Research Article: Two New Pimelic Diphenylamide HDAC Inhibitors Induce Sustained Frataxin Upregulation in Cells from Friedreich’s Ataxia Patients and in a Mouse Model

Date Published: January 21, 2010

Publisher: Public Library of Science

Author(s): Myriam Rai, Elisabetta Soragni, C. James Chou, Glenn Barnes, Steve Jones, James R. Rusche, Joel M. Gottesfeld, Massimo Pandolfo, Antoni L. Andreu.

Abstract: Friedreich’s ataxia (FRDA), the most common recessive ataxia in Caucasians, is due to severely reduced levels of frataxin, a highly conserved protein, that result from a large GAA triplet repeat expansion within the first intron of the frataxin gene (FXN). Typical marks of heterochromatin are found near the expanded GAA repeat in FRDA patient cells and mouse models. Histone deacetylase inhibitors (HDACIs) with a pimelic diphenylamide structure and HDAC3 specificity can decondense the chromatin structure at the FXN gene and restore frataxin levels in cells from FRDA patients and in a GAA repeat based FRDA mouse model, KIKI, providing an appealing approach for FRDA therapeutics.

Partial Text: Friedreich’s ataxia (FRDA) is the most common of the early-onset autosomal recessive ataxias in Caucasians. In addition to progressive neurological disability, FRDA causes a hypertrophic cardiomyopathy and an increased risk of diabetes mellitus. Skeletal abnormalities such as kyphoscoliosis and pes cavus are common. The first symptoms usually are noticed around the time of puberty [1], [2]. As is the case for almost all neurodegenerative diseases, no proven treatment that can stop the progression of FRDA is now known. FRDA is caused by severely reduced levels of frataxin [3], a highly conserved mitochondrial protein, that result from a large GAA triplet repeat expansion within the first intron of the frataxin gene (FXN). In vitro and in vivo in bacterial plasmids the expanded repeat can adopt a triple helical structure that directly interferes with transcriptional elongation [4]. However, the discovery that long GAA repeats suppress the expression of a nearby reporter gene in transgenic mice in a manner similar to position effect variegation (PEV) observed in Drosophila pointed to a role of epigenetic mechanisms in the pathogenesis of FRDA [5], [6], [7]. PEV results in the silencing of a gene located near a heterochromatic region because of the spreading of heterochromatin into the gene itself. This phenomenon does not occur in all cells, hence the term “variegation”, but nevertheless leads to an overall downregulation of the involved gene at the tissue and organ level. In agreement with this hypothesis, the typical marks of heterochromatin, such as DNA methylation and histone deacetylation, are found near the expanded GAA repeat both in FRDA patients’ cells and in mouse models [6], [7], [8], [9]. Based on these observations, we speculated that histone deacetylase (HDAC) inhibitors (HDACIs) might reverse FXN silencing by directly increasing histone acetylation on the FXN gene, leading to chromatin decondensation and active transcription. The dynamic interplay between histone acetylation, performed by histone acetyltransferases (HATs) and deacetylation, catalyzed by HDACs, is indeed a central mechanism to regulate gene expression, with increased acetylation associated with an open chromatin conformation and active genes [10]. Eighteen HDACs (more strictly, protein deacetylases) have been identified in the human genome, including the zinc-dependent HDACs (class I, class II, and class IV), and the NAD+-dependent protein deacetylase enzymes (class III, or sirtuins) [11]. Accordingly, a diverse class of compounds that inhibit HDACs has been developed. Despite a potential widespread effect of HDAC inhibition on key processes as cellular differentiation and development, many of these compounds are well tolerated in humans and some of them show therapeutic promise in a wide range of diseases, including cancer, metabolic and neurological diseases [12], [13]. Target specificity and kinetic properties of HDACIs probably define the spectrum of genes whose expression they affect, explaining why a catastrophic general deregulation of gene expression is not observed with their use.

We generated new pimelic diphenylamide HDACIs to maximize efficacy in upregulating frataxin and to optimize pharmacological properties in view of a future translation to the clinic. The new compounds show the same type of inhibition kinetics and HDAC3 specificity as the previously reported molecules. We selected two of these compounds, 109 and 136, that differ for overall potency and degree of specificity for HDAC3, both being higher for compound 109 (Figure 1), and tested them in FRDA patients’ PBMC and in the KIKI mouse model. We then determined in both systems the duration of effect of these drugs on frataxin mRNA and protein, and on total and local (upstream of the GAA repeat) histone acetylation.



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