Computational assessment of pharmacological similarity between Class A GPCRs

Though G protein-coupled receptors (GPCRs) are the target of 30% of the clinically-approved drug market, these drugs target just ~25% of the non-olfactory GPCR superfamily (108). Almost half of the remaining untargeted GPCRs are “orphans” with no known endogenous ligand(s), representing a rich and underutilised source of pharmacotherapeutic targets. To facilitate ligand identification, the Kufareva and Smith research groups previously developed GPCR-Contact-Informed Neighbouring Pocket (GPCR-CoINPocket). It is a metric of pharmacological similarity between Class A GPCRs, incorporating sequence similarity and structurally-observed ligand interaction patterns derived from liganded GPCRs in the Pocketome, an annotated encyclopaedia of liganded protein structures. In this thesis, I re-evaluate previous GPCR-CoINPocket predictions and develop an upgraded and improved version of GPCR-CoINPocket. In Chapter 2, I re-investigated the pharmacology of the orphan receptor, GPR37L1. In the previous iteration of GPCR-CoINPocket, GPR37L1 was used as a prototypical orphan for the prospective validation of the metric. However, previous retractions from our lab meant that clear evidence was once again lacking for the signalling pathways and activity of GPCR-CoINPocket-predicted ligands at GPR37L1. In the present study, I found no evidence of ligand-dependent or constitutive Gαi- or GαS-directed agonism in HEK293 or CHO-K1 cells, leaving the pharmacological toolbox of GPR37L1 once again empty. In Chapter 3, I developed a method for quantifying the similarity of ligand sets and generated a benchmark of pharmacologically similar and dissimilar Class A GPCRs for the assessment of updates and upgrades to GPCR-CoINPocket. My method formalises the intuitive concept of pharmacological similarity by deriving receptor similarity scores from high quality ChEMBL-annotated receptor:ligand binding data via an approximation of the number of shared unique chemotypes. In Chapter 4, I used my benchmarking set to evaluate four areas of potential improvement for GPCR-CoINPocket: the orthosteric contact fingerprints defining receptor pockets, the inclusion (or exclusion) of ECL2 fingerprints, the logic of score aggregation, and the amino acid similarity matrix used to score sequence similarity. The evaluation of these upgrades led to the development of GPCR-CoINPocket v2, which outperformed transmembrane similarity and GPCRdb-defined orthosteric pocket similarity in the discrimination of pharmacologically similar from dissimilar Class A GPCRs. The primary upgrade responsible for this improvement was the replacement of the Gonnet amino acid similarity matrix with a chemically-informed matrix developed in this thesis that directly (and solely) reflected structurally-observed ligand binding pattern similarities between amino acids. In Chapter 5, I prospectively validated GPCR-CoINPocket v2 using the human β2-adrenoceptor as the prototypical target. I identified 4 compounds typically binding to the OT, MCH1, and SST2 receptors with nanomolar affinity/potency that unexpectedly had affinity for the β2-adrenoceptor in competitive radioligand binding assays. The key output of this thesis is GPCR-CoINPocket v2, a direct upgrade to the original orthosteric pocket-based metric of pharmacological similarity between Class A GPCRs. I have validated it both retrospectively and prospectively, illustrating one use of the method that allows it to complement virtual and physical high-throughput screening technologies. Further applications and limitations are discussed in Chapter 6, but it is hoped that the methods and tools I have developed here can be easily adopted and aid in elucidating orphan GPCR physiology and pharmacology

Design of small-molecule inhibitors of sulfatase 2 and ERK5

PhD ThesisModern targeted cancer therapeutics are directed against components of cell signalling pathways, responsible for driving tumour progression. In this thesis, chemical tools and inhibitors of two enzymes involved in cell signalling, namely sulfatase 2 (Sulf-2) and extracellular signal-regulated kinase 5 (ERK5), have been investigated. Sulfatase 2 is a heparan sulfate (HS) processing enzyme, which has been implicated in the progression of several cancers including hepatocellular carcinoma (HCC). In HCC patients, high Sulf-2 mRNA expression correlates with a poor prognosis. The first published small-molecule Sulf-2 inhibitors were monosaccharide glucosamine derivatives, bearing a sulfamate at the O6-position. The most potent analogue from this series, was 5, having a reported IC50 against Sulf-2 of 130 μM. Although 5 was not sufficiently potent for use in target validation studies, its discovery provided encouragement that viable Sulf-2 inhibition was possible with low molecular weight compounds. In the absence of a published crystal structure and of a biological assay suitable for a highthroughput screening campaign, the structure of the endogenous substrate was considered as a potential starting point for identification of probe compounds, for use in target validation studies. Biphenyl A and biphenyl ether B sulfamates were designed in an attempt to identify a non-saccharide scaffold, which could mimic the spatial arrangements of groups believed to be important for binding of the endogenous substrate to Sulf-2. Access to these targets was facilitated by the development of a sulfamate protecting group strategy, which enabled a more flexible approach to the synthesis of phenolic O-sulfamates. v Preliminary sulfatase inhibition data have been generated, indicating that biphenyl 162 and biphenyl ether sulfamates 197 exhibited better potency against Sulf-2 than monosaccharide glucosamine 5 in our assay format. Extracellular signal-regulated kinase 5 (ERK5) is a member of the protein kinase superfamily, which plays an essential role in the transduction of extracellular signals to intracellular effectors. Activation of the ERK5 signalling pathway is associated with cell survival, proliferation, and differentiation, and ERK5 over-expression has been implicated in tumour development. An ERK5 inhibitor, XMD8-92 218, which was reported in the literature, displayed selectivity for ERK5 when tested against a panel of 402 kinases in an ATP-site competition binding assay (ERK5 IC50 = 300 nM) and showed reasonable activity in HeLa cells (GI50 = 1.5 μM). This compound also inhibited the growth of two human tumour xenograft models (HeLa cells and Lewis lung cells), highlighting that ERK5 is a valid target for anti-cancer therapy. A pyrrole carboxamide series of ERK5 inhibitors was optimised from lead compounds such as 318, which had good absorption in the Caco-2 assay but suffered from low solubility and medium clearance in mouse liver microsomes in vitro, and translated to poor oral bioavailability in vivo. The objective of the optimisation studies was to improve ERK5 inhibitory activity, both in the biochemical and cellular assays, and improve pharmacokinetic parameters to deliver compounds suitable for in vivo efficacy studies. Three areas were identified for investigation i.e. alkylation of the amide nitrogen; replacement of the meta-chloro on the aroyl ring; and substitution on the heteroaromatic amide ring at the ortho and para positions. vi Docking of compounds into a recently published co-crystal structure of an analogue of XMD8-92 bound to ERK5 has provided information on the possible binding mode of the pyrrole carboxamide series and was used to guide the design of inhibitors (Figure 1). Extension of the carboxamide heteroaromatic amine at the para position, with aliphatic and cyclic aliphatic side-chains bearing a basic centre, resulted in significantly improved ERK5 inhibitory activity (IC50 < 30 nM) as exemplified by 406, which achieved a 3-fold improvement in cellular inhibitory activity, and combined excellent microsomal stability with high solubility. However all the compounds with a basic centre suffered from a high efflux ratio and low membrane permeability in the Caco-2 assay, and as a result, had poor in vivo oral bioavailability in mouse PK studies. The work presented in this thesis has extended the SARs around the pyrrole carboxamide core but did not allow to deliver a new in vivo tool.Cancer Research UK, the Medical Research Council and Astex Pharmaceutical

THE CONCISE GUIDE TO PHARMACOLOGY 2017/18: G protein-coupled receptors.

The Concise Guide to PHARMACOLOGY 2017/18 provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.13878/full. G protein-coupled receptors are one of the eight major pharmacological targets into which the Guide is divided, with the others being: ligand-gated ion channels, voltage-gated ion channels, other ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2017, and supersedes data presented in the 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature Committee of the Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate