针对趋化因子受体CCR5的SPA-WGA药物筛选方法

目的建立针对趋化因子受体CCR5的药物筛选SPA-WGA法[35S]GTPγS结合实验方法。方法通过降低SPA-WGA小球用量和对其他各种因素进行优化,如细胞膜用量、GDP浓度、体系孵育时间和样品连续测量时间,建立了SPA-WGA法[35S]GTPγS结合实验方法。结果最后优化得到实验条件为:100μl反应体系,0.1 mg SPA-WGA小球,10μgCHO-CCR5细胞膜,10μmol.L-1GDP和0.3 nmo.lL-1[35S]GTPγS,室温孵育1.5 h并且在1 200 m in之内完成所有样品测量。该法与传统抽滤法比较,测量得到RANTES的EC50分别为:1.40 nmol.L-1和2.90 nmol.L-1,测量得到SCH-C的IC50分别为:2.95 nmo.lL-1和2.73 nmo.lL-1,测量结果相似,均呈现出较好的剂量效应关系。利用该法筛选54个化合物,筛到3个IC50在1~10 nmol.L-1的化合物。这3个化合物是否具有H IV-1进入抑制剂的潜在开发价值,还有待体外抗H IV-1实验的进一步验证和筛选。结论此方法操作简便,灵敏性和重现性好,且可高通量和自动化,该法适用于能与Gi家族蛋白偶联的GPCR的高通量药物筛选。

Drug screening model targeted to CCR5 based on [35S] GTPγS scintillation proximity assay

Aim To develop a high throughput screening model targeted to CCR5 based on [()~(35)S]GTPγS scintillation proximity assay.Methods Membranes expressing hCCR5 were incubated for 60 min with GDP,[()~(35)S]GTPγS and SPA-WGA beads with agonist or antagonist at room temperature.Then the beads were centrifuged and radioactivity was measured with scintillation counter.The assay,which measures the level of G protein activation following RANTES occupation of CCR5,can be used to determine the degree of agonism and the potency of compounds acting at CCR5.Result A new in vitro assay was established for the high throughput screening of CCR5 after minimizing of the amount of SPA-WGA beads and optimizing of other factors including the amount of CCR5 membranes,the concentration of GDP,the incubation time course and the counting time course.The EC_(50) value of RANTES or the IC_(50) value of SCH-C gained by using [()~(35)S]GTPγS scintillation proximity assay is good and is similar to using [()~(35)S]GTPγS filtration assay.Among 54 small molecular compounds screened by using [()~(35)S] GTPγS scintillation proximity assay,3 compounds with IC_(50) values among 1～10 nmol·L~(-1) will be further testified on their anti-HIV activities by using HIV-1 Neutralization Assays.Conclusion The[()~(35)S] GTPγS scintillation proximity assay is easily and safely operated,automatable,economical and repeatable.And the assay is feasible by high throughput screening for the receptors coupling to G_i protein such as CCR5