Development of a homogeneous calcium mobilization assay for high throughput screening of mas-related gene receptor agonists

• Published in: Acta pharmacologica Sinica Vol. 28; no. 1; pp. 125 - 131
• Main Authors: Zhang, Rui, Yan, Pang-Ke, Zhou, Cai-Hong, Liao, Jia-Yu, Wang, Ming-Wei
• Format: Journal Article
• Language: English
• Published: United States Nature Publishing Group 2007
• Subjects: Animals; beta-Alanine - pharmacology; Calcium - metabolism; Cell Line; CHO Cells; Cricetinae; Cricetulus; Cyclic AMP - metabolism; Drug Evaluation, Preclinical - methods; Dynorphins - pharmacology; Humans; Plasmids - genetics; Receptors, G-Protein-Coupled - agonists; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Transfection; 基因受体; 收缩筋; 生物活性; 营养缺乏
• ISSN: 1671-4083; 1745-7254
• DOI: 10.1111/j.1745-7254.2007.00451.x

Aim： To develop homogeneous calcium mobilization assay for high-throughput screening （HTS） of mas-related gene （Mrg） receptor agonists. Methods： CHO-K 1 cells stably expressing the full-length MrgD receptor and a calcium-sensitive dye were used to develop an HTS assay based on intracellular calcium influx. This method was applied to large-scale screening of a library containing 8000 synthetic compounds and natural product extracts, cAMP measurements were carried out to verify the bioactivities of the hits found by the calcium mobilization assay. Similar approaches were also employed in the identification of the MrgA1 recep- tor agonists following HTS of 16 000 samples. Results： EC50 values of the positive control compounds （β-alanine for MrgD receptor and dynorphin A for MrgA1 receptor） determined by the calcium mobilization assay were consistent with those reported in the literature, and the Z＇ factors were 0.65 and 0.50 for MrgD and MrgA1 receptor assay, respectively. About 31 compounds for the MrgD receptor and 48 compounds for the MrgA 1 receptor showing 〉20% of the maximal agonist activities found in the controls were initially identified as hits. Secondary screening confirmed that 2 compounds for each receptor possessed specific agonist activities. Intracellular cAMP level measurements indicated that the 2 confirmed hits displayed the functionality of the MrgD receptor agonists. Conclusion： A series of validation studies demonstrated that the homogeneous calcium mobilization assay developed was highly efficient, amenable to automation and a robust tool to screen potential MrgD and MrgA1 receptor agonists. Its application may be expanded to other G-protein coupled receptors that mobilize calcium influx upon activation.