Rational selection of small molecules that increase transcription through the GAA repeats found in Friedreich's ataxia

Abstract

Friedreich's ataxia (FRDA) is an autosomal recessive trinucleotide repeat disease with no effective therapy. Expanded GAA repeats in the first intron of the FRDA gene are thought to form unusual non-B DNA conformations that decrease transcription and subsequently reduce levels of the encoded protein, frataxin. Frataxin plays a crucial role in iron metabolism and detoxification. To discover small molecules that increase transcription through the GAA repeat region in FRDA, we have made stable cell lines containing a portion of expanded intron 1 fused to a GFP reporter. Small molecules identified using the competition dialysis method were found to increase FRDA-intron 1-reporter gene expression. One of these compounds, pentamidine, increases frataxin levels in patient cells. Thus our approach can be used to detect small molecules of potential therapeutic value in FRDA.

Lateral-flow immunoassay for the frataxin protein in Friedreich’s ataxia patients and carriers

“…Therefore, the correlation between GAA repeat number and disease is estimated to account for only approximately 50-70% of the variance in age of onset of FA [7,[10][11][12]. An additional significant shortcoming of genomic testing is that the tests cannot be used to monitor molecular efficacy of new therapies designed to treat or prevent onset of FA by boosting levels of the frataxin protein [26,27,29,30].…”

“…One key for potential curative therapies is that the genetic defect is in an intron and not in a coding sequence of the frataxin gene. This opens the possibility of using small molecule drugs to boost frataxin concentrations by increasing transcription of the unaltered, normal coding sequence and recent experimental advances have demonstrated the feasibility of this approach in vitro [26][27][28][29]. In addition, other compounds, such as recombinant human erythropoietin, have been shown to increase frataxin protein levels in vitro by a presently unknown mechanism [30].…”

Small molecules affecting transcription in Friedreich ataxia

“…These authors fused part of the first intron of FXN, containing either 15 or 148 GAA·TTC repeats, to the coding sequence for EGFP (in the Clontech plasmid pEGFP-N3), under control of the human cytomegalovirus immediate early promoter, and established stable HeLa cell lines. Both fluorescence microscopy and western blotting with antibodies to GFP demonstrated reduced levels of GFP expression in the [GAA·TTC] 148 cell line, compared to the [GAA·TTC] 15 cell line (Grant et al, 2006), showing that these are appropriate cell lines in which to screen for compounds that alleviate GAA·TTC-mediated silencing.…”

“…Hebert and colleagues have generated an additional GFP reporter cell line that will be useful for compound screening (Grant et al, 2006). These authors fused part of the first intron of FXN, containing either 15 or 148 GAA·TTC repeats, to the coding sequence for EGFP (in the Clontech plasmid pEGFP-N3), under control of the human cytomegalovirus immediate early promoter, and established stable HeLa cell lines.…”

“…Rational selection and screening of potential therapeutic molecules is one approach to identify such molecules (Grant et al, 2006;Herman et al, 2006;Sarsero et al, 2005), or alternatively, compound libraries might be screened in a high throughput fashion with the fluorescent reporter cell lines described above. To date, high throughput screens have not been reported; however, this approach might lead to the identification of molecules that increase FXN promoter activity, or elongation through the FXN gene, or act at other steps in the transcription process.…”