Crystallography in Fragment-based drug discovery


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

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

 

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

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

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

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

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

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

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

Blog written by Xiangrong Chen

References

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

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

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

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

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

Zika virus: A neglected disease with no specifically designed drugs


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

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

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

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

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

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

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

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

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

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Table 1. List of potential compounds to tes.t

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

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

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

Blog written by: Jose Gascon

References

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

 

Beer versus Alzheimer!


While casually browsing for updates on Alzheimer’s Disease (AD), apolipoprotein E4 (ApoE4) genotype and amyloid beta (Aβ) burden, one title strongly caught the attention of the author of this blog:

Beer Drinking Associates with Lower Burden of Amyloid Beta Aggregation in the Brain: Helsinki Sudden Death Series.

Beer drinking against dementia? This study, published by Kok and colleagues (1) in Alcoholism: Clinical & Experimental Research, at least deserved reading! The authors investigate in this paper the association between the consumption of different alcoholic beverages (spirits, wine, beer) and Aβ aggregation in the brain. Their results rather surprising: beer drinking decreased the prevalence of Aβ-immunoreactivity in brain sections of autopsy cases investigated.

Before looking into details of the study, first a brief background on Aβ and AD. Amyloid plaques or senile plaques are extracellular deposits of the Aβ peptide and are one of the microscopically hallmarks of AD. The Aβ peptide is derived by sequential proteolytic cleavage of the β-amyloid precursor protein (APP) and plaques seem to spread hierarchically throughout the brain in patients with AD (figure 1). AD is a complex disease and little is known about its pathophysiology and cause. Also studies have shown a substantial genetic component in AD, the pattern of inheritance seems far more complex in which genetic risk factors such as ApoE4 work together with environmental factors and life exposure (i.e. life style).

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Figure 1: Characteristic progression of Aβ deposition. Senile plaques (left) seem to spread hierarchically throughout the brain in patients with AD (right). Modified from Jucker et al(2)

Kok et al (1) investigated on how alcohol consumption may influence Aβ burden and analysed for this brain sections (frontal cortex) of 125 cases that were known to consume alcohol and of which Aβ data was available. Only beer drinking was negatively associated with the presence of Aβ-immunoreactivity (figure 2); spirits and wine did not show any correlation, and age, as well as ApoE4 genotype were, as expected, strongly associated with Aβ burden.

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Figure 2: Aβ-immunoreactivity prevalence in beer drinkers versus non-beer drinkers (unadjusted analyses, n= 125).

The mechanism on how beer consumption may influence Aβ aggregation remains speculative. Regular beer consumers were shown to have increased levels of B vitamins and folate, as well as reduced levels of total homocysteine. High blood levels of homocysteine (hyperhomocysteinemia) seem to increase the risk for endothelial cell injury that may result in stroke and are associated with a wide range of diseases including thrombosis and AD (for review see (3)). However, how this links to Aβ burden clearly needs further investigation. Nevertheless, the study by Kok et al (1) is unique and is the first study that assesses the effects of alcohol consumption on post-mortem Aβ aggregation.

The author of this blog wants to emphasize that after reading this blog it is not advisable to drink more beer now. As most are surely aware, excessive use of alcohol can lead to cognitive impairment and many other undesirable things.

 

Blog written by: Lucas Kraft

References

  1. Kok, E. H., Karppinen, T. T., Luoto, T., Alafuzoff, I., and Karhunen, P. J. (2016) Beer Drinking Associates with Lower Burden of Amyloid Beta Aggregation in the Brain: Helsinki Sudden Death Series. Alcohol. Clin. Exp. Res. 10.1111/acer.13102
  2. Jucker, M., and Walker, L. C. (2013) Self-propagation of pathogenic protein aggregates in neurodegenerative diseases. Nature. 501, 45–51
  3. Morris, M. S. (2003) Homocysteine and Alzheimer’s disease. Lancet. Neurol. 2, 425–8

 

 

Who Needs Lab-Based Synthetic Chemists?


Back in 2013 I was a postdoc at the University of Tokyo – when I wasn’t working hard in the laboratory, eating sushi or participating in karaoke, I encountered a paper that fascinated me; ‘Combining 3D printing and liquid handling to produce user-friendly reactionware for chemical synthesis and purification’ was the title of the article.1 The group of Lee Cronin at The University of Glasgow described the fabrication of a multi-chamber reaction vessel, with each region having different catalysts printed onto its surface – in this case Montmorillonite K10 and Pd/C (Figure 1). Building up around the loaded base layer and subsequent sealing of the entire unit allowed, after 90 degree rotations, the synthesis of 3a and 3b (Figure 3) via an initial acid-catalysed Diels-Alder cyclisation, imine formation and subsequent reduction over palladium on carbon with triethylsilane.

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Figure 1: Fabrication of Reaction Vessel1

A schematic of the reaction vessel can be seen in Figures 2 & 3. The first split chamber contains solutions of the initial reactants. When rotated by 90o, the two solutions combine in a second chamber that has been layered with Montmorillonite K10. Once the first reaction is complete (5 h), the box is rotated again by 90o in order to initiate imine formation. A third rotation by 90o passes the solution over a printed surface of palladium on carbon in the presence of triethylsilane, which acts to reduce the imine to the corresponding amine. The crude mixture is finally passed through a silica plug to give the desired final products in yields similar to those utilising traditional glassware (32% vs 40% for 3a, 30% vs 38% for 3b).

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Figure 2: Schematic of Reaction Vessel1

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Figure 3: Rotation and Reaction in Separated Chambers1

 

So, with the above in mind, do we really need lab-based synthetic chemists? Probably – the current range of reactions applicable to this technology is rather limited to robust and well established chemistry; mixtures that are happy to be left under atmospheric conditions, in the presence of water and without proper temperature regulation. However, although there are currently quite a few limitations, I do feel that perhaps after optimisation by a trained scientist, people unfamiliar with advanced synthesis techniques or liquid handling will be able to synthesise pure compounds (medicinally relevant or not) by picking up a box from the shelf and following instructions like one might do with piece of flat-packed furniture from one of the world’s favourite Swedish stores – surely nothing can go wrong with that?

 

Blog written by: Mark Honey

 

References

  1. P.J Kitson,   M.D. Symes,   V. Dragone and  L. Cronin  Chem. Sci., 2013,4, 3099-3103

 

 

 

 

Green and Sustainable Medicinal Chemistry – 25 years later


What is Green Chemistry?

In short, Green Chemistry aims to prevent pollution through waste minimisation and by avoiding toxic and hazardous substances in the production and application of chemical products.  This is achieved by substitution of undesirable chemical products and processes by cleaner, safer and environmentally friendlier alternatives.1

In the 1990s the 12 Principles of Green Chemistry were established (Figure 1).

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Figure 1: 12 Principles of Green Chemistry.  Image taken directly from source.1

 

Green Chemistry Metrics and Tools to assist Medicinal Chemists

Atom economy (AE)

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Environmental factor (E factor)

The E-factor is the amount of waste (kg) produced when 1 kg of desired product is made, taking into account all of the auxiliary components – yield, reagents, waste solvents, waste from chemicals used in work-up and fuel (although difficult to quantify) and can be applied to a multistep process.1

Solvent Selection guides

Solvent selection guides exist that give information about the hazards of particular solvents (Table 1).2  Solvents represent at least half of the material used in the synthesis of pharmaceuticals and a significant amount of the waste per kg of desired compound.2

Recently, solvents have been ranked as follows:2

– Recommended (or preferred): solvents to be tested first in a screening exercise, if of course there is no chemical incompatibility in the process conditions.

– Problematic: these solvents can be used in the lab or in the Kilolab, but their implementation in the pilot plant or at the production scale will require specific measures, or significant energy consumption.

Hazardous: the constraints on scale-up are very strong. The substitution of these solvents during process development is a priority.

Highly hazardous: solvents to be avoided, even in the laboratory.

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Table 1: CHEM21 solvent guides (both taken from directly from source)2

Reagent guides

Members of the ACS GCI Pharmaceutical Roundtable have access to a Reagent guide that’s aim is to encourage chemists to choose ‘greener’ reaction conditions.  At the moment I have not come across a similar guide for non-members.  For the time being it is up to the individual to search in Reaxys or Scifinder, look at reaction conditions and choose greener alternatives.

Green Chemistry and Pharma

Roger Sheldon’s E(fficiency) factor highlighted that Pharma in particular produces vast amounts of waste per kg of desired product (Table 2).3

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Table 2:  The E factor (taken directly from source)3

However, it is important to note that the E factor does not take into account that the implicit molecular complexity of pharmaceuticals is twinned with limited engineering flexibility of reaction set-up.  This puts a significant responsibility for synthetic efficiency directly upon the medicinal chemist compared to those producing Bulk chemicals.  It also does not recognise that some waste is more hazardous than other waste products.4

Over the past two decades the Pharma industry has made great strides in incorporating Green Chemistry and engineering philosophies, especially into process development (read on for highlights).1

Advances in In Silico drug design and increased understanding of physicochemical properties (e.g. Lipinkski’s Rule of 5) has undoubtedly reduced waste through better and more targeted design.4  Many companies and academic institutions (including the SDDC) are documenting experiments in electronic lab notebooks (ELN) rather than using paper lab books thus reducing waste.

The ACS GCI Pharmaceutical Roundtable (12 globally leading pharmaceutical companies and 3 associate members) was established in 2005 specifically to encourage the integration of green chemistry and green engineering in the pharmaceutical industry.

Process Chemistry highlights

MSD and Codexis’ collaboration on the greener synthesis of Sitagliptin increased the overall yield by 50% and reduced the amount of waste by 80%.  This was achieved by discovering that the amino group of a key enamine intermediate did not need to be protected prior to enantioselective hydrogenation (pathway b).5  Codexis then went one step further and through directed evolution and biocatalysis were able to streamline the process.  High-pressure hydrogenation, rhodium catalyst, and the chiral purification steps were eliminated, providing a 13% increase in overall yield to 87% (pathway c).6

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Scheme 1:  Greener synthesis of Sitagliptin (image taken from source)7

The telescoping synthesis of Imatinib (Gleevec) by the Ley group furnished pure product in 32% overall yield and > 95% purity (Scheme 2).  The apparatus engineered employed solid supported scavengers packed into a series of columns to minimise the waste associated with work-ups and purifications between steps.  In-situ reaction monitoring and infrequent handling of potentially hazardous intermediates was also advantageous.8

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Scheme 2:  Ley’s greener synthesis of Gleevec (image taken from source)8

The Boots synthesis of Ibuprofen is a classic in Green Chemistry.  The original synthesis (Scheme 3) devised in the 1960s produced a vast amount of waste as a consequence of poor AE.

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Scheme 3: 1960s Boots synthesis of Ibuprofen

In 1991 a three-step synthesis was devised by Boots-Hoechst-Celanese (BHC) that had high AE thus eliminating most of the waste by products (Scheme 4).  Hydrofluoric acid, Raney nickel and palladium catalysts were recycled, again reducing waste and making the process even more efficient.

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Scheme 4:  1991 BHC greener synthesis of Ibuprofen

Recent ‘Green’ reaction highlights

Looking through the ‘most read’ articles of high impact journals there are an increasing number of Green and Sustainable chemistries reported.  Below I have picked some interesting examples of ‘greener’ reactions that caught my eye and may be attractive to other medicinal chemists.

Li’s photoinduced trifluoromethylation of inactivated arenes avoids the use of expensive metal and toxic metal catalysts (Figure 2)9.  The same group also reported a photoinduced metal-free aromatic Finklestein reaction and Sonogashira coupling.11
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Figure 2:  Li et al Photoinduced trifluoromethylation of inactivated arenes (image taken directly from source)9

The Stahl group showed that a copper(I) salt and TEMPO (2,2,6,6-tetramethylpiperidinyl-N-oxyl) selectively oxidise a range of primary alcohols to aldehydes at room temperature with ambient air as the oxidant (Figure 3).12

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Figure 3:  Stahl’s air oxidation of alcohols (image taken directly from source)12

Mechanistic studies encouraged the same group to develop a new catalyst system that exhibits broader scope and efficiently oxidizes both primary and secondary alcohols (Figure 4).12

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Figure 4:  Stahl’s air oxidation of alcohols (image taken directly from source)12

The main advantages of this reaction are the lack of stoichiometric reagents used (other than oxygen) which greatly simplifies product isolation and reduces waste. Also chlorinated solvents, which are commonly needed with other classes of oxidation reactions, are not required.12

During my project I have had some experience in using T3P® in amide couplings.  In general its use has shortened my reaction times and improved yields over more conventional coupling reagents.  The main advantages reported for T3P® are lower levels of epimerisation, easy work-up as only water-soluble by-products are formed (Scheme 5).13  T3P® is also less hazardous compared to some conventional coupling reagents such as HOBt (now classed as explosive).13

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Scheme 5:  T3P® amide coupling (image taken from source)13

T3P® has also been used in the synthesis of Denagliptin (Scheme 6), initially for amide bond formation and then subsequent dehydration of the primary amide to form the nitrile.13  Thus avoiding the use of hazardous reagents such as phosphorus oxychloride or thionyl chloride.

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Scheme 6:  T3P® amide dehydration (image taken from source)13

Conclusion

Green Chemistry begins with good synthetic route design by the medicinal chemist.  There are now a number of tools and metrics in place to help medicinal chemists to choose the most attractive route both in terms of reaction efficiency and safety.  It is obvious that reducing waste at the source (the lab) will minimise the cost of its treatment and will strengthen economic competitiveness through more efficient use of raw materials.

The increased adoption and publication of ‘greener’ reactions will populate databases such as Reaxys which in turn will encourage chemists to use ‘greener’ reagents and procedures for their own safety.  Ultimately, it is up to the individual chemist to find and use ‘greener’ reactions and it is important that these ‘greener’ reactions are just as good as conventional reactions in terms of reaction efficiency.

It will be interesting to note how future investments and government policy on sustainable and green processes will be shaped by the falling price of fossil fuel derived feedstocks, as a result of lifting sanctions on Iran and the current Shale Gas revolution.

Hopefully, one day we will move towards a greener circular economy rather than the traditional ‘take-make-use-dispose’ economy.  It is promising (at least for the time-being for a Brit!) that Principle 2 (Figure 1) and this green philosophy that encapsulates it, forms the basis of the European Commission’s “Roadmap to a resource efficient Europe”.1

 

Blog written by: Scott Henderson

References

  1. A. Sheldon. Green Chem., 2016, 18, 3180.
  2. Prat et al. Green Chem., 2016, 18, 288.
  3. A. Sheldon. Chem. Ind., 1992, 903.
  4. L. Tucker. Organic Process Research & Development 2006, 10, 315.
  5. B. Hansen et al J. Am. Chem. Soc. 2009, 131, 8798.
  6. https://www.organicdivision.org/ama/orig/Fellowship/2013_2014_Awardees/Essays/Payne.pdf
  7. https://en.wikipedia.org/wiki/Process_chemistry
  8. G. Newman and K.F. Jensen. Green Chem., 2013, 15, 1456.
  9. Li et al J. Am. Chem. Soc. 2016, 138, 5809.
  10. Li et al J. Am. Chem. Soc., 2015, 137, 8328.
  11. Li et al. Nature Communications, 2015, 6, 6526.
  12. S. Stahl and B. L. Ryland. Angew. Chem. Int. Ed. 2014, 53, 8824.
  13. http://www.euticals.com/attachments/article/20/EUTICALS_T3P.pdf

 

 

Big Pharma and their adoption of Orphan Drug Research


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Blog written by: Kamlesh Bala

 

References

  1. https://globalgenes.org
  2. http://csdd.tufts.edu/news/complete_story/pr_tufts_csdd_2014_cost_study
  3. http://www.fda.gov/ForIndustry/DevelopingProductsforRareDiseasesConditions/ucm2005525.htm
  4. https://www.shire.com/-/media/shire/shireglobal/shirecom/pdffiles/newsroom/2016/shire-to-combine-with-baxalta-pr-1-11-16-final.pdf?la=en
  5. http://www.pfizer.co.uk/content/rare-disease-consortium
  6. http://www.pfizer.co.uk/latest-news/2015-11-26-pfizer-hires-professor-michael-linden-lead-new-gene-therapy-research-centre
  7. https://www.novartis.com/stories/education-awareness/why-research-rare-diseases-matters
  8. http://www.roche.com/media/store/releases/med-cor-2015-01-16.htm
  9. http://www.roche.com/media/store/releases/med-cor-2015-01-12.htm
  10. http://www.gsk.com/en-gb/media/press-releases/2016/gsk-receives-positive-chmp-opinion-in-europe-for-strimvelis-the-first-gene-therapy-to-treat-very-rare-disease-ada-scid/
  11. https://www.astrazeneca.com/media-centre/press-releases/2016/AstraZeneca-launches-integrated-genomics-approach-to-transform-drug-discovery-and-development-22042016.html
  12. Meekings KN, Williams CSM, and Arrowsmith JE. Orphan drug development: an economically viable strategy for biopharma R&D. Drug Discovery Today 2012; 17 (13/14):660-664.
  13. http://www.evaluategroup.com/public/reports/EvaluatePharma-Orphan-Drug-Report-2015.aspx

 

 

Manic Mice: A Model Of Bipolar Disorder


 

Introduction

Bipolar disorder falls into a category of psychopathological conditions known as affective disorders. These conditions manifest themselves with  inappropriate and disproportionate exaggerations in mood state. Bipolar disorder itself consists of the swinging between major depressive and manic episodes. The manic episodes of bipolar disorder are the distinguishing factor that enable diagnosis. In many individuals however, manic episodes occur infrequently making diagnosis challenging. As a result, many patients are first diagnosed with depression and this leads to problems with treatment.

Circadian Rhythms and Bipolar Disorder

Patients with bipolar disorder suffer from disturbances in their circadian rhythms. In depressive episodes insomnia or hypersomnia are common factors, and in manic episodes there is a decreased “need for sleep”. The rhythm of sleep is also altered, in particular REM and slow wave stages of sleep. The normal rhythms of activity, hormonal secretions and appetite are also affected. Indeed the normalization of the sleep/wake cycle and exogenous zeitgebers plays an important part in treating many individuals suffering with the illness (Plante 2008). Recently SNPs (signal nucleotide polymorphisms) have been identified in genes that encode crucial components of our endogenous pacemakers associated with mood disorders (Benedetti 2008).  In addition to this, lithium and valproate, the most common mood stabilizing treatments both have known targets and effects in circadian rhythm biology. Lithium for example has been shown in many organisms to lengthen the circadian day and valproate to alter the expression of several circadian proteins.

Animal model of mania

CLOCK is a protein that is thought to play an important part in the molecular feedback mechanisms that make up our endogenous pacemaker. Described in this publication is a mouse model in which CLOCK was mutated to inhibit its interaction with BMAL1, its transcription regulatory complex partner. In the original paper several tests were employed to demonstrate that the mice exhibited different diagnostic criteria of mania (Roybal 2007).  One symptom of mania in humans is feelings of extreme euphoria. In mice this was examined by assessing helplessness in forced swim experiments. Positively the results indicated less helplessness in the mutants. Anxiety tests were also utilized and results were indicative of this second symptom of mania. The mutant mice also show a greater preference for rewarding stimuli, similar to the manic states in bipolar. This was examined by assessing the level of intracranial self-stimulation to the medial forebrain bundle. Finally disrupted circadian rhythms, hyperactivity and decreased sleep were also observed in the mice. Perhaps the most intriguing part of this model is the lithium sensitivity. Treatment with lithium was shown to ameliorate the mood-related effects of the mutation to wild type levels (in the helplessness and anxiety tests).

Evaluation (My Thoughts)

One major weakness in this model is the identification of CLOCK null mutant mice that exhibit normal circadian functioning (Jason 2006). This suggests that the CLOCK 19 mice dysfunction may result from new functions of the mutant CLOCK or a desynchronization of function or parallel function of CLOCK, with the mutations failing to completely inhibit CLOCKs function.

There is no evidence that suggests that mutations in the circadian system are required for the development of bipolar disorder in humans and therefore it could be argued that this model lacks any relevance to the human condition.

Bipolar disorder is more complex that the behavior displayed by these mice, which exhibit no cycling between mood states. These mice are at best a model of constant mania but their utility in predicting therapeutic efficacy remains unclear. At worst this model has no biological relevance for bipolar disorder. The observed influence of lithium in alleviating the effects of the mutation may be attributed to its intrinsic polypharmacology. In addition, in this study lithium was not able to restore all behavioral phenomena to wild type levels.

Despite these reservations it must be acknowledged that circadian rhythm dysfunction is an important factor in bipolar disorder and models that can be used in the development of drugs to “normalize” this are useful.

 

Blog written by: James Noble

References 

  1.  Benedetti F, Serretti A, Colombo C, Barbini B, Lorenzi C, Campori E, 
Smeraldi E Am J Med Genet B Neuropsychiatr Genet, 2003123:23–26. 

  2. David T. Plante, John W. Winkelman, Sleep Disturbance in Bipolar Disorder: Therapeutic Implications Am J Psychiatry 2008; 165:830–843
  3.  Jason P. DeBruyne, Elizabeth Noton, Christopher M. Lambert, Elizabeth S. Maywood, David R. Weaver, Steven M. Reppert, A Clock Shock: Mouse CLOCK Is Not Required for Circadian Oscillator Function, Neuron, 2006, 50 (3), 465-477
  4. Kole Roybal, David Theobold, Ami Graham, Jennifer A. DiNieri, Scott J. Russo, Vaishnav Krishnan, Sumana Chakravarty, Joseph Peevey, Nathan Oehrlein, Shari Birnbaum, Martha H. Vitaterna, Paul Orsulak, Joseph S. Takahashi, Eric J. Nestler, William A. Carlezon, Jr, and Colleen A. McClung,Mania-like behavior induced by disruption of CLOCK, PNAS 2007, 104 (15), 6406-6411

 

The Sharpless Asymmetric Aminohydroxylation


Looking for methods to generate protected vicinal α-amino-β-alcohols, it is difficult to miss the work of Prof. Barry Sharpless who won the Nobel Prize in 2001 “for his work on chirally catalysed oxidation reactions”.1

In 1998 Sharpless and Reddy reported the osmium catalysed asymmetric aminohydroxylation (AA) reaction, which provided either (R)- or (S)-α-aryl-N-Cbz- or N-Boc- protected (R)- or (S)-α-amino-β-alcohols (R)- or (S)-(2) from styrenes (1) (Scheme 1).2  The enantioselectivities were generally excellent and a subsequent oxidation step yielded the corresponding α-arylglycine derivatives (R)- or (S)-(4).

 

victor1

Scheme 1: Asymmetric aminohydroxylation (AA) reaction of substituted styrenes by Sharpless and Reddy

It was observed that the regioselectivity was highly dependent on the nature of the styrene (1) as well as the choice of ligand, solvent, and ligand-solvent combination. Phthalazine ligands such as (DHQ)2PHAL or (DHQD)2PHAL in n-PrOH strongly favoured the benzylic amine (R)- or (S)-(2) over the benzylic alcohol (R)- or (S)-(3) regioisomer.

Investigating the extensions of this methodology I found a paper reporting the conversion of α,β-unsaturated esters to the corresponding β-hydroxyamides.As an example, Sharpless et al reported a synthesis of protected (2R,3S)-3-phenylisoserine (2R,3S)-(6), a precursor for the side chains of the anticancer drugs Taxol and Taxotere (Scheme 2).4

victor2

Scheme 2: Synthesis of (2R,3S)-(6)

Another important discovery was that anthraquinone ligands (DHQ)2AQN and (DHQD)2AQN imposed a regioselectivity pattern different from that seen with their more commonly used PHAL analogs (Scheme 3).5

victor3

Scheme 3: Regioselectivity of asymmetric aminohydroxylation (AA) reaction

Sharpless reported that the substrate orientation within the binding pockets of these two different ligand classes is obviously altered in such a way that opposite regioselection results, but remarkably, without affecting the sense or the degree of the enantiofacial selectivity. Most importantly the major product was obtained in the ration of at least 4:1 over unwanted regioisomer.

Sharpless demonstrated that versatility and great control in this reaction outweigh the toxicity and costs of the reagents. Although osmium tetroxide is well known for causing severe damage to the eyes, potassium osmate is a commercially available stable solid, which is a lot easier and safer to handle than osmium tetroxide. While catalyst and chiral ligand cost around £100 per gram, the loading is minimal (~ 5 mol%).

This quickly has become one of my favourite reactions due to its reliability and practicality. However I do still remember the first time I had to use osmium tetroxide reagent as an undergraduate student, and my boss telling me: “Stop messing around, just do the reaction with your eyes closed!” Still not sure if he was joking…

Blog written by: Victor Zdorichenko 

References

  1. Nobelprize.org. Nobel Media AB 2014. <http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2001/>
  2. Reddy, K. L.; Sharpless, K. B.; J. Am. Chem. Soc. 1998, 120, 1207–1217
  3. Demko, Z. P.; Bartsch, M.; Sharpless, K. B.; Org. Lett. 2000, 2, 2221–2223
  4. Bruncko, M.; Schlingloff, G.; Sharpless, K. B.; Angew. Chem. Int. Ed. Engl. 1997, 36, 1483–1486
  5. Tao, B.; Schlingloff, G.; Sharpless, K. B.; Tetrahedron Lett. 199839, 2507-2510

 

TMEM16A: A new road or a secret gate?


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

Factors at play in TMEM16A activation

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

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

 

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

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

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

sarahq

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

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

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

Blog written by Sarah Lilley

References:

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

Fragment Screen to Market Approval


Here (http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm495253.htm) is a landmark announcement from the FDA approving a new drug, Venclexta, for the treatment of patients with chronic lymphocytic leukaemia (CLL). Venclexta (Venetoclax) is a first in class BCL-2 inhibitor, but it is no ordinary small molecule. Not only is it the first example of a small molecule inhibitor of a protein-protein interaction designed from a fragment screen to reach FDA approval, but it also possesses physicochemical properties a long way from classical Lipinski space for an oral drug. This recent article in Nature reviews (http://dx.doi.org/10.1038/nrc.2015.17) provides a timely perspective on translating studies on the mechanisms of cell apoptosis into novel chemotherapeutics, and the challenges facing the drug discovery project team at AbbieV to bring Venetoclax to the market.

Clinical data for ABT-263, an earlier first generation inhibitor, was reported in 2010. Much has been published about the discovery of ABT-263, but it is still worth reflecting on the many achievements of the drug discovery team. With its origins in early fragment based drug discovery, the work stands as an unrivalled example of the power of fragment screening.

darren1

The molecule was assembled from linking together two small fragments in proximal ligand binding sites that were identified from a pioneering 15N HSQC NMR fragment screen of a 10,000 fragment library. The hits had weak affinity that could not be measured by biochemical assays, so the team pioneered the use of NMR to develop structure activity relationships for fragment optimisation. The work culminated in the discovery of ABT-737 and then finally after further optimisation, ABT-263. It is fascinating to see the guidelines for discovering orally bioavailable drug candidates to be so completely disregarded; a Mw of 974, a Log P of <<5, three basic centres, an aniline, two sulfonyl groups including an acyl sulphonamide and a phenyl thiol.  The acidity of the acyl sulphonamide should further impede permeability, though this may be tempered by the existence of a zwitterionic species formed from the morpholine and piperazine groups. Surely a compound with this profile would struggle to penetrate the lipid membranes of cells, let alone permeate the GI tract and survive oxidative metabolism in the liver! Surprisingly the compound has potent cellular activity, albeit several fold lower than the activity measured in the biochemical assay. But not only that, the compound achieved a successful clinical outcome in phase I human trials in respect to compound exposure and clinical efficacy.

Unfortunately, not everything went the teams way. Dose limiting toxicity of Navitoclax (ABT-236) prevented escalation to levels of exposure required for maximal efficacy. The compound is unselective against BCL-XL, another member of the BCL family highly expressed in platelets and crucial for their survival. Preclinical studies highlighted the potential of thrombocytopenia caused by BAX and BAK mediated platelet cell death that was confirmed in clinical studies, with the MTD limited to substantially below the predicted efficacious dose. After 25 years of research the team had to go back to the drawing board and design a selective BCL-2 inhibitor over BCL-XL.

This would seem a daunting task but for a fascinating observation in the X-ray crystal structure of a close analogue of ABT-263. The work published in this nature paper (http://www.nature.com/nm/journal/v19/n2/full/nm.3048.html) shows the X -ray crystal structure of an analogue of ABT-263 bound to BCL-2 with an intercalating tryptophan (shown in purple) from a neighboring BCL-2 molecule undergoing a p-p stacking interaction with the aryl sulfonamide, while at the same time hydrogen bonding to an aspartic acid residue. Essential to the observation was that the aspartic acid was one of thedarren few residues that differed between BCL-2 and BCL-XL, with the latter having a glutamic acid. The strategy was to attach the indole to the scaffold in such a position as to mimic the intercalating tryptophan on the X-ray crystal structure with the hope of achieving selectivity. Incredibly this was achieved with an azaindole linked via an ether to the central benzamide ring to give ABT-199 that was 1000 fold selective for BCL-2 over BCL-XL in a TR-FRET completion binding assay, albeit reducing to 65 fold in a cellular assay.

ABT-199 was granted break through therapy designation in 2015. In fact the compound was so efficacious in the phase I clinical trial that apoptotic cell death of cancer cells lead to tumour lysis syndrome in some patients, so the dose escalation schedule had to be adjusted to slow the onset of the drug.

The FDA approval brings to the market a first in class medicine to CLL patients that directly targets the apoptotic programme. Of the many achievement of this programme, it is the bravery of the medicinal chemists to push against all the boundaries, guidelines and rules in drug design and yet still reach market approval that gets my admiration. If anything, it clearly emphasises that there are no rules in the design of new drugs, just guidelines.

Blog writted by Darren Le Grand