Identifying Nonselective Hits from a Homogeneous Calcium Assay Screen

Abstract

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.

Comparison on Functional Assays for Gq-Coupled GPCRs by Measuring Inositol Monophospate-1 and Intracellular Calcium in 1536-Well Plate Format

“…The original IP-One assay was developed in 96/384-well plate formats and requires a medium change for adherent cells or use of freshly detached suspension cells [14,18]. The additional cell wash step is obviously not suitable for HTS especially in 1536-well plate format.…”

“…Medium removing or cell wash was suggested previously for the IP-One assay in order to reduce to potential interference from serum and phosphates present in the cell culture medium, reducing the liquid volume in wells, and maintaining the final lithium concentration [14,18]. We performed an assay with a cell wash step (cell culture medium was changed to buffer) to compare with that without a cell wash using adherent cells cultured overnight in 1536-well plates.…”

Monitoring Gq-coupled receptor response through inositol phosphate quantification with the IP-One assay

“…The assay was successfully miniaturized to 384-and 1536-well plates, using adherent cells [56,[63][64]. The reported Z¢ values ranged from 0.5 to 0.8, indicating that the assay was suitable for screening campaigns.…”

“…This gives high assay robustness and consequently minimizes the rate of falsepositive compounds. As an example, the IP-One assay was able to identify false-positive compounds previously identified as agonists in a screening campaign based on a 'no wash' calcium assay [64]. These assays do not include the washing step usually performed after the loading of calcium-sensitive dyes, and tend to be more sensitive than conventional calcium assays to compound auto-fluorescence and to quenching by highly colored compounds.…”

“…This assay is therefore now used at all stages of the screening process. In primary screening, its use has enabled new hits to be identified [56], while in secondary screening the IPone assay has been implemented to confirm the activity of hits revealed during screening campaigns [64].…”