title: Novel in vitro protein fragment complementation assay applicable to high-throughput screening in a 1536-well format.
abstract: Protein-protein interactions (PPIs) play key roles in all cellular processes and hence are useful as potential targets for new drug development. To facilitate the screening of PPI inhibitors as anticancer drugs, the authors have developed a high-throughput screening (HTS) system using an in vitro protein fragment complementation assay (PCA) with monomeric Kusabira-Green fluorescent protein (mKG). The in vitro PCA system was established by the topological formation of a functional complex between 2 split inactive mKG fragments fused to target proteins, which fluoresces when 2 target proteins interact to allow complementation of the mKG fragments. Using this assay system, the authors screened inhibitors for TCF7/beta-catenin, PAC1/PAC2, and PAC3 homodimer PPIs from 123,599 samples in their natural product library. Compound TB1 was identified as a specific inhibitor for PPI of PAC3 homodimer. TB1 strongly inhibited the PPI of PAC3 homodimer with an IC(50) value of 0.020 microM and did not inhibit PPI between TCF7/beta-catenin and PAC1/PAC2 even at a concentration of 250 microM. The authors thus demonstrated that this in vitro PCA system applicable to HTS in a 1536-well format is capable of screening for PPI inhibitors from a huge natural product library.
authors.affiliation: Biomedicinal Information Research Center (BIRC), Japan Biological Informatics Consortium (JBIC), Koto-ku, Tokyo, Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Hashimoto
authors.forename: Taku
authors.initials: T
authors.lastname: Watanabe
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Seki
authors.forename: Satoshi
authors.initials: S
authors.lastname: Karasawa
authors.forename: Miho
authors.initials: M
authors.lastname: Izumikawa
authors.forename: Tomoe
authors.initials: T
authors.lastname: Seki
authors.forename: Shun-Ichiro
authors.initials: S
authors.lastname: Iemura
authors.forename: Tohru
authors.initials: T
authors.lastname: Natsume
authors.forename: Nobuo
authors.initials: N
authors.lastname: Nomura
authors.forename: Naoki
authors.initials: N
authors.lastname: Goshima
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
authors.forename: Motoki
authors.initials: M
authors.lastname: Takagi
authors.forename: Kazuo
authors.initials: K
authors.lastname: Shin-Ya
mesh_terms: D000970:Antineoplastic Agents / Q000493:pharmacokinetics; D001667:Binding, Competitive; D002474:Cell-Free System; D004354:Drug Screening Assays, Antitumor / Q000295:instrumentation / Q000379:methods; D057166:High-Throughput Screening Assays / Q000295:instrumentation* / Q000379:methods*; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods; D008954:Models, Biological; D009842:Oligopeptides; D010446:Peptide Fragments / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D054730:Protein Interaction Domains and Motifs / Q000187:drug effects / Q000502:physiology; D025941:Protein Interaction Mapping / Q000379:methods*; D054852:Small Molecule Libraries / Q000032:analysis
chemical_list: D000970:Antineoplastic Agents; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D054852:Small Molecule Libraries; C085706:FLAG peptide