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: To illustrate the method, we attempted to detect the NF-κB (p50) interaction with DNA. Nuclear factor-κB plays a pivotal role in the coordinated transcription of multiple inflammatory genes and is a probable drug target.(14–16) Two probes, NF-D1 and NF-D2, were prepared to test quantitative detection of p50 binding to DNA. Their double-stranded regions are identical and include an NF-κB binding sequence, d(GGGACTTTCC). These probes interact with each other through their single-stranded tails and are then involved in a strand exchange reaction. Each strand of NF-D1 was labeled with 6FAM and DABCYL at the double-stranded terminus. NF-D1, various concentrations of recombinant p50, and NF-D2 were mixed in a half-area 96-well microplate and changes in fluorescence were measured. Time courses of these changes are shown in Figure 2. The fluorescence signal of NF-D1 increased rapidly within 30 min after addition of NF-D2 and was fivefold higher than that of NF-D1 alone at 60 min in the absence of p50. Fluorescence elevation was suppressed in a p50 concentration-dependent manner by half at 40 nM p50 and almost completely at 320 nM p50.
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.