The Development and Validation of the Luminescence Based Functional Assay for Calcium Sensing Receptor

The age-related imbalance between the increased bone resorption and decreased formation results in the bone loss and osteoporosis. Recently, as the functional role of the seven transmembrane spanning extracellular calcium sensing receptor (CaS) in bone cells has been discovered, the possibility that the bone CaS could potentially be targeted by antagonists to control the bone remodeling has been raised. Though the therapeutic potential of CaS antagonists in bone cells is hampered by their effects on the CaS in nonskeletal tissues, seeking various modulators for CaS in vitro can provide diverse research tools to find the best possible therapeutics for the treatment of osteoporosis. Accordingly, the high-throughput screening of chemical libraries for the searching of CaS antagonists, which specifically interferes with the action of extracellular calcium onto the CaS, may be valuable in finding those possibilities for osteoporosis. To screen the small molecule compound library against CaS in an aequorin based functional assay system, the CaS was transiently expressed in the parental HEK 293 cell with mitochondrially targeted aequorin. With this luminescence assay system, the Z' value was achieved at 0.8016 and the signal to noise ratio of 7.47 for the pilot library screening campaign of the most diverse compound set consisting of 6,800 compounds. These results suggest that the current aequorin based CaS functional assay system is a reliable tool for the screening of CaS antagonists.