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.


A pre-antibiotic apocalypse?

A number of years back I undertook a project researching novel small molecule inhibitors for bacterial RNA polymerase as antibiotic agents. Back in 2009 there was huge worry for the increased bacterial resistance to marketed drugs as there had been for many years prior. I had decided to have a look at recent publications and developments in antibacterial drug research for the purpose of this blog until last Thursday morning when I awoke to the media in a frenzy.

The mainstream newspapers, radio and TV were all discussing antibacterial resistance and the “pre-antibiotic apocalypse” that loomed. Surely, this has been in discussion for years? I realised that I too had perhaps become complacent to the advancement of antibiotics in recent years. So to sidestep the more sensationalist media stories I decided look at the issues and some of the facts behind the story.

The recent media storm comes from comments made by England’s Chief Medical Officer Prof Dame Sally Davies who warned that antibiotic resistance could spell the “end of modern medicine” and unless something was undertaken to address the problem a potential “pre-antibiotic apocalypse” could occur making currently routine operations very risky and put a stop to transplant medicine [1] [2].

These are strong comments indeed however to be honest an opinion that I feel the scientific community have held for a long time. When commencing the antibacterial project 8 years ago methicillin resistant staphylococcus aureus (MRSA) was big news with patients contracting infections in hospitals due to these methicillin resistant strains. The office for national statistics reported that in 2006 the number of death certificates mentioning S.aureus was 2,150 and of those 1,652 reported MRSA strains [3]. Although the number reported deaths due to MRSA fell in following years this was put down to improved policy, hygiene and protocol in hospitals rather than a reduction in the prevalence of the resistant strains or advancement in antibiotics.

Of course, this is only one example of resistant bacterial strains with pneumonia, gonorrhoea and perhaps more worryingly tuberculosis, HIV and malaria amongst other infections that are now increasingly harder to treat due to drug resistant strains. It’s estimated that around 700,000 people die per year worldwide due to infections of drug resistant strains, which include HIV, tuberculosis and malaria [5]. An AMR review: Tackling Drug resistant Strains Globally goes on to say that if the problem is not addressed a forecast prediction of mortality in 2050 indicates the number of deaths due to drug resistant pathogens could be 10 million a year engulfing the predicted deaths by cancer [5]. The World Health organisation (WHO) states that antibiotic resistance is “one of the biggest threats to global health, food security and development today” even hosting an World Antibiotic Awareness week (13-19 Nov 2017) to educate on safe use of antibiotics and the impact of misuse [4].

The antibacterial resistance issue is incredibly complex ranging from inability to create new innovative bacterial classes to the poor potential for economic return in research investment. Many factors have created the problem we now see today and these as need to be addressed for the situation to improve.

The issues of new antibacterial agents are underpinned by the need to create new innovative and novel drugs to counteract the potential for antibacterial resistance. In the past the pipeline of new antibiotics focused on modifying existing antimicrobial classes however this is simply a temporary fix and fail to address multiple resistance mechanisms.

In May of this year the World Health Organisation released a review of Antibiotic agents currently in the pipeline and specified the need for innovative products without cross resistance to existing antibacterial classes [6]

For a drug to be considered “innovative” it must fulfil one criteria of the following; being non cross-resistance to existing antibacterial class, a new chemical class, a new chemical target or a new mechanism of action. The review found that out of 33 drugs currently in the pipeline for priority indications 9 belong to 5 new antibiotic classes.

However the WHO review concluded that the current pipeline is “insufficient to mitigate the threat of microbial resistance” pointing to vast changes in policy, education and research to address the root cause of antibacterial resistance as well as the growing need for new drugs.

Antibacterial resistance has been reported for virtually all drugs currently on market and as well as multi drug resistant strains. This has largely been the blame of overuse and misuse of antibiotics in both humans and agriculture. As new classes of drugs are brought to market, without the education of patients and doctors, changes to policy of governments and organisations it is likely the issue will remain. As the bacteria evolution is at such a high rate it’s likely that resistance to new drug classes will be seen quickly if inappropriately used.

The AMR review: Tackling Drug resistant Strains Globally gives an in depth look at the complex nature we face with combating antimicrobial resistance with one key issue being the necessity for investment following years of under investment by companies and governments.

Between 2003 and 2013 only 5% of the total venture capital of pharmaceutical research and development was spent in antimicrobial research [5]. This equates to $1.8 billion of the total $38 billion spent. Pharmaceutical company’s no longer see antimicrobial research as attractive or that investment would provide an appropriate return for the risk.

Movement away from this research area can also be quantified by the lack drugs in the pipeline. In 2014 there were close to 800 oncology drugs in the pipeline in comparison with the 33 that are currently moving though for antibacterials’. Both the AMR and WHO reviews discuss the initiatives currently in place to encourage private and public investors into antibacterial research in an attempt to improve the attractiveness of the potential risk.

The problem of antibiotic resistance we face is incredibly complex and very real. Following on from last weeks news reports I believe the comments from Dame Sally, although illustratively put, to be nothing further then the truth. It seems little has moved on to truly battle the problem of bacterial resistance and only a combined effort of governments, healthcare professionals, pharmaceutical companies and individual people will get us away from the potential pre-antibiotic era that not only is frightening but a reality if we do nothing.

I started this blog with the best of intentions discuss the very important issue of antibacterial resistance in relation to the comments made in the media however I perhaps underestimated the highly difficult nature of the problem and the complex factors that drive it. So if you are interested I highly recommend the following articles for more detail:

WHO Review: Antibacterial agents in clinical development. May 2017

AMR review: Tackling drug resistant strains globally, May 2016

Blog written by Alex Ashall-Kelly


[1] http://www.independent.co.uk/life-style/health-and-families/health-news/antibiotics-resistance-apocalypse-warning-chief-medical-officer-professor-dame-sally-davies-drugs-a7996806.html

[2] https://www.theguardian.com/society/2017/oct/13/antibiotic-resistance-could-spell-end-of-modern-medicine-says-chief-medic

[3] https://www.ons.gov.uk/peoplepopulationandcommunity/birthsdeathsandmarriages/deaths/bulletins/deathsinvolvingmrsa/2013-08-22

[4] http://www.who.int/mediacentre/factsheets/antibiotic-resistance/en/

[5] AMR review: Tackling drug resistant strains globally, May 2016


[6] WHO Review: Antibacterial agents in clinical development. May 2017 http://apps.who.int/iris/bitstream/10665/258965/1/WHO-EMP-IAU-2017.11-eng.pdf?ua=1

19th RSC/SCI Medicinal Chemistry Symposium

I recently attended the 19th RSC/SCI Medicinal Chemistry Symposium held at Churchill College, University of Cambridge (UK) – this was the first medicinal chemistry symposium I have attended since joining the field approximately 3 years ago, and it certainly didn’t disappoint. The symposium was held over 4 days, with the first day dedicated to a medicinal chemistry workshop for early career researchers (those with 5 years or less experience within medicinal chemistry). I found the medicinal chemistry workshop a good learning experience – we started from a series of hits and had to try to progress at least one compound into a preclinical candidate. The team that I was working in came 2nd out of 7, just being pipped to the post for 1st place.

Mark 1

Introduction to Technical Programme

 The technical programme started on the second day, and from the outset the talks were quite diverse with projects from many therapeutic areas being discussed, with several first disclosures being presented. There was a dedicated Neglected Tropical Diseases session that was webcast live through the RSC website, highlighting a range of diseases that require much more attention than they currently receive. Although a lot of great medicinal chemistry was discussed, it was a talk by Gianni Chessari from Aztex Pharmaceuticals that I found particularly fascinating. Gianni described a fragment-based screening approach using PyramidTM for the discovery of inhibitors of apoptosis proteins (IAP). He described structure-based hit optimization utilizing computational and NMR studies – by analysing the confirmation of their hit compounds, they were able to increase binding affinity and subsequently develop a potent non-peptidic IAP antagonist ASTX660 that is currently being tested in a Phase 2 clinical trial.

Overall I found this conference a great learning experience and a good opportunity to meet like-minded individuals from both academia and industry – I particularly enjoyed the opportunity to network at the Gala Dinner in the impressive surroundings of St John’s College!

Mark 2

Gala Dinner at St John’s College

Blog written by Mark Honey





Alzheimer’s Disease – Dopamine first

Alzheimer’s disease (AD) is a progressive neurodegenerative brain disorder that causes a significant disruption of normal brain structure and function. At the cellular level, AD is characterized by a massive neuronal loss that primarily affects the hippocampus and cortex, mainly due to the accumulation of intracellular neurofibrillary tangles and extracellular amyloidal neuritic plaques.


Fig.1 -Schematic representation of AD-related mechanisms (Medicinal Research Reviews · 2013)

The hippocampus is a critical brain structure for memory development and damages in this area are believed to be the primary cause for memory loss in AD patients. However, progressive structural alterations in different brain areas may play a pivotal role in the worsening of memory and cognitive dysfunctions. Consistent with these observations, several alterations in the dopaminergic system have been reported in AD patients, together with reduced levels of dopamine (DA) and its receptors. Moreover, DA is a well-recognized modulator of hippocampal synaptic plasticity and its binding to dopaminergic receptors in the dorsal hippocampus is a major determinant of memory encoding.

A recent study published on Nature Communications (DOI: 10.1038/ncomms14727) highlighted how, in a mouse model of AD, at a stage when no Ab-plaque deposition, hyperphosphorylated tau tangles or any sign of neuronal loss in cortical and hippocampal regions has yet occurred, a specific apoptotic process is taking place in the dopaminergic neuronal population in ventral tegmental area. The loss of dopaminergic neurons is paralleled by a reduced outflow of DA in the hippocampus, thus contributing to the deficits of hippocampus-dependent memory and synaptic plasticity, as well as impairment in reward processing. These symptoms are improved by stimulating the dopaminergic system with the administration of L-DOPA or the reduction of its endogenous degradation.

Although the abovementioned process has been observed in an experimental model of AD, it might provide an interesting explanation to recent evidences in AD patients, indicating that the clinical diagnosis of dementia is associated with early non-cognitive symptoms, such as depression and apathy. Based on that, changings in the mood of AD patients would be not a consequence of this pathology but rather an alarm for an early stage development of AD, confirming the strict correlation between depression and subsequent loss of memory.

This picture is somehow symmetrical to what researchers involved in Parkinson’s disease and although the molecular mechanisms underlying early dopaminergic neuron degeneration in the ventral tegmental area remain to be elucidated, these results suggest DA as an important player to consider in the context of AD.

Blog written by Samuele Maramai

A Word to the Wise about Ketamine

Major depressive disorder (MDD), also known simply as depression, and its impact on functioning and well-being has been compared to that of other chronic medical conditions such as diabetes. The World Health Organization estimates depression as the fourth highest burden of disease in the world1 .

Sri 1

Figure 1: Proposed neurobiological model of depression2

Prolonged stress and depression alter prefrontal glutamate release and reduce glutamate uptake, leading to increased extracellular glutamate and excitotoxicity3. High levels of extracellular glutamate precipitates neuronal atrophy through dendritic retraction4, reduced dendritic arborization5, and reduced synaptic strength. An example of the effect of prolonged stress on dendritic arborization and length in rats is shown on the right (Fig. 1).

Research on antidepressants has achieved little success in developing fundamentally novel antidepressant mechanisms, leaving psychiatrists with relatively few pharmacologic options. Several antidepressants that are currently in use are mainly targeting the monoaminergic system where substantial numbers of patients are failing to achieve a sustained remission6. Moreover, conventional antidepressants are only beneficial when prescribed over a long- term period. It is clear that we are in urgent need to find a rapidly acting antidepressant with robust efficacy in patients who are resistant to traditional antidepressants.

Sri 2Ketamine is a drug used illicitly as a hallucinogen and was first tested in humans in 1964. It was approved 1970 in the USA as a surgical anaesthesia that was used in Vietnam War due to its safety7. Ketamine can be a prototype for the new generation of antidepressants by showing good efficacy in patients who are refractory to the existing treatments. Low dosages of ketamine reduce depression symptoms within 4 hours of intravenous administration in severely treatment-resistant depressed patients8.

Ketamine exhibits promising antidepressant effects even in patients with bipolar disorder and patients with severe symptoms that did not respond to ECT9. A critical obstacle to the broader study and implementation of ketamine treatment for depression is the lack of clarity as to how to sustain its antidepressant effects. Pilot studies suggest that ketamine may be sustained by repeated intermittent administration with persistence of the antidepressant effects for longer periods in some patients10.

Briefly, ketamine works by enhancing synaptic plasticity (mechanism through which neural circuits regulate their excitability and connectivity) through regulating AMPA and NMDA receptors.

Sri 3

Figure 2: Prefrontal synaptic connectivity during normal mood, depression, and after remission (Abbreviations: ⦿, activate; ∅, block; , decrease; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; BDNF, brain-derived neurotrophic factor; GABA, γ-aminobutyric acid; mTORC1, mammalian target of rapamycin complex 1.)

There have been some clinical trials where ketamine shows acute efficacy 11 in treating TRD (treatment resistant depression) , bipolar depression12 and major depressive disorder with suicidal ideation13, but the number of subjects in these trials ranges from 20 to 57 patients. Inverse-Frequency Analysis of eight million reports from the FDA Adverse Effect Reporting System (FAERS) revealed that patients who received ketamine had a significantly lower frequency of reports of depression than patients who took any other combination of drugs for pain14.

It is encouraging that FAERS makes a case for study of ketamine as a psychiatric drug but there are financial and ethical obstacles for a larger scale clinical trial to validate further the safety and efficacy of Ketamine. To date, Ketamine is not only the most extensively studied NMDA antagonist, with 12 published randomized clinical trials, but is the only NMDA antagonist to date consistently demonstrating antidepressant efficacy across multiple trials15

While Ketamine efficacy and safety are under caution, forthcoming ketamine research should continue to examine three major concerns: 1) elucidating ketamine’s mechanism of action; 2) understanding the administration profile necessary to provide a sustained therapeutic benefit; and 3) examining ketamine’s safety profile, particularly with repeated and likely low-dose administration. Knowing Ketamine can be a drug of abuse, it is difficult to argue its future as a potential drug to treat depression.

Blog written by Srini Natarajan

Nitrogen-Centered Radicals: A Forgotten Species?

In the late 1990s I was working on a chemistry project involving cascade reactions of C-centred radicals (CCRs). At that time, N-centered radicals (NCRs) were little used compared to their C-centered relatives and I was wondering if things had changed much since then.

Considering that the first example of the Hofmann-Löffler-Freytag reaction was reported in 18831 it seems that the development of the chemistry of NCRs has been fairly slow. More recently, in 2008, Zard2 described NCRs as a forgotten species with significant synthetic potential in a comprehensive review of intramolecular NCR cyclisation. NCRs have suffered from limitations because traditional methods for their generation have relied on the synthesis of N-X precursors and required harsh conditions for bond homolysis (Scheme 1, path A). New developments in metal catalysis (path B) and visible-light photocatalysis (path C) have resulted in greater use of NCR chemistry (for C-N bond formation) and advances in this field were reviewed by Zhang3 last year.

Micheal 1

 Scheme 13 Strategies in C-N bond formation based on NCR chemistry

Generation of NCRs using visible-light photocatalysis is particularly attractive due to low catalyst loading and mild conditions, with most reactions occurring at room temperature without the need for highly reactive radical initiators. This technology has been widely applied to radical amination chemistry in the last three years. An interesting example of this approach entitled ‘’Visible-Light-Mediated Generation of Nitrogen Centered Radicals: Transition Metal Free Hydroimination and Iminohydroxylation Cyclisation Reactions’’ was published by Leonori.4 One of the transformations described is a 5-exotrig cyclisation of iminyl radicals to give pyrrolines without the use of a transition metal catalyst (Scheme 2). A diverse range of oximes were converted to the corresponding pyrrolines, in good yields, and bicyclic heterocycles were also prepared using this methodology.


Micheal 2 

Scheme 24 Visible-light mediated hydroimination

The mechanism of this reaction involves single electron transfer (SET) reduction of the aryl oxime ether (A) by a visible-light exited photocatalyst (*PC), followed by N-O bond fragmentation to give iminyl radical (D) which can then cyclise onto the pendant alkene (Scheme 3). The role of cyclohexadiene (CHD) is to reduce the intermediate radical (E), followed by a second reduction of the photocatalyst (PC), which is then irradiated (PC®PC*) completing the catalytic cycle. Cyclic voltammetry studies suggested that that the key single electron transfer (SET) reduction, by excited state organic dye eosin Y, would only be efficient when using nitro-substituted aryl oxime ethers (1a-1d) with suitable reduction potentials. The 2,4-dinitro-aryl oxime (1a) was found to be the best substrate.

Micheal 3.png

Scheme 34 Proposed photoredox cycle and electrochemical studies. EY = eosin Y, ppy = 2-phenylpyridine

It was found that a different activation mode could be used to generate the iminyl radical without the presence of a photocatalyst (Scheme 4). When a solution of the aryl oxime ether (2a) and triethylamine in acetonitrile was irradiated with visible-light in the presence of CHD the previously observed hydroimination product (3a) was obtained, and also an unexpected iminoalcohol (4a). When CHD was not in the reaction mixture iminoalcohol (4a) was the main product. The mechanism proposed for this process involves formation of an unusual electron donor-acceptor complex (5) which undergoes SET giving the dipolar species (6). This species can then fragment, and undergo 5-exo-trig cyclisation to give radical (7). If the radical is not reduced by CHD it can be oxidised by attacking the nitro group (8) which after N-O bond homolysis, and hydrogen atom abstraction, gives the iminoalcohol (4a). Evidence for the source of the oxygen was provided by generation of 2-NO-4-NO2C6H3OH (10) from the reaction which was in contrast to 2,4-dinitrophenol that was produced from the original hydroimination.

Micheal 4

Scheme 44 Initial findings and proposed reaction mechanism for the iminohydroxylation

The chemistry of NCRs has been developing in recent years, but they still remain less well used than CCRs. Significant problems, such a functional group compatibility, need to be solved before they can become routinely used for C-N bond construction alongside traditional ionic chemistry. Visible-light photocatalysis seems very likely to play a role in future developments.

Blog written by Michael Annis


1. J. L. Jeffrey and R. Sarpong, Chem. Sci., 2013, 4, 4092

2. S. Z. Zard, Chem. Soc. Rev., 2008, 37, 1603

3. T. Xiong and Q. Zhang, Chem. Soc. Rev., 2016, 45, 3069

4. J. Davies, S. G. Booth, S. Esaffi, R. A. W. Dryfe and D. Leonori, Angew. Chem. Int. Ed. 2015, 54, 14017

Chemical tools on trial: recent misleading information cases

For this week’s blog, I have picked it a still-hot and enjoyable Nature article by Monya Baker.1

How many times have you had screening results which make no sense to you end in frustration and unexplicable conclusions? How many times have you encountered that your chemical tools (hits) are neither as you thought, behaved oddly or even conducted regular quality controls on them and enough analytical tests? Read more to recognise unexpected setbacks.

The author goes through a few examples where researchers have encountered, for example, that chemical probes hitting specific targets suddenly became inactive when alternatives suppliers came onto stage. A futher realisation that one isomer was presented in different proportions if it was mixed with its other enantiomer (mirror-image like compounds sharing the same molecular formula) altering its activity leading to discover that sometimes, researchers do not know what they have in their hands if too much trust is put on commercial sources or the lack of further structural corroborations.2

When screening large compound libraries and identifying hits, the presence of false positives, impurities, degradation, mixed-up and messed-up stocks could also lead to infructouos crusades. Especially when liquid stocks are kept for months or years, at variable temperatures, the effect of poor solubility, side-reaction impurities, solvent effects and cockails of natural product mixtures amongst others. 3

Suggested online resources like: Chemical Probes Portal, Probe Miner and Probes and Drugs Portal 4 have been created to be used as a good first port of call toolbox with advice and information on which chemicals or drugs to use as well as public assessments.

The infamous case of BOSUTINIB, an approved cancer drug which was sold by nearly 20 suppliers with the chemical substituents wrongly located (please see ‘Spot the difference’ cartoon) has created havoc. jose

Both bosutinib and the other mis-identified isomer target cell-signalling proteins but with different potencies putting under trial numerous papers which reported data on the isomer of bosutinib rather on itself.5

Another researcher, Kim Janda caused an uproar when in its group they could not synthesise a molecule described to boost cell production of a powerful natural antitumor protein, TRAIL. The distributors had perpetuated the mistake in the original publication and after even clinical trials and patents were issued, he filled a patent with the proposed new active version of the TIC10 structure. 6

Another case emphasises how activity showed a big drop after small changes in contrast with bigger changes. Scientists realised that the present of Zn in their preparation was responsible for this activity without effect from the organic molecule whatsoever. Copper or Palladium from its use in catalytic reactions have been reported showing false positives.7

Even the present of NMP (N-methyl-2-pyrrolidone) a polar solvent used in some cases for keeping chemical probes in solution has recenlty showed anticancer activity in control tests, with potential implications in misleading the potency of the substances contained on it. 8

A list of common-sense leading practises to suppress these errors are given in the paper:

  • Use and buy chemicals by their CAS number (unique identification to chemical structures)
  • Request a detailed quality control certificate from external suppliers
  • Place enquires about the synthetic method used
  • Perform independent or in-house analysis upon receipt of the starting chemicals and reagents
  • Structural forms, chiral compounds, potential ambiguity in their chemistry substitution, then determine their optical purity and apply chiral chromatography techniques to isolate single forms.
  • If possible, previous in-house synthesis and determination with structural characterization.

Mismatching results, not-well documented suppliers, dubious chemical insertions (not well validated) as well as poor purities and the presence of contaminants (even at low concentrations) are always to blame for cases like those described in the article over the years. Albeit the final responsibility lies on the chemist’s team who need to be reassured by exploring and conducting further unequivocal tests.

The views represented in this blog are the author’s own.

Blog written by Jose Gascon


  1. https://www.nature.com/nature/journal/v548/n7668/pdf/548485a.pdf
  2. Huber, K. V. M. Nature, 508, 222–227 (2014).
  3. Bisson, J. et al. J. Med. Chem. 59, 1671–1690 (2016)
  4. See: Chemical Probes Portal (www.chemicalprobes.org ), Probe Miner (www.probeminer.icr.ac.uk) and Drugs Portal (www.probes-drugs.org )
  5.  https://www.chemistryworld.com/news/pfizers-response-to-compound-fraud-spotlights-quality-issues-/9234.article
  6. https://phys.org/news/2014-05-chemists-cancer-drug-candidate.html ,Oncotarget, 2014, 5(24): 12728–12737 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4350349/
  7. Hermann, J. C. et al. ACS Med. Chem. Lett. 4, 197–200 (2013)
  8.  Shortt, J. et al. Cell Rep. 7, 1009–1019 (2014).









Pronucleotides Assemble! A multifunctional catalyst designed to stereoselectively synthesise prodrugs

Nucleosides have emerged as a key chemical class in successful antiviral and anticancer drugs with nearly half of currently marketed drugs possessing these cores.1 However, the biologically active nucleoside phosphates, which are generated in vivo by phosphorylation, are poor drug candidates due to issues of permeability and stability. One pronucleotide strategy developed to address these challenges has been described by McGuigan. His ProTide platform introduces a 5’-arlyoxy phosphoramidite to the drug candidate which can result in improved cell permeability and rate of phosphorylation compared with non-phosphoramidite containing nucleosides.2,3

However the introduction of the phosphoramadite in the prodrug can have a significant effect on the potency, toxicity and rate of metabolism, effects which can be associated with the stereochemistry at the phosphoramidite phosphorus. Unlike for carbon chemistry, where stereocontrol is a sophisticated branch of asymmetric catalysis, p-chiral chemistry is far less developed. Although methods do exist to steer products towards a preferred p-chiral isomer, such as dynamic kinetic asymmetric transformation (DYKAT) using chiral auxiliaries or desymmetrisation of achiral species,3 these approaches have significant drawbacks with the former suffering from poor selectivities and low catalytic turnovers, whilst the latter is often a complicated multistep synthesis.

In addition to the stereo-isomeric challenges during prodrug assembly there also remains the challenge of chemoselectivity for 5’ versus 3’ phosphoramidation. With these challenges in mind, I wanted to highlight an excellent paper published by a team from the process research and development group of Merck & Co., USA, where they report the first good example of a catalyst being designed to address the issue of stereo- and chemoselectivity in the synthesis of pronucleotide prodrug candidates. In their paper they focused on a hepatitis C virus RNA polymerase inhibitor currently in late stage clinical trials (MK-3682, Figure 1).4

Figure 1Figure 1: General phosphoramidation scheme and the best in-class catalysts developed to effect the coupling. Yield is the total yield of phosphoramidite isolated, chemoselectivity for 5’ vs 3’ is represented by ratio 5’:3’and d.r is the ratio of P(R) to P(S).

Using mechanistic studies, computational modeling and an understanding about the enzymatic mechanism of P-O bond formation in the phosphorylation of nucleosides, the team successfully developed several small molecule organic catalysts that mimic the concomitant series of activation modes used by enzymes to effect the P-O bond formation. Early studies identified carbamates as a privileged class for controlling stereo- and chemoselectivities with catalyst (R)-B being the best of the first generation catalysts developed. Using computational modeling the carbamate was theorized to carryout 3 roles; leaving group activation, general base catalysis and oxyanion stabilization via a pentavalent transition state, giving rise to a 2.3kcal/mol differentiation between the desired R-stereochemistry and the S-stereochemistry at the phosphoramidite phosphorus (Figure 2). Catalyst I, the best of the catalysts reported, evolved from a conscious effort to increase the transition state differentiation by decreasing the entropy of the system via linkage of catalyst (R)-B. As highlighted in Figure 1 the linked catalyst, Catalyst I, was able to achieve excellent yields and high selectivities for both the desired 5’ product (99:1 in favour of the 5’ product) and with a d.r (of 99:1) in favour of the desired P(R) isomer.

Figure 2Figure 2: Transition state model showing multiple catalyst modes of action. Reproduced from reference 4.

This work demonstrates an excellent step forward in the controlled synthesis of pronucleotide prodrugs by continuing to employ rational design beyond the discovery phase SAR, well into the late stage development of the prodrug. Moreover the published work is an elegant example of the power of using an interdisciplinary approach to solve chemical problems via a rational design cycle.


Written By Jason A. Gillespie



  1. P. Jordheim, D. Durantel, F. Zoulim, C. Dumontet, Nat. Rev. Drug Discov. 12, 447–464 (2013).
  2. Cahard, C. McGuigan, J. Balzarini, Mini Rev. Med. Chem. 4, 371–381 (2004).
  3. J. Sofia et al., J. Med. Chem. 53, 7202–7218 (2010).
  4. A. DiRocco et al., Science, 356, 426–430 (2017).