Discovery of Pranlukast in a High‐Throughput Screen for Novel Inhibitors of LRRC8 Volume Regulated Anion Channels

Abstract

Cells critically regulate their volume in response to hypotonic cell swelling by transporting chloride and small organic osmolytes out of the cell through volume regulated anion channels (VRACs) encoded by members of the recently defined LRRC8 gene family. VRACs are ubiquitously expressed in mammalian cells and have been implicated in diverse cellular functions in addition to cell volume regulation, including endothelial cell calcium signaling and pancreatic beta cell insulin secretion. The lack of potent and specific pharmacological tool compounds represents a critical barrier to evaluating the integrative physiology and therapeutic potential of VRACs. We therefore carried out a high‐throughput screen (HTS) of 1,184 of FDA approved drugs using a fluorescence‐based assay for novel inhibitors of native VRACs expressed in HEK‐293 cells. The assay measures the quenching of intracellular YFP by iodide influx through VRACs activated by hypotonic‐induced cell swelling. In this screen, the cysteinyl leukotriene receptor 1 (CysLT1R) antagonist, Pranlukast, was identified as a novel inhibitor of VRAC. In YFP quenching assays and patch clamp electrophysiology experiments, Pranlukast inhibits HEK‐293 cell VRAC reversibly and dose‐dependently with an IC50 of ~6 μM and ~239 nM, respectively. Interestingly, the CysLT1R pathway has been implicated in activation of VRAC in other cell types, raising the possibility that Pranlukast inhibits VRAC indirectly via the CysLT1R pathway. Using a series of fluorescence assays and qPCR we show that CysLT1R is not functionally expressed in HEK‐293 cells. Additionally, using a structurally distinct CysLT1R inhibitor, Zafirlukast, did not inhibit VRAC. These studies provide clarifying insights into the putative role of CysLT1 receptor signaling in regulation of VRACs and identify Pranlukast as a structurally novel small‐molecule inhibitor of LRRC8 channels.Support or Funding InformationRuth L. Kirschstein National Research Service Award F31 (1F31DK120225‐01)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.