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High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels
Published on: January 27, 2013
Systematic Identification of MCU Modulators by Orthogonal Interspecies Chemical Screening
Daniela M Arduino1, Jennifer Wettmarshausen1, Horia Vais2
1Gene Center/Department of Biochemistry, Ludwig-Maximilians Universität München, Munich 81377, Germany; Institute for Diabetes and Obesity, Helmholtz Zentrum München, Neuherberg 85764, Germany.
Abstract:
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.
Insights
Researchers developed a new assay to find drugs targeting the mitochondrial calcium uniporter complex (MCU). They identified mitoxantrone as a selective MCU inhibitor, offering a new tool for mitochondrial drug discovery.
Area of Science:
- Mitochondrial biology
- Pharmacology
- Biochemistry
Background:
- The mitochondrial calcium uniporter complex (MCU) is crucial for cellular calcium (Ca2+) regulation, impacting bioenergetics, cell death, and signaling.
- Dysfunction of the MCU is implicated in various human diseases, yet specific pharmacological modulators are lacking.
Purpose of the Study:
- To develop a high-throughput screening assay for identifying human MCU-specific small-molecule modulators.
- To discover novel inhibitors of MCU activity for therapeutic applications.
Main Methods:
- A novel high-throughput assay was established using isolated yeast mitochondria reconstituted with human MCU, EMRE, and aequorin.
- A bioenergetic shunt (D-lactate and mannitol/sucrose) was employed to minimize false-positive hits during screening.
- Over 600 clinically approved drugs were screened against the reconstituted MCU system.
Main Results:
- Mitoxantrone was identified as a direct and selective inhibitor of the human MCU from the screen of approved drugs.
- The identified inhibitor, mitoxantrone, was validated in orthogonal mammalian cell-based assays.
- The screening approach proved effective and robust for MCU-specific drug discovery.
Conclusions:
- The developed assay is a powerful tool for identifying MCU-specific modulators and compounds targeting mitochondrial functions.
- Mitoxantrone represents a promising lead compound for further investigation into MCU-targeted therapies.
- This work advances the potential for pharmacological intervention in diseases associated with MCU dysfunction.
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