title: 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.affiliation: Laboratory of Cancer Prevention, Gene Regulation Section, National Cancer Institute-Frederick, Frederick, MD, USA.
authors.forename: Katie M
authors.initials: KM
authors.lastname: Ruocco
authors.forename: Ekaterina I
authors.initials: EI
authors.lastname: Goncharova
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Young
authors.forename: Nancy H
authors.initials: NH
authors.lastname: Colburn
authors.forename: James B
authors.initials: JB
authors.lastname: McMahon
authors.forename: Curtis J
authors.initials: CJ
authors.lastname: Henrich
mesh_terms: D000970:Antineoplastic Agents / Q000032:analysis* / Q000494:pharmacology*; D001681:Biological Assay / Q000379:methods*; D005188:False Negative Reactions; D005189:False Positive Reactions; D006801:Humans; D010936:Plant Extracts / Q000032:analysis / Q000494:pharmacology; D015203:Reproducibility of Results; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors*; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D010936:Plant Extracts; D018808:Transcription Factor AP-1; D001618:beta-Lactamases