RESUMO
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 IC50 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.
