Establishment Of High Throughput Screening System Of5-HT_1A Receptor Agonists And The Crystallization Of FABP₄

Establishment of high throughput screening system of5-HT1A receptor agonistsDepression is one of the most common and disabling psychiatric disorders around the world. The clinical manifestations are low emotion, pessimistic, sleep disorder and someone even will have suicidal impulse. It is thought that many reasons will cause depression, for example the low content of some monoamine neurotransmitters like NE,5-HT and DA. Now many persons are suffered from depression because of the increasing pressure and some hereditary factors. Above all depression is a severe issue in the world.5-HT1A receptor is the first cloned subtype among the5-HT receptor family. It is indicated that5-HT1A receptor is concerned with a series of psychiatric disorders, such us anxiety, depression, schizophrenia, and the agonists of5-HT1A receptor will improve these disorders. Therefore, it is significant to explore and research the agonists of5-HT1A receptor.To research the agonists, we established a high throughput screening system of5-HT1A receptor by detecting the activity of reporter gene. The pcDNA3.1-h5-HT1AR plasmid and pcDNA3.1-pCRE-luc plasmid which harbored a luciferase reporter gene were co-transfected into cells, and we studied and optimized the conditions like the cell, the ratio of plasmids and so on. During the assay, we selected HEK293as the cell of this assay according to the signal efficacy; when the plasmid ratio between pcDNA3.1-h5-HT1AR and pcDNA3.1-pCRE-luc was1:3, we got the best signal-to-noise ratio (S/N); the best incubation time of compound was6hours in this assay; as to four common positive compounds,5-HT.HC1, Flibanserin,8-OH-DPAT and Lorcaserin (APD356) we studied respectively, and the result showed that APD356was the best positive compound in this screening system. The signal was stable after the cell line was cultured for twelve generations, and we screened some compounds and finally got a compound. Above all, a cell line that stably expressed5-HT1A receptor was established, assay conditions were optimized, and at last, it was proved that the cell line was suitable for high throughput screening of5-HT1A receptor agonists. It was of great importance to research antidepressive drugs. Expression, purification and crystallization of FABP4FABP4is the first FABPs to be discovered in the world, and it is widely expressed especially in adipocytes and macrophages. The defect of FABP4may improve insulin resistance as well as the inhibition of artherosclerosis. Above all, FABP4is an important target to cure diabetes and artherosclerosis. Now small molecular inhibitors of FABP4are explored by researchers around the world. In our laboratory, we find some small molecular inhibitors of FABP4. To study the modes of compounds and FABP4, we express and purify protein, and finally crystallize the protein and compounds.