Identifying nonselective hits from a homogeneous calcium assay screen.
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 AequoScreen 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 AequoScreen technology, IP-ONE HTRF, and multiplex assays are unique, orthogonal technologies to identify nonspecific hits.
Cassutt
Kelly J
KJ
Hamamatsu Corp, Bridgewater, NJ, USA.
Orsini
Michael J
MJ
Abousleiman
Mojgan
M
Colone
Dean
D
Tang
Weimin
W
D000331:Aequorin / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D002118:Calcium / Q000032:analysis*; D002460:Cell Line; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes; D016257:Fura-2; D049408:Luminescent Agents / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D013053:Spectrophotometry; D014162:Transfection
D005456:Fluorescent Dyes; D049408:Luminescent Agents; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D000331:Aequorin; D002118:Calcium; D016257:Fura-2