Anticancer Effect of Hemin through ANO1 Inhibition in Human Prostate Cancer Cells.

Anoctamin1 (ANO1), a calcium-activated chloride channel, is overexpressed in a variety of cancer cells, including prostate cancer, and is involved in cancer cell proliferation, migration, and invasion. Inhibition of ANO1 in these cancer cells exhibits anticancer effects. In this study, we conducted a screening to identify novel ANO1 inhibitors with anticancer effects using PC-3 human prostate carcinoma cells. Screening of 2978 approved and investigational drugs revealed that hemin is a novel ANO1 inhibitor with an IC50 value of 0.45 µM. Notably, hemin had no significant effect on intracellular calcium signaling and cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP (cAMP)-regulated chloride channel, and it showed a weak inhibitory effect on ANO2 at 3 µM, a concentration that completely inhibits ANO1. Interestingly, hemin also significantly decreased ANO1 protein levels and strongly inhibited the cell proliferation and migration of PC-3 cells in an ANO1-dependent manner. Furthermore, it strongly induced caspase-3 activation, PARP degradation, and apoptosis in PC-3 cells. These findings suggest that hemin possesses anticancer properties via ANO1 inhibition and could be considered for development as a novel treatment for prostate cancer.

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.

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.

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.

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.

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.

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.

TMEM16A Ca2+-Activated Cl- Channel Regulates the Proliferation and Migration of Brain Capillary Endothelial Cells.

The blood-brain barrier (BBB) is essential for the maintenance of homeostasis in the brain. Brain capillary endothelial cells (BCECs) comprise the BBB, and thus a delicate balance between their proliferation and death is required. Although the activity of ion channels in BCECs is involved in BBB functions, the underlying molecular mechanisms remain unclear. In the present study, the molecular components of Ca2+-activated Cl- (ClCa) channels and their physiological roles were examined using mouse BCECs (mBCECs) and a cell line derived from bovine BCECs, t-BBEC117. Expression analyses revealed that TMEM16A was strongly expressed in mBCECs and t-BBEC117 cells. In t-BBEC117 cells, whole-cell Cl- currents were sensitive to the ClCa channel blockers, 100 µM niflumic acid and 10 µM T16Ainh-A01, and were also reduced markedly by small-interfering RNA (siRNA) knockdown of TMEM16A. Importantly, block of ClCa currents with ClCa channel blockers or TMEM16A siRNA induced membrane hyperpolarization. Moreover, treatment with TMEM16A siRNA caused an increase in resting cytosolic Ca2+ concentration ([Ca2+]cyt). T16Ainh-A01 reduced cell viability in a concentration-dependent manner. Either ClCa channel blockers or TMEM16A siRNA also curtailed cell proliferation and migration. Furthermore, ClCa channel blockers attenuated the trans-endothelial permeability. In combination, these results strongly suggest that TMEM16A contributes to ClCa channel conductance and can regulate both the resting membrane potential and [Ca2+]cyt in BCECs. Our data also reveal how these BCECs may be involved in the maintenance of BBB functions, as both the proliferation and migration are altered following changes in channel activity. SIGNIFICANCE STATEMENT: In brain capillary endothelial cells (BCECs) of the blood-brain barrier (BBB), TMEM16A is responsible for Ca2+-activated Cl- channels and can regulate both the resting membrane potential and cytosolic Ca2+ concentration, contributing to the proliferation and migration of BCECs. The present study provides novel information on the molecular mechanisms underlying the physiological functions of BCECs in the BBB and a novel target for therapeutic drugs for disorders associated with dysfunctions in the BBB.

Pacemaker function and neural responsiveness of subserosal interstitial cells of Cajal in the mouse colon.

KEY POINTS: Rhythmic action potentials and intercellular Ca2+ waves are generated in smooth muscle cells of colonic longitudinal muscles (LSMC). Longitudinal muscle excitability is tuned by input from subserosal ICC (ICC-SS), a population of ICC with previously unknown function. ICC-SS express Ano1 channels and generate spontaneous Ca2+ transients in a stochastic manner. Release of Ca2+ and activation of Ano1 channels causes depolarization of ICC-SS and LSMC, leading to activation of L-type Ca2+ channels, action potentials, intercellular Ca2+ waves and contractions in LSMC. Nitrergic neural inputs regulate the Ca2+ events in ICC-SS. Pacemaker activity in longitudinal muscle is an emergent property as a result of integrated processes in ICC-SS and LSMC. ABSTRACT: Much is known about myogenic mechanisms in circular muscle (CM) in the gastrointestinal tract, although less is known about longitudinal muscle (LM). Two Ca2+ signalling behaviours occur in LM: localized intracellular waves not causing contractions and intercellular waves leading to excitation-contraction coupling. An Ano1 channel antagonist inhibited intercellular Ca2+ waves and LM contractions. Ano1 channels are expressed by interstitial cells of Cajal (ICC) but not by smooth muscle cells (SMCs). We investigated Ca2+ signalling in a novel population of ICC that lies along the subserosal surface of LM (ICC-SS) in mice expressing GCaMP6f in ICC. ICC-SS fired stochastic localized Ca2+ transients. Such events have been linked to activation of Ano1 channels in ICC. Ca2+ transients in ICC-SS occurred by release from stores most probably via inositol trisphosphate receptors. This activity relied on influx via store-operated Ca2+ entry and Orai channels. No voltage-dependent mechanism that synchronized Ca2+ transients in a single cell or between cells was found. Nitrergic agonists inhibited Ca2+ transients in ICC-SS, and stimulation of intrinsic nerves activated nitrergic responses in ICC-SS. Cessation of stimulation resulted in significant enhancement of Ca2+ transients compared to the pre-stimulus activity. No evidence of innervation by excitatory, cholinergic motor neurons was found. Our data suggest that ICC-SS contribute to regulation of LM motor activity. Spontaneous Ca2+ transients activate Ano1 channels in ICC-SS. Resulting depolarization conducts to SMCs, depolarizing membrane potential, activating L-type Ca2+ channels and initiating contraction. Rhythmic electrical and mechanical behaviours of LM are an emergent property of SMCs and ICC-SS.

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.

TMEM16A is indispensable for basal mucus secretion in airways and intestine.

Transmembrane member 16A (TMEM16A) is the Ca2+-activated chloride channel in airways and intestine. It has been associated with goblet cell metaplasia, as expression of TMEM16A is strongly up-regulated in cystic fibrosis and asthma during mucus hypersecretion. However, the possible role of TMEM16A for mucus production or mucus secretion remains obscure, and whether TMEM16A controls the function of intestinal goblet cells is entirely unknown. Basal mucus secretion in lungs occurs through low levels of ATP in the airway surface liquid. Here, we report for the first time that TMEM16A is essential for basal secretion of mucus in airways and intestine. Airway-ciliated and intestinal epithelial-specific knockout of TMEM16A ( TMEM16Aflox/floxFoxJ1, TMEM16Aflox/floxVil1) leads to accumulation of mucus in airway club (Clara) cells and intestinal goblet cells, respectively. Acute ATP-induced mucus secretion by airway club cells is inhibited when TMEM16A is knocked out in ciliated cells, possibly as a result of compromised release of prosecretory cytokines. Knockdown or inhibition of TMEM16A in human Calu3 airway epithelial cells indicates compromised IL-8 release. In intestinal goblet cells lacking expression of TMEM16A, mucus accumulates as a result of compromised ATP-induced secretion. In contrast, cholinergic mucus secretion by compound exocytosis is independent of TMEM16A. The data demonstrate a previously unrecognized role of TMEM16A for membrane exocytosis and describe a novel, ATP-driven pathway for intestinal mucus secretion. We conclude that ATP-dependent mucus secretion in both airways and intestine requires TMEM16A. The present results may form the basis for a novel, therapeutic approach for the treatment of mucus hypersecretion in inflammatory airway and intestinal disease.-Benedetto, R., Cabrita, I., Schreiber, R., Kunzelmann, K. TMEM16A is indispensable for basal mucus secretion in airways and intestine.

Arctigenin, a novel TMEM16A inhibitor for lung adenocarcinoma therapy.

TMEM16A plays critical roles in physiological process and may serve as drug targets for diverse diseases. Recently, TMEM16A has started to be regarded as potential primary lung adenocarcinoma targets. Here, we identified that arctigenin, a natural compound, is a novel TMEM16A inhibitor, and it can suppress lung adenocarcinoma growth through inhibiting TMEM16A both in vitro and in vivo. Our data also showed that the IC50 of actigenin to TMEM16A whole-cell current was 19.29 ± 4.69 µM, and the putative binding sites of arctigenin in TMEM16A were R515 and R535. Arctigenin concentration-dependently inhibited the proliferation and migration of LA795, however, the inhibition effect can be abolished by knockdown of the endogenous TMEM16A with shRNA. Further, we injected arctigenin on xenograft mouse model which exhibited significant antitumor activity with no adverse effect. At last, western blotting results showed the mechanism of arctigenin inhibiting lung adenocarcinoma was through inhibiting MAPK pathway. In summary, TMEM16A is a novel drug target for lung adenocarcinoma treatment. Arctigenin can be used as a lead compound for the development of lung adenocarcinoma therapy drugs.

Inhibition of TMEM16A Ca2+-activated Cl- channels by avermectins is essential for their anticancer effects.

Transmembrane member 16A (TMEM16A) encoded Ca2+-activated Cl- channels were found to be involved in tumorigenesis. Previous studies suggest the effect of TMEM16A gene amplification on tumorigenic proliferation is exerted through its channel function. TMEM16A-specific and potent small molecule inhibitors have been proposed to potentially be useful for the treatment of cancer. Thus, we screened six analogues of avermectin for their inhibitory activities on TMEM16A mediated currents. A whole-cell patch technique was used to record the currents. The IC50 and Emax values for TMEM16A inhibition of five tested avermectins (avermectin B1, ivermectin, doramectin, selamectin, and moxidectin) were 0.15-1.32 µM and 65-87 %, respectively. In addition, these avermectins significantly inhibited endogenous TMEM16A mediated currents and thus, the proliferation, migration, inducing apoptosis of LA795 cancer cells. Eprinomectin (4"-(acetylamino)-4"-deoxy-avermectin B1) and two other important macrolides (erythromycin and azithromycin), which have minimal or no TMEM16A inhibitory effects, were used as negative control drugs. These drugs were found to have limited effects on the proliferation, migration, and apoptosis of LA795 cells. Finally, avermectin B1 and ivermectin dramatically inhibited the growth of xenograft tumors in mice. These data demonstrate that avermectins are novel TMEM16A inhibitors and are potentially useful in specific cancer therapies. These findings also provide a new opportunity to develop TMEM16A modulators.

Four new sucrose diesters of substituted truxinic acids from Trigonostemon honbaensis with their anoctamin-1 inhibitory activity.

Truxinic acid sucrose diesters analogs possess interesting chemical structure by the presence of cyclobutane-ring and macrocyclic sucrose diesters moieties which are rarely found from natural sources. This paper describes the isolation and structural elucidation of four new sucrose diesters of substituted truxinic acids, trigohonbanosides A-D (1-4), from the leaves of Trigonostemon honbaensis. Their chemical structures were elucidated by HR-ESI-MS, NMR, and CD spectroscopic methods. At a concentration of 30 µM, compounds 1-4 moderately inhibited ANO-1 activity with inhibitory percentages of 27.7 ± 1.10%, 35.6 ± 0.92%, 43.7 ± 1.61%, and 40.8 ± 1.25%, respectively.

Diversity-oriented generation and biological evaluation of new chemical scaffolds bearing a 2,2-dimethyl-2H-chromene unit: Discovery of novel potent ANO1 inhibitors.

Chemical territory bearing a 2,2-dimethyl-2H-chromene motif was expanded by utilizing an o-hydroxy aldehyde group of 5-hydroxy-2,2-dimethyl-2H-chromene-6-carbaldehyde as a synthetic handle to install distinctive morphology and functionality of each scaffold. Cell based assays and in silico docking analysis led us to discover that these new compounds exhibit inhibitory effect on anoctamin1 (ANO1). ANO1 is amplified and highly expressed in various carcinomas including prostate cancer, esophageal cancer, breast cancer, and pancreatic cancer. Biological assays revealed that (E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(1H-pyrrol-2-yl)prop-2-en-1-one (3n, Ani-FCC) is a novel, potent and selective ANO1 inhibitor with an IC50 value of 1.23 µM. 3n showed 144 times stronger activity on ANO1 inhibition than ANO2 inhibition and did not alter the chloride channel activity of CFTR and the intracellular calcium signaling. Notably, 3n strongly decreased cell viability of PC-3 and FaDu cells expressing high levels of ANO1 with a decrease in ANO1 protein levels. In addition, 3n significantly enhanced apoptosis via activation of caspase 3 and cleavage of PARP in PC-3 and FaDu cells. This study shows that a novel ANO1 inhibitor, 3n, can be a potential candidate for the treatment of cancers overexpressing ANO1, such as prostate cancer and esophageal cancer.

The Traditional Medicine Banhasasim-Tang Depolarizes Pacemaker Potentials of Cultured Interstitial Cells of Cajal through M3 Muscarinic and 5-HT3 Receptors in Murine Small Intestine.

BACKGROUND: Banhasasim-tang (BHSST) is a classic herbal formulation in traditional Chinese medicine widely used for gastrointestinal (GI) tract motility disorder. We investigated the effects of BHSST on the pacemaker potentials of cultured interstitial cells of Cajal (ICCs) in small intestine in vitro and its effects on GI motor functions in vivo. METHODS: We isolated ICCs from the small intestines and recorded pacemaker potentials in cultured ICCs with the whole-cell patch-clamp configuration in vitro. Intestinal transit rates (ITR%) were investigated in normal mice and GI motility dysfunction (GMD) mouse models in vivo. RESULTS: BHSST (20-50 mg/mL) depolarized pacemaker potentials and decreased their amplitudes in a concentration-dependent manner. Pretreatment with methoctramine (a muscarinic M2 receptor antagonist) did not inhibit BHSST-induced pacemaker potential depolarization. However, when we applied 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; a muscarinic M3 receptor antagonist), BHSST-induced effects were blocked. Pretreatment with Y25130 (a 5-HT3 receptor antagonist) blocked BHSST-induced effects in ICCs. In addition, when we applied 4-DAMP and Y25130 together, BHSST-induced effects were completely blocked. Pretreatment with Ca2+-free solution or thapsigargin inhibited BHSST-induced effects. Moreover, BHSST blocked both the transient receptor potential melastatin (TRPM) 7 and voltage-sensitive calcium-activated chloride (anoctamin-1, ANO1) channels. In normal mice, ITR% values were significantly increased by BHSST in a dose-dependent manner. The ITR% of GMD mice was significantly reduced relative to those of normal mice, which were significantly reversed by BHSST in a dose-dependent manner. CONCLUSION: These results suggested that BHSST depolarizes the pacemaker potentials of ICCs in a dose-dependent manner through the M3 and 5-HT3 receptors via internal and external Ca2+-dependent and TRPM7- and ANO1-independent pathways in vitro. Moreover, BHSST increased ITR% in vivo in normal mice and GMD mouse models. Taken together, the results of this study showed that BHSST had the potential for development as a prokinetic agent in GI motility function.

TMEM16A: An Alternative Approach to Restoring Airway Anion Secretion in Cystic Fibrosis?

The concept that increasing airway hydration leads to improvements in mucus clearance and lung function in cystic fibrosis has been clinically validated with osmotic agents such as hypertonic saline and more convincingly with cystic fibrosis transmembrane conductance regulator (CFTR) repair therapies. Although rapidly becoming the standard of care in cystic fibrosis (CF), current CFTR modulators do not treat all patients nor do they restore the rate of decline in lung function to normal levels. As such, novel approaches are still required to ensure all with CF have effective therapies. Although CFTR plays a fundamental role in the regulation of fluid secretion across the airway mucosa, there are other ion channels and transporters that represent viable targets for future therapeutics. In this review article we will summarise the current progress with CFTR-independent approaches to restoring mucosal hydration, including epithelial sodium channel (ENaC) blockade and modulators of SLC26A9. A particular emphasis is given to modulation of the airway epithelial calcium-activated chloride channel (CaCC), TMEM16A, as there is controversy regarding whether it should be positively or negatively modulated. This is discussed in light of a recent report describing for the first time bona fide TMEM16A potentiators and their positive effects upon epithelial fluid secretion and mucus clearance.

Novel ANO1 Inhibitor from Mallotus apelta Extract Exerts Anticancer Activity through Downregulation of ANO1.

Anoctamin1 (ANO1), a calcium-activated chloride channel, is frequently overexpressed in several cancers, including human prostate cancer and oral squamous cell carcinomas. ANO1 plays a critical role in tumor growth and maintenance of these cancers. In this study, we have isolated two new compounds (1 and 2) and four known compounds (3-6) from Mallotus apelta. These compounds were evaluated for their inhibitory effects on ANO1 channel activity and their cytotoxic effects on PC-3 prostate cancer cells. Interestingly, compounds 1 and 2 significantly reduced both ANO1 channel activity and cell viability. Electrophysiological study revealed that compound 2 (Ani-D2) is a potent and selective ANO1 inhibitor, with an IC50 value of 2.64 µM. Ani-D2 had minimal effect on cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity and intracellular calcium signaling. Notably, Ani-D2 significantly reduced ANO1 protein expression levels and cell viability in an ANO1-dependent manner in PC-3 and oral squamous cell carcinoma CAL-27 cells. In addition, Ani-D2 strongly reduced cell migration and induced activation of caspase-3 and cleavage of PARP in PC-3 and CAL-27 cells. This study revealed that a novel ANO1 inhibitor, Ani-D2, has therapeutic potential for the treatment of several cancers that overexpress ANO1, such as prostate cancer and oral squamous cell carcinoma.

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.

Volume-regulated Cl- current: contributions of distinct Cl- channels and localized Ca2+ signals.

The swelling-activated chloride current (ICl,swell) is induced when a cell swells and plays a central role in maintaining cell volume in response to osmotic stress. The major contributor of ICl,swell is the volume-regulated anion channel (VRAC). Leucine-rich repeat containing 8A (LRRC8A; SWELL1) was recently identified as an essential component of VRAC, but the mechanisms of VRAC activation are still largely unknown; moreover, other Cl- channels, such as anoctamin 1 (ANO1), were also suggested to contribute to ICl,swell. In this present study, we investigated the roles of LRRC8A and ANO1 in activation of ICl,swell; we also explored the role of intracellular Ca2+ in ICl,swell activation. We used a CRISPR/Cas9 gene editing approach, electrophysiology, live fluorescent imaging, selective pharmacology, and other approaches to show that both LRRC8A and ANO1 can be activated by cell swelling in HEK293 cells. Yet, both channels contribute biophysically and pharmacologically distinct components to ICl,swell, with LRRC8A being the major component. Cell swelling induced oscillatory Ca2+ transients, and these Ca2+ signals were required to activate both the LRRC8A- and ANO1-dependent components of ICl,swell. Both ICl,swell components required localized rather than global Ca2+ for activation. Interestingly, while intracellular Ca2+ was necessary and sufficient to activate ANO1, it was necessary but not sufficient to activate LRRC8A-mediated currents. Finally, Ca2+ transients linked to the ICl,swell activation were mediated by the G protein-coupled receptor-independent PLC isoforms.