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Functional characterisation of orphan g protein-coupled receptors and their validation as drug targets


ChemR23 is an orphan G protein-coupled receptor that was cloned in the partnership a few years ago. It was shown to be expressed in dendritic cells (DCs) and macrophages. A biological activity specific for ChemR23 was detected in a human ascitic fluid. This activity was purified to homogeneity by several steps of reverse phase HPLC. Mass spectrometry analysis was performed on purified samples, allowing determining the presence in the sample of a protein encoded by the gene Tig-2, but poorly characterized functionally. However, the C-terminal peptide was not tryptic, lacking the last six aminoacids of the predicted protein. This observation suggested that the active compound might result from the proteolytic processing of the encoded precursor. The active protein was named chemerin. Human chemerin cDNA was cloned and expressed in CHO-K1 cells. The bioactive recombinant protein was purified to homogeneity from conditioned medium, and analysed by mass spectrometry and SDS/PAGE, which confirmed C-terminal truncation. A monoclonal antibody, generated against a C-terminal peptide, was used to purify to homogeneity from CHO-K1 conditioned medium, an unprocessed form of the protein (prochemerin), which was confirmed by mass spectrometry to retain the six C-terminal aminoacids. It was inferred that over 90% of prochemerin released by CHO-K1 cells was enzymatically processed into chemerin. Comparison of the biological activity of the two purified proteins assayed in parallel on CHO-K1 cells expressing human ChemR23 demonstrated that processed chemerin was about a hundred fold more active (EC50: 4.5 +/- 0.7nM) than unprocessed prochemerin (EC50: 393+/-116nM). We have shown that prochemerin is processed extracellularly. Although the protease responsible for this processing is not known, the regulation of this enzyme activity is expected to control as well the generation of active chemerin in the extracellular medium in vivo. The signalling pathways activated by ChemR23 were investigated. Stimulation of these cells by human chemerin at low nanomolar concentrations resulted in the release of intracellular calcium and inhibition of cAMP accumulation, as well as phosphorylation of the p42 and p44 MAP kinases. All these effects were inhibited by Pertussis toxin pretreatment, demonstrating the involvement of Gi family members. The biological function of chemerin was further investigated on leukocyte populations ex vivo. Human recombinant chemerin was chemotactic in vitro for immature, but not mature, dendritic cells, with a maximal activity at 1 nM. We investigated the presence of prochemerin and ChemR23 transcripts in various human tissues and leukocyte populations by real-time RT-PCR. In addition to immature dendritic cells, ChemR23 transcripts were found primarily in spleen, lymph nodes and lung, and at lower levels in a number of other tissues. Abundant chemerin transcripts were found in liver, lung, pituitary and ovary, and lower levels could be detected in most tissues. Interestingly however, no expression of chemerin was found in peripheral blood leukocyte populations. Monoclonal antibodies generated against human ChemR23 by genetic immunisation. High levels of ChemR23 immunoreactivity were found on monocyte-derived immature dendritic cells, and ChemR23 was downmodulated following maturation of the cells as a result of LPS or CD40L stimulation. Similarly, ChemR23 immunoreactivity was observed at the surface of monocyte-derived human macrophages. This novel chemoattractant protein is abundant in a diverse set of human inflammatory fluids, including ascitic fluid from neoplasic origin, or articular fluid from rheumatoid arthritis patients. It is however absent in articular fluid resulting from osteoathrosis. Chemerin is structurally and evolutionary related to the cathelicidin precursors (anti-bacterial peptides), cystatins (cysteine protease inhibitors) and kininogens. Altogether, chemerin appears as a potent chemotactic factor for antigen-presenting cells, largely expressed in human diseases involving an inflammatory component. The expression pattern suggests that this new system contributes greatly to many human diseases involving strong inflammatory responses. This novel protein and its receptor constitute therefore interesting candidates as therapeutic tools and targets for future development. ChemR23 constitutes a candidate target for the treatment of various inflammatory diseases and cancer. The partnership will pursue this program by developing chemical agonists and antagonists. Partnering and licensing will be considered at a later stage of development of the program.
Short chain fatty acids (SCFAs), including acetate, propionate and butyrate, are produced at high concentration by bacteria in the gut, and subsequently released into the bloodstream. Basal acetate concentrations in the blood (about 100mM) can further increase to mM concentrations following alcohol intake. It was known previously that SCFAs could activate leukocytes, particularly neutrophils. In our screening, we identified GPR43-expressing cells as responding functionally to solutions containing acetate. The determination of concentration-action curves allowed estimating the EC50 of GPR43 for sodium acetate to 268 +/- 26µM. We also found that GPR41, which shares 39% amino acid identity with GPR43, was also activated by acetate although to a lower extent. The natural coupling properties and the intracellular signaling pathways activated by GPR43 and GPR41 were investigated. Both receptors coupled negatively to adenylyl cyclase through a Pertussis toxin sensitive G protein (Gi/o class). Stimulation of GPR41 and GPR43 also resulted in the release of intracellular calcium, with a similar rank order of potency. In this latter assay, PTX abolished the response of GPR41, but not of GPR43, suggesting a unique Gi/o coupling for GPR41, and a dual coupling through the Gi/o and Gq families for GPR43. Stimulation of GPR43 and GPR41 expressed in CHO-K1 cells with propionate, induced phosphorylation of p42 and p44 MAP kinases (ERK1/ERK2). The pharmacology of both receptors was extended by assaying a number of short carboxylic acids and related molecules in a cAMP accumulation assay, using CHO-K1 cells, expressing stably GPR41 or GPR43, and stimulated with forskolin. The tested compounds included carboxylic acids containing 1 to 8 carbons, either linear or branched. Among the compounds displaying an agonist activity on GPR43, acetate, propionate and butyrate were characterized by a similar micromolar potency. The rank order of potency was propionate > acetate = butyrate > isobutyrate > caproate > isovalerate > valerate> formate > pivalate > L-OH-butyrate, caprylate and heptanoate, which were active only at concentrations of 10 mM. For human GPR41, the rank order of potency showed notable differences as compared to GPR43. The order was propionate > isobutyrate > butyrate > valerate > isovalerate > caproate > pivalate > acetate. Formate was totally inactive on this receptor. Other molecule including acetaldehyde, acetamide, acetate esters, acetone, ethanol, pyruvate, lactate, glyoxylate, were tested and found to be inactive on both receptors. The distribution of transcripts encoding the two receptors was investigated by RT-PCR in human peripheral tissues, brain areas, lymphoid organs and leukocyte populations. The broad expression profile of GPR41 in a large number of tissues did not allow inferring clear hypotheses regarding its biological functions. In contrast, GPR43 expression was characterized by a more restricted pattern. Transcripts were found at high level in polymorphonuclear cells (PMN) and at lower levels in peripheral blood mononuclear cells (PBMC), as well as in purified monocytes. GPR43 transcripts were also detected in bone marrow and spleen. The highly selective expression of GPR43 in leukocytes, particularly polymorphonuclear cells, suggests a role in the recruitment of these cell populations toward sites of bacterial infections. SCFAs have been described as displaying activities on leukocyte populations. We investigated the effects of SCFAs on human leukocyte, particularly PMN cells. Stimulation of PMN cells by propionate or acetate resulted in a transient increase of intracellular calcium. Concentration-action curves were established for propionate and acetate, demonstrating that both molecules were equally active on PMNs. Polymorphonuclear cells were also tested for their chemotactic response to sodium acetate and sodium propionate. Both SCFAs resulted in a classical bell-shaped dose-response curve with an optimal concentration of 1 mM. The pharmacological profile of GPR43 and its specific expression in neutrophils suggest an early role in the induction of immune and inflammatory responses, with a possible involvement in inflammatory bowel diseases as well as in alcoholism-associated disease susceptibility, two situations characterized by the generation of high levels of acetate and/or other SCFAs. GPR43 might therefore constitute a target allowing modulating immune responses in these pathological situations. Additional studies, including in vivo pharmacology and the generation of knockout models, will be necessary for identifying further the precise roles of these two receptors, and their potential applications as therapeutic targets. The partnership will pursue this program by developing chemical agonists and antagonists, and by studying further the function of this receptor. Partnering and licensing will be considered at a later stage of development of the program.
GPR7 and GPR8 are two structurally related orphan G-protein coupled receptors, presenting high similarities with opioid and somatostatin receptors. Two peptides, L8 and L8C, derived from a larger precursor, were recently described as natural ligands for GPR8. L8 is a 23 amino acid peptide, while L8C is the same peptide with a C-terminus extension of 7 amino acids, running through a dibasic motif of proteolytic processing. Using as query the amino acid sequence of the L8 peptide, we have identified in DNA databases a human gene predicted to encode related peptides, and its mouse ortholog. By analogy with L8 and L8C, two peptides, named L7 and L7C could result from the processing of a 125 amino acid human precursor, through the alternative usage of a dibasic amino acid motif. The ability of L7, L7C, L8 and L8C to activate GPR7 and GPR8 was tested using an aequorin-based functional assay. In this assay, L7C was the most potent agonist of GPR7 (EC50= 50 +/- 11nM) followed by L7, L8 and L8C. L8 was the most potent agonist of GPR8 (EC50= 58 +/- 5nM) followed by L8C, L7C and L7. A C-terminally truncated form of human L7 (WYKPAAGHSSYSVGRAAGLL) was active on both GPR7 and GPR8, but with a lower potency than the L7 or L7C forms. L8 and L7 peptides were iodinated, and binding experiments were performed on membranes obtained from CHO-K1 cells expressing GPR7 or GPR8. In competition binding experiments, L7C and L8C were both more potent than the shorter L7 and L8 peptides on GPR7, L7C displaying the highest affinity. In contrast, GPR8 displayed the highest affinity for L8, followed by L8C, L7 and L7C. To determine the natural coupling of the receptor to intracellular signalling pathways, CHO-K1 cell lines stably expressing GPR7 or GPR8, in the absence of exogenous transduction protein, were generated. Significant inhibition of forskolin-induced cAMP accumulation was observed for low concentrations of the four peptides in CHO-K1-GPR7 cells. Inhibition of cAMP accumulation was also observed in CHO-K1-GPR8 cells. The effect of L7 and L8 peptides on each receptor was strongly inhibited by pertussis toxin. No modification of phosphatidylinositol turnover was observed in COS-7 cells transiently expressing GPR7 or GPR8. The tissue distribution of the two peptide precursors, preproL7 (ppL7) and preproL8 (ppL8), was investigated by RT-PCR in peripheral tissues and central nervous system regions. Specific ppL7 transcripts were found at high levels in adult and fetal brain, substantia nigra, spinal cord, placenta and colorectal adenocarcinoma. Transcripts encoding the ppL8 precursors were detected at high levels by RT-PCR in the substantia nigra, lymphoblastic leukemia, fetal kidney, colorectal adenocarcinoma and trachea. Tissue distribution of the receptor transcripts was determined in parallel. Transcripts encoding GPR7 were detected at high levels in hippocampus, amygdala, trachea and lung carcinoma. GPR7 transcripts were also detected at moderate levels in fetal brain, pituitary gland and prostate. GPR8 transcripts were detected at high levels in caudate nucleus, hippocampus, amygdala, and at moderate levels in the adult brain, thalamus, parietal cortex, pituitary gland, adrenal gland, lymph nodes and lymphoblastic leukemia. The ppL7 and ppL8 genes encode therefore the precursors of a class of peptide ligands, active on two receptor subtypes, GPR7 and GPR8. The mild preference of each receptor for specific peptide species, and the distinct tissue distribution of the receptor and peptide precursors suggest each ligand and receptor has partially overlapping but also specific roles in this signalling system. Given the distribution of GPR7 and its ligands, this receptor constitute a candidate target for the treatment of various diseases and disorders, including appetite disorders and pain. The partnership will pursue this program by developing chemical agonists and antagonists. Partnering and licensing will be considered at a later stage of development of the program.