Background. DNA polymerase gamma is the only DNA replicase identified in animal and fungal mitochondria able to replicate mitochondrial DNA (mtDNA). To date, more than 300 pathogenic mutations have been found in the human catalytic subunit, called POLG, and are associated to several human mitochondrial diseases characterized by multiple deletions and/or depletion of mtDNA. There are no therapies for POLG-related disorders, and no clinical trials have been performed. The yeast Saccharomyces cerevisiae, which possess an orthologous gene, called MIP1, has been proven to be an excellent model to study consequences of POLG mutations and, recently, to find compounds which could be potentially used for the treatment of POLG-related diseases.
Objectives. The main objective of this project is the identification and characterization of bioactive compounds with a potential therapeutic effect on POLG-related mitochondrial disorders using yeast models of MIP1 mutations. Through a screening of two FDA-approved chemical libraries we have already identified 7 drugs, called MRS1,8-13, able to rescue the oxidative growth phenotype of a thermosensitive mip1 mutant strain and of increasing the mtDNA stability, which will be further characterized in this project
Aims. The project has two aims. The first one is a deeper characterization of the MRS1,8-13 effects i) on the copy number of mtDNA; ii) on the Mip1 protein levels; iii) on mtDNA the replication fidelity. Moreover, it will be evaluated weather MRS molecules, tested in combination, have a synergistic effect. The second aim is the identification of new MRS molecules through a new high throughput screening of 1000 novel FDA-approved drugs, followed by the characterization of their effects as done for the previous drugs.
Methods. Different methodologies will be applied to analyse the mitochondrial metabolism in presence or absence of the MRS. The mtDNA maintenance will be evaluated through the measurement of the frequency of petite mutants and the replication fidelity through the measure of the EryR point mutability; the mtDNA levels will be quantified by qPCR, the respiratory activity by oxygraph measurement and the Mip1 stability by Western blot. The chemical library will be screened by a yeast-based assay for the ability of a drug to suppress the respiratory growth defect of a thermosensitive mip1 mutant.
Expected results. We expect to find some positive hits, which in turn, at the end of the project, will be tested in a vertebrate model, specifically in a polg1 mutant model of zebrafish, in collaboration with the Zebrafish Facility at the University of Padua. We expect also to publish a paper on an Open Access Journal in order to disseminate the results.