Abstract
Nuclear translocation is an important step in glucocorticoid receptor (GR) signaling and assays that measure this process allow the identification of nuclear receptor ligands independent of subsequent functional effects. To facilitate the identification of GR-translocation agonists, an enzyme fragment complementation (EFC) cell-based assay was scaled to a 1536-well plate format to evaluate 9,920 compounds using a quantitative high throughput screening (qHTS) strategy where compounds are assayed at multiple concentrations. In contrast to conventional assays of nuclear translocation the qHTS assay described here was enabled on a standard luminescence microplate reader precluding the requirement for imaging methods. The assay uses β-galactosidase α complementation to indirectly detect GR-translocation in CHO-K1 cells. 1536-well assay miniaturization included the elimination of a media aspiration step, and the optimized assay displayed a Z of 0.55. qHTS yielded EC50 values for all 9,920 compounds and allowed us to retrospectively examine the dataset as a single concentration-based screen to estimate the number of false positives and negatives at typical activity thresholds. For example, at a 9 μM screening concentration, the assay showed an accuracy that is comparable to typical cell-based assays as judged by the occurrence of false positives that we determined to be 1.3% or 0.3%, for a 3σ or 6σ threshold, respectively. This corresponds to a confirmation rate of ∼30% or ∼50%, respectively. The assay was consistent with glucocorticoid pharmacology as scaffolds with close similarity to dexamethasone were identified as active, while, for example, steroids that act as ligands to other nuclear receptors such as the estrogen receptor were found to be inactive.
Keywords: Qhts, HTS, EFC, PubChem, glucocorticoid receptor, nuclear translocation, suspension cells
Combinatorial Chemistry & High Throughput Screening
Title: A Miniaturized Glucocorticoid Receptor Translocation Assay Using Enzymatic Fragment Complementation Evaluated with qHTS
Volume: 11 Issue: 7
Author(s): Ping Jun Zhu, Wei Zheng, Douglas S. Auld, Ajit Jadhav, Ryan MacArthur, Keith R. Olson, Kun Peng, Hyna Dotimas, Christopher P. Austin and James Inglese
Affiliation:
Keywords: Qhts, HTS, EFC, PubChem, glucocorticoid receptor, nuclear translocation, suspension cells
Abstract: Nuclear translocation is an important step in glucocorticoid receptor (GR) signaling and assays that measure this process allow the identification of nuclear receptor ligands independent of subsequent functional effects. To facilitate the identification of GR-translocation agonists, an enzyme fragment complementation (EFC) cell-based assay was scaled to a 1536-well plate format to evaluate 9,920 compounds using a quantitative high throughput screening (qHTS) strategy where compounds are assayed at multiple concentrations. In contrast to conventional assays of nuclear translocation the qHTS assay described here was enabled on a standard luminescence microplate reader precluding the requirement for imaging methods. The assay uses β-galactosidase α complementation to indirectly detect GR-translocation in CHO-K1 cells. 1536-well assay miniaturization included the elimination of a media aspiration step, and the optimized assay displayed a Z of 0.55. qHTS yielded EC50 values for all 9,920 compounds and allowed us to retrospectively examine the dataset as a single concentration-based screen to estimate the number of false positives and negatives at typical activity thresholds. For example, at a 9 μM screening concentration, the assay showed an accuracy that is comparable to typical cell-based assays as judged by the occurrence of false positives that we determined to be 1.3% or 0.3%, for a 3σ or 6σ threshold, respectively. This corresponds to a confirmation rate of ∼30% or ∼50%, respectively. The assay was consistent with glucocorticoid pharmacology as scaffolds with close similarity to dexamethasone were identified as active, while, for example, steroids that act as ligands to other nuclear receptors such as the estrogen receptor were found to be inactive.
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Cite this article as:
Zhu Jun Ping, Zheng Wei, Auld S. Douglas, Jadhav Ajit, MacArthur Ryan, Olson R. Keith, Peng Kun, Dotimas Hyna, Austin P. Christopher and Inglese James, A Miniaturized Glucocorticoid Receptor Translocation Assay Using Enzymatic Fragment Complementation Evaluated with qHTS, Combinatorial Chemistry & High Throughput Screening 2008; 11 (7) . https://dx.doi.org/10.2174/138620708785204045
DOI https://dx.doi.org/10.2174/138620708785204045 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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