Nickel-Catalyzed Cross-Coupling of Redox-Active Esters with Boronic Acids

Whilst looking for sp2-sp3 cross coupling conditions I came across this interesting paper (Angew. Chem. Int. Ed. 2016, 55, 9676) from the Baran group titled “Nickel-Catalyzed Cross-Coupling of Redox-Active Esters with Boronic Acids”. This paper expands the use of N-hydroxyphthalimide (NHPI) esters that they had published earlier in the year (J. Am. Chem. Soc. 2016, 138, 2174−2177) in which they coupled aryl zinc reagents with alkyl esters of N-hydroxyphthalimide.

This paper uses this discovery from the Baran laboratory that N-hydroxy-tetrachlorophthalimide (TCNHPI) esters are able to accept an electron from a low-valent metal in a single electron transfer based thermal process. Using moderate temperatures thermal decarboxylative radical formation was achieved and this radical was immediately captured by a transition metal (Ni). This new cross-coupling reaction allows for the facile coupling of activated alkyl carboxylic acids and boronic acids, figure 1.


Baran gives a snapshot into the optimisation of the reaction conditions but explains they were arrived at by extensive experimentation and some of the empirical observations are poorly understood, figure 2.


It was found that DMF was necessary as a co-solvent in 1,4-dioxane for the reaction to proceed in a reasonable yield. Triethylamine was the best base tested and an optimal metal to ligand ratio of 1:1 for the NiCl2 : 4,4′-di-tert-butyl-2,2′-dipyridyl (BBBPY) system was described. Activated TCNHPI esters were used in placed of the previously used NHPI. The activated NHPI esters were more electron rich and incompetent coupling partners under these reaction conditions. All of the reagents for this reaction are commercially available and reasonably priced.

There are over 30 cross-coupling examples given in this paper which cover primary and secondary alkyl carboxylic acids, heteroaromatic boronic acids and show the tolerance for various functional groups. Baran has also shown that this reaction can be telescoped with the in situ generation of the activated ester, figure 3.


The experimental ease of this reaction was demonstrated by using wet solvents and a flask open to the air whilst still achieving a 65% isolated yield. The reaction was also performed on a gram scale with a 61% yield, figure 4.


A mechanism for this reaction has been proposed based on prior mechanistic investigations of Ni-catalysed reactions alkyl halides and Baran’s previous studies using organozinc reagents. Initially Ni complex I undergoes a base and water aided transmetalation with an arylboronic acid to give complex II. Reduction of the activated TCNHPI ester by complex II gives intermediate III which fragments to give the alkyl radical and phthalimide anion. This radical and anion combine with complex IV to yield complex V. The desired product is formed upon reductive elimination along with regenerating the catalytically active species I.


Although the scope of this reaction in very general Baran does highlight a few examples when diminished yields are observed. These include when an ortho-methoxy group is present on a boronic acid or if the activated ester is labile to hydrolytic cleavage.

This short communication describes a very simple and mild reaction that uses cheap and readily available reagents. It is tolerant of a range of functional groups and offers an attractive route to rapidly synthesise an array of compounds using a sp2-sp3 bond formation.

Blog written by Lewis Pennicott



How does ketamine alleviate depression?

Ketamine, which was initially evaluated on prisoners in 1965 (see here) was introduced in the 1960s as a dissociative aesthetic that was preferable to phencyclidine (PCP). It is also widely used in veterinary medicine as a horse tranquiliser and is used non-medically as a psychoactive drug that has earned the moniker Special K. The effects of ketamine are widely attributed to its antagonism of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, which are well-known to perform a number of critical functions within the central nervous system. When Berman and colleagues initially reported in 2000 that single i.v. infusions of subanaesthetic doses of ketamine produced rapid antidepressant effects in depressed patients (see here), there was surprise and amazement in equal measure. The surprise was that a field that had previously been dominated by the hypothesis that depression is caused by an imbalance in monoamine neurotransmitters now had to incorporate a completely novel mechanism, namely hyperfunction of NMDA receptors. The amazement was derived from the size and speed of onset of the antidepressant effect, which was in the region of tens of minutes rather than the weeks required to manifest the effects of “traditional” antidepressants. Initially, these data appeared to be too good to be true and were greeted with a healthy degree of scepticism. Accordingly, the scientific community set about trying to reproduce these data and sure enough, carefully controlled studies replicated these initial observations (see here and here) to the point that the antidepressant effects of ketamine are now generally accepted.

Ketamine is a mixture of two mirror-image molecules (enantiomers); (R)- and (S)-ketamine and Janssen, the pharmaceutical arm of the global healthcare giant Johnson & Johnson, recently reported that (S)-ketamine (esketamine) was able to produce antidepressant effects (see here). The collective data for ketamine and the presumed NMDA receptor antagonism mechanism of action have resulted in a number of NMDA-related drugs being pursued as novel antidepressants (Table 1, see also here) with the commercial interest in this area being reflected by the $560 million that Allergan paid to acquire Naurex and their GLYX-13 (Rapastinel) and NRX-1074 assets (see here).


However, as with all good scientific stories, recent data has provided a surprising twist. Hence, recent data from Zanos and colleagues (see here) suggests that not only might (R)-ketamine be more biologically active that (S)-ketamine, but that the biological activity may actually be associated with the active metabolite (2R,6R)-hydroxynorketamine (HKN) with effects being mediated via AMPA rather than NMDA receptors.



Figure showing the metabolism of racemic (R,S)-ketamine into corresponding (R) and (S) enantiomers of norketamine (norKET) and the (2S,6S) and (2R,6R) enantiomers of hydroxyketamine (HK) and hydroxynorketamine (HNK).

For example, ketamine produced a greater antidepressant-like effect in female mice compared to male mice which corresponded to higher levels of the (2S,6S;2R,6R)-HNK in female compared to male mouse brains. In addition, deuteration of (R,S)-ketamine not only reduced the production of 2S,6S;2R,6R)-HNK but also prevented deuterated ketamine having any antidepressant-like effects in either the mouse forced-swim or learned helplessness tests. More detailed analysis of 2R,6R-HNK showed that this metabolite produced a much greater antidepressant-like effect in mouse models of depression relative to the 2S,6S metabolite.


Left hand panel shows that (2R,6R)-hydroxynorketamine (HNK) has greater efficacy than (2S,6S)-HNK in the mouse learned helplessness test. Right-hand panel shows that (2R,6R)-HNK is not self-administered in mice whereas racemic ketamine is, indicating that compared to the parent, the metabolite has no rewarding properties and is therefore unlikely to be a drug of abuse.

More remarkable, however, were the observations that (2R,6R)-HNK (and also (2S,6S)-HNK) did not affect NMDA-induced currents in a rat hippocampal slice model but rather increased AMPA-mediated excitatory postsynaptic currents. The in vivo antidepressant like effects of (2R,6R)-HNK could be blocked by prior treatment of mice with the AMPA receptor blocker NBQX and the longer-lasting (i.e., 24-h postdose) antidepressant effects of (2R,6R)-HNK were associated with an increased expression of the GluA1 and GluA2 subunits of the AMPA receptor.

Is it actually important that we understand the mechanism of ketamine’s antidepressant effects? Well, the simple answer is no and yes; no from the patient’s point of view since he/or she is not too concerned about how they have got better, only that they have got better; and yes, because despite ketamine’s remarkable effects, the therapeutic use of ketamine (or esketamine) is associated with significant issues related to its mechanism-related dissociative side-effects and its route of administration (intravenous or possibly intranasal). Accordingly, we need to understand the mechanism of action to develop new and improved drugs that retain the antidepressant effects but are devoid of the issues associated with ketamine itself. Irrespective of what the actual mechanism of action of ketamine turns out to be, there is little doubt that we are on the verge of a major breakthrough in the treatment of treatment-resistant depression, and possibly the broader population of depressed patients. This in turn could not only revolutionise the treatment of depression but could reenergise the whole field of psychiatric drug discovery that has become a backwater of pharmaceutical company drug discovery over the last decade or so.

Blog written by John Atack


The importance of using the right tool for the job


As with many jobs that we have to do both in and out of work, it is important to have the right tools for the right job.  However, many research efforts are being undermined by the quality of the tools employed – particularly when it comes to small molecule chemical tools.  One of the major challenges when it comes to unravelling the complexities of the biology underpinning any disease is the ability to ask precise questions and to effectively test hypotheses– as both are entirely dependent on the quality of the reagents employed.  Over the recent years, there has been a growth in the range of specific, available molecular biology reagents to control and quantify gene, transcript or protein production as well as an increased awareness of the need for quality control and rigorous experimental conditions to ensure that any data generated are meaningful.  However, this vigilance has been less evident in the use of small molecule chemical ligands in target validation studies – and in particular, many groups have put their faith in molecules that are either reported in top peer-reviewed journals as being selective inhibitors, or are sold as such.  This has led to many papers reporting findings which are fundamentally unsound, and leads to a cascade of wasted time and effort over many years.  There are many challenges to be faced in initiating and prosecuting drug discovery projects and it is imperative that we do not make the journey any more difficult by using inappropriate tools in key validation studies.

Unfortunately, although the experienced community has done much to shout about this problem – both regarding the lack of reproducibility of published data (links to – and and and also the need for high quality chemical probes ( and and and and, the wider problem of the continued use of sub-optimal molecules continues.  There are many reasons why small molecule inhibitors might be non-specific, or indeed actually false positives, but the experience to understand these data often doesn’t reside in the groups that are needing to use the tools for complex disease / pathway mechanism studies.

To rise to this challenge, a new website has been created ( which aims to increase the quality and reproducibility of biomedical research using chemical probes.


The website contains fundamental information for the wider community on the properties required for a chemical probe, details on common pan-assay interference compounds (link – and recommended best practice covering the use of chemical probes in target validation experiments.  Working behind the scenes at this new portal is a team of experienced scientists from across the globe that have enormous collective experience in the fields of assay technologies, medicinal chemistry, chemical biology, target validation and drug discovery & development.  This team is systematically working through published ‘chemical probes’ and providing expert commentary on their suitability for use for both in vitro and in vivo experiments.    The concept is still in its early phase – there are lots of probes already evaluated and their reviews are available for anyone to read – and many open calls are drawing in probes for a wide range of new target classes.  Hopefully, with the support of both the user and reviewer communities, this effort will start to make a big impact on the quality of disease target validation and hence on the choice of early drug discovery projects in the future.

Blog written by Simon Ward




The antibiotics crisis…..

Microorganisms have always been able to develop resistance to antibiotic through various mechanisms, however, this process has been hugely accelerated by their misuse and overuse in particular in controlling bacterial infection in intensive animal farming. This has recently culminated with the isolation of bacterial strains resistant to colistin, an antibiotic used as a last resort for life-threatening infections. The WHO issued several warnings, however no strict actions were undertaken, and as a consequence we are getting closer to a point of no-return, the so called post-antibiotic era.

Antibiotic resistance is now a major issue for public health and is leading to increased mortality (>50000 in Europe and US alone every year), longer hospital stays and as a consequence having a huge impact on national health care budgets. If no action is taken, it is estimated that by 2050 10 million people worldwide will die every year from antibiotic resistant bacterial infections. Despite the urge for new and efficacious antibiotics able to fight resistant bacterial strains, only two new classes of antibiotics have reached the market in the last thirty years.

Why is it so difficult to develop antibiotics with a novel mechanism of action? There are two main reasons why pharmaceutical companies are not developing new antibiotics: one is economical (low investment return) as the newly approved drugs will be used only against multi-resistant strains (small number of patients); and the second is technical (identification of new targets and chemical matter), has proven to be quite challenging. Genomics have allowed the identification of new validated targets to start antibacterial drug discovery programs, with >60 targets being screened by 34 companies. A huge effort was made by GSK (running 70 HTS screens) and AstraZeneca (running 65 HTS screens), which have led to the identification of very few lead series. However, the biochemical activity of these compounds was not translated in cellular assays, with potential antibiotics not being able to penetrate across the bacterial membrane. One of the main reasons behind this attrition is down to physicochemical properties of the chemical libraries used, which nowadays are finely honed to follow Lipinski’s rule of five, rules that are not strictly followed by antibiotics.

In this context, Innovative Medicines Initiative (a partnership between the European Union and the European Federation of Pharmaceutical Industry and Associations) has launched a €700 million programme “New Drugs 4 Bad Bugs” to tackle antimicrobial resistance as a collaboration between industry, academia and biotech organization.  Of particular interest from an early stage drug discovery point of view, are the projects TRANSLOCATION (Molecular basis of the bacterial cell wall permeability), aimed to better understand the complex machinery behind drug transport and efflux in gram-negative bacteria; and ENABLE (a drug-discovery platform for antibiotics) aimed at lead-optimisation of promising compounds and progressing them into drug candidates. The aim is to progress at the least one compound into phase 1 clinical trials by 2020. Other projects that are part of this programme include: COMBACTE (creating a pan-European network of clinical sites), COMBACTE-CARE (taking on the most dangerous resistant bacteria), COMBACTE-MAGNET (help on healthcare-associated infections), iABC (new treatments to help cystic fibrosis patients), DRIVE-AB (New economic models for antibiotic development). In 2015, the Centers for Disease Control and Prevention (US) has launched their strategy to combat antibiotic resistance (National Strategy for Combating Antibiotic-Resistant Bacteria (CARB))

Several initiatives and research programmes are currently trying to solve this antibiotics crisis; in the meantime the WHO provides guidelines on how everyone in society, from the general public to doctors, pharmacists, veterinarians and farmers, can help to slow down the process of antibiotic resistance.

Blog written by Marco Derudas


Sensing a breakthrough….

Touch is one of the five key senses that allow humans to effectively interact with their external environment. It’s also important for proprioception, the body’s sensing of the relative positioning of neighbouring body parts and the strength of effort being employed in movement. The ability to sense force, scientifically termed mechanosensation, has been the subject of much scientific endeavour. Despite this our understanding of this fundamental process at the molecular level has until recently been lacking. Electrical changes in cells and tissues in response to mechanical deformation have been widely reported and characterised, but it wasn’t until 2010 that Ardem Patapoutian and colleagues identified the Piezo proteins (Coste et al 2010). This small family (2 members at present) of unique structural proteins (Piezo1 & Piezo2) give rise to mechanically activated cation channels.

Since their discovery there have been a number of reports of human diseases arising from genetic mutations in the Piezo channels (table 1). These Piezo associated channelopathies result in significant and severe musculoskeletal phenotypes but have surprisingly not been associated with any defect in touch or proprioception.

Author, year Channel Disease Description
Zarychanski et al 2012;

Bae et al; 2013

Piezo1 Hereditary xerocytosis Hemolytic anaemia characterised by erythrocyte dehydration; gain of function (exhibit slowed inactivation)
Lukacs et al 2015 Piezo1 Congenital lymphatic dysplasia Loss of function (reduced channel surface expression levels)
Coste et al 2013

McMillin et al 2014

Okubo et al 2015

Piezo2 Distal arthrogryposis type 3 & type 5 Bone developmental malformations (cleft palate) and contractures; gain of function (inactivation changes)

Table 1: recent papers describing human mutations in Piezo channels

However two recent papers (Chesler et al 2016; Mahmud et al 2016) have described for the first time loss of function mutations in Piezo2. These patients, in common with the Piezo2 gain of function mutations, have skeletal abnormalities including contractures. However both papers report a loss of certain elements of both touch sensation and proprioception consistent with Piezo2 being key to these processes. Specifically patients had selective loss of discriminative touch perception parameters (touch, pressure and vibration) but responded to types of gentle stimulation (slow brushing) on hairy skin. In contrast non-hairy skin (glabrous skin) did exhibit a sensory defect. Affected patients also displayed profoundly decreased proprioception leading to ataxia (disorder of balance and co-ordination) and dysmetria (inability to judge distance or scale characterised by undershoot or overshoot with position of limbs).

These papers have provided clinical insight into touch and proprioception and a key role for Piezo2.  Whether Piezo2 or its close family member, Piezo1, can be modulated to deliver therapeutic benefit remains to be seen. However the discovery of the first low molecular weight modulator of Piezo1, Yoda1 was recently published (Syeda et al 2015), potentially lowering this hurdle. This compound, the output of a large high throughput screening campaign, is an agonist of both human and mouse Piezo1 and optimistically will help clarify further the role of these enigmatic channels in human health and disease

Figure 1: chemical structure of Yoda1


Blog written by Martin Gosling


Chesler et al (2016) The role of PIEZO2 in human mechanosensation. New England Journal of Medicine doi: 10.1056/NEJMoa1602812

Mahmud et al (2016) Loss of the proprioception and touch sensation channel PIEZO2 in sibling with a progressive form of contractures. Clinical Genetics doi: 10.1111/cge.12850

Syeda et al (2015) Chemical activation of the mechanotransduction channel Piezo1. ELife doi: 10.7554/eLife.07369.

Kainate receptors in epilepsy: A tale of two subunits (?)

Kainate is a potent neurotoxin derived from a seaweed, Digenea simplex. The toxin has a high affinity for and lends its name to a distinct genetic family of ion channel receptors that are normally targeted by glutamate, the principal excitatory neurotransmitter in the brain.1 While broadly distributed, kainate receptors (KARs) are less abundant than the other glutamatergic receptor families, however they have long been implicated in the development and progression of epileptic seizures.2

Exposure to kainate induces symptoms and patterns of brain remodelling similar to that seen in temporal lobe epilepsy (TLE). While this has been used in the past to develop anti-epileptic drugs, until relatively recently none were targeted at the KARs themselves.3 Therapeutic options currently available for TLE and other forms of epilepsy, work by reducing the overall levels of neuronal excitability in the brain and often come with unwanted side-effects but progress in this compelling field of research has been hampered by a lack of highly selective agents with which to probe KAR function.

There are 5 subunits (GluK1-5) that can combine to form KARs. GluK1-3 may form functional channels on their own or in combination with the others, while GluK4-5 can only do so in combination with GluK1-3.1 Due to the highly similar genetic make-up of these subunits, most of what is known of them individually comes from studying each subunit in isolated cell expression systems.3 The most well studied KAR subunits are GluK1 and GluK2, which are highly expressed in and around the hippocampus, a key region of the brain involved in TLE. Under normal circumstances, GluK1 and 2 appear to play opposite roles to one another, with GluK2 present mostly on excitatory “principal” neurones in the hippocampal network, while GluK1 is more abundant on inhibitory “interneurons” that help regulate the activity of the former. In TLE, there is a major shift in the balance of signalling that results in GluK2 having a far greater influence with the result being a greater risk of epileptic patterns of activity.2

With this information, a first and obvious avenue for treatment might be to target the GluK2 receptors, however, in addition to the problems of selectivity, the picture is somewhat complicated by the presence of pre-synaptic GluK2 on inhibitory interneurons that actually enhance their inhibitory effects, inhibition of which would probably work against a blanket blockade of GluK2 receptors. Similarly, GluK1 is also present on principal cells and can reduce the regulatory effects mediated by cannabinoid receptors. 2

A way around these problems might be the development of allosteric modulators, drugs that positively or negatively alter receptor function, instead of just switching it off or on. By targeting specific GluK subunits in this manner, it may be possible to achieve better, targeted regulation of neuronal excitability. A development which may make this approach even more interesting is the report by Fisher that the different subunits in heteromeric KARs may independently influence the function of the whole channel without disrupting the normal functionality of the companion subunits.4 This is particularly intriguing from a drug development perspective as compounds developed on simpler, homomeric, receptors may still retain their expected functions at the more complex receptor mixes found on native neurones.

There may be much more to this tale yet.

  1. Traynelis, S.F., Wollmuth, L.P, McBain, C.J., Menniti, F.S., Vance, K.M., Ogden, K.K., Hansen, K.B., Yuan, H., Myers, S.J., Dingledine, R., and Sibley, D. (2010), Glutamate Receptor Ion Channels: Structure, Regulation, and Function. Pharmacological Reviews 62(3), 405-496
  2. Crépel, V. (2013), Kainate receptors in epilepsy. WIREs Membrane Transport and Signaling, 2: 75–83.
  3. Matute, C. (2011). Therapeutic Potential of Kainate Receptors. CNS Neuroscience & Therapeutics, 17(6), 661–669.
  4. Fisher, J. L. (2014). The neurotoxin domoate causes long-lasting inhibition of the kainate receptor GluK5 subunit. Neuropharmacology. 85, 9–17

Blog written by Iain Barrett


Searching for a cost effective alternative to Primary Human Hepatocytes…………still a way to go!!!

As our fledgling DMPK group looks to expand our portfolio of assays one of the main driving factors when evaluating a new method is the cost effectiveness of bringing a method in house as compared to employing a CRO.  We run a microsome stability screen as one of a panel of assays in our initial triage of compounds which upon progression are then assessed in a broader range of (generally more expensive) assays including CYP isoform inhibition, MDCK-MDR1 and Hepatocyte stability among others.  The average cost among four of the leading CROs offering the hepatocyte stability assay is ≈£600 per compound per species.  To purchase cryo-preserved hepatocytes and run the assay in the same format as the CROs would be ≈£100 per compound per species, a significant saving but still an expensive assay.  In addition, primary human hepatocytes (PHHs) are subject to inter-individual phenotypic variability; whilst suspensions of freshly isolated hepatocytes demonstrate near in-vivo levels of metabolic competence this is significantly reduced within 2-3 hours limiting the utility of assessing low metabolised compounds; rapid loss of canalicular and basolateral efflux transport in freshly isolated PHHs may also affect metabolic outcomes.  The metabolic competence of PHHS is therefore not an intrinsic property but dependent upon the culture environment.  Despite these drawbacks clearance intrinsic clearance in PHH suspensions is by the far the most widely used format and still represents the significantly more accurate indicator of intrinsic hepatic in vivo clearance when compared to data derived from rat/dog hepatocytes/allometric scaling or hepatocyte subcellular fractions.

The development of a more practical, stable, sustainable and less costly model of hepatocyte metabolism is therefore highly desired.  The ideal alternative cell line for intrinsic clearance in suspension would therefore (apart from being cheaper than PHH) stably express the entire array of CYPs, Phase II enzymes and (as much at least as freshly isolated suspension PHH) transporter proteins at physiologically relevant levels.  Immortalised and hepatoma derived cell lines including HepG2, Huh7 and Fa2N-4 have been investigated as such alternatives but all lack significant expression for large swathes of Liver-specific function.  HepaRG cells demonstrate significant advantage over these cell lines and recent papers (1,2) suggest interest in using these cells in the pharmaceutical setting may be growing especially with the recent availability of pre-differentiated cryo-preserved HepaRG cells.

Originally described by Gripon et al (3) HepaRG cells are a hepatoma cell line that can be differentiated into a phenotype that closely resembles mature hepatocyte which exhibit cellular interactions, drug metabolism/transport, and drug induction responsiveness comparable to PHH cultures.  After 6 weeks under differentiating conditions in culture (2 % DMSO, 50 µM hydrocortisone hemisuccinate) the expression of the main phase I & II enzymes and transport proteins averages between 20-100% of levels exhibited in freshly isolated PHH (with the notable exceptions of CYP7A1, GSTA1, MDR1, MPR1 which demonstrate a >5x increase (4)) but which closely mirror the levels expressed in sandwich/3D cultured PHH.  CYPs 2C9, 2D6 and 3A5 also all contain poor metaboliser alleles but this reflects the typical Caucasian allotype of the original donor.  Chr 22 (which encodes 2D6) also only has one copy in HepaRG cells contributing further to low expression levels.  Additionally HepaRG cells demonstrate cytosolic sequestering of the transcription activator CAR and translocation to the nucleus upon activation by phenobarbital, a crucial PHH hallmark lacking in most hepatic cell lines.

Whilst differentiated HepaRG cells demonstrate a reasonable level of hepatic function and morphology after ≈10 days in culture (compared to cultured PHH) such that their utility as a model for hepatotoxicity has been clearly demonstrated (5) they still fall short of being a suitable substitute for performing intrinsic clearance studies in suspension.  One problem is that the cells need to be maintained in 2% DMSO to enable adequate differentiation to a hepatocyte phenotype via activation by, for instance, AP-1 (induces cell cycle arrest and differentiation) which also exerts transcription regulation across a broad array of metabolic genes.  Efforts are currently being made to develop methods to differentiate HepaRG cells without the use of DMSO (6) and with the emergence of CRISPR as a means to rapidly and easily modify genes (e.g. replacing low metabolic alleles) perhaps it won’t be too long before we have cost effective, stable, widely available and consistent source of cells with which to perform reliable Intrinsic clearance assays.

  1. Ferguson, S et al (2016) ‘Contextualising Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures’ Drug Metabolism and Disposition 44: 1463-1479
  2. Plevris, J et al (2016) ‘Human Hepatic HepaRG Cells Maintain an Organotypic Phenotype with High Intrinsic CYP450 Activity/Metabolism and Significantly Outperform Standard HepG2/C3A Cells for Pharmaceutical and Therapeutic Applications’ Basic & Clinical Pharmacology & Toxicology 15 JUL 2016, DOI: 10.1111/bcpt.12631
  3. Gripon, P et al (2002) ‘Infection of a Human Hepatoma Cell Line by Hepatitis B Virus’ Proceedings of the National Academy of Sciences of the United States of America, 99 (24): 15655–15660
  4. Andersson, T et al (2008) ‘Evaluation of HepaRG Cells as an in Vitro Model for Human Drug Metabolism Studies’ Drug Metabolism and Disposition 36 (7): 1444–1452
  6. Sakharov, D (2016) ‘Maintenance of High Cytochrome P450 Expression in HepaRG Cell Spheroids in DMSO-Free Medium’ Bulletin of Experimental Biology and Medicine 161 (1): 138-142

Blog written by Marcus Hanley

New Single-Molecule Patch-Clamp FRET Assay

The single channel patch clamp technique is a powerful tool that provides a direct measure of ion channel function in living cells. When the single channel technique is combined with fluorescence techniques (particularly FRET microscopy technology), it becomes a more powerful tool to study single molecules, and in which conformational domain (ie open-intermediate-close state) they bind. Conformational changes play a critical role in the activation, deactivation, and open–closed activities of ion channels and conformational dynamics is often extremely difficult to be directly characterized by spectroscopic imaging or by single channel patch-clamp alone.  In a recent publication in journal of American Chemistry Society (JACS) by Sasmal & Lu (2016), they have developed a new and combined technical approach, single-molecule patch-clamp FRET microscopy (FRET anisotropy microscopy), to probe ion channel conformational dynamics in living cell by simultaneous and correlated measurements of real-time single-molecule FRET spectroscopic imaging. They used the ligand gated ion channel NMDA to investigate how NMDA and glycine ligands bind to the binding domain. The method is capable of simultaneously recording single-molecule FRET efficiency and conformational changes of single NMDA receptor channels. This allows accurate characterisation of ligand binding events (binding and unbinding of ligands) in direct relation to changes in the receptor/channel conformation.

Schematic diagram showing the FRET microscopy technique – this is able to record single channel events and four channel fluorescence measurements simultaneously allowing correlation of: 1. electrically on and off state by patch clamp, 2. optically determined conformational close and open state by FRET and, 3. binding and unbinding state of glycine ligand by anisotropy measurement at the LBD of GluN1 subunit.

A                                                                                                     B.




J Am Chem Soc. 2016 Jul 20;138(28):8789-801.

The report provides new insight into some of the functional states of NMDA receptors, in this case recombinantly expressed in HEK-293 cells. Specifically the authors shows that seemingly identical electrically off states (ie channel is closed with no ions flowing) are associated with multiple conformational states. On the basis of experimental results, they have proposed a multistate clamshell model to interpret the NMDA receptor open–close dynamics (Diagram B). Thus single molecule FRET technique is a powerful and sensitive approach for probing the conformational intermediate state of protein and ion channel receptors.

C                                                                                              D


J Am Chem Soc. 2016 Jul 20;138(28):8789-801.

In conclusion, this study has demonstrated that binding kinetics as well as ion channel conformation dynamics and the method has the ability to detect the intermediate states in addition to electrical on and off states.


 1) Sasmal DK1Yadav R1Lu HP1 (2016) J Am Chem Soc. 2016 Jul 20; 138(28):8789-801. doi: 10.1021/ jacs.6b03496. Epub 2016 Jul 12.



Crystallography in Fragment-based drug discovery

Fragment-based drug discovery (FBDD) serves as a starting point for the development of drug candidates to generate high affinity ligands. X-ray crystallography, a structural method, can be used to map the interactions of small molecules with proteins, rapidly and efficiently increasing the development of drug discovery. Early FBDD projects utilizing crystallography method as primary screening methods, can directly discover truly positive fragments, although crystal structures of protein should have a high diffraction value.

The process of fragment screening based on crystallography is illustrated in Fig.1.[1, 2] The purpose of this method is to expose protein to fragments and solve the crystal structures of the complexes. It, in most cases, involves growing crystals of the target protein and soaking them in solutions of the fragments, either as single compounds or as cocktails of compounds.[2]



Figure 1.Typical flow chart for high-throughput ligand screening using crystallography. [1, 2]

Fragments are followed by “rule of three” [3], i.e. molecular weight < 300 Da; clogP ≤ 3; number of hydrogen-bond donors ≤ 3; number of hydrogen-bond acceptors ≤ 3. Compared with small molecules, fragments have a lower binding affinity, but a higher hit rate to the target protein. Because of its low binding affinity feature to the target protein, a relatively high concentration of compounds are used in crystallography, in the region of 25–100 mM.[2]

The crystal structure of a protein with a high resolution is important for this method. It must be robust, stable under soaking conditions and diffract to beyond about 2.5 Å resolution—sufficient to place fragments unambiguously in electron density.[4] Besides, it is necessary to generate crystals of a similar size and quality on a large scale for the fragment screening.

From previous researches, crystallography for fragment-based screening has been successfully used to discover inhibitors, especially some difficult targets such as b-secretase[5], as well as inhibitors of a wide range of other enzymes: hPNMT, Phosphodiesterase 4A, Hsp90, Bromodomain, Adenosine A2 receptor, etc.. (Table1) One classic example is the discovery of inhibitors of β-secretase.

Murray and co-workers[5] screened a library containing 347 fragments in cocktails containing six compounds. Two hits found to have nearly identical interactions with Beta secretase-1(BACE-1), forming hydrogen bonds with catalytic aspartate residues D32 and D228. Then, they identified further fragments to access to two important regions which were important for substrate peptide binding by molecular docking and crystallography for fragment-based screening.

Article title Target protein Primary/secondary
FBDD screening
Application of Fragment Screening by X-ray
Crystallography to the Discovery of
Aminopyridines as Inhibitors of -Secretase
β-Secretase X-ray crystallography Fluorescence-based
activity assay
Missing fragments: detecting cooperative
binding in fragment-based drug design
hPNMT X-ray crystallography ITC/Molecular
dynamics free energy
Fragment-based screening for inhibitors of
PDE4A using enthalpy arrays and X-ray
Fragment-Based Drug Discovery Applied to
Hsp90. Discovery of Two Lead Series with
High Ligand Efficiency
Hsp90 NMR/X-ray
Fragment-Based Discovery of Bromodomain
Inhibitors Part 1: Inhibitor binding modes and implications for lead discovery
Bromodomain Fluorescence anisotropy
anisotropy assay
Fragment-Based Discovery of Bromodomain
Inhibitors Part 2: Optimization of
Phenylisoxazole Sulfonamide
AcK pocket
Fluorescence anisotropy
assay/Modelling X-ray
SPR/Thermal shift
Structure-based design of potent and
ligand-efficient inhibitors of CTX-M class A
based bioassays/
Antibacterial activity
Discovery of 1,2,4-triaine derivatives as
adenosine A2A antagonists using structure
based drug design
Adenosine A2
Discovery and Optimization of New
Benzimidazole- and Benzoxazole-Pyrimidone
Selective PI3Kβ Inhibitors for the Treatment
of Phosphatase and TENsin homologue
(PTEN)-Deficient Cancers
PI3K In vitro enzyme
assay/Cell based assay
X-ray crystallography
In vitro enzyme
Synthesis, Structure–Activity Relationship
Studies, and X-ray Crystallographic Analysis
of Arylsulfonamides as Potent Carbonic
Anhydrase Inhibitor
Stopped-flow kinetic
Implications of Promiscuous Pim-1 Kinase
Fragment Inhibitor Hydrophobic Interactions
for Fragment-Based Drug Design
Pim-1 Kinase Docking/X-ray
Mobility shift assay

Table 1. Some examples of fragment-based screening.[4]

Blog written by Xiangrong Chen


[1] I. Tickle, A. Sharff, M. Vinkovic, J. Yon, H. Jhoti, High-throughput protein crystallography and drug discovery, Chemical Society Reviews, 33 (2004) 558-565.

[2] H. Jhoti, A. Cleasby, M. Verdonk, G. Williams, Fragment-based screening using X-ray crystallography and NMR spectroscopy, Current Opinion in Chemical Biology, 11 (2007) 485-493.

[3] M. Congreve, R. Carr, C. Murray, H. Jhoti, A ‘Rule of Three’ for fragment-based lead discovery?, Drug Discovery Today, 8 (2003) 876-877.

[4] Z. Chilingaryan, Z. Yin, A.J. Oakley, Fragment-Based Screening by Protein Crystallography: Successes and Pitfalls, International Journal of Molecular Sciences, 13 (2012) 12857-12879.

[5] M. Congreve, D. Aharony, J. Albert, O. Callaghan, J. Campbell, R.A.E. Carr, G. Chessari, S. Cowan, P.D. Edwards, M. Frederickson, R. McMenamin, C.W. Murray, S. Patel, N. Wallis, Application of Fragment Screening by X-ray Crystallography to the Discovery of Aminopyridines as Inhibitors of β-Secretase, Journal of Medicinal Chemistry, 50 (2007) 1124-1132.

Zika virus: A neglected disease with no specifically designed drugs

I was shocked to see in recent weeks how a potential connection between a virus infection and  pregnant women have led to a global concern for the association with a high number of babies being born with microencephaly, initially in Brazil but with other cases shown worldwide, and it has even been associated to the rare Guillain-Barré Syndrome (1). Children with these syndromes are likely to have shorter life expectancies. A recent opinion article (2) with extensive research on this particular virus is analised in this blog.

After the epidemic outbreak of the Ebola virus in 2014-2015 which killed thousands of people in Africa and risked to generate a pandemic crisis, mobilising the World Health Organisation (WHO) and Governments, we have been exposed to another case of unprepared health concern with serious global implications.

Zika virus (ZIKV) is a virus from the Flaviviridae family with genetically similarities with the ones responsible for the Dengue Fever and the Yellow Fever. ZIKV was isolated and reported over 60 years ago and since then the small number of publications, the lack of a crystal structure, the abscence of reports of molecules having been screened either in vitro or in vivo in animal models, and the lack of patents covering drugs targeting ZIK virus (although there are some focused in compounds addressing the Dengue Fever), has left it as an undoubtedly case of a “Neglected Disease”.

So, despite of having some knowledge of the virus, little if not nothing seems to have been done in order to understand its risks, exposing under this circumstances, right now, its danger after the outbreak. The WHO has had to move faster than 2 years ago with the Ebola epidemic, and has  issued a Public Health Emergency of International Concern (PHEIC).

Ekins and co-workers(2), after extensive research on related viruses and taking into consideration antivirals and non-related drugs, suggest to create a fast-track plan of action in order to tackle the problem with more urgency, leadership and preparation. This could be applicable to other future outbreaks and put us in a better situation to fight future epidemics.

With the information we have on other closely related virus like the Dengue, the immediately plan of action against this outbreak should have to start with the use of already FDA-approved antivirals profiled against related viruses (with a safety and efficacy profile proved) as a starting point in fighting the ZIKV, and test other drugs, non antivirals and compounds from commercial sources (eg. Libraries) in a descendent order of priority as shown in Fig 1.


Figure 1. Compounds and chemical libraries suggested to be tested against Zika virus

Without much further do, the authors propose the following Drug Discovery plan:

  1. To develop a cell or ZIKV-target based in vitro assay, which might have to be done in special protective environments, limiting the number of companies or organisations capable of such as assays.
  2. The immediate test of all kind of available drugs (up to 48 FDA approved known antivirals) into the previously generated and validated assay, capable to produce some results against the absence of any other treatment, as reflected in Table 1.
  3. To study and understand the genome of the ZIKV and how a chemotherapy approach could lead to effectively target the virus.
  4. To develop and use “homology models” showing the suggested protein sequence based in similar/ related viruses with known molecule action-modes using target prediction software, such as SWISS-MODEL.
  5. To establish a pharmacological profile with a suitable/ ethical animal model


Table 1. List of potential compounds to tes.t

Equally important and without leaving it off the schedule, the scientific community should undertake efforts to reveal the virus structure, its function and physiology and establish the relationship between the infection and the human neurological abnormalities.

Despite of getting even better co-ordinating resources through management organisations like WHO, more should be addressed in an emergency outbreak from National Governments and Health Institutions through funding (eg. from the FDA repurposing approved drugs, from big pharmaceutical companies through the donation of chemicals to be tested, from biotechs investing in the development of ZIKV-based in vitro assays, from ground-field-experienced organisations like Medecins Sans Frontieres, etc…)

We need to retain alert against highly likely-to happen diseases lurking upon us at any time and learn from past actions to make us better prepared to fight them.

Blog written by: Jose Gascon


  1. Oehler E, Watrin L, Larre P, Leparc-Foffart I, Lastere S, Valour F, Baudouin L, Mallet HP, Musso D, Ghawche; Zika virus infection complicated by GuillainBarre syndrome–case report, French Polynesia, December 2013. Euro Surveill. 2014; 19(9): 20720
  2. Ekins S, Mietchen D, Coffee M, Stratton TP, Freundlich JS, Freitas-Junior L, Muratov E, Siqueira-neto J, Williams AJ, Andrade C; Open drug discovery for the Zika virus. F1000Research 2016, 5:150