Establishment And Application Of A High-Throughput- Screening Model For Medium-Chain Fatty Acid-Sensing Receptor GPR84

G protein coupled receptor 84(GPR84) is medium-chain fatty acid-sensing receptor(C9-C14). It plays an important role in metabolism of fatty acid and immune response, and it’s also deeply involved in inflammatory immune diseases such as inflammatory bowel disease, multiple sclerosis and endotoxemia. As GPR84 is a proinflammatory receptor, it may provide an effective method to treat these diseases by finding GPR84 antagonists. It’s important to construct a cell line stably expressing human GPR84 for screening its ligands and researching diseases. Nowadays, drug screening methods for GPCRs are radioligand binding and some other assays based on their downstream signal pathways (cAMP and Ca2+). Gal 6 as a member of Gαq family, receptors being activated could induce release of intracellular Ca2+ by second messagers IP3. In this research, we transfected plasmids encoding GPR84 and Gα16 proteins into HEK293 cells. After selection with antibiotics, we picked out monoclones which stably expresses GPR84 to form cell lines. The permanent expression of exogenous GPR84 proteins in HEK293 cells was detected by RT-PCR, immunofluorescence staining, calcium mobilization and cAMP assay. Western blot and FACS were conducted to examine the activity of GPR84. Above all the results demonstrate that GPR84 receptor has excellent biology activity. Based on cell lines stably expressing GPR84, we conducted high-throughput assay for the screening of GPR84 ligands and found new GPR84 antagonists. We can further research on the biological functions of GPR84 and immune diseases targeted GPR84 with these antagonists.Construction of HEK293/GPR84/Gal 6 cell line. Use electroporation transfected Plasmids into HEK293 cells, which encoding GPR84 and Gα16 proteins. After 28 days selection with antibiotics, we picked out monoclones to form cell lines and determination expression of exogenous proteins by RT-PCR and immune-fluorescence staining.Detection of GPR84’s activity on HEK293/GPR84/Gα16 cell line. Calcium mobilization,cAMP assay and Western blot were conducted to examine the activity of GPR84.Calcium mobilization, FACS and immunofluorescence staining were conducted to detect the desensitization and Internalization of GPR84 induced by 6-OAU. GPR84 could be activated by its agonist 6-OAU in a dose-dependent way. It demonstrates that the HEK293/GPR84/Ga16 cell line has excellent biology activity.Establishment and application of Calcium mobilization assay for high-throughput screening. (1) Calcium mobilization assay was divided into agonist model and antagonist model. In the agonist model of the Calcium mobilization assay, 6-OAU that can active receptors GPR84, could induce Ca2+ signals which then be recorded by fluorescent dyes Fluo-4 and detected by a machine FDSS. But in the antagonist model, first, compounds are incubated with cells for 10 min. If drugs could block Ca2+ signals, They may have antagonism effect to receptors. (2) A series of compounds library in The Chinese National Compound Library were evaluated with the screening model for antagonists, and the compound named ZQ-02 was finally found for further study.