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Journal of Biomolecular Screening
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12/2/285    most recent
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Identifying Nonselective Hits from a Homogeneous Calcium Assay Screen

Kelly J. Cassutt

Hamamatsu Corp., Bridgewater, NJ

Michael J. Orsini

Mojgan Abousleiman

Dean Colone

Weimin Tang

Pharmaceutical Research and Development, Johnson and Johnson, Raritan, NJ

The authors used a homogeneous calcium dye kit with a cell line transfected using a recombinant protein construct to screen a 50,000 compound library for G-protein coupled receptor (GPCR) agonists. Only 1 of the 365 primary hits activated Gq-coupled GPCRs, as shown using IP-ONE HTRF®. Furthermore, an agonist screen against the entire compound library and same heterologous cell line using AequoScreenTM technology generated no false positives and identified the same positive hit. Next, a multiplex assay composed of both Fluo-3 and Fura-2–loaded cells identified 1 false positive and the same true-positive hit out of the 365 primary hits. Finally, rescreening the 365 primary hits against the parental cell line loaded using the homogeneous calcium dye kit confirmed the specificity of the same true-positive hit only. In summary, the results suggest that AequoScreenTM technology, IP-ONE HTRF®, and multiplex assays are unique, orthogonal technologies to identify nonspecific hits.

Key Words: G-protein-coupled receptor • homogeneous calcium assay • aequorin • functional drug screening system • Fura-2 • Fluo-3 • IP-ONE HTRF®

This version was published on March 1, 2007

Journal of Biomolecular Screening, Vol. 12, No. 2, 285-287 (2007)
DOI: 10.1177/1087057106298538
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