DSE ‐ FRET : A new anticancer drug screening assay for DNA binding proteins

Nuclear factor‐κB ( NF ‐κB) is a key regulator of cancer progression and the inflammatory effects of disease. To identify inhibitors of DNA binding to NF ‐κB, we developed a new homogeneous method for detection of sequence‐specific DNA ‐binding proteins. This method, which we refer to as DSE ‐ FRET , is based on two phenomena: protein‐dependent blocking of spontaneous DNA strand exchange ( DSE ) between partially double‐stranded DNA probes, and fluorescence resonance energy transfer ( FRET ). If a probe labeled with a fluorophore and quencher is mixed with a non‐labeled probe in the absence of a target protein, strand exchange occurs between the probes and results in fluorescence elevation. In contrast, blocking of strand exchange by a target protein results in lower fluorescence intensity. Recombinant human NF ‐κB (p50) suppressed the fluorescence elevation of a specific probe in a concentration‐dependent manner, but had no effect on a non‐specific probe. Competitors bearing a NF ‐κB binding site restored fluorescence, and the degree of restoration was inversely correlated with the number of nucleotide substitutions within the NF ‐κB binding site of the competitor. Evaluation of two NF ‐κB inhibitors, Evans Blue and dehydroxymethylepoxyquinomicin ([−]‐ DHMEQ ), was carried out using p50 and p52 (another form of NF ‐κB), and IC 50 values were obtained. The DSE ‐ FRET technique also detected the differential effect of (−)‐ DHMEQ on p50 and p52 inhibition. These data indicate that DSE ‐ FRET can be used for high throughput screening of anticancer drugs targeted to DNA ‐binding proteins.