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.



Epigenetics: The sins of the father

In the recent paper in Nature (2014, vol 507, p. 22-24), Virginia Hughes reports the experiments carried out by Dr Dias and Dr Ressler from the University of Atlanta in recent years. They have studied the involvement in inheritance imprints in mice as a result of a fear-based reaction associated to acetophenone. As a result, they found a larger than normal expression of M71 glomeruli receptors in their offspring’s noses. These receptors are encoded by a single gene, known as Olfr151.

This elegant, but still inconclusive cause-effect mechanism approach, brings a possible explanation to a controversial observation back to the 19th century when French biologist Jean-Baptiste Lamark pointed out the pass of acquired traits to future generations. Since then, scientists have observed this phenomenon in plants, animals and even humans.

Although some scientists are still sceptical about the transmitance method, nobody denies the phenomenon. Finding an explanation to this complex problem would involve a deeper study on reproductive biology and to study both mother and father lines over few generations.

The strong suggestion that this heriditary transmission of environmental factors is due to epigenetics, a concept introduced in the 2000’s, where there are some changes in the way that DNA is packed and expressed without altering its sequence, is one of the strong lines of thought, where chemical tags (methylation) on DNA can turn genes on and off.

But even if epigenetics is directly involved in the inheritance, through marks on the material contained in the sperm, the first question to be addressed would be to understand how the effects of environmental/ health legacy get embedded into the animal’s germ cells.

Epigenetics is still unable to explain how this observed phenomenon gets passed down through multiple generations, surviving several rounds of genetic re-programing. Other suggested agents might involve histones (proteins which has been observed that they can be passed down through generations) or short RNA molecules which role would be to latch on DNA and affect further into gene expression.

Scientists are optimistic about finding a cause-effect relationship in the years to come for a phenomenon which has proved elusive for researchers in the past hundred years.

solid and air stable 2‑Pyridylzinc reagents – Real alternative to 2-pyridyl boronates?

A collaborative publication from Buchwald and Knochel trying to solve the ever recurring issue faced when using 2-pyridyl boronate. From past experience, 2-pyridyl boronates have always been problematic when used in ‘Suzuki’ type cross coupling and often prone to fast protodeborylation.

The recent development of Burke’s MIDA boronate is neat but far from widely applicable. Here Buchwald & Knochel are taking on solid and air-stable 2-pyridylzinc reagents

Their initial examples are forming a range of simple Air Stable 2-Pyridylzinc Pivalates by lithium or magnesium (Turbo Grignard) halogen exchange followed by transmetalation to Zn(OPiv)2



Very simple process but the scope is rather limited. The 2-Pyridylzinc Pivalates are reported as ‘free-flowing solids, indefinitely stable when stored under an inert atmosphere, and comparable in reactivity to organozinc halides in Negishi reactions’ which they clearly demonstrate in the later part of their publication.

The second part of the article focuses on alternative means to produce air stable and solid 2-pyridylzinc reagents. The small reported ‘screen’ identified dioxane as a stabilizing ligand forming a dioxanate complex, retaining reasonable air stability over a 24 hour period.

MP2Overall, an interesting and readily applicable alternative methodology to the problematic 2-pyridyl boronate by forming solid and air stable solid 2-Pyridylzinc reagents.

Evaluating drug binding to target proteins in cells and tissues – the CETSA technique

Most drugs bind directly to a target protein usually at a functional site and can either inhibit or activate the target protein to elicit the desired clinical effect. How effective the drug is (efficacy) is dependent on how effectively it binds to the target protein (target engagement). Target engagement is determined by the local concentration of a drug as well as its binding efficiency, both of which can be variable within and between patients. Drug efficacy has traditionally been monitored by evaluation of downstream cellular responses as drug binding has not been able to be directly measured in cells. Martinez-Molina et al., have recently developed  a novel technique to measure target engagement inside cells and tissues.

Thermal shift assays (TSA) are used on purified proteins to measure the extent of unfolding of the protein over a range of temperatures. Binding of drug ligand to the purified protein can ‘shift’ the temperature at which the protein unfolds. Here, a protocol has been developed in which soluble protein fractions from  cell lysates are separated into multiple aliquots and heated to different temperatures. The heated lysates are then centrifuged to separate soluble fractions from precipitates. The soluble fractions are resolved by SDS-PAGE followed by western blot analysis.   Thermal melt curves for each target protein are then constructed from the data. Known drugs were added to cell lysates and shifts in melting curves of target proteins detected. Due to the similarities of this method to TSA, the authors named this technique ‘cellular thermal shift assay (CETSA)’. They  correlated the CETSA data with TSA data from purified proteins, with similar melting temperatures obtained for the unliganded protein. They demonstrated thermal melt shifts of target protein with the addition of compound using both CETSA and TSA methods. They obtained dose response curves termed ‘Isothermal dose-response fingerprints’ by treating lysates with different concentrations of drug whilst keeping temperature and heat exposure time constant.

The authors were also able to acquire thermal melt dose response curves using intact cells by exposing the cells to drugs before the preparation of lysates and demonstrated relative binding differences between intact cells and lysates. The use of dye exclusion experiments confirmed that cellular membranes remained intact up to a temperature of 65˚C. Further, they established that CETSA can be used to determine target selectivity. A CDK inhibitor selectively engaged with CDK4 and CDK6 but not CDK2 or CDK9, confirming results from activity assays.

CETSA can be used to monitor the in vivo engagement of drugs with their targets. After dosing of animals with a compound inhibiting MetAP2, the group were able to demonstrate that thermal melt profiles of MetAP2 could be produced from lysates from frozen organs.

This technique therefore potentially has wide spread applications in preclinical drug development, such as assessment of target engagement and specificity, validation of clinical drug candidates  and estimation of drug efficacy in patients.


Martinez Molina D, Jafari R, Ignatushchenko M, Seki T, Larsson EA, Dan C, Sreekumar L, Cao Y, Nordlund P. Science. 2013 Jul 5;341(6141):84-7. doi: 10.1126/science.1233606.

The impact of aromatic ring count on compound developability: further insights by examining carbo- and hetero-aromatic and -aliphatic ring types

In this article from 2011, Ritchie et al. review the detrimental effects of increasing aromatic ring count on several developability measures such as solubility, lipophilicity, protein binding, P450inhibition and hERG binding.

The fewer the number of aromatic rings contained in an oral drug candidate, the more developable that candidate is likely to be, and that more than three aromatic rings in a molecule correlate with poorer compound developability (increased risk of compound attrition)

The sum of aromatic ring count andclogD7.4 has now been used as a simple but effective indicator of the solubility category. A new parameter, aromatic atom count – sp3 atom count (Ar-sp3), which describes aromatic/aliphatic balance, has also been introduced recently in an analysis of oral drugs and patented medicinal chemistry compounds.


Carboaromatics and and benzo-fused ring systems are inherently lipophilic and have strong deleterious effects on developability by

  • lowering aqueous solubility
  • increasing lipophilicity (CHI log D 7.4)
  • increasing HSA and AGP binding.
  • negative effects on CyP 3A4, 2C9, 2C19 and hERG inhibition.
  • A small beneficial effect (a reduction) was observed in CyP 1A2 binding as the carboaromatic ring count increased, presumably because of the size limitation of the narrow, planar active site in this enzyme

The consequences of increasing heteroaromatic ring count, although not as dramatic as seen with carboaromatics, resulted in:

  • lower aqueous solubility,
  •  increased HSA binding
  • increased CyP 3A4 and 2C9 inhibition.
  • no effect on AGP binding, CyP 2C19 inhibition or hERG binding
  • reduction in CHI logD7.4;this might be expected to result in higher solubility but in reality this appears to be negated by the addition of a planar ring.

Increasing carboaliphatic ring count had little impact on developability measures, although there was a modest increase in lipophilicity.

Increasing heteroaliphatic ring count improved developability by

  • increasing solubility
  • lower lipophilicity,
  •  lower HSA binding
  • reduce CyP, 2c9, 2c19 and 1a2inhibition.
  • no effect on AGP binding, CyP 3A4 inhibition

sl2The replacement of heteroaromatic rings with carboaromatic rings:

  • increases molecular weight (MW) (presumably owing to the increase in six-membered rings and the additional positions available for substitution on carbon rings)
  • increase lipophilicity (DaylightclogP and CHI logD7.4),
  • reduced aqueous solubility, higher protein binding and CyP450 inhibition
  •  hERG binding also increased although the effects were less dramatic but still statistically significant, except for CyP 1A2 inhibition.

When this analysis was repeated for compounds with a total of four aromatic rings, a similar pattern of deterioration in developability was observed as the proportion of carboaromatic content increased relative to heteroaromatic content.

Thus, based on this analysis the authors recommend a replacement of carboaromatic rings with heteroaromatic rings where possible. A recent survey also suggested that many synthetically tractable heteroaromatic ring systems have yet to be synthesized.

Predicting cancer targets modulated by Ayurvedic medicines

The recent availability of databases that provide both phenotypic descriptions and the chemical structures of the constituent compounds in traditional Chinese and Indian medicines, have enabled Bender et al  (J. Chem. Inf. Model. 2013 (53) 661 – 673, DOI: 10.1021/ci3005513 , to develop a cool algorithm to predict the mode of action (MOA) of these compounds and to predict novel protein targets for cancer therapies.

Traditional medicine has been utilised by human for thousands of years and normally viewed as complementary or alternative to mainstream therapies.  However, both Chinese and traditional Indian medicine (Ayervedic) have provided us with important drugs for instance Artemisinin an antimalarial drug and reserpine an antihypertensive drug.

From 1981 to 2007, 67% of the pharmaceuticals or new molecular entities (NMEs) introduced into the market were natural product based or a derivative there of.  These natural products often have desirable properties which make them good drugs; they are soluble despite breaking Lipinski’s Rule of Five, they embody privileged structures that are more frequently found to bind a variety of proteins in different organisms, and they are safe and well-tolerated, often having been commonly used for centuries.

However, there are major challenges that need to be resolved that enable the development of a new drug from a traditional medicine.  These include the isolation of the active constituents, the synthesis of the active constituents, the elucidation of the mode of action and finally the development of the compound as a “drug”.

The recent availability of databases that provide chemical structures and their corresponding phenotypes have enabled Bender et al to predict the MOA of compounds found in TCM and Ayurveda addressing one of these major challenges.  First they developed a classifier using bioactive compounds from the ChEMBL database, ChEMBL biological targets, ECFP_4 fingerprints for each compound and a Naïve Bayes classifier.


Figure 1: The compounds were represented using the Extended Connectivity Fingerprints, with a diameter of 4 bonds ECFP_4.  The ECPF is derived from the Morgan algorithm and was implemented in Sitegic’s Pipeline Pilot (Accelrys Inc).  Each atom identifier contains topological information on the atom that includes the number of immediate heavy atoms, the atom’s mass, its charge, the number of hydrogens attached, the valance minus the number of hydrogens and whether it is part of a ring.

This was used to predict which compound (fingerprint) would inhibit each protein target.  Then by creating fingerprints for each traditional medicine compound they could predict which protein targets they would hit.  For example they predicted the protein targets for some of the active ingredients of Panaz ginseng

FP5Figure 2:  Predicted targets for some of the active ingredients in Panax Ginseng

Next they correlated different proteins targets with different phenotypes. Predicting which molecular targets were modulated by the compounds in each different phenotype.  This enabled them to identify the protein targets most frequently modulated by Ayurvedic medicines, with possible anti-tumour effects.  The 10 most enriched protein targets are shown in the table below. The progesterone receptor currently has over 10 inhibitors with FDA approval.  Other proteins identified by this methods include regulators of other well-known cancer targets.


Figure 3:  Top 10 cancer targets in predicted to be inhibited by Ayurvedic medicines.

Novel Rat model for Alzheimer’s disease

It is stating the obvious that having good animal models is critical to the success of any drug discovery program. In many more complex diseases however, good animal models are not available. The ‘gold standard’ animal models for Alzheimer’s disease, Aβ-overproducing transgenic AD mice; do not demonstrate robust tauopathy and subsequent neuronal loss without the addition of genes not linked to familial AD.

In a recent paper Cohen et al., (1) have generated transgenic rats bearing human mutant APP (amyloid precursor protein) and PS1 (presenilin 1). These animals appear to manifest the full spectrum of age-dependent Alzheimer’s disease pathologies alongside cognitive disturbances. They have age-dependent β-amyloid deposition as well as intraneuronal Aβ1-42 and soluble Aβ oligomers. Many mouse models do present with some tauopathy, however, they do not present with neurofibrillary tangles (NFT) as observed in human AD. In this rat model however, they identified striking tauopathy. As well as hyperphosphorylated Tau, structures reminiscent to NFTs were identified close to β-amyloid plaques in aged rats. In addition immunostaining revealed structures consistent with NFTs in 16 month old rats. These NFT-like structures were also frequently observed in areas without plaques, as is found in human AD.

In concert with the molecular pathology, these transgenic rats exhibited neuronal loss and neuronal degeneration that was progressive and age-dependent. There was also an inverse correlation between the neuronal numbers and Aβ1-42 abundance. TUNEL staining indicated the presence of nicked DNA and measurements of active caspase-3 suggested the neurons were apoptosing.  This neuronal loss paralleled changes in behavioural characteristics such as novel object recognition (which is a hippocampal-dependent measure of working memory) that was significantly impaired in older transgenic animals. This was repeated in the Barnes maze, where there were no difference between wild-type and transgenic animals at 6 months, but after 15 months the transgenic animals made significantly more errors than wild-type.

With recent late-stage failures of treatments for Alzheimers this new animal model opens up the possibility to test novel therapeutics in a more human disease-like model.

Bexarotene in Alzheimer’s Disease: A case of lack of replication, lack of replication, lack of replication

In a previous blog (see here) we briefly discussed drug repositioning as an alternative approach to identifying novel therapies for Alzheimer’s disease. This strategy follows in the wake of the recent failures last year of the large Phase 3 trials with Bapineuzumab and Solineuzumab. At the forefront of drug repositioning has been Baxter International’s Gammagard which is one of a number of IVIG (intravenous immunoglobulin; obtained from the pooled plasma of healthy volunteers) treatments approved for boosting the immune system. Based on an initial small, 18-month, 24-patient study in which Alzheimer’s patients appeared to respond to the therapy (see here), Phase 3 studies were initiated. However, hopes were never especially high given that the proposed mechanism – infused antibodies would help clear amyloid peptide and/or tau protein – was vague at best and was not based upon preclinical mechanistic studies. Unfortunately these doubts proved warranted when in early May, a press release from Baxter (see here) disclosed that Gammagard had failed it’s primary endpoints and as a result it became yet another casualty in the Alzheimer drug development mine field.

In contrast to Gammagard, for which there is a marked absence of preclinical data to support its use in Alzheimer’s disease, a paper from Cramer and colleagues made quite a splash when it was published in Science in early 2012 (see here). They described how the Eisai skin cancer drug Bexarotene (marketed as Targretin) enhanced the clearance of amyloid and improved cognitive performance in preclinical animal models. Bexarotene was approved by the FDA in December of 1999 for the treatment of refractory cutaneous T-cell lymphoma and in the aftermath of the Science publication, doctors were inundated with requests for “off-label” prescriptions of Bexarotene for the treatment of Alzheimer’s patients (see here, for example). Although perfectly legal, the off-label prescribing of Bexarotene raises separate issues of patient safety and ethics (as discussed in the New England Journal of Medicine), especially since the clamour for Bexarotene was based on preliminary evidence from a single study. Indeed, recent studies have failed to replicate important aspects of the initial report (see here, here, here and here, with the authors response here). Most notably, although some inconsistent effects on amyloid peptide concentrations and cognitive improvements were observed, there was a failure to replicate the reduction in amyloid plaques reported in the initial study. These studies highlight the difficulty in reproducing preclinical data, a topic illustrated very well by the Bayer group (see here). Obviously, the publication of a single article, albeit in an august journal such as Science, should be treated with caution until it is replicated. Nevertheless, clinical studies have commenced and the effects of Bexarotene are being studied in the clinic in a Phase Ib biomarker study in healthy young adults as well as in Alzheimer’s disease patients in a Phase 2 [18F]AV-45 amyloid imaging trial. It is a sobering reflection on the large unmet need in Alzheimer’s disease that despite all the caveats and with the limitations highlighted even within the popular press (e.g. “A huge caveat here is that many promising drugs seem to work in mice but fail when used in humans”, Forbes magazine, 10th of February, 2012), a single publication can create a demand for the off-label use of a drug that is has significant side-effects but for which there is, as yet, no evidence of clinical efficacy in Alzheimer’s disease.

Facile strategy for the creation of complex and diverse compounds

High-throughput screening (HTS) of synthetic chemical libraries, containing mainly small molecules, is widely used in drug discovery programmes, both in industry and academia.

HTS has provided many drug leads, but mainly for biological targets that can be modulated by low molecular weigh and planar compounds. For more complex biological targets, HTS will fail due to the nature of the composition of the screening library. A recent study (J. Med. Chem. 2011, 6405) has shown that medicinal chemists have been synthesizing, over the last 50 years, compounds with lower than ideal Fsp3 (fraction of sp3-hybridised carbons) values and higher than ideal ClogP values, the former attributed to the increasing ease of sp2-sp2 coupling reactions. Therefore there is an interest in creating new libraries of complex compounds with better “druglike” features.

Hergenrother and co-workers (Nature Chemistry, 2013, 195) describe a ring-distortion strategy to rapidly (≤5 synthetic steps) generate collections of complex and diverse small molecules from readily available polycyclic natural products. An important consequence of starting with natural products is that all the intermediates generated are complex structurally and worth of inclusion in the final library.

They demonstrate this strategy for three complex natural products, gibberellic acid, adrenosterone and quinine using combinations of ring-cleavage, ring expansions, ring-fusions and ring rearrangements reactions (Fig 1-3).

cv4Figure 1. Ring-distortion approach on gibberellic acid.

cv5Figure 2. Ring-distortion approach on adrenosterone.

cv6Figure 3. Ring-distortion approach on quinine.

The average Fsp3 values for Hergenrother compounds was found to be 0.59, considerably higher than the 0.23 average found for a ChemBridge commercial collection of 150,000 compounds, while the ClogP was 2.90, 1.1 log units lower that that in the commercial collection, corresponding to a 12-fold reduction in hydrophobicity. Moreover, a chemoinformatic analysis (Tanimoto coefficients) revealed very low similarity between all of the compounds synthesized in this way which is a much superior derivatisation strategy than the conventional modification of peripheral functionalities.

Screening this library should definitely be of great interest to medicinal chemists.

Synthesis of Pyrazoles and Indazoles by Cu/O2 catalysed C-H activation

In a recent paper by Huanfeng Jiang a new practical synthesis of both pyrazoles and indazoles was described. By using a simple copper / oxygen catalytic system direct C-H bond amination was achieved.

Huanfeng started investigating this reaction by varying the copper catalyst, additive, solvent and reaction temperature to attempt to optimise the reaction conditions (table 1).

lp table 1Table 1: Optimisation of reaction conditions

With the optimal condition of Cu(OAc)2 (10 mol %) and DABCO (30 mol %) in DMSO at 100°C under O2 (1 atm) the scope of this reaction was explored (table 2 and 3).

As can be seen in table 2 the reaction gave pyrazoles in a >80 % yield where R1 = aromatic / olefinic, R2 = aromatic / olefinic / alkyl and R3 = aromatic / olefinic / alkyl. A wide range of functionalities were tolerated including fluorides, chlorides, bromides, nitriles, trifluoromethyls, carbonyls, sulphonamides and ethers. The only functionality investigated that was not tolerated was a free NH2. Huanfeng states that the geometry of the hydrazone double bond in the starting material is not important and believes that at elevated temperature in the presence on DABCO this would isomerise.

lp table 2Table 2: Synthesis of Pyrazoles

 After further optimisation of the reaction conditions it was found that the addition of 1 equivalent K2CO3 and an increased temperature of 120°C gave the best yields of indazoles. This reaction, as expected, tolerated the same functionalities as the pyrazole series. However, the potential to produce, presumably difficult to separate, mixtures of regioisomers makes this synthesis less desirable. It was noted that in the case of unsymmetrical aryl group that electron-rich substrates were favoured in the insertion step and that steric factors might also affect the regioselectivity.

lp table 3Table 3: Synthesis of Indazoles

The methodology to synthesise pyrazoles was then extended further to start from an aryl enone (scheme 1). Yields for this one-pot procedure (85 %) were comparable to the one step synthesis from a hydrazone (92 %).

lp scheme 1Scheme 1:  One-pot Pyrazole synthesis

To further the applicability of this chemistry Huanfeng functionalised a pyrazoles with NBS and used this building block in a Sonogashira reaction, Suzuki-Miyaura reaction and an amidation reaction (scheme 2).

lp scheme 2Scheme 2: Pyrazole derivatisation

To probe the mechanism of the C-H amidation several experiments were conducted using stochiometric quantities of an electron-transfer scavenger (1,4-dinitrobenzene), a radical clock (diallyl ether), or a radical inhibitor (hydroquinone, TEMPO) (scheme 3). The reaction proceeded in each case and when using diallyl ether no cyclised product was observed. These results suggest that this is not a radical mediated transformation.

lp scheme 3Scheme 3: Mechanism probing experiments

Based on these preliminary mechanistic studies Huanfeng has postulated the following mechanism (scheme 4). Initially olefinic hydrazone 1 or aryl hydrazone 3 would react with Cu(OAc)2 to form an Cu-N adduct A. The nitrogen from this Cu-N adduct could then undergo an intramolecular electrophilic substitution followed by aromatisation via C to give the pyrazole / indazole. The reduced copper species (Cun-2) could then be reoxidised by oxygen to complete the catalytic cycle.  Alternatively metallacycle B could be formed by electrophilic metalation or C-H bond activation followed by reductive elimination and aromatisation.


lp scheme 4Scheme 4: Proposed catalytic cycle

In conclusion this synthesis uses cheap and readily available reagents and has been shown to proceed in high yields even when preformed in air (72 % yield, table 1 entry 14).  This methodology circumvents the need to prefunctionalise starting materials with (pseudo) halides or directing groups and therefore has the potential to reduce the step count and open up previously more challenging or unavailable pyrazoles or indazoles.