1. An Adaptable High-Throughput Technology Enabling the Identification of Specific Transcription Modulators.
- Author
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Bergbrede T, Hoberg E, Larsson NG, Falkenberg M, and Gustafsson CM
- Subjects
- Adenosine Triphosphate antagonists & inhibitors, Adenosine Triphosphate biosynthesis, DNA-Binding Proteins antagonists & inhibitors, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, DNA-Directed RNA Polymerases antagonists & inhibitors, DNA-Directed RNA Polymerases genetics, DNA-Directed RNA Polymerases metabolism, HeLa Cells, Humans, Kinetics, Methyltransferases antagonists & inhibitors, Methyltransferases genetics, Methyltransferases metabolism, Mitochondria genetics, Mitochondria metabolism, Mitochondrial Proteins antagonists & inhibitors, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Recombinant Proteins genetics, Recombinant Proteins metabolism, Small Molecule Libraries chemistry, Transcription Factors antagonists & inhibitors, Transcription Factors genetics, Transcription Factors metabolism, Fluorescence Resonance Energy Transfer methods, High-Throughput Screening Assays, Mitochondria drug effects, Oxidative Phosphorylation drug effects, Small Molecule Libraries pharmacology, Transcription, Genetic drug effects
- Abstract
Mitochondria harbor the oxidative phosphorylation (OXPHOS) system, which under aerobic conditions produces the bulk of cellular adenosine triphosphate (ATP). The mitochondrial genome encodes key components of the OXPHOS system, and it is transcribed by the mitochondrial RNA polymerase, POLRMT. The levels of mitochondrial transcription correlate with the respiratory activity of the cell. Therefore, compounds that can increase or decrease mitochondrial gene transcription may be useful for fine-tuning metabolism and could be used to treat metabolic diseases or certain forms of cancer. We here report the establishment of a novel high-throughput assay technology that has allowed us to screen a library of 430,000 diverse compounds for effects on mitochondrial transcription in vitro. Following secondary screens facilitated by the same assay principle, we identified 55 compounds that efficiently and selectively inhibit mitochondrial transcription and that are active also in cell culture. Our method is easily adaptable to other RNA or DNA polymerases and varying spectroscopic detection technologies.
- Published
- 2017
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