BAP Certificate in Non-Clinical Psychopharmacology


At the beginning of the month, I was lucky enough to attend a residential course held by the British Association for Psychopharacology (BAP) in Cambridge. The training, which was held over four days, provided an overview of many major techniques used in this area of scientific research, as well as advances within the field.

We heard about cutting-edge research, from experts in academia, industry and the heath care sector. Our first lecture started with the basic concepts of genomics, and went on to the difficulties involved in interpreting genome-wide association studies (GWAS), an approach which is sometimes used to identify candidate genes for genetically complex neurological disorders. Another talk covered techniques including optogenetics and designer G-protein-coupled receptors (DREADDs). We went over the advantages of both these methods, which are used to precisely control neural activity, but also touched upon some of the limitations that still exist with these technologies. Other talks covered application of imaging methods and behavioural models.

Lectures were broken up by workshops on statistics and experimental design, as well as a group project. In a workshop focusing on PK/PD calculations, I was introduced to the concept of counter clockwise hysteresis plots. This is when you see two different response levels at a given drug concentration, the result of a delayed effect of the drug at the target. During this session, we spoke about the importance of considering these factors when designing a study as to avoid producing misleading data.

For our group project, we were given the task to form a Drug Discovery leadership team, where we had to choose a drug target for a neurodegenerative disorder, which we deemed to be the strongest candidate. With this target in mind, we put together a plan outlining why it is a worthy target and how we would go about identifying a molecule to take to clinic. Our conclusions were pitched later in the week in a “Dragons’ Den” like situation to see if our case was strong enough to get funding.

As part of the course, we took a trip to the Addenbrook’s hospital where we had the opportunity to take a tour around The Wolfson Brain Imaging Centre. Here we were able to see their clinical and pre-clinical imaging facilities, which included Positron Emission Tomography and Magnetic Resonance Imaging and heard about some of the ongoing research in the department.

After the intensive days, we all had the chance to sit down as a group for dinner and talk to the individuals who had presented throughout the day and had after dinner talks including one from the president of BAP. On the final evening, we headed down to Queens’ College, where we were presented with our certificates in Non-clinical psychopharmacology, which was a perfect way to finish off the course.

As someone who is relatively new to the area of research, I took away a lot from the course. I would definitely recommend this programme and believe that it could be beneficial for individuals at any stage of their career. The programme provided a fantastic platform to network and interact with others from many different area of psychopharmacology. I am excited to attend the BAP summer meeting 2018 to hear more from world leading scientist in both clinical and non-clinical psychopharmacology, and attend the evening disco, which has even been described as legendary!

Blog written by Olivia Simmonds

 

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Development of Ultra-Rapid Insulins


The goal of insulin therapy for diabetic patients is to mimic closely the physiologic pattern of insulin release by the pancreas in order to maintain normoglycaemia.

Available as the beef/pig pancreas derived hormone since 1922, the first human recombinant insulin was developed by Genentech and marketed by Eli Lilly in 1982.

Standard 2 Zinc-insulin (which is hexameric) must be injected ~30 minutes before a meal to allow for disassembly in the subcutaneous depot into dimers and monomers (the active species).

At the turn of the millenium, to facilitate more accurate dosing, the principles of protein engineering were applied to destabilize the dimer and hexamer interfaces and produce rapid-acting insulin analogs (Fig. 1). Raj 1

Figure 1. Amino acid compositions of the rapid-acting insulin analogues (1).

Lispro/Humalog (Eli Lilly), Aspart/Novalog (Novo Nordisk) and Glulisine/Apidra (Sanofi-Aventis) can all be injected 5 to 15 minutes before a meal (Fig. 2).

Raj 2

Figure 2. 4 hour physiological plasma insulin profiles plotted together with pharmacokinetic profiles for insulin lispro and human insulin in type 1 diabetes (2).

Attention has more recently focused on the development of ultra-rapid insulins for dosing at (or even after) the time of the meal, for the benefit of children, insulin pump users and for highly insulin-resistant type 2 diabetics. Current approaches to speeding up the onset of absorption include modification of excipients and enabling of tissue diffusion.

Affreza

Powdered insulin delivered by a nebulizer into the lungs is absorbed more rapidly than subcutaneous insulin and absorption is of short duration. Exubera, developed by Inhale Therapeutics (then Nektar) was the first hexameric inhaled insulin product to be marketed by Pfizer in 2006. Unfortunately a 2007 study concluded that Exubera “appears to be as effective, but no better than injected short-acting insulin”. Exubera was dispensed using a bulky device (Fig. 3) with little dosing flexibility and poor sales led to its withdrawal in 2007. More recently, Afrezza, a monomeric inhaled insulin developed by Mannkind was approved by the FDA in 2014. Afrezza is delivered using a small device about the size of an asthma inhaler (Fig. 3), peaks at ~15-20 minutes and is eliminated from the body within ~2-3 hours. Raj 3

Figure 3.

The rapid absorption and decreased duration of Afrezza closely resembles physiological insulin release (Figs. 2 & 4).

Raj 4

 Figure 4. Pharmacokinetic profiles for inhaled Afrezza and SC insulin lispro (in type 1 DM patients) and for inhaled Exubera and SC human insulin (in type 2 DM patients)(4).

Biochaperone Lispro

An alternative to engineering the insulin aggregation interfaces is to introduce biotechnological enhancers. Eli Lilly has complexed Lispro insulin with French biotech company Adocia’s proprietary BioChaperone (BC) to accelerate absorption (licensed in 2014) (Fig. 5).

Raj 5

Figure 5. Adocia’s BioChaperone technology is based on polymers, oligomers and organic compounds. The BC-insulin complex forms spontaneously in water, protecting it from enzymatic degradation and enhancing absorption after injection (5).

BC Lispro promoted a statistically significant 63% increase in metabolic effect over the first hour in comparison with Novolog, having previously been demonstrated to outperform Eli Lilly’s Humalog (Fig. 6).

Raj 6

Figure 6. Comparison of mean blood glucose profiles after subcutaneous injection of lispro and BC lispro (6).

Despite results from 6 clinical studies indicating that BC Lispro performs better than Humalog, Eli Lilly decided to terminate its collaboration with Adocia in 2017 (possibly because of the costly failure of its Alzheimer’s drug solanezumab). Lilly is now developing its own ultra-rapid Lispro in house (LY900014 currently in phase 3), formulated with two new excipients, treprostinil (a vasodilator) and citrate (a vascular permeabilizer). The rights to BC Lispro reverted back to Adocia from Eli Lilly at no cost and the company is currently seeking a new partner to shoulder the costs of phase 3 clinical trials, regulatory and marketing hurdles.

Fiasp

Novo’s Faster-acting insulin aspart (FIASP), the first ultra-rapid insulin to be approved and marketed (Fig. 7), is an innovative formulation containing Vitamin B3 (niacinamide) to increase the speed of absorption, and the naturally occurring amino acid (L-Arginine) for stability.

FIASP was sidelined by the FDA in October 2016 but approved in September 2017 following clarification of immunogenicity and clinical pharmacology data.

Raj 7

Figure 7. Fiasp FlexTouch Prefilled Pens -100 units/mL (7).

 FIASP can be injected from 2 minutes before to up to 20 minutes into a meal and acts twice as fast as Novolog/Aspart (Fig. 8).

rAJ 8

Figure 8. Blood insulin Aspart concentration after subcutaneous injection of Fiasp and Novolog in patients with type 1 diabetes (8).

This was achieved without a significant difference in the overall rate of severe or confirmed hypoglycemia. Clinical trial data showed that FIASP gave a lower post-meal spike and that patients also lowered their A1C levels.

Novo Nordisk is very keen to expand the use of ultra-rapid acting FIASP in an artificial pancreas setting and it is already approved for use in insulin pumps in Europe.

Given that Novolog was the third best selling diabetes medication in 2015 with $3.03 billion in global sales (9), Fiasp is well poised to become the leader in this enormous market segment.

Raj 9

http://www.doctablet.com

References

  1. https://www.diapedia.org/management/8104096115/short-acting-insulin-analogues
  2. Home, P.D. (2015) Plasma insulin profiles after subcutaneous injection: how close can we get to physiology in people with diabetes? Diabet. Obes. Metab. 17, 1011-1020.
  3. http://pharmamkting.blogspot.co.uk/2015/02/look-ma-no-bong-afrezza-inhaled-insulin.htmlAl-Tabakha, M.M. (2015) Future prospect of insulin inhalation for diabetic patients: The case of Afrezza versus Exubera. J. Control. Release 215, 25-38.
  4. https://www.adocia.com/technology/biochaperone-technology-2/
  5. http://www.diyabetimben.com/ultra-hizli-etkili-insulin-biochaperone-lispro/
  6. https://online.pharmacy/product/fiasp-insulin-3/
  7. https://www.fiasppro.com/the-fiasp-story/onset-of-appearance.html
  8. pharmaceutical-technology.com

Blog written by Raj Gill

Reducing attrition in drug discovery: the AstraZeneca 5R-framework


The high attrition in drug discovery is responsible for the extremely high cost of developing a new medicine. Some suggestions aimed at reducing the attrition have been put forward such as: improving efficacy and safety profiles, reducing toxicity, improving preclinical models, better understanding of mechanism (Nat Rev Drug Discov. 2004, 3, 711-716) and shifting the attrition to earlier phases (Nat. Rev. Drug Discov. 2010, 9, 203–214).

Based on this, scientists at AstraZeneca have established a five-dimensional framework (5R framework) (Nat. Rev. Drug Discov. in 2014) aimed at improving the low success rates in the process. The framework includes five determinants: right target, right tissue, right safety, right patient and right commercial potential, identified as key features in the drug discovery process (summary depicted in Figure 1).

Marco 1

Figure 1. The 5R framework (from Nat. Rev. Drug Discov. 2018).

In addition to the “5R framework”, AstraZeneca scientists decided to reduce their diseases portfolio and to potentiate their capabilities for target selection and validation (better understanding of biology and mechanism of the disease, stronger target rational).

Furthermore they improved their lead generation strategy (expansion of their compounds library including library sharing, and integration with other screening approaches), and their pharmacokinetic/pharmacodynamics modelling, patient stratification and biomarkers. In a more recent paper published in Nat. Rev. Drug Discov. in 2018, they show how the application of these guidelines have led to an increase in project success rates (Fig. 2) and to a reduction of cycle time and cost (Fig. 3).

Marco 2

Figure 2. Project success rates for the AstraZeneca (AZ) portfolio.

The overall project success rates have increased from 4% (2005-2010) to 19% (2012-2016); the cost to reach clinical proof of concept has decreased by 31% when comparing the two time cohorts, and by 42% compared to the industry average.

Marco 3

 

Figure 3. Metrics for projects costs (a. first good laboratory practice dose; b. clinical proof of concept) and cycle times.

With regard to cycle times, they observed a considerable reduction of the length for phase II (50% shorter than industry average) although this may return in line to the average in the future.

To further support how the introduction of the 5R framework has influenced AstraZeneca pipelines, Table 1 reports the new molecular entities and new biologics that entered phase III in 2012-2016, highlighting how their progression was influenced by these guidelines. The 5R framework can be used at any stage of the process, as it has been done for Olaparib (selected as drug candidate before 2011) resulting in the initiation of novel clinical studies. (Readers are encourage to check Box 1 in the paper for an example of the 5R framework applied to Osimertinib).

Marco 4

Table 1. Influence of 5R framework for 15 projects new molecular entities and new biologics entering phase III.

Although these guidelines have clearly improved productivity, 81% of the projects still failed at some stage of the process, but it is clear they are moving the process in the right direction and it will be interesting to see how these guidelines may affect the R&D strategy of other companies.

Blog written by Marco Derudas

Serine Racemase Showdown: Clash of the Crystal Structures


Having been sitting on an X-ray crystal structure and half-finished manuscript for quite some time now, imagine my chagrin when during a casual perusal of the PDB, I found an extremely similar structure had just been deposited to the one I was aiming to publish.

The deposited structure (5X2L) is for wild-type human serine racemase (SR) and the associated paper focuses on in silico screening and medicinal chemistry, while my structure contains two cysteine-to-aspartate point mutations (C2D, C6D) with a (speculative) paper that explores the crystallography and biophysical side. As well as briefly summarising the findings of the study, I thought it would be fun to compare our two crystal structures in what I have dubbed…

Clash of the Crystals

Chloe 1

But firstly, some background on the contestant/s: 5X2L (published) vs. CRK1 (mine, unpublished).

In the blue corner…(and the red corner)…weighing in at 37.4 kDa, we have a pyridoxial-5’-phosphate (PLP)-dependent enzyme that catalyses the racemisation of L-serine to D-serine, as well as the α,β-elimination of water from L-serine. This forebrain-localised enzyme produces about 90% of D-serine in the brain, and because D-serine is a co-agonist of N-methyl-D-aspartate glutamate receptors (NMDARs), SR inhibition has been touted as an up-and-coming approach to indirectly modulate NMDAR activity. This is a potential game-changer for treating disorders underpinned by NMDAR overactivation, such as neuropathic pain, neurodegenerative disorders, and epileptic states.

So let’s hear it for…serine racemase! [Thunderous applause]

 Round 1: Paper summary

Anyone well versed in SR literature will know the paper in question, ‘Design, synthesis, and evaluation of novel inhibitors for wild-type human serine racemase’ by Takahara et al. (2018)1 is an additional chapter to an ongoing story. Several groups have previously tried to identify new SR inhibitors that are potent, selective, and structurally distinct from the countless amino acid analogue inhibitors that are already well-described2, and for many this has proved to be a challenging endeavour.

The status quo shifted when a series of dipeptide-like inhibitors with a clear structural motif and slow-binding kinetics was identified by Dixon et al. (2006)3, which later provided the query molecule for an in silico screen performed by the same group behind 5X2L4. The resulting inhibitors contained an essential central amide structure with a phenoxy substituent, and substitution of parts of the structure for heavier halogen atoms such as bromine and iodine produced derivatives with improved inhibitory activity (comparable to classical SR inhibitors), binding affinity, and ligand efficiency. The Mori group took their explorations even further by testing the most potent derivative in vivo to demonstrate the SR inhibitor suppressed neuronal activity-dependent Arc expression to regulate NMDAR overactivation5.

The current paper expands on these studies by firstly, solving the crystal structure of wild-type SR for molecular docking and in silico screening; secondly, using these methods to identify new SR inhibitors related to their previously described peptide compounds; and thirdly, testing these inhibitors in an in vitro assay. The team synthesised 15 derivatives, of which one showed relatively high inhibitory activity, making a nice addition to their growing rolodex of peptide SR inhibitors.
Chloe 2

Figure 1. Structure and binding pocket of the novel peptide SR inhibitor derivative identified by Takahara et al.

Round 2: Clash of the Crystals!

Both contestants were crystallised using the sitting-drop vapour diffusion method in very similar experimental conditions (Table 1). Both structures were determined to a highly respectable resolution of 1.8 Å, and organised into a large domain and small domain connected by a flexible loop region (Fig. 2). The PLP cofactor (Fig. 2; yellow sticks), on which SR is dependent for its catalytic activity, can be seen covalently linked to Lys56.

Table 1. Summary of key features of 5X2L and CRK1.

Feature 5X2L CRK1
Crystallisation conditions 10% PEG 8000, 5 mM MgCl2, 0.1 M Bis-Tris pH 6.0, 10% ethylene glycol, 20 °C 15% PEG 3350, 250 mM MgCl2, 0.1 M Bis-Tris pH 6.5, 20 °C
Resolution 1.8 Å 1.8 Å
Space group P212121 P21
a, b, c (Å)

α, β, γ (°)

80.1   112.6   88.0

90.0   90.0   90.0

69.0   53.8  79.4

90.0   106.1   90.0

Crystal system Orthorhombic Monoclinic
No. residues resolved 305/340 321/340
Ligands PLP, Mg2+ PLP, Mg2+

 

SR belongs to the fold-type II family of PLP-dependent enzymes, meaning it contains a β-sheet core surrounded by α-helices, with the active site located in a cleft between the two domains. Accordingly, both domains of 5X2L and CRK1 contain a parallel-stranded β-sheet surrounded by nine α-helices in the large domain and three in the small domain. A magnesium ion (pink sphere) that helps to stabilise protein folding and increase maximal activity6 is octahedrally coordinated by three water molecules, the acid groups of Glu210 and Asp216, and the carbonyl oxygen of Ala214.

Chloe 3

Figure 2. Overall X-ray crystal structure of the human SR holoenzyme CRK1. Residues are coloured from red to violet, N-terminus to C-terminus, and all helices are numbered 1–12 based on the order they occur in the polypeptide sequence. Each SR monomer comprises a large domain (helices 1–3 and 7–12) and small domain (helices 4–6) connected by a flexible loop region.

 

CRK1 boasts good ordering of residues, with only a few not well-defined: 1–3, 132–135, and 339–340. Aside from those at the C- and N-terminus, which are often poorly resolved during structure solution anyway, the only other undefined residues (132–135) were localised to the top of helix 5 in the highly-mobile small domain. 5X2L shows similarly undefined residues at the termini (1–2, 318–340) although in addition it is also missing residues of the flexible loop region (67–76) that connects the two domains.

Solvent-exposed loops are notorious for being tricky to model due to their high occupancy. The loop may be visible in CRK1 but not 5X2L because CRK1 has the help of its (symmetry) mates. By viewing the symmetry partners in the crystal lattice, the loop residues 69–73 are seen to be stabilised by a water-mediated interaction between Leu72 in one monomer and Lys221 in an adjacent monomer.

These favourable contacts may not occur for both structures because 5X2L crystallised in the orthorhombic space group P212121 while CRK1 crystallised in the monoclinic space group P21. Both possess 2-fold symmetry, but differences in molecular packing would have influenced whether the loop region would be suitably positioned to receive stabilising crystal lattice contacts.

Round 3: Best [Super]Pose!

A superposition of 5X2L and CRK1 (Fig. 3) revealed that, unsurprisingly, the two structures were well aligned with a Cα RMSD of 0.55 Å. Any remaining conformational differences are likely to result from the unresolved loop region, the missing helix and polypeptide strand that make up residues 318–340, and random structural variations. So hardly a ‘clash’ but at least it makes for a nice picture.

Chloe 4

Figure 3. Superposition of the X-ray crystal structures of 5X2L (blue) and CRK1 (red).

By now there is no doubt you are wondering who the champion is of the undeniably riveting Clash of the Crystals.

The answer is both, and neither, because any discovery that contributes to scientific advancement is a champion in my book J

Yes, even when said discovery beats me to the punch.

Blog written by Chloe Koulouris

References

  1. Takahara S, Nakagawa K, Uchiyama T, Yoshida T, Matsumoto K, Kawasumi Y et al. Design, synthesis, and evaluation of novel inhibitors for wild-type human serine racemase. Bioorg Med Chem Lett. 2017 Dec 13.
  2. Jirásková-Vaníčková J, Ettrich R, Vorlová B, Hoffman HE, Lepšík M, Jansa P et al. Inhibition of human serine racemase, an emerging target for medicinal chemistry. Curr Drug Targets. 2011 Jun; 12(7):1037-55.
  3. Dixon SM, Li P, Liu R, Wolosker H, Lam KS, Kurth MJ et al. Slow-binding human serine racemase inhibitors from high-throughput screening of combinatorial libraries. J Med Chem. 2006 Apr; 49(8):2388-97.
  4. Mori H, Wada R, Li J, Ishimoto T, Mizuguchi M, Obita T et al. In silico and pharmacological screenings identify novel serine racemase inhibitors. Bioorg Med Chem Lett. 2014 Aug; 24(16):3732-5.
  5. Mori H, Wada R, Takahara S, Horino Y, Izumi H, Ishimoto T et al. A novel serine racemase inhibitor suppresses neuronal over-activation in vivo. Bioorg Med Chem. 2017 Jul 15; 25(14):3736-45.
  6. De Miranda J, Panizzutti R, Foltyn VN, Wolosker H. Cofactors of serine racemase that physiologically stimulate the synthesis of the n-methyl-d-aspartate (nmda) receptor coagonist d-serine. Proc Natl Acad Sci U S A. 2002 Oct; 99(22):14542-7.

 

Large Scale Study Shows Antidepressants are More Effective than Placebo


This week an article in the Lancet has shed light on the controversy surrounding antidepressants.[1] Psychiatric disorders account for 22.8% of the global burden of disease, of which depression is the leading cause. In 2016 there were over 64 million prescriptions issued for antidepressants, which is more than double the amount issued ten years previously. Until now there has been much debate regarding the effectiveness of antidepressant drugs in treating this debilitating disorder. This study has been pivotal in providing evidence in addressing this controversy, as previous studies have not adequately examined the long term effects of antidepressants.

The study looked into 522 trials involving 116,477 patients and found that all antidepressants investigated were more effective than placebo. However, they weren’t all equally effective: It found that the drugs ranged from being a third more effective than a placebo to more than twice as effective. Interestingly, the findings showed that escitalopram, mirtazapine, paroxetine, agomelatine, and sertraline were the most efficacious in adult patients. On the other hand, reboxetine, trazodone, and fluvoxamine were found to be the least effective of the drugs tested, with effects diminishing over time. Many patients stop using antidepressants after only a few weeks, which may have contributed towards skewed results in previous investigations as many of the drugs tested are only effective after long term use. However, researchers noted that most of the data in the meta-analysis covered eight weeks of treatment, although it did not take into account problems that may emerge from longer term us of the drugs.

Overall, clinicians consider this study to be an important piece of evidence in encouraging patients to pursue treatment options for depression, including antidepressants where necessary. Hopefully, this seminal article will help remove the stigma surrounding depression and the use of antidepressants. More than ever, mental health is being acknowledged as a topic for serious discussion and in light of this article more awareness needs to be made about the treatment options for these disorders which affect one in four adults in the UK.[2]

Blog written by Rachael Besser

References

1 Cipriani, Andrea et al. Comparative efficacy and acceptability of 21 antidepressant drugs for the acute treatment of adults with major depressive disorder: a systematic review and network meta-analysis

2 McManus, S., Meltzer, H., Brugha, T. S., Bebbington, P. E., & Jenkins, R. (2009). Adult psychiatric morbidity in England, 2007: results of a household survey. The NHS Information Centre for health and social care.

 

Case placement in Emeryville


I have recently returned from my PhD Case placement at the Novartis Institutes for Tropical Disease (NITD) who recently relocated to Emeryville California. NITD drug discovery is focused in 3 areas; malaria, cryptosporidiosis and kinetoplastid diseases.

My PhD project has been to identify drug-like inhibitors that inhibit an enzyme that has been identified as a target for the kinetoplastid pathogen that causes African sleeping sickness, trypanosoma brucei. Whilst on my placement at NITD I was fortunate to work alongside scientists that have been involved with identifying drug candidates against the same pathogen, and have parasite assays in place to examine the effects of drug molecules against them. I was able to culture the live parasite and it was exciting to see that the compounds that I had designed and synthesised during my PhD inhibited the growth of the parasite.
During my time at NITD I was also able to work in the research chemistry laboratories, synthesising further analogues against my enzyme target, it was a great opportunity to work in a cutting edge research environment with a variety of instruments that facilitated day to day chemistry experiments. Over the course of my placement I was able to identify new chemical analogues that showed improved potency against the parasite, that I am currently following up back at the Sussex Drug Discovery Centre.

Ryan 1

At the weekends I was able to do a bit of travelling around California, some of the highlights were visiting Yosemite national park (above), cycling over the Golden Gate Bridge and visiting Alcatraz.

Blog written by Ryan West

Beware Greeks bearing gifts


The publication that I wanted to discuss was bought to my attention whilst reading Derek Lowe’s superb blog “in the Pipeline”. I would wholeheartedly recommend readers of this particular website to read the original blog discussion of the publication there. The link to in the Pipeline article is here http://blogs.sciencemag.org/pipeline/archives/2017/10/05/beware-of-zinc-and-of-other-stuff and the link to the original article is as follows https://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.7b01071

In the publication a group at Dundee University were exploring a fragment based approach, against an enzyme which has a role in ubiquitin conjugating called Ube2T. The team had been successful in identifying a fragment hit in an earlier publication. In this first publication they had utilised differential scanning fluorimetry (otherwise known as a thermal shift assay) and followed up with biolayer interferometry, as an orthogonal technique to confirm primary hits from the DSF primary screen. To further generate confidence, ligand observed NMR was carried out.

From this work a specific fragment was chosen as the top hit and was then measured with 15N- labelled NMR and ITC measurements against which at this stage showed positive binding. At this point analogues were ordered around this fragment and this is where the project hit some rough water. None of the analogues seem to show any significant SAR, and the limited number of analogues that did bind were much weaker in potency than the original hit

In parallel, co-crystallization of the fragment against Ube2T was attempted. While this was unsuccessful in showing specific binding, it did highlight a rearrangement of the protein, and the presence of a metal ion close to the catalytic residue, this metal ion was then identified as Zn2+

As no zinc was present in the co-crystallization buffer system, the team investigated the original fragment hit compound, running a zincon colorimetric assay on the sample which was gave a positive result for the presence of zinc. The team re-ran the original ITC experiment with the fragment hit in the presence of EDTA (which should chelate all zinc present), this showed that all binding was lost. In summary, the fragment hit must have contained Zn2+ ions which was the cause of the activity.

Gareth 1

Figure A showing ITC binding experiment of Ube2T with the original fragment compound with and without EDTA present. ZnCl2 against Ube2T is also shown as a comparison.

Taken from: F. E. Morreale et al., Mind the Metal: A Fragment Library-Derived Zinc Impurity Binds the E2 Ubiquitin-Conjugating Enzyme Ube2T and Induces Structural Rearrangements. Journal of Medicinal Chemistry60, 8183–8191 (2017).

The authors did point out that this hit was from a commercial library and one of the actions they undertook was to ask for the QC data from the supplier, at the point when the related analogues of the fragment compound did not show any activity. This obviously did not highlight the presence of the Zn2+

I did wonder when reading this publication if a different screening cascade may have identified this type of false positive, before having to get to the step of a very labour intensive co-crystallization process. For example a cell based assay might by more resilient to the presence of metal ions. However with a fragment based project, this may not be possible due to the poor affinity of fragments and also obtaining an effective concentration within the reduced DMSO tolerance of a cell based format compared to a biochemical assay. The only recommendation for this specific case, would be carry out further purification of samples, and then re-assay. It would also appear that the data from commercial suppliers is lacking and maybe that can be changed.

The authors should be commended for this work and putting it in the public domain. Drug discovery is a hard, long and quite often an unsuccessful process, and anything we can do to reduce the time following red herrings the better.

Blog written by Gareth Williams

 

 

Circular dichroism for protein characterisation


During my PhD project, I was working on a bacterial protein involved in iron transport. The secondary and tertiary information about this protein was limited and function was not well known at that time. In order to shed more light on this, I was looking for various techniques and came across ‘Circular Dichroism’ [CD], a widely used technique for analysing the secondary structure content of the protein of interest in solution. Circular dichroism measures the difference in the absorption of left and right circularly polarised light by chiral molecules. It is also a great technique to study the protein-ligand interactions.

The secondary structure composition is associated with characteristic spectra based on the absorption due to the peptide bond in the far ultraviolet [UV] CD region (180-260 nm) whereas tertiary structure features are influenced by the spectrum in the near UV region (260-320 nm) with absorption being due to aromatic residues [2]. In this category, synchrotron radiation circular dichroism spectroscopy [3] [SRCD] also became popular which provided more structural information with the available options for measuring the spectra to lower wavelengths, with improved signal to noise ratio levels.

I came across an article in Nature Scientific Reports indicating more advancement in this area by the introduction of High-throughput SRCD [HT-SRCD] using multi-well plates [1] which was interesting. The paper describes the features provided by Beamline B23 of Diamond Light Source, Oxford, UK for setting up the HT-SRCD using multi-well plates. It also describes examples for high-throughput measurements carried out using multi-well systems. In short, this HT-SRCD could be a potential resource for studying the protein folding, conformational changes in protein structure induced by ligands, buffers and other components as well as secondary structure determination on a high-throughput scale.

Blog written by Mohan Rajasekaran

  1. Hussain, R., Javorfi, T., Rudd, T. R., and Siligardi, G. (2016) High-throughput SRCD using multi-well plates and its applications. Scientific reports 6, 38028
  2. Kelly, S. M., Jess, T. J., and Price, N. C. (2005) How to study proteins by circular dichroism. Biochimica et Biophysica Acta (BBA) – Proteins and Proteomics 1751, 119-139
  3. Wallace, B. A., and Janes, R. W. (2010) Synchrotron radiation circular dichroism (SRCD) spectroscopy: an enhanced method for examining protein conformations and protein interactions. Biochemical Society transactions 38, 861-873

 

 

 

 

Citizen scientists


Tesh 1Many moons ago while I was an postgrad in Dundee, when a computer screen was the size of a medium to large beach ball, i.e. the late-90s, I came across a curious project over the then relatively nascent internet called SETI@home.

The project was conceived by the Berkley SETI Research Centre as an initiative to engage with the general public to help address a specific (and possibly the ultimate) question, “Is there anybody out there?”. The premise being that if there are other (extra-terrestrial) intelligent life forms out there in the universe, they may be broadcasting (knowingly or otherwise) their presence and thus should be detectable by simply listening in.

A vast amount of data was collected, consisting of recorded radio signals across a chunk of the spectrum, which then needed to be analysed. The scientific community could not fund/carry out the analysis and needed an “out of the box” solution. Step in SETI@home, where the idea was to make use of the processing power of the vast numbers of PCs that were starting to take up residence within the public domain. The strategy was not particularly interactive and solely relied in the ability of PCs to crunch through the data when not in use by their owner. All you had to do was register and packets of data were sent out to your PC (the University of Dundee’s in my case, ahem!) and your PC did the rest. There was something hypnotic, ethereal and satisfying as the screen presented how much data had been processed and how much more there was to go. Even as someone who only has a passing interest in space related matters, wondering if there were any “curious” signals in the data, how long it would take to get a positive answer and if enough bandwidth had been covered to account for a negative outcome, was exciting and alluring. Though it was not particularly interactive, it did capture the imagination of the wider public and made use of their individual resources to try and answer an important scientific question.

A decade or so later I came across another project (planethunter.org) that pricked my curiosity and again it was a space related project. The premise was as simple as it was unimaginable – spot planets as they traverses across the face of their host star(s) in the line of site from Earth from up to ~1500 light years away. Tesh 2

This however was made possible by the recently launched Kepler space telescope by NASA, which is a space based observatory with the sole purpose of peering at a patch of the sky continuously and recording (every 30 minutes) the light emitted by the stars within its field. Every 3 months or so the data would be downloaded by NASA and distributed to its collaborators within the scientific community for analysis. This involved dealing with an incredible volume of data, for which data processing and analysis pipelines had to be set up for.

Though these were in themselves incredibly successful, it was recognised fairly early on that engaging with the public in a meaningful way would be advantageous, not only to prevent public apathy (which seems to haunt many of these large projects) but also in recognising that any analysis pipeline set up would ultimately be limited.

Analysis of any large data sets requires assumptions, which are usually applicable across the majority of instances but not where there are deviations from the “expected scenario”. The ability of the human eye to perceive subtle differences and patterns was seen as an advantage here, and which, with the right approach could potentially be tapped. Step in planethunter.org (hosted by zooinverse.org) and citizen scientists.

At its simplest level (below), the platform asks the public if there is a periodic signal within the light curve of a particular star. However, the level of analysis could be more complex if desired, e.g. identifying the star type, accessing the unprocessed/raw data and links to information about a stars’ age, its metallicity, a deep visual look into its neighbourhood and so on. There were forums set up to discuss the data in general, reoccurring glitches in the data, individual stars, analysis pipelines for larger bespoke batch analyses and much more.

Tesh 3So did these citizen scientists find anything? Yes, being the clear answer. To date there have been 10 peer reviewed publications and more will no doubt follow

(https://www.zooniverse.org/about/publications).

These types of endeavours, where the public can be engaged in a meaningful way to answer specific and scientifically inspiring questions, are important on a number of levels:

  1. Access to a “free” and potentially vast resource.
  2. There are important (and sometimes unexpected) discoveries to be made.
  3. It prevents public apathy.
  4. Exposure.
  5. Funding (e.g. from the greater exposure of the project)

The question arises that in this age of big data, particular with the explosion in cell biology and disease related big data projects, why do they not also have such endeavours? For such well-funded scientific areas, for there to be only one such endeavour on zooniverse.org (Etch a Cell) is if nothing else sad. Etch a Cell is an initiative led by the Francis Crick Institute where the aim is to engage with the public to help build 3D models of the nuclear envelope from electron micrographs. This is of interest to me and (possibly some) other people in my field of research but it hardly captures the ziet geist as Planethunters and SETI@home did and continues to do so.

Blog written by Tesh Patel