RRC ID 44055
Author Tsuganezawa K, Nakagawa Y, Kato M, Taruya S, Takahashi F, Endoh M, Utata R, Mori M, Ogawa N, Honma T, Yokoyama S, Hashizume Y, Aoki M, Kasai T, Kigawa T, Kojima H, Okabe T, Nagano T, Tanaka A.
Title A fluorescent-based high-throughput screening assay for small molecules that inhibit the interaction of MdmX with p53.
Journal J Biomol Screen
Abstract A fluorescent-based high-throughput screening (HTS) assay for small molecules that inhibit the interaction of MdmX with p53 was developed and applied to identify new inhibitors. The assay evaluated the MdmX-p53 interaction by detecting the quenching of the fluorescence of green fluorescent protein (GFP) fused to the MdmX protein, after its interaction with a p53 peptide labeled with a fluorescence quencher. In this report, the developed HTS assay was applied to about 40 000 compounds, and 255 hit compounds that abrogated the GFP quenching were selected. Next, the obtained hits were reevaluated by other assays. First, their effects on the diffusion time of a fluorescently-labeled p53 peptide after incubation with the MdmX protein were tested by measuring the diffusion time using fluorescence correlation spectroscopy, and six stable hit compounds with IC(50) values less than 5 µM were selected. Next, we further confirmed their inhibition of the MdmX-p53 interaction by surface plasmon resonance. To indicate the efficacy of the hit compound as a candidate anticancer drug, we showed that the hit compound triggered apoptosis after p53 and p21 accumulation in cultured MV4;11 leukemia cells. Thus, the new HTS assay is effective for obtaining novel MdmX-p53 interaction inhibitors that are valuable as candidate compounds for cancer treatment.
Volume 18(2)
Pages 191-8
Published 2013-2-1
DOI 10.1177/1087057112460729
PII 1087057112460729
PMID 22989451
MeSH Binding, Competitive / drug effects Cell Line, Tumor Drug Screening Assays, Antitumor / methods High-Throughput Screening Assays / methods* Humans Protein Binding / drug effects Proto-Oncogene Proteins c-mdm2 / metabolism* Small Molecule Libraries* Spectrometry, Fluorescence* Tumor Suppressor Protein p53 / metabolism*
IF 2.297
Times Cited 14
WOS Category BIOTECHNOLOGY & APPLIED MICROBIOLOGY CHEMISTRY, ANALYTICAL BIOCHEMICAL RESEARCH METHODS
Resource
Human and Animal Cells Wi-38