A dynamic and screening-compatible nanoluciferase-based complementation assay enables profiling of individual GPCR-G protein interactions.

G protein-coupled receptors (GPCRs) are currently the target of more than 30% of the marketed medicines. However, there is an important medical need for ligands with improved pharmacological activities on validated drug targets. Moreover, most of these ligands remain poorly characterized, notably because of a lack of pharmacological tools. Thus, there is an important demand for innovative assays that can detect and drive the design of compounds with novel or improved pharmacological properties. In particular, a functional and screening-compatible GPCR-G protein interaction assay is still unavailable. Here, we report on a nanoluciferase-based complementation technique to detect ligands that promote a GPCR-G protein interaction. We demonstrate that our system can be used to profile compounds with regard to the G proteins they activate through a given GPCR. Furthermore, we established a proof of applicability of screening for distinct G proteins on dopamine receptor D2 whose differential coupling to Gαi/o family members has been extensively studied. In a D2-Gαi1versus D2-Gαo screening, we retrieved five agonists that are currently being used in antiparkinsonian medications. We determined that in this assay, piribedil and pergolide are full agonists for the recruitment of Gαi1 but are partial agonists for Gαo, that the agonist activity of ropinirole is biased in favor of Gαi1 recruitment, and that the agonist activity of apomorphine is biased for Gαo We propose that this newly developed assay could be used to develop molecules that selectively modulate a particular G protein pathway.

Activation of the Orphan G Protein-Coupled Receptor GPR27 by Surrogate Ligands Promotes ß-Arrestin 2 Recruitment.

G protein-coupled receptors are the most important drug targets for human diseases. An important number of them remain devoid of confirmed ligands. GPR27 is one of these orphan receptors, characterized by a high level of conservation among vertebrates and a predominant expression in the central nervous system. In addition, it has recently been linked to insulin secretion. However, the absence of endogenous or surrogate ligands for GPR27 complicates the examination of its biologic function. Our aim was to validate GPR27 signaling pathways, and therefore we sought to screen a diversity-oriented synthesis library to identify GPR27-specific surrogate agonists. To select an optimal screening assay, we investigated GPR27 ligand-independent activity. Both in G protein-mediated pathways and in ß-arrestin 2 recruitment, no ligand-independent activity could be measured. However, we observed a recruitment of ß-arrestin 2 to a GPR27V2 chimera in the presence of membrane-anchored G protein-coupled receptor kinase-2. Therefore, we optimized a firefly luciferase complementation assay to screen against this chimeric receptor. We identified two compounds [N-[4-(anilinocarbonyl)phenyl]-2,4-dichlorobenzamide (ChemBridge, San Diego, CA; ID5128535) and 2,4-dichloro-N-{4-[(1,3-thiazol-2-ylamino)sulfonyl]phenyl}benzamide (ChemBridge ID5217941)] sharing a N-phenyl-2,4-dichlorobenzamide scaffold, which were selective for GPR27 over its closely related family members GPR85 and GPR173. The specificity of the activity was confirmed with a NanoLuc Binary Technology ß-arrestin 2 assay, imaging of green fluorescent protein-tagged ß-arrestin 2, and PathHunter ß-arrestin 2 assay. Interestingly, no G protein activation was detected upon activation of GPR27 by these compounds. Our study provides the first selective surrogate agonists for the orphan GPR27.

Superconserved receptors expressed in the brain: Expression, function, motifs and evolution of an orphan receptor family.

GPR27, GPR85 and GPR173 constitute a small family of G protein-coupled receptors (GPCR) that share the distinctive characteristics of being highly conserved throughout vertebrate evolution and predominantly expressed in the brain. Accordingly, they have been coined as "Superconserved Receptors Expressed in the Brain" (SREB), although their expression profile is more complex than what was originally thought. SREBs have no known validated endogenous ligands and are thus labeled as "orphan" receptors. The investigation of this particular category of uncharacterized receptors holds great promise both in terms of physiology and drug development. In the largest GPCR family, the Rhodopsin-like or Class A, around 100 receptors are considered orphans. Because GPCRs are the most successful source of drug targets, the discovery of a novel function or ligand most likely will lead to significant breakthroughs for the discovery of innovative therapies. The high level of conservation is one of the characteristic features of the SREBs. We propose herein a detailed analysis of the putative evolutionary origin of this family. We highlight the properties that distinguish SREBs from other rhodopsin-like GPCRs. We present the current evidence for these receptors downstream signaling pathways and functions. We discuss the pharmacological challenge for the identification of natural or synthetic ligands of orphan receptors like SREBs. The different SREB-related scientific questions are presented with a highlight on what should be addressed in the near future, including the confirmation of published evidence and their validation as drug targets. In particular, we discuss in which pathological conditions these receptors may be of great relevance to solve unmet medical needs.

Nanoluciferase-Based Complementation Assay to Detect GPCR-G Protein Interaction.

G protein-coupled receptors (GPCR) are one of the principal class of membrane proteins and around 30% of the currently marketed drugs act on one of them. The efficacious detection of ligands with the desired pharmacological profile remains a challenge of paramount importance in the GPCR drug discovery and pharmacological research. Recent evidences demonstrate that GPCR ligands can stabilize distinct receptor conformation and trigger various signaling pathways with different efficacies and/or potencies. This phenomenon called functional selectivity or biased signaling may lead to improved drugs with fewer side effects. Most receptors are promiscuous and can couple to more than one G protein family. To enable the discovery of biased ligands able to selectively trigger one G protein pathway over another, simple and efficient screening procedures are needed. The traditional assays aiming at detecting G protein activation monitor the generation of second messengers ([Ca2+]i, cAMP, IP1) or active G proteins (with GTP-g-S for instance). While these approaches have proven sensitive and robust, they are not suited for the detection of a single GPCR-G protein interaction. Here, we present in detail a method to assess directly the interaction between the receptor and the G protein. It permits the profiling of a receptor or a ligand toward G protein interactions and is compatible with high-throughput screening.

Structure-activity relationships of agonists for the orphan G protein-coupled receptor GPR27.

GPR27 belongs, with GPR85 and GPR173, to a small subfamily of three receptors called "Super-Conserved Receptors Expressed in the Brain" (SREB). It has been postulated to participate in key physiological processes such as neuronal plasticity, energy metabolism, and pancreatic ß-cell insulin secretion and regulation. Recently, we reported the first selective GPR27 agonist, 2,4-dichloro-N-(4-(N-phenylsulfamoyl)phenyl)benzamide (I, pEC50 6.34, Emax 100%). Here, we describe the synthesis and structure-activity relationships of a series of new derivatives and analogs of I. All products were evaluated for their ability to activate GPR27 in an arrestin recruitment assay. As a result, agonists were identified with a broad range of efficacies including partial and full agonists, showing higher efficacies than the lead compound I. The most potent agonist was 4-chloro-2,5-difluoro-N-(4-(N-phenylsulfamoyl)phenyl)benzamide (7y, pEC50 6.85, Emax 37%), and the agonists with higher efficacies were 4-chloro-2-methyl-N-(4-(N-phenylsulfamoyl)phenyl)benzamide (7p, pEC50 6.04, Emax 123%), and 2-bromo-4-chloro-N-(4-(N-phenylsulfamoyl)phenyl)benzamide (7r, pEC50 5.99, Emax 123%). Docking studies predicted the putative binding site and interactions of agonist 7p with GPR27. Selected potent agonists were found to be soluble and devoid of cellular toxicity within the range of their pharmacological activity. Therefore, they represent important new tools to further characterize the (patho)physiological roles of GPR27.

The G protein-coupled receptors deorphanization landscape.

G protein-coupled receptors (GPCRs) are usually highlighted as being both the largest family of membrane proteins and the most productive source of drug targets. However, most of the GPCRs are understudied and hence cannot be used immediately for innovative therapeutic strategies. Besides, there are still around 100 orphan receptors, with no described endogenous ligand and no clearly defined function. The race to discover new ligands for these elusive receptors seems to be less intense than before. Here, we present an update of the various strategies employed to assign a function to these receptors and to discover new ligands. We focus on the recent advances in the identification of endogenous ligands with a detailed description of newly deorphanized receptors. Replication being a key parameter in these endeavors, we also discuss the latest controversies about problematic ligand-receptor pairings. In this context, we propose several recommendations in order to strengthen the reporting of new ligand-receptor pairs.

Identification and pharmacological characterization of succinate receptor agonists.

BACKGROUND AND PURPOSE: The succinate receptor (formerly GPR91 or SUCNR1) is described as a metabolic sensor that may be involved in homeostasis. Notwithstanding its implication in important (patho)physiological processes, the function of succinate receptors has remained ill-defined because no pharmacological tools were available. We report on the discovery of the first family of potent synthetic agonists. EXPERIMENTAL APPROACH: We screened a library of succinate analogues and analysed their activity on succinate receptors. Also, we modelled a pharmacophore and a binding site for this receptor. New agonists were identified based on the information provided by these two approaches. Their activity was studied in various bioassays, including measurement of cAMP levels, [Ca2+ ]i mobilization, TGF-α shedding and recruitment of arrestin 3. The in vivo effects of activating succinate receptors with these new agonists was evaluated on rat BP. KEY RESULTS: We identified cis-epoxysuccinic acid and cis-1,2-cyclopropanedicarboxylic acid as agonists with an efficacy similar to that of succinic acid. Interestingly, cis-epoxysuccinic acid was 10- to 20-fold more potent than succinic acid on succinate receptors. For example, cis-epoxysuccinic acid reduced cAMP levels with a pEC50  = 5.57 ± 0.02 (EC50  = 2.7 µM), compared with succinate pEC50  = 4.54 ± 0.08 (EC50  = 29 µM). The rank order of potency of the three agonists was the same in all in vitro assays. Both cis-epoxysuccinic and cis-1,2-cyclopropanedicarboxylic acid were as potent as succinate in increasing rat BP. CONCLUSIONS AND IMPLICATIONS: We describe new agonists at succinate receptors that should facilitate further research on this understudied receptor.

Forskolin-free cAMP assay for Gi-coupled receptors.

G protein-coupled receptors (GPCRs) represent the most successful receptor family for treating human diseases. Many are poorly characterized with few ligands reported or remain completely orphans. Therefore, there is a growing need for screening-compatible and sensitive assays. Measurement of intracellular cyclic AMP (cAMP) levels is a validated strategy for measuring GPCRs activation. However, agonist ligands for Gi-coupled receptors are difficult to track because inducers such as forskolin (FSK) must be used and are sources of variations and errors. We developed a method based on the GloSensor system, a kinetic assay that consists in a luciferase fused with cAMP binding domain. As a proof of concept, we selected the succinate receptor 1 (SUCNR1 or GPR91) which could be an attractive drug target. It has never been validated as such because very few ligands have been described. Following analyses of SUCNR1 signaling pathways, we show that the GloSensor system allows real time, FSK-free detection of an agonist effect. This FSK-free agonist signal was confirmed on other Gi-coupled receptors such as CXCR4. In a test screening on SUCNR1, we compared the results obtained with a FSK vs FSK-free protocol and were able to identify agonists with both methods but with fewer false positives when measuring the basal levels. In this report, we validate a cAMP-inducer free method for the detection of Gi-coupled receptors agonists compatible with high-throughput screening. This method will facilitate the study and screening of Gi-coupled receptors for active ligands.