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

Sarah 1

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 Necessary Nitrogen Atom

In the modern drug discovery process, medicinal chemists strive for high-impact design elements during multiparameter optimisation of lead compounds into efficacious drug candidates. One of such well-known design elements, often referred to as “the magic methyl effect”, “the methyl walk” or “the methyl scan”, involves a replacement of a H atom with a Me group and, as the result, can lead to profound potency improvement (>100-fold).

The authors of the recently published perspective (DOI: 10.1021/acs.jmedchem.6b01807) discuss another common design element for multiparameter optimisation: substitution of a CH group with a N atom in aromatic and heteroaromatic ring systems. Such replacement, when going from a simple benzene ring to pyridine, results in profound changes in molecular and physicochemical properties, such as distribution of electron density, polar surface area, basicity, which in turn affects lipophilicity and solubility. In more complex chemical structures, these changes can impact upon a number of intra- and intermolecular, orbital, steric, electrostatic, and hydrophobic interactions, such as lone pair, dipole−dipole, hydrogen bonding, metal coordination, van der Waals, σ-hole, σ*S−X, and π-system interactions, which in turn can translate into modified pharmacological profiles.

The authors then illustrate an extensive number of drug discovery case studies from the recent literature, where a replacement of a CH group with a N atom resulted in ≥10-fold improvement in at least one key pharmacological parameter, as shown, for example, in Figure 1. The replacement of the C7 CH group in (1) with a N atom to give (2) resulted in a 300-fold improvement in biochemical potency, Cdc7 IC50 = 2700 and 9.0 nM for (1) and (2), respectively. This potency improvement was attributed to the different conformational preferences of the two analogues. The biaryl dihedral angle of >150° in indole (1) greatly differs from that of aza-indole (2) (dihedral angle of 0°), facilitated by a steric clash of H7 and H6′ in (1) and lone pair repulsion between N7 and N2′ in (2).


Figure 1. An example of a necessary nitrogen atom on potency improvement, likely due to different conformational preferences.

The effects of such replacement on basicity, lipophilicity, polar surface area, and hydrogen bonding capacity are relatively predictable; whereas effects on aqueous solubility, passive permeability, efflux profiles, active transport, protein binding, and metabolic stability can be more whimsical and counterintuitive. In some examples, no rational explanation could be given for the observed effect.

With regards to changes in potency, as the authors are cautious to point out, there is an approximately equal probability of increasing or decreasing potency by exchanging CH groups and N atoms, based on a matched molecular pair analysis (MMPA) of available data. However, these findings are very similar to those discussed for the magic methyl effect.

This perspective is not a manual for what pharmacological improvements will be realized upon the substitution of a CH group with a N atom, but rather as an extensive exemplar of the dramatic improvements that can be achieved under certain circumstances. The systematic N atom scan (N-scan) should be exploited where appropriate, particularly in cases where the preferred binding pose of the ligand is not known.

Some recommendations for the N-scan tactics are summarised below. The newly installed N atom might:

  1. Engage in a hydrogen bond with specific residues of the target receptor or receptor-bound water molecules that need to be satisfied
  2. Remove unfavorable van der Waals interactions the replaced CH group made with the target receptor
  3. Form unfavorable electrostatic interactions with an antitarget receptor
  4. Have a positive effect on the binding conformation of the ligand
  5. Mask a hydrogen bond donor in the ligand
  6. Reduce the basicity or HBA strength of an existing N atom in the ligand
  7. Evenly distribute the polar surface area of the ligand
  8. Properly tune the lipophilicity of the ligand
  9. Be shielded by other substituents or functionality in the ligand
  10. Stabilize chemically labile functionality in the ligand
  11. Replace a metabolically labile CH group in the ligand

Medicinal chemists have to constantly juggle the design of biologically active molecules with drug-like properties according to the rules of Lipinski and Veber, in order to achieve good permeability and absorption levels, which in turn lead to high oral bioavailability. These properties are particularly important when considering therapeutic targets located in the central nervous system (CNS) behind the blood−brain barrier (BBB). Keeping the number of hydrogen bond acceptors down is one of such requirements; however, in some cases, it may turn out that the introduction of one (or two) extra N atoms may be necessary.

Blog written by Irina Chuckowree


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

Crystallography in Fragment-based drug discovery

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

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



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

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

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

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

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

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

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

Blog written by Xiangrong Chen


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

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

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

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

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

Zika virus: A neglected disease with no specifically designed drugs

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

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

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

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

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

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


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

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

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


Table 1. List of potential compounds to tes.t

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

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

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

Blog written by: Jose Gascon


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


Big Pharma and their adoption of Orphan Drug Research

A rare disease/disorder is defined as such when it affects fewer than 200,000 (USA) or fewer than 2000 (Europe) of the population at any given time. To date, over 7000 rare diseases have been identified, affecting more than 350 million people globally. In the EU, as many as 30 million people may be affected, with the same number affected in the USA. Fifty percent of those affected are children, with approximately 52 million of these children not living to see their 5th birthday.1 Considering that only 5% of rare diseases have an FDA approved drug, how much research is being carried out by Pharmaceutical companies towards orphan diseases?

Drug discovery is an expensive and time-consuming business, with strict regulatory standards and high attrition rates. According to the Tufts Center for the Study of Drug Development, as of 2014 the cost of developing a single drug stood at approximately $2.9 billion.2 Traditionally, big Pharma focused on developing drugs aimed at “common diseases” with a large patient population to maximize the possibility of recovering research and development costs, with rare diseases pushed aside because they provided little financial incentive. These rare diseases were said to be “orphaned”, and can also include common diseases that have been ignored because they are far more prevalent in developing countries than in the developed world.

In 1983, Congress passed the Orphan Drug Act which provided manufacturers with three attractive primary incentives.

  • Federal funding of grants and contracts to perform clinical trials of orphan products
  • Tax credit of 50% of clinical testing costs
  • An exclusive right to market the orphan drug for 7 years from the date of marketing approval

The Food and Drug Administration commissioned the Office of Orphan Products Development (OOPD) to dedicate its mission to promoting the development of products that demonstrate promise for the diagnosis and/or treatment of rare diseases or conditions. In fulfilling that task, the OOPD interacts with the medical and research communities, professional organizations, academia, governmental agencies, and the pharmaceutical industry, as well as rare disease groups. The OOPD also administers the Orphan Products Grants Program which provides funding for clinical research that tests the safety and efficacy of drugs, biologics, medical devices and medical foods in rare diseases or conditions.3 The program has successfully enabled the development and marketing of more than 400 drugs and biologic products for rare diseases since 1983. In contrast, the decade prior to 1983 saw fewer than ten such products come to market.

So, with this in mind, how has research into orphan drugs progressed?

One biotech company stands out. Genzyme was founded in 1981, producing modified enzymes to test in clinical trials. In 1991, Genzyme won FDA approval for Ceredase for the treatment of Gaucher’s disease, the success of which allowed Genzyme to concentrate research into recombinant human enzymes to treat enzyme deficient conditions. To overcome supply constraints, Cerezyme quickly replaced Ceredase a few years later, and in 2010 Genzyme became the fourth largest American biopharmaceutical company with a revenue of $4 billion. In 2011 Sanofi acquired Genzyme for approximately $20 billion making Genzyme its global center for excellence in rare diseases.

The marriage between Shire and Baxalta early this year has made them the Global leaders in rare diseases, creating the number one rare diseases platform in revenue and pipeline depth.4 The combined portfolio will have over 50 programs that address rare diseases. Shire anticipates more than thirty recent and planned product launches from the combined pipeline, contributing approximately $5 billion in annual revenues by 2020.

Big pharma now wanted a slice of the pie, and drug research into rare diseases was not exclusive to Biotechs.

In 2014 Pfizer joined with the Global Medical Excellence Cluster (GMEC), a group of six leading UK universities to form a Rare Disease Consortium, focusing on exploring the human genome to treat hematologic, neuromuscular and pulmonary rare diseases.5 Professor Michael Linden (Kings College London) joined Pfizers newly formed Genetic Medicine Institute in 2015 to evaluate the viability of producing effective, clinical grade gene therapy viruses.6

In 2015, Dr. Mark Fishman, President of the Novartis Institutes for BioMedical Research (NIBR) stated that “Our focus on rare diseases flows from our desire to help patients underserved by today’s medicines.” Novartis scientists have investigated treatments for more than 40 rare diseases, with the US Food and Drug Administration granting Novartis dozens of “orphan drug” designations. They also have more than 15 medicines approved for these conditions.7

In 2015 Roche bought the French pharma company Trophos and its lead drug for the rare neuromuscular disease spinal muscular atrophy (SMA) for €470 million.8 They have also taken on board the genomics firm Foundation Medicine  as well as a collaboration with genetic testing specialist 23andMe in Parkinson’s disease.9

GSK has established its own research unit exclusively devoted to seeking cures for rare diseases. GSKs partnership with Ospedale San Raffaele Telethon Institute for Gene Therapy and Fondazione Telethon (Telethon) has resulted in a recent European approval for Strimvelis, the first ex-vivo stem cell gene therapy to treat patients with the very rare disease, ADA-SCID.10

In April this year, Astra Zeneca announced that along with MedImmune, a genomics initiative has been launched to transform drug discovery and development across its entire research and development pipeline. AstraZeneca has partnered with research institutions including the Wellcome Trust Sanger Institute (UK), Human Longevity Inc. (US), and The Institute for Molecular Medicine (Finland) in the hope to unearth rare genetic sequences that are associated with disease and with responses to treatment.11

The emergence of patient advocacy groups has improved the understanding of how debilitating these diseases can be, with the non-profit organisation Global Genes® being one of the leading rare disease patient advocacy organizations in the world, promoting the needs of the rare disease community.1 Passing of the FDA’s Orphan Drug Act (and similar legislation in other countries) paved the way for biotech companies to carry out research into rare diseases, offering government incentives, shorter development timelines, and exclusive markets rights. Smaller clinical trials with a reduced patient pool and FDA fast track designation has led to an average time of 3.9 years from Phase II to launch, compared to 5.4 years for non-orphan drugs.12

Research into rare diseases by smaller companies was also made possible through funding by non-profit organisations such as the Cystic Fibrosis Foundation, operating like a venture capital firm. Big Pharma can support expensive research costs, especially when orphan drugs can bring in attractive revenues – Cerezyme has an annual treatment cost of  $300,000 per patient! According to the EvaluatePharma Orphan Drug Report 2015, worldwide sales are forecast at $178 billion by 2020, with the market growing by 11% each year.13

However, with more orphan drugs coming to market and the high costs per patient, will healthcare systems be able to continue to pay/subsidise for them?

Blog written by: Kamlesh Bala



  12. Meekings KN, Williams CSM, and Arrowsmith JE. Orphan drug development: an economically viable strategy for biopharma R&D. Drug Discovery Today 2012; 17 (13/14):660-664.



TMEM16A: A new road or a secret gate?

As ion channels go, TMEM16A are busy ones. As one of a number of channels responsible for chloride conduction at the cell surface, their activity has implications for both water movement and transmembrane potential. They are found in the cells of epithelial and smooth muscle tissue throughout the body, and their functional diversity encompasses secretion, cell proliferation, cardiac excitability, smooth muscle contraction and the prevention of polyspermy. With such a broad range of locations and potential functions, it stands to reason that their control mechanism might be complex. Indeed, if you look them up in the ion channel and receptor guide published by the BJP, they are not readily categorised as either ‘ligand-gated’ or ‘voltage-gated’, but languish under the heading ‘other’ alongside several other recent additions to the chloride channel family (ClC, CFTR and volume-regulated channels). Since their molecular identification in 2008, investigation into their gating control has generated a complex and sometimes confused picture involving both ligand and voltage mechanisms. A recent paper by Contreras-Vite and colleagues2 attempts to integrate experimental evidence gained over the last 8 years in the proposal of an updated model of TMEM16A gating.

Factors at play in TMEM16A activation

There are several well-established factors controlling the conduction of chloride ions through TMEM16A channels. Primarily:

  1. TMEM16A is a chloride channel, activated directly by intracellular calcium
  2. Activation by calcium is strongly influenced by membrane potential
  3. Speed of opening/closing is influenced by the concentration and nature of the permeant anion


These first two factors are inextricably linked. Under ‘resting’ physiological conditions of intracellular Ca2+ concentration (0.1 uM) and membrane potential (-40 to -60 mV, for example), these channels appear to be closed despite the presence of calcium.  Depolarisations above the chloride equilibrium potential begin to elicit a TMEM16A current, conduction increasing with increasing depolarisation, giving TMEM16A its classic ‘outward-rectifier’ profile. However, when intracellular calcium concentration increases beyond 1 uM, voltage sensitivity appears to be lost, and TMEM16A conduction is seen at negative and positive membrane potentials alike. There is also evidence to suggest that the intracellular side of the channel has the capacity to bind 2 Ca2+ ions. In terms of gating speed, both fast and slow gating kinetics have been seen (whole-cell and patch recordings) depending on the duration of membrane depolarisation. This speed also appears to be influenced by the level of extracellular chloride, with the slow component most markedly affected (slowed further) by increasing extracellular chloride levels from 30 to 140 mM. More permeant anions (SCN, I, NO3) promote/accelerate opening and slow channel closure when applied extracellularly.

So how do you bring these factors together in order to model TMEM16A gating? In the present study2, Contreras-Vite and his colleagues look at their own experimental findings combined with published information, presenting for example the novel observation that in zero intracellular calcium, TMEM16A conduction is still possible, but requires strong depolarisations beyond +100 mV. They also show that reducing extracellular chloride reduces channel open probability, and any ‘fast’ gating kinetics are entirely lost when the channel is maximally activated by high levels of intracellular calcium, and state that intracellular chloride level appears to have no effect on channel activation.

They use these findings to calculate the open-probability of the channel under the influence of these different factors, and define the rate constants governing the transitions between discreet ‘open’ (O) and ‘closed’ (C) states when 0, 1 or 2 Ca2+ ions are bound to the channel in the presence or absence of 1 external Cl ion. By using these to simulate steady-state activation properties and comparing these to their experimentally-derived activation and closure (tail-current) data, they came up with the following 12-state Markov chain model:


Essentially, ‘open’ channel states are represented in the right half of the model, ‘closed’ in the left, concentric levels represent calcium binding – from the outer level in which both putative Ca2+ binding sites are occupied, the centre representing the channel with no calcium bound; each state being linked by a rate constants representing parameters listed fully in the paper, most of which are voltage-dependent, some being fast and some being slow (indicated in the diagram key).

Using this model, the authors demonstrate that they can reproduce the activation and deactivation kinetics shown by their experimental data, although they themselves admit that the quality of the fit begins to decrease under extreme levels of intracellular calcium and voltage. They do, however, successfully use it to predict that calcium binding affinity does not change with varying extracellular chloride. They then show experimentally that this does appear to be the case.

The basis of this latest gating model comes from evidence which is only briefly summarised here. There are, of course, other factors which have been proposed to influence TMEM16A channel activity under physiological conditions, such as the binding of calmodulin and inhibition of activation by intracellular protons. Whether this model proves to be correct, time will tell. But in targeting drugs to this channel, knowing how stable and long-lasting some of these conformations may be under various physiological conditions might lead to more efficient, state-dependent drug pharmacology.

Blog written by Sarah Lilley


  1. “Still round the corner there may wait, A new road or a secret gate.” J R R Tolkein
  2. Contreras-Vite JA, Cruz-Rangel S, De Jesús-Pérez JJ, Figueroa IA, Rodríguez-Menchaca AA, Pérez-Cornejo P, Hartzell HC, Arreola J. (2016) Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models. Pflugers Arch. 2016 May 2. [Epub ahead of print]
  3. Cruz Rangel S, De Jesús Pérez JJ, Contreras Vite JA, Pérez Cornejo P, Hartzell H, Arreola J (2015) Gating modes of calcium-activated chloride channels TMEM16A and TMEM16B. JPhysiol 24:5283–98. doi:10.1113/JP271256, PMID: 2672843
  4. Ni YL, Kuan AS, Chen TY (2014) Activation and inhibition of TMEM16A calcium-activated chloride channels. PLoS One 9:e86734. doi:10.1371/journal.pone.0086734, PMID:24489780
  5. Ferrera L, Caputo A, Galietta L. (2010) TMEM16A protein: A new identity for Ca2+-dependent chloride channels. Physiology. 2010, DOI: 10.1152/physiol.00030.2010, PMID: 21186280

The remyelination dogma

The myelin sheath is a major component of the white matter of the vertebrate brain and spinal cord.  It serves as an electrical insulator along nerve axons that accelerates conduction and enables higher brain function [1].  This specialised lipid-rich structure maintains axonal integrity by providing metabolic and trophic support, whilst allowing for efficient nerve conduction by minimising energy consumption [1].  However, characteristic to a number of CNS disease states is the process of demyelination [2].  For example, in Multiple Sclerosis (MS), where white and grey matter brain lesions occur throughout the brain and spinal cord, there is defective saltatory signal conduction and axonal damage, which then manifests itself in clinical symptoms such as motor weakness [3].  Interestingly though, in the adult CNS a quiescent pool of precursor cells exists which can differentiate into new oligodendrocytes capable of replacing lost myelin sheath [4].  In fact remyelination is a natural repair mechanism believed to protect against progressive axonal damage in MS.  Therefore, the focus of current therapeutic research has recently shifted from preventative to reparative in respect to existing white matter lesions.

The beneficial effects of remyelination are functional recovery via the restoration of both axonal conduction and trophic axonal support [5], and the additional neuroprotective properties of reducing the cell’s energy consumption.  This is done via recruitment of resident precursor cells to generate new oligodendrocytes [6].  However, the extent of this regeneration is limited. Although in vivo models to study MS show efficient and extensive remyelination, myelin sheaths generated in the adult brain of MS patients are generally thinner with shorter internodes. In MS, the process of remyelination is insufficient in about 80% of lesions and fails to counteract the accumulation of permanent axonal damage [7].  Successful remyelination may require the existence of oligodendrocytes, but initially needs the necessary factors that allow for the sufficient migration and differentiation of these precursor cells [8].  It is these factors that are thought to limit its success in the MS brain, although the underlying cellular and molecular mechanisms remain poorly understood.

But even so, do therapies focusing on enhancing this remyelination process inevitably incur neuroprotection?  One example which suggests they may not be so intimately related is the drug Fingolimod, approved by the FDA in 2010 to treat relapsing-remitting MS (RRMS).  Although this drug has been shown to reduce brain atrophy in RRMS patients [9], The Lancet this year reported that the INFORMS study, which used this drug in primary-progressive MS (PPMS) patients, failed to show any improvement in the neurodegenerative process [10].  So, why are we failing to find treatment for PPMS?  Is remyelination really the answer?  There is evidence from animal studies which shows that remyelination and neuroprotection may actually occur independently of each other.  After completion of remyelination the mice showed initial recovery of locomotor performance; however 6 months post completion, performance then began to decline compared to age-matched controls [11].  These studies highlighted that axonal damage continues long after remyelination, and can still accumulate over time to result in functional impairment.  Remyelination alone cannot compensate for the stress that demyelination has on a neuronal cell, and therefore neuroprotective strategies that do not rely solely on restoration of myelin must be explored.

Translating both remyelinating and neuroprotective strategies from bench to bedside, however, relies on appropriate in vitro and in vivo experimental settings for the development of new drug targets.  Focusing on remyelination in MS, the most commonly used animal models are toxin models, whereby a focal injection or systemic administration of a toxin, for example lysolecithin or cuprizone respectively, induces demyelination and successive remyelination [12].  A benefit of these models is that they exhibit endogenous remyelination with a predictable spatial and temporal distribution.  However, because remyelination is certain in these models, they are unsuitable for accessing the ability of a pharmaceutical compound to induce remyelination and can only be used to study the acceleration of it.  Additionally, the lesions in these models do not develop much, if any, autoimmune reaction, and inflammation is one reason thought to be behind the failure of remyelination in the MS brain.  This may be one reason behind some of the discrepancies that are seen between lab and clinic.  Having said that, remyelination in both the lysolecithin and cuprizone model is hampered in aged animals the chronic EAE model exhibits limited remyelination and a substantial input from the immune system [12].  These paradigms may provide a way of better studying the induction of remyelination in a compromised environment.

In conclusion, two factors must be addressed for research in this are to proceed: firstly the experimental setting most appropriate to study drug targets for remyelination; and secondly, the simultaneous neuroprotective strategies that should be employed to prevent the accumulation of irreversible grey matter damage.

Blog written by Victoria Miller



  1. Morrison.B, “Oligodendroglia: metabloic supporters of axonsTrends in Cell Biology, vol. 23, pp. 644-651, 2013.
  2. Bercury.K, “Dynamics and mechanisms of CNS myelinationDevelopmental cell, vol. 32, pp. 447-458, 2015.
  3. Chang.A, “Cortical remyelination: a new target for repair therapies in multiple sclerosisAnnual of Neurology, vol. 346, pp. 165-173, 2012.
  4. R. F.-C. C. Franklin, “Remyelination in the CNS: from biology to therapy,” Nature Reviews: Neuroscience, vol. 9, pp. 839-855, 2008.
  5. Honmou.O, “Restoration of normal conduction properties in demeylinated spinal cord axons in the adult rat by transplantation of exogenous Schwann cellsJounral of Neuroscience, vol. 16, no. 10, pp. 3199-3208, 1996.
  6. ElWaly.B, “Oligodrendogenesis in the normal and pathological central nervous systemFrontiers Neuroscience, vol. 8, p. 145, 2014.
  7. Frischer.J, “Clinical and pathological insights into the dynamic nature of the white matter multiple sclerosis plaqueAnnual of Neurology, vol. 78, pp. 710-721, 2015.
  8. Kuhlmann.T, “Differentiation block of oligodendrogial progenitor cells as a cause for remyelination failur in chronic multiple sclerosisBrain, vol. 131, pp. 1749-1758, 2008.
  9. Ingwersen.J, “Fingolimod in multiple sclerosis: mechanisms of action and clinical efficacyClinical Immunology, vol. 142, pp. 15-24, 2012.
  10. Lublin.F, “Oral Fingolimod verus placebo in primary progressive multiple sclerosis: results of INFORMS, a large phase III, randomised, double-blind, placebo-controlled trialLancet, 27 January 2016.
  11. Manrique-Hoyos.N, “Late motor decline after accomplished remyelination: impact for progressive multiple sclerosisAnnual of Neurology, vol. 19, pp. 227-244, 2012
  12. Blakemore.W, “Remyelination in experimental models of toxin-induced demyelinationCurrent Topics in Microbiological Immunology, vol. 318, pp. 193-212, 2008.

Smuggling drugs into the brain: old and new tricks


Figure 1. Proposed mechanisms of transport across the blood-brain barrier

Every medicinal chemist involved in neuroscience drug discovery has experienced the joys and pains of the blood brain barrier (BBB), classically defined as the system of tight junctions between the epithelial cells of the brain capillaries that strictly regiment the access of molecules into the CNS.

As medicinal chemists, we usually picture the BBB as a more impenetrable version of other biological interfaces and consequently we design our CNS-penetrant molecules applying more rigid physicochemical filters. Additionally, we use in vitro brain permeability models that tend to focus only on passive diffusion and efflux.

In reality big and polar molecules, antibodies and viruses have the ability of crossing or eluding the BBB using a number of ‘side entrances’.

In the last 30 years the understanding of the BBB mechanisms has increasingly gained clarity and accordingly many new opportunities for drug delivery into the brain have been tested. These new opportunities usually exploit existing mechanisms utilised by endogenous molecules that need to gain access to the brain (e.g. nutrients, aminoacids, regulatory blood proteins) or tricks invented by pathogens. Old and new ways of crossing the BBB have been recently reviewed by William A. Bank in the April issue of Nature Reviews Drug Discovery (doi:10.1038/nrd.2015.21).

Some of the most interesting and overlooked pathways include:

Access via influx (blood-to-brain) transporters – this is an old strategy for drug delivery (e.g. L-dopa, gabapentin which use transporters for neutral aminoacids). More recently this mechanism has been considered for selective delivery to targeted areas of the brain.

‘Trojan Horse strategies  –  where a therapeutic agent (cargo) is conjugated to a ligand (Trojan Horse) of a particular influx transporter expressed on the luminal membrane (blood-side). The complex in usually routed on the abluminal membrane (brain-side) by transcytosis.

Absorptive transcytosis – another vesicle-based pathway often used by penetrating peptides and antibodies fragments.

Extracellular pathways or functional leaks – these are anatomically defined areas of the brain that are deficient in blood brain barrier and as such allow controlled access to small amount of serum proteins including albumin and immunoglobulins. It has been suggested that antibodies – with low volume of distribution and high circulating half-life – can enter the CNS using this way.

Many small molecules and biologics that exploit these or similar tricks are being validated in the clinic.

Nevertheless, these mechanisms are quite difficult to predict and permeability models available to medicinal chemists for rational design are unfortunately still very rudimental…


Figure 1 adapted from: Smuggling Drugs into the Brain: An Overview of Ligands Targeting Transcytosis for Drug Delivery across the Blood–Brain Barrier; Julia V. Georgieva et al. Pharmaceutics 2014, 6, 557-583; doi:10.3390/pharmaceutics6040557


Blog written by Alessandro Mazzacani

The ‘Pathogen Box’

Last year I (Ryan West) attended the ISNTD-D3 2015 conference where I saw a presentation by Julio Martin from GSK Tres Cantos. He gave an interesting talk about open source drug discovery focusing on kinetoplastid diseases. One of the highlights of the talk for me was the introduction of three 200 compound collections that have been phenotypically identified as inhibitors of trypanosoma brucei, trypanosoma cruzi or leshmania donovani.

The collections were generated by screening a 1.8 million compound library from GSK against the pathogens in question. Hits were filtered to remove cytotoxic compounds and undesired structural features. The compound collections have been made available to public and private partners to facilitate research into neglected tropical diseases. Efficacy and cytotoxicity data has been published on ChEMBL and highlighted in Nature Scientific Reports 5: 8771 doi:10.138/srep08771.

Recently there has been a move towards encouraging and facilitating research into neglected tropical diseases. Medicines Malaria Venture (MMV) released a box of 400 compounds in 2011 that were active against the blood stage form of the P. falciparum parasite. Again, these compounds were freely available to the research community for further scientific investigation. This resulted in more than 40 publications, the depositing of publicly accessible data from over 20 screens and the initiation of at least 3 medicinal chemistry programs for a range of neglected tropical diseases.

Continuing from the success of the ‘Malaria Box’ initiative MMV have been awarded a further grant from the Bill and Melinda Gates foundation to collate another library of compounds. This time the molecules have been identified phenotypically against a broader range of 8 unique pathogens which are responsible for a large proportion of the world’s neglected tropical diseases. The collection has been available from December 2015, and as part of my project I have been able to acquire one of the kits for testing. We are interested in an enzyme target responsible for the blood stream respiration of the parasite that causes human African trypanosomiasis. We hope to publish our results in the near future.

This open source approach of sharing compound collections along with generated experimental data will boost research and aid the discovery of novel molecules for treating neglected tropical diseases. It will be interesting to see the progress of these open source endeavours and if they will be more widely adopted by other disease areas.


Blog written by Ryan West