An Overview of High Throughput Screening at G Protein Coupled Receptors

Abstract

Technologies used for high throughput screening (HTS) at G protein coupled receptors (GPCRs) comprise two major approaches; those generally conducted measuring signal intensity changes using a microtiter plate format, and those measuring cellular protein redistribution via imaging-based analysis systems. Several homogeneous assays, i.e. those without wash and fluid phase separation steps, measure changes of second messenger signaling molecules including cAMP, Ins P3 and calcium. Imaging based assays determined the translocation of GPCR associated proteins such as β arrestin, or internalization of the receptor labeled with fusion tags. Generally, the former assays are used in a primary screening campaign, whilst the latter are used in secondary screening and lead optimization. However, increasing use of automated confocal imaging systems and prevalence of modified cell lines has expanded use of protein redistribution assays. Finally, radiometric techniques are widely used, frequently to measure GPCR ligand binding, using a scintillation proximity assay format. In this paper, the various assay methods used for HTS at G protein coupled receptors are compared and contrasted. G PROTEIN COUPLED RECEPTORS AND HIGH THROUGHPUT SCREENING G protein coupled receptors (GPCRs) are a proven class of targets for drug discovery and are frequent targets entering high throughput screening (HTS) laboratories. Modern HTS is a highly automated approach to compound identification using robotic, fluid dispensing systems and sensitive signal detection instruments [1]. Several assay techniques for GPCR screening employ non-radiometric assay platforms. Radiometric techniques dominate in assays in which ligand binding to the GPCR per se is measured, [2]. There is increased adoption of functional assays in which the effects of the activated GPCR on function are determined, with concomitant development and implementation of cell based assay technologies, and automated cell culture and dispensing [3]. Compounds active at GPCRs have therapeutic benefit in many diseases ranging from central nervous system disorders, including pain, schizophrenia and depression, and metabolic disorders, such as cancer, obesity or diabetes [5]. GPCRs are considered a highly ‘druggable’ class of proteins, with over 40% of marketed drugs (such as Zyprexa, Clarinex, Zantac and Zelnorm) acting to modulate their function. Interestingly, approximately 9% of global pharmaceutical sales are realized from drugs targeted *Corresponding author: Tel: 510 979 1415 x 103; Fax: 510 979 1650; E-mail: reglen@discoverx.com 98 Frontiers in Drug Design & Discovery, 2005, Vol. 1 Richard M. Eglen against only 40-50 well-characterized GPCRs. As there are between 800-1000 genes in the human genome belonging to the GPCR superfamily, it is likely that many more GPCRs remain to be validated as drug targets. Furthermore, endogenous ligands have been identified for only 200 GPCRs, even though the human genome contains many more GPCR genes. When the sensory classes of GPCRs are excluded, many of the remaining GPCRs are ‘orphan’ in nature, in that no ligand or function is presently known. There is therefore, intense interest in identifying novel ligands for orphan GPCRs, both as potential therapeutics, or as pharmacological probes to refine physiological function [6]. The primary function of GPCRs is to transduce extracellular stimuli into intracellular signals. GPCRs are a large, diverse and highly conserved class of membrane-bound proteins. On the basis of homology with rhodopsin, they possess a single, serpentine, polypeptide chain with seven transmembrane helices, comprising of three extracellular loops and three intracellular loops. The amino terminal is located extracellularly, and the carboxy terminal intracellularly. GPCRs are divided into three broad classes, based upon the similarity of the transmembrane sequences and the nature of their ligands. Class 1 includes rhodopsin-like receptors, and the ligands that activate them are biogenic amines, chemokines, prostanoids, and neuropeptides. Class 2 includes secretin-like receptors, and are activated by ligands including secretin, parathyroid hormone, glucagon, calcitonin gene related peptide, adrenomedullin, calcitonin, etc. Class 3 includes metabotropic-glutamate-receptor-like and calcium sensing receptors [7]. Agonist binding to the GPCR promotes a conformational change in the protein, specifically an ionic interchange between the 3 and 4 transmembrane domain. This induces coupling of the GPCR to the G protein, initiating signaling to the cell interior. Although a diverse protein class, the diversity of GPCR pathways activated in the cell is relatively small, and often involves modulation of two membrane bound enzymes, adenylyl cyclase and phospholipase C. GPCR signaling induces coupling of the liganded receptor to a heteromeric G protein. These are composed of α, β and γ subunits, also a diverse protein group, comprising 18 Gα, 5 Gβ and 11 Gγ subunits. The GPCR/G protein interaction accelerates exchange of guanosine triphosphate (GTP) for guanosine diphosphate (GDP) on the α subunit, leading to the dissociation of the complex from the βγ subunits. The free α or βγ subunits then interact with second messengers; the precise nature of which is dependent upon the GPCR subtype, and the G protein subunits mobilized [8]. GPCR coupling to Gαs and Gαi /o proteins activate or inhibit, respectively, adenylate cyclase, the enzyme responsible for converting adenosine triphosphate (ATP) to 3’ 5’ cAMP and inorganic pyrophosphate. cAMP then acts at several downstream targets including ion channels, kinases that modulate gene transcription and cell metabolism. GPCRs coupling to Gαq/o proteins, alternatively, activate phosphoinositol phospholipase Cβ, which hydrolyzes phosphatidylinositol 4, 5 bisphosphate (PIP2) forming sn 1, 2 diacylglycerol and inositol 1, 4, 5 trisphosphate (Ins P3). Ins P3 binds and opens endoplasmic Ins P3 gated calcium channel, causing release of bound calcium into the cytosol. Metabolic products of Ins P3 also modulate cellular function including inositol (1, 3, 4, 5) P4 (Ins P4), which acts to facilitate Ins P3, mediating calcium release synergistically [8]. An Overview of High Throughput Screening Frontiers in Drug Design & Discovery, 2005, Vol. 1 99 In the presence of continued agonist activation of the GPCR, signaling is attenuated by a coordinated process of desensitization, inactivation and internalization occurs, resulting in GPCR phosphorylation by specific GPCR kinases (GRKs), and subsequent association with the adapter molecule, β arrestin. This complex then initiates the internalization and recycling processes of the receptor: ligand complex [9]. The protein, β arrestin, also facilitates formation of multimolecular complexes, and provides the means by which liganded GPCRs influence numerous cell pathways. By this means, GPCRs activate interconnected pathways, including those involving MAP kinases, nonreceptor tyrosine kinases, receptor tyrosine kinases, phosphatidylinositol 3-kinases, and JNKs [10]. In the context of assays for HTS, several aspects of GPCR signaling described above have been utilized in various assay formats. Thus, ligand binding, guanine nucleotide exchange, second messenger mobilization, ancillary protein recruitment and receptor internalization have all been used as the basis for HTS assays. In the present review, various high throughput approaches for GPCR screening are assessed and are summarized in Table (1). Several reviews on this subject have been published, and the reader is referred to these for additional references [1-4]. Cellular Expression of GPCRs The cell background imposes phenotypic selectivity on GPCR ligand pharmacology [11], which implies that the cell type used to express the receptor for screening is critical. Phenotype-specific pharmacology arises from several causes. Firstly, several GPCRs, particularly those from classes 1 and 3, form dimers or higher order oligomers at the plasmamembrane. Although GPCRs dimerize early in the synthetic pathway, the addition of agonists or antagonists influences formation of higher order oligomers [12]. It is now evident that GPCR oligomer formation depends on both the cell type, as well as the ligand. [13] Secondly, ancillary proteins expressed in the cell also modulate receptor function, and therefore, ligand pharmacology. Specifically, GPCRs interact with several transmembrane and soluble proteins, collectively termed GPCR-interacting proteins or GIPs. These GIP complexes couple to the carboxy terminal of the receptor, linking GPCR activation to a diverse network of pathways termed ‘receptorsomes’; the co-localization of which provides defined spatial and temporal motifs that are highly cell dependent [14]. One example of the effect of such ancillary proteins are the RAMP (receptor activity modifying proteins) class of proteins, the nature of which determines the level and pharmacology of GPCR activity. Thirdly, the cell phenotype influences the action of allosteric GPCRs ligands. Such ligands, in contrast to surmountable agonists and antagonists, act at sites additional to the ligand binding site on the GPCR. They thus modulate receptor function, while maintaining important temporal aspects of cell signaling [15]. Allosteric ligands can have high selectivity between GPCR subtypes, and may even exert actions in their own right by modulating constitutive receptor activity. Since neither all allosteric ligands block receptors similarly, nor all agonists are blocked equally, the allosteric function of the GPCR ligand also varies according to the cellular phenotype [15]. Overall, compound efficacy, previously considered to be an exclusive property of GPCR and its ligand, is now also an essential property of the cell or tissue in which the receptor is expressed [16]. These considerations highlight the importance of the choice of cell for us