Discovery and Characterization of Small-Molecule Potentiators of K<sub>ir</sub>4.1/5.1
October 22, 2020 | 10:00 AM - 12:00 PM
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Discovery and Characterization of Small-Molecule Potentiators of Kir4.1/5.1
- Mechanisms of Kidney and Vascular Disease
October 22, 2020 | Location: On-Demand
Abstract Time: 10:00 AM - 12:00 PM
Category: Hypertension and CVD
- 1403 Hypertension and CVD: Mechanisms
- Mcclenahan, Samantha J., Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Denton, Jerod S., Vanderbilt University Medical Center, Nashville, Tennessee, United States
Samantha J. Mcclenahan,
Jerod S. Denton,
Heterotetrameric inward rectifier potassium (Kir) channels composed of Kir4.1 (KCNJ10) and Kir5.1 (KCNJ16) play key roles in regulation of sodium, potassium, and water balance by the distal convoluted tubule of the renal tubule. Loss-of- function mutations in KCNJ10 lead to EAST syndrome, which is characterized by neurological dysfunction and renal salt wasting. Here we describe the discovery and characterization of small-molecule potentiators of Kir4.1/5.1 that could potentially be used to rescue the function of EAST syndrome mutant channels.
We established a monoclonal HEK-293 cell line that stably expresses human Kir4.1 and Kir5.1 from a bicistronic vector, and developed and validated a fluorescence-based thallium-flux assay of Kir4.1/5.1 channel function in 384-well format. This assay was used to screen small-molecules from the Vanderbilt Chemistry Institute Library and confirmed with whole-cell electrophysiology.
To date, we have screened more than 60,000 compounds using this assay and identified 420 putative inhibitors and 354 putative potentiators of Kir4.1/5.1. Forty-five of these potentiators increase Kir4.1/5.1-mediated thallium flux by greater than 50% and are selective for Kir4.1/5.1 over homomeric Kir4.1 channels. Fourteen (14) potentiators are active at low single micromolar concentrations with EC50 values less than 5 μM, while six (6) compounds appear to be highly efficacious and increase Kir4.1/5.1-mediated thallium flux by more than 100%. Importantly, we have also verified with patch clamp electrophysiology the activity of one of the first-in-class activator of Kir4.1/5.1, termed VU206. In thallium flux assays, VU206 potentiates thallium flux dose-dependently with an EC50 of 1.9 μM and maximal efficacy of ~50% above baseline between 10-30 μM. In whole-cell patch clamp experiments, VU206 led to maximal activation of whole-cell currents at 10 μM, complementing the thallium assay. Analysis of current-voltage relationships showed robust activation at both negative and positive test potentials without a depolarizing shift in the reversal potential suggesting VU206 potentiates Kir4.1/5.1 activity without affecting ion selectivity.
Findings from this study provide a novel pharmacological tool for exploring renal Kir4.1/5.1 channel integrative physiology and therapeutic potential of Kir4.1/5.1 potentiators for the treatment of EAST syndrome.
- NIDDK Support