TMEM16A: Closing the circle

Raimund Dutzler and his lab have been the first group to solve the crystal structure of the mammalian mouse TMEM16A ion channel using cryo-electron microscopy (Paulino et al., 2017). This study is the first essential step to resolving the controversies which has raged in the ion channel community since the discovery of this protein responsible for the calcium activated chloride channel back in 2008 by 3 independent groups (Caputo et al; Schroeder et al; Yang et al., 2008). More recently Brunner et al. (2014) was the first to solve the crystal structure for the related fungal protein Nectria haematococca TMEM16 (nhTMEM16), a lipid scramblase.  Their hypothesis being that the TMEM16 protein family, consisting of ten genes (TMEM16A-k, missing out I) split from a common ancestral lipid scramblase and evolved in mammals to produce the only 2 known chloride ion channels, TMEM16A and TMEM16B, in the TMEM16 family. Up until this point functional studies had shown Ca2+ activation, voltage dependency and through mutagenesis which amino acids had an impact on chloride conductance in TMEM16A. Brunner et al’s X-ray structure, to a 2.6 Å resolution, gave rise to a number of profound questions about the chloride ion channels TMEM16A & B.

Their structure of the TMEM16 homodimer did not conform to ion channel dogma. Based on the homology model of the lipid scramblase X-ray structure for nhTMEM16A, Whitlock and Hartzel (2016) proposed that the calcium activated chloride ion conductance protein TMEM16A shared structural similarities to the lipid scramblases i.e. it was also an homodimer and each dimer had a subunit cavity or pore, but the chloride permeation channel was made up of half lipid, half protein and the Ca2+ binding site was located within the transmembrane domain of the subunit cavity of the protein. In June 2017 (TMEM16A: 2pores, or not 2 pores) I discussed 2 studies (Lim et al. 2016 & Jeng et al. 2016) using covalently linked mouse TMEM16A subunits with one of the two subunits carrying mutations, which confirmed that the TMEM16A homodimer had 2 independent chloride pores.

Raimund Dutzler’s lab are the first to report the structure of mammalian TMEM16A ion channel with cryo-electron microscopy of mouse TMEM16A at a resolution of 6.6Å (Paulino et al., 2017). They are proposing that the putative pore region of mTMEM16A is now an enclosed aqueous proteinaceous pore with a large intracellular vestibule that narrows to a chloride conducting pathway surrounded by alpha –helices (Figure 1). They show this is brought about by a realignment of helices, when compared to the nhTMEM16A X-ray structure, helices 4 and 6 move from the edges of the subunit cavity, in to enclose the aqueous proteinaceous pore and closing it off to membrane lipids (Figure 1). However, a drawback of this study is the resolution of the cyro-electron microscopy technique, as crucially it does not give the locations of amino acids side chains or the pitch of helices within the protein. We will await the increased resolution of an X-ray protein structure to confirm these important initial findings. Also, the protein was purified in the presence of high levels of Ca2+ and therefore could represent a non-conducting form of mTMEM16A.

Roy 1

Figure 1. Mechanistic relationships within TMEM16 family.

(A) Depiction of the mTMEM16A pore. The molecular surface of the pore region is shown as grey mesh. The boundaries of hydrophobic (black) and polar regions (grey) of the membrane are indicated by rectangular planes. The positions of positively charged residues affecting ion conduction are depicted as blue and bound Ca2+ ions as green spheres. Hypothetical Cl ions (radius 1.8 Å) placed along the pore are displayed as red spheres. (B) Schematic depiction of features distinguishing lipid scramblases (left) from ion channels (right) in the TMEM16 family. The view is from within the membrane (top panels) and from the outside (bottom panels). The helices constituting the membrane accessible polar cavity in scramblases have changed their location in channels to form a protein-enclosed conduit. A and B, Permeating ions and lipid headgroups are indicated in red. (Paulino et al. (2017) eLife;6: e26232).

They tested their hypothesis with functional studies, mutating basic amino acids for neutral Alanines in both the vestibule and within the pore region of the mTMEM16A to see what impact this had on chloride conductance. They found, as predicted, that altering the charge in the vestibule had little impact on chloride conductance whereas, altering the charge in the pore had a more pronounced effect.

The Brunner et al. (2014) X-ray structure and the homology models that arose from it, gave rise to the controversy in the literature, trying to reconcile ion channel dogma with the available TMEM16A functional data and the nhTMEM16 X-ray structure. Paulino et al. (2017) has with this essential first mTMEM16A structure, albeit with the cryo-electron microscopy technique at 6.6Å resolution which needs to be confirmed with the increased resolution of an X-ray structure, resolved our understanding of how chloride ions pass through TMEM16A. And in mTMEM16A anyway, we still await elucidation of the human structure, this has come with the realignment of helices 4 and 6 closing the circle of the subunit furrow to form an aqueous chloride conducting pore.

Blog written by Roy Fox


  1. Paulino et al. (2017) eLife; 6:e26232
  2. Caputo et al. (2008) Science 322:590–594.
  3. Schroeder (2008) Cell 134:1019–1029.
  4. Yang et al. (2008) Nature 455:1210–1215.
  5. Brunner et al. (2014) Nature 516:207–212.
  6. Whitlock JM and Hartzell HC (2016) Eur. J. Physiol. 468: 455-473.
  7.  Lim et al. (2016) J. Gen. Physiol. 148:5, 375-392.
  8.  Jeng et al. (2016) J. Gen. Physiol. 148:5, 393-404.

Rare Activating Mutations in the Calcium Sensing Receptor (CaSR), and Resultant Autosomal Dominant Hypocalcemia (ADH): Current and Future Pharmacological Intervention


Activating mutations of the G protein-coupled receptor, calcium-sensing receptor (CaSR), cause the rare and difficult to treat autosomal dominant hypocalcemia (ADH) and Bartter syndrome type 5 (BS5).

These mutations lower the set-point for extracellular Ca2+ sensing. This leads to increased parathyroid hormone (PTH) secretion. Normally, PTH stimulates Ca2+ reabsorption in the kidney and inhibits disposal, in addition to regulating skeletal Ca2+ release and uptake in bone. PTH effects are mediated via the type-1 PTH receptor (PTH1R) that couples to the small G protein Gs and activates cAMP signalling. PTH also stimulates the 1/-hydroxylation of vitamin D compounds in the kidney, and indirectly stimulates Ca2+ uptake in the gut. The effects of PTH and vitamin D in kidney, bone, and gut elevate serum Ca2+ levels and complete the classic regulatory feedback loop of the Ca2+ homeostasis. Patients with activating mutations have absent or reduced parathyroid hormone (PTH) owing to CaSR mediated hypocalcaemia and suppression of PTH secretion, and development of hypercalciuria. The mechanism of this regulation and the involvement of [Ca2+]]i , ERK1/2, or other signalling mechanisms is under investigation but is not completely understood. Nephrocalcinosis and nephrolithiasis, an increased risk of calcifications of the basal ganglia and cataract formation and osteoporosis are frequent findings 1.

Current Therapies

There are several established clinically effective therapeutic alternatives for the hypercalcemic disorders e.g. Familial Hypercalciuric Hypercalcaemia (FHH), as a result of inactivating mutations of the CaSR, however the hypocalcemic disorders are difficult to treat.

Vitamin D analogs and Ca2+ supplementation is standard treatment to raise serum Ca2+ levels in ADH patients. However, this inevitably aggravates the hypercalciuria which frequently limits attempts to normalize serum Ca2+. Several other treatments e.g. hydrochlorothiazide was found to temporarily reduce hypercalciuria, and replacement of PTH1–34 (teriparatide) was generally effective in reducing symptoms and/or raising serum Ca2+. Although urinary Ca2+ excretion declined or was constant despite rising serum Ca2+ levels in most patients, nephrocalcinosis can develop despite continuous PTH therapy. In ADH the kidney responds with increased Ca2+ excretion to an increased filtered Ca2+ load produced by any rise of serum Ca2+ levels. This most likely further elevates the risk of renal complications. The effects of CaSR predominate the effects of PTH on tubular reabsorption of Ca2+ in the kidney. For this reason, the activated CaSR in ADH and BS5 reduces the desired effects, and increases the adverse effects, of conventional therapeutic attempts 2.

At present then most patients remain symptomatic despite intensive therapy with all available strategies. At best, the available therapeutic options reduce symptoms without further increasing the elevated risk of tissue complications. Complete symptom relief and the reduction of long-term complications caused by the underlying disease itself are currently not achievable and none of the current therapeutic strategies corrects the underlying pathophysiology.

Future Pharmacological Intervention

There is a need to target the defect in CaSR to correct the underlying pathophysiology, and functional modulation of CaSR can be achieved by allosteric modulators. A positive allosteric CaSR modulator, the calcimimetic cinacalcet is presently used to treat FHH. Negative allosteric modulators or calcilytics directly inhibit CaSR and have been developed to stimulate endogenous PTH secretion, as an alternative to injections, to promote bone formation in osteoporosis. These drugs may be useful in reducing excessive CaSR activity in ADH and BS5 patients3.

Negative Allosteric Modulators in vitro and in rodents

To date only wild-type receptors or enzymes have been studied in vitro. To pharmacologically correct the molecular cause of ADH and BS5, the function of altered CaSR or G protein/11 (GNA11), will need to be rectified and the efficacy of calcilytics needs to be established. Over 70 activating CaSR and GNA11 mutations that cause ADH or BS5 have been described and functional in vitro tests with calcilytics have been reported for 32 activating CaSR and two activating GNA11 point mutants, and all were sensitive to at least one calcilytic in vitro. Four of these mutants (A840 V, Q245R, E228K, and E228A) were also sensitive in vivo4.

Studies to date have used amino-alcohol compounds Ronacaleret (SB-751689); NPSP795 (SB- 423562); SB-423557 (prodrug of NPSP795); MK-5442 (JTT-305) and finally the quinazolinone AXT914.

The in vivo effects of calcilytics have been studied in CaSR C129S and A843E knock-in mouse models. These models mimic the human ADH phenotype, with decreased serum Ca2+ and PTH, and increased serum phosphate levels. These mice also showed hypercalciuria and reduced urinary cAMP excretion. MK-5442 or NPS 2143 increased serum Ca2+ and PTH but stabilized or even decreased urinary Ca2+ excretion. MK-5442 also increased urinary cAMP excretion, and administration for 3 months reduced Ca2+ excretion and prevented renal calcification. By contrast, long-term subcutaneous PTH injections also increased serum Ca2+ but failed to reduce Ca2+ excretion and did not prevent renal calcification 4,5.

Negative Allosteric Modulators in Clinical Trial

In man, phase I and II clinical trials with calcilytics with a particularly short serum half-life have been developed to stimulate endogenous PTH secretion, as an oral alternative to PTH injections, to enhance bone formation in osteoporosis in postmenopausal women. Serum Ca2+ levels were normal at baseline in these participants and therefore they were presumed to have wild-type CaSR. Serum Ca2+, phosphate, and PTH levels were determined, as well as renal Ca2+ excretion 6.

All drugs were administered orally once daily, with the exception of NPSP795, which was given intravenously. These trials for were relatively unsuccessful for osteoporosis and no alteration in bone mineral density was appreciated. However, a dose-dependent increase in serum Ca2+ levels was consistently observed. Calcilytics are so far generally well tolerated with most common adverse effects being mild i.e. fatigue, headache, constipation, diarrhea, nausea, and dyspepsia.

These studies performed in wild-type CaSR patients provide valuable insight into the possible therapeutic use in ADH patients.

Calcilytics in ADH patients

A small Phase II trial with the calcilytic NPSP795 for the treatment of five ADH patients harbouring the A840 V, Q245R, E228K, and E228A mutations have been reported recently. Here, serum Ca2+ levels were maintained despite fasting and no Ca2+ or vitamin D supplementation with one participant developing hypercalcemia 7. No clinical trials have been performed in patients with BS5.

All calcilytic trials discussed reported a short-lived dose-dependent increases in PTH and Ca2+, followed by a return to baseline 6–12 h after administration. These results are in line with the rapid pharmacokinetic profiles of ronacaleret 6 and AXT914 (tmax 1–2 h, t½ 4–5 h) 8 and NPSP795 (t½ < 1h after i.v. administration, tmax 2-3h, t½ 1.4-3.7h after oral administration of the prodrug SB423557).

The results of these clinical trials are promising. Considering that different calcilytics from different chemical classes (amino-alcohol and quinazolinones) with different modes of action exert similar biochemical effects, implies that these effects are specific to calcilytics, and are not a single compound or chemical class effect, broadening the opportunity for calcilytic development. Taken together, there is still an unmet need for ADH patients and the repurposing of calcilytics for in this population is a promising approach for these difficult to treat diseases.

Blog written by Elizabeth Owen

  1. Mayr, B.M. et al. (2015) Genetics in endocrinology: gain and loss of function mutations of the calcium sensing receptor and associated proteins: current treatment concepts. Eur. J. Endocrinol. 174, R189–R208
  2. Mitchell, D.M. et al. (2012) Long-term follow-up of patients with hypoparathyroidism. J. Clin. Endocrinol. Metab. 97, 4507–4514
  3. Hu, J. and Spiegel, A.M. (2007) Structure and function of the human calcium-sensing receptor: insights from natural and engineered mutations and allosteric modulators. J. Cell. Mol. Med. 11, 908–922
  4. Dong, B. et al. (2015) Calcilytic ameliorates abnormalities of mutant calcium-sensing receptor (CaSR) knock-in mice mimicking autosomal dominant hypocalcemia (ADH). J. Bone Miner. Res. 30, 1980–1993
  5. Hannan, F.M. et al. (2015) The calcilytic agent NPS 2143 rectifies hypocalcemia in a mouse model with an activating calcium-sensing receptor (CaSR) mutation: relevance to autosomal dominant hypocalcemia type 1 (ADH1). Endocrinology 156, 3114–3121
  6. Caltabiano, S. et al. (2013) Characterization of the effect of chronic administration of a calcium-sensing receptor antagonist, ronacaleret, on renal calcium excretion and serum calcium in postmeno- pausal women. Bone 56, 154–162
  7. Ramnitz, M. et al. (2015) Treatment of autosomal dominant hypocalcemia with the calcilytic NPSP795. J. Bone Miner. Res. 30 (Suppl. 1), SA0002
  8. John, M.R. et al. (2014) AXT914 a novel, orally-active parathyroid hormone-releasing drug in two early studies of healthy volunteers and postmenopausal women. Bone 64C, 204–210



Breaking Convention with Sulfoximines – from Lab Oddity to Clinical Candidate

In 2013 a bold decision taken by Lücking et al[1] to incorporate a sulfoximine group into a lead series overcame all of the fundamental issues of the project and lead to the discovery of BAY 1000394 as a clinical CDK inhibitor (Fig 1) – who would have thought it?

The sulfoximine strategy was just one of the concepts that the team from Bayer explored to remove carbonic anhydrase (CA) activity whilst maintaining CDK activity. Fortuitously, the inclusion of the sulfoximine moiety (BAY 1000394) abolished CA activity completely and also improved many of the pharmacokinetic properties of the lead (Fig 1).

Scott 1

Fig 1: Initial HTS hit (1); para-sulfonamide (2); clinical pan-CDK inhibitors ZK304709 and BAY 10003941

Perhaps Lücking et al could have been forgiven for not pursuing sulfoximine approach – when reading this paper I’ve got to ask myself a) “would I have thought of that?”; b) “would I have gone to the trouble of making the sulfoximine and how would I have made it?”; c) “why would I make the sulfoximine?”; d) “are there any sulfoximines on the market today?”………..

  1. “Would I have thought of that?”

Short answer – No.

Long answer – I don’t think I had ever heard of a sulfoximine prior to reading this paper.  If somebody had drawn the structure on my fumehood perhaps I would have been sceptical about the functional group being drug-like.  Reading into the subject, it seems that sulfoximines were discovered recently (in terms of functional groups)[2] and that sulfoximine chemistry has been carried out by a handful of research groups, making it quite a niche area.  Traditional applications of sulfoximines have been centred on their use as chiral auxiliaries and ligands for asymmetric catalysis.2 So maybe it’s not too surprising that I would not have thought of that.

  1. “Would I have gone to the trouble of making the sulfoximine and how would I make it?”

OK, let’s assume I said yes to a).

Sulfoximines are not commercially available so ‘analogue bashing’ might be slow as this is unlikely to be a one-stepper! As sulfoximine chemistry has been (until recently) a niche area, synthetic methods have been limited and have various safety concerns.  One method that forms in-situ hydrazoic acid from the reaction of sodium azide, diphenyl sulfoxide and polyphosphoric acid has triggered an explosion in the past.[3]  If this was the only method then maybe I would not have bothered to make the sulfoximine.

Luckily over the last decade new and safer synthetic strategies have been established and are outlined in a recent paper by Tota et al who also report the first direct synthesis of NH-sulfoximines from sulfides[4] (Fig 2).  Work like this has helped to make sulfoximines more synthetically tractable.

Scott 2

Fig 2. Strategies and conditions for preparation of sulfoximines from sulfides4

  1. “Why would I make the sulfoximine analogue?”

Fortunately, Lücking has composed a mini-review on sulfoximines[5] and summarises nicely the chemical properties and versatility of the sulfoximine functional group (Fig 3).  The sulfoximine group is asymmetric at sulfur, stable, small, hydrophilic and offers another point of diversification at the nitrogen when compared to the sulfone analogue.5 BAY 1000394 exemplifies why the sulfoximine anlalogue should be made in cases where the sulfonamide analogue is active but requires further iteration to improve PK properties and off-target profile.

Scott 4

Fig 3. Sulfoximines and their chemical versitility5

  1. “Are there any sulfoximine drugs on the market today?

Short answer: No. However, in 2013 the EPA approved the first commercially available insecticide containing the sulfoximine moeity, Sulfoxaflor, to Dow Chemical Corporation.5

Scott 5

Fig 4. Dow Chemical Corporation’s Sulfoxaflor5

Long answer: Although there are no sulfoximine drugs on the market at the moment, at least three have reached the clinic.  All are kinase inhibitors for the treatment of cancer (Fig 5).5 BAY1143572 (Atuveciclib) is the first highly selective inhibitor of CDK9 to reach the clinic.  Guess what – it is another recent contribution by Lücking and co.[6]

Scott 6

Fig 5. Clinical sulfoximinesfor the treatment of cancer5

Recently, Sirvent and Lücking have incorporated the sulfoximine group into a range of marketed drugs with promising results in terms of improving PK properties whilst maintaining activity.2 Studies such as those outlined above are helping to raise the profile of sulfoximine chemistry, further developments in synthetic methodology and an increase in commercial sulfoximine building blocks would accelerate the likelihood of seeing the sulfoximine group in a drug molecule.2 Watch this space.

Blog written by Scott Henderson

[1] U. Lücking et al.  ChemMedChem 2013, 8, 1067

[2] J.A Sirvent and U. Lücking;  ChemMedChem 2017, 12, 487

[3] G. Satzinger, P. Stoss, Arzneim.-Forsch. 1970, 20, 1214

[4] A. Tota et al. Chem. Commun., 2017, 53, 348

[5] U. Lücking. Angew. Chem. Int. Ed. 2013, 52, 9399

[6] U. Lücking et al.  ChemMedChem 2017, 12, 1776.


Silent but deadly: Understanding the importance of ‘silent’ mutations in the Cystic Fibrosis gene

Cystic fibrosis results from the mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene1. This mutation causes decreased chloride ion transport across the surface epithelia and dehydration of the airway surface liquid leading to the accumulation of thick viscous mucus which can trap pathogens setting up chronic bacterial infections.

More than 2,000 mutations in the CFTR gene have been identified1 – approximately 1,700 of which cause cystic fibrosis. An individual will inherit CF if both parents carry a mutation in the CFTR gene. These two faults may be the same or they could consist of two different mutations. The severity of disease varies between individuals and is partly due to how the inherited mutations affect the manufacture of the CFTR protein and how well it works within the cell.

The remaining 270 mutations identified include synonymous single nucleotide polymorphisms (sSNPs) or ‘silent’ mutations2. Due to the degenerative nature of the genetic code – i.e. more than one codon specifies each amino acid, sSNPs were considered to have no effect on the folding and therefore function of the protein produced. It should be noted that sSNPs do not cause disease by themselves, but are more common in patients with the most severe disease. Genome-wide association studies have linked sSNPs with over 50 human diseases so how can such ‘silent’ mutations be having such a detrimental effect?

This question was addressed by several teams, one led by Professor Igatova from the University of Hamburg and Professor Sheppard at the University of Bristol with help from colleagues in the Netherlands and the USA3.

The group at the University of Hamburg identified an sSNP, T2562G, which modifies the local translation speed of the CFTR, leading to detrimental alterations in the protein folding and function3. T2562G is one of the most common SNPs in the CFTR gene, with a prevalence of 34% in the general population4. This sSNP does not cause CF by itself but is it commonly found in patients with CFTR-related disorders5.

Professor Sheppard’s group at the University of Bristol showed that this inaccurate folding caused a narrowing in the final CFTR chloride ion channel which slowed the movement of the ions through the cell membrane3.

T2562G was found to introduce a codon pairing to a low-abundance tRNA which is rare in human bronchial epithelia but interestingly, not in other human tissues. The low abundance of the tRNA coded for by T2562G resulted in a far slower translocation speed at the Thr854 codon leading to vital changes in CFTR stability and function (Fig. 1B). The folding and function of T2562G-CFTR could be rescued by increasing the cellular concentration of the tRNA cognate to the mutant ACG codon (Fig. 1C).

This work illustrates how the function of the CFTR can be influenced by mutations in the CFTR gene that are not in themselves CF-causing. When these ‘silent’ mutations occur in conjunction with CF-causing mutations they can greatly alter the severity of disease. Understanding the effects of these silent changes on protein folding and function will help to understand the root cause of disease and perhaps ultimately find new treatments.

HollyFig. 1

The synonymous single nucleotide polymorphism (sSNP) T2562G inverts local translation speed in CFTR mRNA, which can be rescued by tRNAThr(CGU).

(A) Thr-854–encoding codon ACT in wild-type CFTR is translated fast, as its cognate tRNAThr(AGU) is relatively abundant. (B) T2562G sSNP converts the ACT triplet to ACG codon, which is read by the rare cognate tRNAThr(CGU) and reduces local ribosomal speed. Stochasticity in the delivery of tRNAThr(CGU) cognate to the ACG codon creates variations in the intimate translation speed of each ACG codon at this position and hence generates 2 distinct CFTR channel populations, one with wild-type–like (wtl) CFTR properties and a second with a reduced conductance and a more compact structure (small-conductance [sc] population). (C) Increase of the cellular level of tRNAThr(CGU) pairing to the mutated ACG codon restores ribosome speed at the rare Thr-ACG codon and rescues the CFTR conductance defect.

Blog written by Holly Charlton


  1. Cutting GR. Cystic fibrosis genetics: from molecular understanding to clinical application. Nat Rev Genet. 2015;16: 45–56.
  2. Veit G, Avramescu RG, Chiang AN, Houck SA, Cai Z, Peters KW, et al. From CFTR biology toward combinatorial pharmacotherapy: expanded classification of cystic fibrosis mutations. Mol Biol Cell. 2016;27: 424–33.
  3.  Kirchner S, Cai Z, Rauscher R, Kastelic N, Anding M, Czech A, Kleizen B, Ostedgaard LS, Braakman I, Sheppard DN, Ignatova Z. Alteration of protein function by a silent polymorphism linked to tRNA abundance. PLoS Biol. 2017 May 16;15(5):e2000779.
  4. Cuppens H, Marynen P, De Boeck C, Cassiman JJ. Detection of 98.5% of the mutations in 200 Belgian cystic fibrosis alleles by reverse dot-blot and sequencing of the complete coding region and exon/intron junctions of the CFTR gene. Genomics. 1993;18: 693–7.
  5. Steiner B, Truninger K, Sanz J, Schaller A, Gallati S. The role of common single-nucleotide polymorphisms on exon 9 and exon 12 skipping in non-mutated CFTR alleles. Human Mutat. 2004;24: 120–9.