DSE-FRET: A new anticancer drug screening assay for DNA binding proteins.
Bottom Line: 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.
Affiliation: 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.Show MeSH
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Mentions: A 10-point dose–response experiment with Evans Blue (EB) was also carried out. One hundred μM Evans Blue inhibits NF-κB binding to DNA by EMSA and has been suggested to bind non-covalently to the p50 DNA binding region by molecular modeling.(17) In DSE-FRET, 10 μM EB showed little effect on p50, but 30 μM EB inhibited p50 completely (Fig. 4). Evans Blue also inhibited p52 in a similar fashion. The IC50 values of EB for p50 and p52 inhibition were 12.9 and 12.8 μM, respectively. We also showed that our method can be used for evaluation of an uncompetitive inhibitor, (−)-DHMEQ, which binds covalently to a specific Cys residue of Rel family proteins to inhibit their DNA binding.(18,19) We detected an inhibitory effect of (−)-DHMEQ on p50 and p52 by DSE-FRET (Fig. 5). As shown previously,(19) (−)-DHMEQ was less potent against p52 (IC50 62.5 μM) compared to p50 (IC50 8.8 μM).
Affiliation: 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.