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Novel In Vitro Protein Fragment Complementation Assay Applicable to High-Throughput Screening in a 1536-Well Format

Biomedicinal Information Research Center (BIRC), Japan Biological Informatics Consortium (JBIC), Tokyo, Japan

Taku Watanabe

Biomedicinal Information Research Center (BIRC), Japan Biological Informatics Consortium (JBIC), Tokyo, Japan, Amalgaam Co., Ltd., Tokyo, Japan

Tatsuya Seki

Biomedicinal Information Research Center (BIRC), Japan Biological Informatics Consortium (JBIC), Tokyo, Japan, Amalgaam Co., Ltd., Tokyo, Japan

Satoshi Karasawa

Biomedicinal Information Research Center (BIRC), Japan Biological Informatics Consortium (JBIC), Tokyo, Japan, Amalgaam Co., Ltd., Tokyo, Japan, Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, Saitama, Japan

Miho Izumikawa

Biomedicinal Information Research Center (BIRC), Japan Biological Informatics Consortium (JBIC), Tokyo, Japan

Tomoe Seki

Biomedicinal Information Research Center (BIRC), Japan Biological Informatics Consortium (JBIC), Tokyo, Japan

Shun-Ichiro Iemura

Biomedicinal Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan

Tohru Natsume

Biomedicinal Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan

Nobuo Nomura

Biomedicinal Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan

Naoki Goshima

Biomedicinal Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan

Atsushi Miyawaki

Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, Saitama, Japan

Motoki Takagi

Biomedicinal Information Research Center (BIRC), Japan Biological Informatics Consortium (JBIC), Tokyo, Japan, motoki-takagi{at}aist.go.jp

Kazuo Shin-Ya

Biomedicinal Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan

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/β-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 µM and did not inhibit PPI between TCF7/β-catenin and PAC1/PAC2 even at a concentration of 250 µM. 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. ( Journal of Biomolecular Screening 2009:970-979)

Key Words: protein fragment complementation assay • bimolecular fluorescence complementation • monomeric Kusabira-Green protein • protein-protein interaction • high-throughput screening

This version was published on September 1, 2009

Journal of Biomolecular Screening, Vol. 14, No. 8, 970-979 (2009)
DOI: 10.1177/1087057109341406


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