Do we really need a new pill for anxiety disorders?


Many treatments are available on prescription for anxiety disorders, so why are we still interested in studying new medications? This is the first question I asked myself when I started working in this field a couple of years ago.

First of all, I believe that long-term treatments for any sort of anxiety disorder should not be pharmacological.

GPs should encourage patients to start some process of self-understanding based on psychotherapy or cognitive behavioural therapy as soon as possible. This is particularly true for children and young individuals who have more chances to learn how to control these debilitating and long lasting conditions.

So again, why do we need a new pill for anxiety?

Short and medium-term control of anxiety symptoms is another important theme to consider.

Recommended first-line pharmacological treatment are second generation antidepressants, whose efficacy in treating many anxiety disorders has been proven. Nevertheless antidepressants generally need 2 to 4 weeks to show benefits and this make them rather ineffective for symptomatic treatment of acute episodes such as panic attacks.

As a result of this, the heavily demonised benzodiazepines are still routinely prescribed by GPs with 1 million people in the UK alone identified as long-term prescribed benzodiazepine users, an average of 180 per general practice. In reality benzodiazepines should not be prescribed for more than 4 weeks to avoid the risk of addiction and the terrible withdrawal syndrome associated with sudden discontinuation after long-term uninterrupted use.

So in conclusion, I believe that a new medication for anxiety is needed. As a scientist, I am honoured to be able to contribute to this long lasting search.

In my next blog I will describe what I envision this new medication for anxiety should look like.

 Blog written by Alessandro Mazzacani

 


					

Molecular Dynamics simulations a gained technique for protein structure elucidation


Molecular dynamics (MD) simulations are widely used in different fields, particularly in structure-based drug discovery. MD simulations have provided detailed information on the variations and conformational changes of proteins. This method has been used to investigate the structure, dynamics and thermodynamics of biological molecules and their complexes and is an important method for structure prediction and model refinement for proteins.

A recent paper of Bermudez et al. (2016) gives an overview of the applications and impact of MD simulations on structural studies, including protein folding prediction, protein assembly processes and sampling conformational space (Fig.1).

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The MD simulations used different molecular parameters, such as the energy function force field (FF) which is used to calculate the potential energy of a system of atoms or coarse-grained particles. Some various commonly used molecular simulation computational programs for molecular dynamics are the Assisted Model Building with Energy Refinement (AMBER), the Chemistry at Harvard Macromolecular Mechanics (CHARMM), the Groningen Molecular Simulation (Gromos) and the Optimized Potentials for Liquid Simulations (OPLS). These software can provide close enough approximation to estimate a wide range of protein conformations that can be studied for a specific biological function.

Protein folding is one of the most important parameters to study in structural biology, therefore there is a need for improved computational methods in protein folding predictions. MD simulations can provide complementary information with high structural resolution. The Blue Gene supercomputer developed by IBM was built for protein folding predictions, as well as Folding@home in which computers all around the world provide computer processing time to perform MD simulations for protein folding and to examine the causes of protein misfolding. In a study of Lindorff-Larsen et al. a supercomputer system for MD simulation called Anton (D.E. Shaw Research) was used to predict the folding of 12 structurally diverse proteins (Chignolin, Trp-cage, BBA, Villin, WW domain, NTL9, BBL, Protein B, Homeodomain, Protein G, α3D and λ-repressor).

To predict protein folding and investigate protein folding pathways MD simulations can also be used with some alternative approaches that are able to give more information on the characteristics of the folding process. Some recent applications of these methods include the coarse-graining (CG) method, implicit solvent models, enhanced sampling algorithms or accelerated molecular dynamics. For example Miao et al., used dual boost accelerated MD to capture the folding of four fast-folding proteins (chignolin, Trp-cage, villin headpiece and WW domain) identifying distinct conformational states (unfolded and intermediate). These examples demonstrated the ability of MD simulations to understand and predict the protein-folding pathways.

MD simulations also are useful for elucidating protein assemblies and relating protein-protein interfaces. One method commonly used for this is course graining (CG), in which a small group of atoms are treated as single particles, to reduce the number of particle-particle interactions to be calculated, however this method might lack detailed molecular information. Dimerization of transmembrane (TM) proteins is usually investigated by CG models. For example Han et al. used coarse-grained and atomistic simulations to model the homodimerization of the synaptobrevin-2 (sybII) transmembrane domain protein and selected TM domain mutants.  Another exceptional example is the study by Zhao et al. in which using different techniques combined such as cryo-EM, cryo-electron tomography and MD simulations to reveal the tubular assembly of HIV-1 capsid protein hexamers, resulting in all atom models for the entire HIV-1 capsid. The structural information gained is promising for rational drug design to target the HIV capsid.

Another useful application of MD simulation is for the exploration of the conformational space of macromolecules. MD based sampling is used to obtain functional and dynamic models of pharmacologically targeted proteins to obtain the conformational flexibility and the conformational space of the protein. Perdih et al. used a drug design strategy using multiple protein structures of the MurD for the identification of novel MurD ligase inhibitors using targeted molecular dynamic simulation and the Off-Path simulations (OPS)

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Figure 2. Inhibitor design strategy based on an enzyme structural flexibility, bacterial MurD Ligase. Perdih et al. (2014).

In another study performed by Calimet et al. they proposed an atomic resolution mechanism using MD simulation and also a possible model for the gating process of a eukaryotic chloride channel gated by glutamate (GluCL) that can possibly apply for other pentameric ion channels. All these studies showed gainful and effective examples of the application of MD simulation in combination with various other techniques to investigate structural information of pharmacological proteins. It is important to mention that MD simulations cannot completely capture the detailed molecular composition of biological systems. New computational methods and software need to be developed with the improvement of hardware and sampling algorithms.

References

Bermudez, M. et al. (2016) More than a look into a crystal ball: protein structure elucidation guided by molecular dynamics simulations. Drug Discov Today 21, 1799-1805.

Dill, K.A. and MacCallum, J.L. (2012) The protein-folding problem, 50 years on. Science 338, 1042–1046.

Lindorff-Larsen, K. et al. (2011) How fast-folding proteins fold. Science 334, 517–520.

Miao, Y. et al. (2015) Accelerated molecular dynamics simulations of protein folding. J. Comput. Chem. 36, 1536–1549.

Han, J. et al. (2015) Synaptobrevin transmembrane domain dimerization studied by multiscale molecular dynamics simulations. Biophys. J. 109, 760–771.

Zhao, G. et al. (2013) Mature HIV-1 capsid structure by cryo-electron micetroscopy and all-atom molecular dynamics. Nature 497, 643–646.

Perdih, A. et al. (2014) Inhibitor design strategy based on an enzyme structural flexibility: a case of bacterial MurD ligase. J. Chem. Inform. Model. 54, 1451–1466.

Calimet, N. et al. (2013) A gating mechanism of pentameric ligand-gated ion channels. Proc. Natl. Acad. Sci. U. S. A. 110, E3987–E3996.

Blog written by Thalia Carreno Velazquez

Can we have too many fluorines?


The incorporation of fluorine atoms in drug like molecules has become increasingly prevalent over the last few decades. A publication from Merck in 2008 reported on how the total number of fluorine-containing drugs had significantly increased since the late 50s (Figure 1).

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The strategy of incorporating fluorine atoms impart on a wide range of properties, from increasing potency, impacting on lipophilicity and permeability across cells. A more prevalent strategy for the medicinal chemist relies on the incorporation of fluorines for the modulation of drug metabolism and improvements on the physicokinetic properties. Fluorines are frequently used as bioisoteres of carbonyl-containing moieties and can reduce metabolism by directly replacing a proton prone to oxidation. Gillis et al. recently reviewed some instances where the incorporation of fluorine atoms had a key impact on metabolism and pharmacokinetic properties but also played a unique role in influencing molecular conformational. However, an interesting aspect of fluorine atoms comes from their use of labelled ligands in Positron Emission Tomography (PET) imaging. PET imaging is used as a non-invasive technique to demonstrated target exposure in clinical studies and as a clinical tool for cancer diagnosis. In such cases, short lived radionucleotides such as [18F]-fluorines are incorporated at a late-stage in tracer molecules, hence the need for clean and efficient late-stage fluorination methodologies.

There has been tremendous progress in organo-fluorine chemistry over the last decades where fluorines are introduced efficiently and some recent reviews can be found here and here. The methods are too numerous to describe or list. For example nucleophilic fluorination reactions, and in particular [18F]-fluorination, do still remain widely used but yields can be low (Figure 2).

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Metal-Free Oxidative Fluorination of Phenols with [18F]Fluoride; Gouverneur et al.; Angew. Chem. 2012, 124, 6837

However, electrophilic fluorination have become more widely popular due to their reactivity toward a wide range of functional group but are largely limited to a very small selection of reagents, namely SelectFluor, NFSI and N-F-pyridinium (Figure 3).

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From a drug discovery perspective, could these late-stage fluorination methodologies be applied to rapidly explore the fluorination of a scaffold that could otherwise be lengthy to prepare? Could improved potency be gained by a late stage fluorination? What about improving metabolism by a simple last step fluorination? The synthetic methodology is available and a recent article from the Ritter group ‘Late-stage fluorination: Fancy Novelty or useful tool?’ seems to suggest that recent advances have enabled such strategies.

References:

The Many Roles for Fluorine in Medicinal Chemistry; William K. Hagmann; J. Med. Chem., 2008, 51 (15), pp 4359–4369; DOI: 10.1021/jm800219f

ADMET rules of thumb II: A comparison of the effects of common substituents on a range of ADMET parameters; Paul Gleeson, Gianpaolo Bravi, Sandeep Modi, Daniel Lowe; Bioorganic & Medicinal Chemistry, 2009, 17(16), pp 5906-5919; DOI: 10.1016/j.bmc.2009.07.002

Applications of Fluorine in Medicinal Chemistry; Eric P. Gillis, Kyle J. Eastman, Matthew D. Hill, David J. Donnelly, and Nicholas A. Meanwell; J. Med. Chem., 2015, 58 (21), pp 8315–8359; DOI: 10.1021/acs.jmedchem.5b00258.

Metal-Free Oxidative Fluorination of Phenols with [18F]Fluoride; Gao, Z.; Lim, Y. H.; Tredwell, M.; Li, L.; Verhoog, S.; Hopkinson, M.; Kaluza, W.; Collier, T. L.; Passchier, J.; Huiban, M.; Gouverneur, V. Angew. Chem. 2012, 124, 6837

Introduction of Fluorine and Fluorine-Containing Functional Groups; Theresa Liang, Constanze N. Neumann, Tobias Ritter; Angewandte Chemie International Edition 2013, 52 (32), pp 8214–8264; DOI: 10.1002/anie.201206566

Modern Carbon–Fluorine Bond Forming Reactions for Aryl Fluoride Synthesis; Michael G. Campbell, Tobias Ritter; Chem. Rev., 2015, 115 (2), pp 612–633; DOI: 10.1021/cr500366b

Late-Stage Fluorination: Fancy Novelty or Useful Tool?; Constanze N. Neumann and Tobias Ritter; Angewandte Chemie International Edition, Special Issue: 150 Years of BASF, 2015, 54 (11), pp 3216-3221; DOI: 10.1002/anie.201410288

Blog written by Michael Paradowski

 

 

Advances in Cryo-Electron Microscopy


Back in August this year I attended the EFMC-ISMC in Manchester, a really enjoyable conference with a plethora of great talks covering a variety of medicinal chemistry topics. In the emerging topics session there was a fantastic presentation from Dr Neil Ranson from the University of Leeds on the recent advances in the field of cryo-electron microscopy (cryo-EM) and its utility in structural biology. For me it was a new technique that I hadn’t seen much on before.

Recent technological advances with electron microscopes by using direct electron detectors for imaging molecules and using refined software packages that translate two dimensional sets of images into three dimensional models have pioneered the generation high resolution near atomic (< 4Å) protein structures. Reviewed nicely by Bai et al.

In his talk Dr Ranson highlighted the advantages and identified some limitations with the technique. One advantage that cryo-EM has over X-ray crystallography is that there is no prior need to crystallise the protein, reducing the time spent on optimisation of crystallisation conditions and soaking experiments. This also highlights the advantage that it is possible to obtain structural data on proteins that are notoriously difficult to crystallise, for example membrane proteins. Progress over the past couple years in this area has been significant with structures of the ion channel TRPV1 and the integral membrane protein gamma secretase being solved with cryo-EM. Of particular interest with the TRPV1 publication the ion channel is imbedded into a lipid nanodisc infrastructure, allowing the generation of more structurally relevant data of proteins in conditions that are analogous to their native biological environment. This methodology will be instrumental in the solution of further membrane protein structures, which could provide a similar impact to research as X-ray crystallography had with soluble proteins.

One of the major hurdles that cryo-EM faces is that small (<150kDa) proteins are not as static when bombarded by electrons as larger proteins or protein complexes are. This currently leads to poor data generation for small or flexible proteins and further technological development or technique refinement will be vital in progressing this area. As the technique is receiving a renewed interest from a variety of researchers, progress over these hurdles will hopefully in time be overcome.

In summary, the progress made with cryo-EM over the past few years has been substantial. Future data generated with this technique will certainly deliver structural insight into membrane proteins and protein complexes aiding the discovery of new chemical tools for these biologically important targets.

References

Review on cryo-EM – Bai et al. Trends in Biochemical Sciences, January 2015, Vol. 40, No. 1, p 49-57

TRPV1 publication – Gao et al. Nature, 16 June 2016, Vol 534, p 347-351

Gamma secretase publication – Bai et al. Nature, 10 September 2015, Vol 525, p 212-217

Blog written by Ryan West

TMEM16A: Function follows form


In their recent review for Current Opinion in Structural Biology, Brunner et al. (2016) have followed on from the groups break through TMEM16 protein structure paper, putting forward an hypothesis of potential mechanisms of activation of TMEM16 scramblase activity.  But is this applicable to the 2 confirmed ion channels, within the TMEM16 protein family, TMEM16A and TMEM16B?

Obviously, their hypothesis is based on their nhTMEM16 crystal structure with TMEM16 proteins forming homodimers with 10 transmembrane helices in each protein (Figure 4 of review below).  Mutational studies have identified Cl binding sites which align to the entrance of the proposed subunit cavity, along with Ca2+ binding residues that activate TMEM16 protein added to the functional data (Yu et al. 2012) which helps to link the ion channel to the subunit cavity of TMEM16A and B.  This is given further strength by Yu et al. (2015) who produced a chimeric TMEM16A protein, with a substituted 15 amino acid domain from TMEM16F, which gained scrambling activity and that this substitution was located within the subunit cavity.  This model, along with homology models based on it, seems now to be the accepted structure but does not explain the gating mechanism of the close cousins the chloride channels.  Further family member’s X-ray structure will need to be elucidated to confirm Brunner et al.’s archetypal structure.

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Figure 4.   Potential mechanisms for Ca2+ activation. Inactive proteins are shown on the left, active proteins on the right. Ca2+ is indicated as blue spheres. Top, ‘clogging’, activation proceeds by a conformational change in the subunit cavity. Center, ‘plug’, the protein is activated by a movement of the cytoplasmic domains that block the path in the inactive conformation. Bottom, ‘electrostatic gate’, the Ca2+ neutralizes the excess negative net charge in the binding site (red) and this removes an electrostatic barrier for lipids traversing the cavity. This effect could be general for phospholipids (the negative charge of the phosphate at the lipid headgroup is indicated).

Brunner et al. Current Opinion in Structural Biology 2016, 39: 61-70

In figure 4 of their review (above) Brunner et al. speculate about potential activation mechanisms of TMEM16; first, a structural conformational change in the hydrophilic groove of the subunit cavity, which might take place to block the passage of lipids (‘clogging’).  Second, the N-terminus of TMEM16 might move through conformational changes within the protein to occlude the cytosolic entry of the subunit cavity, therefore ‘plugging’ the cavity/pore.  And third, no large conformational changes take place, but instead the negatively charged amino acids in the Ca2+ binding site act as an electrostatic gate which is nullified on binding Ca2+.  However, scrambling of ceramides (that do not contain a negative charge) still requires activation of the scramblase by Ca2+ (Suzuki et al. 2013).  All are conceivable along with other combinations of the outlined mechanisms of activation of TMEM16 proteins.

The Brunner et al. structure is easily perceived for scramblase activity with the scramblase ‘pore’ having access to membrane lipid via the proposed subunit cavity.  But the question is, can these proposed mechanisms be transferred to the TMEM16A and TMEM16B chloride channels, raising the fundamental question do they have the same structure, is the subunit cavity the ion channel pore, and what is the gating mode?  As alluded to mutational evidence suggests that the location of the ion channel might be within the subunit cavity proposed by Brunner et al. (2016) but how any of the TMEM16 family proteins activate scrambling/ion permeation is currently unknown.

An alternative to Brunner et al.’s hypothesis is put forward by Ma et al. (2016) who clearly and precisely review the evidence for TMEM16A as a chloride channel; the Ca2+ dependent properties, voltage-dependent properties and regulation by other molecules.  In figure 6 of their review (below) they propose a different gating model of TMEM16A regulated by Ca2+, the cartoon of their model indicates the ion channel is proteinaceous in nature and that at high intracellular Ca2+ concentrations the Ca2+ binding site moves outside the membrane, which they suggest is consistent with the evidence that high intracellular Ca2+ concentrations causes TMEM16A inactivation.  But this would not explain the sensitivity of TMEM16A and TMEM16B to the presence of chloride or their voltage-dependency each of which might confer subtle conformational changes which regulate ion channel activity.

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Figure 6.  Proposed gating models of TMEM16A regulated by Ca2+. TMEM16A currents activated by zero Ca2+, low Ca2+ (<1 µM) and high Ca2+ (>1 µM) exhibit different electrophysiological properties in terms of outward rectification, activation kinetics, and deactivation kinetics, as well as rundown. The crystal structure of nhTMEM16 reveals two Ca2+ ions in each monomer binding site located within the membrane (Brunner et al., ). We propose that only one Ca2+ binds to the monomer binding site at low Ca2+ concentrations, whereas two Ca2+ ions bind to the binding site at high Ca2+ concentrations. Therefore, the channel is proposed to have at least four states: “0 Ca” state, “low Ca” state, “high Ca” state, and “rundown” state. In the “0 Ca” state, the channel can be activated by strong depolarization in the absence of Ca2+. In the “low Ca” state, only one Ca2+ ion binds to the monomer binding site, and depolarization facilitates Ca2+ binding to its binding site, thus increasing channel activity. In the “high Ca” state, two Ca2+ ions binds to the monomer binding site, and induce a great conformational change of the channel. The Ca2+ binding site moves out of the membrane, and thus the channel does not respond to membrane depolarization. In the “rundown” state, the channel is closed even in the presence of high Ca2+ concentrations.

Neither model suggests where and what regulatory protein interactions do to confirmation of the ion channel in TMEM16A and TMEM16B, although potentially pull down experiments for associated proteins would give us the answer.  One such protein, calmodulin has been proposed although this is still controversial.

How do we go about reconciling these two models of TMEM16 protein, ultimately we need more structural information principally on vertebrate/human TMEM16 proteins, with the required resolution to see TMEM16 crystal structure in either the ‘open’ or ‘closed’ state or potentially even the more subtle conformational changes in crystal structure generated in chloride free conditions.  This would confirm one or the other proposed structures that makes up the lipid permeation/ion channel pore and gain insight into the gating mode of the TMEM16 protein family and therefore gain an understanding of the function following form.

References

Brunner JD, Schenck S, and Dutzler R. 2016. Structural basis for phospholipid scrambling in the TMEM16 family. Curr. Op. Structural Biol. 39: 61-70.

Ma K, Wang H, Yu J, Wei M and Xiao Q. 2016. New insights on the regulation of Ca2+-activated chloride channel TMEM16A. J. Cell. Physiol. 9999: 1-10.

Suzuki J, Fujii T, Imao T, Ishihara K, Kuba H and Nagata S. 2013. Calcium-dependent phospholipid scramblase activity of TMEM protein family members. J. Biol. Chem. 288: 13305-13316.

Yu K, Duran C, Qu Z and Hartzell HC. 2012. Explaining calcium-dependent gating of anoctamin-1 chloride channels requires a revised topology. Cir. Res. 110: 990-999.

Yu K, Whitlock JM, Lee K, Ortlund EA, Yuan Cui Y and Hartzell HC 2015. Identification of a lipid scrambling domain in AN06/TMEM16F. eLife. 4.

Blog written by Roy Fox