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


 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


Towards understanding Ionic interactions with Aromatic Rings

This blog article refers to the very recent work of András Perczel and colleagues in the paper Four Faces of the Interaction between Ions and Aromatic Rings (D. Papp, P. Rovó, I. Jákli, A. G. Császár, A. Perczel J. Comput. Chem. 2017, DOI: 10.1002/jcc.24816). This work is particularly interesting as it uses a mixture of data driven approaches from crystallography and structural biology as well as high level Quantum Mechanical (QM) calculations to answer a question that is raised fairly regularly in molecular design in structurally enabled projects – that of how do we optimise interactions between ionically charged species and aromatic systems.

Biology uses ionic-to-aromatic (IAr) interactions to stabilise macrostructure of proteins and other biological ensembles. Often aromatic residues such as phenylalanine (PHE), tryptophan (TRP) and tyrosine (TYR) interact with charged residues (e.g. negative charged residues (asparagine (ASP) and glutamate (GLU)) or positively charged residues (arginine (ARG) and Lysine (LYS)) to energetically stabilise proteins and peptides. Fundamentally this is the interaction of the charge of the ion and the quadrupole moment of the ring. If we understand this, and the correct vectors and applications of electron density, then we can use it to improve the interactions of aromatic rings in our drug molecules versus charged residues in a target. Take, for instance, a kinase; There are charged catalytic residues in the pocket which are key to activity. Can we use the understanding of these interactions to better get our aromatic rings in our inhibitors to bind to them / disrupt them?

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Fig 1: The interaction preferences of a cation (CP), or an anion (AP) either co-planar (ǁ) or perpendicular () to the ring. The darker green represents the most favoured vectors.

The authors investigated the Protein Databank (PDB) and the Cambridge Structural Database (CSD) to pull information on evidence-based interaction vectors, before engaging in ab initio calculations using Quantum Chemical approaches to attempt to quantify the kinds of energies involved. Below you can see the typical angles and distances of interaction between various ions and aromatic residues from the PDB.

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Fig 2: Occurrences in the PDB vs. the plane angles of interactions between various residues. Plots on the right demonstrate also the distances of these interactions.

This crystallographic information can help demonstrate which vectors and distances are preferred when designing interaction partnerships in your ligands.

The authors also use high level computational methods (FPA, NBO Hartree-Fock) to demonstrate complex electronics situations of electron-rich and deficient-rings in both small molecule and single point ions to give a semi-quantitative value of interactions (in kCalmol-1):

CP (23–37) > AP (14–21) > CPǁ (9–22) > APǁ (6–16)

Notes from the blogger (who’s thoughts are his own)

Aside from the computational chemistry calculations, the authors have demonstrated how a simple search of available databases such as the CSD and PDB can be used to mine meaningful incidental information for drug design. There are implications of using PDB data however in that the mass of crystallography was shot using various conditions, including salt and pH variations between structures. This may weaken the interaction strength between solvent accessible residues across the structures – this wholesale big data approach should be taken with slight caution for this reason.

The information gathered is quite intuitive to the med chemist, but helps to cement in ideas when designing ligands – either how to enable their rings to better make use of charged interactions, or, more subtly, if the rotamers of an aromatic ring is stabilised by one such charge, how best to use the stabilised vectors to go after other things in the pocket.

Their calculations help set up a semi-quantitative design rules, which may help drive interaction priorities, but as for the actual values, well, they may need to be taken with a pinch of something ionic…

Blog written by Ben Wahab

Who inspires you?

As my close friends, family and colleagues are probably aware, due to the presence of a gigantic (500ml) bottle of Gaviscon (Figure 1), I have been suffering from a condition known as GERD (Gastroesophageal reflux disease).

Figure 1. Ranitidine (H2 receptor blocker) & Gaviscon (500 ml) prescribed for GERD

Admittedly there are far worse diseases to be afflicted with, however, the symptoms include chronic sore & inflamed throat, heartburn and chest pain which can be rather unpleasant. One of the medications I have been prescribed is ranitidine (a H2 receptor blocker, Figure 1), which is thankfully giving me some relief! The development of ranitidine was in response to the first in class H2 receptor blocker, Cimetidine discovered by Sir James Black at Smith, Kline & French. Sir James Black had an impressive career and is credited for the discovery of both Adrenergic β-blockers & H2 receptor blockers. This was obviously an incredible achievement for which he won the Nobel prize in 1988. How did he do it? more specifically, how did he successfully develop H2 blockers?

After discovering Adrenergic β-blockers Black noted the parallels between the pharmacology of both histamine and adrenaline. By making analogues of histamine one would certainly be able to find histamine β receptor antagonists. The physiological role for histamine was ambiguous at the time however Black observed that patients with peptic ulcers showed increased acid production in response to histamine, in fact it was the basis for diagnosis. Like any drug discovery programme, it wasn’t always straightforward. The medicinal chemists got to work on making antagonists based on the structure of histamine, Black thought making ring analogues of histamine would do the trick as this had worked previously for adrenergic β-blockers. After considerable effort by the chemists, testing in a variety of bioassays, no active compounds were found. It has been stated that the chemists were accused of being ‘’unimaginative’’ (as if that would ever be the case!).

After 4 years of chemical synthesis and no antagonism achieved things were not looking good. Black had a change of heart. Perhaps the chemists should have been spending more time investigating the amino acid side chains than substituting ring structures. Scanning back through earlier data, the sixth compound to be synthesized, Nα-gyanylhistamine, a side chain variant, showed a low level of inhibition and was previously missed because it was only a partial agonist (Table 1).  The side chain of Nα-gyanylhistamine was lengthened resulting in 3-[4(5)-imidazolyl]propylguanidine (Table 1) this compound showed an ~6 fold increase in potency but it was still only a partial agonist (Figure 2). Black was eager to find a full antagonist as a partial agonist at a low dose would only stimulate acid production and not block it.

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Figure 2. Enhanced potency of the partial agonist 3-[4(5)-imidazolyl]propylguanidine (91488) in guinea-pig right atrium assay.

One significant challenge for the programme was the fact that the compounds synthesized, namely; guanidines, carboxyamidines, isoureas and isothioureas, are all strong bases so at physiological pH would be protonated and therefore not easily absorbed. When the chemists synthesized a set of non-basic compounds both the agonist and antagonist effects were lost, except for one compound, a thiourea analogue (Table 1, PA2= 3.45), which displayed weak, but full, antagonist action in the in vitro guinea pig right atrium assay. Increasing the side chain produced burimamide (Table 1, PA2= 5.11). As had been seen for the guanindine compound this significantly increased the potency, finally they had a selective H2 histamine receptor antagonist. Burimamide took a year to synthesize.

Burimamide was tested in healthy volunteers and shown to inhibit gastric acid secretion confirming the transferability between the animal models and human disease. Burimamide was still not potent enough to be given orally so further compound optimisation was required to develop an even more potent antagonist. Tautomerism and alteration of electronic effects on the imidazole ring bought the chemists to Metiamide (Table 1). Metiamide increased the rate of ulcer healing in 700 patients, however a few suffered from granulocytopenia toxicity. The chemists came to the rescue again, this time replacing the thiourea moiety with cyanoguanidine, and in the process producing the safe drug Cimetidine (Table 1), the first in class H2 receptor blocker.

These drugs have saved the lives of millions of people with heart disease and peptic ulcers. At the time there were few treatment options for patients with peptic ulcers. The only cure was via surgical intervention. Sir James Black and his team are a definite inspiration, just remember his ‘’three C’s for effective drug discovery: Collaboration, Concentration and Commitment’’.

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Table 1. Lead optimisation of the first H2 blocker (Source: Personal reflections on Sir James Black (1924-2010) and histamine by C. Robin Ganellin)

Blog written by Jess Booth

To hear James Black in person follow this link: https://www.webofstories.com/play/james.black/12;jsessionid=F3B2C475B86A0B279E9FFEA3119B9C22


Personal reflections on Sir James Black (1924-2010) and histamine by C. Robin Ganellin

Dimaprit-[s-[3-(N, N-dimethylamino)propyl] isothiourea] – a highly specific histamine H2-receptor agonist. Part 2. Structure-activity considerations. Agent Actions. Durant, GJ et al. 1977;7:38-43.

Perspectives in Drug Development and Clinical Pharmacology: The Discovery of Histamine H1 and H2 Antagonists by Alan Wayne Jones

Putting Theory into Practice: James Black, Receptor Theory and the Development of the Beta-Blockers at ICI, 1958–1978 by Viviane Quirke