The importance of knowing how long drugs stick around bound to their targets

A really useful summary published recently by Robert Copeland in Nature Reviews Drug Discovery looks back at the concept of drug residence time.  The core principle, that the drug-protein target lifetime or residence time is the key to the observed downstream pharmacological effect has been developed, discussed (and variously challenged) over time, and this paper contains many useful references to the wide data sets published.  (The contradictory views are not really commented upon – although in my opinion most of these relate to concerns of the over-simplification or generalisation of the principles outlined, rather than fundamental disbelief in the ideas).

All of us who work on drug discovery projects are very familiar with the principles of thermodynamic equilibrium assay readouts – XC50s, Kis etc., which are run under closed conditions within a simplified biochemical assay paradigm.  We are also all too aware that the real-life situation is considerably more challenging and that there exist many factors which govern drug concentration at the desired site of action.

In considering the lifetime of a drug bound to its protein, we’re basically looking at one of two simplistic models for all these discussions – either a straightforward model in which a molecule binds directly to protein as  b below or a molecule binds, causes a conformation change in the protein to induce a better fit – c below.


From: Nature Reviews Drug Discovery 15, 87–95 (2016) doi:10.1038/nrd.2015.18


Whichever is appropriate, the Koff is key to the residence time and generally the downstream pharmacology.   The great strength of this review is the number of examples and references that are included, which make it really helpful as a resource to get (back) into this area.  The overall message is that SAR should be built up using both Kd values and equilibrium affinity and, that technologies, such as SPR, are now able to provide these data in a timely and sufficient way.

Useful examples:

Measuring the kinetics of saquinavir binding to HIV1 protease and associated resistant mutants, found that the downstream IC50 for viral replication correlated well with Koff (or residence time), although the Kon was found to only vary by 2x across the molecule set.

A series of COX inhibitors was found to have COX2 over COX1 selectivity.  The COX2 binding was found to model well to an induced fit model and the COX1 binding to the one step model.  A slow value for k4 in picture c above was then found to fit with a greater effect seen for binding to COX2 than COX1.

A series of FabI enoyl reductase inhibitors demonstrated a striking correlation between the numbers of animals surviving infection with the given bacterium and residence time, with no correlation at all present to Ki for the target protein.

Similarly, a series of A2a receptor agonists demonstrated a clear correlation of efficacy with residence time and not with Kd.

A very nice study published on BTK (kinase) inhibitors exploited an active site cysteine to prepare a range of molecules as reversible inhibitors with varied electrophilicity.  These inhibitors achieved residence times from minutes to a week which correlated excellently with cellular residence and downstream efficacy.

An ex vivo assay of drug-target residence for HSP90 occupancy identified that longer residence times at HSP90 protein translated into greater duration of action in PD models.


Blog writted by Simon Ward

What’s new in the market: the 2015 FDA drugs approval

Another year has gone and forty-five new drugs have been approved by FDA. A nice analysis has been published by Asher Mullard in Nat Rev Drug Disc 2016, 15, 73-76, providing the statistic related to these new drugs (for the complete list see Table 1 in the paper).

2015 was a very prolific year with double approval rate with respect to the period 2005-2009 and with rejections at an all-time low, with only two drugs not been approved.

Twenty-five drugs were filed with a priority review (FDA will take action within six months from the filing compared to ten months for the standard filing), ten with a breakthrough status (status given to speed up the development and approval of drugs with significant advances over approved therapy), six with an accelerated approval (status given to drugs used to treat unmet medical need disease).

Figure 1 reports the percentage of approvals for each therapeutic area. Oncology played a big role with 1/3 of the approved drugs indicated for this therapeutic area.

Figure 1

Figure 1. Pie chart indicating the newly approved drugs divided by therapeutic area.

Of particular interest is Palbociclib, a first-in-class CDK4/CDK6 inhibitor developed by Pfizer for the treatment of hormone-receptor-positive advanced breast cancer, which has received accelerated and breakthrough designation status; forecast sales are predicted to be in the region of $5 billion per year by 2020; other predicted blockbusters are indicated in Figure 2, including two monoclonal antibodies: Alirocumab and Evolocumab, first-in-class inhibitors of PCSK9, a protein involved in the regulation of blood cholesterol level. Forecast sales for these two drugs are expected to be over $2 billion per year. In total over a third of the approved drugs are expected to be blockbusters.

Figure 2

Figure 2. Forecast blockbusters.

Twenty-one of the approved drugs have an indication for orphan diseases, of which about 50% are anticancer drugs, indicating the success in developing new and more selective anticancer agents, including four drugs to treat multiple myeloma.

Looking at 2016 potential new approval, forty New Molecular Entity and Biologics Licence Application were filed in 2015. A selection of these potential new drugs is reported in Figure 3.

Figure 3

Figure 3. New drugs seeking approval in 2016

This includes Eteplirsen, a first exon-skipping oligonucleotide for the treatment of Duchenne muscular dystrophy and Venetoclax, a first-in-class BCL-2 inhibitor for the treatment of chronic lymphocytic leukaemia.

We will find out their destiny during this year; keep a eye on our blog!

Blog written by Marco Derudas



Aptamers as positive modulators

What to do when you want to validate an assay for drug discovery, but there are little or no literature tools available?

Well according to a publication from a group at Astra Zeneca, the use of Aptamers could be one way to solve this problem. Aptamers are lengths of DNA or RNA, generally 20-100 bases, which could be used in the same manner as small molecule tools, binding to targets of interest.

Using the historically difficult target glycine receptor as a model system (GlyRα1), the authors  generated aptamer libraries of RNA using the SELEX methodology (systematic evolution of ligands via exponential enrichment). This process involves the exposure of an aptamer library to your target, which has been immobilised (here the team used a biotin/streptavidin interaction), any unbound material is washed away and bound material is collected and amplified by RT-PCR. This is repeated across a number of iterations, improving the overall success rate.

In this case the SELEX process was run with a variety of different sources of the glycine receptor.  The active molecules generated from this method were then further validated using a radioligand filter binding assay. This resulted in eight aptamers being selected for scale up and further profiling in a variety of glycine receptor assays.

Selective binding of the aptamers was shown using SPR using immobilized glycine receptor, this was further supported by immunofluorescence of fixed cells and live cell imaging experiments.  Functional profiling of the aptamers occurred using a membrane potential dye assay supported with patch clamp electrophysiology.

The SPR measurements revealed all eight aptamers had Kd values in the low nanomolar range.  Interestingly two of the eight aptameters had slow on and off rates of binding .The cellular locations from the imaging experiments showed that the majority of the aptamer was present in the cytosol, and to a lesser extent at the plasma membrane. The authors suggested the cytosol accumulation may be due to interactions with Golgi and endoplasmic reticulum.

The functional assays highlighted some interesting findings. Using the membrane potential dye assay, five of the aptamers gave results suggesting they were positive modulators of the glycine receptor.  When this was further explored with one of the aptamers (c2) in a single cell patch clamp it was shown to be a positive modulator.

Overall the publication uses a variety of different supporting techniques to identify a positive modulator aptamer of the glycine receptor.

Could these molecules have a brighter future than just tools and become an alternative to small molecule therapeutics?  The issue of stability and delivery of the treatment have to be solved in each case, but the answer is yes.  As the authors point out, the FDA has approved the aptamer Macugen used for the treatment of age related macular degeneration.  The significant drawback of this medication is the requirement that it has to be injected into the eye of the patient.  So for the moment, tools seem to the current use for aptamers, however other clinical uses will be developed.


Figure showing the positive modulation of glycine receptor using aptamer C2

Shalaly, N. D., Aneiros, E., Blank, M., Mueller, J., Nyman, E., Blind, M., Dabrowski, M. A., et al. (2015). Positive Modulation of the Glycine Receptor by Means of Glycine Receptor-Binding Aptamers. Journal of Biomolecular Screening, 20(9), 1112-1123. Retrieved from


Blog written by Gareth Williams

Drug discovery- a child’s play

Last Sunday I took my, almost 10 year old, daughter to our  first ever visit to the Brighton Science Festival.

The decision to go came after finding out that PhD students from the Sussex Drug Discovery Centre (SDDC), where having a workshop aiming to introduce children from 7 to 14 to the concept of drug discovery.

We arrive at Hove Park School, a secondary school in Hove where the event was taking place, around lunch time. We were hoping that arriving in the middle of meal time, the place would be less crowded, but we found out that families were still eager to visit the exhibitions with empty stomachs. As  soon as we arrived we headed to the class room where the SDDC where having their display, but we did not even attempt to wait in the queue. It seemed that our PhD students were attracting lots of attention, and later found out that the Saturday queues were even longer. So we went around the school and visited other stands. Navigating around the school was not easy, lots of confusing corridors, staircases and rooms with little or poor indication. On our third attempt to get into the room where the Drug Discovery  demonstration was taking place, we decided to wait in the 15min queue. Once in the queue kids received a lab coat, a pair of goggles a pen and a little notebook. Kids were getting excited even before starting their tour around the room.

Fifteen minutes later the group moved into the first stall where the children were received by James; James is a PhD student in Biochemistry and despite on his second day at the fair, and having countlessly repeated the same words James was enthusiastically talking about proteins. He had prepared an experiment to show how proteins work by demonstrating how a piece of liver (full of proteins) would destroy a H2O2 (a “nasty chemical” according to what kids would remember after the show)… and with a bit of liquid soap in the mixture the kids were screaming of excitement watching a big lump of foam expanding in a measuring cylinder.

Next was Katie’s turn to helps kids design a new drug!! Katie is doing her PhD in Chemical Biology also at the SDDC. The group of kids were given a chemistry model kit and a piece of paper, representing a bacteria’s protein, with a big white section, representing the pocket where the ligand binds. The children were given total freedom to attempt to create a molecule that could fit in the white pocket. Some of the designs were quite imaginative; while most of the kids tried to fit the pocket with a flat molecule, some other very creative kids went beyond that and build a 3D structure…we might see them working at the SDDC in the future!!  Once the kids had finished designing their drugs, they had to test whether their models would fit into the bacteria’s protein pocket made of papier mâché, and also check that it would not fit into a similar protein found in humans. What a clever way of explaining to kids what side effects of drugs are.


Tom and Hayley, both Chemistry PhD students at the University of Sussex, had the kids doing a real chemistry experiment!! Wasn’t that fun!! The intention of the demonstration was not to know which chemicals were being mixed and which reaction was taking place, but that by adding one chemical to the another a chemical reaction could occur. The PhD students had carefully planned the reaction by choosing one that would form a yellow precipitate when adding one colourless solution to another colourless one, resulting in even more screams of excitement when the yellow powder appeared inthe kids’ Erlenmeyer flasks. It was then demonstrated the purification method of re-crystallisation, which was performed by the demonstrators as it involved heating up the reaction flasks. Nevertheless, the children were carefully following up the discussion on how a chemical dissolves at high temperatures and precipitates again when cooled down.

Last but not least, Lucas, also a Biologist PhD student at the SDDC, went on to explain about biochemical assays. In kids’ words, if the drug they had made was good or not. This was again a hands on experiment where they added a solution of the drug into a solution of the protein, followed by the addition of the “detection reagent”. If the solution turned pink, Eureka!! the drug was doing its job.
Overall, the kids had a great time at the SDDC workshop, they had a chance to do some real experiments and they were even dressed up as proper scientists!! A success!!

I am sure next year the SDDC PhD students will be asked to repeat the job, however, I am also sure those involved will ask for a few more pair of hands. It was full-time non-stop two day job with no time for breaks in order to satisfy the eager of the little ones to discover the world of “Making Marvellous Medicines”.

Blog written by Carol Villalonga-Barber



“Druglike” space – an artefact of the reactions we (medicinal chemists) use?

Scientists from AstraZeneca have recently carried out an analysis of the impact of organic synthesis on medicinal chemistry programs and revealed that, with a few exceptions, the most common reactions used in 1984 are still used in 2014, and that the chemical space generated with those few more frequently used reactions is composed of structurally similar compounds and therefore maybe biasing the drugs that are emerging.

In the article, Brown and Boström compare the most frequent reactions in Medicinal Chemistry in 2014 versus 1984 (see Figure 1) and found out that only a few newer reactions have taken a space in the top 20 list, such as the Suzuki and Buchwald cross-coupling reactions. While the latter was first published in 1994, and therefore could not have been used a decade before, the Suzuki-Miyaura reaction was first published in 1981 but the impact of this reaction was not seen in 1984. Similarly, newly developed synthetic reactions have yet to show an impact in recent drug discovery programmes.


Figure 1. Occurrence of a particular reaction, plotted as percentage of which it shows up in at least one manuscript (Figure taken from DOI: 10.1021/acs.jmedchem.5b01409)

Instead, comparing the reactions used by medicinal chemists to those used by chemists working towards the synthesis of natural products shows a completely different picture (see Figure 2). While the former tend to make amide bonds, aryl-aryl bonds, and amino-aryl bonds, the latter concentrate on functionalising oxygen atoms, set stereocentres and make carbon-carbon bonds.

Although carbon-carbon bond formation is a common practice, the type of reactions used by medicinal chemists and natural product chemists differs significantly. Medicinal chemists tend to used Suzuki coupling reactions while in natural product production it varies with aldol, Wittig and Grignard reactions.


Figure 2. Occurrence of a particular reaction type plotted as percentage of which it shows up in at least one medicinal chemistry manuscript versus natural product papers (Figure taken from DOI: 10.1021/acs.jmedchem.5b01409)

The authors question whether the type of reactions selected by medicinal chemists is done out of convinence (e.g because of efficiency and chemoselectivity of the reaction), therefore leading to libraries of compounds  with similar shape, or whether it is indeed there where the drug space is more abundant. To address this question they examine the frequency of biphenyl fragments (normally achieved through Suzuki reaction) in an AstraZeneca collection and found that over time there has been a 6-fold increase on the appearance of this fragment.  Using an in-house database (IBEX) they further examined the possible substitution patterns of mono– and disubstituted biphenyl structures.   For monosubstituted biphenyl fragments it was found that the para substituion was preferred while for the bisubstituted compounds it was the paraortho arrangement (Fig. 3). These preferences result in a high density of linear and disk shape molecules (see Figure 4 green and blue dots), whether the less frequent substitution patterns lead to more diverse molecular shapes (see Figure 4 red dots).


Figure 3. Frequency population of various biphenyl regioisomers in the IBEX (Figure taken from DOI: 10.1021/acs.jmedchem.5b01409)


Figure 4.Population analysis of representative biphenyl compounds illustrating the geometrical diversity of para-para (green), meta-para(blue), and ortho-ortho (pink) compounds (Figure taken from DOI: 10.1021/acs.jmedchem.5b01409)

Brown and Boström point out that, despite the reactions being used by medicinal chemists today still rely on chemistry discovered decades ago, scientific and technological innovations are still of great influence in medicinal chemistry laboratories. Automation, microwave and supercritical fluid chromatography are just some examples of technological advances  and recent reaction improvements have provided advantages to the classical reactions conditions discovered decades ago. However, the impact of recently developed methodologies, such as ring-closing metathesis, C-H bond activation, selective fluorination, biocatalysis etc… is still to be seen in medicinal chemistry laboratories.


Blog written by Carol Villalonga-Barber

Functional Analysis of Schizophrenia Risk Genes: Complements on an Elegant Piece of Work

Schizophrenia (see here) is a debilitating disorder comprising of three symptom domains: 1. The so-called positive (psychotic) symptoms of auditory hallucinations (hearing voices), paranoia and disorganised behaviour; 2. Negative symptoms, such as a lack of motivation and the loss of pleasure in activities that were formerly pleasurable; and 3. Cognitive deficits, reflected by a lack of mental agility and a general “brain fog”. Although the positive symptoms are relatively well-controlled by first- and second-generation antipsychotic drugs, such as haloperidol, risperdol, olanzapine, paliperidone, and aripiprazole, all of which antagonise dopaminergic D2 receptors, the negative symptoms and cognitive deficits are poorly treated. Unfortunately, the search for new treatments for schizophrenia is hampered by the lack of an understanding of the underlying pathological mechanisms. However, recent genetic data has begun to shine light onto some of the mysteries of the disorder and have implicated a role for complement component 4 (C4) genes (see here).

Prior to the advent of genome-wide association (GWA) studies, candidate gene studies implicated a number of different genes in the pathogenesis of schizophrenia (such as COMT, DISC1, DTNBP1 and NRG1) yet due to a lack of statistical power, these have failed to provide meaningful insights into the pathology of the disease (see here). More recently, the use of genome-wide association (GWA) studies, which links disease risk to specific regions of the genome, has begun to identify genetic variants associated with schizophrenia. Hence, the Schizophrenia Working Group of the Psychiatric Genomics Consortium reported at least 108 different regions of the genome associated with a risk of schizophrenia (see here). These data highlighted associations between the dopamine D2 receptor and genes involved in glutamate neurotransmission, consistent with the well-described dopamine and glutamate dysfunction hypotheses of schizophrenia. Intriguingly, a number of other genes were associated with the immune system, of which the strongest were those associated with the major histocompatibility complex (MHC), a region containing 18 polymorphic human leukocyte antigen (HLA) genes. The genetic links between the immune system and schizophrenia is consistent with epidemiological data suggesting, for example, that over a third of cases could be prevented if infection in pregnant women was prevented (see here).

Fig 1Complex association

The MHC is a complicated region of Chromosome 6 and is divided into three classes (MHC I, II and III; see figure 1, taken from here). Recent work from Sekar and colleagues (see here) has focussed upon the most strongly associated markers in the Class III region and more specifically the C4 gene. The C4 gene has the added complexity of existing as C4A and a C4B isotypes, both of which vary in structure and copy number and both have a long and short form, differentiated by the present or absence, respectively, of a human endogenous retroviral (HERV) insertion, which lengthens the gene but not the protein sequence. To cut a long and complicated story short, the different C4 alleles resulted in widely varying levels of C4A and C4B expression in the brain (Figure 2 – see Sekar et al) with greater levels of C4A expression being related to a greater risk of schizophrenia.


Figure 2. Expression levels of C4A RNA


In the immune system, C4 activates C3, which in turn covalently attaches to its targets and triggers engulfment by phagocytic cells and in the developing mouse brain, C3 is important for synaptic pruning, a crucial process in neurodevelopment. Accordingly, Sekar and colleagues showed that mice deficient in the C4 gene had deficits in synaptic remodelling (reduced synaptic pruning) similar to those observed in C3-deficint mice. Hence, the increased expression of C4 protein in schizophrenia may well result in increased synaptic pruning that might in turn be related to the cortical thinning and reduction in synaptic organisation that have been reported in schizophrenia. Although such studies will not directly lead to novel therapeutics, they nevertheless demonstrate an elegant translation of genetic information into functional studies and provide the basis for hypotheses that might possibly transform the treatment of this disorder.


Blog writted by John Atack

Scratching away at pain ?

We’ve all felt pain. And although we have different thresholds and differing definitions of what pain is we commonly all have one thing in common – we don’t like it. Although there are a variety of analgesics available to us they have their limitations with respect to either efficacy (they don’t manage the pain very well) or they have unwanted side effects (CNS disturbances, addiction potential etc).

Although well acknowledged within the drug discovery community that there is an acute need for novel pain therapeutics, particularly for chronic neuropathic pain, the lack of clinical translatability of preclinical pain models has led many large companies to exit or avoid this area. It is perhaps this historical backdrop that makes the NaV1.7 (SCN9A) story so interesting. The role of voltage-gated sodium channels (NaVs) in generating the upstroke of action potentials, and hence controlling excitability in nerves, has been pharmacologically exploited for decades with local anaesthetics. Most of us have experienced them first hand on a trip to the dentist with a pain numbing lidocaine injection. If you have, then you’ve also experienced some of the downsides of a non-specific NaV blocker, in this case loss of sensation. Knowing which of the 9 NaV family members (NaV1.1 – NaV1.9) to target to deliver effective analgesia whilst not impacting on other important NaV channel mediated functions was a conundrum for many years. This changed in the mid-2000s when genetic studies looking at patients with recurrent pain (primary erythermalgia, paroxysmal extreme pain disorder) and those with an inability to sense pain (congenital insensitivity to pain) identified the SCN9A gene, which encodes the NaV1.7 channel, as the culprit. With both loss and gain of function mutations in humans giving opposing phenotypes, constituting arguably the highest level of pre-clinical target validation, considerable attention turned to this channel and developing selective blockers. It has not however been a straight forward journey and ten years down the line there are blockers entering early clinical development, albeit with varying degrees of success and varying selectivity profiles (see table below). And it is the latter, selectivity, that has provided the considerable challenge from a drug discovery perspective – the degree of conservancy between the 9 family members is extremely high, particularly in the pore region of the channels where most sodium channel blockers bind.


From Martz, L. SciBX 7(23); doi:10.1038/scibx.2014.662 (2014)


Peptide toxins which profoundly affect the gating (activation and/or inactivation) of NaV channels have attracted attention as they can demonstrate isoform selectivity. These target the voltage sensing domains (VSDs) of the NaV channels, an area which not surprisingly based upon the differing activation and inactivation profiles exhibited by the NaV family members has the highest sequence divergence. In 2013 a low molecular weight compound, PF-04856264, was reported1 to display selectivity for NaV1.7 over other isoforms. PF-04856264 was also reported to bind to the 4th VSD domain (VSD4), the conclusion based upon a series of comprehensive functional studies using chimeric channels. In a recent paper published by investigators from Genentech and Xenon (Ahuja et al2) the binding of this class of compounds (aryl sulphonamides) to VSD4 has not only been confirmed buts its structural basis for isoform selectivity explained. The group describe an elegant strategy of generating crystal structures of a chimeric human/bacterial channel to overcome the challenge of expressing full length NaV1.7. The chimera retained the key pharmacological properties of the channel and importantly produced high levels of protein to facilitate the crystallographic studies. The structural information outlined in the paper is consistent with the aryl sulphonamides binding to VSD4 and stabilising it in an activate state. Furthermore mutational analysis of the VSD4 receptor site identified the key motifs required for aryl sulphonamide binding and identified the key structural motifs that are responsible for the isoform selectivity of this class of compounds.drug binding site

From Ahuja et al 2015

At the Sussex Drug Discovery Centre we are big fans of structurally enabled targets and anticipate that the structural information determined by Ahuja et al will make a pivotal contribution to the design of improved NaV blockers for pain. However in common with many other drug discovery stories the path from target identification to delivery of a drug can be long and irritating………….. interestingly NaV1.7 is also implicated in itch3.


  1. McCormack et al (2013) Voltage sensor interaction site for selective small molecule inhibitors of voltage gated sodium channels. PNAS;
  2. Ahuja et al (2015) Strcutral basis of NaV1.7 inhibition by an isoform-selective small molecule antagonist. Science 350(6267), 5464-1 – 5464-8
  3. Devigili et al (2014) Paroxysmal itch caused by a gain-of-function NaV1.7 mutation. Pain 155(9), 1702 – 1707

Blog written by Martin Gosling

Chemical Litmus Test for Aldehyde Oxidase

Everybody working within drug discovery is acutely aware of the high attrition rate of small molecule drug candidates throughout the drug discovery process – this becomes especially troublesome during late stage development as the time and money already invested to progress these candidates is significant.

As a synthetic medicinal chemist, I strive to develop metabolically stable molecules that show a desired effect against a particular target – avoiding known problematic functional groups, substitution patterns and alteration of the electronic properties of compounds can reduce potential liabilities that plague late stage candidate progression. One (of many) increasingly recognised problematic areas is that of aldehyde oxidase (AO) metabolism of heteroaromatic compounds; current predictive tools have proven difficult to refine, and so medicinal chemists are required to submit individual compounds for biotransformation testing, which is both costly and time consuming. Therefore, a quick and robust chemical test indicating the propensity for a compound to undergo AO metabolism could prove to be an early warning sign for medicinal chemists.

Figure 1

Figure 1: Aldehyde oxidase and proposed litmus test

The mechanism by which AO is proposed to operate is via nucleophilic attack of the carbon adjacent to the heteroaromatic nitrogen by a molybdenum bound oxygen (Figures 1 and 2A). This reactivity is similar to the Authors developed method for direct C-H functionalisation of heteroarenes using alkylsulfinate radicals – that being nucleophilic attack of the aromatic carbon and subsequent C-H cleavage. Due to this similar reactivity, the Authors decided to apply their chemistry to create a Litmus Test for AO metabolism. In order to make this Litmus Test easily accessible to medicinal chemists, the following conditions needed to be met:

  • Must use readily available reagents that are not moisture or air sensitive
  • Easy to handle and analyse
  • The conditions must tolerate a plethora of commonly encountered functional groups
  • Not overly sensitive to stoichiometry of substrates/reagents (tip of a spatula accuracy)

Figure 2

Figure 2: A) AO and Litmus Test mechanism of action; B) Indication of substrate susceptibility to AO

After extensive optimisation (as highlighted by the Baran Groups blog) using Bis(((difluoromethyl)sulfinyl)oxy)zinc (DFMS) as the radical source, the addition of a new LCMS peak with a  mass of substrate +50 would indicate a positive result, and therefore that compound is potentially an AO sensitive substrate (Figure 2B).

In order to validate their Litmus Test, the Authors initially subjected known AO substrates to their newly developed conditions (Table 1) – it is clear to see that these five compounds appear positive in their chemical test.

scheme 1

table 1

Table 1: Optimised Litmus Test with five known AO substrates

As AO is an enzyme, subtle structural changes of a molecule can alter substrate susceptibility, and as it is difficult for chemical reactions to mimic such a sensitive environment (and so the potential for false positives), the Authors took structurally related substrates to compound 5 – some of which are known to be AO resistant, and investigated their reactivity (Table 2). On the whole, the Litmus Test proved predictive of AO susceptibility, with only one false positive being encountered amongst the compounds described (compound 13, a ketolide antibiotic).

table 2

Table 2: Predictive accuracy of Litmus Test

This is by no means a fall-proof method to gauge AO substrate susceptibility, neither is it designed to replace biotransformation tests as false positives are possible due to the non-enzymatic nature of the chemical reaction. However, when used with caution, it is both a quick and cheap early warning sign that trouble may lie ahead with your otherwise promising compound.

Blog written by Mark Honey


Rejuvenating Sirtuins

So what is it about those so called life span extending enzymes? It was in the late 1990’s when the Silent Information Regulator 2 (SIR2) gene was shown to extend the life span of yeast (1), an effect that was reported in worms and flies shortly after (2, 3). Since then, SIR2-like genes (named sirtuins) were identified in many other species (4, 5) and spurred studies about their life span extending effects in mammals. The possibility of small-molecule sirtuin activators that would increase human health span (or even life span) captivated many researchers and dramatically increased attention towards this enzyme family. But what are sirtuins? And what has red wine got to do with them?

Sirtuins are a highly conserved class of NAD+-dependent lysine deacylases that belong to the family of histone deacetylases (HDACs). Seven different sirtuins are identified in mammals (sirtuin 1 to sirtuin 7) that mostly catalyse a deacetylation reaction using NAD+ as cofactor (6). As the NAD+/NADH ratio represents the energy state of a cell, sirtuins readily react to an altered metabolism and in doing so strongly influence and regulate the metabolic state of the cell (7). Thus, it is not surprising that the identification of the role of sirtuin in metabolic disorders and how sirtuins can be targeted respectively have received much interest. Most research focused on the first member of the sirtuin family, sirtuin 1, but also on sirtuin 3 that is localized in mitochondria. For instance, reduced levels of sirtuin 1 has been associated with type 2 diabetes in humans and mice (8, 9). Activation of these enzymes can therefore have favourable physiological effects and not surprisingly numerous sirtuin activators (STACs) have been described and synthesised. And here is where our short story begins.

Lucas 3-2-16 Picture 1

Figure 1 | Potential physiological benefits of STACs in the treatment of age associated diseases. Picture from (10).

It was in 2003, under Harvard investigator David Sinclair, when resveratrol was identified as sirtuin 1 activator (11). Resveratrol, which is a natural compound found in red wine reportedly increased DNA stability in yeast and extended their life span by (stunningly) 70 % through activation of Sir2 (as mentioned, the yeast sirtuin analogue). For wine enthusiasts this was delightful news, unfortunately, the biochemical assay used to identify resveratrol was shortly after called into question. The original study used an enzymatic assay that contained a fluorescently labelled peptide substrate (Fluor de Lys) and that demonstrated activation of sirtuin 1 by the red wine polyphenol. However, other studies reported activation only in the presence of covalently bound fluorophore on the substrate and not by resveratrol itself (12, 13). Not until 2013 this issue should be resolved and be demonstrated that sirtuin 1 can actually be activated by resveratrol (under certain conditions). In the meanwhile, Sirtris Pharmaceuticals, Inc. was founded in 2004 by David Sinclair and bought in 2008 by GlaxoSmithKline (GSK) for $720 million. This move of GSK seemed rather surprising as the development of sirtuin activators as drugs had no strong foundation and even GSK internal Scientists failed to substantiate Sirtris’s claims (14). Especially after the (pharma giant) Pfizer report in 2010 (15) that concluded that resveratrol, as well as other STACs such as SRT1720 do not directly activate sirtuin 1, schadenfreude on the part of pharma watchers about GSK’s investment can be pictured.

Now, do resveratrol and other STACs actually activate sirtuin 1? This controversy should be resolved in 2013 by two studies, one led by David Sinclair (16) and the other by Clemens Steegborn (17). Sinclair’s et al. hypothesis was that the bulky and hydrophobic fluorophore used in the enzymatic assay mimics endogenous substrates required for sirtuin 1 activation by STACs (16). In fact, the fluorophore covalently bound to the peptide lowered the peptide Michaelis constant Km in response to sirtuin 1 activation by STACs. Substitution of the fluorophore with naturally occurring hydrophobic amino acids still supported activation. Based on their results, Sinclair’s group suggested an “assisted allosteric mechanism” in which STACs activate sirtuin 1 only with unique peptide substrates and concluded that “allosteric activation of SIRT1 by STACs remains a viable therapeutic intervention strategy for many diseases associated with aging” (16). Conveniently, Sirtris Pharmaceuticals gets integrated into GSK’s R&D in the same year of the Sinclair’s publication (14).

Lucas 3-2-16 Picture 2

Figure 2 | Assisted allosteric activation by STACs requires a glutamic acid residue (Glu230) in proximity to the catalytic core of sirtuin 1 and a specific hydrophobic motif of the endogenous substrate such as found in peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α). In detail, deacetylation by sirtuin1 is dependent on the presence of hydrophobic amino acids at positions +1 and +6 relative to the acetylated lysine on PGC-1α.

This brings us up to date and with interest the progress of Sirtris Pharmaceutical within GSK will be followed, as well as the outcome of their clinical trials. Activation of sirtuins in the treatment of age associated and metabolic disorders such as type 2 diabetes by small molecules is a promising approach. Interestingly, the general public seems in this regard already one step ahead and identified these rejuvenating enzymes for themselves. Sirtuin gene activating diets praise a “revolutionary plan for health & weight loss” (see “the sirt food diet” by Aidan Goggins), however, if these diets activate sirtuins is questionable. Same counts for resveratrol in red wine that in given concentrations probably does not activate sirtuins in humans (18). Nevertheless, we (wine enthusiasts – the author of this blog included) may continue to believe so. Cheers!

Blog written by Lucas Kraft


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New Year, New Chemistry – “Any-stage functionalisation” via strain-release amination

The Baran group at The Scripps Research Institute ( have recently reported an interesting reagent-based methodology to enable “any-stage functionalisation” of both simple and complex amines with small cyclic motifs such as bicyclo[1.1.1] pentanes, azetidines and cyclobutanes [1].

The strategy employs a turbo-amide to break a “spring-loaded” C-C or C-N bond, and in doing so directly aminates a strained species (Fig 1), enabling simpler syntheses and expanding retrosynthetic logic of some traditionally challenging targets.

Scott 01-02-2015 Picture 1

Fig 1. A – Strain-release amination using turbo amide and “strain-release reagent”, propellane, to afford bicyclo[1.1.1]pentan-1-amine on large scale after deprotection. B – Strain-release amination using turbo amide of late stage intermediate and “strain release” reagent, propellane, to afford a “propellerized” tertiary amine. Image taken directly from [1] without permissions

These small heterocyclic and bicyclic motifs can serve as bioisosteres in medicinal chemistry with the potential to bypass structural liabilities and navigate intellectual property space [2]. The incorporation of such structures into molecules using traditional methods (Fig 2), and from a late stage intermediate, can involve multiple FGIs and may involve the synthetic route being altered significantly to accommodate the small cyclic structure.

Scott 01-02-2015 Picture 2

Fig 2. Conventional and ‘new’ retrosynthetic analysis to obtain Pfizer’s precursor, bicyclo[1.1.1]pentan-1-amine. Image adapted from [3] and [1] without permissions

Pfizer’s urgent requirement of the costly precursor, bicyclo[1.1.1]pentan-1-amine (3 kg ~ $150 k)(Fig 2), in kilogram quantities for the synthesis of a clinical candidate, was the driving force behind this innovative methodology. Fortuitously, the strategy developed was then successfully demonstrated on a variety of secondary amines including commercial drugs, highlighting the applicability of strain-release amination. The ‘strain-release reagent’, propellane, used in direct amination was then substituted to include azetidines and cyclobutanes (B and C from Fig 3 respectively).

Scott 01-02-2015 Picture 3

Fig 3. The concept of strain-release amination: using the turbo amide of a lead with “strain-release reagent” A, B or C to append a strained ring system at “any stage” of synthesis onto the lead. Image taken directly from [1] without permissions

It will be interesting to see how this methodology develops; what other “strain-release reagents” can be employed, and how strain-release amination will be realised by academia and industry.

For a more complete account of the development of strain-release amination, further applications and in-depth details of the methodology (> 400 pages of supporting info!) visit and read the primary reference [1].

Blog written by Scott Henderson


  1. Gianatassio, J. M. Lopchuk, J. Wang, C.-M. Pan, L. R. Malins, L. Prieto, T. A. Brandt, M. R. Collins, G. M. Gallego, N. W. Sach, J. E. Spangler, H. Zhu, J. Zhu, P. S. Baran. Science. 351, 6270, 241 (2016)
  2. A. Meanwell. J. Med. Chem. 54, 2529 (2011)
  3. D. Bunker, N. W. Sach, Q. Huang, P.F. Richardson. Org. Lett. 13, 4746 (2011).