Development And Application Of New Models For Chemokine Receptor Modulator's Screening With Calcium Mobilization Detection

Chemokine receptors belong to G protein coupled receptor(GPCR) superfamilies,which are important targets for drugs.Nowadays,the commonly used assays for GPCR are radioligand binging and some other assays based on their signal pathways.Gα16,as a member of Gq family,could induce release of intracellular calcium ion by second messagers after receptors being activated.In this paper,we developed new high-throughput models for CCR5 and CXCR4 modulator's screening by detecting calcium mobilization based on co-expression of receptor and Gα16.1.Development and application of a new model for CCR5 modulator's screening,which contains three sections.Section 1:Construction of CHO/CCR5/Gα16 cell line.Plasmids encoding CCR5 and Gα16 proteins were transfected into CHO cells by electroporation.After selection with antibiotics for 20 days,monoclones were picked out to form cell lines and the expression of exogenous proteins were conformed by RT-PCR.Section 2:Determination of CCR5's activity on CHO/CCR5/Gα16 cell line.[³⁵S]-GTPγS binding assay was conducted to examine the activity of CCRS.CCR5 could be activated by its agonist RANTES in a dose-dependent way.And maraviroc,which is the antagonist to CCR5,could inhibit RANTES-induced activation of CCR5 in a dose-dependent way.Section 3:Construction and application of Calcium mobilization assay.(1) Calcium mobilization assay was divided into two models,agonist model and antagonist model.In the agonist model,compounds that can active receptors,could induce calcium signals which then be recorded by fluorescent dyes Fluo-4 and detected by a machine Flexstation.Oppositely in the antagonist model,compounds are incubated with cells first.Those have antagonism effect to receptors could block calcium signals.(2) Effect of RANTES and maraviroc to CCR5 were examined using the two models respectively.Agonists and antagonists could be exactly distinguished.And the EC₅₀ value or IC₅₀ value of different ligands decided by the two models, were consistent with the results of[³⁵S]-GTPγS binding assay.(3) A series of compounds from CCR5 antagonist lead compound library were evaluated with the constructed screening model.We obtained 44 compounds,whose inhibitory effect were more than 80%as to the positive control maraviroc.And their IC₅₀ values were close to maraviroc,which was then conformed by [³⁵S]-GTPγS binding assay.2.Development of a new model for CXCR4 modulator's screening,which also contains three sections.Section 1:Construction of CHO/myc-CXCR4/Gα16 cell line.Plasmids encoding myc-CXCR4 and Gα16 proteins were transfected into CHO cells by electroporation.After selection with antibiotics for 20 days,monoclones were picked out to form cell lines and the localization of expressed CXCR4 on the cell membrane was conformed by Immunofluorescence.Section 2:Determination of CXCR4's activity on CHO/myc-CXCR4/Gα16 cell line. [³⁵S]-GTPγS binding assay and Calcium mobilization assay were conducted to examine the activity of CXCR4.On the two assays,CXCR4 could be both activated by its agonist SDF-1 in a dose-dependent way.Section 3:Futher study about CXCR4 on CHO/myc-CXCR4/Gα16 cell line.SDF-1-induced internalization of CXCR4 was detected by Immunoflurescence.The desensitization of CXCR4 upon the sthnulation of SDF-1 for different time was detected by calcium mobilization assay.SDF-1 could induce CXCR4 desensitization in a few minutes,which was one and all with previous studies.We then examined the expression level of CXCR4 upon the stimulation of SDF-1 for different time,and we found that CXCR4 was downregulated after being stimulated with SDF-1 for 3 hours.Conclusions:1.Two cell lines,CHO/CCR5/Gα16 and CHO/myc-CXCR4/Gα16,were constructed.On the constructed cell lines,CCR5 and CXCR4 were of excellent activity.2.Calcium mobilization assay was divided into two models,agonist model and antagonist model,which could exactly distinguish agonists and antagonists.3.Evaluated with the antagonist model,44 compounds which have intensive inhibitory effect on CCR5,were picked out for further study.4.Futher studies about CXCR4 indicated the functional integrity of CXCR4 on CHO/myc-CXCR4/Gα16 cell line.