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

¹ Laboratory of Cancer Prevention, Gene Regulation Section, National Cancer Institute-Frederick, Frederick, MD.
² Molecular Targets Development Program, National Cancer Institute-Frederick, Frederick, MD.; Data Management Services, Inc., National Cancer Institute-Frederick, Frederick, MD.
³ Molecular Targets Development Program, National Cancer Institute-Frederick, Frederick, MD.
⁴ Molecular Targets Development Program, National Cancer Institute-Frederick, Frederick, MD.; Basic Research Program, SAIC-Frederick, Inc., National Cancer Institute- Frederick, Frederick, MD.

^* To whom correspondence should be addressed. E-mail: henrichc{at}ncifcrf.gov.

	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 -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 -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, -lactamase

First published on December 14, 2006, doi:10.1177/1087057106296686

Journal of Biomolecular Screening 2007;12:133.

A more recent version of this article appeared on February 1, 2007


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