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A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors
Published on: December 2, 2022
Identifying nonselective hits from a homogeneous calcium assay screen
Kelly J Cassutt1, Michael J Orsini, Mojgan Abousleiman
1Hamamatsu Corp, Bridgewater, NJ, USA.
Journal of Biomolecular Screening
|February 10, 2007
Summary
Screening a 50,000 compound library identified a single G-protein coupled receptor (GPCR) agonist. Orthogonal technologies like AequoScreen and IP-ONE HTRF effectively distinguished specific hits from nonspecific ones.
Area of Science:
- Biochemistry
- Pharmacology
- Cell Biology
Background:
- G-protein coupled receptors (GPCRs) are crucial drug targets.
- High-throughput screening (HTS) is vital for identifying GPCR modulators.
- Distinguishing specific agonists from false positives is a key challenge in HTS.
Purpose of the Study:
- To screen a large compound library for G-protein coupled receptor (GPCR) agonists.
- To evaluate the utility of orthogonal assay technologies in validating screening hits.
- To identify specific GPCR agonists with high confidence.
Main Methods:
- A homogeneous calcium dye assay was used to screen 50,000 compounds against a transfected cell line.
- Hits were validated using IP-ONE HTRF, AequoScreen technology, and multiplex assays (Fluo-3/Fura-2).
- Rescreening against the parental cell line confirmed hit specificity.
Main Results:
- Only one specific Gq-coupled GPCR agonist was identified from 365 primary hits.
- AequoScreen technology yielded no false positives, confirming the single true hit.
- Multiplex assays identified one false positive and the same true-positive hit.
- Rescreening confirmed the specificity of the single identified agonist.
Conclusions:
- AequoScreen technology, IP-ONE HTRF, and multiplex assays are effective orthogonal methods for hit validation.
- These technologies help in identifying specific GPCR agonists and eliminating nonspecific hits.
- The study highlights the importance of orthogonal validation in drug discovery screening.
