Abstract
BackgroundPharmacological high-throughput screening (HTS) represents a powerful strategy for drug discovery in genetic diseases, particularly when the full spectrum of pathological dysfunctions remains unclear, such as in Friedreich ataxia (FRDA). FRDA, the most common recessive ataxia, results from a generalized deficiency of mitochondrial and cytosolic iron-sulfur cluster (ISC) proteins activity, due to a partial loss of frataxin function, a mitochondrial protein proposed to function as an iron-chaperone for ISC biosynthesis. In the absence of measurable catalytic function for frataxin, a cell-based assay is required for HTS assay.MethodsUsing a targeted ribozyme strategy in murine fibroblasts, we have developed a cellular model with strongly reduced levels of frataxin. We have used this model to screen the Prestwick Chemical Library, a collection of one thousand off-patent drugs, for potential molecules for FRDA.ResultsThe frataxin deficient cell lines exhibit a proliferation defect, associated with an ISC enzyme deficit. Using the growth defect as end-point criteria, we screened the Prestwick Chemical Library. However no molecule presented a significant and reproducible effect on the proliferation rate of frataxin deficient cells. Moreover over numerous passages, the antisense ribozyme fibroblast cell lines revealed an increase in frataxin residual level associated with the normalization of ISC enzyme activities. However, the ribozyme cell lines and FRDA patient cells presented an increase in Mthfd2 transcript, a mitochondrial enzyme that was previously shown to be upregulated at very early stages of the pathogenesis in the cardiac mouse model.ConclusionAlthough no active hit has been identified, the present study demonstrates the feasibility of using a cell-based approach to HTS for FRDA. Furthermore, it highlights the difficulty in the development of a stable frataxin-deficient cell model, an essential condition for productive HTS in the future.
Highlights
Pharmacological high-throughput screening (HTS) represents a powerful strategy for drug discovery in genetic diseases, when the full spectrum of pathological dysfunctions remains unclear, such as in Friedreich ataxia (FRDA)
Experimental data from the conditional FRDA mouse models demonstrated that an increased superoxide production could not explain by itself the FRDA pathology [22] and that the mitochondrial iron accumulation is a late event in the disease [23]
Partial loss of frataxin leads to a proliferation defect in frataxin ribozyme cell lines To generate viable FRDA cellular models with reduced frataxin level, we used a ribozyme antisense strategy in cells derived from mice carrying the conditional allele [23]
Summary
Pharmacological high-throughput screening (HTS) represents a powerful strategy for drug discovery in genetic diseases, when the full spectrum of pathological dysfunctions remains unclear, such as in Friedreich ataxia (FRDA). Friedreich's ataxia (FRDA), the most common autosomal recessive ataxia associating spinocerebellar ataxia and cardiomyopathy [1,2], is most often due to a (GAA)n repeat expansion within the first intron of the gene encoding the mitochondrial protein frataxin [3,4] This frequent mutation leads to a severely reduced level of frataxin as a consequence of transcriptional silencing either through heterochromatin formation or through the formation of a triplex helix [5,6,7]. Studies showed iron deposits in cardiac tissue of FRDA patients [8] and in the yeast strain deleted for frataxin (ΔYFH1) thereby linking impaired iron homeostasis to the disease [9] This led to the hypothesis that elevated levels of mitochondrial iron, as a consequence of frataxin deficiency, could generate cell-damaging superoxide and hydroxyl radicals through Fenton reaction. Frataxin has been demonstrated to interact with the ISC biosynthesis scaffold complex IscU/Nfs1/ISD11 [31,32,33,34,35]
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