CysLT1 receptor antagonists pranlukast and zafirlukast inhibit LRRC8-mediated volume regulated anion channels independently of the receptor
CysLT1 receptor antagonists pranlukast and zafirlukast inhibit LRRC8-mediated volume regulated anion channels independently of the receptor
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DOI：
10.1152/ajpcell.00281.2019
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发表时间：
2019-10-01
影响因子：
5.5
通讯作者：
Denton, Jerod S.
中科院分区：
文献类型：
--
作者：
Figueroa, Eric E.;Kramer, Meghan;Denton, Jerod S.
由富含亮氨酸重复序列8(LRRC8)基因家族编码的容量调节阴离子通道(VRAC)在多种细胞过程中发挥重要作用，可能是药物作用的靶点。由于缺乏可用于研究vrac生理学的药理化合物，我们对美国食品和药物管理局批准的1184种药物进行了高通量筛选，以寻找新型vrac调节剂。我们发现半胱氨酰白三烯受体1(CysLT1R)拮抗剂普鲁司特是一种新的内源性vrac抑制剂，在人胚胎肾293(HEK293)细胞中表达。普鲁司特以独立、可逆和剂量依赖的方式抑制vrac，最大有效率仅为50%。在其他类型的细胞中，CysLT1R通路与vrac的激活有关，这促使我们测试普鲁司特是否需要CysLT1R来抑制vrac。定量聚合酶链式反应分析表明，在HEK293细胞中几乎检测不到CYSLTR1mRNA。此外，CysLT1R激动剂白三烯D4对vrac活性没有影响，也不能刺激G(Q)偶联受体信号转导。CysLT1R重组LTD4-CysLT1R-G(Q)-钙信号在HEK293细胞中的异源表达，但对普鲁司特抑制vrac无影响。最后，我们证明了CysLT1R拮抗剂扎鲁司特抑制vrac的IC50接近17u M，并且完全有效。我们的数据表明，普鲁司特和扎鲁司特都可能是独立于CysLT1R发挥作用的直接通道抑制剂。这项研究阐明了白三烯信号在vrac调控中的可能作用，并确定了两种新的化学支架，可用于开发更有效和更特异的vrac抑制剂。
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 similar to 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 G(q)-coupled receptor signaling. Heterologous expression of the CysLT1R reconstituted LTD4-CysLT1R- G(q)-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 similar to 17 mu M 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.