An Adaptable High-Throughput Technology Enabling the Identification of Specific Transcription Modulators.
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.
1 Lead Discovery Center GmbH, Dortmund, Germany.
Tim
T
Bergbrede
2 Institute of Biomedicine, University of Gothenburg, Goteborg, Sweden.
Emily
E
Hoberg
3 Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne, Germany.
Nils-Göran
NG
Larsson
2 Institute of Biomedicine, University of Gothenburg, Goteborg, Sweden.
Maria
M
Falkenberg
2 Institute of Biomedicine, University of Gothenburg, Goteborg, Sweden.
Claes M
CM
Gustafsson
D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010085:Oxidative Phosphorylation / Q000187:drug effects*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*
D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; C499631:TFAM protein, human; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D008780:Methyltransferases; C462443:TFB2M protein, human; D012321:DNA-Directed RNA Polymerases; C519567:POLRMT protein, human
Antiviral drugs; assays; binding or purification; cancer and cancer drugs; enzyme assays or enzyme kinetics; fluorescence methods; labeling; nucleic acid chemistry