Expansion of GAA x TTC triplets within an intron in FXN (the gene encoding frataxin) leads to transcription silencing, forming the molecular basis for the neurodegenerative disease Friedreich's ataxia. Gene silencing at expanded FXN alleles is accompanied by hypoacetylation of histones H3 and H4 and trimethylation of histone H3 at Lys9, observations that are consistent with a heterochromatin-mediated repression mechanism. We describe the synthesis and characterization of a class of histone deacetylase (HDAC) inhibitors that reverse FXN silencing in primary lymphocytes from individuals with Friedreich's ataxia. We show that these molecules directly affect the histones associated with FXN, increasing acetylation at particular lysine residues on histones H3 and H4 (H3K14, H4K5 and H4K12). This class of HDAC inhibitors may yield therapeutics for Friedreich's ataxia.

Histone deacetylase (HDAC) inhibitors, including various benzamides and hydroxamates, are currently in clinical development for a broad range of human diseases, including cancer and neurodegenerative diseases. We recently reported the identification of a family of benzamide-type HDAC inhibitors that are relatively non-toxic compared with the hydroxamates. Members of this class of compounds have shown efficacy in cellbased and mouse models for the neurodegenerative diseases Friedreich ataxia and Huntington disease. Considerable differences in IC 50 values for the various HDAC enzymes have been reported for many of the HDAC inhibitors, leading to confusion as to the HDAC isotype specificities of these compounds. Here we show that a benzamide HDAC inhibitor, a pimelic diphenylamide (106), is a class I HDAC inhibitor, demonstrating no activity against class II HDACs. 106 is a slow, tight-binding inhibitor of HDACs 1, 2, and 3, although inhibition for these enzymes occurs through different mechanisms. Inhibitor 106 also has preference toward HDAC3 with K i of ϳ14 nM, 15 times lower than the K i for HDAC1. In comparison, the hydroxamate suberoylanilide hydroxamic acid does not discriminate between these enzymes and exhibits a fast-on/fast-off inhibitory mechanism. These observations may explain a paradox involving the relative activities of pimelic diphenylamides versus hydroxamates as gene activators.The link between post-translational modifications by reversible histone acetylation and deacetylation and mRNA transcription has been shown to be one of the key mechanisms of epigenetic gene regulation (1). Acetylation of histone lysine residues, controlled by the histone acetyltransferases and histone deacetylases (HDACs), 2 has been a subject of intense recent research (2-5). Generally, histone hypoacetylation causes transcriptional silencing, whereas histone hyperacetylation results in transcriptional activation of various genes (6 -8). Eighteen HDACs have been identified in the human genome, including the zinc-dependent HDACs (class I, class II, and class IV), and the NAD ϩ -dependent enzymes (class III or sirtuins) (9, 10). HDACs 1, 2, 3, and 8 belong to class I, showing homology to the yeast enzyme RPD3. Class II is further divided into class IIa (HDACs 4, 5, 7, and 9) and IIb (HDAC 6 and 10), according to their sequence homology and domain organization. HDAC11 is the lone member of class IV (9, 11). The sirtuins (class III) are related to the yeast Sir2 protein and are involved in regulation of metabolism and aging (10).To date, a number of small molecule inhibitors of the zincdependent HDACs have been identified (12). These compounds can be broadly grouped in four chemical classes: the hydroxamates, the benzamides, butyrate analogs, and cyclic peptides, such as depsipeptide and related compounds (12, 13). Hydroxamate-based inhibitors, such as trichostatin A (TSA) and suberoylanilide hydroamic acid (SAHA; Fig. 1A) are believed to be pan-HDAC inhibitors (14 -16); however, recent studies have shown that TSA and SAHA...

Transcriptional dysregulation has emerged as a core pathologic feature of Huntington's disease (HD), one of several triplet-repeat disorders characterized by movement deficits and cognitive dysfunction. Although the mechanisms contributing to the gene expression deficits remain unknown, therapeutic strategies have aimed to improve transcriptional output via modulation of chromatin structure. Recent studies have demonstrated therapeutic effects of commercially available histone deacetylase (HDAC) inhibitors in several HD models; however, the therapeutic value of these compounds is limited by their toxic effects. Here, beneficial effects of a novel pimelic diphenylamide HDAC inhibitor, HDACi 4b, in an HD mouse model are reported. Chronic oral administration of HDACi 4b, beginning after the onset of motor deficits, significantly improved motor performance, overall appearance, and body weight of symptomatic R6/2 300Q transgenic mice. These effects were associated with significant attenuation of gross brain-size decline and striatal atrophy. Microarray studies revealed that HDACi 4b treatment ameliorated, in part, alterations in gene expression caused by the presence of mutant huntingtin protein in the striatum, cortex, and cerebellum of R6/2 300Q transgenic mice. For selected genes, HDACi 4b treatment reversed histone H3 hypoacetylation observed in the presence of mutant huntingtin, in association with correction of mRNA expression levels. These findings suggest that HDACi 4b, and possibly related HDAC inhibitors, may offer clinical benefit for HD patients and provide a novel set of potential biomarkers for clinical assessment.rologic disorder caused by a CAG repeat expansion within the coding region of the HD gene (Htt), resulting in a mutant protein (htt) with a lengthened polyglutamine tract (1). Mutant htt protein has been shown to disrupt transcription by multiple mechanisms, but it is unclear which are most important to pathology (2-4). By interacting with specific transcription factors, htt can alter the expression of clusters of genes controlled by those factors. For example, several genes driven by Sp1, which has been shown to interact with htt (5, 6), show decreased mRNA expression in human HD and in mouse models of HD (7). Alternatively, htt may have more global effects on transcription by disrupting core transcriptional machinery (8, 9) or by altering posttranslational modifications of histones, resulting in condensed chromatin structure (10-13). Understanding the basis for transcriptional dysregulation is important for choosing appropriate drug-discovery strategies.Manifestations of transcriptional dysregulation are evident from several gene-profiling studies, which have revealed alterations in the expression of large numbers of genes in the brains of different HD mouse models and in human subjects with HD (7, 14-16). Many of the expression changes in mouse models are observed in early stages of illness before the onset of symptoms, suggesting that gene expression alterations may be pathogenic.Because o...

Chemical modulation of histone deacetylase (HDAC) activity by HDAC inhibitors (HDACi) is an increasingly important approach to modify the etiology of human disease. Loss-of-function diseases arise as a consequence of protein misfolding and degradation leading to system failures. The ΔF508 mutation in cystic fibrosis transmembrane conductance regulator (CFTR) results in the absence of the cell surface chloride channel and a loss of airway hydration, leading to premature lung failure and reduced lifespan responsible for cystic fibrosis (CF). We now show that the HDACi suberoylanilide hydroxamic acid (SAHA) restores surface channel activity in human primary airway epithelia to levels that are 28% of wild-type CFTR. Biological silencing of all known class I and II HDACs reveals that HDAC7 plays a central role in restoration of ΔF508 function. We suggest that the tunable capacity of HDACs can be manipulated by chemical biology to counter the onset of CF and other human misfolding disorders.

BackgroundFriedreich ataxia, an autosomal recessive neurodegenerative and cardiac disease, is caused by abnormally low levels of frataxin, an essential mitochondrial protein. All Friedreich ataxia patients carry a GAA⋅TTC repeat expansion in the first intron of the frataxin gene, either in the homozygous state or in compound heterozygosity with other loss-of-function mutations. The GAA expansion inhibits frataxin expression through a heterochromatin-mediated repression mechanism. Histone modifications that are characteristic of silenced genes in heterochromatic regions occur at expanded alleles in cells from Friedreich ataxia patients, including increased trimethylation of histone H3 at lysine 9 and hypoacetylation of histones H3 and H4.Methodology/Principal FindingsBy chromatin immunoprecipitation, we detected the same heterochromatin marks in homozygous mice carrying a (GAA)230 repeat in the first intron of the mouse frataxin gene (KIKI mice). These animals have decreased frataxin levels and, by microarray analysis, show significant gene expression changes in several tissues. We treated KIKI mice with a novel histone deacetylase inhibitor, compound 106, which substantially increases frataxin mRNA levels in cells from Friedreich ataxia individuals. Treatment increased histone H3 and H4 acetylation in chromatin near the GAA repeat and restored wild-type frataxin levels in the nervous system and heart, as determined by quantitative RT-PCR and semiquantitative western blot analysis. No toxicity was observed. Furthermore, most of the differentially expressed genes in KIKI mice reverted towards wild-type levels.Conclusions/SignificanceLack of acute toxicity, normalization of frataxin levels and of the transcription profile changes resulting from frataxin deficiency provide strong support to a possible efficacy of this or related compounds in reverting the pathological process in Friedreich ataxia, a so far incurable neurodegenerative disease.