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Activating alternative chloride channels to treat CF: Friends or Foes?

Report on the Meeting of the Basic Science Working Group in Dubrovnik, Croatia
Open ArchivePublished:October 29, 2019DOI:https://doi.org/10.1016/j.jcf.2019.10.005

      Highlights

      • Report of the 2019 ECFS Basic Science Working Group Meeting.
      • Topic ``Activating Alternative Chloride Channels to Treat CF: Friends or Foes?''

      Abstract

      The European Cystic Fibrosis Society (ECFS) Basic Science Working Group (BSWG) organized a session on the topic “Activating Alternative Chloride Channels to Treat CF: Friends or Foes?”, within the 16th ECFS Basic Science Conference gathering ∼200 researchers from CF Basic Science. The session was organized into two round tables, each chaired by Margarida Amaral (BioISI, University of Lisboa, Portugal) and Jeffrey Beekman (University Medical Centre Utrecht, Netherlands) as Chair and Vice-Chair of the BSWG, respectively. The purpose of this session was to bring attention of participants of the ECFS Basic Science Conference to alternative chloride channels as important therapeutic targets for CF which may treat all individuals with CF, independently of their CFTR genotype. The session had various speakers who had presented abstracts to the conference on this topic and discussants with different views. Here we try to convey the presentations as well as ideas that emerged during the discussion.

      Keywords

      1. Introduction

      The BSWG organized a session within the 16th ECFS Basic Science Conference in Dubrovnik, Croatia, 27–30 March 2019 on the topic “Activating Alternative Chloride Channels to Treat CF: Friends or Foes?”, which took place on Thursday, 28 March (18:00–19:45).
      The purpose of this session, which counted with ∼200 researchers who participated in this annual conference, was to bring attention of participants in the ECFS BS Conference to chloride channels as good candidates for drug discovery in CF in the short/medium term, namely for individuals with CF who are not eligible for the approved CFTR modulator drugs, but in fact for any individual with CF, irrespective of the CFTR genotype.
      The session comprised two roundtables, the first on the topic “Can we regulate TMEM16A independently of Ca2+? And TMEM16A /SLC26A9 independently of CFTR?” and it was chaired by Jeff Beekmann (NL) and co-chaired by Karl Kunzelmann (DE). The second roundtable which focussed on “TMEM16A and mucus”, was chaired by Margarida Amaral (PT) and co-chaired by Luis Galietta (IT).
      Abstracts submitted to the conference on these two topics (3 on the first topic and 4 on the second) were presented as very short talks (3 min each) to trigger discussion. Both round tables had a panel of invited discussants, selected among the invited speakers and participants in the Conference: 7 on the first topic and 4 on the second.

      2. Can we regulate TMEM16A independently of Ca2+? and TMEM16A /SLC26A9 independently of CFTR?

      There was an introduction to the topic describing “Facts about TMEM16A/ANO1 by Karl Kunzelmann (DE) whose research in recent years has focussed on the regulation of proteins in the TMEM16 family, namely TMEM16A (anoctamin 1, ANO1).
      The first presentation by Henry Danahay (UK) addressed the question “Can we regulate TMEM16A independently of Ca2+?” (Abstract 32) and data was presented that support the argument that it is possible to regulate TMEM16A function independently of Ca2+. Compounds have been identified which act as TMEM16A potentiators which activate channel activity independently of a change in Ca2+. These same potentiators were shown to enhance UTP-stimulated anion secretion in primary cultures of CF human bronchial epithelial (HBE) cells grown at air-liquid interface (ALI) and again with no effect on intra-cellular Ca2+ levels. Data were also presented on the pharmacology of Eact, a N-aroylaminothiazole compound previously proposed to be a TMEM16A activator [
      • Namkung W.
      • Yao Z.
      • Finkbeiner W.E.
      • Verkman A.S
      Small-molecule activators of TMEM16A, a calcium-activated chloride channel, stimulate epithelial chloride secretion and intestinal contraction.
      ]. Under conditions where intra-cellular Ca2+ levels were tightly buffered, there was no evidence of TMEM16A activation by Eact. In contrast, when Ca2+ was not buffered, Eact induced an increase in intra-cellular Ca2+ levels in both FRT-TMEM16A cells and in primary CF-HBE (ALI) cells. These data suggest that Eact is not a TMEM16A activator but rather an inducer of intra-cellular Ca2+, thus activating TMEM16A via the Ca2+.
      The second short talk was by Roberta Benedetto (DE) and focussed on “Niclosamide repurposed for the treatment of inflammatory airway disease” (Abstract 111) and raised the possibility that TMEM16A inhibition, rather than activation, is needed to decrease mucus in CF airways [
      • Benedetto R.
      • Cabrita I.
      • Schreiber R.
      • Kunzelmann K
      TMEM16A is indispensable for basal mucus secretion in airways and intestine.
      ]. The role of TMEM16A in asthma was exploited based on the observation that asthma-induced TMEM16A+/+ mice exhibit concomitant enhanced expression of TMEM16A and mucins in metaplastic club cells and airway smooth muscle, suggesting that this channel contributes to mucus hypersecretion and bronchoconstriction. Mucus accumulation was not observed for TMEM16A−/− mice under the same asthma-inducing conditions. Data presented also showed that treatment with the non-specific inhibitor of TMEM16 proteins niflumic acid (NFA), led to a decrease in mucus production and bronchoconstriction. Similar results were shown for TMEM16A+/+ asthmatic mice, but not with the anthelminthic drug niclosamide (also a potent inhibitor of TMEM16A and TMEM16F proteins). These data suggested that the FDA-approved drug niclosamide could potentially be used for the treatment of asthma, and possibly also CF.
      The third and last short talk on this round table was by Madalena Pinto (PT) and dealt with the “Identification of novel ANO1/TMEM16A regulators as alternative therapeutic targets for Cystic Fibrosis” (Abstract 34). The use of an assay that monitors TMEM16A traffic was described in a high-throughput siRNA screen using a library of siRNAs targeting 691 genes previously identified as CFTR regulators or CFTR-interacting proteins (Amaral lab, unpublished data). Primary screen data revealed 179 genes regulating TMEM16A PM traffic and these were involved in diverse pathways such as regulation of transcription, G-protein coupled receptor (GPCR) signalling, phosphorylation, inflammatory response, and proliferation. A secondary screen using the halide-sensitive (HS)-YFP assay in HEK cells identified among those 179 hits 30 that modulate the ATP-induced YFP-quenching, either positively or negatively. Among these 30 genes, 8 were further selected for functional validation through whole-cell patch-clamp, based on their biological functions and 2 GPCRs were found to be the most potent TMEM16A regulators. These results will possibly reveal alternative drug targets to compensate for the absence of CFTR function from which all individuals with CF could benefit, independently of their genotype.
      Following these 3 presentations there was discussion of the topic. To this end, there were several discussants invited among ECFS Conference participants who presented on this topic at other Conference sessions, namely: Raimund Dutzler (CH); Yukiko Sato (CA); Nicoletta Pedemonte (IT); Martin Gosling (UK); Renaud Beauwens (BE); Michele Genovese (IT); John Hanrahan (CA).
      Throughout, there was very little discussion on SLC26A9. Indeed, there was presentation on this round table focused on it and consequently the lively discussion that followed was dominated by TMEM16A/ANO1, likely because of the lack of consensus on this topic among discussants. Not all of the discussion that took place can be captured in this short review.Nevertheless, at the end of it, the audience was called to participate by electronic voting (cell phone based), according to their opinion to give their opinion to a number of key questions which are listed in Table 1.
      Table 1Replies to the electronic poll carried out at Roundtable (average n of participants =36 (Max = 55; Min=19).
      Regarding the question “What would be the expected result of inhibiting TMEM16A in the airways?”, the audience was completely divided in their responses, with no more than 5% on a single reply. These varied from “Reduction in ASL fluid”; “Reduction of mucus secretion and reduce chloride secretion”; “Thickening of mucus” to “Maybe bronchodilation”; “Less fluid secreted”; “Less mucus” but also “No drastic effect under normal conditions”.

      3. TMEM16A and Mucus?

      Some studies have described that TMEM16A/Ano1 is predominantly expressed in goblet (mucus producing) cells [
      • Sondo E.
      • Caci E.
      • Galietta L.J.V
      The tmem16a chloride channel as an alternative therapeutic target in cystic fibrosis.
      ,
      • Kondo M.
      • Tsuji M.
      • Hara K.
      • Arimura K.
      • Yagi O.
      • Tagaya E.
      • et al.
      Chloride ion transport and overexpression of TMEM16A in a guinea-pig asthma model.
      ] and that its overexpression under IL-4/IL-13 results from airway goblet cell metaplasia [
      • Benedetto R.
      • Cabrita I.
      • Schreiber R.
      • Kunzelmann K
      TMEM16A is indispensable for basal mucus secretion in airways and intestine.
      ,
      • Scudieri P.
      • Caci E.
      • Bruno S.
      • Ferrera L.
      • Schiavon M.
      • Sondo E.
      • et al.
      Association of TMEM16A chloride channel overexpression with airway goblet cell metaplasia.
      ]. Thus the key question is whether TMEM16A plays a role in mucus production and consequently its activation might increase mucus production, with very negative consequences for individuals with CF. Nevertheless, other studies have described its expression also on ciliated cells [
      • Lérias J.R.
      • Pinto M.C.
      • Botelho H.M.
      • Awatade N.T.
      • Quaresma M.C.
      • Silva I.A.L.
      • et al.
      A novel microscopy-based assay identifies extended synaptotagmin-1 (ESYT1) as a positive regulator of anoctamin 1 traffic.
      ] and indeed a conditional TMEM16A−/− knock-out mouse was generated using the Foxj1 promoter, which is specifically inactivating this channel in ciliated cells [
      • Benedetto R.
      • Cabrita I.
      • Schreiber R.
      • Kunzelmann K
      TMEM16A is indispensable for basal mucus secretion in airways and intestine.
      ]. Since this mouse has a phenotype, namely accumulation of mucus in airway cells [
      • Benedetto R.
      • Cabrita I.
      • Schreiber R.
      • Kunzelmann K
      TMEM16A is indispensable for basal mucus secretion in airways and intestine.
      ], it can be concluded that TMEM16A/Ano1 expression clearly occurs also in ciliated cells.
      This round table counted with 4 short talks, again selected among the submitted abstracts to the conference on this topic.
      The first short talk was again by Henry Danahay (UK) and focussed on “TMEM16A channel function does not influence goblet cell numbers in the human airway epithelium” (Abstract 10). Data using quantitative immunohistochemistry were shown for both positive and negative regulators of TMEM16A channel function to test the hypothesis that potentiating/inhibiting TMEM16A channel activity in cultured HBE (ALI) cultures can modify goblet cell formation (as suggested in the published literature). Data presented did not show any changes in airway goblet cell formation (i.e. numbers of goblet cells) by either increasing or decreasing TMEM16A channel function. In parallel, IL-13 treatment was shown to cause a significant increase in goblet cells, but this increase was unaffected by inhibition of TMEM16A channel function. In parallel, 24 h incubation of CF-HBE (ALI) with TMEM16A potentiator compounds did not increase the secretion of mucins (either MUC5AC or MUC5B), thus suggesting that TMEM16A channel function does not regulate mucin secretion/ exocytosis in airway goblet cells.
      In the second short talk, Filipa Simões (PT) presented data indicating that “Cell proliferation upregulates TMEM16A in mucus cell hyperplasia” (Abstract 52). The aim of this study was to assess how epithelial differentiation influences ion transport and mucus production during differentiation and inflammation. Levels of TMEM16A and mucin MUC5AC were determined during differentiation of human airway Basal Cell Immortalized Non-Smoker (BCi-NS1.1) cell line [
      • Walters M.S.
      • Gomi K.
      • Ashbridge B.
      • Moore M.A.S.
      • Arbelaez V.
      • Heldrich J.
      • et al.
      Generation of a human airway epithelium derived basal cell line with multipotent differentiation capacity.
      ] and an inverse correlation was found: while TMEM16A is almost absent in differentiated cells, MUC5AC is abundantly expressed. Moreover, the upregulation of TMEM16A induced by IL-4 occurred with a concomitant increase in cell proliferation (assessed by Ki-67) and this TMEM16A induction was inhibited by the proliferation blocker mitomycin C. Altogether, data presented were suggestive of TMEM16A upregulation upon pro-inflammatory cytokines being specific to mucus cell hyperplasia (i.e., proliferation), but not metaplasia. Moreover, MUC5AC induction appears to occur independently of TMEM16A expression.
      Since it has been demonstrated that normal mucus formation requires both cAMP-dependent HCO3 secretion and Ca2+-mediated mucin exocytosis [
      • Yang N.
      • Garcia M.A.S.
      • Quinton P.M
      Normal mucus formation requires cAMP-dependent HCO3- secretion and Ca2+-mediated mucin exocytosis.
      ], understanding the putative role of Ca2+ in mucus is an important topic in CF. Accordingly, Dalia Fakih (SE) gave the third short talk which addressed how “The attached stratified mucus in obstructive airway disease is detached by calcium removal” (Abstract 81). Dalia first described previous work showing that Ca2+ removal is required for normal mucus unfolding and expansion of the mucin polymers [
      • Ermund A.
      • Recktenwald C V.
      • Skjåk-Braek G.
      • Meiss L.N.
      • Onsøyen E.
      • Rye P.D.
      • et al.
      OligoG CF-5/20 normalizes cystic fibrosis mucus by chelating calcium.
      ], so as to facilitate its clearance. This was the rationale to design potential mucus-detaching drugs based on a novel Ca2+-chelating peptide to treat the elastase-exposed airways. Data was presented showing that this Ca2+-chelating peptide was effectively able to detach the mucus layer and significant reduction of airway obstruction. Common osmotic agents (hypertonic saline or mannitol) which were used as controls, while still decreasing airway mucus obstruction, failed to detach the attached mucus layer.
      The fourth and final short talk by Arianna Venturini (IT) was focussed on the “Physiological role and therapeutic importance of TMEM16A chloride channel in the airway epithelium” (Abstract 31). Previously published data had shown that modification of TMEM16A carboxy-terminus results in channels that are active even at low intracellular Ca2+ concentrations [
      • Scudieri P.
      • Musante I.
      • Gianotti A.
      • Moran O.
      • Galietta L.J.V
      Intermolecular interactions in the TMEM16A dimer controlling channel activity.
      ]. So the goal was to express a genetically-activated version of TMEM16A (GA-TMEM16A) which included such C-terminus modification in CF airway epithelial cells (using a lentiviral vector) to understand if this would result in correction of CF abnormal ion transport. As a control, a genetically-inactivated version of TMEM16A (GITMEM16A) was used. Short-circuit current recordings of the cell lines expressing these variants found that the activity of GA-TMEM16A was quite persistent after stimulation with low concentration of Ca2+-elevating agents in contrast to the wt-TMEM16A channel which typically exhibits only transient activity and GI-TMEM16A had no function. Epithelia expressing GA-TMEM16A also evidenced increased ASL height in comparison to wt-TMEM16A or control CF epithelia.
      Following these 4 short presentations there was again extensive discussion of this topic. To this end, additional discussants were invited among ECFS Conference participants who presented on this topic at other Conference sessions, namely: Mike Gray (UK); Pamela Millar-Büchner (DE); and Martial Delion (BE).
      Again, after the extensive discussion, the audience was called to participate by electronic voting (cell phone based), according to their opinion to give their opinion to a number of key questions which are listed in Table 2.
      Table 2Replies to the electronic poll carried out at Roundtable (average n of participants = 36 (Max = 55; Min = 19).

      4. Concluding remarks

      Pharmacological modulation of alternative chloride channels may offer benefits to CF patients, and TMEM16A, a calcium-activated chloride channel, is generally considered as one of the more promising drug targets in this area. High consensus amongst participants was observed for the ability to modulate TMEM16A activity. Modulation of TMEM16A activity may involve compounds that act as channel potentiators that do not raise intracellular calcium, which would thus avoid potential calcium-dependent side effects. Additionally, modulating plasma membrane trafficking of TMEM16A may also represent a way to modulate TMEM16A activity.
      The consensus was lower on the physiological role of TMEM16A in human airway disease. Consensus was observed at the level of TMEM16A function in human in vitro cell models. These models support that TMEM16A activity would enhance airway ion and fluid secretion, and thus that activation may compensate for the absence CFTR ion transport and thus be beneficial for CF lung disease. However, the function of TMEM16A in the airways may be more complex as data also suggest additional functions in e.g. cytokine-induced airway cell proliferation. In contrast with a beneficial effect of TMEM16A activation, observations from TMEM16A knock-out mice and asthma-like models suggest that TMEM16A may actually increase mucus obstruction and bronchoconstriction. Should such observations be relevant for humans, modulators of TMEM16A could potentially further aggravate CF lung disease.
      In conclusion, whereas modulation of TMEM16A without raising intracellular calcium appears feasible, the biological function of TMEM16A and other alternative chloride channels in human airways still remains unclear. Opposing views on TMEM16A may result from the use of different model systems and the lack of selective tool compounds to activate or inhibit specific ion channels. Studies with advanced in vitro models namely primary human gene-edited airway epithelial cells and potentially also non-epithelial cells, as well as animal models that more closely resemble human airways as compared to mouse are needed. These may play a decisive role in defining the role of these channels, to define mode-of-action of pharmacological modulators and to predict the in vivo effects of modulators in human airway disease.

      Declaration of Competing Interest

      The authors have no conflict of interest related to this manuscript.

      Acknowledgments

      The authors would like to thank all participants and discussants in this session and the audience at the Basic Science conference for participating in this session for making it so lively as well as Bertrand Kleizen for making the electronic poll possible. Work in MDA lab is supported by UID/MULTI/04046/2013 centre grant from FCT, Portugal (to BioISI) and “DIFFTARGET” grant (FCT/POCTI-PTDC/BIM-MEC/2131/2014). The authors are also grateful to Simão Luz for the analysis of responses to electronic polls

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