Development of novel therapeutic approaches for the treatment of Friedreich's ataxia targeting the biochemical process of iron-sulfur cluster assembly

Friedreich's ataxia (FA) is the most common hereditary ataxia, with 1 in 30,000 people affected worldwide. It is a genetic, neurodegenerative and cardiac disease caused by defective expression of frataxin (FXN), a mitochondrial protein stimulating the biosynthesis of iron-sulfur (Fe-S) clusters, which are metallo-cofactors of proteins involved in a multitude of essential biological functions. The thesis project aims to develop drugs for the treatment of this disease by combining biochemical and biophysical techniques, in vitro screening and tests in animal models.
We have previously shown that FXN acts by stimulating the supply of sulfur to the Fe-S cluster biosynthesis system. More recently, we have discovered and extremely fine cross-regulation between FXN and ferredoxin-2 (FDX2), an enzyme intervening at the next step to that catalyzed by FXN. Our data show that these two enzymes compete with each other for binding to the Fe-S cluster biosynthesis complex, thereby repressing each other's activities. Hence, an small excess of FDX2 relative to FXN decreases the efficiency of the reaction, suggesting that a decrease in FDX2 levels could increase the efficiency of Fe-S cluster synthesis in FXN-deficient conditions such as in FA patients. We were able to validate this hypothesis in vivo in a drosophila model of FA, showing that decreasing FDX2 levels improves fly survival. Our data suggest that FDX2 could be used as a novel therapeutic target for FA. In parallel, we have identified compounds by high-throughput screening that stimulate Fe-S cluster biosynthesis and we suspect that they act by alleviating FDX2 repression. The objectives of this project are to elucidate the mode of action of these compounds, better understand the cross-regulation between FXN and FDX2 and test the molecules in the AF drosophila model in order to evaluate their potential as drug candidates. By targeting the primary defect of AF, i.e. the defect in the synthesis of Fe-S clusters, we hope to obtain drugs with high therapeutic potential.
This project is positioned in the CEA's "Biotechnology of Tomorrow" strategic axis, at the interface of life sciences and engineering to address a public health issue. This project combines biology and technologies for the development of a new axis in biotherapy. It is based on a previous CFR thesis funded by the CEA (K. Want 2021-2024), which enabled the development of an anaerobic in vitro screening platform, unique in France, and the identification of active molecules. This thesis also generated the first results showing the existence of cross-regulation between FXN and FDX2, which are the basis of this new project. The continuation of this project thus meets the CEA's objective of building on work already underway, to intensify their developments with the aim of ensuring clinical transfer and scaling up for industrial transfer. Furthermore, this project will rely on the CEA's expertise in modelling and on the I2BC biophysics platforms. It therefore seems important that this project be supported and funded by the CEA.

Innovative pharmacological strategy to counter biohazard toxins

THESIS OBJECTIVE. Develop PROTAC molecules for proteasome-mediated degradation of toxins internalized in host cells, and propose drug candidates for in vivo studies at the end of the thesis.

BACKGROUND AND CHALLENGES. Plant and bacterial toxins are among the most toxic natural substances, and are responsible for fatal diseases such as botulism and tetanus. Once the toxin is internalized in the target cells, immunotherapy is ineffective, and there are no curative treatments for these biomolecules. One way of achieving a major breakthrough in the development of medical countermeasures would be to target the toxin directly into the cytoplasm of host cells using PROTAC molecules. PROTACs are heterobifunctional degraders that specifically eliminate targeted proteins by hijacking the cell's ubiquitin-proteasome system. This recent therapeutic strategy represents an attractive technology for new drug discovery.

METHODOLOGY. To carry out this project, the thesis student will carry out in silico screening campaigns to identify ligands for a toxin and improve their affinity. Key validation experiments will require recombinant production of a toxin fragment, and will be carried out in E. coli. From the most promising optimized ligands, targeted libraries of PROTAC molecules directed against the toxin will be synthesized in collaboration with a team of chemists. The student will evaluate the ability of these molecules to interact with and eliminate the internalized toxin in cultured cells using different approaches, in order to propose drug candidates for in vivo studies at the end of the thesis.