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


Besides the 150 presently characterised G protein-coupled receptors (Gapers), unequal number of orphan receptors awaits the characterisation of their legend and function. We will build on the expertise of the partners in the frame of GPCRresearch in order to transform these orphan receptors into validated targets for drug development. Cell lines expressing orphan receptors will be used in a high throughput functional assay in order to identify their natural legends from tissue extracts and other biological samples. Distribution of the receptors in normal and diseased tissues will be studied, as well as their function in mouse knock out models, and their potential involvement in human diseases. Surrogate legends will be searched in chemical libraries, as a first step toward drug development.
Most characterized and non characterized receptors have been made available in expression vectors. About a hundred orphan receptors have been expressed in cell lines allowing high throughput screening for agonists in extracts from natural sources or in peptide or chemical libraries. This assay, based on the luminescence of aequorin has been adapted to 384 wells plate format. An inducible system, based on the tet-on technology was set up in order to overcome expression problems with some receptors. We have constructed a microarray dedicated to GPCRs, which has been used for generating the expression profile of receptors in a number of cell populations and tissues. It was however found that the sensitivity of this system is not high enough to detect all receptors functionally relevant. As a consequence, we have limited the amplitude of the expression database generated using this approach, as compared to what was planned initially. We have also constructed large arrays for human genes that allowed to determine the transcriptional program activated by selected receptors. We have developed softwares designed to handle microarray data in general, and have developed a cell microarray technique that will allow to test in a microarray format thousands of genes or interfering RNAs for their functional activity in a cell based assay. Broad distributions of orphan GPCRs have also been collected by using RT-PCR, while more detailed studies have been performed, using in situ hybridization, immunohistochemistry of FACS analysis, following the generation of monoclonal antibodies generated by DNA immunization. A large collection of fractions of tissue extracts and biological fluids has been constructed, from a wide range of porcine or human tissues, and by using various extraction protocols dedicated to different classes of molecules. This collection was complemented by expression libraries, random peptide libraries, and collections of agents known or suspected to display biological activities through G protein-coupled receptors.

From these various types of screening, the natural ligands of seven orphan receptors have been characterized, either through the purification of the natural peptidic ligand, or through the matching with a known molecule. These receptors are GPR54, OR143/NPFFR2, GPR86/P2Y13, GPR7, GPR8, GPR43 and ChemR23. In four situations, this led to the filing of a patent application. Among these patented receptors, one (GPR7) is expressed in the central nervous system, and responds to a novel neuropeptide. The second (ChemR23) is expressed in antigen presenting cells and its ligand is a new chemoattractant protein present in a variety of human inflammatory fluids. The third (GPR43) is expressed mainly in neutrophils and respond to short chain fatty acids. The last receptor (GPR86/P2Y13) is a novel purinergic receptor responding to ADP. Two additional biological activities were found and have been purified and analyzed by mass spectrometry, but the precise nature of these two ligands could not be determined precisely at this stage. This characterization will be pursued in the follow-up of this program. Detailed distributions have been performed for these new receptors, using Taqman analysis, monoclonal antibodies, FACS analysis and immunohistochemistry. We have characterized the pharmacology and structure/function aspects of deorphanized receptors (and their ligands), including GPR7, GPR8, ChemR23 and GPR86/P2Y13. Several knock out models have been studied in detail. The knock out model of GPR10 identified the receptor as playing a key role in the control of stress responses. Three other models have been tested in a broad range of assays, but will still require additional studies. A knock out model for ChemR23 was obtained, and its study has started. Finally, we have initiated the screening of chemical libraries using as targets three of the receptors recently characterized in the consortium, as well as a few orphan receptors. Agonists and antagonists have been identified for two of these receptors. Altogether, the program has reached most of the expected achievements. The number of receptors functionally characterized is beyond the expected achievement. This is the direct consequence of the focus placed from the initiation of the program onto the development of diverse and efficient screening assays, and the construction of a large collection of tissue extracts. The program will be pursued after termination of this EU contract, particularly the deorphanization process using the available collection of cell lines, ligands and extracts, the functional characterization of deorphanized receptors, the characterization of established knock out models, and the development of chemical ligands.

Funding Scheme

CSC - Cost-sharing contracts


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Participants (4)

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171 77 Stockholm
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