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Identification of the molecular target of clofilium tosylate, a drug rescuing mitochondrial defects due to pathological mutations in the mitochondrial DNA polymerase

Progetti locali
Programma di ricerca
FIL2016, quota incentivante
Ente finanziatore
UniversitÓ di Parma
Settore ERC
LS2_6 - Molecular genetics, reverse genetics and RNAi
15/04/2017 - 14/04/2018
Prof. Enrico Baruffini

Aree / Gruppi di ricerca

Partecipanti al progetto

Descrizione del progetto

In humans, the nuclear gene POLG(1) encodes for the catalytic subunit of DNA polymerase gamma, or Pol gamma, the only replicase present in mitochondria. Currently, more than 180 pathological mutations have been identified in POLG, associated to several mitochondrial diseases with infantile, juvenile or adult onset, and characterized by multiple deletions and/or depletion of mitochondrial DNA (mtDNA) in affected tissues. To date, no therapies are available for mitochondrial pathologies, including those caused by mutations in POLG.

Pol gamma is present in all the animals and fungi. In the yeast Saccharomyces cerevisiae, the mtDNA polymerase is encoded by MIP1 and displays a sequence similarity higher than 50% with human Pol gamma. Thanks to the high similarity between human Pol gamma and Mip1, and to the fact that yeast can survive and grow with large deletions of mtDNA or in absence of it,  yeast has been used in our  and in others laboratories as a model organism useful to understand whether a novel mutation in POLG is responsible for  a disease and to understand the mechanisms through which the mutation affects the enzymatic activity.

Recently, in collaboration with researchers at the University Paris-Sud, France, we have screened a collection of molecules approved by the FDA and EMA to search drugs able to restore the oxidative growth of a mip1 mutant strain. Out of 13 positive molecules, one, clofilium tosylate (CLO), has been studied more deeply. We showed that in yeast CLO rescues different phenotypes associated to a large number of mip1 mutations. CLO also rescues the phenotypes due to hetero- or homozygous deletion of polg in the worm Caenorhabditis elegans and, most important, increases the levels of mtDNA in fibroblasts of a patient having two heterozygous mutations in POLG. Therefore, CLO has been identified as a putative therapeutic molecule for POLG-related defect.

In this context, the focal point of the present research is to determine the mechanism though which CLO exerts its rescuing activity, and in particular to identify its molecular target. To achieve this aim, we will use the following experimental approaches:

1) We will study the role of fission and MICOS complex in the rescue by CLO, based on a chemogenomic analysis previously performed, which showed that some of the mutants which are more sensitive to CLOs are deleted in genes involved in mitochondrial metabolism. These genes are FIS1 and DNM1, which both encode for mitochondrial proteins involved in mitochondrial fission and MIC60, which encodes for a subunit of the MICOS complex.

2) We will identify genes that, when mutated, confer resistance to CLO.

3) We will identify genes that, when overexpressed, confer resistance to CLO.

The last two approaches, complementary to each other, are based on the hypothesis that if the alteration of expression of a gene confers resistance to a molecule, this gene may be the target, or may interact with the target.

4) We will establish an interactomic network among the identified genes, in order to propose the mechanism through which CLO rescues the mitochondrial deficiencies and to identify its molecular target.

The identification of a target which is bound by a drug is not only important from a theoretical point of view, but also from a practical one, especially in order to obtain information useful to identify or design ex novo molecules able to bind the target with a higher affinity.

Ultimo aggiornamento: 10/04/2017 14:16
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