Arduino Daniela M, Wettmarshausen Jennifer, Vais Horia, Navas-Navarro Paloma, Cheng Yiming, Leimpek Anja, Ma Zhongming, Delrio-Lorenzo Alba, Giordano Andrea, Garcia-Perez Cecilia, Médard Guillaume, Kuster Bernhard, García-Sancho Javier, Mokranjac Dejana, Foskett J Kevin, Alonso M Teresa, Perocchi Fabiana
Gene Center/Department of Biochemistry, Ludwig-Maximilians Universität München, Munich 81377, Germany; Institute for Diabetes and Obesity, Helmholtz Zentrum München, Neuherberg 85764, Germany.
Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Mol Cell. 2017 Aug 17;67(4):711-723.e7. doi: 10.1016/j.molcel.2017.07.019.
The mitochondrial calcium uniporter complex is essential for calcium (Ca) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca signal transduction. Despite its involvement in several human diseases, we currently lack pharmacological agents for targeting uniporter activity. Here we introduce a high-throughput assay that selects for human MCU-specific small-molecule modulators in primary drug screens. Using isolated yeast mitochondria, reconstituted with human MCU, its essential regulator EMRE, and aequorin, and exploiting a D-lactate- and mannitol/sucrose-based bioenergetic shunt that greatly minimizes false-positive hits, we identify mitoxantrone out of more than 600 clinically approved drugs as a direct selective inhibitor of human MCU. We validate mitoxantrone in orthogonal mammalian cell-based assays, demonstrating that our screening approach is an effective and robust tool for MCU-specific drug discovery and, more generally, for the identification of compounds that target mitochondrial functions.
线粒体钙单向转运体复合物对于所有哺乳动物组织线粒体摄取钙（Ca）至关重要，它在线粒体中调节生物能量学、细胞死亡和钙信号转导。尽管其与多种人类疾病有关，但目前我们缺乏针对单向转运体活性的药物。在此，我们介绍一种高通量检测方法，可在初级药物筛选中筛选出针对人类MCU的小分子调节剂。利用分离的酵母线粒体，将其与人MCU、其必需调节因子EMRE和水母发光蛋白重组，并利用基于D-乳酸和甘露醇/蔗糖的生物能量分流来极大地减少假阳性结果，我们在600多种临床批准药物中鉴定出米托蒽醌是人类MCU的直接选择性抑制剂。我们在基于正交哺乳动物细胞的检测中验证了米托蒽醌，表明我们的筛选方法是用于MCU特异性药物发现的有效且强大的工具，更广泛地说，是用于鉴定靶向线粒体功能的化合物的有效工具。
