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 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.
Department of Cellular and Molecular Biology, Graduate School of Biomedical & Health Science, Hiroshima University, Hiroshima, Japan; Japanese Red Cross Kanto-koshinetsu Block Blood Center, Tokyo, Japan.
Toru
T
Miyagi
Bunsyo
B
Shiotani
Ryuya
R
Miyoshi
Takuya
T
Yamamoto
Takanori
T
Oka
Kazuo
K
Umezawa
Takahiro
T
Ochiya
Mikihisa
M
Takano
Hidetoshi
H
Tahara
D001549:Benzamides / Q000494:pharmacology; D001665:Binding Sites; D003512:Cyclohexanones / Q000494:pharmacology; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D005070:Evans Blue / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D020128:Inhibitory Concentration 50; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D001549:Benzamides; D003512:Cyclohexanones; D015342:DNA Probes; D004268:DNA-Binding Proteins; D016328:NF-kappa B; D011994:Recombinant Proteins; C464444:dehydroxymethylepoxyquinomicin; D005070:Evans Blue
Antineoplastic agents; DNA-binding proteins; NF-κB; dehydroxymethylepoxyquinomicin; fluorescence resonance energy transfer