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ESR 8 Project - Molecular insights into inhibition mechanisms of the mitochondrial transcription

Work Package 6: Molecular mechanisms of mitochondrial transcription and RNA processing.

Partner: Claes Gustafsson

Institution: University of Gothenburg, Sweden

Duration: 36 months

Objectives: There are a number of different drugs that induce cancer cell death by targeting components in mitochondria necessary for inducing apoptosis. One example is the arsenite compounds that are used to treat hematological malignancies. Mitochondrial gene expression has also been directly targeted in cancer therapy. Tigecycline selectively kills leukemia stem and progenitor cells, and functions via mitochondrial translation inhibition. In fact, cancer cells may be extra sensitive to inhibitors of mitochondrial expression, since these cells are rapidly dividing, utilize energy inefficiently, and are often found in a hypoxic environment. Indeed, a recent report demonstrated that tumor cells in metabolically compromised microenvironments have a limited ability to respond to decreased mitochondrial function, a characteristic which may be used to target quiescent populations of tumor cells (Nat Commun. 2014 Feb 18;5:3295). We have collaborated with the Lead Discovery Center (LDC) in Dortumnd, Germany and performed a screen to identify small molecular substances that selectively inhibit mitochondrial transcription and thereby decrease mitochondrial function. After a screen of 172,000 substances, we have now identified a family of chemical compounds that act as selective inhibitors of mitochondrial transcription, with an IC50 of down to 10 nM. In the present project, we will identify the mechanism of action of these recently described chemical compounds and explain in molecular detail how they can act as selective inhibitors of mitochondrial transcription. To this end we will use reconstituted in vitro systems and standard biophysical techniques (e.g. surface plasmon resonance and microscale thermophoresis) to identify how our drugs target the RNA polymerase and how these interactions translate into functional consequence on a molecular level. We will also collaborate with the Hällberg laboratory at the Karolinska Institutet, to solve the structures of mitochondrial RNA polymerase - inhibitor co-complexes by X-ray crystallography. Finally, mitochondrial transcription and DNA replication are linked events. We will address how inhibition of mitochondrial transcription affects the stability of mitochondrial DNA in cell culture.

Expected results: The proposed set of experiments will help us to solve the mechanism of action of the family of small molecules that we recently identified. The experiments will also provide a deeper understanding of the mitochondrial RNA polymerase and ist molecular mechanisms of action. As a result, the identified compounds may later be evaluated as potent inhibitors for mitochondrial transcription, hence mitochondrial function, which may be used to target quiescent populations of tumor cells.