The Applications Of SPA Technology In High Throughput Screening Of Receptors For Drugs

In high throughput drug screening, the SPA technology has been widely used overseas. The objects of this thesis were to apply SPA technology in HTS of both nuclear and membrane receptors. The principal contents of this thesis including: (1) expression of the full-length PPARγby BEVS. At first, the full-length PPARγgene was subcloned into plasmid pFastBac1 using molecular biology technologies, the reconstructed plasmid pFastBac1-PPARγwas transformed into E. Coli DH10Bac, the bacmid transfected into insect Sf-9 cell to express PPARγ. Ligand binding assays suggested that the receptor was bioactive; (2) with PPARγ,RXRα,[3H]BRL49653,biotin-PPRE and SPA beads, an "ABCDE"SPA technology was established to screen PPARγmodulators, following an HTS campaign of 16000 compounds, 12 were confirmed "hit", including two whose IC50 values were below 0.5 μM; (3) [3H]cytisine, α4β2-nAChR and FlashBlue beads were employed to develop a binding assay based on SPA technology, which was used to screen 32000 compounds, and 17 "hit"were confirmed with IC50 values were below 10 μM. Conclusions: (1) BEVS was successfully used to express the full-length nuclear receptor PPARγ; (2) it was the first use of the SPA technology in HTS in China which was applied to both nuclear and membrane receptors, and resulted in the discovery of a series of "hit"; (3) compared with conventional HA methods, the SPA technology has obvious...