2,3-Substituted Indole synthesis by Regioselective Electrophilic Trapping


The selective synthesis of indoles is an important area of organic chemistry, largely due to their prevalence in countless natural products, medicines, materials etc. Numerous methods have been described for the synthesis of substituted indoles, with many requiring the pre-instillation of functional groups by incorporating them within the initial starting material. Late stage functionalisation is also possible, but in order for this to be achieved additional steps are often required, with some involving harsh conditions or the use of expensive transition metal catalysts. With this in mind, the report described by Eiichi Nakamura from the University of Tokyo could be of interest to people wishing to make a small library of substituted indoles from one common building block.1

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Scheme 1

Within this communication, Nakamura describes the synthesis of indoles via dimetalated intermediates – although the synthesis via 2,3-dimetalloindoles has previously been reported, the selective introduction of electrophiles has, to the best of my knowledge, not previously been achieved. The problem of obtaining such selectively is shown in Scheme 2 – the dimetalated intermediates A and B react without selectivity, and so a mixture of isomers are obtained. In comparison to other dimetalated indoles,2 the Authors show that dizincioindoles are stable and relatively easily formed, suggesting a dimeric structure (shown in Figure 1) to rationalise this stability.

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Scheme 2

 

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Figure 1

The Authors show that when using of ZnCl2, electrophiles can be introduced at C-3 when simple electrophiles are used, but this can be switched C-2 when transition metals are introduced. When ZnPhBr is used, reactivity is generally more selective for C-3, unless a stannane is used, with differing reactivity probably due to a change in mechanism from simple nucleophilic reactions to that occurring through charge transfer (please see reference 1 for detailed substrate scope).

Although the above chemistry is not completely selective, it is certainly a good starting point for further investigation, as the selective synthesis of indoles remains and important task within many disciplines. I suppose the next question is – what other heterocyclic systems can this chemistry be applied to next?

Blog by Mark Honey

All Schemes and Figures were taken from J. Am. Chem. Soc. 2017, 139, 23-26

References

  1. J. Am. Chem. Soc. 2017, 139, 23-26
  2. Organometallics, 1998, 17, 2906

 

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“Post-antibiotic era”


In 2013, the Centers for Disease Control and Prevention (CDC) published a report on the burden and threats of antibiotic-resistant germs (CDC, 2013a). Bacteria resistant to different classes of antibiotics rapidly emerge worldwide and endanger the efficacy of antimicrobial. This resistance crisis can mostly be attributed to the overuse and misuse of antibiotics, but also to the lack of innovation and new drug development. Antibiotic research is not any longer attractive to the pharmaceutical industry, higher profits are made with new anti-cancer therapies and with other chronic conditions such as diabetes. “If we’re not careful, we will soon be in a post antibiotic era” (CDC, 2013b). What are the reasons for the decline in this field of research and are there any advancements in antibiotic drug research and development?

The era of antibiotics started in 1928 with the discovery of penicillin by Sir Alexander Fleming (Fleming, 1929). They should revolutionize modern medicine and increase general life expectancy. However, shortly after the introduction of penicillin, penicillin resistance in bacteria emerged and should become a substantial clinical problem. Discovery of new beta-lactam antibiotics such as methicillin in the 1960s only temporarily restored confidence, as antibiotic related resistance developed shortly after; the first case of methicillin-resistant Staphylococcus aureus (MRSA) in 1962 in the UK. This mêlée of antibiotic introduction and antibiotic resistance should go on for decades to come and still is happening at present day.

Causes of antibiotic resistance are diverse. Mostly, they are a result of overuse and misuse. Often, wrong antibiotics are prescribed and duration of antibiotic therapy is faulty. Overuse of antibiotics in agriculture to promote growth of farm animals and to prevent infectious diseases additionally contributes to increased resistance. Unfortunately, we are standing against this resistance with only few new antibiotics. The development of new antibiotics by pharmaceutical companies and academia slowed over the past decade due to funding cuts and due to being considered unprofitable. While 19 new antibiotics were approved in between 1980 and 1984, only 6 were approved by the Food and Drug Administration (FDA) since 2010. Many big pharma companies pulled out of research and the companies left didn’t do much progress. Thus, a combination of poor prospect and profit, as well as difficulties in getting regulatory approval slowed down the field.

Nevertheless, there has been a recent revive in antibiotic R&D and several antibiotics are in the pipeline for clinical trials. These include a variety of classes of antibiotics such as aminoglycosides, tetracyclines and beta-lactamase inhibitors. But also new approaches are being investigated (e.g. inhibiting endotoxin production by germs), as well as new sources of natural antibiotics. Teixobactin for instance was isolated from Eleftheria terrae and represents a new class of antibiotic that inhibits cell wall synthesis (Ling et al., 2015).

References, recommended articles in bold

Blog written by Lucas Kraft Continue reading

Screening the DEC (DNA-encoded chemistry)


A 2016 paper by workers at GSK, published in J. Med. Chem., really grabbed my attention. GSK appear to have achieved the difficult feat of discovering a molecule that exhibits monokinase selectivity!  Intriguingly, the initial hit was discovered not through screening the GSK kinase inhibitor set, nor through the HTS screening of the GSK compound collection (~2 million compounds).  The novel benzoxazepinone series was actually found by screening GSK’s DNA-encoded library.[1]

In its simplest form a DNA-encoded library is constructed with chemical building blocks that are individually ‘tagged’ with DNA fragments.  The DNA serves as an amplifiable genetic barcode.  Millions (or billions) of compounds can be produced in weeks using ‘routine’ combichem approaches (Fig 1).  The resulting libraries can be screened in a single test tube against the target.  Hits are read/identified by PCR amplification of DNA barcodes – Fantastic!  A smart and efficient way of getting around the arduous deconvolution involved with combinatorial libraries.[2]

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Figure 1 taken from a fantastic review of GSK’s contribution to the field of DNA-encoded libraries (Med. Chem. Commun., 2016 016, 7, 1898–1909)

An advantage of screening DNA-encoded libraries is that they enable a more thorough exploration of chemical space (4 – 5 orders of magnitude more!) than is achievable by traditional HTS methods.[3]  To give you an idea of the space explored, just one of the many DNA-encoded libraries that GSK screened to find the benzoxazepinone hit had a ‘total warhead diversity of approx. 7.7 billion’.[1] 

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Figure 2 taken from paper (J. Med. Chem. 2016, 59, 2163−2178)

 The benzoxazepinone series was selected for further investigation because it appeared to be far removed from what is perceived as the ‘same old’ kinase motif.  Fortunately for the team on the project, this line of enquiry furnished a novel chemotype with tractable SAR.  The initial hit matter was progressed to a lead that had desirable properties for a kinase inhibitor (i.e. excellent potency, exquisite kinase specificity, oral bioavailability etc. etc.) and also appears to be competitive with ATP whilst making no interactions with the hinge of the kinase at all! (a.k.a. Type III kinase inhibitor).  The atypical binding mode of the inhibitor was elucidated using a raft of techniques including photoaffinity labeling, hydrogen-deuterium exchange analysis and co-crystallography.[1]

DNA-encoded libraries are an accessible screening platform to both academic groups (whilst undertaking my Master’s project I saw the creation of (very similar) PNA-encoded libraries) and industry. The reason for an academic group to screen via this method is obvious – the combinatorial platform enables them to amass vast compound libraries that would have taken a substantial cash injection and multiple (hu)man hours to produce.  For industry, the DNA-encoded libraries represent another screening platform to churn out novel hit matter, casting the net deep into chemical space.

The construction and screening of DNA-encoded libraries is a relatively young field (~ 10 years). Reports of high-affinity hits for biological targets are becoming more common.[3] Ultimately the quality of the library and its output will depend on the diversity/novelty of each building block and the diversity of reactions employed in its construction.  For sure DNA-encoded libraries (and other encoded libraries) represent a step forward from the usual solid-phase mix and split OBOC libraries of the past.  Improvements in methodology (encoding and selection methods), library construction (arrays of 3D- and diverse building blocks, reaction diversity etc.), library curation and data analysis are all possible growth areas in the future.

GSK’s paper also serves as a useful reminder that choosing an unusual starting point – especially in the Kinase field where many privileged kinase motifs exist in the public domain – may guide you into hitherto unexplored chemical space.  The benefits of a proprietary starting point are obvious – the DNA-encoded library screening platform employed in this paper was critical to the discovery of novel, selective and potent hit matter.[1]

For recent reviews read [2], [3] and [4].

 Blog written by Scott Henry Henderson

References

[1] Harris et al., J. Med. Chem. 2016, 59, 2163

[2] Zimmermann and Neri., Drug Discovery Today. 2016, 21, 11, 1828

[3] Goodnow, Dumelin and Keefe., Nature Reviews Drug Discovery (2016) [doi:10.1038/nrd.2016.213]

[4] Arico-Muendel., Med. Chem. Commun., 2016 016, 7, 1898–1909

5 key points of a new drug for anxiety


In my previous blog on anxiety, I discussed how the treatments available for anxiety disorders have modest efficacy or severe side-effects. Furthermore many patients do not respond to first line pharmacological treatments (principally antidepressants). Psychological approaches (such as cognitive-behavioural therapy) are often limited in availability. As a result of this, benzodiazepines are frequently used for short-term management of anxiety symptoms. It can be argued that benzodiazepines are probably the most effective and well tolerated medications for anxiety; nevertheless, their long-term use is not recommended due to the unfavourable risk profile.

At the Sussex Drug Discovery Centre with the support of the Medical Research Council (MRC) we are currently studying a new generation of anxiolytic drugs.

These drugs, similarly to benzodiazepines, act as positive modulators of GABAA receptors, the main inhibitory mechanism in the mammalian brain, but are selective for the ‘anxiolytic’ subpopulation of the receptor. This approach may result in a drug with significantly less liabilities and ultimately improved risk-benefit profile.

I will focus on 5 key characteristics that a new ideal anxiolytic drug should possess to be superior to existing first-line treatments and benzodiazepines.

  1. Be active on all anxiety disorders and over the range of severity.The most common anxiety disorders are generalised anxiety disorder, social anxiety disorder, panic disorder, obsessive-compulsive disorder and post-traumatic stress disorder1. They all share common psychological and physical symptoms but differ in having characteristic features. The ideal drug should be active on all diseases and be effective in reducing both psychological and physical symptoms including the acute presentation of symptoms, which are common in all anxiety disorders.
  2. Have a rapid onset of action. Antidepressants, currently the first line treatment for anxiety, typically need 2 to 4 weeks to show benefits and this has obvious repercussions on the management of acute symptoms. On this point, the ideal anxiolytic drug should be similar to fast acting benzodiazepines which are effective in less than half an hour.
  3. Do not interfere with daily life. Antidepressants are associated with a number of physical side-effects: abdominal pain, weight gain, loss of libido, headache and nausea. Interestingly, anxiety is also included in the list of potential side-effects for antidepressants, in particular in the first weeks of treatment.                           Benzodiazepines at anxiolytic doses may induce sedation, dizziness and memory impairment. We believe that our approach which targets the anxiolytic subpopulation of GABAA receptors will result in a drug with a clean anxiolytic profile devoid of sedation side-effects2.
  4. Not be associated with the development of physical dependence or tolerance.  Problems upon discontinuation of treatment after long-term use are common to antidepressant and benzodiazepines. Up to a third of people who stop SSRIs and SNRIs have withdrawal symptoms which can last between 2 weeks and 2 months. These include a number of physical symptoms and additionally the possibility of developing rebound anxiety. Benzodiazepine long-term use has been associated with both physical dependence and tolerance (the need to increase the dose to obtain similar therapeutic effect). Sudden discontinuation from benzodiazepine treatment is even more problematic with a withdrawal syndrome that again includes rebound anxiety as a common symptom. The mechanism underpinning benzodiazepine dependence and tolerance is still a matter of discussion but there is general consensus that it probably involves some sort of neuroadaptation of the GABAergic synapses which constitute one-third of the synapses of the whole human brain. It has been argued that selective GABAA modulators may be superior in avoiding these neuroadaptation processes3, but in reality more studies are necessary to characterise selective modulators in models of dependence and tolerance.
  5. Not have a potential for addiction and abuse. It has been estimated that approximately 0.1–0.2% of the adult population abuse or are dependent upon benzodiazepines. Individuals with potential for abuse tend to cluster into two categories: alcohol or drugs-dependent outpatients and people suffering from anxiety disorders. Many studies – including drug self-administration models in several species and studies with human connoisseurs ‘familiar’ with recreational drugs – have indicated that selective GABAA modulators targeting only the anxiolytic subtype may again be superior in avoiding addiction and abuse4. Interestingly, studies on the neural bases of the addictive properties of benzodiazepines seems to confirm the results in animal models5.

 

 

Blog written by Alessandro Mazzacani

References

  1. Anxiety disorders, post-traumatic stress disorder, and obsessive–compulsive disorder, D. S. Baldwin et al., Medicine, 2016, 44 (11), pages 664–671.
  2. TPA023 [7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine], an Agonist Selective for α2- and α3-Containing GABAA Receptors, Is a Nonsedating Anxiolytic in Rodents and Primates, J. Atack et al., The Journal of Pharmacology and Experimental Therapeutics, 2006, 316(1), pages 410-422
  3. Mechanisms Underlying Tolerance after Long-Term Benzodiazepine Use: A Future for Subtype-Selective GABAA Receptor Modulators?, C. H. Vinkers and B. Oliver, Advances in Pharmacological Sciences, 2012, doi:10.1155/2012/416864
  4. Beyond classical benzodiazepines: novel therapeutic potential of GABAA receptor subtypes, Uwe Rudolph and Frédéric Knoflach, NATURE REVIEWS DRUG DISCOVERY, 2011, 10, pages 685-697
  5. Neural bases for addictive properties of benzodiazepines, M. Brown et al., NATURE, 2010, 463, pages 769-775

 

 

Antibodies and ion channels: potential therapies in airway disease?


A recent (and rather heroic) review of molecular drug targets1 identifies ion channels as one of four privileged families of proteins – alongside GPCRs, nuclear receptors and kinases – which continue to dominate drug discovery. However, the rate of drug approvals in this particular target class between 1991 and 2015 appears to be slowing. One possible reason may be the notorious difficulty in producing ‘clean’ small molecules for ion channel targets given the close structural homology between family members, and the very real (and costly) risk of off-target effects involving ion channels of the heart and the disruption of normal cardiac rhythm. A solution to improve target specificity may arise from another current growth area in drug discovery: the use of monoclonal antibodies and antibody-drug conjugates.

The field of antibody therapy has already brought about treatments for cancer, cardiovascular, inflammatory and ophthalmic diseases, and the prevention of transplant rejection. This field is currently weighted towards targeting circulating cytokines, growth factors, inflammatory mediators and their receptors – all protein targets which have extracellular domains readily accessible for antibody generation. Until recently the same has been true in the area of respiratory research, but a recent article by Douthwaite et al2 reviews progress in targeting more complex membrane-bound proteins that are key effectors in respiratory disease processes, namely ion channels and GPCRs.

So what governs the prospect of ion channels as antibody targets in lung disease? The potential advantages of antibody specificity are clear, beyond that achieved by small molecule approaches to drug design. The challenges are two-fold: firstly, with multiple trans-membrane domains, these proteins have complex topology with limited extracellular domains for antigen generation. Secondly, their close association with the lipid bilayer of a cell membrane makes isolating the proteins with the correct structural conformation difficult – the same problem that has hampered efforts to generate their crystal structures over the years. Yet progress has been made using a synthetic peptide system known as CLiPS (chemical linkage of peptides to scaffolds), resulting in functional antibodies to the complex G-protein receptor structure CXCR2. Combining this finding with reports of successful polyclonal antibody generation to the third extracellular loop of a number of ion channels (a region common to calcium, potassium, sodium and TRP channels) the authors suggest that the same approach may be extended to ion channels for monoclonal antibody generation. Other means of targeting antigens (cell-based over-expression systems and viral methods) are also reviewed in the article and have recently led to the generation of a monoclonal antibody antagonist of the TRPA1 ion channel – a promising target for asthma and airway inflammation. Its functional effectiveness was assessed by measuring calcium uptake in response to TRPA1-mediated cellular stimulation by mustard oil, cold temperature and osmotic pressure. Although efficacy was less than that of current small molecule antagonists (reducing cellular signal by 50% as opposed to full block), the trial proved the effectiveness of the particular antibody targeting and generation strategy used and provides a good basis on which to test measures to improve efficacy.

As the authors point out, respiratory drug targets are usually members of complex networks of mediators and receptors with functional redundancy. Within these networks spatial or temporal interplay may be a factor in target activation and subsequent biological effect. Coupled with phenotypically diverse patient populations seen with asthma, CF and COPD, picking a single molecular target such as an ion channel or GPCR may not provide a ‘one-size-fits-all’ therapy. It might, however, provide an effective low-risk component of a combination approach. Inhalation would give ready access to membrane-bound ion channel targets of the airway mucosa (vital in the maintenance of mucus viscosity and airway surface liquid height), and with a naturally long half-life antibodies may bring the advantage of low dosing frequency – particularly beneficial in chronic conditions. Offering a variety of mechanisms of action (inhibition, agonism, receptor internalisation, cell depletion, state-dependent recognition/interaction) alongside the possibility of drug conjugation, there is much potential in the relationship between monoclonal antibodies and ion channels, providing significant opportunity in the area of respiratory research.

Blog by Sarah Lilley

References

1 Santos et al (2017)

http://www.nature.com/nrd/journal/v16/n1/full/nrd.2016.230.html

2Douthwaite et al (2017)

http://www.sciencedirect.com/science/article/pii/S0163725816300596

The future for membrane protein structural studies


High-throughput structure determination with X-ray crystallography has demonstrated its value to accelerate drug discovery for over a decade. It can facilitate not only the optimisation of lead compounds and target identification, but also lead discovery through increasing screening capabilities. But despite the progress in genome sequencing, robotics and bioinformatics, the structures and functional mechanisms of membrane proteins are still relatively equivocal. Membrane proteins account for approximately 30% of all proteins (Wallin & Heijne, 1998) and are targeted by an estimated 60% of all drugs (Overington et al., 2006).  Their crystal structures, however, only comprise ~3% of those in the Protein Data Bank (PDB).  So, what challenges remain in their structural determination, and will the recent resurgence in cryo electron microscopy (cryo-EM) be critical to furthering our insight into these proteins?

To begin with we must consider the hurdles which must be overcome for the crystallisation of a protein. These lie firstly in the overexpression of the protein (Seddon et al., 2004); secondly in the extraction of this protein out of the membrane in a non-aggregated, stable state; and finally, in making sufficient crystal contacts with the protein crystal for structural determination, a problem augmented by membrane proteins due to their tendency to exhibit inherent flexibility (Bill et al., 2011).  Strategies have been employed to overcome some of these problems, including mutagenic or chimeric approaches (Abdul-Hussein et al., 2013; Caffrey, 2015), with the overall intention to improve membrane stability, increase crystal contacts and fix conformational states.  However, these are not universally applicable and many membrane proteins have proven to be resistant to crystallisation irrespective of focused efforts.

So when does it become necessary to look for an alternative? Cryo-EM may not have provided one previously as high resolution protein structures were limited by both EM hardware and smaller membrane proteins’ tendency to aggregate. Yet, this method had always evaded some of the major challenges of crystallography, for example reducing the amount of protein needed in comparison to crystal studies (μg vs. mg scale). And, with existing advancements in the EM hardware, in terms of detectors and microscopes, the use of this technique for the study of membrane protein structure is being readily adopted.  Crucially, cryo-EM specimens are made by fast freezing biological samples from the solution directly in liquid nitrogen temperature.  This ability to maintain the protein complex in its soluble state permits the structure to be examined in a state much more closely resembling its native state.  Cryo-EM can now obtain near-atomic resolution structures of macromolecular complexes up to several MDa in size and, in combination with image classification algorithms, not only are high resolution 3D maps now possible, but also the evaluation of several conformational states of the same sample.

This changing landscape in recent years has permitted the high resolution structural determination of a number of membrane proteins. One key group are the ryanodine receptors (RyR’s), which facilitate the intracellular release of Ca2+, integral to muscle contraction.  They are implemented in cardiac arrhythmias and have surfaced as potential therapeutic targets for heart failure (Betzenhauser & Marks, 2010; Anderson & Marks, 2010).  More specifically the RyR1 channel, a tetrameric channel with a molecular mass >2.2 MDa, lends itself to EM structural determination over x-ray crystallography.  Nevertheless, until a couple of years ago the highest resolution structure obtained was a mere ~1 nm resolution (Ludtke et al., 2005), which could only give an indication of overall channel architecture.  This channel has since been determined to a much higher ~4 Å (Yan et al., 2015), an impressive feat when considering that it is the largest of all known ion channels.  From this, several new important functional domains and changes in channel conformation have been identified, helping to deduce potential channel gating mechanisms.

It must not be overlooked that cryo-EM does pose its own, new challenges. However, the exciting opportunities offered by cryo-EM has motivated a focused effort to improve application, from sample preparation, to data collection and processing, to the modelling of the 3D maps.  Some challenges still remain: one of the major factors preventing the attainment of even higher resolution structures is radiation damage caused by exposure to high energy electrons (Glaeser & Taylor, 1978).  Even at very small electron doses (~3 e2) damage has been shown on some charged side chains  (Grant & Grigorieff, 2015a) and damage to the specimen caused by doses above ~10  e2 result in a loss of diffraction from 2D and 3D crystals (Baker et al., 2010). It is therefore thought that the early frames of a direct detector movie contain the highest resolution signal, however at present are not able to be recovered due to limitations in the hardware.

Still in its early stages, cryo-EM is unlikely to replace X-ray crystallography entirely and it is also unlikely that there will be a method unanimous to all membrane proteins.  It may be that a combination of techniques could be used in conjunction with each other.  Examples of this include the docking of X-ray crystallographic structures within cryo-EM maps, or solving the phases of X-ray crystallographic diffraction data using cryo-EM maps.  Although X-ray crystallography has proved to be a powerful tool, cryo-EM is enabling the structural study of some membrane targets which had previously been deemed unattainable. And with the advent of new technologies, the future for membrane protein structural studies is filled with potential.

Blog written by Victoria Miller

References

Abdul-Hussein S, Andréll J, Tate CG. 2013. Thermostabilisation of the serotonin transporter in a cocaine-bound conformation. J Mol Biol 425:2198-2207

Anderson DC & Marks AR. 2010. Fixing ryanodine receptor Ca leak – a novel therapeutic strategy for contractile failure in heart and skeletal muscle. Drug Discov Today Dis Mech 7:e151-157

Baker LA, Smith EA, Bueler SA, Runimsteim JL. 2010. The resolution dependence of optimal exposures in liquid nitrogen temperature electron cryomicroscopy of catalase crystals. J Struct Biol 169:431-437

Betzenhauser MJ & Marks AR. 2010. Ryanodine receptor channelopathies. Pflugers Arch 460:467-480

Bill RM, Henderson PJF, Iwata S, Kunji ERS, Michel H, Neutze R, et al. 2011. Overcoming barriers to membrane proteins in vitro. Methods Mol Biol 601:219-245

Caffrey M. 2015.  A comprehensive review of the lipid cubic phase or in meso method for crystallising membrane and soluble proteins in vitro. Methods Mol Biol 601:219-245

Glaeser RM & Taylor KA. 1978. Radiation damage relative to transmission electron microscopy of biological specimens at low temperature: a review. J Microscopy 112:127-138

Grant T & Grigorieff N. 2015a. Measuring optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6. eLife 4:e06980

Ludtke SJ, Serysheva II, Hamilton SL, Chiu W. 2005. The pore structure of the closed RyR1 channel. Structure 13:1203-1211

Overington JP, Al-Lazikani B, Hopkins AL. 2006. How many drug targets are there? Nat Rev Drug Discov 5:993-996

Seddon AM, Curnow P, Booth PJ. 2004.  Membrane proteins, lipids and detergents: not just a soap opera.  Biochem Biophys Acta 1666:105-117

Wallin E & Heijne von, G. 1998. Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Protein Sci 7:1029-1038

Yan Z, Bai X-C, Yan C, Wu J, Li Z, Xie T, et al. 2015.  Structure of the rabbit ryanodine receptor RyR1 at near-atomic resolution. Nature 517:50-55

 

Redressing the balance – Understanding the disproportionate impact of Cystic Fibrosis on Women



Cystic fibrosis is a hereditary recessive disease which is the result of mutations in the Cystic Fibrosis Transmembrane Conductance (CFTR) gene. Mutation of the CFTR leads to decreased chloride transport across the surface epithelia and consequent dehydration of the airway surface liquid (ASL) which lines the airway. This dehydration causes the formation of a thick viscous mucus which traps pathogens and cannot be cleared by ciliary transport.

Improving therapies have increased the mean life expectancy of patients with CF from less than 5 years of age in the 1950s to almost 40 years of age today1. However, even with the advent of more advanced and specific therapies, CF continues to impact women to a greater degree than men. A study carried out by Raksha Jain et al in 2014 demonstrated that women with CF have a decreased median life expectancy (36.0 years), compared to men (38.7 years). They showed that female gender was a significant risk factor for death (hazard ratio 2.22, 95% CI 1.79-2.77), despite taking in to account many variables which a known to influence CF mortality2.

Given these gender differences, several groups have looked at the possible influence of sex hormones on the mucociliary apparatus, which is composed of mucus, cilia and the ASL. These studies showed that sex hormones were able to influence the airway epithelial cell apical sodium and chloride transport3,4, ASL volume5 and the beat frequency of cilia6 as well as affecting the inflammatory mediators predisposing women to increased infection and colonisation7.

Van Horn et al published a study in Science Advances in September 2016 which integrated NMR, molecular dynamics, homology modeling and protein structure predictive modeling to investigate the role of oestrogen in CF at a molecular level. Specifically, they studied the KCNE3 protein which modulates the voltage-gated potassium channel KCNQ1, removing voltage-dependant gating producing a constitutively open leak channel. In this way KCNE3 regulates the recycling of potassium ions (K+) which in turn impacts the flow of chloride ions (Cl) across epithelial tissues.

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Van Horn et al showed that oestrogen induces the phosphorylation of KCNE3 Ser82 residue resulting in the disruption of its complex with KCNQ1, thereby decreasing K+ ion recycling and subsequent Cl secretion leading to a further reduction in ASL volume and increased mucus viscosity. This in turn results in decreased mucociliary clearance, higher rates of infection and colonisation and increased morbidity in female CF patients.

It is hoped that the advent of new techniques in the imaging and structural analysis of membrane-protein interactions will not only help to redress the balance of prognosis between the sexes but ultimately make CF more treatable in the future.

References

  1. Marshall BC. Cystic Fibrosis Foundation Patient Registry: 2013. Annual Data Report. Available at http://cff.org
  2. Harnness-Brumlley CL., Elliot, AC., Rosenbluth DB., Raghavan D., Jain R. Gender Differences in Outcomes of Patients with Cystic Fibrosis. Journal of Women’s Health 2014;Dec 1;23(12) 1012-1020
  3. Sweezey NB., Ghibu F. Gagnon S. Sex hormones regulate CFTR in developing fetal rat lung epithelial cells. Am J Physiol 1997;272:L844–851
  4. Singh AK., Schultz BD., Katzenellenbogen JA, et al. Estrogen inhibition of cystic fibrosis transmembrane conductance regulator-mediated chloride secretion. J Pharmacol Exp Ther 2000;295:195–204
  5. Coakley RD., Sun H., Clunes LA, et al. 17beta-Estradiol inhibits Ca2+-dependent homeostasis of airway surface liquid volume in human cystic fibrosis airway epithelia. J Clin Invest 2008;118:4025–4035
  6. Jain R., Ray JM., Pan JH. Brody SL. Sex hormone-dependent regulation of cilia beat frequency in airway epithelium. Am J Respir Cell Mol Biol 2012;46:446–453
  7. Chotirmall SH., Smith SG., Gunaratnam C, et al. Effect of estrogen on pseudomonas mucoidy and exacerbations in cystic fibrosis. N Engl J Med 2012;366:1978–1986

Blog written by Holly Charlton