Using Biochemical Light-switches to Illuminate Ion Channel Activity

There’s no doubting that optogenetics is an important recent development within the field of neuroscience. Channelrhodopsins, non-mammalian proteins that conduct ions in response to specific wavelengths of light, have now been inserted into various neuronal pathways to demonstrate the use of light to control modalities as diverse as vision, hearing, pain and motor control. In November 2017, The Scientist reported on progress in human clinical trials using channelrhodopsins in combination with viral vectors to restore a degree of function in damaged sensory neurons in response to light1. In a study conducted by Allergan, patients suffering from retinitis pigmentosa were injected with virus carrying the genetic signal to express channelrhodopsins specifically in retinal ganglion cells, bypassing the damaged light-detecting cells of the retina, enabling a rudimentary sense of light-detection in patients that were previously totally blind. Although primarily a safety study, it has shown promising progress in the field which may soon see developments towards treating hearing loss and chronic pain using a similar approach.

However, alongside the use of non-mammalian light-activated proteins to control neuronal activity, an alternative light-based approach has been developing which has direct and immediate usefulness as a tool in the field of drug discovery. The use of light to reversibly deliver ligand to a native protein receptor or ion channel, or ‘optopharmacology’, is the subject of an interesting recent mini-review by Bregestovski, Maleeva and Gorostiza2. For drug discovery, the use of these chemical photo-switches enables the rapid, and most importantly, reversible activation of ion channel function in response to light. Ligand-gated channels are the subject of this particular review, but much work with voltage-gated channels and G-protein-coupled receptors has now also been published. This approach provides a huge step forward from the previous use of caged-compounds in flash photolysis – often used, for example, to study synaptic transmission, but would leave the preparation awash with ligand that was slow to clear by re-uptake/diffusion, meaning difficult and slow ‘one-hit’ experimentation.

At the Ion Channel Modulation Symposium in Cambridge last year (2016), Dirk Trauner spoke about the development of these tools and demonstrated their use in research conducted both within his group and in conjunction with collaborators around the world, showing examples (see figures 1 & 2 below) of the use of photochromic ligands in both their soluble (PCL) and tethered (PTL) forms.

These two forms are defined by the nature of their interaction with target proteins – PCLs are designed to mimic the ligand of a specific receptor but are freely diffusible and may not exhibit sub-type specificity within a tissue. PTLs, as their named suggests, become covalently tethered to their target, usually via naturally-occurring or genetically modified cysteine residues, conferring a much higher degree of selectivity. On exposure to specific wavelengths of light, the molecules photoisomerize between cis-and trans- states, enabling ligand-receptor interaction that triggers either activation or deactivation of the target protein.

Of the two types, PCLs are the simplest to use. Figure 1 shows the impressive degree of temporal control of gained over the function of the capsaicin receptor TRPV1 in the presence of 1uM of a ‘photolipid’ PCL (here, an azobenzene combining the vanilloid head-group of capsaicin with photoswitch-containing fatty-acid chain (AZCA derivatives)) in response to simply varying light stimulation between 350 and 450 nm.

Figure 1: reproduced from Frank et al (2015)3

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Having proved the principle in TRPV1-expressing HEK cells, optical switching in the presence of this PCL was applied to isolated mouse DRG neurones, and c-fibre nociceptive neurones in saphenous nerve preparations, both of which contain native TRPV1 receptors. All showed rapidly reversible non-selective cation conductance in response to the shorter wavelengths of light, translating into nerve depolarisation and action potential firing in native neurones – responses which were absent in TRPV1-/- knockout mice.

Trauner also presented work using PTLs, and show-cased developments made in collaboration with US colleagues to extend the chemical tether to reinforce its chemical stability and reduce the chance of off-target attachment. Their new ‘PORTL’ (photoswitchable orthogonal remotely tethered ligand), shown in figure 2a, was used to demonstrate dual optical control of mGluR2 and GluK2 expressed in the same cell, differentially responding to the specified wavelengths of light (figure 2b).

Figure 2: reproduced from Broichhagen et al (2015)4                 Sarah 2

This elegant chemistry is a powerful tool for studying ion channel-mediated physiology. Its use, for example, to selectively activate or silence particular neurones, or sub-populations of heteromeric channels containing a common tagged subunit, with a degree of spacial and temporal control unachievable with perfusion, enables more qualitative assessment of the interaction between  new possible therapeutic compounds and their target proteins. And like channelrhodopsins, the use of these photochromic ligands as therapies in their own right is also possible and currently being investigated.

Blog by: Sarah Lilley


1 The Scientist Nov 16 2017 article by Shawna Williams

2Bregestovski, P., Maleeva, G., and Gorostiza, P. (2017) Light-induced regulation of ligand-gated channel activity. British J Pharm, doi: 10.1111/bph.14022.

3Frank, JA., Moroni, M., Moshourab, R., Sumser, M., Lewin, GR., and Trauner, D. (2015) Photoswitchable fatty acids enable optical control of TRPV1. Nature Comms 6, doi:10.1038/ncomms8118

 4Broichhagen, J., Damijonaitis, A., Levitz, J., Sokol, KR., Leippe, P., Konrad, D., Isacoff, EY., and Trauner, D. (2015) Orthogonal optical control of a G protein-coupled receptor with a SNAP-tethered photochromic ligand. ACS Cent Sci 1 383-393, doi: 10.1021/acscentsci.5b00260

The Silicon switch in Drug Discovery

Hit-to-Lead optimisation is a crucial step in Drug Discovery. It implies the wise modification of hit molecules by following specific pharmacologic and pharmacokinetic parameters. Many strategies can be employed to tackle this challenge, one of them is bioisosterism. Bioisosters are moieties or atoms that show the same physicochemical properties and biological activity. Thus, medicinal chemists can rely on a large chemical toolbox, for example, by changing an amide bond to an oxazole or shielding a carboxylic acid with a tetrazole. This all depends how we want to drive the series (in terms of physicochemical properties) through the bottleneck of the Drug Development. Bioisosterism is also widely used for the IP space expansion of chemical libraries.

In this light, I would like to discuss the “big brother” of carbon, the silicon. Within the third row of the periodic table, silicon is located below the carbon; they share the same valency of 4 and commonly forms tetrahedral molecules, the most common silicon linkage being Si-C and Si-O. The replacement of carbon by silicon within bioactive compounds could therefore yield new compounds with different properties for lead optimization.1 Small chemical differences exist between silicon and carbon. Indeed, it is known that the C-Si bond is 20% larger than the C-C bond – this observation has consequences on the shape and conformation of the molecule, which in turn leads to different interactions with the biological system. Silicon compounds are also more lipophilic than their carbon congeners. Therefore, switching from carbon to silicon could improve cell penetration, which is very important for compounds targeting the central nervous system for example. Nevertheless it also creates solubility and metabolic clearance issues that could be mitigated, depending where we want to put the cursor in terms of DMPK. A “hidden” feature of silicon is that it can form hexacoordinated compounds in comparison with carbon: that has great significance in Medicinal Chemistry since many potent transition state mimics containing silanediols have been developed. Finally, silicon is more electropositive than carbon, which leads to a difference in bond polarity and ultimately to a different biological outcome, one good example is the ammonium/silicon exchange found in Zifrosilone (acetylcholinesterase inhibitor).

A lot of work have been produced recently towards the pharmacological evaluation of new silicon-containing molecules (Figure 1), however none of these progresses has yet yielded a marketed drug. As said recently by the blogger Derek Low,2 silicon stays in the shadows, despite the huge potential offered by this element in balancing physicochemical properties with DMPK and lowering compound attrition during the lead optimization phase.

I believe that a new era for silicon in Drug Discovery will come soon; we cannot neglect this element any longer.

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Figure 1. Examples of some bioactive silicon-containing molecules with enhanced pharmacology and DMPK

Blog written by Mohamed Benchekroun


(1)          Ramesh, R.; Reddy, D. S. Quest for Novel Chemical Entities through Incorporation of Silicon in Drug Scaffolds. J. Med. Chem. 2017.

(2)          Lowe, D. Silicon Stays in the Shadows (accessed Dec 7, 2017).

Mutational Signatures in Cancer

A cancer carries thousands of somatic mutations, most of which provide no selective advantage to clones which acquire them. Much research is focussed on the few driver mutations that do confer such an advantage; how such mutations enable cancer development and whether they can be targeted as cancer therapies.  However, the recent reduction in cost of large scale sequencing (either whole exome or whole genome sequencing), has allowed mutational information to be used in a different way.  The study of mutational signatures can provide information about the mutagens to which a patient has been exposed to over a lifetime, any DNA repair mechanisms malfunctioning within the tumour, and potential therapeutic agents to which the tumour may be sensitive (Helleday, Eshtad et al. 2014).

Following large scale sequencing, signatures are generated by categorising base pair substitutions into 6 categories (C.G→A.T, C.G→G.C, C.G→T.A, T.A→A.T, T.A→C.G, T.A→G.C) and also taking into account the bases on each side of the substituted base. This gives 96 possibilities and each of these can be scored, giving an image such as that below.  This is a signature common in colorectal cancers, and is a product of defects in the mismatch repair (MMR) pathway.

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Figure 1. An example of a mutational signature from a MMR defective tumour. Taken from

Sequencing can also provide information about other types of mutation.  Indels, which are insertions or deletions can be quantified, together with their average size.  This provides further information about the damage/repair environment within the tumour.  For example, indels between 4 and approximately 50 bases, surrounded by mircohomology can be indicative of tumours relying on non-homologous end joining due to a defect in homologous recombination (HR).  Information about gross chromosomal rearrangements, such as tandem duplications, translocations and karyotypic variations can be integrated with base substitution information.  This can be viewed in a circos plot.  Below are examples of these plots from a tumour defective in homologous recombination (A) with large numbers of rearrangements and another defective in MMR (B) with large numbers of substitutions.

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Figure 2. Circos plots of tumours with different repair defects.  A is HR defective and B is MMR defective.  Adapted from (Davies, Morganella et al. 2017).

So what is the value of studying these mutational signatures? Since they give a historical perspective on the damage accrued by the genome, they can be used to monitor historical exposure to various agents.  For example, the presence of a particular mutational signature associated with exposure to Aristolochic acids (present in traditional medicines) has been identified to be present in a large number hepatocellular carcinomas in Asia (Ng, Poon et al. 2017). This in turn can inform policy on the availability of such agents.

Additionally study of mutational signatures allows the improved targeting of therapies. HR defective tumours present a distinct mutational signature and such tumours are generally sensitive to PARP inhibitors. MMR defective tumours carry a high mutational load and respond well to immune checkpoint inhibitors such as anti-PD-1 antibodies (Le, Durham et al. 2017). Although colorectal tumours are routinely examined for MMR status to allow specific treatment, MMR defective tumours in other cancer types are likely missed. A recent study showed that a small percentage of breast cancers carry MMR defects without germline mutation in MMR genes (Davies, Morganella et al. 2017). These tumours may respond well to immune checkpoint blockade. Therefore, the classification of tumours by mutational signatures could provide a broader but more highly targeted use for several existing therapies. Although currently these are only identified in a research setting, the falling cost of sequencing may allow such characterisation to be part of the process in treatment of cancer patients.

Blog written by Jess Hudson


Davies, H., S. Morganella, C. A. Purdie, S. J. Jang, E. Borgen, H. Russnes, D. Glodzik, X. Zou, A. Viari, A. L. Richardson, A. L. Borresen-Dale, A. Thompson, J. E. Eyfjord, G. Kong, M. R. Stratton and S. Nik-Zainal (2017). “Whole-Genome Sequencing Reveals Breast Cancers with Mismatch Repair Deficiency.” Cancer Res 77(18): 4755-4762.

Helleday, T., S. Eshtad and S. Nik-Zainal (2014). “Mechanisms underlying mutational signatures in human cancers.” Nat Rev Genet 15(9): 585-598.

Le, D. T., J. N. Durham, K. N. Smith, H. Wang, B. R. Bartlett, L. K. Aulakh, S. Lu, H. Kemberling, C. Wilt, B. S. Luber, F. Wong, N. S. Azad, A. A. Rucki, D. Laheru, R. Donehower, A. Zaheer, G. A. Fisher, T. S. Crocenzi, J. J. Lee, T. F. Greten, A. G. Duffy, K. K. Ciombor, A. D. Eyring, B. H. Lam, A. Joe, S. P. Kang, M. Holdhoff, L. Danilova, L. Cope, C. Meyer, S. Zhou, R. M. Goldberg, D. K. Armstrong, K. M. Bever, A. N. Fader, J. Taube, F. Housseau, D. Spetzler, N. Xiao, D. M. Pardoll, N. Papadopoulos, K. W. Kinzler, J. R. Eshleman, B. Vogelstein, R. A. Anders and L. A. Diaz, Jr. (2017). “Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade.” Science 357(6349): 409-413.

Ng, A. W. T., S. L. Poon, M. N. Huang, J. Q. Lim, A. Boot, W. Yu, Y. Suzuki, S. Thangaraju, C. C. Y. Ng, P. Tan, S. T. Pang, H. Y. Huang, M. C. Yu, P. H. Lee, S. Y. Hsieh, A. Y. Chang, B. T. Teh and S. G. Rozen (2017). “Aristolochic acids and their derivatives are widely implicated in liver cancers in Taiwan and throughout Asia.” Sci Transl Med 9(412).