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Tools for GPCR Drug Discovery npg Acta Pharmacologica Sinica (2012) 33: 372–384 © 2012 CPS and SIMM All rights reserved 1671-4083/12 www.nature.com/aps Review Tools for GPCR drug discovery Ru ZHANG1, Xin XIE1, 2, * 1Shanghai Key Laboratory of Signaling and Disease Research, Laboratory of Receptor-based Bio-medicine, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China; 2State Key Laboratory of Drug Research, the National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China G-protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most success- ful therapeutic targets for a broad spectrum of diseases. The design and implementation of high-throughput GPCR assays that allow the cost-effective screening of large compound libraries to identify novel drug candidates are critical in early drug discovery. Early functional GPCR assays depend primarily on the measurement of G-protein-mediated 2nd messenger generation. Taking advantage of the continuously deepening understanding of GPCR signal transduction, many G-protein-independent pathways are utilized to detect the activity of GPCRs, and may provide additional information on functional selectivity of candidate compounds. With the combination of automated imaging systems and label-free detection systems, such assays are now suitable for high-throughput screening (HTS). In this review, we summarize the most widely used GPCR assays and recent advances in HTS technologies for GPCR drug discovery. Keywords: G-protein-coupled receptors (GPCRs); high-throughput screening; high-content screening; functional assay; G-protein- dependent assay; G-protein-independent assay; label-free assay; functional selectivity Acta Pharmacologica Sinica (2012) 33: 372–384; doi: 10.1038/aps.2011.173; published online 23 Jan 2012 Introduction activate phospholipase Cβ (PLCβ), which catalyzes the forma- G-protein-coupled receptors (GPCRs), also known as 7 trans- tion of diacylglycerol (DAG) and inositol-1,4,5-trisphosphate membrane receptors, are the largest family of cell surface (IP3). IP3 then binds and opens the endoplasmic IP3-gated receptors and account for approximately 4% of the protein- calcium channel, causing the release of calcium into the cyto- [1] coding human genome . They are activated by a wide variety plasm. GPCRs coupling to Gα12/13 further activate the guanine of stimulants, including light, odorant molecules, peptide and nucleotide exchange factor RhoGEF, which in turn activates non-peptide neurotransmitters, hormones, growth factors and the small G protein RhoA. lipids, and control a wide variety of physiological processes In the presence of continuous agonist stimulation, GPCRs including sensory transduction, cell–cell communication, neu- are phosphorylated by specific GPCR kinases (GRKs), and ronal transmission, and hormonal signaling. the recruitment of β-arrestins to the phosphorylated GPCRs After agonist binding, activated receptors catalyze the eventually terminates G protein signaling and leads to a coor- exchange of guanidine diphosphate (GDP) for guanidine dinated process of receptor desensitization, inactivation and triphosphate (GTP) on the α-subunit of heterotrimeric G internalization[3]. The β-arrestins also facilitate the forma- proteins (composed of α-, β-, and γ-subunits), which in turn tion of multi-molecular complexes and provide a means for engages conformational changes that lead to the dissociation G protein-independent signaling of GPCRs, including those of Gα from the dimeric Gβγ subunits[2]. GPCRs coupled to involving mitogen-activated protein (MAP) kinases, recep- Gαs and Gαi/o proteins activate or inhibit, respectively, adeny- tor and non-receptor tyrosine kinases, phosphatidylinositol late cyclase, the enzyme responsible for converting adenosine 3-kinases (PI3K) and others[4]. triphosphate (ATP) to 3’,5’-cyclic adenosine monophosphate Given their importance in health and disease, together with (cAMP). cAMP serves as a second messenger that activates their potential for therapeutic intervention via using small protein kinase A (PKA) and other downstream effectors (pre- molecules as regulators, GPCRs represent the largest family of [3] viously reviewed ). GPCRs coupled to Gαq/o alternatively druggable targets. These receptors are the target of >50% of the current therapeutic agents on the market, including more than a quarter of the 100 top-selling drugs, with profits in the * To whom correspondence should be addressed. [5] E-mail [email protected] range of several billion US dollars each year . Therefore, Received 2011-09-30 Accepted 2011-11-15 GPCR assay development and GPCR ligand screening remain www.chinaphar.com npg Zhang R et al 373 the major focus of drug discovery research worldwide. His- ulators)[10]. The traditional radioligand binding assays require torically, radioligand binding assays with receptor-containing washing and filtration steps, which can only be scaled down membranes were used to identify compounds that target to a 96-well format. GPCRs. However, binding affinity data do not tell us whether A homogenous scintillation proximity assay (SPA), which the compound is an agonist or an antagonist, or more impor- can be easily scaled down and automated for HTS applica- tantly, the overall potency of a compound under physiological tions, was developed more recently (reviewed in[11]). In SPA, conditions. Efforts have been made in the past few decades only the radiolabeled molecules binding to the GPCR immobi- to develop signaling-dependent cell-based functional assays lized on the surface of SPA beads can activate the scintillation to provide more accurate and comprehensive data of the com- beads, which produce photons detectable with a scintillation pounds targeting GPCRs. counter. SPA thus allows binding reactions to be tested with- An ideal assay for GPCR ligand screening should be simple, out washing or filtration steps. Although radioligand binding nonradioactive, robust, homogenous, and easily adapted to gives a clear, unmistakable signal, radioligands are relatively a microtiter plate format (96-, 384-, or 1536-well) for robotic expensive, problematic to dispose of, and some isotopes have automation. Since GPCR signaling consists of a series of spa- inconveniently short half-lives. These drawbacks have led to tial and temporal events, another important consideration is the creation of highly sensitive nonradioactive alternatives. whether to measure a proximal or distal signaling step after Many new binding assays are based on time-resolved fluo- GPCR stimulation. Measurement of events proximal to recep- rescence resonance energy transfer (TR-FRET) technology, tor activation will reduce the incidence of false positives[6]; such as DELFIATM TRF from PerkinElmer, the LanthaScreenTM however, moving down the signal transduction cascade will system from Invitrogen, and the Tag-liteTM system from Cisbio. enhance the signal-to-noise ratio due to signal amplifica- Tag-liteTM is a newly developed homogeneous time-resolved tion. Many GPCRs also activate multiple signaling pathways. fluorescence (HTRF) technology[12] for assaying GPCR ligand Biased signaling, a phenomenon in which certain agonists binding in HTS format. A suicide enzyme (either SNAP- or display better efficacies in activating one pathway over oth- CLIP-tag) is fused to the N-terminus of a GPCR without affect- ers, is another critical issue to consider in functional screen ing its binding and activity, and a non-permeant substrate development[4, 7]. If a functional assay capturing only one labeled with terbium cryptate fluorophore (Lumi4-Tb) is used signaling pathway is selected for screening compound librar- to specifically and covalently label the receptors expressed ies, potentially valuable compounds could be missed if the on the cell surface. The ligands are labeled with red or green compound does display biased activity. Therefore, multiplex- acceptors. The long fluorescence lifetime of the terbium ing of signaling pathways or assays representing an overall cryptate allows a time-resolved measurement of FRET emis- cellular response may be used to resolve such problems. In sion when all natural fluorescence disappears. The assay is this review, we summarize the most widely used GPCR assays carried out in a “mix and measure” format, which can be used and recent advances in high-throughput screening (HTS) tech- not only for ligand binding studies but also for receptor activ- nology for GPCR drug discovery (Table 1). ity analysis and GPCR dimerization assessment (as discussed below). The availability of Tagged-GPCR expressing cell lines Receptor binding assay and the fluorophore-labeled ligands are some of the limita- Receptor binding assay can be used to characterize in great tions in this approach. detail the interaction between receptor and its ligands, such as the intrinsic affinity of ligands to the receptor, association/dis- G-protein dependent functional assays sociation rates, and the density of receptor in tissues or cells[8]. Ligand-binding assays are useful to identify new compounds Receptor binding assay is a cell-free method theoretically suit- that target GPCRs. Further analysis of the biological responses able for any GPCR screening without involving downstream after compound binding will help complete the whole picture signaling from the receptor. This type of assay can also obtain concerning the overall characteristics of the compound. Upon
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