VI-116, A Novel Potent Inhibitor of VRAC with Minimal Effect on ANO1

Abstract

Volume-regulated anion channel (VRAC) is ubiquitously expressed and plays a pivotal role in vertebrate cell volume regulation. A heterologous complex of leucine-rich repeat containing 8A (LRRC8A) and LRRC8B-E constitutes the VRAC, which is involved in various processes such as cell proliferation, migration, differentiation, intercellular communication, and apoptosis. However, the lack of a potent and selective inhibitor of VRAC limits VRAC-related physiological and pathophysiological studies, and most previous VRAC inhibitors strongly blocked the calcium-activated chloride channel, anoctamin 1 (ANO1). In the present study, we performed a cell-based screening for the identification of potent and selective VRAC inhibitors. Screening of 55,000 drug-like small-molecules and subsequent chemical modification revealed 3,3'-((2-hydroxy-3-methoxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (VI-116), a novel potent inhibitor of VRAC. VI-116 fully inhibited VRAC-mediated I^- quenching with an IC₅₀ of 1.27 ± 0.18 μM in LN215 cells and potently blocked endogenous VRAC activity in PC3, HT29 and HeLa cells in a dose-dependent manner. Notably, VI-116 had no effect on intracellular calcium signaling up to 10 μM, which completely inhibited VRAC, and showed high selectivity for VRAC compared to ANO1 and ANO2. However, DCPIB, a VRAC inhibitor, significantly affected ATP-induced increases in intracellular calcium levels and Eact-induced ANO1 activation. In addition, VI-116 showed minimal effect on hERG K⁺ channel activity up to 10 μM. These results indicate that VI-116 is a potent and selective VRAC inhibitor and a useful research tool for pharmacological dissection of VRAC.

Keywords: ANO1; ANO2; VI-116; VRAC; inhibitor.

Figure 1

Identification of novel VRAC inhibitors. (A) Principle of high−throughput screening assay. (B) Representative YFP fluorescence traces for VRAC activity. LN215 cells were treated with indicated concentrations of DCPIB for 5 min. VRAC was activated by application of hypertonic solution for 5 min. (C) Representative YFP fluorescence traces for VRAC inhibitors and inactive compounds. (D) Chemical structures of novel VRAC inhibitors (VI-101, VI-201, and VI-301) and DCPIB.

Figure 2

Inhibitory effects of VI-101, VI-201 and VI-301 on VRAC and ANO1. (A–C) Representative YFP fluorescence traces for VRAC activity. LN215 cells were treated with the indicated concentrations of VI-101, VI-201 and VI-301 with hypotonic solution for 5 min. (D) Dose-response curve of VRAC inhibition (mean ± S.E., n = 4). (E–G) Representative YFP fluorescence traces for ANO1 activity. FRT−ANO1−YFP cells were treated with the indicated concentrations of VI-101, VI-201 and VI-301 for 10 min. ANO1 was activated and inhibited by ATP (100 μM) and Ani9 (10 μM, dashed line), respectively. (H) Dose-response curve of ANO1 inhibition (mean ± S.E., n = 4).

Figure 3

Effect of VI-116 and DCPIB on VRAC activity and cell viability. (A) Structure of VI-116. (B) Representative YFP fluorescence traces. The inhibitory effects of VI-116 on VRAC activity were determined using YFP fluorescence assay in LN215 cells. VRAC was activated by application of hypertonic solution for 5 min. (C) Summary of VRAC dose-response (mean ± S.E., n = 4). (D) Summary of VRAC dose−response with 10% FBS (mean ± S.E., n = 4). (E,F) NIH3T3 cells were treated with the indicated concentrations of VI-116 or DCPIB for 24 h and cell viability was measured by MTT assay (mean ± S.E., n = 4), ** p < 0.01.

Figure 4

VI-116 potently blocks VRAC−mediated chloride current in HEK293T and LN215 cells. (A) Whole-cell membrane currents were recorded at a holding potential of −50 mV by voltage steps ranging from −120 to 120 mV (in steps of 20 mV) in HEK293T cells. VRAC was activated by application of hypertonic solution for 3 min, and then the cells were treated with indicated concentrations of VI-116. (B) Current/voltage plot of mean currents at the middle of each voltage pulse (n = 3). (C) Whole-cell VRAC currents were recorded at a holding potential of −50 mV by voltage steps ranging from −100 to 100 mV (in steps of 20 mV) in LN215 cells. (D) Current/voltage plot of mean currents at the middle of each voltage pulse (n = 3).

Figure 5

Effects of VI-116 on ANO1 and ANO2 chloride channel activity. (A) Representative Fluo4 fluorescence traces from 3 independent experiments in FRT cells. Cells were pretreated with VI-116 for 10 min, and P2Y receptors were activated with 100 μM ATP. (B,C) Representative apical membrane currents from 3–4 independent experiments in FRT cells expressing human ANO1. (B) Cells were treated with indicated concentrations of VI-116 and DCPIB for 10 min, and ANO1 was fully activated by 100 μM ATP and completely blocked by 10 μM Ani9. (C) ANO1 was activated with 3 μM E_act and the indicated concentrations of VI-116 were applied, and remaining ANO1 channel activity was completely blocked by 10 μM Ani9. (D) Representative YFP fluorescence traces in FRT cells expressing human ANO2. Cells were treated with the indicated concentrations of VI-116 for 10 min. ANO2 was fully activated by 100 μM ATP and completely blocked by 30 μM T16A_inh-A01. (E) Representative Fluo4 fluorescence traces from 3 independent experiments in FRT cells. Cells were pretreated with DCPIB for 10 min followed by application of 100 μM ATP. (F) Representative apical membrane currents in FRT cells expressing human ANO1. ANO1 was activated with 3 μM E_act and then the indicated concentrations of DCPIB were applied.

Figure 6

Effects of VI-116 and DCPIB on CFTR and hERG channel activity. (A,B) Representative apical membrane currents from 5 independent experiments in FRT cells expressing human CFTR. CFTR was fully activated by 20 μM forskolin (FSK) and inhibited with indicated concentrations of VI-116 and DCPIB. CFTR was completely blocked by 20 μM CFTR_inh−172. (C) Summary of dose-response (mean ± S.E., n = 5). (D,E) The effect of VI-116 and DCPIB on hERG activity was measured using a thallium flux assay in HEK293T cells expressing hERG. Cells were treated with the indicated concentrations of VI-116 and DCPIB for 10 min. hERG channel was activated by application of stimulus buffer and was inhibited by 50 μM cisapride. (F) Summary of dose−response (mean ± S.E., n = 6).

Figure 7

Effects of VI-116 and DCPIB on VRAC activity in PC3, HT29 and HeLa cells. (A,B) Representative YFP fluorescence traces for the inhibitory effects of VI-116 and DCPIB in PC3 cells. VRAC was activated by application of hypertonic solution for 5 min. (C) Summary of dose-response (mean ± S.E., n = 4). (D) LRRC8A−E mRNA expression levels in PC3 cells. (E,F) Inhibitory effects of VI-116 and DCPIB in HT29 cells. VRAC was activated by application of hypertonic solution for 5 min. (G) Summary of dose-response (mean ± S.E., n = 4). (H) LRRC8A−E mRNA expression levels in HT29 cells. (I,J) Inhibitory effects of VI-116 and DCPIB in HeLa cells. VRAC was activated by application of hypertonic solution for 5 min. (K) Summary of dose-response (mean ± S.E., n = 4). (L) LRRC8A−E mRNA expression levels in HeLa cells. mRNA expression levels of LRRC8A−E were determined by qRT−PCR and normalized to β−actin (mean ± S.E., n = 4).