A high-throughput cell-based assay to identify specific inhibitors of transcription factor AP-1.

Abstract

The oncogenic transcription factor AP-1 (activator protein-1) is required for tumor promotion and progression. Identification of novel and specific AP-1 inhibitors would be beneficial for cancer prevention and therapy. The authors have developed a high-throughput assay to screen synthetic and natural product libraries for noncytotoxic inhibitors of mitogen-activated AP-1 activity. The cell-based high-throughput screen is conducted in a 384-well format using a fluorescent resonance energy transfer (FRET) substrate to quantify the activity of a beta-lactamase reporter under the control of an AP-1-dependent promoter. The ratiometric FRET readout makes this assay extremely robust and reproducible, particularly for use with natural product extracts. To eliminate false positives due to cell killing, a cytotoxicity assay was incorporated. The AP-1 beta-lactamase reporter was validated with inhibitors of kinases located upstream of AP-1 and with known natural product inhibitors of AP-1 (nordihydroguaiaretic acid and curcumin). The assay was able to identify other known AP-1 inhibitors and protein kinase C modulators, as well as a number of chemically diverse compounds with unknown mechanisms of action from natural products libraries. Application to natural product extracts identified hits from a range of taxonomic groups. Screening of synthetic compounds and natural products should identify novel AP-1 inhibitors that may be useful in the prevention and treatment of cancers.

• Authors: Katie M Ruocco, Ekaterina I Goncharova, Matthew R Young, Nancy H Colburn, James B McMahon, Curtis J Henrich
• Journal: Journal of biomolecular screening (J Biomol Screen) Vol. 12 Issue 1 Pg. 133-9 (Feb 2007) ISSN: 1087-0571 [Print] United States
• PMID: 17175522 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, N.I.H., Intramural)

Chemical References

• Antineoplastic Agents
• Plant Extracts
• Transcription Factor AP-1
• beta-Lactamases

Topics

• Antineoplastic Agents (analysis, pharmacology)
• Biological Assay (methods)
• False Negative Reactions
• False Positive Reactions
• Humans
• Plant Extracts (analysis, pharmacology)
• Reproducibility of Results
• Transcription Factor AP-1 (antagonists & inhibitors)
• beta-Lactamases (metabolism)