Systematic Identification of MCU Modulators by Orthogonal Interspecies Chemical Screening

Abstract

SUMMARY The mitochondrial calcium uniporter complex is essential for calcium (Ca2+) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca2+ 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.

Progress in understanding mitochondrial calcium uniporter complex‐mediated calcium signalling: A potential target for cancer treatment

“…In HEK293 cells, 10 μM mitoxantrone significantly inhibits (~85%) MCU-mediated Ca 2+ currents via its charged side arms binding to acidic residues in the selectivity filter of MCU (Arduino et al, 2017). Mitoxantrone has a concentration-dependent inhibitory effect on MCU.…”

“…Certain MCU-dependent pro-oncogenic events are thought to be more sensitive to mitoxantrone, such as the increased metastatic abilities of MCF-7 cells and HCC cells, as well as the MCU-dependent chemotherapy resistance in multiple myeloma cells (Arduino et al, 2017;Ren et al, 2017;Song et al, 2013;Yu et al, 2017). Certain MCU-dependent pro-oncogenic events are thought to be more sensitive to mitoxantrone, such as the increased metastatic abilities of MCF-7 cells and HCC cells, as well as the MCU-dependent chemotherapy resistance in multiple myeloma cells (Arduino et al, 2017;Ren et al, 2017;Song et al, 2013;Yu et al, 2017).…”

Pharmacological modulation of mitochondrial calcium homeostasis

“…Finally, we address recent progresses on pharmacological targeting of mitochondrial Ca 2+ homeostasis and present a newly established high‐throughput chemical screening approach for the systematic identification of modulators of the mitochondrial Ca 2+ uniporter (Arduino et al . ).…”

“…These insights have been recently exploited for in silico modelling of MCU and binding of mitoxantrone, a novel inhibitor of the uniporter channel (Arduino et al . ).…”

“…) and D261A MCU (Arduino et al . ) mutants conferred reduced RuR sensitivity, highlighting a possible role in RuR‐dependent MCU inhibition. Second, expression of pure MCU in planar lipid bilayers was shown to be sufficient to reconstitute ion channel activity, generating a single channel Ca 2+ current consistent with that of the uniporter (De Stefani et al .…”