title: A fluorescent-based high-throughput screening assay for small molecules that inhibit the interaction of MdmX with p53.
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.
authors.affiliation: RIKEN Systems and Structural Biology Center, Yokohama, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Tsuganezawa
authors.forename: Yukari
authors.initials: Y
authors.lastname: Nakagawa
authors.forename: Miki
authors.initials: M
authors.lastname: Kato
authors.forename: Shigenao
authors.initials: S
authors.lastname: Taruya
authors.forename: Fumio
authors.initials: F
authors.lastname: Takahashi
authors.forename: Morio
authors.initials: M
authors.lastname: Endoh
authors.forename: Rei
authors.initials: R
authors.lastname: Utata
authors.forename: Masumi
authors.initials: M
authors.lastname: Mori
authors.forename: Naoko
authors.initials: N
authors.lastname: Ogawa
authors.forename: Teruki
authors.initials: T
authors.lastname: Honma
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Hashizume
authors.forename: Masaaki
authors.initials: M
authors.lastname: Aoki
authors.forename: Takuma
authors.initials: T
authors.lastname: Kasai
authors.forename: Takanori
authors.initials: T
authors.lastname: Kigawa
authors.forename: Hirotatsu
authors.initials: H
authors.lastname: Kojima
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Okabe
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Nagano
authors.forename: Akiko
authors.initials: A
authors.lastname: Tanaka
mesh_terms: D001667:Binding, Competitive / Q000187:drug effects; D045744:Cell Line, Tumor; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D011485:Protein Binding / Q000187:drug effects; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D054852:Small Molecule Libraries; D013050:Spectrometry, Fluorescence; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D054852:Small Molecule Libraries; D016159:Tumor Suppressor Protein p53; D051736:Proto-Oncogene Proteins c-mdm2