Zhu Ting, Fang Li-yan, Xie Xin
The National Center for Drug Screening, Chinese Academy of Sciences, Shanghai 201203, China.
Acta Pharmacol Sin. 2008 Apr;29(4):507-16. doi: 10.1111/j.1745-7254.2008.00775.x.
To develop a universal high-throughput screening assay based on Galpha15/16- mediated calcium mobilization for the identification of novel modulators of Gprotein- coupled receptors (GPCR).
In the present study, CHO-K1 or HEK293 cells were co-transfected with plasmids encoding promiscuous G-protein Galpha15/16 and various receptors originally coupled to Galphas, Galphai, or Galphaq pathways. Intracellular calcium change was monitored with fluorescent dye Fluo-4.
We found out for all the receptors tested, Galpha15/16 could shift the receptorso coupling to the calcium mobilization pathway, and the EC50 values of the ligands generated with this method were comparable with reported values that were obtained using traditional methods. This assay was validated and optimized with the zeta-opioid receptor, which originally coupled to Galphai and was recently found to play important roles in neurodegenerative and autoimmune diseases. A largescale screening of 48 000 compounds was performed based on this system. Several new modulators were identified and confirmed with the traditional GTPgammaS binding assay.
This cell-based calcium assay was proved to be robust and easy to automate, and could be used as a universal method in searching for GPCR modulators.
开发一种基于Gα15/16介导的钙动员的通用高通量筛选测定法，用于鉴定G蛋白偶联受体（GPCR）的新型调节剂。
在本研究中，将编码通用G蛋白Gα15/16和最初与Gαs、Gαi或Gαq途径偶联的各种受体的质粒共转染到CHO-K1或HEK293细胞中。用荧光染料Fluo-4监测细胞内钙变化。
我们发现，对于所有测试的受体，Gα15/16可将受体偶联转移至钙动员途径，且用该方法产生的配体的半数有效浓度（EC50）值与使用传统方法获得的报道值相当。用ζ-阿片受体对该测定法进行了验证和优化，ζ-阿片受体最初与Gαi偶联，最近发现其在神经退行性疾病和自身免疫性疾病中起重要作用。基于该系统对48000种化合物进行了大规模筛选。通过传统的GTPγS结合测定法鉴定并确认了几种新的调节剂。
这种基于细胞的钙测定法被证明是可靠且易于自动化的，可作为寻找GPCR调节剂的通用方法。
