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 β-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 β-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. (Journal of Biomolecular Screening 2007:133-139)

Microwave-Based Reaction Screening: Tandem Retro-Diels–Alder/Diels–Alder Cycloadditions ofo-Quinol Dimers

“…Both enantiomers of synthesized bicyclo[2.2.2]octenones and cis -decalins were subjected to screening at 20 μM for inhibition in three biological assays at the National Cancer Institute (NCI): AP-1 (activator protein-1, an oncogenic transcription factor), 33 TRAIL (Tumor necrosis factor-α-related apoptosis-inducing ligand) sensitization, 34 and HIF-2 (hypoxia inducible factor 2). 35 Compound (−)- 31 (Figure 5, inset) showed selective inhibition against AP-1 at 4 μM concentration with luciferase reporter assays in HEK293 cells.…”

Unraveling the Anticancer Effect of Curcumin and Resveratrol

“…In this assay, curcumin has shown inhibiting AP-1 in the dose-dependent manner with IC 50 values of 100 μM [51]. …”

Matching the power of high throughput screening to the chemical diversity of natural products

“…In the case of targeted assays, the HTS readout typically reflects an enzymatic activity such as luciferase for reporter gene assays or enzymatic conversion, accumulation, or depletion of target-specific substrates. Examples of reporter gene assays reflecting modulation of signaling pathways and applied to natural product screening include AP1, 122 HIF1, 123 HIF2α, 124 interferon regulatory factor 1, 125 PPARγ, 126 and heat shock response. 127 Reporter constructs have also been used to monitor stability of target proteins.…”

“…This can be assessed by running a cytotoxicity assay in parallel with the reporter assay. 122,124 Alternatively, cell toxicity in reporter assays can be assessed by inclusion of a second reporter as exemplified by a dual luciferase assay to simultaneously assess cytostatic and cytotoxic effects of samples (subsequently applied to natural product extracts) on NF1-null astrocytoma cells. 147 Cataloging libraries for cytotoxic activity has also been applied to identify generally toxic extracts or fractions.…”

“…101 Careful choice of detection systems can also alleviate non-specific effects. For example, the AP1 assay referenced above 122 employed a β lactamase reporter rather than the usual luciferase. The choice of a fluorescent ABCG2 substrate, pheophorbide a, with a wide Stokes shift reduced the probability of interference, but since pheophorbide a is a plant-derived chlorophyll derivative, there was significant interference from similar compounds in plant extracts.…”