences in blood pressure, although results are not consistent.…”

Next-generation inward rectifier potassium channel modulators: discovery and molecular pharmacology

“…There are a number of neurological drugs (e.g., selective serotonin reuptake inhibitors and tricyclic antidepressants) that inhibit homomeric Kir4.1 channels; however, they block Kir4.1 at concentrations in the tens of micromolar (140)(141)(142). As a first step toward developing more potent and specific small-molecule tools for probing the physiology and therapeutic value of Kir4.1, we screened 76,574 compounds from the VICB library for modulators of homomeric Kir4.1 channels using a thallium flux assay (143). Putative inhibitors and potentiators of Kir4.1 were identified in the screen.…”

Screening Technologies for Inward Rectifier Potassium Channels: Discovery of New Blockers and Activators

“…HEK293 cells expressing either Kir4.1 or Kir6.2/SUR1 channels were screened in 386-well format using the Tl + -sensitive dye Thallos-AM. 85,86 VU0134992 (IC 50 = 1 µM), the most potent Kir4.1 inhibitor identified in the screen, was 30-fold more selective for Kir4.1 channels over Kir1.1, Kir2.1, and Kir2.2 channels, but showed equal activity toward GIRK and Kir4.2 channels. 85 Through the use of whole-cell patch clamping, glutamate and isoleucine residues lining the pore of the Kir4.1 channel were found to be critical for the activity of VU0134992.…”