Fractionation of a Herbal Antidiarrheal Medicine Reveals Eugenol as an Inhibitor of Ca2+-Activated Cl− Channel TMEM16A

The Ca2+-activated Cl− channel TMEM16A is involved in epithelial fluid secretion, smooth muscle contraction and neurosensory signaling. We identified a Thai herbal antidiarrheal formulation that inhibited TMEM16A Cl− conductance. C18-reversed-phase HPLC fractionation of the herbal formulation revealed >98% of TMEM16A inhibition activity in one out of approximately 20 distinct peaks. The purified, active compound was identified as eugenol (4-allyl-2-methoxyphenol), the major component of clove oil. Eugenol fully inhibited TMEM16A Cl− conductance with single-site IC50∼150 µM. Eugenol inhibition of TMEM16A in interstitial cells of Cajal produced strong inhibition of intestinal contraction in mouse ileal segments. TMEM16A Cl− channel inhibition adds to the list of eugenol molecular targets and may account for some of its biological activities

TITLE PAGE α-AMINOAZAHETEROCYCLIC-METHYLGLYOXAL ADDUCTS DO NOT INHIBIT CFTR CHLORIDE CHANNEL ACTIVITY

ABSTRACT Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have potential applications in the therapy of secretory diarrheas and polycystic kidney disease. Recently, several highly polar α-aminoazaheterocyclic-methylglyoxal adducts were reported to reversibly inhibit CFTR chloride channel activity with IC50 values in the low picomolar range (Routaboul et al. J. Pharmacol. Exp. Ther. 322:1023-1035, more than 10,000-fold better than that of thiazolidionone and glycine hydrazide CFTR inhibitors identified previously by highthroughout screening. Here, we resynthesized and evaluated the α-aminoazaheterocyclicmethylglyoxal adducts of Routaboul et al. reported to have high CFTR inhibition potency (compounds 5, 7 and 8). We verified that the reported synthesis procedures produced the target compounds in high yield. However, we found that these compounds did not inhibit CFTR chloride channel function in multiple cell lines at up to 100 µM concentration, using three independent assays of CFTR function including short-circuit current analysis, whole-cell patch-clamp and YFPfluorescence quenching. As positive controls, near 100% CFTR inhibition was found by thiazolidionone and glycine hydrazide CFTR inhibitors. Our data provide direct evidence against CFTR inhibition by α-aminoazaheterocyclic-methylglyoxal adducts

The SLC26A9 inhibitor S9‐A13 provides no evidence for a role of SLC26A9 in airway chloride secretion but suggests a contribution to regulation of ASL pH and gastric proton secretion

The solute carrier 26 family member A9 (SLC26A9) is an epithelial anion transporter that is assumed to contribute to airway chloride secretion and surface hydration. Whether SLC26A9 or CFTR is responsible for airway Cl− transport under basal conditions is still unclear, due to the lack of a specific inhibitor for SLC26A9. In the present study, we report a novel potent and specific inhibitor for SLC26A9, identified by screening of a drug-like molecule library and subsequent chemical modifications. The most potent compound S9-A13 inhibited SLC26A9 with an IC50 of 90.9 ± 13.4 nM. S9-A13 did not inhibit other members of the SLC26 family and had no effects on Cl− channels such as CFTR, TMEM16A, or VRAC. S9-A13 inhibited SLC26A9 Cl− currents in cells that lack expression of CFTR. It also inhibited proton secretion by HGT-1 human gastric cells. In contrast, S9-A13 had minimal effects on ion transport in human airway epithelia and mouse trachea, despite clear expression of SLC26A9 in the apical membrane of ciliated cells. In both tissues, basal and stimulated Cl− secretion was due to CFTR, while acidification of airway surface liquid by S9-A13 suggests a role of SLC26A9 for airway bicarbonate secretion

A synthetic ion transporter that disrupts autophagy and induces apoptosis by perturbing cellular chloride concentrations

Perturbations in cellular chloride concentrations can affect cellular pH, autophagy and lead to the onset of apoptosis. With this in mind synthetic ion transporters have been used to disturb cellular ion homeostasis and thereby induce cell death; however, it is not clear whether synthetic ion transporters can also be used to disrupt autophagy. Here we show that squaramide-based ion transporters enhance the transport of chloride anions in liposomal models and promote sodium chloride influx into the cytosol. Liposomal and cellular transport activity of the squaramides is shown to correlate with cell death activity, which is attributed to caspase-dependent apoptosis. One ion transporter was also shown to cause additional changes in the lysosomal pH which leads to impairment of lysosomal enzyme activity and disruption of autophagic processes. This disruption is independent of the initiation of apoptosis by the ion transporter. This study provides the first experimental evidence that synthetic ion transporters can disrupt both autophagy and induce apoptosis

PAR4-Mediated PI3K/Akt and RhoA/ROCK Signaling Pathways Are Essential for Thrombin-Induced Morphological Changes in MEG-01 Cells

Thrombin stimulates platelets via a dual receptor system of protease-activated receptors (PARs): PAR1 and PAR4. PAR1 activation induces a rapid and transient signal associated with the initiation of platelet aggregation, whereas PAR4 activation results in a prolonged signal, required for later phases, that regulates the stable formation of thrombus. In this study, we observed differential signaling pathways for thrombin-induced PAR1 and PAR4 activation in a human megakaryoblastic leukemia cell line, MEG-01. Interestingly, thrombin induced both calcium signaling and morphological changes in MEG-01 cells via the activation of PAR1 and PAR4, and these intracellular events were very similar to those observed in platelets shown in previous studies. We developed a novel image-based assay to quantitatively measure the morphological changes in living cells, and observed the underlying mechanism for PAR1- and PAR4-mediated morphological changes in MEG-01 cells. Selective inhibition of PAR1 and PAR4 by vorapaxar and BMS-986120, respectively, showed that thrombin-induced morphological changes were primarily mediated by PAR4 activation. Treatment of a set of kinase inhibitors and 2-aminoethoxydiphenyl borate (2-APB) revealed that thrombin-mediated morphological changes were primarily regulated by calcium-independent pathways and PAR4 activation-induced PI3K/Akt and RhoA/ROCK signaling pathways in MEG-01 cells. These results indicate the importance of PAR4-mediated signaling pathways in thrombin-induced morphological changes in MEG-01 cells and provide a useful in vitro cellular model for platelet research

Novel Amino-Carbonitrile-Pyrazole Identified in a Small Molecule Screen Activates Wild-Type and ∆F508 Cystic Fibrosis Transmembrane Conductance Regulator in the Absence of a cAMP Agonist

Cystic fibrosis (CF) is caused by loss-of-function mutations in the CF transmembrane conductance regulator (CFTR) Cl⁻ channel. We developed a phenotype-based high-throughput screen to identify small-molecule activators of human airway epithelial Ca²⁺-activated Cl⁻ channels (CaCCs) for CF therapy. Unexpectedly, screening of ∼110,000 synthetic small molecules revealed an amino-carbonitrile-pyrazole, C(act)-A1, that activated CFTR but not CaCC Cl⁻ conductance. C(act)-A1 produced large and sustained CFTR Cl⁻ currents in CFTR-expressing Fisher rat thyroid (FRT) cells and in primary cultures of human bronchial epithelial (HBE) cells, without increasing intracellular cAMP and in the absence of a cAMP agonist. C(act)-A1 produced linear whole-cell currents. C(act)-A1 also activated ΔF508-CFTR Cl⁻ currents in low temperature-rescued ΔF508-CFTR-expressing FRT cells and CF-HBE cells (from homozygous ΔF508 patients) in the absence of a cAMP agonist, and showed additive effects with forskolin. In contrast, N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (VX-770) and genistein produced little or no ΔF508-CFTR Cl⁻ current in the absence of a cAMP agonist. In FRT cells expressing G551D-CFTR and in CF nasal polyp epithelial cells (from a heterozygous G551D/Y1092X-CFTR patient), C(act)-A1 produced little Cl⁻ current by itself but showed synergy with forskolin. The amino-carbonitrile-pyrazole C(act)-A1 identified here is unique among prior CFTR-activating compounds, as it strongly activated wild-type and ΔF508-CFTR in the absence of a cAMP agonist. Increasing ΔF508-CFTR Cl⁻ conductance by an "activator," as defined by activation in the absence of cAMP stimulation, provides a novel strategy for CF therapy that is different from that of a "potentiator," which requires cAMP elevation

Novel 5-substituted benzyloxy-2-arylbenzofuran-3-carboxylic acids as calcium activated chloride channel inhibitors

Transmembrane protein 16A (TMEM16A) channels are recently discovered membrane proteins that functions as a calcium activated chloride channel (CaCC). CaCCs are major regulators of various physiological processes, such as sensory transduction, epithelial secretion, smooth muscle contraction and oocyte fertilization. Thirty novel 5-substituted benzyloxy-2-arylbenzofuran-3-carboxylic acids (B01-B30) were synthesized and evaluated for their TMEM16A inhibitory activity by using short circuit current measurements in Fischer rat thyroid (FRT) cells expressing human TMEM16A. IC(50) values were calculated using YFP fluorescence plate reader assay. Final compounds, having free carboxylic group displayed significant inhibition. Eight of the novel compounds B02, B13, B21, B23, B25, B27, B28, B29 exhibit excellent CaCCs inhibition with IC(50) value <6 μM, with compound B25 exhibiting the lowest IC(50) value of 2.8 ± 1.3 μM. None of the tested ester analogs of final benzofuran derivatives displayed TMEM16A/CaCCs inhibition

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