Comparison of Variability and Sensitivity between Nuclear Translocation and Luciferase Reporter Gene Assays

Abstract

High-content screening (HCS), a technology based on subcellular imaging by automated microscopy and sophisticated image analysis, has emerged as an important platform in small-molecule screening for early drug discovery. To validate a subcellular imaging assay for primary screening campaigns, an HCS assay was compared with a non-image-based readout in terms of variability and sensitivity. A study was performed monitoring the accumulation of the forkhead transcription factor of the O subfamily (FOXO3a) coupled with green fluorescent protein in the nucleus of human osteosarcoma (U-2 OS) cells. In addition, the transcription of a luciferase gene coupled with a FOXO3a-responsive promoter was monitored. This report demonstrates that both assay formats show good reproducibility in primary and concentration response screening despite differences in statistical assay quality. In primary screening, the correlation of compound activity between the 2 assays was low, in contrast to the good correlation of the IC 50 values of confirmed compounds. Furthermore, the high-content imaging assay showed a mean shift of 2.63-fold in IC 50 values compared with the reporter gene assay. No chemical scaffold was specifically found with 1 of the technologies only, however these results validate the HCS technology against established assays for screening of new molecular entities. (Journal of Biomolecular Screening 2009:59-65)

A High-Content Screen Identifies Inhibitors of Nuclear Export of Forkhead Transcription Factors

“…This result is consistent with previous work showing the poor correlation between FOXO1 localization and transactivation activity. 18 …”

“…18 Unterreiner et al showed a poor correlation between hits from image-based screening of FOXO3 activators and those from reporter-based screening. 18 Kau et al identified inhibitors of FOXO1 nuclear export, but whether these inhibitors affect the transcriptional activity of FOXO1 was not reported. 19 Our data are consistent with the interpretation that nuclear translocation is only one mechanism of FOXO1 regulation.…”

“…this result is consistent with previous work showing the poor correlation between fOxO1 localization and transactivation activity. 18 dIScuSSIon fOxO1 is an attractive and challenging target for smallmolecule screens because of its many roles in regulating cell signaling. this study describes the development of an imagebased high-content screen to identify small molecules that enrich fOxO1 in the nucleus.…”

“…Of the previous studies examining chemical regulators of fOxO1 or fOxO3 translocation, only one has compared localization of these proteins to their transcriptional activation. 18 Unterreiner et al 18 showed a poor correlation between hits from image-based screening of fOxO3 activators and those from reporter-based screening. kau et al 19 identified inhibitors of fOxO1 nuclear export, but whether these inhibitors affect the transcriptional activity of fOxO1 was not reported.…”

Image-Based High-Content Screening in Drug Discovery

“…Both assays were equally reproducible, but the HCS assay had a better statistical quality. The HCS assay was more sensitive than the RGA although it was not able to identify additional chemical scaffolds as hits (Unterreiner et al, 2009). However, this study represents only one HCS assay and one RGA, and the picture might change when comparing more assays.…”