CrispR technology: Designer babies and Swine Donors


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The last decade has seen tremendous progress in ‘gene-editing’ techniques to make them more accurate, fast and replace the potential hit-and-miss methods of genetic engineering over the past few decades. The new technique called CrispR (pronounced Crisper!!) that emerged from pioneering work in bacterial cells in the early 2000s has evolved over the years to find applications in biotechnology, fundamental research and medical science.

The preliminary CrispR toolkit involved a RNA molecule called gRNA (guide-RNA) that is homologous to the area of DNA you wish to modify and a nuclease Cas9. The gRNA, as the name suggests, guides Cas9 to the region of interest to create a double strand break (DSB). The native cellular repair machinery: NHEJ (Non-homologous end joining) or HR (Homologous recombination), then repairs and introduces the necessary changes using a template DNA. Once the basic methodology of this technique was established, CrispR was used widely for gene-editing in almost every living organism from cells to bacteria, insects to plants and animals to now human embryos Doudna & Charpentier 2014; Sander & Joung 2014).

Nisha 2

Two studies published in the recent months show promising results with CrispR-mediated gene editing in embryos. The first study at Oregon Health & Science University (OHSU) and Salk Institute in the US, focuses on using CrispR for genome-correction to avoid transmitting genetic disorders in human embryos (Ma et al. 2017). The authors were keen on targeting genetic disorders that manifest in late-adulthood such as MYCBP3 gene mutation, which leads to hypertrophic cardiomyopathy (HCM). This genetic disease occurs in one in 500 and is a common cause of death in young athletes. The scientists CrispR-ed the mutated allele of MYCBP3 in embryos from IVF donors and replaced it with the wild-type maternal allele instead of a synthetic DNA template. By injecting the Crisp-R-Cas-9 cocktail along with the sperm (from a HCM male patient) into a wild-type oocyte (source of maternal DNA), they achieved increased correction rates and avoided mixed populations in the resulting embryos. Additionally, whole genome sequencing identified very few off-target effects of the gene editing, promising a potential break-through in germ line gene therapy. For ethical reasons these embryos could not be implanted into a uterus to develop a baby.

Yet another promising venture for CrispR was in the organ-transplant arena, where scientists in China have successfully CrispR-ed the retroviral gene in pigs (Niu et al. 2017). The study has attracted huge media attention as it promises xeno-transplants from pigs to humans without the fear of an immune attack. Traditionally such transplants are rejected in humans, as the PERV gene from pigs leads to retroviral infection in humans. The scientists attempted to target 25 copies of the PERV-gene (Porcine Endogenous Retrovirus) all at once using CrispR in adult cells, that would be later fused with ova (female reproductive cell) to grow into embryos and implanted in sows. Although, they met with disappointment in their initial trails due to ‘shredded-genomes’ in the targeted cells, Laika – the PERV inactivated piglet, was born after using a specific cocktail that kept the Crisp-R cells alive despite the aggressive gene-editing. Many other similar studies are underway exploring organ transplants from swine donors, by modifying their genome to ‘humanise’ them for successful transplants (Petersen et al. 2016; Martens et al. 2017) . While controversies continue in the field, if gene editing is required to replace anti-viral drugs in such a transplant, CrispR surely shows immense potential in the clinic.

Owing to the recent discoveries this gene-editing tool is now under the scrutiny of ethical committees and policy makers, as they fear a domino effect and the advent of ‘designer babies’. Nature, earlier this month published a report that marked down this technology due to the unintended mutations it caused in-vivo in a mice experiment (Schaefer et al. 2017). The study asserts the importance whole genome sequencing if these tools were to be applied in ‘real-people’. Although geneticists are not unaware of the potential pitfalls in genome editing, if Crisp-R will survive these hurdles only time will tell.

Blog written by Nisha Peter

References

Doudna, J.A. & Charpentier, E., 2014. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science (New York, NY), 346(6213), pp.1258096–1258096.

Ma, H. et al., 2017. Correction of a pathogenic gene mutation in human embryos. Nature, 72, p.1117.

Martens, G.R. et al., 2017. Humoral Reactivity of Renal Transplant-Waitlisted Patients to Cells From GGTA1/CMAH/B4GalNT2, and SLA Class I Knockout Pigs. Transplantation, 101(4), pp.e86–e92.

Niu, D. et al., 2017. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science (New York, NY), p.eaan4187.

Petersen, B. et al., 2016. Efficient production of biallelic GGTA1 knockout pigs by cytoplasmic microinjection of CRISPR/Cas9 into zygotes. Xenotransplantation, 23(5), pp.338–346.

Sander, J.D. & Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology, 32(4), pp.347–355.

Schaefer, K.A. et al., 2017. Unexpected mutations after CRISPR-Cas9 editing in vivo. Nature Methods, 14(6), pp.547–548.

Fabulous Fluorine in Medicinal Chemistry


Since the FDA approved the steroid fludrocortisone, the first fluorine-containing drug, in 1955, the number of fluorine-containing drugs appearing on the market has rapidly risen to approximately 25% of drugs.1 These include the blockbuster drugs Prozac, Lipitor and Prevacid, Fig. 1. This may seem like an unusual trend considering fluorine-containing natural products are quite rare, so why is it that fluorine is so abundant in drugs? When you consider the unique chemical properties that fluorine has, the reason for a drug designer’s love of fluorine becomes clearer.

Catherine 1

Fig. 1: Examples of fluorine-containing drugs

Fluorine is a small, highly electronegative and lipophilic atom. The incorporation of fluorine into a molecule is an increasingly popular strategy to improve drug potency. Fluorine substitution can enhance potency and impact target selectivity by affecting pKa, modulating conformation, hydrophobic interactions and lipophilicity. Fluorine is also frequently used to improve drug metabolism. More recently, radiolabelled fluorine drugs have been used in positron emission tomography (PET) for imaging and diagnosis purposes in medicine.2

Catherine 2.JPG

Fig. 2: Effects of fluorine in medicinal chemistry3

Fluorine is much more lipophilic than hydrogen, so incorporating fluorine atoms in a molecule will often make it more fat soluble. This means permeability across membranes is increased resulting in a higher bioavailability. Because of the strong electron withdrawing ability of fluorine, it’s inclusion in a molecule has a very strong effect on the acidity or basicity of proximal functional groups. Altering the pKa can strongly modify the binding affinity and the pharmacokinetic properties of a pharmaceutical agent. Often, fluorine is introduced to lower the basicity of a compound which aids in permeability.4

Fluorine can play an important and unique role in influencing molecular conformation. Sterically, fluorine is similar in size to a hydrogen atom, but the high electronegativity of fluorine results in a highly polarised C-F bond with a strong dipole moment and a low-lying C-F anti-bonding orbital available for hyperconjugative donation.2 This can cause fluorine-containing molecules to adopt a different preferential conformation compared to the non-fluorinated molecule which may result in increased binding affinity to a receptor.

One of the major challenges in drug discovery is that of low metabolic stability of compounds. Lipophilic compounds are susceptible to oxidation by liver enzymes, in particular cytochrome P450. Fluorine substitution at the metabolically labile site or at adjacent sites to the site of metabolic attack is a common strategy to improve metabolic stability. The inductive effect of fluorine should result in decreased susceptibility of adjacent groups to metabolic attack by cytochrome P450. Additionally, fluorine can modulate lipophilicity and restrict conformation, which may afford improved metabolic stability.3

PET imaging using 18F tracers is a rapidly developing area in medicinal chemistry. PET scans show biological processes which can give invaluable metabolic information. PET is used as a diagnostic tool, particularly in oncology, but also as an in vivo pharmacological imaging tool in drug development, especially in the areas of biodistribution and drug occupancy studies.4

One drawback of introducing fluorine substituents into drugs is that fluorination can be rather difficult and many processes create challenges in the manufacturing process. However, numerous new, safe and mild fluorinating reagents have been invented in recent years, many of which are commercially available, making the process much simpler.5,6

Blog written by Catherine Tighe

References:

  1. Wang, J.; Sánchez-Roselló, M.; Aceña, J. L.; del Pozo, C.; Sorochinsky, A. E.; Fustero, S.; Soloshonok, V. A.; Liu, H. Chemical Reviews 2014, 114, 2432.
  2. Zhou, Y.; Wang, J.; Gu, Z.; Wang, S.; Zhu, W.; Aceña, J. L.; Soloshonok, V. A.; Izawa, K.; Liu, H. Chemical Reviews 2016, 116, 422.
  3. Gillis, E. P.; Eastman, K. J.; Hill, M. D.; Donnelly, D. J.; Meanwell, N. A. Journal of Medicinal Chemistry 2015, 58, 8315.
  4. Purser, S.; Moore, P. R.; Swallow, S.; Gouverneur, V. Chemical Society Reviews 2008, 37, 320.
  5. Yerien, D. E.; Bonesi, S.; Postigo, A. Organic & Biomolecular Chemistry 2016, 14, 8398.
  6. Campbell, M. G.; Ritter, T. Organic Process Research & Development 2014, 18, 474.

 

 

Letting the public loose in our labs


On Sunday 25th June 2017, University of Sussex hosted its first Community Festival. The local public were invited to explore the Falmer campus and get involved with a number of hands-on activities across the various schools and departments. There were taster activity sessions at the Sussex Sport facilities, nature walks around the campus, live jazz, talks and more. Thousands of people turned up for the day (1).

 

Sussex Drug Discovery Centre was represented on the programme by a delegation of our group – Trudy Myers (SDDC Co-ordinator), Jess Booth (Assay Development and Screening Biologist), Kay Osborn (Biology Technician), Lucas Kraft (PhD Student) and myself. Those who signed up to our activity were taken on a whistle-stop tour through the drug discovery process.

First of all, Jess gave an overview of what the drug discovery process entails (see Figure 1) and where the SDDC fits into that process.

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Figure 1 : the drug discovery process (2)

After this introduction, our guests donned lab coats and safety glasses and were given a tour of the group’s protein purification suite. Kay explained how the term “protein” is used in the context of drug discovery to describe tiny molecular machines in our bodies that carry out a variety of functions, and in some cases, cause disease symptoms. She showed the group some incubating flasks containing E. coli which is often used to produce a large quantity of these disease-causing protein. Then she explained how that protein can be later isolated from the bacteria cells via a technique called FPLC (fast protein liquid chromatography) (3) which separates proteins out by their different sizes.

Fiona 4

Next, the newly-briefed scientists were taken into the main biology lab where they were able to run their own biochemical assay. While some took to the pipettes quicker than others, all had fun pipetting reagents onto plates that had lots of different drug compounds in them to see if they got any hits – wells that turned pink indicated a successful compound that had bound to the protein and inhibiting its function while blue ones were unsuccessful ones.

Fiona 5

During the last part of our activity I showed our participants around the oncology chemistry lab. I pointed out many of the similar pieces of kit that they would find in their kitchen. We discussed how the results from the assay they had just run in the biology lab informs the work that is carried out by chemists and how we vary the procedures (or “recipes”) we use to make new drug compounds.

The feedback for the activity was overall very positive and our enthused guests went away knowing a lot more about the drug discovery process than when they arrived, which was our aim.

Fiona Scott is a first year PhD student at the SDDC.

References

  1. University welcomes local residents for fun-filled day of discovery. University of Sussex News. [Online] [Cited: 1st August 2017.] http://www.sussex.ac.uk/newsandevents/index?page=5&id=40722.
  2. NMT Pharmaceuticals. [Online] [Cited: 1st August 2017.] http://nmtpharma.com/en/drug-development-stages/.
  3. Dermot Walls, Sinéad T. Loughran. Protein Chromatography Methods and Protocols. New York : Humana press, 2011. pp. 439-447.

 

Malaria taking control – increasing its chances for reinfection?


This week I attended the ISNTD Bites conference 2017 at the Institute of Child Health, London. I was really impressed with one of the talks given by Ailie Robinson on the work that she had conducted for her PhD at the London School of Hygiene and Tropical Medicine. Ailie has been investigating the influence of Plasmodium infection on the human volatile odour profiles in an endemic setting.

What caught my interest was something that I hadn’t previously considered with vector borne pathogenic diseases; which was how pathogens can affect its host in order to improve the likelihood of its host to be bitten by its vector and thus completing its life cycle.

Ailie presented her work where she had analysed the volatile odour profiles from three groups of participants with varying asymptomatic infections of malaria. The three groups investigated were: low density malaria (Plasmodium falciparum) infection, high density malaria infection or negative infection as a control.

Interestingly the gas-chromatographic-mass-spectrometry (GCMS) analysis of the volatile odour profiles from the people in the three groups showed that the high density infection group had significant increases in 3 organic compounds over the other 2 groups. To further investigate this interesting observation Ailie went on to present her work using gas-chromotography-electroantennography (GC-EAG); which is a technique that can be used to determine if the malaria causing mosquito (Anopheles gambiae) is attracted to a certain chemical; to show that all three of the observed molecules that were significantly increased in the high density infection group were highly attractive to the female mosquito. Inferring that the malaria parasite is by someway (either by expressing these chemicals itself or via a host response to infection) maximising its chances for reinfection.

Another interesting outcome from these experiments is the potential for the simple detection of asymptomatic malaria infection by using a breath test. See Ailie Robinson’s article here – (http://www.scidev.net/global/malaria/news/malaria-breath-test-CSIRO.html). I look forward to seeing her published data soon.

Blog written by Ryan West

Spirocycles in Drug Discovery


Medicinal chemists are constantly in search of molecules that explore new chemical space whilst looking for novelty amongst the plethora of patented molecules. Three-dimensionality is also key in maintaining solubility of new drug compounds.

Oxetanes have long been known in medicinal chemistry as very useful bioisosteres or surrogates for carbonyl or gem-dimethyl motifs. They reduce lipophilicity and therefore increase solubility when compared to their gem-dimethyl counterparts and reduce metabolism and are less likely to be covalent binders in comparison with the corresponding carbonyls. They have also been shown to be excellent hydrogen bond acceptors,   Penny 1

Whilst oxetanes have been thoroughly explored in drug discovery as a single motif, their composition in spirocycles has been relatively unexplored. In 2008, Carreira et al compared parent amino heterocycles (eg. azetidines, pyrrolidines and piperidines) with the corresponding spirocycle incorporating oxetanes.

Penny 2

In all cases, the spirocycles were stable at pH 1-10 and were considerably less basic than their parent compounds due to their conformation. All have lower logD than their gem-dimethyl and carbonyl analogues. In the cases shown above, the spirocycles also exhibited far lower intrinsic clearance than the carbonyl or parent compounds.

Morpholine moieties are also regularly found in drug discovery programs and in final drug candidates, however, the spirocyclic surrogate seems to fall under the radar despite it being much more soluble, less lipophilic and more metabolically stable.

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In addition, the oxygen lone pair is shown to be 1.3 angstroms further out than in morpholine and can therefore be regarded as an elongated morpholine (similar to piperidone but much more metabolically stable). This spirocycle could be used to probe chemical space within a binding site or could in fact have a stronger interaction within the binding site due to probing deeper within a given pocket of the site.                          Penny 4

Carreira et al later looked at the differences between morpholines, piperidines, piperizines and thiomorpholines compared with their spirocylic analogues in terms of their physico and biochemical properties. In general, the spirocyclic compounds had higher solubility, lower logD and were intrinsically more stable in human and liver microsomes compared to the parent compounds.

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The authors proceeded to synthesise the antibacterial compound Ciprofloxacin with the piperazine motif replaced with either a piperizine-like spirocycle (Compound A) or the morpholine-like spirocycle (Compound B). Both compounds showed comparable MIC and most interestingly, neither A nor B showed any sign of metabolism in human microsomes whereas Ciprofloxacin showed slight metabolism.

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Mykhailiuk et al published an interesting paper very recently which is what initially sparked this blog. They comment on the distinct lack of spirocyclic compounds in FDA approved molecules despite the number of patents surrounding such molecules slowly increasing in recent years. The authors describe spirocycles as an “overlooked motif for drug discovery”.  Their work surrounds the replacement of 2-substituted piperidines with 2-azaspiro[3.3]heptane surrogates. These compounds are not synthetically challenging as one might imagine and they originate from commercially available, relatively cheap starting materials with synthesis easily scalable to 50g of product in a single batch. They also demonstrate that the synthesis has scope and can tolerate a wide range of substituents.

The synthesis of the spirocyclic analogue of FDA-approved local anaesthetic Bupivacaine was completed by this group to compare this motif in a drug discovery setting. The results showed that despite their analogue exhibiting slightly higher plasma protein binding and in-vitro metabolism, in-vivo, their analogue showed faster onset and similar duration to Bupivacaine with lower systemic toxicity than the FDA approved drug.                               Penny 7Whilst spirocycles remain unrepresented in FDA approved molecules, it is clear that interest both within academia and industry is growing. With studies like these being published more frequently demonstrating the improved drug-like properties of these compounds when compared to their monocyclic counterparts, it is likely that it won’t be long before these motifs are seen in approved drugs.

Blog written by Penny Turner

References:

Angew. Chem. Int. Ed., 2010, 49: 9052-9067

Angew. Chem. Int. Ed., 2008, 47: 4512-4515

Angew. Chem. Int. Ed., 2010, 49: 3524-3527

Angew. Chem. Int. Ed., 2017, 56: 8665-8869

Dopaminergic neurons display antigens: autoimmunity in Parkinson’s disease pathology


Parkinson’s disease (PD) is a chronic, progressive degenerative disorder of central nervous system.  Its pathology is characterised by selective loss of dopaminergic neurons in the nigrostrial pathway, and clinical manifestations are exhibited as motor impairments, including resting tremor, bradykinesia, and rigidity.  Current medications offer symptomatic relief but, to date, do not address the dopaminergic neuronal death. The lack of understanding of the etiology of this selective cell death still remains the major stumbling block in the development of neuroprotective therapies. Current research implicates a number of key molecular mechanisms compromising the function and survival of this specific subset of neurons, and these involve abnormal protein accumulation and phosphorylation, mitochondrial dysfunction, oxidative damage and deregulated kinase signalling.  Although the current hypothesis focuses on the toxic aftermath of α-synuclein protein deposits, an alternative theory pioneered by Dr. Sulzer’s group within the Department of Neuorology at Columbia University, implies a role of the immune system in PD pathology, more specifically suggesting that Parkinson’s is in fact an autoimmune disease.

As mentioned, pathological features of PD include the loss of nigrostriatal dopamine neurons and the formation of Lewy bodies rich in fibrillar α-synuclein. This 140-amino-acid protein is abundantly expressed at a relatively high level throughout the brain (Iwai et al., 1995) and is thought to play physiological roles in the regulation of the dopamine transporter (Sidhu et al., 2004). However, misfolding of α-synuclein into protofibrils and higher-order oligomers (Uversky et al., 2002) leads to a toxin gain of function (Martin et al., 2006), which is associated with the pathogenesis of neurodegeneration (Giasson et al., 2000). Furthermore, this protein has been genetically linked to the early onset of familial PD (Kruger et al., 1998). However, the mechanism by which α-synuclein causes neurodegeneration remains unclear.

In addition to α-synuclein dysfunction, PD pathology is also characterised by a sustained microglial reaction throughout the disease progression Imamura et al., 2003). Microglial cells are the resident immune cells in brain and play a major part in the neuroinflammatory response (Soulet and Rivest, 2008). On an epidemiological level, the contribution of an inflammatory response in neurodegeneration is evidenced by the decreased risk of falls in PD patients on administering the non-steroidal anti-inflammatory drug (NSAID), ibuprofen (Gagne and Power, 2010). On a cellular and molecular level, the significant elevation in inflammatory cytokines has been found in both the cerebrospinal fluid and postmortem brain of PD patients (Mogi et al., 1994). These cytokines have been reported to induce the death of dopaminergic cells (Vivekanantham et al., 2015), and thus facilitating neurodegeneration in PD.

Dr. Sulzer’s group have recently identified the presence of antigens displayed on dopaminergic neurons in post-mortem brain tissues. It has long been argued that brain cells are safe from immune cell attack because they do not display these molecular markers for immune cell target recognition.  However, these new findings indicate that they can in fact be targeted.  Abnormal processing of self-proteins can produce epitopes, which are presented by major histocompatibility complex (MHC) proteins to be recognised by specific T cells that have escaped tolerance during thymic selection (Marrack and Kappler, 2012). Such actions by the acquired immune system have been implicated in autoimmune disorders, including type-1 diabetes. Although PD has not before been linked to autoimmunity, it does demonstrate altered protein processing. As previously described, activation of microglia and elevated cytokine levels have described in PD patients, indicating a role of the innate immune system.  But what evidence is there to implicate the acquired immune system?

Rationale for targeting the adaptive arm of the immune system as a therapeutic strategy in PD was initially provided by Brochard, et al (2009). It was found that CD8+ and CD4+ T cells, but importantly not B cells, infiltrate the substantia nigra (SN) in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD during the course of neuronal degeneration, which is consistent with postmortem human PD specimens. Further investigation concluded that T cell-mediated dopaminergic toxicity is almost exclusively arbitrated by CD4+ T cells (Brochard et al., 2009).  The Sulzer group from Columbia University, more recently, reported antigen presentation by MHC class I expression in dopamine neurons in the SN of adult human PD brains (Sulzer et al., 2017).  This was the result of activation by cytokines released from microglia.  CD8+ T cells kill neurons that present the appropriate combination of MHC class I and peptide (Cebrián et al., 2014).  On comparison of PD patients to age-match healthy controls, the group then identified two antigenic regions in α-synuclien (Fig. 1a).  The first near the N terminus elicited an apparent class II restricted IL-5 and IFNγ response (Fig. 1b–d). The second antigenic region was near the C terminus required phosphorylation of amino acid residue S129 that resulted in a markedly higher IL-5 responses in patients with Parkinson’s disease than in healthy controls. The Y39 antigenic region is noticeably close to the α-synuclien mutations that cause Parkinson’s disease (A30P, E46K, H50Q, G51D, A53T; Hernandez et al., 2016), and phosphorylated S129 residues, found in the second antigenic region, are present at high levels in Lewy bodies of patients with Parkinson’s disease (Fujiwara et al., 2002). Finally, blood tests have revealed that people with Parkinson’s show an immune response to these antigens, while people who don’t have the condition do not (Sulzer et al., 2017).

Victoria

Figure 1α-synuclein autoimmune responses are directed against two regions. a, Sequence of α-synuclein. Antigenic regions are highlight with dashed line with amino acids Y39 and S129 shown in bold. b-d, Magnitude of responses expressed as SFC per 106 PBMC’s per peptide and participant combination. Left, response to all overlapping native α-synuclein 15-mer peptides in patients with PD (n=733) and control (n=372).  Right, response against specific 15-mers.  Grey shading indication the antigenic region containing Y39. e-g,  Magnitude of responses. Left, response to all native phosphorylated S129 α-synuclein 15-mer peptides in patients with PD (n=150) and control (n=72). Right, response against specific S129 peptides. Closed circles, patients with PD (n=19, † indicates peptides; n=25, all other peptides); open circles, control (n=12).  Two-tailed Mann-Whitney U-test; NS, not significant.

These findings are the first time the immune system has been associated with a major pathological role in Parkinson’s. They present an argument for the classification of PD as an autoimmune disorder.  However, what isn’t clear is which comes first: does the immune response directly causes neuron death, or does the disease result in a heightened immune response?  If suppression of this autoimmune response does indeed stop disease progression, these findings could provide an attractive target for therapeutic intervention.

Blog written by Victoria Miller

References

Brochard et al., J. Clin. Invest. (2009) 119, 182–192.

Cebrián et al., Nat. Commun. (2014) 5, 3633.

Fujiwara et al., Nat. Cell Biol. (2002) 4, 160–164.

Gagne & Power, Neurology (2010), 74, 995–1002.

Giasson et al., Science (2000), 290, 985–989.

Hernandez et al., J. Neurochem. (2016) 139, 59–74.

Imamura et al., Acta Neuropathol. (2003), 106, 518–526.

Iwai et al., Neuron (1995), 14, 467–475.

Kruger et al., Nat. Genet.(1998), 18, 106–108.

Marrack & Kappler, Cold Spring Harb. Perspect. Med. (2012) 2, a007765.

Martin et al., J. Neurosci. (2006), 26, 41–50.

Mogi et al., Neurosci. Lett. (1994), 165, 208–210.

Sidhu et al., FEBS Lett. (2004), 565, 1–5.

Soulet & Rivest, Curr. Biol. (2008), 18, R506–508.

Sulzer, Nature (2017) 0.

Uversky et al., J. Biol. Chem. (2002), 277, 11970–11978.

Vivekanantham et al., Int. J. Neurosci. (2015), 125, 717–725.

The Necessary Nitrogen Atom


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

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

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

Irina

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

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

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

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

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

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

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

Blog written by Irina Chuckowree

 

TMEM16A: 2 pores, or not 2 pores


Two groups (Lim et al. 2016 & Jeng et al. 2016) with companion papers in the Journal of General Physiology have tried to answer the question, does TMEM16A have 1 or 2 Cl conducting pores. They have done this with functional studies using covalently linked mouse TMEM16A (mTMEM16A) subunits over expressed in HEK293T cells with one of the two subunits carrying mutations that change the functional properties of TMEM16A ion channels.

Functional members of the TMEM16 family were known to consist of 2 identical subunits (Fallah et al., 2011; Sheridan et al., 2011; Tien et al., 2013) however, with Brunner et al. (2014) solving the crystal structure of the phospholipid scramblase TMEM16 family member from the fungus Nectria haematococca (nhTMEM16) confirmed that TMEM16 molecules adopt a homodimeric architecture. With each subunit harbouring a hydrophilic groove, the “subunit cavity”, located at the periphery of the dimer that is exposed to the lipid bilayer (Figure 1A). The location of the Ca2+ binding site in the hydrophobic part of the phospholipid bilayer offers a plausible explanation for the observed voltage dependence of calcium activation in TMEM16A ion channels, as Ca2+ has to cross part of the transmembrane electric field to reach their binding site.

However, because of the unique architecture of the subunit cavity, forming a half-channel that is exposed to lipids on one side, a potential alternative arrangement of subunits in ion channels of TMEM16A and B was envisioned. In this alternative arrangement, the 2 exposed half-channels can theoretically form a single enclosed aqueous pore that would be completely surrounded by protein residues, akin to other know channel architecture (Figure 1B). In such an arrangement, the Ca2+ binding site and the residues lining the ion conduction path would be in close proximity, and it could thus be expected that any changes in the pore or the Ca2+ binding site in one of the subunits may affect the activation and conduction properties of the entire protein. In contrast, in the case of the separated 2 pore ion conduction pathways, the same mutation may only affect activation and conduction in one of the 2 pores (Figure 1A).

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Figure 1. A) Schematic representation of TMEM16A containing two pores that are independently regulated by Ca2+. B) Hypothetical alternative arrangement of subunits resulting in a single pore. Lim et al. 2016 J. Gen. Physiol. 148:5, 375-392.

In CLC proteins, this question was addressed by kinetic analysis of single channel recordings which are known to consist of 2 independent ion transport pathways (Miller 1982). But, because the low single-channel conductance of TMEM16A precludes such a strategy, Lim et al. (2016) studied macroscopic currents of the over expressed, covalently linked subunits of mTMEM16A in HEK293T cells with one of the 2 subunits carrying mutations that change the functional properties of the channel and therefore attempting to answer whether mTMEM16A ion channels consist of 1 or 2 pores, and if 2, whether the 2 pores function independently.

Recordings were performed in the inside-out patch configuration and not whole cell, which under some of their test conditions was potentially non-physiological. They showed that these linked proteins were stable and dimeric, and that the covalent link between the 2 subunits does not significantly alter the functional properties of the TMEM16A protein. The covalent linked dimers showed the established Ca2+ and voltage dependent gating, Ca2+ binding cooperativity and chloride selectivity of TMEM16A ion channels. However, they have also shown a biphasic Ca2+ activation is evident upon careful correction of the irreversible rundown that becomes more severe at higher Ca2+ concentrations with a predominant Ca2+ activation at low and a second shallow step at high Ca2+ concentration, for the linked WT-WT dimer the EC50 for Ca2+ activation was 0.209 µM and 724µM respectively. The second activation lacks any voltage dependence and might thus reflect the interaction of Ca2+ with an unknown low-affinity site located at the cytoplasmic part of the channel. Their results suggest that exposure to 1mM Ca2+ does not change the high anion over cation selectivity of the channel, nor its conductance, but that it results from an increase in the open probability.

More importantly they have also demonstrated that both subunits act independently with respect to Cl permeation and gating characteristics. They have shown WT linked dimers have the same properties as wild type TMEM16A channels and when they link WT-WT and WT-mutated channels, the signature of the linked dimer retains the functional signature of each subunit of the dimer, inferring 2 independent conducting pores in the dimer. Also, besides the unaltered anion selectivity and conductance of the construct containing only a single activatable subunit, provides additional evidence for the spatial separation of both pores. Functional independence is also corroborated by experiments on constructs where 2 subunits show different potency of Ca2+ activation and where each activation step retains the signature of the non-concatenated counterparts (Figure 2).

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Figure 2. Rundown-corrected concentration-reponse relation of the WT-E702Q concatemer at 80mV. The solid line is the best fit to the triphasic Hill equation. Dashed lines indicate the first activation of WT (green) and E702Q (orange) at 80mV. Lim et al. 2016 J. Gen. Physiol. 148:5, 375-392.

Their experiments with mutant containing dimers thus provide strong functional evidence for independent activation of 2 separate ion conduction pores in the covalently linked dimeric mTMEM16A channel. Although the results presented in this study suggest that activation of different subunits of TMEM16A opens distinct pores, the exact mechanism of TMEM16A activation is still a subject of much speculation. Although, their evidence implies that the TMEM16A ion channel may contain two pores, and Ca2+ activation of individual subunits opens the pore associated with that activated subunit (Figure 3).

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Figure 3. Cartoon summarizing the functional properties of TMEM16A. Ion conduction pores in the dimeric protein are indicated in light blue. Ca2+ is displayed as dark blue, and Cl- is displayed as red spheres. Lim et al. 2016 J. Gen. Physiol. 148:5, 375-392.

As yet no structural information on either mouse or human TMEM16A protein has been published, neither do we have, at the moment, the resolution to see TMEM16A crystal structure in either the open or closed state. However, Lim et al. (2016) have provided functional evidence that mTMEM16A ion channel has 2 independently functional pores, at high Ca2+ concentrations increases their open probability, potentially giving us a condition for high resolution crystallography to confirm whether TMEM16A has 2 pores, or not 2 pores.

Blog written by Roy Fox

References

Brunner et al. (2014) Nature 516:207-212.

Fallah et al. (2011) Mol. Cell. Proteomics. 10:M110.004697.

Jeng et al. (2016) J. Gen. Physiol. 148:5, 393-404.

Lim et al. (2016) J. Gen. Physiol. 148:5, 375-392.

Miller et al (1982) Proc. Natl. Acad. Sci. 299:401-411.

Ni et al. (2014) PLoS One. 9:e86734.

Sheridan et al. (2011) Exp. Physiol. 97:177-183.

Tien et al., (2013) Proc. Natl. Acad. Sci. USA. 110:6353-6357.

Lower cholesterol with a vaccine?


Coronary heart disease is the most common cause of death worldwide. It is caused by the narrowing of coronary arteries by the build-up of fatty material, atheroma, within the artery walls. Chest pain, owing to narrowing of coronary arteries, is known as angina, and complete blockage of the artery can cause heart attack (British Heart Foundation). Familial hypercholesterolemia (FH) is one of the main risk factors of coronary heart disease and is usually caused mutations in genes which encode proteins which are responsible for removing low density lipoprotein from circulation (Sjouke et al., 2011).

 One of the main genes, identified as causative of FH in an autosomal dominant manner, is proprotein convertase subtilisin/kexin 9 (PCSK9) (Taranto et al. 2015). Mutations in this gene cause a gain of function. Serum levels of PCSK9 are positively associated with low-density lipoprotein (LDL) concentration, i.e. hypercholesterolemia, as well as phenotypic severity of coronary heart disease (Melendez et al., 2017).

 Drug discovery for inhibitors of PCSK9 has led to the generation of 2 prominent drugs from Amgen, Repatha, and from Sanofi/Regeneron, Praluent. More recently a vaccine to inhibit PCSK9, AT04A has been developed which has been effective in mice (Laufs & Ference, 2017). This would be a more convenient mode of coronary heart disease prevention as just an annual booster vaccine would be required, rather than monthly dosing as with the aforementioned drugs. The molecule supplied in the vaccine stimulates the production of antibodies against the enzyme, which blocks PCSK9 and allows clearance of LDL, which lowers cholesterol. Mice induced with hypercholesterolemia and atherosclerosis from their diet displayed a 53% decrease in total cholesterol following subcutaneous injection of the vaccine. Now the vaccine is in phase I trials – which involves testing on 72 human volunteers and is due to be completed by the end of the year.

 Ultimately this study has further proved that lowering cholesterol reduces the risk of coronary heart disease and therefore the importance of healthy lifestyle in conjunction with cholesterol reducing medications. The vaccine AT04A may be the way forward for lowering cholesterol and reducing the vast incidences of coronary heart disease in humans.

 Blog written by Rachael Besser

 References

 Laufs & Ference, European Heart Journal (2017) 0, 1-3

 Melendez et al., Archives of Biochemistry and Biophysics (2017) 625-626, 39-53

 Sjouke et al., Curr. Cardiol. Rep., (2011) 13, 527-536

 Taranto et al., Nutrition, Metabolism & Cardiovascular Diseases (2015) 25, 979-987

 British Heart Foundation: https://www.bhf.org.uk/research/heart-statistics

 

 

 

A alternative approach in drug development: Targeted protein degradation


The concept of targeted protein degradation as an alternative approach to small molecule protein inhibition has many attractive potentials. The catalytic nature of protein degraders means that a lower systemic exposure may be needed to achieve the desired therapeutic effect when compared to a small molecule inhibitor – which may need a higher concentration to maintain occupancy of a binding site. These lower systemic exposures can have the benefit of a reduced risk of off-target and toxic side effects. Another advantage of targeted protein degradation is that the protein ligand need not bind at a site that inhibits the protein. This approach could now open the door to investigate desirable targets that were previously thought to be undruggable.

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Figure 1

In order to achieve targeted protein degradation a ligand would need to be designed that binds to the protein to be degraded. This ligand is then attached via a linker to an E3 ligase ligand (Figure 1). When a target protein ligand has a linker of an optimal composition/length, and is also attached at position that does not affect binding to the target protein, protein degraders with picomolar activity can be synthesised. An excellent review on targeted protein degradation was published at the end of 2016 by Craig Crews (doi:10.1038/nrd.2016.211).

Although the concept of targeted protein degradation has been around since the 1990’s it wasn’t until 2008 when the first small molecule E3 ligase ligand (nutlin-3a recruiting MDM2) was able to show cell penetration that many more groups became interested in this area. In 2013 Arvinas was started to exploit their Proteolysis-Targeting Chimera (PROTAC) technology and this was followed in 2015 by C4 Theraputics using their Degronimid platform. Large pharma have also shown their interest in this area with the signing of multiple large deals with these two companies.

I recently attended an SCI conference “Targeting the Ubiquitin – Proteasome Pathway” where there was excellent presentations from Craig Crews who likened the PROTAC technology to a chemical equivalent to CRISPR. Craig also mentioned that at Arvinas they have also been able to achieve CNS penetration with their PROTACS but wouldn’t describe how this had been achieved. Another presentation that was also very interesting was by Tom Heightman from Astex Pharmaceuticals where he highlighted their protein degradation technology CLIPTACs (figure 2). This approach uses two cell permeable ligands which undergo a cycloaddition reaction in the cell to form a CLIPTAC.  This technology is has the advantage of guaranteeing cell permeability which can be much harder to achieve when using the PROTAC / Degronimid approach.

Lewis 2.JPG

Figure 2

The number of publications in the area of protein degradation has noticeably increased over the past 12 months. In recognition of this increase in popularity a special edition devoted to “Inducing Protein Degradation as a Therapeutic Strategy” is due to be published by the Journal of Medicinal Chemistry imminently which I am very much looking forward to reading.

Blog written by Lewis Pennicott