Antibodies and ion channels: potential therapies in airway disease?

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

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

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

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

Blog by Sarah Lilley


1 Santos et al (2017)

2Douthwaite et al (2017)

Kinase inhibitors for CNS penetration

The need to develop new brain cancer treatments using targeted molecular therapy was recognised over a decade ago. Glioblastoma—the most common form of malignant primary brain tumour – is the leading cause of cancer death in children and it also accounts for a high proportion of cancer deaths in adults. Currently, there are only two FDA approved chemotherapeutics for the treatment of glioblastoma multiform: the alkylating agents temozolomide and the carmustine-based Gliadel wafer. The success of kinase inhibitors in treating various malignancies suggests that it is highly desirable to identify a kinase inhibitor, capable of effectively crossing the blood-brain barrier (BBB). This necessity also arises from the risk factor when metastasis of tumour to the central nervous system (CNS) occurs as a mechanism of emergent resistance, if the inhibitor does not freely penetrate CNS.

While the importance of free BBB penetration for drugs targeting brain cancer is well understood, it is also essential to correctly assess the extent of this BBB penetration (as opposed to just achieving a target free concentration in the brain), for which a comparison of free brain concentrations to free plasma concentrations is needed (Kp,uu). The values of <0.1 are considered to be low (limited CNS penetration), whereas the values of >0.3 demonstrate a significant degree of free BBB penetration. The principal requirement for any small molecules to achieve the adequate Kp,uu values is that the molecules are not the substrates of the efflux transporters, such as P-gp or Bcrp, which are highly expressed at the BBB interface, and to possess the required for BBB permeability physicochemical properties. These properties for the CNS drug design have been well reviewed and summarised by Zoran Rankovic, the most critical being the topological polar surface area (TPSA) of the molecule and the number of hydrogen bond donors (HBDs).

In the recent review on kinase inhibitors for the treatment of brain cancer, Tim Heffron has analysed known small molecule kinase inhibitors with reported CNS penetration data and compared their physicochemical properties with those of the approved CNS drugs. Typically, the kinase inhibitors utilise multiple hydrogen bond interactions to achieve effective binding to the catalytic site of a kinase. As the result, the median TPSA values for the approved kinase inhibitors are double of that of the approved CNS drugs (Table 1). Interestingly, for two categories of kinase inhibitors, the first – with limited brain penetration and the other – with evident CNS penetration, there is remarkable similarity in the median values of cLogP, cLogD7.4, TPSA, HBD, and MW. The only notable difference was in the calculated pKa median values, where CNS penetrating kinase inhibitors have a lower median pKa than either kinase inhibitors that do not cross the BBB or CNS drugs.


Table 1. Comparison of median values of physicochemical properties for kinase inhibitors that are reported or predicted (based on efflux transport data) to have limited CNS penetration or reported to have significant free CNS penetration and/or no significant P-gp or Bcrp efflux.

It is worth noting that the quality of the data set for this comparison and, therefore, additional differentiation in the properties between the groups might be affected by a lack of data on free-brain-to-free-plasma drug concentration ratios (Kp,uu) for most molecules. In addition, there are limitations to the use of calculated physical properties that might conceal actual differences between molecules, and a potential for species differences to affect the interpretation of reported data for P-gp efflux.

Besides, the common medchem strategies to improve CNS penetration and to reduce efflux transport, such as utilisation of intramolecular hydrogen bonds to effectively mask HBDs and reduction in number of rotatable bonds, would not be accounted for in the calculated properties of those molecules.

The research into CNS penetrant kinase inhibitors is a fairly new direction, and to date only a few kinase inhibitors have been reported that are designed to be BBB permeable. This demonstrates that success in this area can be achieved, even if the physicochemical properties of kinase inhibitors and those of CNS drugs at first appear at odds. Of course, many additional variables impact evaluation of CNS penetrant kinase inhibitors clinically (e.g., PK, selectivity profile, safety, extent of free brain penetration, etc.). However, the significant unmet medical need for such inhibitors and the appreciation for what constitutes meaningful (free) brain penetration are driving the current R&D efforts in the discovery of kinase inhibitors for the treatment of brain cancer.

Blog written by Irina Chuckowree

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


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.

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.

Novel method for assessing cardiotoxicity

One of the most common reasons for failure of compounds in drug discovery programmes is cardiac toxicity. Therefore assessing this in the early stages of any drug discovery programme is key. Currently the earliest indicator for cadio-tox is interaction between compounds and a voltage gated potassium channel hERG (the human Ether-à-go-go-Related Gene) channels. Generally electrophysiological techniques are used in hERG, although fluorescent-based kits are now available.  Although hERG is the most common interaction for cardiotoxicity, this does not measure other potential interactions that could result in cardiotox. Sirenko et al. have taken advantage of the recent emergence of inducible pluripotent stem cells (iPSC) to explore the effects of compounds on cardiomyocytes in more detail.

Culture of iPSC is not as straightforward as immortalised cells, however the cells were obtained from a commercial source (Cellular Dynamics) saving the differentiation process and these cells are able to be reproducibly differentiated into a large quantity and cryopreserved until use. The use of iPSC derived cardiomyocytes for toxicity testing has the clear advantage that these cells express full cardiomyocyte functionality and even have the beating characteristic expected of primary cardiomyocytes. Since this beating is analogous to the beating of a heart, and ion channel block can present as drug-induced arrhythmias, it is not hard to see how this can be used to identify cardiotoxic compounds. At this point most groups would have struggled to have the technology available to analyse these beatings, but since the authors were from Molecular Devices, they conveniently had a FLIPR tetra and image xpress high content screening (HCS) platform along with the technical knowledge to enable these beatings to be quantified.

Sirenko et al. were able to quantify these beatings on both systems. On the HCS, they used time-lapse images and quantified the beating using their own differential algorithm that measured the number of beats per minute. On the FLIPR, they used the Calcium 5 Assay Kit which enabled the beating to be measured using Ca2+ influx in beats/min. Since the HCS could not acquire images as quickly as the FLIPR, the HCS approach was less sensitive than using the FLIPR and less work was performed using this method, however, they were still able to obtain IC50’s for known agonists adrenaline and isoproterenol.

The increased sensitivity and throughput possible using the FLIPR, enabled more studies to be performed using it. As such compound-induced dose-dependent atypical beating patterns induced by known cardio-toxic compounds such as hERG, Ca2+ and Na+ channel blockers were measured. They were also able to validate an automated analysis algorithm that was used to calculate the effect of a variety of compounds in high-throughput format, with positive or negative chronographic effects such as digoxin and propranolol. Furthermore, due to the increased sensitivity of the FLIPR it was also possible to quantify components of each individual Ca2+ peak (i.e. amplitude, peak width, decay time etc.) rather than just the number of beats. This meant they were able to measure more compound specific effects that have more physiological deviations such as QT prolongation, i.e. cisapride that increased both peak spacing and peak width.

A large number of drug withdrawals from the market as well as late stage clinical trials are from cardiac toxicity. These withdrawals are clearly very expensive, but more importantly potentially very damaging to health. The use of hERG testing clearly identifies many cardiotoxic compounds, however, the use of iPSC cardiomyocytes and measuring Ca2+ influx and beating enables many more facets of cardiac toxicity to be measured. The study by Sirenko demonstrates the use of this technique to identify compounds already known to effect different facets of cardiac toxicity. The real test, however, would be to put compounds that were withdrawn from the market, or clinical trials due to cardiac toxicity that were missed by other tox screens. If the technique set out by Sirenko et al., were to have picked up this toxicity it would demonstrate a step forward in early determination.

Faster metabolism

However well new compounds in development perform in vitro, the real confirmation is if they have desired effect in the body, and without major side effects. A key parameter in this understanding is the effect of the body’s metabolism on the compounds. It is highlighted and discussed in this article , where the authors have developed an early method to determine the functional effect of the metabolites formed on the drug target.

The Authors took human H4 receptor ligands which had been well characterised as active inverse agonists in a 384 well functional cell based assay using H4 receptor linked  to a reporter gene (β-Galactosidase ) and incubated them with liver microsomes (containing the cytochrome enzymes). The cytochrome enzymes converted the compounds into their respective metabolites (as would occur in the liver). The metabolites were then separated and identified using a LC/MS (electrospray ionization in positive ion mode). The individual metabolites were then collected and reformatted into separate wells in a microtitre plates. A freeze drying process was employed to remove organic solvents such as acetonitrile and formic acid which were required by the liquid chromatography, and the metabolites were re-solubilised in DMSO. One concern the authors did address is obtaining a full solubilisation of the freeze dried metabolite in the DMSO solution, however the metabolites that they were using had a number of protonated nitrogen atoms and were relatively polar so poor solubility was not an issue in this case.  The authors however suggested if dealing with very non-polar compounds, 10% DMSO could be added before the freeze drying step, which would dry into a DMSO film which would aid with re-solubilisation step. Another suggestion would be to use further analytical techniques such as ELSD (evaporative light scattering detection), to determine the true concentration of the metabolite preparation and therefore correct any activity measurements determine the true concentration curve for the metabolite

Once the metabolites were re-solubilised in DMSO, the author’s re- tested them in the 384 well cell based reporter gene assay.  This allowed determination of the functional response of the metabolites in comparison to parent compounds. With the optimisation of the fraction collection procure, two individual compounds, with a full profiling run from the LS/MS can be screened on a 384 plate. This allows key compounds from structure activity profiled in a timely manner to be profiled.

The results from this work were quite interesting, the first finding was that there was a contaminant in all four of the preps of the compounds, and this contaminant was a histamine receptor antagonist underlining the importance of QC on compounds that you are testing in any drug discovery programme, otherwise structure activity relationships could be mislead. When the individual metabolites were tested, one was shown to be a competitive antagonist compared to its parent compound being an inverse agonist. Again this is important to determine to drive further optimisation of your lead compound.  Other metabolites appeared to be inactive or still have the same functional response as their parent compound.

The key from this study is the process development which allows a fast turnaround of key series in the same assay format used for SAR studies and can be integrated into a screening cascade. That can only help in assisting the drug design process.

Antibacterial drug discovery is hard work

The area has rightly acquired a reputation as a graveyard for drug hunters. With many big pharma research groups exiting the area over the last 15 years those remaining are to be applauded because undoubtedly the need for new, effective antibacterials remain high.

This seems to be an area prone more than most to the ‘latest idea’. Ten years ago genomics was going to allow us to identify all the essential targets and then prosecute them in a logical manner. The widespread failure of this “pile ‘em high/screen ‘em quick” approach (including the high-profile demise of the GSK group) has pushed many of the remaining companies back in the opposite direction towards screening natural product collections or libraries of dross DOS compounds. So it’s encouraging to find groups who have had the courage to retain their heads whilst all around were losing theirs.  Whilst AZ senior management have not covered themselves with glory over the last few years, in this case their persistence is to be applauded.

What marks this publication ( ) out is the successful progression of a novel anti-bacterial target from target identification, through to validating the target with lead molecules in an in vivo model. The authors suggest this is only the second time this has been done in the last 10 years.

Thymidylate kinase (TMK) is the enzyme that transfers phosphate from ATP to thymidine monophosphate to form thymidine diphosphate. It is essential for survival of cells because blockade stops DNA replication. TMK sits at the junction of the de novo and salvage metabolic pathways and has been explored as an antiviral and antimycobaterial target in the past. However, the difficulty of identifying good quality molecules has prevented TMK from being validated in vivo.

Unlike protein kinases, where the ATP molecule is buried in a lipophilic cleft, in TMK the ATP site is relatively solvent exposed meaning the most ‘druggable’ site is that which binds thymidine monophosphate. This also has the advantage of being the site that shows most difference when compared to the human orthologue and hence holds out the potential for achieving selectivity. Taking thymine as a starting point the authors made a series of heterocyclic analogues, attempting to mimic the sugar ring system with additional liphophilic substituents trying to pick up interactions with an identified hydrophobic pocket. TK-924 was their starting point for optimisation.

The authors describe an optimisation programme which made over 1000 compounds, each synthesised via a 10- to 15-step synthetic route-a truly Herculean effort! One wonders how many of the compounds were made at AZ and how many were made on contract (and perhaps sometimes the efforts of the CRO are never appropriately acknowledged!). Nevertheless optimisation led to TK-666. The synthesis is described in the supplementary material but for those of you who like to see such things here is the route-

The compound showed good enzyme activity against Gram positive bacteria, excellent selectivity vs. the human orthologue and good antibacterial activity (<1ug/ml) vs. pathogenic Gram-positive bacteria (including resistant strains). In the standard thigh model the compound showed efficacy at 100mg/kg by administration of a single intraperitoneal dose. Perhaps disappointingly the dosing route and activity point towards improvements that still need to be addressed by the medicinal chemistry programme.

Nevertheless an interesting new set of antibacterial compounds worthy of further exploration.

Cancer stem cells- a new target to inhibit tumour growth

One of the keys for drug discovery is to be able to target the diseased pathways/cells without affecting the healthy cell/pathways. This is particularly evident in cancer, where the challenge is to target the cancerous cells without affecting healthy cells. Three recent publications have raised the bar of this for drug discovery. These studies have identified stem cells at the heart of the tumour which appear to be the drivers for certain types of cancer that are resistant to current chemotherapy. Simultaneously providing potentially revolutionary novel targets for cancer, whilst also increasing the challenge to hit these cells without targeting the healthy cells.

To put these findings in context, for some time there has been debate as to whether stem cells sit at the heart of cancers and the role that these cells may have. Up until this point, the evidence for stem cells has mainly been from immunohistochemical staining/FACs sorting and assaying them in vitro. The problem with using these methods is whether the in vivo phenotype is being altered by in vitro culturing. However, by use of lineage tracing, three separate groups (Luis Parada at the South Western Medical School, University of Texas; Cedric Blanpain of the Free University of Brussels and Hans Clevers at the Hubrecht Institute in Utrecht) in different tumours, in the brain, gut and skin, have demonstrated in each, a subpopulation of stem cells that may propagate and spread the cancer.

Glioblastoma multiforme (GMB) is an aggressive tumour that initially responds to chemotherapy however, the cancer nearly always returns. As such GBM is considered incurable and has a median survival of 15 months. In the first of these studies Chen et al., (1) used mice bred to develop GBM, in which they labelled healthy adult neural stem cells, but not their descendants, with a genetic marker. They found all the tumours again contained at least a few labelled cells, along with the unlabelled cells. Chemotherapy with temozolomide killed the unlabelled cells, but the tumours returned. When the animals were tested again, the tumours contained unlabelled cells that came from the labelled stem cells. When they used the chemotherapeutic treatment alongside a technique to supress the labelled stem cells they found the tumours shrank back to “residual vestiges” that bore no resemblance to GBM. Hence, they identified a chemotherapeutically resistant tumour cell that behaved more like stem cells. These cells themselves do not rapidly divide; however, they give rise to rapidly dividing progeny that are susceptible to current chemotherapy.

In a similar way Schepers et al., (3) used genetically engineered mice to label healthy gut cells and stem cells in benign intestinal tumours, a precursors of cancer. These labels carried a drug-inducible marker, that, when activated, fluoresce one of four colours. They found that even though the tumours consist of many different cell types, each tumour fluoresced the same colour, suggesting they arose from one single stem cell. To double check this, the researchers added a lower dose of the drug that caused the stem cells to fluoresce a different colour. In doing so, they demonstrated the stem cells were consistently producing progeny of different cell types.

In the final study Driessens et al., (2) also used linage tracing and identified distinct proliferative cell compartments within a benign papilloma. The majority of the cells had only limited proliferative potential, however a fraction had the capacity to persist long term. The former population gave rise to a terminally differentiated cell population; however, the more persistent population had stem cell-like characteristics and produced many progeny. In addition, as the tumours became more aggressive they were also more likely to produce new stem cells and less likely to produce terminally differentiated cells.

These studies present clear evidence of the existence of cancer stem cells, explaining the re-occurrence of some cancers after successful chemotherapy, but also critically providing new lines of research for drug targeting. Clearly research will now be focussed on killing these cells to eradicate the cancer, however, targeting the stem cell’s proliferative capacity, or encouraging them to differentiate into non-dividing cells may also be as effective.  Since the numbers of stem cells within the tumours appear to be so small and readily able to differentiate, isolating them to study in vitro is unlikely to be fruitful. However, the labelling and tracking the stem cells and their progeny in vivo demonstrated by these groups enable these cells to be studied in vivo and will be vital for drug discovery programmes. This will enable micro-dissection; genomic sequencing and micro-array analysis to be more easily performed on a pure population of cancer stem cells to be used for target identification/validation. In addition by enabling the tracking of these cells and their progeny in vivo the efficacy of any compounds targeted to either kill the stem cells, or prevent the production of progeny could be assed relatively easily.

The challenge of targeting a small cell population that relatively little is known about, without damaging healthy tissue-resident stem cells will be great. However, the research provides great tools to aid this process together with a new way in to provide novel therapeutic agents for cancer therapy.

1.           Chen J, Li Y, Yu T-S, McKay RM, Burns DK, Kernie SG, Parada LF. A restricted cell population propagates glioblastoma growth after chemotherapy. Nature (August 1, 2012). doi: 10.1038/nature11287.

2.           Driessens G, Beck B, Caauwe A, Simons BD, Blanpain C. Defining the mode of tumour growth by clonal analysis. Nature (August 1, 2012). doi: 10.1038/nature11344.

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