Conference Report: RSC BMCS Mastering MedChem IV, 14th March 2018


Fiona

On Wednesday 14th March 2018 I attended the RSC Biomedical and Medicinal Chemistry Sector’s 4th Mastering MedChem Symposium. The opening session chair, Professor Joe Sweeney (University of Lancaster) described the event as the sector’s “most successful new conference”. This year it was being held at my almer mater, University of Strathclyde in Glasgow.

The day was split into three sessions where a series of talks from a mixture of speakers across academia and industry shared stories of good practice in drug discovery. A variety of disease areas were covered. Prof. Stuart Conway (University of Oxford) gave a useful overview of epigenetics and bromodomain ligands. Malaria was covered by Prof. Ian Gilbert of the University of Dundee’s Drug Discovery Unit, although I was surprised to learn the project had progressed as far as it had given no mode of action for their compounds had been elucidated due to the complex nature of the malaria disease pathway.

During the lunchbreak and afternoon coffee session I was able to chat to attendees about the poster I had brought which summarised some of my PhD project involving the development of kinase inhibitors to target cancer via a synthetic lethality strategy. It was interesting discussing with other students attending with posters how they had prepared similar benzimidazole compounds to mine but for entirely different disease targets.

While I was a tad concerned there would be no conversation around kinases given the general nature of a number of the talk titles, Dr. Iain Simpson of AstraZeneca shared the story of the optimisation of a compound for a kinase project that, serendipitously, had the same core as one of my own series. It was very useful to speak to him afterwards to compare notes on dialling in selectivity for our respective targets.

There were also more generic talks about the industry and techniques for improving the efficiency of the laborious drug discovery process. Prof. Adam Nelson (University of Leeds) commented on the small pool of reactions most medicinal chemists use for preparing libraries (amide formation, Pd-catalysed couplings, alkylations etc.). He also shared a way of finding bioactive compounds faster without having to purify every single reaction run in the lab by assaying crude reaction mixtures and then only scaling up and purifying reactions with bioactive components, which he termed “activity-directed synthesis”, a rather intriguing method but must have limitations given the toxicity of many chemical reagents!

Dr. Craig Johnstone (Evotec) gave examples of case studies where focussing on multiple drug parameters at once had increased the productivity of a number of his company’s projects, instead of the scenic route most projects take to optimise individual physiochemical properties. He spoke about the potential of artificial intelligence to predict what reactions would work before heading into the lab and he also took an opportunity to justify his theory that every good drug candidate has a logD of around 2.

To round up the day, the memoriam McGuigan Lecture was given by Prof. Chris Schofield (University of Oxford) who took us on a whistle-stop tour of the development of inhibitors of serine and Zn(II) dependent beta-lactamases and their involvement in combating bacterial resistance. Due to the unfortunate absence of Dr. Nicole Hamblin from Charles River laboratories (the only female speaker in the programme I might add), an alternative Q&A was held with a panel of the speakers from the day. Attendees asked a number of questions around the feasibility of artificial intelligence in drug discovery, the importance of specialisation for career progression and a particularly useful discussion point in their careers often have to keep moving around the country, jumping from project to project, before a rare long-term lectureship or industry post arises, if at all. The panel sympathised with this view and mainly came to the conclusion that the way research is funded needs to change to give scientists more sustainable livelihoods.

I found the symposium to be a very useful experience, particularly as it was aimed at early career researchers such as myself. It was nice to be back in Scotland for a bit and I would hope to attend future Mastering MedChem meetings.

Blog written by Fiona Scott, PhD researcher, Sussex Drug Discovery Centre

 

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Homocysteine


This publication written by Dong-Mei Zhang et al 1 describes the investigation of serum homocysteine and its role in cognitive impairment.

The authors investigate hyperhomocysteinemia (increased levels of serum homocysteine) and its association with an increased risk of cognitive impairment. According to Sudha Seshadri et al 2, an increased plasma homocysteine level is a strong independent risk factor for the development of dementia and Alzheimer’s disease, where they say that the risk of developing Alzheimer disease doubles with a plasma homocysteine level greater than 14 μmol/L, as well as being at major risk of suffering from coronary artery disease. Homocysteine levels increase with age, with common adult levels in Western populations being 10 to 12 μmol/L.

The graph below is taken from Axis-Shield 5 who carry out in-vitro diagnostic testing. The graph shows that the higher the homocysteine level, the greater the risk of developing coronary artery disease/Alzheimer’s disease.

Kamlesh 1

Homocysteine is a homologue of the amino acid cysteine, with an extra methylene bridge. It is biosynthesised in the body through metabolism of methionine (S-demethylation). Metabolism of homocysteine is aided by vitamin B12, folic acid and vitamin B6. As well as other factors, deficiencies in these vitamins may increase serum homocysteine levels. Hyperhomocysteinemia can contribute to a greater risk of developing diseases such as cardiovascular diseases/retinal vascular disease as well as neuropsychiatric diseases. B vitamins have therefore been considered as a possible option to reduce the risk of Alzheimer’s and dementia.

The authors wanted to address the discrepancy in the existing literature on the therapeutic effect of vitamin B and folates in patients with significant cognitive deficits, secondary to Alzheimer’s disease or dementia. They carried out a meta-analysis of randomized controlled trials in elderly patients with poor cognitive ability secondary to Alzheimers or dementia, who received homocysteine lowering B vitamins supplements and had serum homocysteine levels reported.

Existing evidence on vitamin B supplement induced reduction of cognitive decline by lowering homocysteine levels is conflicting. The authors mention other studies where daily folic acid supplements were taken by people with folate deficency (Durga et al 3, 800μg/d for 3 years and Fioravanta et al 4 15mg/d for 60 days), showing an improved cognitive performance. These results were not included in the meta-analysis because the serum homocysteine levels were not reported.

The overall results suggested that folate in combination with vitamin B12 and/or B6 supplements failed to offer any significant advantage in slowing down or preventing the progression of cognitive decline, although Vitamin B supplements were shown to significantly reduce homocysteine levels.

Blog written by Kamlesh Bala

1 Dong-Mei Zhang et al, Journal of Geriatric Psychiatry and Neurology 2017, Vol 30 (1) 50-59

2 Seshadri S, Beiser A, Selhub J et al, N Engl J Med 2002; 346:476-483

3. Durga J et al, Lancet. 2007; 369 (9557): 208-216

4. Fioravanti M et al, Arch Gerontol Geriatr. 1998; 26 (1): 1-13

5. http://www.homocysteine.co.uk/measuring-homocysteine/

Keywords: Hyperhomocysteinemia, homocysteine, cognitive decline

Modular C(sp2)-C(sp3) radical cross-coupling with PT-sulfones


The construction of sp2-sp3 carbon-carbon bonds is sometimes not trivial with standard two-electron coupling reactions such as Suzuki, Heck and Negishi. To offer chemists alternative options to form these types of carbon-carbon bonds the Baran Lab have been working on metal-catalysed radical cross-coupling (RCC) reactions. I have previously written about some of their earlier work in this area. In their latest paper (Science 360, 75 –80 (2018)) they write about their discovery that a redox-active phenyl-tetrazole (PT) sulfone could be used in these RCC reactions (figure 1).

Lewis 1

Figure 1

Some PT-sulfone reagents are commercially available but others such as (3f) can be easily made from phenyl-tetrazole (6) by sulfur-carbon bond formation (alkyl halide displacement or Mitsunobu) and then oxidation of the resulting sulphide (mCPBA or ammonium molybdate/hydrogen peroxide). These sulfones are useful building blocks and can be used in RCC reactions as they are or further functionalised such as α-alkylation or α-fluorination (figure 2).

Lewis 2

Figure 2

A small set of fluorinated sulfone building blocks (8-11) were used to introduce mono/di-fluoromethyl and mono/di-fluoroethyl moieties onto a selection of aromatics (figure 3). Unfortunately, these reaction conditions are not able to install a trifluoromethyl group. Sulfones (7-11) are not currently commercially available but 100-500 mg quantities can be requested directly from the Baran lab via this link.

Lewis 3

Figure 3

Baran ran a series a competition experiments and under these reaction conditions observed the following reactivity trend Cl/Br < SO2PT < NHPI/TCNHPI (figure 4). This observation was tested with sequential chemoselective RCC reactions. Firstly, a decarboxylative cross-coupling (DCC) was performed followed by a desulfonylative cross-coupling (SCC) (figure 5).

Lewis 4

Figure 4

Lewis 5

Figure 5

These new reagents and chemoselective reaction conditions offer a simple and general method to add to the tool box of sp2-sp3 carbon-carbon bond forming reactions. The ability to diversify and fluorinate a common building block will increase the interest from medicinal chemists as will the ability to introduce simple alkyl fluorides without the use of harsh reaction conditions or toxic reagents.

Blog written by Lewis Pennicott

 

A Brief Comparison of Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC)


  1.  MST

MST assay is based on thermophoresis, the directed movement of molecules in a temperature gradient induced by an infrared laser. Thermophoresis is highly sensitive to all types of binding-induced changes, such as size, charge, hydration shell or conformation, which allows for a precise quantification of molecular events. (Jerabek-Willemsen, André et al. 2014) Initial, the molecules are distributed homogenously with an initial fluorescence signal. When the IR laser is activated, the fluorescent signal is decreased as a ‘T-Jump’ form. With the turnoff of IR-laser, the molecule diffusion is back, solely driven by mass diffusion. The trace difference between a fluorescent molecule binding with or without non-fluorescent ligands indicates a binding signal. (Figure 1)

MST can handle weak conformation change on binding of two molecules in different buffer system, such as biological liquids. Another advantage is low sample consumption and MST can measure dissociation constants from pM to mM. (Wienken, Baaske et al. 2010, Jerabek-Willemsen, André et al. 2014) However, in most of cases, the sample should be labeled with hydrophobic fluorophores which would probably cause non-specific binding effects. (Table 1)

 Tina 1

Figure 1. MST setup and experiments. A. The machine Monilith NT. 115 from NanoTemper Technologies GmbH. B. Schematic representation of MST optics. C. Typical signal of a MST experiment. D. Typical binding experiment.(Jerabek-Willemsen, André et al. 2014)

  1. ITC

ITC is a biophysical technique to measure the heat exchange associated with molecular interactions at a constant temperature. (Duff Jr, Grubbs et al. 2011, Milev 2013) It directly determines the binding affinity (Ka), enthalpy changes (ΔH), and binding stoichiometry (n) of the interaction between two or more molecules in solution. The experimental methodology involves performing several titrant injections from a syringe (usually the ligand) into the solution (usually the macromolecule) in the cell, while maintaining the system at isobaric, quasi-isothermal conditions. When the ligands are injected to the cells, the ligands bind to macromolecules and the machine detects the heat upon binding. With several injections, ligands bound to protein continually. However, when the target protein becomes saturated with the ligand, less binding occurs and the heat change starts to decrease. If the macromolecule is saturated with ligand, no more binding occurs, and only heat of dilution is observed.

ITC has been widely applied as a major tool in drug discovery fields, validating and optimizing the hits (Leavitt and Freire 2001, Peters, Frasca et al. 2009) and also in binding studies, such as protein-protein, protein-DNA, small molecule-protein interactions(de Azevedo, Walter et al. 2008, Liang 2008). ITC is a fast and straight way to detect binding affinity of two molecules by the change of binding enthalpy. However, some complexes may exhibit rather small binding enthalpies that are not suitable for the ITC measurement. (Table 1)

MST ITC
Advantages ²   Small sample size

²   Immobilization free

²   Minimal contamination of the sample

²   Ability to measure complex mixtures

²   Wide size range for interactants (ions to MDa complexes)

²   Ability to determine thermodynamic binding parameters in a single experiment

²   Modification of binding partners are not required

 

Disadvantages ²   Hydrophobic fluorescent labelling required, may cause non-specific binding

²   No kinetic information

²   Highly sensitive to any change in molecular properties

²   Large sample quantity needed

²   Kinetics cannot be determined

²   Limited range for consistently measured binding affinities

²   Non-covalent complexes may exhibit rather small binding enthalpies since signal is proportional to the binding enthalpy

²   Not suitable for HTS

Table 1. Advantages and disadvantages of ITC and MST.

  1. Ligand-protein binding affinities detected by ITC and MST

One example is about comparing the biophysical data of small molecules with Protein kinase CK2 using both MST and ITC assays.(Winiewska, Bugajska et al. 2017) In this paper, the interactions of four halogenated benzotriazoles with the catalytic subunit of human protein kinase CK2 had been investigated. Among the four compounds, only one compound (5-BrBt) had a consistent binding affinity data in both MST and ITC assays, the solubility of which substantially exceeded the ligand concentration. For another three compounds, when the compounds titrated to the protein solution for ITC measurement, the binding affinities determined by ITC were around 10-folded weaker than by MST. The main problem was the limited titrant solubility that resulted in the formation of nano-aggregates. The issue was ignored by titrating the protein to the compound solution as the protein was soluble enough. (Figure 2) The protein-ligand affinities that derived from ITC may be underestimated because of the compound solubility problem, while the problem can be avoided by MST. Tina 2

Figure 2. Correlation between MST- and ITC-derived binding affinities determined for complexes of halogenated benzotriazoles with hCK2α. Kd(ITC), were obtained with ITC experiment, in which either inhibitor (red) or protein (blue) was used as a titrant. Vertical and horizontal bars represent standard deviation (MST) and 67% confidence intervals (ITC), respectively.(Winiewska, Bugajska et al. 2017)

Blog written by Xiangrong (Tina) Chen

de Azevedo, J., F. Walter and R. Dias (2008). “Experimental approaches to evaluate the thermodynamics of protein-drug interactions.” Current drug targets 9(12): 1071-1076.

Duff Jr, M. R., J. Grubbs and E. E. Howell (2011). “Isothermal titration calorimetry for measuring macromolecule-ligand affinity.” J Vis Exp 55: e2796.

Jerabek-Willemsen, M., T. André, R. Wanner, H. M. Roth, S. Duhr, P. Baaske and D. Breitsprecher (2014). “MicroScale Thermophoresis: Interaction analysis and beyond.” Journal of Molecular Structure 1077: 101-113.

Leavitt, S. and E. Freire (2001). “Direct measurement of protein binding energetics by isothermal titration calorimetry.” Current opinion in structural biology 11(5): 560-566.

Liang, Y. (2008). “Applications of isothermal titration calorimetry in protein science.” Acta biochimica et biophysica Sinica 40(7): 565-576.

Milev, S. (2013). “Isothermal titration calorimetry: Principles and experimental design.” General Electric 9.

Peters, W. B., V. Frasca and R. K. Brown (2009). “Recent developments in isothermal titration calorimetry label free screening.” Combinatorial chemistry & high throughput screening 12(8): 772-790.

Wienken, C. J., P. Baaske, U. Rothbauer, D. Braun and S. Duhr (2010). “Protein-binding assays in biological liquids using microscale thermophoresis.” Nature communications 1: ncomms1093.

Winiewska, M., E. Bugajska and J. Poznański (2017). “ITC-derived binding affinity may be biased due to titrant (nano)-aggregation. Binding of halogenated benzotriazoles to the catalytic domain of human protein kinase CK2.” PloS one 12(3): e0173260.

 

 

 

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

 

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.