Development of a homogeneous calcium mobilization assay for high throughput screening of mas-related gene receptor agonists
Abstract
Aim: To develop homogeneous calcium mobilization assay for high-throughput
screening (HTS) of mas-related gene (Mrg) receptor agonists. Methods: CHO-K1
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 receptor
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 MrgA1 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.
Keywords:
screening (HTS) of mas-related gene (Mrg) receptor agonists. Methods: CHO-K1
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 receptor
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 MrgA1 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.