构建多巴胺传感器检测多巴胺受体激动剂和拮抗剂

The screening model for dopamine receptor agonists by a dopamine sensor

帕金森病(Parkinson’s disease,PD)是一种黑质致密部多巴胺能神经元缺失或死亡的中枢神经系统退行性疾病。临床上,左旋多巴是治疗PD最有效也最常用的药物。然而,长期服用左旋多巴易出现运动并发症以及因外周多巴胺累积导致的其他不良反应,成为PD治疗亟待解决的问题。多巴胺受体激动剂与多巴胺功能相似,能直接刺激突触后多巴胺受体,尽可能延缓左旋多巴的使用,减少长期服用左旋多巴引起的并发症。因此,筛选有效的多巴胺受体激动剂成为研究和治疗PD的关键问题。为建立快速、稳定、可靠的多巴胺受体激动剂筛选方法,本研究将人2型多巴胺受体(dopamine receptor 2,DRD2)基因与环状绿色荧光报告基因(circular permuted EGFP,cpEGFP)融合构建重组基因,用慢病毒载体包装,该载体置换基因编码的G蛋白偶联受体(G protein-coupled receptor,GPCR)激活传感器的胞内3环(the third intracellular loop,ICL3)内部跨膜结构域,在不改变GPCR活性的基础上,通过多巴胺受体激动剂与GPCR相互作用介导的构象变化调节GPCR融合的cpEGFP荧光。病毒载体感染HEK293T细胞,通过嘌呤霉素筛选后,获得高表达DRD2的细胞株,并用多巴胺受体激动剂作为阳性药(包括多巴胺、甲磺酸溴隐亭、卡麦角林、普拉克索)摸索最佳筛选和检测条件,建立稳定的多巴胺受体激动剂筛选模型。结果显示,本研究建立的多巴胺受体激动剂筛选模型能够检测到多梯度细胞接种密度下的药物活性,而且在多巴胺、甲磺酸溴隐亭、卡麦角林和普拉克索浓度很低(最低约0.1μmol·L^(-1))的情况下也能检测到阳性信号。经多次实验显示,本研究构建的模型最佳筛选条件:细胞接种数为7×10^(4)个,多巴胺受体激动剂阳性药的有效浓度为1~100μmol·L^(-1)均有较好的检测效果。此外,本研究先加入10μmol·L^(-1)多巴胺受体拮抗剂(包括盐酸氯普噻吨、多潘立酮、舒必利),然后再加入10μmol·L^(-1)多巴胺受体激动剂后,则无法检测到荧光阳性信号,这表明多巴胺受体拮抗剂阻断了多巴胺受体激动剂的活性,因而无法引起多巴胺受体变构,说明该模型具有较好的特异性,同时也能用于多巴胺受体拮抗剂的筛选和检测。总之,该研究构建了稳定的多巴胺传感器检测体系,可有效筛选潜在的多巴胺受体激动剂,而且操作简便,检测系统稳定,可实现快速、大规模的筛选模式,大大提高了药物筛选的效率,为以DRD2为靶点的药物开发和PD治疗提供了关键的方法体系。

Parkinson’s disease(PD)is a degenerative disease of the central nervous system due to the loss or death of dopaminergic neurons in the substantia nigra.Clinically,levodopa is the most effective and commonly used drug for PD treatment.However,long-term levodopa therapy is prone to motor complications and other side effects caused by excessive peripheral dopamine production,which has become an urgent problem to be solved in PD treatment.Dopamine receptor(DR)agonists are similar to dopamine.They can directly stimulate postsynaptic dopamine receptors,produce the same effect as dopamine,delay the application of levodopa as much as possible,and reduce complications caused by long-term use of levodopa.Therefore,screening effective dopamine receptor agonists has become a key issue in the study and treatment of PD.In order to establish a rapid,stable and reliable method for dopamine receptor agonist screening,this study used the human dopamine receptor 2(DRD2)gene fused with a circular permuted EGFP(cpEGFP)to construct a recombinant gene,packaged with lentiviral vector,and the vector replaced the parted inner transmembrane domain of the third intracellular loop(ICL3)of geneticallyencoded GPCR-activation based(GRAB)sensors.The fluorescence of GPCR-fused cpEGFP is regulated by conformational changes mediated by the interaction of dopamine receptor agonists with GPCRs without altering GPCR activity.The HEK293T cells were infected with viral vector,screened by puromycin to select highly expressed cells.Dopamine receptor agonists(including dopamine,bromocriptine mesylate,cabergoline,pramipexole)were used as positive drugs to explore the best screening and detection conditions,establishing a stable model to evaluate the dopamine receptor agonist.The results showed that the optimal filter for the dopamine receptor agonist in this study was the cell seeding count of 7×10^(4),and the effective concentration of the positive drug was 1-100μmol·L^(-1).In addition,pretreated with 10μmol·L^(-1)dopamine receptor antagonists(including chlorprothixol hydrochloride,domperidone,and sulpiride),the positive fluorescence signal of overexpressed DRD2-cpEGFP HEK293T cells could not be detected when exposed to 10μmol·L^(-1)dopamine receptor agonists,which proved that dopamine receptor antagonists could block the activity of dopamine receptor agonists,so they cannot activate dopamine receptor allosteric,indicating that the model has good specificity and can also be used for the screening and detection of new dopamine receptor antagonists.In summary,the study constructs a stable dopamine sensor detection system,which can effectively screen potential dopamine receptor agonists.The operation procedures are simple and rapid.And it can be used for a large-scale screening providing a fundamental methodology for drug development and PD treatment targeted on DRD2.