Effect of Calcium Supplementation and TMEM16A Inhibition on Endoplasmic Reticulum Stress Induced by Dental Fluorosis in Mice.

BACKGROUND: Dental fluorosis is a discoloration of the teeth caused by the excessive consumption of fluoride. It represents a distinct manifestation of chronic fluorosis in dental tissues, exerting adverse effects on the human body, particularly on teeth. The transmembrane protein 16a (TMEM16A) is expressed at the junction of the endoplasmic reticulum and the plasma membrane. Alterations in its channel activity can disrupt endoplasmic reticulum calcium homeostasis and intracellular calcium ion concentration, thereby inducing endoplasmic reticulum stress (ERS). This study aims to investigate the influence of calcium supplements and TMEM16A on ERS in dental fluorosis. METHODS: C57BL/6 mice exhibiting dental fluorosis were subjected to an eight-week treatment with varying calcium concentrations: low (0.071%), medium (0.79%), and high (6.61%). Various assays, including Hematoxylin and Eosin (HE) staining, immunohistochemistry, real-time fluorescence quantitative polymerase chain reaction (qPCR), and Western blot, were employed to assess the impact of calcium supplements on fluoride content, ameloblast morphology, TMEM16A expression, and endoplasmic reticulum stress-related proteins (calreticulin (CRT), glucose-regulated protein 78 (GRP78), inositol requiring kinase 1α (IRE1α), PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6)) in the incisors of mice affected by dental fluorosis. Furthermore, mice with dental fluorosis were treated with the TMEM16A inhibitor T16Ainh-A01 along with a medium-dose calcium to investigate the influence of TMEM16A on fluoride content, ameloblast morphology, and endoplasmic reticulum stress-related proteins in the context of mouse incisor fluorosis. RESULTS: In comparison to the model mice, the fluoride content in incisors significantly decreased following calcium supplements (p < 0.01). Moreover, the expression of TMEM16A, CRT, GRP78, IRE1α, PERK, and ATF6 were also exhibited a substantial reduction (p < 0.01), with the most pronounced effect observed in the medium-dose calcium group. Additionally, the fluoride content (p < 0.05) and the expression of CRT, GRP78, IRE1α, PERK, and ATF6 (p < 0.01) were further diminished following concurrent treatment with the TMEM16A inhibitor T16Ainh-A01 and a medium dose of calcium. CONCLUSIONS: The supplementation of calcium or the inhibition of TMEM16A expression appears to mitigate the detrimental effects of fluorosis by suppressing endoplasmic reticulum stress. These findings hold implications for identifying potential therapeutic targets in addressing dental fluorosis.

Discovery of ANO1 Inhibitors based on Machine learning and molecule docking simulation approaches.

Calcium-activated chloride channels (CaCCs) are chloride channels that are regulated according to intracellular calcium ion concentrations. The channel protein ANO1 is widely present in cells and is involved in physiological activities including cellular secretion, signaling, cell proliferation and vasoconstriction and diastole. In this study, the ANO1 inhibitors were investigated with machine learning and molecular simulation. Two-dimensional structure-activity relationship (2D-SAR) and three-dimensional quantitative structure-activity relationship (3D-QSAR) models were developed for the qualitative and quantitative prediction of ANO1 inhibitors. The results showed that the prediction accuracies of the model were 85.9% and 87.8% for the training and test sets, respectively, and 85.9% and 87.8% for the rotating forest (RF) in the 2D-SAR model. The CoMFA and CoMSIA methods were then used for 3D QSAR modeling of ANO1 inhibitors, respectively. The q2 coefficients for model cross-validation were all greater than 0.5, implying that we were able to obtain a stable model for drug activity prediction. Molecular docking was further used to simulate the interactions between the five most promising compounds predicted by the model and the ANO1 protein. The total score for the docking results between all five compounds and the target protein was greater than 6, indicating that they interacted strongly in the form of hydrogen bonds. Finally, simulations of amino acid mutations around the docking cavity of the target proteins showed that each molecule had two or more sites of reduced affinity following a single mutation, indicating outstanding specificity of the screened drug molecules and their protein ligands.

Trans-ε-viniferin as an inhibitor of TMEM16A preventing intestinal smooth muscle contraction.

TMEM16A regulator is an important tool to study the physiological functions and pathogenesis related to TMEM16A. In the present study, trans-ε-viniferin (TV) was identified as a TMEM16A inhibitor with inhibitory activity against TMEM16A mediated Cl- currents, which was reversible, without affecting intracytoplasmic Ca2+ concentration and TMEM16A protein expression. TV inhibited intestinal peristalsis and prolonged gastrointestinal transport time. TV could inhibit autonomic and Eact-stimulated intestinal contractility, and was equally effective in ACh- and HA-induced high contractile states. The results indicate that TV significantly inhibits the intestinal smooth muscle contraction, which may be applied in the treatment of TMEM16A-related intestinal dynamic abnormalities.

Zafirlukast inhibits the growth of lung adenocarcinoma via inhibiting TMEM16A channel activity.

Lung cancer has the highest mortality among cancers worldwide due to its high incidence and lack of the effective cures. We have previously demonstrated that the membrane ion channel TMEM16A is a potential drug target for the treatment of lung adenocarcinoma and have identified a pocket of inhibitor binding that provides the basis for screening promising new inhibitors. However, conventional drug discovery strategies are lengthy and costly, and the unpredictable side effects lead to a high failure rate in drug development. Therefore, finding new therapeutic directions for already marketed drugs may be a feasible strategy to obtain safe and effective therapeutic drugs. Here, we screened a library of over 1400 Food and Drug Administration-approved drugs through virtual screening and activity testing. We identified a drug candidate, Zafirlukast (ZAF), clinically approved for the treatment of asthma, that could inhibit the TMEM16A channel in a concentration-dependent manner. Molecular dynamics simulations and site-directed mutagenesis experiments showed that ZAF can bind to S387/N533/R535 in the nonselective inhibitor binding pocket, thereby blocking the channel pore. Furthermore, we demonstrate ZAF can target TMEM16A channel to inhibit the proliferation and migration of lung adenocarcinoma LA795 cells. In vivo experiments showed that ZAF can significantly inhibit lung adenocarcinoma tumor growth in mice. Taken together, we identified ZAF as a novel TMEM16A channel inhibitor with excellent anticancer activity, and as such, it represents a promising candidate for future preclinical and clinical studies.

In-vitro and in-vivo anti-allergic effects of magnolol on allergic rhinitis via inhibition of ORAI1 and ANO1 channels.

ETHNOPHARMACOLOGICAL RELEVANCE: Flos Magnoliae (the dried flower buds of Magnolia biondii Pamp, FM) is a known herbal traditional medicine used for the symptomatic relief of nasal congestion and rhinorrhea caused by rhinitis and sinusitis. Magnolol, a neolignan from the magnolia family, is a secondary metabolite known to have anti-allergic and anti-inflammatory effects. However, the underlying mechanisms and therapeutic effect of magnolol in the treatment of allergic rhinitis (AR) remain elusive. AIMS OF THE STUDY: Anoctamin 1 (ANO1), a calcium-activated anion channel, mediates mucus and electrolyte secretion in nasal airway epithelial cells, whereas calcium release-activated calcium channel protein 1 (ORAI1) participates in the activation of T-lymphocytes and mast cells. The aim of our study is to understand the mechanisms of action of magnolol against AR, i.e., whether it acts through the modulation of ANO1 and ORAI1 channels that are expressed in nasal epithelial cells and T-lymphocytes, respectively. MATERIALS AND METHODS: Whole-cell patch clamp was used to record the activity of ORAI1 and ANO1 ion channels in ORAI1 or ANO1 overexpressed HEK293T cells, while the Ussing chamber apparatus was used to measure electrolyte transport via the epithelium, in Calu-3 cells cultured in an air-liquid interface. Additionally, calcium imaging of Jurkat T-lymphocytes was used to assess changes in the intracellular calcium concentration. Magnolol toxicity was assessed using the CCK-8 assay, and its effect on T-lymphocyte proliferation was measured by labeling human primary T-lymphocytes with carboxyfluorescein succinimidyl ester. Finally, OVA-induced Balb/c mice were employed to evaluate the effect of magnolol on nasal symptoms, as well as cytokine and eosinophil infiltration in AR. RESULTS: Magnolol inhibits ORAI1 and ANO1 channels in a concentration-dependent manner. Magnolol (30 µM) inhibits anti-CD3 induced cellular proliferation and production of IL-2 via ORAI1 channels in T-lymphocytes. Further, ATP-induced electrolyte transport mediated by ANO1 channels is significantly inhibited by magnolol in IL-4 sensitized Calu-3 cells. Notably, 300 µM magnolol significantly attenuates cytokine and eosinophil infiltration, thus alleviating AR symptoms in mice OVA-induced AR. CONCLUSION: Magnolol may be a promising therapeutic agent for the treatment and prevention of AR.

Systems Approaches to Unravel Molecular Function: High-content siRNA Screen Identifies TMEM16A Traffic Regulators as Potential Drug Targets for Cystic Fibrosis.

An attractive approach to treat people with Cystic Fibrosis (CF), a life-shortening disease caused by mutant CFTR, is to compensate for the absence of this chloride/bicarbonate channel by activating alternative (non-CFTR) chloride channels. One obvious target for such "mutation-agnostic" therapeutic approach is TMEM16A (anoctamin-1/ANO1), a calcium-activated chloride channel (CaCC) which is also expressed in the airways of people with CF, albeit at low levels. To find novel TMEM16A regulators of both traffic and function, with the main goal of identifying candidate CF drug targets, we performed a fluorescence cell-based high-throughput siRNA microscopy screen for TMEM16A trafficking using a double-tagged construct expressed in human airway cells. About 700 genes were screened (2 siRNAs per gene) of which 262 were identified as candidate TMEM16A modulators (179 siRNAs enhanced and 83 decreased TMEM16A traffic), being G-protein coupled receptors (GPCRs) enriched on the primary hit list. Among the 179 TMEM16A traffic enhancer siRNAs subjected to secondary screening 20 were functionally validated. Further hit validation revealed that siRNAs targeting two GPCRs - ADRA2C and CXCR3 - increased TMEM16A-mediated chloride secretion in human airway cells, while their overexpression strongly diminished calcium-activated chloride currents in the same cell model. The knockdown, and likely also the inhibition, of these two TMEM16A modulators is therefore an attractive potential therapeutic strategy to increase chloride secretion in CF.

TMEM16A, a Homoharringtonine Receptor, as a Potential Endogenic Target for Lung Cancer Treatment.

Lung cancer has the highest rate of incidence and mortality among all cancers. Most chemotherapeutic drugs used to treat lung cancer cause serious side effects and are susceptible to drug resistance. Therefore, exploring novel therapeutic targets for lung cancer is important. In this study, we evaluated the potential of TMEM16A as a drug target for lung cancer. Homoharringtonine (HHT) was identified as a novel natural product inhibitor of TMEM16A. Patch-clamp experiments showed that HHT inhibited TMEM16A activity in a concentration-dependent manner. HHT significantly inhibited the proliferation and migration of lung cancer cells with high TMEM16A expression but did not affect the growth of normal lung cells in the absence of TMEM16A expression. In vivo experiments showed that HHT inhibited the growth of lung tumors in mice and did not reduce their body weight. Finally, the molecular mechanism through which HHT inhibits lung cancer was explored by western blotting. The findings showed that HHT has the potential to regulate TMEM16A activity both in vitro and in vivo and could be a new lead compound for the development of anti-lung-cancer drugs.

Cepharanthine, a novel selective ANO1 inhibitor with potential for lung adenocarcinoma therapy.

Anoctamin-1 (ANO1), also known as transmembrane protein 16A (TMEM16A), is identified as a Ca2+-activated Cl- channel that is expressed in many organs and tissues. It is involved in numerous major physiological functions and especially in tumor growth. By screening 530 natural compounds, we identified cepharanthine as a potent blocker of ANO1 channels with an IC50 of 11.2 ± 0.9 µM and Emax of 92.7 ± 1.7%. The Lys384, Arg535, Thr539, and Glu624 in ANO1 are critical for the inhibitory effect of cepharanthine. Similar to its effect on ANO1, cepharanthine inhibits ANO2, the closest analog of TMEM16A. In contrast, up to 30 µM of cepharanthine showed limited inhibitory effects on recombinant ANO6 and bestrophin-1-encoded Ca2+-activated Cl- currents, but it showed no effects on endogenous volume-regulated anion currents (VRAC). Cepharanthine could also potently suppress endogenous ANO1 currents, significantly inhibit cell proliferation and migration, and induce apoptosis in LA795 lung adenocarcinoma cells. Moreover, animal experiments have shown that cepharanthine can dramatically inhibit the growth of xenograft tumors in mice. The high specificity provided by cepharanthine could be an important foundation for future studies of the physiological role of ANO1 channels, and these findings may reveal a new mechanism of its anticancer effect.

Diethylstilbestrol, a Novel ANO1 Inhibitor, Exerts an Anticancer Effect on Non-Small Cell Lung Cancer via Inhibition of ANO1.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca2+-activated chloride channel (CaCC) overexpressed in various carcinomas. It plays an important role in the development of cancer; however, the role of ANO1 in NSCLC is unclear. In this study, diethylstilbestrol (DES) was identified as a selective ANO1 inhibitor using high-throughput screening. We found that DES inhibited yellow fluorescent protein (YFP) fluorescence reduction caused by ANO1 activation but did not inhibit cystic fibrosis transmembrane conductance regulator channel activity or P2Y activation-related cytosolic Ca2+ levels. Additionally, electrophysiological analyses showed that DES significantly reduced ANO1 channel activity, but it more potently reduced ANO1 protein levels. DES also inhibited the viability and migration of PC9 cells via the reduction in ANO1, phospho-ERK1/2, and phospho-EGFR levels. Moreover, DES induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage in PC9 cells, but it did not affect the viability of hepatocytes. These results suggest that ANO1 is a crucial target in the treatment of NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent.

Advances in Anoctamin 1: A Potential New Drug Target in Medicinal Chemistry.

ANO1, anoctamin 1(also known as TMEM16A), is the molecular basis of calcium-activated chloride channels with ten transmembrane segments which are widely expressed in mammalian cells, including epithelial cells, vascular smooth muscle tissues, electro-excitatory cells, and some tumor cells. To date, multiple studies have shown that many natural and synthetic compounds have regulatory effects on ANO1. Therefore, ANO1 could be a potential new drug target for the treatment of cancer, pain, diarrhea, hypertension, and asthma. In this study, we review the structure of ANO1 and its involvement in cancer, pain, diarrhea, hypertension, and asthma.

Nimodipine inhibits intestinal and aortic smooth muscle contraction by regulating Ca2+-activated Cl- channels.

Nimodipine is a clinically used dihydropyridine L-type calcium channel antagonist that effectively inhibits transmembrane Ca2+ influx following the depolarization of smooth muscle cells, but the detailed effect on smooth muscle contraction is not fully understood. Ca2+-activated Cl- channels (CaCCs) in vascular smooth muscle cells (VSMCs) may regulate vascular contractility. We found that nimodipine can inhibit transmembrane protein 16A (TMEM16A) activity in a concentration-dependent manner by cell-based fluorescence-quenching assay and short-circuit current analysis, with an IC50 value of ~5 µM. Short-circuit current analysis also showed that nimodipine prevented Ca2+-activated Cl- current in both HT-29 cells and mouse colonic epithelia accompanied by significantly decreased cytoplasmic Ca2+ concentrations. In the absence of extracellular Ca2+, nimodipine still exhibited an inhibitory effect on TMEM16A/CaCCs. Additionally, the application of nimodipine to CFTR-expressing FRT cells and mouse colonic mucosa resulted in mild activation of CFTR-mediated Cl- currents. Nimodipine inhibited basolateral CCh-activated K+ channel activity with no effect on Na+/K+-ATPase activity. Evaluation of intestinal smooth muscle contraction showed that nimodipine inhibits intestinal smooth muscle contractility and frequency, with an activity pattern that was similar to that of non-specific inhibitors of CaCCs. In aortic smooth muscle, the expression of TMEM16A in thoracic aorta is higher than that in abdominal aorta, corresponding to stronger maximum contractility in thoracic aorta smooth muscle stimulated by phenylephrine (PE) and Eact. Nimodipine completely inhibited the contraction of aortic smooth muscle stimulated by Eact, and partially inhibited the contraction stimulated by PE. In summary, the results indicate that nimodipine effectively inhibits TMEM16A/CaCCs by reduction transmembrane Ca2+ influx and directly interacting with TMEM16A, explaining the mechanisms of nimodipine relaxation of intestinal and aortic smooth muscle contraction and providing new targets for pharmacological applications.

Ca2+-activated Cl- channel TMEM16A inhibition by cholesterol promotes angiogenesis in endothelial cells.

Introduction: Ca2+-activated Cl- channel TMEM16A is expressed in endothelial cells, and contributes to many diseases such as hypertension, blood-brain barrier dysfunction, and pulmonary hypertension. It remains unclear whether TMEM16A regulates endothelial angiogenesis, which participates in many physiological and pathological processes. Cholesterol regulates many ion channels including TMEM16A, and high cholesterol levels contribute to endothelial dysfunction. It remains to be determined whether cholesterol regulates TMEM16A expression and function in endothelial cells. Objective: This study aimed to investigate whether cholesterol regulated TMEM16A expression and function in endothelial angiogenesis. Methods: Whole-cell patch clamp techniques were used to record Ca2+-activated Cl- currents in human aortic endothelial cells (HAECs) and HEK293 cells transfected with TMEM16A-overexpressing plasmids. Western blot was used to examine the expression of TMEM16A and DNA methyltransferase 1 (DNMT1) in HAECs. CCK-8 assay, would healing assay, and tube formation assay were used to test endothelial cell proliferation, migration and angiogenesis, respectively. Results: TMEM16A mediates the Ca2+-activated Cl- channel in HAECs. Cholesterol treatment inhibited TMEM16A expression via upregulation of DNMT1 in HAECs, and the inhibitory effect of cholesterol on TMEM16A expression was blocked by 5-aza, the DNMT1 inhibitor. In addition, direct application of cholesterol inhibited TMEM16A currents in heterologous HEK293 cells with an IC50 of 0.1209 µM. Similarly, cholesterol directly inhibited TMEM16A currents in HAECs. Furthermore, TMEM16A knockdown increased in vitro tube formation, cell migration and proliferation of HAECs, and TMEM16A overexpression produced the opposite effect. Conclusion: This study reveals a novel mechanism of cholesterol-mediated TMEM16A inhibition, by which cholesterol reduces TMEM16A expression via DNMT1-mediated methylation and directly inhibits channel activities. TMEM16A channel inhibition promotes endothelial cell angiogenesis.

A fungus-derived purpactin A as an inhibitor of TMEM16A chloride channels and mucin secretion in airway epithelial cells.

TMEM16A is a Ca2+-activated Cl- channel involved in mucus secretion in inflamed airways and proposed as a drug target for diseases associated with mucus hypersecretion including asthma. This study aimed to identify novel inhibitors of TMEM16A-mediated Cl- secretion in airway epithelial cells from a collection of compounds isolated from fungi indigenous in Thailand and examine its potential utility in mitigating airway mucus secretion using Calu-3 cells as a study model. Screening of > 400 fungal metabolites revealed purpactin A isolated from a soil-derived fungus Penicillium aculeatum PSU-RSPG105 as an inhibitor of TMEM16A-mediated Cl- transport with an IC50 value of ~2 µM. A consistent inhibitory effect of purpactin A on TMEM16A were observed regardless of TMEM16A activators or in the presence of an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII), a negative regulator of TMEM16A. In addition, purpactin A did not affect cell viability, epithelial barrier integrity and activities of membrane transport proteins essential for maintaining airway hydration including CFTR Cl- channels and apical BK K+ channels. Intriguingly, purpactin A prevented a Ca2+-induced mucin release in cytokine-treated airway cells. Taken together, purpactin A represents the first class of TMEM16A inhibitor derived from fungus, which may be beneficial for the treatment of diseases associated with mucus hypersecretion.

A small molecule inhibitor of the chloride channel TMEM16A blocks vascular smooth muscle contraction and lowers blood pressure in spontaneously hypertensive rats.

Hypertension is a major cause of cardiovascular morbidity and mortality, despite the availability of antihypertensive drugs with different targets and mechanisms of action. Here, we provide evidence that pharmacological inhibition of TMEM16A (ANO1), a calcium-activated chloride channel expressed in vascular smooth muscle cells, blocks calcium-activated chloride currents and contraction in vascular smooth muscle in vitro and decreases blood pressure in spontaneously hypertensive rats. The acylaminocycloalkylthiophene TMinh-23 fully inhibited calcium-activated TMEM16A chloride current with nanomolar potency in Fischer rat thyroid cells expressing TMEM16A, and in primary cultures of rat vascular smooth muscle cells. TMinh-23 reduced vasoconstriction caused by the thromboxane mimetic U46619 in mesenteric resistance arteries of wild-type and spontaneously hypertensive rats, with a greater inhibition in spontaneously hypertensive rats. Blood pressure measurements by tail-cuff and telemetry showed up to a 45-mmHg reduction in systolic blood pressure lasting for four-six hours in spontaneously hypertensive rats after a single dose of TMinh-23. A minimal effect on blood pressure was seen in wild-type rats or mice treated with TMinh-23. Five-day twice daily treatment of spontaneously hypertensive rats with TMinh-23 produced sustained reductions of 20-25 mmHg in daily mean systolic and diastolic blood pressure. TMinh-23 action was reversible, with blood pressure returning to baseline in spontaneously hypertensive rats by three days after treatment discontinuation. Thus, our studies provide validation for TMEM16A as a target for antihypertensive therapy and demonstrate the efficacy of TMinh-23 as an antihypertensive with a novel mechanism of action.

Anoctamin 1 antagonism potentiates conventional tocolytic-mediated relaxation of pregnant human uterine smooth muscle.

BACKGROUND: Currently available tocolytic agents are not effective treatment for preterm labor beyond 48 h. A major reason is the development of maternal side effects which preclude the maintenance of an effective steady-state drug concentration. One strategy that can mitigate these side effects is utilizing synergistic drug combinations to reduce the drug concentrations necessary to elicit a clinical effect. We have previously shown that three anoctamin 1 (ANO1) antagonists mediate potent relaxation of precontracted human uterine smooth muscle (USM). In this study, we aimed to determine whether a combination of sub-relaxatory doses of tocolytic drugs in current clinical use [the L-type voltage-gated calcium channel (VGCC) blocker, nifedipine (NIF); and the ß2-adrenergic (ß2AR) agonist, terbutaline (TRB)] will potentiate USM relaxation with two ANO1 antagonists [benzbromarone (BB) and MONNA (MN)]. OBJECTIVE: This study sought to examine the synergistic potency and mechanistic basis of two ANO1 antagonists with currently available tocolytic drugs. Functional endpoints assessed included relaxation of pre-contracting pregnant human USM tissue, inhibition of intracellular calcium release, and reduction of spontaneous transient inward current (STIC) recordings in human uterine smooth muscle cells. METHODS: Human myometrial strips and primary human USM cells were used in organ bath and calcium flux experiments with different combinations of sub-threshold doses of ANO1 antagonists and terbutaline or nifedipine to determine if ANO1 antagonists potentiate tocolytic drugs. RESULTS: The combination of sub-threshold doses of two ANO1 antagonists and current tocolytic drugs demonstrate a significant degree of synergy to relax human pregnant USM compared to the effects achieved when these drugs are administered individually. CONCLUSION: A combination of sub-threshold doses of VGCC blocker and ß2AR agonist with ANO1 antagonists potentiates relaxation of oxytocin-induced contractility and calcium flux in human USM ex vivo. Our findings may serve as a foundation for novel tocolytic drug combinations.

Volume-regulated chloride channel regulates cell proliferation and is involved in the possible interaction between TMEM16A and LRRC8A in human metastatic oral squamous cell carcinoma cells.

OBJECTIVES: Volume-regulated anion channels (VRACs), expressed in various cells, play an important role in cell volume regulation. Despite being physiologically defined almost half a century ago, only the molecular candidates of VRAC, TMEM16A, LRRC8A, and bestrophin-1 (BEST1), are known. Here, we aimed to explore the functional significance of VRAC in, HST-1, an oral squamous cell carcinoma (OSCC) cell line. METHODS: Cell proliferation assays, RT-PCR, Western blot, and flow cytometry were used to estimate changes in gene expression and cell proliferation. Ion channel activity was recorded using the patch-clamp technique. Specific genes were knocked-down by siRNA assays. RESULTS: VRAC, identified as a hypotonicity-induced current, was highly functional and associated with the proliferation of HST-1 cells but not of HaCaT (a normal keratinocyte) cells. The pharmacological profile of VRAC in HST-1 was similar to that reported previously. DCPIB, a specific VRAC inhibitor, completely inhibited VRAC and proliferation of HST-1 cells, eventually leading to apoptosis. VRAC in HST-1 was attenuated by the knockdown of TMEM16A and LRRC8A, while knockdown of BEST1 affected cell proliferation. In situ proximity ligation assay showed that TMEM16A and LRRC8A co-localized under isotonic conditions (300 mOsM) but were separated under hypotonic conditions (250 mOsM) on the plasma membrane. CONCLUSIONS: We have found that VRAC acts to regulate the proliferation of human metastatic OSCC cells and the composition of VRAC may involve in the interactions between TMEM16A and LRRC8A in HST-1 cells.

Design of Anticancer 2,4-Diaminopyrimidines as Novel Anoctamin 1 (ANO1) Ion Channel Blockers.

Pyrimidine is a privileged scaffold in many synthetic compounds exhibiting diverse pharmacological activities, and is used for therapeutic applications in a broad spectrum of human diseases. In this study, we prepared a small set of pyrimidine libraries based on the structure of two hit compounds that were identified through the screening of an in-house library in order to identify an inhibitor of anoctamin 1 (ANO1). ANO1 is amplified in various types of human malignant tumors, such as head and neck, parathyroid, and gastrointestinal stromal tumors, as well as in breast, lung, and prostate cancers. After initial screening and further structure optimization, we identified Aa3 as a dose-dependent ANO1 blocker. This compound exhibited more potent anti-cancer activity in the NCI-H460 cell line, expressing high levels of ANO1 compared with that in A549 cells that express low levels of ANO1. Our results open a new direction for the development of small-molecule ANO1 blockers composed of a pyrimidine scaffold and a nitrogen-containing heterocyclic moiety, with drug-like properties.

Molecular mechanism of CaCCinh-A01 inhibiting TMEM16A channel.

TMEM16A is a calcium-activated chloride channel that is associate with several diseases, including pulmonary diseases, hypertension, diarrhea and cancer. The CaCCinh-A01 (A01) is widely recognized as an efficient blocker of TMEM16A and has been used as a tool drug to inhibit TMEM16A currents in the laboratory. A01 also has excellent pharmacokinetic properties and can be developed as a drug to target TMEM16A. However, the molecular mechanism how A01 inhibits TMEM16A is still elusive, which slows down its drug development process. Here, calculations identified that the binding pocket of A01 was located above the pore, and it was also discovered that the binding of A01 to TMEM16A not only blocked the pore but also led to its collapse. The interaction model analysis predicted that R515/K603/E623 were crucial residues for the binding between TMEM16A and A01, and the site-directed mutagenesis studies confirmed the above results. The binding mode and quantum chemical calculations showed that the carboxyl and the amide oxygen atom of A01 were the key interaction sites between TMEM16A and A01. Therefore, our study proposed the inhibitory mechanism of TMEM16A current by A01 and revealed how A01 inhibits TMEM16A at the molecular level. These findings will shed light on both the development of A01 as a potential drug for TMEM16A dysfunction-related disorders and drug screening targeting the pocket.

Inhibitory activities of curzerenone, curdione, furanodienone, curcumol and germacrone on Ca2+-activated chloride channels.

Calcium-activated chloride channels (CaCCs) as a kind of widely expressed ion channels play crucial roles in a variety of physiological regulation. TMEM16A has been identified as the molecular basis of CaCCs in numerous cell types and is considered a new drug target for many diseases. Regulating the function of TMEM16A through small molecule modulators has become a new strategy to improve respiratory and digestive dysfunction and even tumor therapy. Herein, we obtained 5 sesquiterpenoids, named curzerenone, curdione, furanodienone, curcumol and germacrone with TMEM16A inhibition and revealed their mechanism of action by fluorescent and electrophysiological assays. Cell-based YFP fluorescence data demonstrated that 5 compounds inhibited TMEM16A-mediated I- influx in a dose-dependent manner. To explore the mechanism of 5 compounds on CaCCs, FRT cells with high expression of TMEM16A, HBE, HT-29 and T84 cells and mouse colons were used in short-circuit current assay. Our results showed that 5 compounds inhibited the Ca2+-activated Cl- currents generated by the Eact, ATP and UTP stimulation, and this inhibitory effect was related not only to the direct inhibition of channel opening, but also the inhibition of intracellular Ca2+ concentration and K+ channel activity. In addition to CaCCs, these 5 compounds also had definite inhibitory activities against cystic fibrosis transmembrane regulator (CFTR) at the cellular level. In summary, these compounds have the potential to regulate the activites of TMEM16A/CaCCs and CFTR channels in vitro, providing a new class of lead compounds for the development of drugs for diseases related to chloride channel dysfunction.

Anti-glioma effects of 2-aminothiophene-3-carboxamide derivatives, ANO1 channel blockers.

Anoctamin1 (ANO1), a calcium-activated chloride ion channel (CaCC), is associated with various physiological functions including cancer progression and metastasis/invasion. ANO1 has been considered as a promising target for cancer therapeutics as ANO1 is over-expressed in a variety of cancers including glioblastoma (GBM) and inhibition of ANO1 has been reported to suppress cell proliferation, migration and invasion in GBM. GBM is one of the most common and aggressive cancers with poor prognosis with median survival for 15 months. Lack of effective treatment options against GBM emphasizes urgent necessity of effective GBM therapeutics. In an effort to discover potent and selective ANO1 inhibitors capable of inhibiting GBM cells, we have designed and synthesized a series of new 2-aminothiophene-3-carboxamide derivatives and performed SAR studies using both fluorescent cellular membrane potential assay and whole-cell patch-clamp recording. We observed that among these substances, 9c and 10q strongly suppress ANO1 channel activities and possess remarkable selectivity over ANO2. Unique structural feature of 10q, a cyclopentane-fused thiophene-3-carboxamide derivative, is the presence of benzoylthiourea functionality which dramatically contributes to activity. Both 9c and 10q suppress more strongly proliferation of GBM cells than four reference compounds including 3, Ani-9 and are also capable of inhibiting much more strongly colony formation than reference compounds in both 2D colony formation assay and 3D soft agar assay using U251 glioma cells. In addition, 9c and 10q suppress far more strongly migration/invasion of GBM cells than reference compounds. We, for the first time, found that the combination of ANO1 inhibitor (9c or 3) and temozolomide (TMZ) brings about remarkable synergistic effects in suppressing proliferation of GBM cells. Our study may provide an insight into designing selective and potent ANO1 inhibitors aiming at GBM treatment.