James L. J. Coleman

Identifying ligands at orphan GPCRs: current status using structure-based approaches

GPCRs are the most successful pharmaceutical targets in history. Nevertheless, the pharmacology of many GPCRs remains inaccessible as their endogenous or exogenous modulators have not been discovered. Tools that explore the physiological functions and pharmacological potential of these ‘orphan’ GPCRs, whether they are endogenous and/or surrogate ligands, are therefore of paramount importance. Rates of receptor deorphanization determined by traditional reverse pharmacology methods have slowed, indicating a need for the development of more sophisticated and efficient ligand screening approaches. Here, we discuss the use of structure‐based ligand discovery approaches to identify small molecule modulators for exploring the function of orphan GPCRs. These studies have been buoyed by the growing number of GPCR crystal structures solved in the past decade, providing a broad range of template structures for homology modelling of orphans. This review discusses the methods used to establish the appropriate signalling assays to test orphan receptor activity and provides current examples of structure‐based methods used to identify ligands of orphan GPCRs.

Orphan receptor ligand discovery by pickpocketing pharmacological neighbors

Understanding the pharmacological similarity of G protein-coupled receptors (GPCRs) is paramount for predicting ligand off-target effects, drug repurposing, and ligand discovery for orphan receptors. Phylogenetic relationships do not always correctly capture pharmacological similarity. Previous family-wide attempts to define pharmacological relationships were based on three-dimensional structures and/or known receptor-ligand pairings, both unavailable for orphan GPCRs. Here, we present GPCR-CoINPocket, a novel contact-informed neighboring pocket metric of GPCR binding-site similarity that is informed by patterns of ligand-residue interactions observed in crystallographically characterized GPCRs. GPCR-CoINPocket is applicable to receptors with unknown structure or ligands and accurately captures known pharmacological relationships between GPCRs, even those undetected by phylogeny. When applied to orphan receptor GPR37L1, GPCR-CoINPocket identified its pharmacological neighbors, and transfer of their pharmacology aided in discovery of the first surrogate ligands for this orphan with a 30% success rate. Although primarily designed for GPCRs, the method is easily transferable to other protein families.

Metalloprotease cleavage of the N terminus of the orphan G protein–coupled receptor GPR37L1 reduces its constitutive activity

The N terminus of the receptor GPR37L1 enables constitutive signaling to Gαs. Turning off a constitutively active GPCR In animals, most G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs) are inactive under basal conditions and require either binding of a ligand, isomerization of their bound ligand by light, or cleavage of a tethered ligand to become activated. Coleman et al. found that GPR37L1, which is a GPCR abundant in the cerebellum, signaled to Gαs and stimulated cyclic AMP signaling in the absence of any exogenously added ligand when expressed in a cultured cell line and assayed in the presence of normal growth medium. Furthermore, cleaved fragments of this receptor are detectable in human cerebral spinal fluid, and biochemical analysis revealed a long and short form of the receptor. The short form, which resulted from cleavage of the N terminus, was inactive and was the predominant form detected in rodent cerebellum. Metalloprotease inhibitors blocked the cleavage of the N terminus, suggesting that signals that regulate the activity of these proteases, rather than ligand-binding events, control the signaling mediated by this GPCR. How widespread this type of GPCR regulatory mechanism is and whether the cleaved fragments have bioactivity remain interesting questions. Little is known about the pharmacology or physiology of GPR37L1, a G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor that is abundant in the cerebellum. Mice deficient in this receptor exhibit precocious cerebellar development and hypertension. We showed that GPR37L1 coupled to the G protein Gαs when heterologously expressed in cultured cells in the absence of any added ligand, whereas a mutant receptor that lacked the amino terminus was inactive. Conversely, inhibition of ADAMs (a disintegrin and metalloproteases) enhanced receptor activity, indicating that the presence of the amino terminus is necessary for GPR37L1 signaling. Metalloprotease-dependent processing of GPR37L1 was evident in rodent cerebellum, where we detected predominantly the cleaved, inactive form. However, comparison of the accumulation of cAMP (adenosine 3′,5′-monophosphate) in response to phosphodiesterase inhibition in cerebellar slice preparations from wild-type and GPR37L1-null mice showed that some constitutive signaling remained in the wild-type mice. In reporter assays of Gαs or Gαi signaling, the synthetic, prosaposin-derived peptide prosaptide (TX14A) did not increase GPR37L1 activity. Our data indicate that GPR37L1 may be a constitutively active receptor, or perhaps its ligand is present under the conditions that we used for analysis, and that the activity of this receptor is instead controlled by signals that regulate metalloprotease activity in the tissue.

The G protein-coupled receptor N-terminus and receptor signalling: N-tering a new era

G protein-coupled receptors (GPCRs) are a vast family of membrane-traversing proteins, essential to the ability of eukaryotic life to detect, and mount an intracellular response to, a diverse range of extracellular stimuli. GPCRs have evolved with archetypal features including an extracellular N-terminus and intracellular C-terminus that flank a transmembrane structure of seven sequential helices joined by intracellular and extracellular loops. These structural domains contribute to the ability of a GPCR to be correctly synthesised and inserted into the cell membrane, to interact with its cognate ligand(s) and to couple with signal-transducing heterotrimeric G proteins, allowing the activated receptor to selectively modulate a number of signalling cascades. Whilst well known for its importance in receptor translation and trafficking, the GPCR N-terminus is underexplored as a participant in receptor signalling. This review aims to discuss and integrate recent advances in knowledge of the vital roles of the GPCR N-terminus in receptor signalling.

Rapid Knockout and Reporter Mouse Line Generation and Breeding Colony Establishment Using EUCOMM Conditional-Ready Embryonic Stem Cells: A Case Study

As little as a decade ago, generation of a single knockout mouse line was an expensive and time-consuming undertaking available to relatively few researchers. The International Knockout Mouse Consortium, established in 2007, has revolutionized the use of such models by creating an open-access repository of embryonic stem (ES) cells that, through sequential breeding with first FLP1 recombinase and then Cre recombinase transgenic mice, facilitates germline global or conditional deletion of almost every gene in the mouse genome. In this Case Study, we describe our experience using the repository to create mouse lines for a variety of experimental purposes. Specifically, we discuss the process of obtaining germline transmission of two European Conditional Mouse Mutagenesis Program (EUCOMM) “knockout-first” gene targeted constructs and the advantages and pitfalls of using this system. We then outline our breeding strategy and the outcomes of our efforts to generate global and conditional knockouts and reporter mice for the genes of interest. Line maintenance, removal of recombinase transgenes, and cryopreservation are also considered. Our approach led to the generation of heterozygous knockout mice within 6 months of commencing breeding to the founder mice. By describing our experiences with the EUCOMM ES cells and subsequent breeding steps, we hope to assist other researchers with the application of this valuable approach to generating versatile knockout mouse lines.

The enantiomers of zacopride: an intra-species comparison of their potencies in functional and anxiolytic models.

1 The 5‐HT3 receptor antagonist, zacopride, and its enantiomers, R(+)‐zacopride and S(−)−zacopride, were examined in three pharmacological models: (i) 5‐HT‐induced depolarizarion of the mouse isolated vagus nerve preparation, (ii) the 5‐HT‐evoked von Bezold‐Jarisch reflex in the mouse, and (iii) the mouse light:dark box model of anxiety. Other standard 5‐HT3 receptor antagonists were also included for comparison in these studies. 2 Racemic zacopride, and both of the enantiomers, displayed potent 5‐HT3 receptor antagonist activity in the isolated vagus nerve and in the von Bezold‐Jarisch model. No 5‐HT3 receptor agonist or partial agonist effects of these compounds were detected. 3 In the isolated vagus nerve, R(+)‐zacopride and ondansetron were surmountable 5‐HT3 receptor antagonists (pA2 values of 9.3 and 8.3, respectively), whereas racemic zacopride, S(—)‐zacopride and tropisetron were insurmountable antagonists, markedly suppressing the maximum response to 5‐HT. 4 In vivo, racemic zacopride, R(+)‐zacopride, S(—)‐zacopride and WAY 100289 were potent antagonists of the 5‐HT‐evoked von Bezold‐Jarisch reflex, with minimum effective doses (lowest dose required to reduce the reflex by ≥85%; MED85) of 1.0, 3.0, 0.3 and 3.0 μg kg−1, s.c, respectively. 5 Racemic zacopride, R(+)‐zacopride and S(—)‐zacopride were active in the mouse light:dark box model of anxiety, with similar potencies (minimum effective dose 1 μg kg−1, s.c.) and similar active dose‐ranges (1–1000 μg kg−1, s.c). 6 The doses of racemic zacopride, R(+)‐zacopride and S(−)−zacopride required to block 5‐HT3 receptors in vivo correlated reasonably well with their potencies in an anxiety model within the same species. In these studies, there was no evidence of a marked difference between the anxiolytic potencies of R(+)‐zacopride and S(—)‐zacopride.

GPCRs in context: sexual dimorphism in the cardiovascular system

Cardiovascular disease (CVD) remains the largest cause of mortality worldwide, and there is a clear gender gap in disease occurrence, with men being predisposed to earlier onset of CVD, including atherosclerosis and hypertension, relative to women. Oestrogen may be a driving factor for female‐specific cardioprotection, though androgens and sex chromosomes are also likely to contribute to sexual dimorphism in the cardiovascular system (CVS). Many GPCR‐mediated processes are involved in cardiovascular homeostasis, and some exhibit clear sex divergence. Here, we focus on the G protein‐coupled oestrogen receptor, endothelin receptors ETA and ETB and the eicosanoid G protein‐coupled receptors (GPCRs), discussing the evidence and potential mechanisms leading to gender dimorphic responses in the vasculature. The use of animal models and pharmacological tools has been essential to understanding the role of these receptors in the CVS and will be key to further delineating their sex‐specific effects. Ultimately, this may illuminate wider sex differences in cardiovascular pathology and physiology.

Using constitutive activity to define appropriate high-throughput screening assays for orphan G protein-coupled receptors.

Orphan G protein-coupled receptors represent an underexploited resource for drug discovery but pose a considerable challenge for assay development because their cognate G protein signaling pathways are often unknown. In this methodological chapter, we describe the use of constitutive activity, that is, the inherent ability of receptors to couple to their cognate G proteins in the absence of ligand, to inform the development of high-throughput screening assays for a particular orphan receptor. We specifically focus on a two-step process, whereby constitutive G protein coupling is first determined using yeast Gpa1/human G protein chimeras linked to growth and β-galactosidase generation. Coupling selectivity is then confirmed in mammalian cells expressing endogenous G proteins and driving accumulation of transcription factor-fused luciferase reporters specific to each of the classes of G protein. Based on these findings, high-throughput screening campaigns can be performed on the already miniaturized mammalian reporter system.