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See detailDEVELOPMENT OF PHARMACOLOGICAL TOOLS FOR THE IDENTIFICATION OF G PROTEIN-COUPLED RECEPTORS LIGANDS
Gilissen, Julie ULg

Doctoral thesis (2016)

G protein-coupled receptors (GPCRs) represent the protein family most successfully targeted for treating human diseases. They couple to G proteins to mobilize second messenger pathways that lead to ... [more ▼]

G protein-coupled receptors (GPCRs) represent the protein family most successfully targeted for treating human diseases. They couple to G proteins to mobilize second messenger pathways that lead to cellular responses and ultimately to physiological changes. However many are poorly characterized with few ligands reported or remain completely orphans. Therefore, there is a growing need for screening-compatible and sensitive assays in order to identify new ligands. The present project aims at developing pharmacological tools to characterize the pharmacology and physiology of GPCRs. Our approach rely on i) development of receptor models and assays for the identification of ligands, ii) screening of chemical and virtual small molecules libraries and iii) analysis of structure-activity relationships study of active molecules. The project has been divided in two parts. To set-up assays for the evaluation of GPCRs activation, we selected the understudied succinate receptor 1 (SUCNR1) that is proposed to affect cellular metabolism and pathophysiology of diseases in multiple organs. Nevertheless the receptor has never been validated as a drug target because very few ligands have been described. So, developing pharmacological tools for SUCNR1 remains of great interest in therapeutic drug discovery. First, we have started by examining SUCNR1 signaling pathways in HEK293 cells. Our investigations have highlighted the efficient coupling to Gαi and thus the negative modulation of intracellular cAMP levels. Consequently we have implemented an assay sensitive to cAMP variations to identify ligands able to induce SUCNR1 activation. However, an important drawback to track agonists for Gαi-coupled receptors is the mandatory stimulation of cAMP levels. Inducers such as forskolin must be used and are sources of variations and errors. In order to avoid these artifacts we have set-up and validated a cAMP-inducer free method based on the GloSensor biosensor. This real time assay was amenable to high-throughput screening for the detection of Gαi-coupled receptors agonists. The strategy monitoring basal cAMP levels compared to the stimulated cAMP levels allowed to decrease recording time and artifcats from forskolin use, leading to the identification of fewer false positives and unidentified false negatives. Although both methods found agonists in the chemical library screened, no active new scaffolds on SUCNR1 were discovered. We infer that this method could facilitate the study and screening of Gαi-coupled receptors for active ligands. Secondly, given the interesting potential of SUCNR1 for promising therapeutic advances, we have carried out the study of the receptor interaction with its natural ligand, succinate. We have optimized the previous three-dimensional model for SUCNR1 binding pocket by means of more detailed structure-activity relationships study of succinate related molecules. The study of structure-activity relationships performed by Pierre Geubelle, in parallel to this work, allowed the deduction of the structural elements required to be active on SUCNR1. Thus we have defined a pharmacophore for activity on the receptor and subsequently evaluated various cycloalkanes. With our cAMP assay, Pierre Geubelle has highlighted the (1R, 2S)-1,2-cyclopropanedicarboxylate to be able to activate SUCNR1. We confirmed the activity of this compound on SUCNR1 capacity to recruit arrestin 3 and determined the pharmacological properties of this new ligand as SUCNR1 agonist, in vitro and in vivo. To confirm our in vitro results, we have also assessed the hypertensive properties of this cyclic analogue. Intravenous addition at the dose of 0.1 mg.kg-1 in rats has been demonstrated to increase blood pressure in the same range as succinate. Consequently we have demonstrated that (1R, 2S)-1,2-cyclopropanedicarboxylate could be regarded as an original synthetic full agonist for SUCNR1. In addition, the pharmacophore for SUCNR1 should help to generate synthetic compounds characterized by an increased potency and/or efficacy compared to succinate. [less ▲]

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See detailTargeted and random mutagenesis of orphan GPCRs of the SREB family
Laschet, Céline ULg; Dupuis, Nadine ULg; Derj, Anouar ULg et al

Poster (2016, January 25)

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See detailInsight into SUCNR1 (GPR91) structure and function
Gilissen, Julie ULg; Jouret, François ULg; Pirotte, Bernard ULg et al

in Pharmacology & Therapeutics (2016)

SUCNR1 (or GPR91) belongs to the family of G protein-coupled receptors (GPCR), which represents the largest group of membrane proteins in human genome. The majority of marketed drugs targets GPCRs ... [more ▼]

SUCNR1 (or GPR91) belongs to the family of G protein-coupled receptors (GPCR), which represents the largest group of membrane proteins in human genome. The majority of marketed drugs targets GPCRs, directly or indirectly. SUCNR1 has been classified as an orphan receptor until a landmark study paired it with succinate, a citric acid cycle intermediate. According to the current paradigm, succinate triggers SUCNR1 signaling pathways to indicate local stress that may affect cellular metabolism. SUCNR1 implication has been well documented in renin-induced hypertension, ischemia/reperfusion injury, inflammation and immune response, platelet aggregation and retinal angiogenesis. In addition, the SUCNR1-induced increase of blood pressure may contribute to diabetic nephropathy or cardiac hypertrophy. The understanding of SUCNR1 activation, signaling pathways and functions remains largely elusive, which calls for deeper investigations. SUCNR1 shows a high potential as an innovative drug target and is probably an important regulator of basic physiology. In order to achieve the full characterization of this receptor,more specific pharmacological tools such as small-molecules modulators will represent an important asset. In this review, we describe the structural features of SUCNR1, its current ligands and putative binding pocket. We give an exhaustive overview of the known and hypothetical signaling partners of the receptor in different in vitro and in vivo systems. The link between SUCNR1 intracellular pathways and its pathophysiological roles are also extensively discussed. [less ▲]

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See detailßarrestin coupling of the orphan GPCR GPR27
Dupuis, Nadine ULg; Gilissen, Julie ULg; Derj, Anouar ULg et al

Poster (2015, January 27)

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See detailIdentification, Design and Evaluation of Pharmacological tools for the orphan GPCR GPR22
Geubelle, Pierre ULg; Gilissen, Julie ULg; Dupuis, Nadine ULg et al

Poster (2015, January 27)

GPCRs are the largest family of membrane receptors and are characterized by 7 transmembrane domains. GPR22 is a GPCR that has no known endogenous ligand and is thus considered "orphan". Its presence ... [more ▼]

GPCRs are the largest family of membrane receptors and are characterized by 7 transmembrane domains. GPR22 is a GPCR that has no known endogenous ligand and is thus considered "orphan". Its presence situated at the heart and brain levels makes it a potential target for new therapeutic pathways. This study consist in the identification of a synthetic ligand of GPR22 receptor to use it as a pharmacological tool in the study of the signaling channels of GPR22 in order to understand its role and to validate it as a new therapeutic target. The initial hypothesis was that GPR22 is coupled to the Gαi protein. [less ▲]

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See detailForskolin-free cAMP assay for Gi-coupled receptors
Gilissen, Julie ULg; Geubelle, Pierre ULg; Dupuis, Nadine ULg et al

in Biochemical Pharmacology (2015)

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See detailIdentification, Design and Evaluation of Pharmacological tools for the orphan GPCR GPR22
Geubelle, Pierre ULg; Gilissen, Julie ULg; Dupuis, Nadine ULg et al

Poster (2014, November 21)

GPCRs are the largest family of membrane receptors and are characterized by seven transmembrane domains. This family of receptors is currently the most successfully targeted protein for therapeutic ... [more ▼]

GPCRs are the largest family of membrane receptors and are characterized by seven transmembrane domains. This family of receptors is currently the most successfully targeted protein for therapeutic purposes. GPR22 is a GPCR that was discovered in 1997. It has no known endogenous ligand and is thus considered "orphan". Its presence situated at the heart and brain levels makes it a potential target for new therapeutic pathways. The only information about its signaling channel could be its coupling with G proteins. This study consist in the identification of a synthetic ligand of GPR22 receptor to use it as a pharmacological tool in the study of the signaling channels of GPR22 in order to understand its role and to validate it as a new therapeutic target. The initial hypothesis was that GPR22 is coupled to the Gαi protein. [less ▲]

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See detailLIGAND-INDEPENDENT IDENTIFICATION OF ORPHAN GPCR ARRESTIN BINDING
Dupuis, Nadine ULg; Gilissen, Julie ULg; Derj, Anouar ULg et al

Poster (2014, June 05)

Detailed reference viewed: 40 (11 ULg)
See detailIdentification of chemical probes and signaling pathways for the orphan GPCR GPR27
Dupuis, Nadine ULg; Gilissen, Julie ULg; Pirotte, Bernard ULg et al

Poster (2013, June 06)

The largest family of membrane receptors is represented by G protein-coupled receptors (GPCRs), which are characterized by 7 transmembrane domains. Even if marketed drugs currently target only 10% of all ... [more ▼]

The largest family of membrane receptors is represented by G protein-coupled receptors (GPCRs), which are characterized by 7 transmembrane domains. Even if marketed drugs currently target only 10% of all GPCRs, they represent more than 30% of all small molecules based therapies. The physiological and pathophysiological role of a GPCR is defined by its expression pattern, signaling pathway and specific ligand[1]. GPCRs which have not yet been associated to a physiological ligand are called orphan GPCRs and represent ~100 of the ~370 human non-odorant GPCRs[2]. This project aims at identifying and developing pharmacological tools for GPR27 (SREB1), one of these orphan receptors. GPR27 has recently been shown to have a role in the regulation of insulin promoter activity and insulin secretion[3]. Nevertheless, the pharmacology of GPR27 remains elusive and the lack of appropriate pharmacological tools dramatically restricts the understanding of its function and its validation as a drug target. Thus, we plan to study its signaling pathway and to develop screening methods that will allow us to identify small molecules able to interact with GPR27. These are important steps toward understanding its function and evaluating GPR27 as a potential drug target, for instance in insulin-related metabolic disorders such as type II diabetes or in other pathologies where it might be involved. References 1) Wise, A., et al. (2002). Drug discovery today, 7, 235 2) Fredriksson, R., et al. (2003). Molecular pharmacology, 63, 1256 3) Ku, G. M., et al. (2012). PLoS genetics, 8, e1002449 [less ▲]

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See detailChemical probes and signaling pathways for the orphan GPCR GPR27
Dupuis, Nadine ULg; Gilissen, Julie ULg; Pirotte, Bernard ULg et al

Poster (2013, January 28)

Detailed reference viewed: 29 (1 ULg)