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

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.


Introduction
Cell volume regulation is an important homeostatic function in living cells, and volume-regulated anion channel (VRAC) is the major Cl − channel in regulatory volume decrease (RVD) [1]. Recently, two research groups revealed that LRRC8A is an essential protein for VRAC through whole-genome siRNA library screening [2,3]. LRRC8A is a major component of swelling-induced chloride current (I Cl, swell ) that occurs during RVD in various cells. Previous studies showed that I Cl, swell and RVD were significantly blocked when LRRC8A was downregulated by siRNA [2]. VRAC is involved not only in volume regulation but also various physiological roles, such as apoptotic volume decrease (AVD), release of excitatory amino acids (EAAs), and taurine transport [4,5]. Interestingly, VRAC is formed by heterohexamers of the LRRC8 protein family (LRRC8A−E) and is ubiquitously expressed in almost all types of mammalian cells [2,3]. Subunit composition of VRAC influences physiological processes such as taurine transport and superoxide production [5,6].
Anoctamin 1 (ANO1), also known as TMEM16A, is highly expressed in epithelial cells and other cell types such as smooth muscle cells and sensory neurons, and is involved in a variety of physiological events, such as fluid secretion, cell proliferation, migration, smooth

Identification of Novel Small Molecule Inhibitors of VRAC
We performed a cell-based HTS to identify potent and selective VRAC inhibitors. To measure VRAC activity, human glioma cells, LN215, were stably transfected with halide sensors YFP-F46L/H148Q/I152L. As shown in Figure 1A, to investigate the effect of compounds on VRAC activity, cells were treated with hypotonic solution (150 mOsm) containing 25 µM of test compounds for 5 min, followed by iodide (70 mM) containing solution. In Figure 1B, we investigated the effect of DCPIB, a VRAC inhibitor, on VRAC activity in LN215 cells expressing YFP-F46L/H148Q/I152L. The YFP fluorescence was significantly decreased by DCPIB with an IC 50 of 3.45 µM in a dose-dependent manner. We screened 55,000 drug-like small-molecules and identified three novel VRAC inhibitors, VI-101, VI-201 and VI-301 ( Figure 1C,D).

Identification of a Potent and Selective VRAC Inhibitor, VI-116
Since there are no VRAC inhibitors that potently inhibit VRAC without effects on ANO1, we further investigated the effects of three novel VRAC inhibitors on VRAC and ANO1 activity. Unfortunately, all three novel VRAC inhibitors also potently blocked both VRAC and ANO1 ( Figure 2). To discover more potent and selective VRAC inhibitors, we performed a chemical modification study based on VI-101, which has the highest efficacy. We observed the effects of 19 analogs of VI-101 on VRAC and ANO1 activity and finally identified VI-116, a potent and selective VRAC inhibitor that inhibits VRAC activity (IC 50 = 1.3 µM) with 13-fold higher potency than ANO1 activity (IC 50 = 39.1 µM) (Table 1). Notably, the most potent analog of VI-101, VI-110, has an IC 50 value of 0.58 µM. However, since VI-110 also potently inhibited the activity of ANO1 (IC 50 = 4.8 µM), VI-116, a relatively potent and most selective VRAC inhibitor, was selected for further study.

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells (Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells (Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells (Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells (Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells (Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells ( Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells ( Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells ( Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells (Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells (Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells (Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells (Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells (Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells (Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells (Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells (Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells (Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells (Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells (Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC50 = 1.28 μM) more potently blocked VRAC activity, compared to DCPIB (IC50 = 3.45 μM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC50 of VI-116 and DCPIB were 6.89 μM and 14.1 μM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 μM, but DCPIB significantly reduced cell viability at 30 μM.
To investigate the effect of VI-116 on ICl, swell, we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated ICl, swell in a dose-dependent manner in both HEK293T and LN215 cells (Figure 4).

VI-116 Potently Blocks VRAC-Mediated Chloride Currents
We further investigated the effect of VI-116 on VRAC activity using YFP quenching assay in LN215 cells. As shown in Figure 3, VI-116 (IC 50 = 1.28 µM) more potently blocked VRAC activity, compared to DCPIB (IC 50 = 3.45 µM). To investigate the effect of VI-116 and DCPIB on VRAC activity in the presence of 10% FBS, LN215 cells were treated with VI-116 and DCPIB in a medium containing 10% FBS for 5 min. In the presence of 10% FBS, the IC 50 of VI-116 and DCPIB were 6.89 µM and 14.1 µM, respectively ( Figure 3D). To determine whether VI-116 and DCPIB are cytotoxic, we observed the effect of VI-116 and DCPIB on cell viability in NIH-3T3 cells. As shown in Figure 3E,F, VI-116 did not affect cell viability up to 30 µM, but DCPIB significantly reduced cell viability at 30 µM.   To investigate the effect of VI-116 on I Cl, swell , we used the whole-cell voltage-clamp technique in HEK293T and LN215 cells. VI-116 completely blocked hypotonic perfusion activated I Cl, swell in a dose-dependent manner in both HEK293T and LN215 cells (Figure 4).

Minimal Effect of VI-116 on Human ANO1, ANO2 and hERG Channel Activity
To investigate whether VI-116 affects ANO1 and ANO2 chloride channel activity, we first measured the effect of VI-116 on intracellular calcium signaling in FRT cells. As shown in Figure 5A, VI-116 did not affect the ATP-induced increase in intracellular calcium levels up to 10 µM and showed a weak inhibitory effect on intracellular calcium signaling at 30 µM. To observe the effect of VI-116 on ANO1 channel activity, apical membrane current of ANO1 was measured in FRT cells expressing human ANO1. Notably, VI-116 did not alter the ANO1 activation by ATP or E act , a specific activator of ANO1 [25], and ANO1 chloride channel was completely blocked by Ani9 ( Figure 5B,C). ANO2 activity was measured using YFP fluorescence quenching assay in FRT cells expressing human ANO2. VI-116 had a minimal effect on ANO2 up to 10 µM in FRT cells expressing human ANO2 ( Figure 5D). (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. Unexpectedly, DCPIB strongly blocked ATP-induced intracellular calcium levels increasing in a dose-dependent manner ( Figure 5E). Interestingly, DCPIB had a unique effect on E act -activated ANO1 chloride channel activity. At 10 µM, ANO1 chloride current activated by E act was increased, but at 100 µM, ANO1 chloride current was almost completely inhibited ( Figure 5F).
To observe the effect of VI-116 and DCPIB on CFTR, a cAMP-regulated chloride channel, we measured the apical membrane current of CFTR in FRT cells expressing human CFTR. As shown in Figure 6A−C, VI-116 and DCPIB blocked CFTR chloride channel with IC 50 values of 12.4 µM and 71.7 µM, respectively. We also observed the effect of VI-116 and DCPIB on human ether-a-go-go related gene (hERG) K + channel, which is a major antitarget of drug discovery and induces long QT syndrome when the channel is blocked [26]. VI-116 showed minimal inhibitory effect on hERG K + channel activity at 10 µM with IC 50 of 89.6 µM, whereas DCPIB significantly blocked hERG K + channel activity at 10 µM with an IC 50 of 11.4 µM ( Figure 6D-F). These results suggest that VI-116 is a potent and selective VRAC inhibitor with useful applications for in vitro and in vivo experiments.

VI-116 Potently Inhibits Endogenous VRAC Activity in PC3, HT29 and HeLa Cells
A heterologous complex of LRRC8A and LRRC8B−E constitutes VRAC, and VRAC channel properties differ depending on the type of heterogeneous complex [5]. Therefore, the characteristics of VRAC also depend on the cell type. Here, we investigated the effect of VI-116 on VRAC activity in three different cell lines, PC3 prostate, HT29 colon, and HeLa cervical cancer cells. As shown in Figure 7, the quantitative real-time PCR (qRT-PCR) analysis showed that PC3 cells had relatively high expression rates of LRRC8A (D and E) but HT26 cells and HeLa cells had relatively high expression rates of LRRC8A and LRRC8D, respectively. VI-116 potently and completely blocked VRAC activity in PC3, HT29, and HeLa cells with an IC50 of 0.63 ± 0.05 μM, 3.14 ± 1.96 μM, and 1.20 ± 0.08 μM, respectively. In the case of DCPIB, it showed weaker potency than VI-116 in PC3, HT29, and HeLa cells with an IC50 of 4.15 ± 1.79 μM, 11.3 ± 4.13 μM, and 3.36 ± 1.04 μM, respectively.

VI-116 Potently Inhibits Endogenous VRAC Activity in PC3, HT29 and HeLa Cells
A heterologous complex of LRRC8A and LRRC8B−E constitutes VRAC, and VRAC channel properties differ depending on the type of heterogeneous complex [5]. Therefore, the characteristics of VRAC also depend on the cell type. Here, we investigated the effect of VI-116 on VRAC activity in three different cell lines, PC3 prostate, HT29 colon, and HeLa cervical cancer cells. As shown in Figure 7, the quantitative real-time PCR (qRT-PCR) analysis showed that PC3 cells had relatively high expression rates of LRRC8A (D and E) but HT26 cells and HeLa cells had relatively high expression rates of LRRC8A and LRRC8D, respectively. VI-116 potently and completely blocked VRAC activity in PC3, HT29, and HeLa cells with an IC 50 of 0.63 ± 0.05 µM, 3.14 ± 1.96 µM, and 1.20 ± 0.08 µM, respectively. In the case of DCPIB, it showed weaker potency than VI-116 in PC3, HT29, and HeLa cells with an IC 50 of 4.15 ± 1.79 µM, 11.3 ± 4.13 µM, and 3.36 ± 1.04 µM, respectively.

Discussion
VRAC is ubiquitously expressed in almost all mammalian cell types and in a wide range of cancer cells [27]. Calcium-activated chloride channel ANO1 is also expressed in various cell types such as smooth muscle, epithelial cells, small sensory neurons, and olfactory-derived cells, and is highly amplified in human cancers such as prostate cancer, breast cancer, esophageal cancer, pancreatic cancer, oral squamous cell carcinoma, and head and neck squamous cell carcinoma [7]. VRAC and ANO1 chloride channels are activated by cell swelling and intracellular calcium increase, respectively. However, previous studies have shown crosstalk between VRAC and ANO1. For example, both LRRC8A and ANO1 are involved in serum-induced VRAC-like currents, and cell swelling and intracellular calcium increase can stimulate both VRAC and ANO1 [28,29]. Therefore, potent and selective inhibitors of VRAC and ANO1 are needed to elucidate the physiological roles of these two chloride channels. For ANO1 inhibitors, as shown in our previous study, Ani9 potently inhibits ANO1 channel activity with only a minimal effect on VRAC [13]. However, all of the previous VRAC inhibitors were nonselective or partial inhibitors [14][15][16][17][18][19][20][21]. Although DCPIB, the most frequently used VRAC inhibitor, did not affect calcium-activated chloride currents in calf pulmonary artery endothelial (CPAE) cells [30], we found that DCPIB potently blocked the ATP-induced intracellular calcium increase associated with the activation of ANO1 ( Figure 5E). In addition, DCPIB may also directly affect ANO1 channel activity as it can alter Eact-activated ANO1 chloride currents ( Figure 5F).

Discussion
VRAC is ubiquitously expressed in almost all mammalian cell types and in a wide range of cancer cells [27]. Calcium-activated chloride channel ANO1 is also expressed in various cell types such as smooth muscle, epithelial cells, small sensory neurons, and olfactory-derived cells, and is highly amplified in human cancers such as prostate cancer, breast cancer, esophageal cancer, pancreatic cancer, oral squamous cell carcinoma, and head and neck squamous cell carcinoma [7]. VRAC and ANO1 chloride channels are activated by cell swelling and intracellular calcium increase, respectively. However, previous studies have shown crosstalk between VRAC and ANO1. For example, both LRRC8A and ANO1 are involved in serum-induced VRAC-like currents, and cell swelling and intracellular calcium increase can stimulate both VRAC and ANO1 [28,29]. Therefore, potent and selective inhibitors of VRAC and ANO1 are needed to elucidate the physiological roles of these two chloride channels. For ANO1 inhibitors, as shown in our previous study, Ani9 potently inhibits ANO1 channel activity with only a minimal effect on VRAC [13]. However, all of the previous VRAC inhibitors were nonselective or partial inhibitors [14][15][16][17][18][19][20][21]. Although DCPIB, the most frequently used VRAC inhibitor, did not affect calcium-activated chloride currents in calf pulmonary artery endothelial (CPAE) cells [30], we found that DCPIB potently blocked the ATP-induced intracellular calcium increase associated with the activation of ANO1 ( Figure 5E). In addition, DCPIB may also directly affect ANO1 channel activity as it can alter E act -activated ANO1 chloride currents ( Figure 5F). DCPIB can activate TREK K + channels in cultured astrocytes and increase basal K + current in neurons in brain slices. In addition, a recent report showed that DCPIB can induce a rapid increase in intracellular Ca 2+ and directly activate BK channels in a calcium independent manner [23,24]. Here, we showed that DCPIB inhibited hERG K + channel activity at an IC 50 of 11.4 µM in HEK293T cells expressing hERG. We used FluxOR™ thallium assay which is widely used for the hERG activity test (Figure 6). This result conflicts with a previous report that DCPIB did not inhibit hERG at 10 µM in Xenopus oocytes overexpressed with hERG [25]. This may be due to differences in cellular properties such as the drug permeability of HEK293T cells and oocytes.
In Figure 7, VI-116 and DCPIB showed inhibition of VRAC activity with different potencies in PC3, HT29, and HeLa cells. These results are consistent with previous reports showing that DCPIB inhibits endogenous VRAC activity with different potency in other cell types [2,31,32]. The difference in potency of VI-116 and DCPIB between cell types is probably due to the different heterogeneous complexes of LRRC8A-E constituting VRAC, and the different drug permeability of each cell type.
Since various cells were cultured in media containing 10% FBS in many in vitro experiments, we measured the inhibitory effect of VI-116 on VRAC activity in the presence of 10% FBS ( Figure 3D). In the solution with 10% FBS, the potency of VI-116 was decreased 5.4-fold compared to the condition without FBS. The cause of this phenomenon is presumed to be that VI-116 binds to plasma proteins such as albumin present in FBS, thereby reducing the potency of VI-116.
In summary, the novel VRAC inhibitor VI-116 strongly inhibited VRAC chloride channel activity with only a minimal effect on ANO1, ANO2, and hERG channel activity at 10 µM which completely inhibits VRAC. In addition, VI-116 potently and completely inhibited endogenous VRAC activity in four different cell lines, LN215, PC3, HT29, and HeLa cells. These results suggest that VI-116 can be used as a useful tool for the pharmacological dissection of VRAC, increasing the efficiency of in vitro and in vivo studies to elucidate the physiological role of VRAC.

Materials and Reagents
DCPIB, T16A inh -A01, and other chemicals were purchased from Merck (St. Louis, MO, USA). Chemical libraries used for screening were purchased from ChemDiv (San Diego, CA, USA).

YFP Fluorescence Quenching Assay
YFP-F46L/H148Q/I152L expressed LN215 cells were cultured in 96-well black-walled microplates (Corning Inc., Corning, NY, USA) at a density of 15,000 cells/well in DMEM supplemented with 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin. Screening was performed using FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany). Each well of a 96-well plate was washed 3 times in PBS (200 µL/wash), kept 50 µL hypotonic solution (in mM): 70 NaCl, 5 KCl, 20 HEPES (170 mOsm/kg). Test compounds (0.5 µL) were added to each well at 25 µM final concentration. After 4 min, 96-well plates were transferred to a plate reader for fluorescence assay. Each well was assayed individually for VRAC-mediated I − influx by recording fluorescence continuously (400 ms per point) for 0.4 s (baseline), then 50 µL of 140 mM I − solution was added at 0.5 s, and then YFP fluorescence was recorded for 5 s. The initial iodide influx rate was calculated from fluorescence data by nonlinear regression.

FluxOR Potassium Ion Channel Assay
HEK293 cells stably expressing human Kv11.1 (hERG) were cultured in 96-well plates. After 48 h, the cells were incubated at 28 • C for 4 h to increase the membrane expression of hERG. The culture medium was replaced with a 80 µL/well of FluxOR (Invitrogen, Waltham, MA, USA) loading buffer and incubated for 1 h at 37 • C in the dark. After removing the loading buffer, 100 µL of assay buffer was added to each well. To measure the effect of VI-116 and DCPIB on hERG channels, the cells were pretreated with test compounds for 10 min. FluxOR fluorescence (excitation/emission: 490/525 nm) was recorded for 4 s before the addition of 20 µL of stimulus buffer containing thallium ions, and the fluorescence was recorded. FluxOR fluorescence was recorded and analyzed using the FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany) and the MARS Data Analysis Software (BMG Labtech, Ortenberg, Germany). All buffers were prepared according to the manufacturer's instructions.

Cell Proliferation Assay
NIH-3T3 cells were plated on 96-well microplates. After 24 h incubation, cells were treated with VI-116 or DCPIB and incubated for 24 h. The culture medium and the compounds were changed every 12 h. To assess cell proliferation, the cells were incubated with MTS for 1 h. The soluble formazan produced by the cellular reduction of MTS was quantified by measuring the absorbance at 490 nm with an Infinite M200 (Tecan, Grödig, Austria) microplate reader. MTS assay was performed using a CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA).

Intracellular Calcium Measurement
FRT cells were plated on 96-well black-walled microplates and loaded with Fluo-4 NW according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). After 1 h incubation, the cells were added with VI-116 or DCPIB and incubated for 10 min. The 96-well plates were transferred to a plate reader for fluorescence assay. Fluo-4 fluorescence (excitation/emission: 485/538 nm) was measured with a FLUOstar Omega microplate reader (BMG Labtech). Intracellular calcium was increased by an application of 100 µM ATP.

Statistical Analysis
The results of the experiments are displayed as the means ± S.E. The statistical analysis was performed with a Student's t-test. A value of p < 0.01 was considered statistically significant.

Conflicts of Interest:
The authors declare no conflict of interest.