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A High-Throughput Cell-Based Assay to Identify Specific Inhibitors of Transcription Factor AP-1

Laboratory of Cancer Prevention, Gene Regulation Section, National Cancer Institute–Frederick, Frederick, MD

Ekaterina I. Goncharova

Molecular Targets Development Program, National Cancer Institute–Frederick, Frederick, MD; Data Management Services, Inc., National Cancer Institute–Frederick, Frederick, MD

Matthew R. Young

Nancy H. Colburn

Laboratory of Cancer Prevention, Gene Regulation Section, National Cancer Institute–Frederick, Frederick, MD

James B. McMahon

Molecular Targets Development Program, National Cancer Institute–Frederick, Frederick, MD

Curtis J. Henrich

Molecular Targets Development Program, National Cancer Institute–Frederick, Frederick, MD; Basic Research Program, SAIC-Frederick, Inc., National Cancer Institute- Frederick, Frederick, MD

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 ß-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 ß-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.

Key Words: AP-1 • high-throughput assay • natural products • ß-lactamase

This version was published on February 1, 2007

Journal of Biomolecular Screening, Vol. 12, No. 1, 133-139 (2007)
DOI: 10.1177/1087057106296686


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