REVIEW ARTICLES Strategies for G-protein coupled receptor deorphanization Sorin Tunaru1* 1Institute for Heart and Lung Research, Department of Pharmacology, Ludwigstr. 43, 61231 Bad Nauheim, Germany (Received 1 March, 2017; accepted 3 May, 2017) Abstract G-protein coupled receptors (GPCRs) form the largest group of transmembrane proteins in mammals, and they are major regulators of multiple cellular processes. Intense research performed in the last decades revealed their implication in pathophysiology and included many of their members as important drug targets. With the advent of genomics a large number of new GPCR genes have been discovered, however many of them still encode receptors for which no ligands and biological roles have yet been discovered. They are termed orphan GPCRs (oGPCRs) and are under intense scientific investigations by the pharma industry and academia to discover ligands and thus to determine their contribution to pathophysiology. Two strategies have been used to identify ligands of GPCRs, known as the reverse and forward pharmacology. Both of them are presented and discussed in this review. Keywords: GPCR, deorphanization, reverse pharmacology, forward pharmacology, drug discovery INTRODUCTION a consequence of their biological importance, more than 40% of the world-wide approved drugs target GPCRs G-protein coupled receptors are the largest superfamily either as agonists or antagonists. Several examples of of transmembrane receptors in eukaryotes. Th eir key commercially approved drugs targeting GPCRs and their structural signature is the presence of a seven transmembrane therapeutic eff ects can be found in the Table 1. Although (7TM) domain which separates the extracellularly located GPCRs are the most prominent drug target, with a N-terminus from the intracellular C-terminus. Another market value of over 65 billion dollars in annual sales (4), property of GPCRs is that they mediate cellular eff ects of a almost 140 genes in human genome still encode receptors cognate ligand by coupling to intracellular heterotrimeric for which neither biological roles nor endogenous ligands guanine nucleotide binding proteins, or G-proteins, have been identifi ed, termed orphan GPCRs (oGPCRs) composed of subunits. Functionally, G-proteins can (5, 6). be grouped into four subtypes based on the structural GPCR deorphanization started in 1986, when the and functional similarities of their -subunit: Gq/11, primary structure of the -adrenergic receptor (-AR) mediating intracellular calcium increase; Gs, activating was characterized. Surprisingly, its amino-acid sequence adenylyl-cyclases (ACs) and increasing cytosolic cyclic not only shared high homology with the sequence of adenosine monophosphate (cAMP) in cells; Gi, negative visual rhodopsin but also shared a common secondary regulators of ACs, thus reducing intracellular cAMP structure, the 7TM domain (7). Th is seminal fi nding led concentration and G12/13, mediating cytoskeletal to the concept that all plasma membrane 7TM proteins remodeling cellular motility (1, 2). which are able to interact with G-proteins, belong to a GPCRs are key mediators in the regulation of a superfamily of receptors. At that time it was already thought variety of physiological processes and also participate that a number of biologically active metabolites, such as in a multitude of diseases. Th ey are required for the acetylcholine, eicosanoids (especially prostaglandins), function of complex senses such as olfaction, taste, or biogenic amines and hormones were inducing eff ects by visual perception. Moreover, their role in the regulation acting as ligands of GPCRs. Newly developed molecular of blood pressure, kidney function, cancer progression biology techniques, such as degenerated PCR and and allergic reactions has been well documented (3). As low-stringency hybridization, were successfully used to *Corresponding author ( Sorin Tunaru, Department of Pharmacology, Max-Planck-Institute for Heart and Lung Research, Ludwigstr. 43, 61231 Bad Nauheim, Germany; Phone: +49-(0)6032-705-1210, Fax: +49-(0)6032-705-1204, E-mail: [email protected]) Molecular Life │ VOLUME 1, No. 1, 2017, 71-79 71 Sorin Tunaru G-protein coupled receptor deorphanization identify new receptors, with diff erent homologies to -AR 1) Loss-of-function studies by genetic deletion or (8). Moreover, with the expansion of genomics at the turn mutational analysis can indicate whether an oGPCR plays of the century, a large number of potential GPCR genes an important role in the regulation of a physiological were identifi ed, reaching almost 900 in human and over process. Several examples support the idea of genetic 1000 in mouse, including olfactory receptors (9-11). Due studies as valuable preliminary steps in the identifi cation to the large number of GPCR genes discovered, some of of potentially relevant oGPCRs: for example, mutations them were abbreviated as “GPR” followed by a number, in the GPR101 gene, which is strongly expressed in the as in the case of GPR81 or GPR37 and so on. However, hypothalamus (13), have been associated with gigantism this terminology does not necessarily refl ect any degree of and acromegaly (14). In the case of GPR149, which shows relatedness among them. expression in granulosa cells and oocytes, the gene-defi cient In the last decades, huge eff orts by the academia and mice showed increased fertility and enhanced ovulation pharma industry have been made to identify endogenous (15). Disruption of another orphan receptor, GPR45, ligands and agonists of oGPCRs and by this to reveal their caused hypothalamic proopiomelanocortin (POMC) role in pathophysiology and to possibly include them expres sion and obesity in mice (16) whereas mice lacking in the druggable targets portfolio. Th e identifi cation of GPR21 receptor, which shows expression in the adipose modulators of oGPCRs can be a tedious and risky process tissue and liver, were resistant to diet-induced obesity and and can reach tremendous costs at the risk of obtaining had increased glucose tolerance and insulin sensitivity modest results, if any at all. Th erapeutically, although the (17). A compelling database of GPCR genes and known endogenous ligand of a potentially important oGPCR associated phenotypes can be found at IUPHAR database: may remain unknown, the identifi cation of a compound http://www.guidetopharmacology.org. with agonistic properties can be considered a major 2) Bioinformatics analysis can be used to compare success because it allows to directly testing its potential sequences of oGPCRs with those of the liganded benefi cial role. receptors to predict cognate ligands based on sequence Two main pharmacological strategies have been used in homology. For example, the orphan receptor GPR105 the last years for deorphanization, known as the reverse (known as P2Y14) which showed signifi cant homology and the forward pharmacology. Both strategies will be with other nucleotide-liganded purinergic receptors was presented in this article discussing their applicability in screened against a library containing nucleotide di- and the orphan receptor research fi eld. triphosphate conjugates to identify UDP-glucose as ligand (18). Another example was the identifi cation of REVERSE PHARMACOLOGY sphingosine-1-phospahte (S1P) as a potent ligand of EDG-1 (S1P1) receptor (15) a fi nding which led to the Th e concept of reverse pharmacology, also known as subsequent discovery of multiple receptors (S1P1 to S1P5) target-based drug discovery, relies on the hypothesis that receptors for S1P, based on sequence homology analyses the modulatory eff ect of a protein (i.e., receptor, ion (19-21). Furthermore, due to the success of resolving the channel, enzyme and so on) will have important biological 1- and 2-AR receptors crystal structures (22), followed consequences which can be of therapeutic importance, by the X-ray structures of 20 receptors in the following thus becoming a pharmacological target (12). In other years, structure-based drug design (SBDD) can be an words, reverse pharmacology aims to identify compounds eff ective tool to design potential agonists or antagonists which can alter a chosen target’s activity. In the case of (23, 24). Several examples where SBDD in conjuction GPCRs, the identifi cation of the endogenous ligand with computer modelling has been successfully employed and/or modulatory compounds, as its primary goal, serves to fi nd new ligands at known or orphan GPCRs are the two convergent objectives: fi rstly, the understanding of identifi cation of alloseric modulators and agonists of the cellular pathway the receptor is integrated into and the proton receptors GPR68 and GPR65 (25). Another its role in the regulation of a particular physiological remarkable example with potential clinical relevance is process and secondly, it gives information on its possible the discovery through SBDD of a novel μ-opioid receptor therapeutic value. Th e most straightforward way to agonist which selectively drives Gi/o-mediated signaling identify ligands at oGPCRs is to ectopically express while minimizing -arrestin-dependent signaling path- them in a cellular system and to measure dependency of ways (26). Furthermore, 3D homology modelling of ligand-induced modulation of a signaling pathway on the GPCRs using as templates crystal structure of numerous presence of the receptor in quest (Fig. 1). In this strategy, GPCRs represents an important tool for ligand prediction the selected receptor is used as bait to identify