The pharmacology of the TMEM16A channel: therapeutic opportunities

The Ca2+-gated Cl− channel TMEM16A is a key regulator of vital physiological functions such as control of vascular tone and local blood flow, and epithelial solute transport. The channel has emerged as a potential target for diseases such as hypertension, stroke, and cystic fibrosis.High-throughput screening efforts led to the identification of potent test compounds, and an activator has reached Phase 1 clinical trial.Recent advances in the TMEM16A structure and its gating mechanisms now open opportunities for rational drug design and may expedite the identification of novel drug-like modulators.Ultimately, preclinical studies of the ion channel biophysics, primary and secondary pharmacology, efficacy, pharmacodynamic and pharmacokinetic properties, toxicology, free fraction, and tissue penetration will be required to enable new molecules to reach clinical trials. The TMEM16A Ca2+-gated Cl− channel is involved in a variety of vital physiological functions and may be targeted pharmacologically for therapeutic benefit in diseases such as hypertension, stroke, and cystic fibrosis (CF). The determination of the TMEM16A structure and high-throughput screening efforts, alongside ex vivo and in vivo animal studies and clinical investigations, are hastening our understanding of the physiology and pharmacology of this channel. Here, we offer a critical analysis of recent developments in TMEM16A pharmacology and reflect on the therapeutic opportunities provided by this target. The TMEM16A Ca2+-gated Cl− channel is involved in a variety of vital physiological functions and may be targeted pharmacologically for therapeutic benefit in diseases such as hypertension, stroke, and cystic fibrosis (CF). The determination of the TMEM16A structure and high-throughput screening efforts, alongside ex vivo and in vivo animal studies and clinical investigations, are hastening our understanding of the physiology and pharmacology of this channel. Here, we offer a critical analysis of recent developments in TMEM16A pharmacology and reflect on the therapeutic opportunities provided by this target. The TMEM16A channel as a drug targetCa2+-activated Cl− channels (CaCCs) encoded by the TMEM16A gene are a class of anion channels activated by a rise of intracellular Ca2+ ([Ca2+]i) typically provoked by the activation of Gq protein-coupled receptors (GqPCRs). The TMEM16A channel thus links intracellular Ca2+ handling with cellular electrical activity [1.Agostinelli E. Tammaro P. Polymodal control of TMEM16x channels and scramblases.Int. J. Mol. Sci. 2022; 23: 1580Crossref PubMed Scopus (1) Google Scholar,2.Hawn M.B. et al.Molecular mechanisms of activation and regulation of ANO1-encoded Ca2+-activated Cl- channels.Channels. 2021; 15: 569-603Crossref PubMed Scopus (3) Google Scholar]. The TMEM16A channel plays a role in a variety of physiological functions including the control of vascular smooth muscle and pericyte tone and the regulation of epithelial cell (EC) secretion (see later, 'Pharmacology of the TMEM16A channel: focus on airways and vasculature') [1.Agostinelli E. Tammaro P. Polymodal control of TMEM16x channels and scramblases.Int. J. Mol. Sci. 2022; 23: 1580Crossref PubMed Scopus (1) Google Scholar,2.Hawn M.B. et al.Molecular mechanisms of activation and regulation of ANO1-encoded Ca2+-activated Cl- channels.Channels. 2021; 15: 569-603Crossref PubMed Scopus (3) Google Scholar].Our detailed understanding of CaCC physiology and pharmacology has long been impeded by not knowing the channel's molecular structure. The identification of TMEM16A as a gene encoding CaCC [3.Caputo A. et al.TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity.Science. 2008; 322: 590-594Crossref PubMed Scopus (967) Google Scholar, 4.Yang Y.D. et al.TMEM16A confers receptor-activated calcium-dependent chloride conductance.Nature. 2008; 455: 1210-1215Crossref PubMed Scopus (988) Google Scholar, 5.Schroeder B.C. et al.Expression cloning of TMEM16A as a calcium-activated chloride channel subunit.Cell. 2008; 134: 1019-1029Abstract Full Text Full Text PDF PubMed Scopus (892) Google Scholar] electrified the ion channel field and triggered a wealth of investigations leading to the determination of the TMEM16A 3D structure [6.Paulino C. et al.Structural basis for anion conduction in the calcium-activated chloride channel TMEM16A.Elife. 2017; 6e26232Crossref PubMed Scopus (97) Google Scholar, 7.Dang S. et al.Cryo-EM structures of the TMEM16A calcium-activated chloride channel.Nature. 2017; 552: 426-429Crossref PubMed Scopus (170) Google Scholar, 8.Paulino C. et al.Activation mechanism of the calcium-activated chloride channel TMEM16A revealed by cryo-EM.Nature. 2017; 552: 421-425Crossref PubMed Scopus (142) Google Scholar], the identification of binding sites for endogenous [9.Le S.C. et al.Molecular basis of PIP2-dependent regulation of the Ca2+-activated chloride channel TMEM16A.Nat. Commun. 2019; 10: 3769Crossref PubMed Scopus (36) Google Scholar,10.Yu K. et al.A network of phosphatidylinositol 4,5-bisphosphate binding sites regulates gating of the Ca2+-activated Cl- channel ANO1 (TMEM16A).Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 19952-19962Crossref PubMed Scopus (0) Google Scholar] and synthetic [11.Dinsdale R.L. et al.An outer-pore gate modulates the pharmacology of the TMEM16A channel.Proc. Natl. Acad. Sci. U. S. A. 2021; 118e2023572118Crossref PubMed Scopus (3) Google Scholar, 12.Cheng Y. et al.Identification of a conserved drug binding pocket in TMEM16 proteins.Res. Sq. 2022; (Published online February 10, 2022)https://doi.org/10.21203/rs.3.rs-1296933/v1Google Scholar, 13.Lam A.K.M. et al.Inhibition mechanism of the chloride channel TMEM16A by the pore blocker 1PBC.Nat. Commun. 2022; 13: 2798Crossref PubMed Google Scholar] ligands or natural products [14.Shi S. et al.Theaflavin binds to a druggable pocket of TMEM16A channel and inhibits lung adenocarcinoma cell viability.J. Biol. Chem. 2021; 297101016Abstract Full Text Full Text PDF Scopus (3) Google Scholar,15.Guo S. et al.The molecular mechanism of ginsenoside analogs activating TMEM16A.Biophys. J. 2020; 118: 262-272Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar], and the elucidation of the role of the channel in a range of cell types [1.Agostinelli E. Tammaro P. Polymodal control of TMEM16x channels and scramblases.Int. J. Mol. Sci. 2022; 23: 1580Crossref PubMed Scopus (1) Google Scholar,2.Hawn M.B. et al.Molecular mechanisms of activation and regulation of ANO1-encoded Ca2+-activated Cl- channels.Channels. 2021; 15: 569-603Crossref PubMed Scopus (3) Google Scholar,16.Oh U. Jung J. Cellular functions of TMEM16/anoctamin.Pflugers Arch. 2016; 468: 443-453Crossref PubMed Scopus (106) Google Scholar]. While research efforts enabled the discovery of potent test compounds (inhibitors and activators), no therapeutic drugs acting on TMEM16A have yet reached use in clinical practice.TMEM16A inhibitors have potential therapeutic uses in conditions such as hypertension (pulmonary and systemic), stroke, and overactive bladder while activators could be used in the treatment of disorders associated with epithelial dysfunction such as CF, chronic obstructive pulmonary disease (COPD), Sjögren's syndrome, and dry eye syndrome. TMEM16A is expressed in more than a single organ or tissue; this might imply that drugs acting on TMEM16A may lead to effects on more than one site. However, several established therapeutic targets have multiple-tissue distributions, a fact that is in some cases a therapeutic advantage (see later, 'Concluding remarks'). In this opinion, we reflect on the therapeutic opportunities and potential concerns for safety pharmacology that might be associated with pharmacological modulation of the TMEM16A channel. Emphasis is given to airways ECs and vascular cells, since there have been prominent recent advances in TMEM16A pharmacology at these sites, where TMEM16A activators and inhibitors could potentially improve the clinical treatment of CF, COPD, and diseases of altered vascular tone, respectively.The TMEM16 family of anion channels and lipid scramblasesCaCC currents were first observed ~40 years ago in Xenopus laevis oocytes [17.Miledi R. A calcium-dependent transient outward current in Xenopus laevis oocytes.Proc. R. Soc. Lond. B Biol. Sci. 1982; 215: 491-497Crossref PubMed Scopus (351) Google Scholar,18.Barish M.E. A transient calcium-dependent chloride current in the immature Xenopus oocyte.J. Physiol. 1983; 342: 309-325Crossref PubMed Google Scholar] and salamander photoreceptors [19.Bader C.R. et al.Voltage-activated and calcium-activated currents studied in solitary rod inner segments from the salamander retina.J. Physiol. 1982; 331: 253-284Crossref PubMed Google Scholar]. An intense search for the genes encoding CaCC led to the proposition of a range of candidates including members of the Ca2+-activated Cl− channel (CLCA) and bestrophin gene families; however, the proteins encoded by these genes mediated currents with electrophysiological properties that did not fully resemble those of the CaCCs originally identified in the previously mentioned cell types [20.Hartzell H.C. et al.Anoctamin/TMEM16 family members are Ca2+-activated Cl- channels.J. Physiol. 2009; 587: 2127-2139Crossref PubMed Scopus (202) Google Scholar]. By contrast, heterologously expressed TMEM16A channels mediated currents that closely matched the native CaCCs [3.Caputo A. et al.TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity.Science. 2008; 322: 590-594Crossref PubMed Scopus (967) Google Scholar, 4.Yang Y.D. et al.TMEM16A confers receptor-activated calcium-dependent chloride conductance.Nature. 2008; 455: 1210-1215Crossref PubMed Scopus (988) Google Scholar, 5.Schroeder B.C. et al.Expression cloning of TMEM16A as a calcium-activated chloride channel subunit.Cell. 2008; 134: 1019-1029Abstract Full Text Full Text PDF PubMed Scopus (892) Google Scholar].The TMEM16A gene is a founding member of a family of ten genes (TMEM16x) in eukaryotes. While sharing high sequence similarity, the ten TMEM16x paralogs have different functions: (i) TMEM16A and B form CaCCs; (ii) TMEM16D, E, F, K, and J function as lipid scramblases, which facilitate bidirectional movement of lipids – especially phosphatidylserine (PS) – across the cell membranes, and may possess nonselective ion channel activity; and (iii) TMEM16C, G, and H have additional cellular roles such as serving as auxiliary subunits for other channels (see [1.Agostinelli E. Tammaro P. Polymodal control of TMEM16x channels and scramblases.Int. J. Mol. Sci. 2022; 23: 1580Crossref PubMed Scopus (1) Google Scholar,2.Hawn M.B. et al.Molecular mechanisms of activation and regulation of ANO1-encoded Ca2+-activated Cl- channels.Channels. 2021; 15: 569-603Crossref PubMed Scopus (3) Google Scholar,21.Pedemonte N. Galietta L.J. Structure and function of TMEM16 proteins (anoctamins).Physiol. Rev. 2014; 94: 419-459Crossref PubMed Scopus (314) Google Scholar] for in-depth reviews of the TMEM16x physiological roles).The TMEM16x proteins were also termed 'anoctamins' [4.Yang Y.D. et al.TMEM16A confers receptor-activated calcium-dependent chloride conductance.Nature. 2008; 455: 1210-1215Crossref PubMed Scopus (988) Google Scholar], because TMEM16A is a primarily anion-selective channel with a predicted topology of eight ('oct') transmembrane TM domains. However, as noted previously, TMEM16x proteins have also functions other than serving as anion channels and their experimentally determined structures (see later) demonstrated the existence of ten TM domains. The term anoctamin is therefore now considered inaccurate by several researchers working in the field. In this opinion, the original nomenclature (TMEM16x) is used.The structure of the TMEM16A channelThe structure–function relationship of TMEM16x proteins was recently reviewed in [1.Agostinelli E. Tammaro P. Polymodal control of TMEM16x channels and scramblases.Int. J. Mol. Sci. 2022; 23: 1580Crossref PubMed Scopus (1) Google Scholar,2.Hawn M.B. et al.Molecular mechanisms of activation and regulation of ANO1-encoded Ca2+-activated Cl- channels.Channels. 2021; 15: 569-603Crossref PubMed Scopus (3) Google Scholar,22.Ji W. et al.TMEM16A protein: calcium-binding site and its activation mechanism.Protein Pept. Lett. 2021; 28: 1338-1348Crossref PubMed Scopus (0) Google Scholar, 23.Kalienkova V. et al.The groovy TMEM16 family: molecular mechanisms of lipid scrambling and ion conduction.J. Mol. Biol. 2021; 433166941Crossref PubMed Scopus (17) Google Scholar, 24.Falzone M.E. et al.Known structures and unknown mechanisms of TMEM16 scramblases and channels.J. Gen. Physiol. 2018; 150: 933-947Crossref PubMed Scopus (61) Google Scholar]; thus, here we provide a succinct account of aspects of particular relevance to pharmacology. The TMEM16A channel is a homodimer with each monomer forming an independent pore [25.Jeng G. et al.Independent activation of distinct pores in dimeric TMEM16A channels.J. Gen. Physiol. 2016; 148: 393-404Crossref PubMed Scopus (51) Google Scholar,26.Lim N.K. et al.Independent activation of ion conduction pores in the double-barreled calcium-activated chloride channel TMEM16A.J. Gen. Physiol. 2016; 148: 375-392Crossref PubMed Scopus (56) Google Scholar] (Figure 1). Each monomer has intracellular N and C termini and it encompasses ten TM domains. TMEM16A residues critical for Ca2+ binding, such as glutamates at positions 702 and 705, were identified prior to the determination of the TMEM16A structure [27.Yu K. et al.Explaining calcium-dependent gating of anoctamin-1 chloride channels requires a revised topology.Circ. Res. 2012; 110: 990-999Crossref PubMed Scopus (136) Google Scholar,28.Tien J. et al.A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity.Elife. 2014; 3e02772Crossref PubMed Google Scholar]. Each monomer contains two high-affinity Ca2+-binding sites that enable channel opening in response to a rise in intracellular Ca2+ [7.Dang S. et al.Cryo-EM structures of the TMEM16A calcium-activated chloride channel.Nature. 2017; 552: 426-429Crossref PubMed Scopus (170) Google Scholar,8.Paulino C. et al.Activation mechanism of the calcium-activated chloride channel TMEM16A revealed by cryo-EM.Nature. 2017; 552: 421-425Crossref PubMed Scopus (142) Google Scholar]. An additional modulatory Ca2+-binding site was also found in TMEM16x proteins [29.Le S.C. Yang H. An additional Ca2+ binding site allosterically controls TMEM16A activation.Cell Rep. 2020; 33108570Abstract Full Text Full Text PDF Scopus (7) Google Scholar,30.Bushell S.R. et al.The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K.Nat. Commun. 2019; 10: 3956Crossref PubMed Scopus (53) Google Scholar]. Pore opening in response to Ca2+ binding requires the rearrangement of a gate comprising a cytoplasm-facing portion of TM6 ('steric gate') [8.Paulino C. et al.Activation mechanism of the calcium-activated chloride channel TMEM16A revealed by cryo-EM.Nature. 2017; 552: 421-425Crossref PubMed Scopus (142) Google Scholar,31.Peters C.J. et al.The sixth transmembrane segment is a major gating component of the TMEM16A calcium-activated chloride channel.Neuron. 2018; 97: 1063-1077Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar] and opening of the outer pore [11.Dinsdale R.L. et al.An outer-pore gate modulates the pharmacology of the TMEM16A channel.Proc. Natl. Acad. Sci. U. S. A. 2021; 118e2023572118Crossref PubMed Scopus (3) Google Scholar,32.Lam A.K.M. Dutzler R. Mechanism of pore opening in the calcium-activated chloride channel TMEM16A.Nat. Commun. 2021; 12: 786Crossref PubMed Scopus (8) Google Scholar]. The Ca2+-binding sites are formed by a series of negatively charged residues; the unoccupied sites produce an electrostatic repulsion to permeating anions ('electrostatic gate') [33.Lam A.K. Dutzler R. Calcium-dependent electrostatic control of anion access to the pore of the calcium-activated chloride channel TMEM16A.Elife. 2018; 7e39122Crossref Scopus (24) Google Scholar]. This electrostatic barrier is lowered following Ca2+ binding and neutralisation of the negative charges of the Ca2+-binding sites [33.Lam A.K. Dutzler R. Calcium-dependent electrostatic control of anion access to the pore of the calcium-activated chloride channel TMEM16A.Elife. 2018; 7e39122Crossref Scopus (24) Google Scholar] (Figure 2).Figure 2TMEM16A channel gating and mechanisms of pharmacological modulation.Show full captionDiagram of a pore of the TMEM16A channel in the closed and open conformations. Binding of Ca2+ to the channel induces opening of the transmembrane 6 TM6 (steric) gate and outer pore (shown as tilts in the pore structure), and attenuation of the electrostatic gate. The green and red backgrounds depict positive and negative electrostatic potentials in the pore, respectively. The broken-line box highlights channel gating in the absence of pharmacological modulators. Inhibitors may act as open channel blockers that bind to the pore to occlude ion permeation (lower diagram). Gating modifiers may lead to closure (inhibitors) or opening (activators) of the pore (upper diagram). Additional mechanisms of action (not shown) may involve a compound binding to and stabilising the open or the closed state of the channel. Created using Microsoft Power Point.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The cryoelectron microscopy (cryo-EM) structures of the TMEM16A channel [6.Paulino C. et al.Structural basis for anion conduction in the calcium-activated chloride channel TMEM16A.Elife. 2017; 6e26232Crossref PubMed Scopus (97) Google Scholar, 7.Dang S. et al.Cryo-EM structures of the TMEM16A calcium-activated chloride channel.Nature. 2017; 552: 426-429Crossref PubMed Scopus (170) Google Scholar, 8.Paulino C. et al.Activation mechanism of the calcium-activated chloride channel TMEM16A revealed by cryo-EM.Nature. 2017; 552: 421-425Crossref PubMed Scopus (142) Google Scholar] show that the pore assumes an hourglass shape that is largely sheltered from the membrane, but detachment of TM4 and TM6 creates a funnel-shaped vestibule that has regions directly exposed to the cytoplasm and the lipid bilayer [23.Kalienkova V. et al.The groovy TMEM16 family: molecular mechanisms of lipid scrambling and ion conduction.J. Mol. Biol. 2021; 433166941Crossref PubMed Scopus (17) Google Scholar,24.Falzone M.E. et al.Known structures and unknown mechanisms of TMEM16 scramblases and channels.J. Gen. Physiol. 2018; 150: 933-947Crossref PubMed Scopus (61) Google Scholar], and it was proposed that lipids may line part of the pore [34.Whitlock J.M. Hartzell H.C. A pore idea: the ion conduction pathway of TMEM16/ANO proteins is composed partly of lipid.Pflugers Arch. 2016; 468: 455-473Crossref PubMed Scopus (49) Google Scholar]. The TMEM16x scramblases such as TMEM16K [30.Bushell S.R. et al.The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K.Nat. Commun. 2019; 10: 3956Crossref PubMed Scopus (53) Google Scholar] and TMEM16F [35.Feng S. et al.Cryo-EM studies of TMEM16F calcium-activated ion channel suggest features important for lipid scrambling.Cell Rep. 2019; 28: 567-579.e564Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar,36.Alvadia C. et al.Cryo-EM structures and functional characterization of the murine lipid scramblase TMEM16F.Elife. 2019; 8e44365Crossref PubMed Scopus (62) Google Scholar] lack a water-filled pore but possess a 'groove' that enables PS scrambling. The TMEM16A pore might have evolved from a partial closure of the scrambling groove. The vicinity of plasmalemmal lipids to the ion permeation pathway suggests that TMEM16A-mediated currents can be directly influenced by lipids, including phosphatidylinositol 4,5-bisphosphate (PIP2) [9.Le S.C. et al.Molecular basis of PIP2-dependent regulation of the Ca2+-activated chloride channel TMEM16A.Nat. Commun. 2019; 10: 3769Crossref PubMed Scopus (36) Google Scholar,37.Ta C.M. et al.Contrasting effects of phosphatidylinositol 4,5-bisphosphate on cloned TMEM16A and TMEM16B channels.Br. J. Pharmacol. 2017; 174: 2984-2999Crossref PubMed Scopus (30) Google Scholar, 38.Jia Z. Chen J. Specific PIP2 binding promotes calcium activation of TMEM16A chloride channels.Commun. Biol. 2021; 4: 259Crossref PubMed Scopus (8) Google Scholar, 39.Ko W. et al.Allosteric modulation of alternatively spliced Ca2+-activated Cl- channels TMEM16A by PI4,5P2 and CaMKII.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 30787-30798Crossref PubMed Scopus (9) Google Scholar] and omega-3 dietary fatty acids [40.Leon-Aparicio D. et al.Oleic acid blocks the calcium-activated chloride channel TMEM16A/ANO1.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2022; 1867159134PubMed Google Scholar].Overview of pharmacological modulators of the TMEM16A channelFor many years, the pharmacology of CaCCs has been limited to compounds with low potency and specificity. For example, the fenamate class of compounds, including niflumic acid, flufenamic acid, and mefenamic acid, are commonly used blockers that are effective at micromolar concentrations; other CaCC blockers, such as 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) and 4-Acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) are even less potent [41.Alexander S.P. et al.The concise guide to pharmacology 2021/22: ion channels.Br. J. Pharmacol. 2021; 178: S157-S245PubMed Google Scholar]. Since the cloning of the TMEM16A channel, several small-molecule modulators have been identified (reviewed in [42.Hao A. et al.Emerging modulators of TMEM16A and their therapeutic potential.J. Membr. Biol. 2021; 254: 353-365Crossref PubMed Scopus (2) Google Scholar, 43.Liu Y. et al.The Ca2+-activated chloride channel ANO1/TMEM16A: an emerging therapeutic target for epithelium-originated diseases?.Acta Pharm. Sin. B. 2021; 11: 1412-1433Crossref PubMed Scopus (5) Google Scholar, 44.Zhong J. et al.Advances in anoctamin 1: a potential new drug target in medicinal chemistry.Curr. Top. Med. Chem. 2021; 21: 1139-1155Crossref PubMed Scopus (2) Google Scholar]). These include synthetic inhibitors acting at submicromolar concentrations, such as N-[(4′-methoxy)-2′-naphthyl]-5-nitroanthranilic acid (MONNA) [45.Oh S.J. et al.MONNA, a potent and selective blocker for transmembrane protein with unknown function 16/anoctamin-1.Mol. Pharmacol. 2013; 84: 726-735Crossref PubMed Scopus (82) Google Scholar], 2-(4-chloro-2-methylphenoxy)-N-[(2-methoxyphenyl)methylideneamino]-acetamide (Ani9) [46.Seo Y. et al.Ani9, a novel potent small-molecule ANO1 inhibitor with negligible effect on ANO2.PLoS One. 2016; 11e0155771Crossref Scopus (90) Google Scholar], and 2-bromodifluoroacetylamino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid o-tolylamide (TMinh-23) [47.Cil O. et al.A small molecule inhibitor of the chloride channel TMEM16A blocks vascular smooth muscle contraction and lowers blood pressure in spontaneously hypertensive rats.Kidney Int. 2021; 100: 311-320Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar] (Figure 3).Figure 3Chemical structures of a range of TMEM16A modulators.Show full captionChemical structures of a range of TMEM16A modulators (synthetic or natural inhibitors, repurposed drugs, or synthetic activators) described in the main text. Created using ChemDraw and Microsoft Power Point. Abbreviations: Ani9, 2-(4-chloro-2-methylphenoxy)-N-[(2-methoxyphenyl)methylideneamino]-acetamide; Eact, 3,4,5-trimethoxy-N-(2-methoxyethyl)-N-(4-phenyl-2-thiazolyl)benzamide; Fact, N-(4-bromophenyl)-3-(1H-tetrazol-1-yl)benzamide; MONNA, N-[(4′-methoxy)-2′-naphthyl]-5-nitroanthranilic acid; TMinh-23, 2-bromodifluoroacetylamino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid o-tolylamide.View Large Image Figure ViewerDownload Hi-res image Download (PPT)MONNA was found not to interact with other anion channels such as CF transmembrane conductance regulator (CFTR), chloride channel-2 (ClC-2), and bestrophin-1 [45.Oh S.J. et al.MONNA, a potent and selective blocker for transmembrane protein with unknown function 16/anoctamin-1.Mol. Pharmacol. 2013; 84: 726-735Crossref PubMed Scopus (82) Google Scholar]. However, the possibility that MONNA modulates other, yet-to-be-identified targets was suggested, based on the observation that MONNA evoked comparable levels of relaxation of isolated rodent arteries – where the TMEM16A channel is abundantly expressed – in the presence of various extracellular Cl− concentrations [48.Boedtkjer D.M. et al.New selective inhibitors of calcium-activated chloride channels – T16A-A01, CaCC-A01, and MONNA – what do they inhibit?.Br. J. Pharmacol. 2015; 172: 4158-4172Crossref PubMed Scopus (0) Google Scholar]. Ani9 appeared to have high selectivity for TMEM16A, since it did not inhibit the closely related TMEM16B channel or other channels such as CFTR, the volume-regulated anion channel (VRAC), and epithelial Na+ channels (ENaCs) and did not interfere with intracellular Ca2+ signalling [46.Seo Y. et al.Ani9, a novel potent small-molecule ANO1 inhibitor with negligible effect on ANO2.PLoS One. 2016; 11e0155771Crossref Scopus (90) Google Scholar]. TMinh-23 is the most potent TMEM16A inhibitor identified thus far, with a concentration causing 50% inhibition (IC50) of ~30 nM for heterologous TMEM16A currents [47.Cil O. et al.A small molecule inhibitor of the chloride channel TMEM16A blocks vascular smooth muscle contraction and lowers blood pressure in spontaneously hypertensive rats.Kidney Int. 2021; 100: 311-320Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar,49.Truong E.C. et al.Substituted 2-acylaminocycloalkylthiophene-3-carboxylic acid arylamides as inhibitors of the calcium-activated chloride channel transmembrane protein 16A (TMEM16A).J. Med. Chem. 2017; 60: 4626-4635Crossref PubMed Scopus (25) Google Scholar]; however, a detailed selectivity profile for this compound was not reported.Drug repurposing screens have led to the identification of additional TMEM16A inhibitors such as cepharanthine (an anti-inflammatory drug) [50.Zhang X. et al.Cepharanthine, a novel selective ANO1 inhibitor with potential for lung adenocarcinoma therapy.Biochim. Biophys. Acta Mol. Cell Res. 2021; 1868119132Crossref Scopus (0) Google Scholar], zafirlukast (an anti-asthmatic drug) [51.Shi S. et al.Zafirlukast inhibits the growth of lung adenocarcinoma via inhibiting TMEM16A channel activity.J. Biol. Chem. 2022; 298101731Abstract Full Text Full Text PDF Scopus (0) Google Scholar], and niclosamide (an anthelminthic) [52.Cabrita I. et al.Niclosamide repurposed for the treatment of inflammatory airway disease.JCI Insight. 2019; 4e128414Crossref PubMed Scopus (32) Google Scholar,53.Miner K. et al.Drug repurposing: the anthelmintics niclosamide and nitazoxanide are potent TMEM16A antagonists that fully bronchodilate airways.Front. Pharmacol. 2019; 10: 51Crossref PubMed Scopus (55) Google Scholar] (Figure 3). These drugs, however, also act on other targets and inhibited the TMEM16A channel with relatively low potency, with the exception of niclosamide for which the IC50 for TMEM16A inhibition was ~0.1 μM at positive membrane potentials (Vm) but activated the current at negative Vm [52.Cabrita I. et al.Niclosamide repurposed for the treatment of inflammatory airway disease.JCI Insight. 2019; 4e128414Crossref PubMed Scopus (32) Google Scholar]. Thus, the net effect of niclosamide on TMEM16A channels in vivo will depend on the Vm and possibly on the free [Ca2+]i of the target cells. A range of natural compounds such as gallotannins [54.Namkung W. et al.Inhibition of Ca2+-activated Cl- channels by gallotannins as a possible molecular basis for health benefits of red wine and green tea.FASEB J. 2010; 24: 4178-4186Crossref PubMed Scopus (0) Google Scholar], evodiamine and rutaecarpine [55.Zhao Z. et al.Identification of evodiamine and rutecarpine as novel TMEM16A inhibitors and their inhibitory effects on peristalsis in isolated Guinea-pig ileum.Eur. J. Pharmacol. 2021; 908174340Crossref Scopus (0) Google Scholar], and theaflavin [14.Shi S. et al.Theaflavin binds to a druggable pocket of TMEM16A channel and inhibits lung adenocarcinoma cell viability.J. Biol. Chem. 2021; 297101016Abstract Full Text Full Text PDF Scopus (3) Google Scholar] are relatively low-potency TMEM16A inhibitors and have other reported targets (Figure 3).Small molecules that enhance TMEM16A activity include N-(4-bromophenyl)-3-(1H-tetrazol-1-yl)benzamide (Fact) (a potentiator) and 3,4,5-trimethoxy-N-(2-methoxyethyl)-N-(4-phenyl-2-thiazolyl)benzamide (Eact) (an activator) that enhance CaCC currents in airways EC lines [56.Namkung W. et al.Small-molecule activators of TMEM16A, a calcium-activated chloride channel, stimulate epithelial chloride secretion and intestinal contraction.FASEB J. 2011; 25: 4048-4062Crossref PubMed Scopus (144) Google Scholar] (Figure 3). Whether Eact acts directly on TMEM16A, however, has been questioned [57.Genovese M. et al.TRPV4 and purinergic receptor signalling pathways are separately linked in airway epithelia to CFTR and TMEM16A chloride channels.J. Physiol. 2019; 597: 5859-5878Crossref PubMed Scopus (24) Google Scholar,58.Liu S. et al.Eact, a small molecule activator of TMEM16A, activates TRPV1 and elicits pain- and itch-related behaviours.Br. J. Pharmacol. 2016; 173: 1208-1218Crossref PubMed Scopus (22) Google Scholar]. Mor