CysLT1 receptor antagonists pranlukast and zafirlukast inhibit LRRC8-mediated volume regulated anion channels independently of the receptor

Abstract

Volume-regulated anion channels (VRACs) encoded by the leucine-rich repeat containing 8 ( LRRC8) gene family play critical roles in myriad cellular processes and might represent druggable targets. The dearth of pharmacological compounds available for studying VRAC physiology led us to perform a high-throughput screen of 1,184 of US Food and Drug Administration-approved drugs for novel VRAC modulators. We discovered the cysteinyl leukotriene receptor 1 (CysLT1R) antagonist, pranlukast, as a novel inhibitor of endogenous VRAC expressed in human embryonic kidney 293 (HEK293) cells. Pranlukast inhibits VRAC voltage-independently, reversibly, and dose-dependently with a maximal efficacy of only ~50%. The CysLT1R pathway has been implicated in activation of VRAC in other cell types, prompting us to test whether pranlukast requires the CysLT1R for inhibition of VRAC. Quantitative PCR analysis demonstrated that CYSLTR1 mRNA is virtually undetectable in HEK293 cells. Furthermore, the CysLT1R agonist leukotriene D4 had no effect on VRAC activity and failed to stimulate Gq-coupled receptor signaling. Heterologous expression of the CysLT1R reconstituted LTD4-CysLT1R- Gq-calcium signaling in HEK293 cells but had no effect on VRAC inhibition by pranlukast. Finally, we show the CysLT1R antagonist zafirlukast inhibits VRAC with an IC50 of ~17 µM and does so with full efficacy. Our data suggest that both pranlukast and zafirlukast are likely direct channel inhibitors that work independently of the CysLT1R. This study provides clarifying insights into the putative role of leukotriene signaling in modulation of VRAC and identifies two new chemical scaffolds that can be used for development of more potent and specific VRAC inhibitors.

A SWELL time to develop the molecular pharmacology of the volume-regulated anion channel (VRAC)

“…Taken together with their additional observation that an inhibitor of 5-lypoxygenase, the major source of inflammatory leukotrienes that cause asthma, also inhibits RVD and taurine release, led the authors to propose a model in which osmotic cell swelling stimulates the release of leukotrienes which, in turn, signal through CysLT1 to promote VRAC activation and RVD. According to their model, antagonists of CysLT1 should inhibit swelling-induced VRAC activation, as observed in our study [ 72 ]. We therefore set out to determine if Pranlukast-dependent inhibition of VRAC in HEK-293 cells is dependent on leukotriene signaling.…”

Zafirlukast is a broad‐spectrum thiol isomerase inhibitor that inhibits thrombosis without altering bleeding times

“…Graphs represent mean ± SEM. Data were analysed by Student's t-test, *P < 0.05 Mackie, & Straiker, 2011;Figueroa, Kramer, Strange, & Denton, 2019).…”

Zinc pyrithione activates the volume-regulated anion channel through an antioxidant-sensitive mechanism

“…Human embryonic kidney 293 (HEK293) cells were cultured in 75-cm 2 flasks with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. HEK293 cells stably transfected with the YFP(F46L/H148Q/I152L) fluorophore (i.e., HEK293-Ozzy cells) (32) were cultured in the same medium supplemented with 700 mg/mL G418 sulfate (Corning, NY). Human colorectal carcinoma 116 (HCT116) cells were cultured in 75-cm 2 flasks with McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS) and 0.5% penicillin/ streptomycin.…”

“…We recently reported the discovery of the CysLT1 receptor antagonist pranlukast as a novel VRAC inhibitor in a screen of the SelleckChem collection of 1,184 FDA-approved drugs (32). The HTS assay used in that screen reports the quenching of the YFP variant Ozzy by iodide as the anion enters Figure 1.…”

“…Many of these studies have highlighted the need for developing potent and specific pharmacological tools for probing the physiology and therapeutic potential of VRAC in human diseases. We recently reported the discovery in a high-throughput screening (HTS) of the cysteinyl leukotriene 1 receptor (CysLT1) antagonist pranlukast that inhibits VRAC independently of the CysLT1 receptor (32). This discovery not only adds to the growing toolkit of inhibitors available for studying VRAC but also demonstrates the utility of modern drug discovery approaches for developing VRAC pharmacology.…”