Development Of Drug Screening Models Targeted To G Protein-Coupled Receptors Based On Scintillation Proximity Assay

Chemokine receptors, which belong to the G protein-coupled receptor(GPCR) family, participate in a variety of physiological activities and become a group of important drug targets. In this thesis, four stable cell lines CHO-CXCR2 ,CHO-CXCR3 , CHO-CCR3 and CHO-CCR4 were established by the MACS technology with over-expressed chemokine receptors on the cell surface. The scintillation proximity assay(SPA) is a homogenous technique, it does not require post-reaction liquid handing steps and is highly automatable. Due to its simplicity and high-through nature the assay has been used widely to study molecular interactions. In this thesis, drug-screening models targeted to GPCRs were developed using SPA technology. Firstly, a SPA-WGA-based [35S]GTPγS binding assay was set up to measure basal and agonist stimulated [35S]GTPγS binding to G-protein in cell membrane preparation. And a SPA-WGA-based ligand-receptor binding assay was developed to detect the affinity of ligand binding. In order to cut the cost, the amount of SPA-WGA beads was minimized and the concentration of membranes and the time of incubation were opimized. These assay are useful tools for GPCR research and drug screening targeted to GPCRs. Secondly, an Anti-G protein SPA -based [35S]GTPγS binding assay was developed, which overcomes the limitations of conventional [35S]GTPγS binding assay that can only detect the activation of the Gi protein family and ligand-receptor binding assay that can not distinguish agonist, antagonist and inversive agonist.