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.

Penny 3

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.

Penny 5

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.

Penny 6

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


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).


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


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).


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).

Roy 1

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).

Roy 2

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).

Roy 3

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


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


 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.

Lewis 1

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?

Ben 1

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.

Ben 2

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




Sometimes the grass isn’t always greener

High content screening methods or automated microscopy based assays, are a more recent development in drug discovery. This technology is rapidly becoming a mainstream tool in profiling compound activities. One clear harbinger of this uptake in high content screening is the numerous different vendors which have brought their platforms to market enabling this work to proceed.

There are many advantages of using automated microscopy assays, compared to conventional assays. One generally assumed advantage is removal of compounds that cause optical interference due to the type of data obtained by a high content readout and the methods used in the assay (wash steps for example)

This assumption was tested in the following publication:


In this article the authors screened 315,000 compounds with a high content assay using the IN cell Analyser 3000 platform, where they were looking for modulators of micro RNA biogenesis pathway using HeLa S3 expressing green fluorescent protein. Active compounds would lead to an increase in expression of the fluorescence signal.

Using a hit threshold of 20% signal increase for the screen, they were able to obtain a hit rate of 0.36% (1130 compounds). The authors were able to retest 836 of these primary screen hits, in both single concentration and concentration response curves with both the original cell line and the parental cell line which did not express the green fluorescent protein. This is where the project hit some trouble.

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Around 22% of hit compounds reconfirmed with the GFP cell line in both the single concentration and concentration response curves, which is not unreasonable. Disappointingly, roughly the same numbers repeated in the cell line which did not contain the GFP expression system. Effectivity all the active compounds were not specific and would suggest that they are all false positives.

These identified false positive compounds were grouped together by structure into four main classes and were listed in the publication by the authors. This could be a useful tool if you want to compare other compounds which you might be working against this list. It should be remembered that (currently) these compounds are only false positives in this assay, with its specific fluorescent wavelengths used. Just because the key compound in your project is a member of one of these classes, it does not mean you need to stop working on them, but it would suggest further investigation would be warranted.

If possible, it would have been interesting to see if the results were repeated with a standard conventional fluorescence assay compared to the automated microscopy method whether the team would have achieved the same results?

So in summary, automated microscopy assay can suffer from optical interference caused by compounds, they are not immune.

However, overall the authors should be commended for releasing this publication; it highlights that all assay formats (including automated microscopy assays) will have a degree of false positive compounds and that you have to use all available methods to ensure your data output is as confident as possible.

Blog written by Gareth Williams


Bacterial immunotherapy: Can Salmonella be used to kill cancer?

I was recently at dinner with a family friend, who has survived stomach cancer, but lost his wife to liver cancer two and a half years ago. He is an interesting man, who worked for a large oil company for many years, travelling Saudi Arabia and other areas of the Middle East before retiring about 20 years ago. He has an incredible wealth of knowledge in almost all matters, and there are very few conversations to which he cannot contribute. So, with this in mind and his own past suffering with cancer, it is safe to say that when he starts talking about a novel cancer therapeutic, he probably has done his research and knows a little about the subject matter. I was told that the Telegraph had printed an article on Salmonella and its ability to flag tumours to the immune system. Stories about novel cancer therapeutics often appear in newspapers and social media, and I rarely give them much thought, but this left me somewhat intrigued so I thought it would be interesting to look into it further.

The use of bacterial preparations to stimulate the immune system in cancer patients has been a contested subject for over a hundred years, since William Coley began routinely injecting streptococcal organisms into bone cancer and sarcoma patients. Prior to this, Coley lost one of his first patients due to widespread metastases, despite a forearm amputation in response to a malignant tumour. This deeply moved him and, having trawled through the literature, he found a correlation between concurrent bacterial infection and tumour regression. Results from his bacterial treatment of cancer patients suggested that treatment with “Coley’s toxin” lead to the regression of tumours. However since the advent of chemotherapy and radiotherapy, the use of Coley’s toxin gradually disappeared (McCarthy, E.F., 2006).

Immunology has progressed quite a lot since Coley carried out this work; the mechanisms involved are now better understood and it is for this reason that interest in this area of research has been reignited.

One of the main drawbacks of chemotherapy is its inability to target tumours specifically; this leads to high off-target toxicity in non-cancerous cells and low tumour penetration with the chemotherapeutic. However, the use of bacteria provides unique mechanisms by which site-specific treatment of tumours may be possible.

The natural ability of bacteria to sense their environment through chemoattractants, and then actively follow chemical gradients whilst crossing biological barriers means they are able to penetrate tumour tissue. Metabolically-active, genetically-modified bacteria are also able to perform specific tasks once at the tumour site, such as the production of immunomodulatory molecules (cytokines) or enzymatic conversion of a pro-drug into an active therapeutic (St Jean, A.T., 2008). Bacterial vectors are also inherently immunostimualtory, as Toll-like receptors (TLRs) expressed by innate immune cells recognise bacterial-expressed virulence factors such as peptidoglycan and LPS. This leads to downstream activation of DCs, which travel from the local tumour environment to draining lymph nodes and activate adaptive immune responses through presenting tumour antigens to T-cells (Chorobik, P., 2013).

So, why is Salmonella a favourable candidate for potential bacterial therapy? Salmonella Typhimurium have been shown to have a high affinity for tumour cells and their facultative anaerobic nature means they can happily infiltrate the hypoxic areas of tumours, but they have also been shown to target non-hypoxic regions and metastases. Salmonella spp. are highly motile and can therefore penetrate into therapeutically-resistant regions of tumours, and have been shown to be preferentially attracted to such areas. Salmonella also displays direct tumour-killing activity, as they compete for nutrients and also stimulate primary and secondary immune responses. Toxins produced by the bacteria may have apoptotic effects on tumour cells, and intracellular infection with Salmonella can lead to cell death through autophagy. The combination of all these attributes can lead to reduction in tumour size (Chang, W.W., 2014).

In order to develop new, Salmonella-based vector strains for the administration of therapies, they must be attenuated/altered to stimulate an appropriate immune response. Both S. Typhimurium and S. Typhi are responsive to attenuation, and roughly 50 genes can be inactivated to produce a specific profile of virulence factors, which lends them to being used as appropriate vectors for therapeutics.

The successful use of Salmonella in reducing tumour size in murine models of cancer has been well documented in the literature. Attenuated Salmonella has been shown to work in combination with cisplatin to demonstrate an additive effect on the reduction of tumour size in mice (Lee, C.H., 2005). These results show the impact of untransformed attenuated bacteria as a result of its inherent ability to augment immune responses. Multiple studies using S. Typhimurium, genetically engineered to express pro-inflammatory mediators (e.g. TNF, IL-18) or chemokines (CCL21) also demonstrate similar success in treating tumours in murine models of cancer (Chorobik, P., 2013).

However, the story is not quite so successful when it comes to looking at similar studies in humans. An attenuated strain of S. Typhimurium (VNP20009) has been tested in a phase I study in which metastatic cancer patients were dosed intravenously with the bacteria. None of the 25 patients experienced cancer regression, significant levels of circulating TNF were measured in the peripheral blood and tumour colonisation with Salmonella was only observed in biopsies from three of the 25 patients (Toso et al., 2002). These results are in contrast with all of the animal models and could be a result of limited tumour-specific targeting by the bacteria.

The more recent developments in this field of research, which prompted the news media to publish articles suggesting that Salmonella can cure cancer, uses a much less virulent strain of bacteria, with a much higher lethal dose. This means that larger concentrations of the bacteria can be used without the side effects observed in the original phase I study by Toso et al. The bacteria are also engineered to overexpress and inducibly secrete Vibrio vulinficus flagellin B (flaB), which stimulates innate immune responses through the TLR 5 pathway and in this way acts as an excellent adjuvant for immunotherapy. Three days post infection, levels of intratumoural bacteria were 10000 fold higher than other organs, and it was at this time point that the FlaB payload was delivered through induction with L-arabinose. As a result of this, the off-target toxic effects are massively reduced and targeted therapy is achieved (Zheng, J.H., et al., 2017). So far, this research utilising Salmonella’s innate ability to target tumours, as well as inducibly secrete the therapeutic looks promising in mice, but we will have to wait and see if this is developed for human trials.

In summary, bacteria have been used as immunomodulators for cancer therapy for a long time, but the more recent advances in immunology and molecular biology mean that we are now able to further tailor microbes to create potentially viable therapeutics. The more recent studies look promising in mice, and perhaps the use of genetically engineered bacteria to deliver therapeutics to tumour sites will be used routinely in the future. However, the only recent study in humans shows that the mouse models are not always indicative of how these therapies will fare in man. The unique ability of bacteria to specifically colonise tumour sites and then deliver their payload means they are ideal candidates for tumour-specific therapy, so advances in this area of research will hopefully lead to novel and viable therapies for cancer in the near future.

Blog written by Will Pearce


Chang, W.W. and Lee, H.C., (2014), Salmonella as an Innovative Therapeutic Antitumor Agent, nt. J. Mol. Sci. 15(8), 14546-14554

Chorobik, P. et al., (2013); Salmonella and cancer: from pathogens to therapeutics, Acta Biochim Pol.  60 (3):285-97

Lee, C.H.; Wu, C.L.; Tai, Y.S.; Shiau, A.L (2005) Systemic administration of attenuated Salmonella choleraesuis in combination with cisplatin for tumor therapy. Mol. Ther.  11, 707–716

McCarthy, E.F., (2006); The Toxins of William B. Coley and the Treatment of Bone and Soft-Tissue Sarcomas, Iowa Orthop J, 26: 154-158

St Jean, A.T., (2008); Bacterial therapies: completing the cancer treatment toolbox, Curr Opin Biotechnol, 19: 511-517

Toso JF1Gill VJHwu PMarincola FMRestifo NPSchwartzentruber DJSherry RMTopalian SLYang JCStock FFreezer LJMorton KESeipp CHaworth LMavroukakis SWhite DMacDonald SMao JSznol MRosenberg SA (2002), Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J Clin Oncol. 2002 Jan 1;20(1):142-52

Zheung, J.H., Nguyen, V.H, Jian., S.N., Park, S.H., Tan, W., Hong, S.H., Shin, M.G., Chung, I.J., Hong, Y., Bom, H.S., Choy, H.E., Lee, S.E., Rhee, J.H., Min, J.J., (2017), Two-step enhanced cancer immunotherapy with engineered Salmonella typhimurium secreting heterologous flagellin, Science Translational Medicine  Vol. 9, Issue 376,