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Investigating new therapeutic approaches to Friedreich's Ataxia

Mid-Term Report Summary - FAST (Investigating new therapeutic approaches to Friedreich's Ataxia)

During the first part of the project we proceeded in the search for frataxin-specific ubiquitin-competing molecules (UCMs), with the aim to identify more effective compounds. Based on the data obtained from computational studies, we have now extended our search to small molecules that could dock to additional, more druggable surfaces on frataxin. Second-generation molecules, selected through this new screening approach, have been validated for their interaction with frataxin, their impact on frataxin ubiquitination and their efficacy in living cells. Direct interaction between recombinant frataxin and the selected compounds was demonstrated in vitro through fluorescence spectroscopy approaches. Moreover, the selected compounds are able to significantly abrogate frataxin ubiquitination in transfected cells in a dose-dependent manner. Importantly, second-generation molecules show an increased efficacy in promoting frataxin accumulation, compared to the previously identified compounds.
Moreover, we made progress toward the identification of the E3 ligase responsible for frataxin ubiquitination. We performed a functional screening of an E3 ligase-restricted siRNA library to search for candidate genes, whose suppression could induce an increase in frataxin levels. The selected candidate siRNA were then validated for their ability to promote frataxin accumulation in different cellular systems. One of the selected genes was able to consistently induce frataxin accumulation, when its expression was suppressed by the corresponding siRNA. This gene is likely to represent the enzyme responsible for frataxin degradation. Accordingly, overexpression of the corresponding cDNA, but not its catalytic inactive variant, induced frataxin ubiquitination in cells, suggesting that the identified E3 ligase is responsible for frataxin ubiquitination.