Watanabe Yuichi, Kawaguchi Kosuke, Saito Syuken, Okabe Takayoshi, Yonesu Kiyoaki, Egashira Shinichiro, Kameya Masafumi, Morita Masashi, Kashiwayama Yoshinori, Imanaka Tsuneo
Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
Drug Discovery Initiative, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Biochem Biophys Rep. 2016 May 7;6:260-265. doi: 10.1016/j.bbrep.2016.05.004. eCollection 2016 Jul.
The glycosome, a peroxisome-related organelle, is essential for the growth and survival of trypanosomatid protozoa. In glycosome biogenesis, Pex5p recognizes newly synthesized glycosomal matrix proteins via peroxisome-targeting signal type-1 (PTS-1) and transports them into glycosomes through an interaction with Pex14p, a component of the matrix protein import machinery on the glycosomal membrane. Knockdown of the or with RNAi has been shown to inhibit the growth of Thus, compounds that inhibit the interaction of Pex5p-Pex14p are expected to become lead compounds in the development of anti-trypanosomal drugs. Here, we report a homogenous time-resolved fluorescence (HTRF) assay for the screening of compounds that inhibit the Pex5p-Pex14p interaction. The binding of GST-Pex14p and Pex5p-His with or without additional compounds was evaluated by measuring the energy transfer of the HTRF pair, using a terbium-labeled anti GST antibody as the donor and an FITC-labeled anti His antibody as the acceptor. The assay was performed in a 384-well plate platform and exhibits a Z'-factor of 0.85-0.91, while the coefficiency of variation is 1.1-7.7%, suggesting it can be readily adapted to a high-throughput format for the automated screening of chemical libraries. We screened 20,800 compounds and found 11 compounds that inhibited energy transfer. Among them, in a pull-down assay one compound exhibited selective inhibition of Pex5p-Pex14p without any Pex5p-Pex14p interaction.
糖体是一种与过氧化物酶体相关的细胞器，对锥虫原生动物的生长和存活至关重要。在糖体生物发生过程中，Pex5p通过1型过氧化物酶体靶向信号（PTS-1）识别新合成的糖体基质蛋白，并通过与Pex14p相互作用将它们转运到糖体中，Pex14p是糖体膜上基质蛋白导入机制的一个组成部分。已证明用RNAi敲低 或 会抑制 的生长。因此，抑制Pex5p-Pex14p相互作用的化合物有望成为抗锥虫药物开发中的先导化合物。在此，我们报告了一种用于筛选抑制Pex5p-Pex14p相互作用的化合物的均相时间分辨荧光（HTRF）测定法。使用铽标记的抗GST抗体作为供体，异硫氰酸荧光素（FITC）标记的抗His抗体作为受体，通过测量HTRF对的能量转移来评估GST-Pex14p和Pex5p-His在有无其他化合物情况下的结合。该测定在384孔板平台上进行，Z'因子为0.85 - 0.91，变异系数为1.1 - 7.7%，表明它可轻松适用于高通量形式以自动筛选化学文库。我们筛选了20,800种化合物，发现11种化合物抑制能量转移。其中，在下拉测定中，一种化合物表现出对Pex5p-Pex14p的选择性抑制，且无任何Pex5p-Pex14p相互作用。
