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

BackgroundFriedreich'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.
Methodology/Principal FindingsIn an effort to further improve the pharmacological profile of pimelic diphenylamide HDACIs as potential therapeutics for FRDA, we synthesized additional compounds with this basic structure and screened them for HDAC3 specificity. We characterized two of these compounds, 136 and 109, in FRDA patients' peripheral blood lymphocytes and in the KIKI mouse model. We tested their ability to upregulate frataxin at a range of concentrations in order to determine a minimal effective dose. We then determined in both systems the duration of effect of these drugs on frataxin mRNA and protein, and on total and local histone acetylation. The effects of these compounds exceeded the time of direct exposure in both systems.
Conclusions/SignificanceOur results support the pre-clinical development of a therapeutic approach based on pimelic diphenylamide HDACIs for FRDA and provide information for the design of future human trials of these drugs, suggesting an intermittent administration of the drug.

背景 弗里德赖希共济失调 (Friedreich's ataxia, FRDA) 是高加索人群中最常见的隐性遗传性共济失调，其病因是frataxin蛋白 (frataxin) 水平严重降低。frataxin是一种高度保守的蛋白质，其水平下降源于frataxin基因 (FXN) 第一内含子内存在大片段GAA三核苷酸重复扩增。在FRDA患者细胞及小鼠模型中，扩增的GAA重复序列附近可观察到异染色质 (heterochromatin) 的典型特征。具备庚二酰二苯胺结构且具有HDAC3特异性的组蛋白去乙酰化酶抑制剂 (Histone deacetylase inhibitors, HDACIs) 可松解FXN基因位点的染色质结构，并在FRDA患者细胞及基于GAA重复序列构建的FRDA小鼠模型KIKI中恢复frataxin蛋白水平，为FRDA治疗提供了极具潜力的策略。
方法/主要发现 为进一步优化具备庚二酰二苯胺结构的HDACIs作为FRDA潜在治疗药物的药理学特性，我们合成了更多该基础结构的化合物，并筛选出具有HDAC3特异性的分子。我们在FRDA患者外周血淋巴细胞 (peripheral blood lymphocytes) 及KIKI小鼠模型中，对其中两种化合物136和109进行了系统性表征。为确定最低有效剂量，我们在一系列浓度梯度下测试了它们上调frataxin表达的能力。随后，我们在两个实验系统中分别测定了这些药物对frataxin mRNA及蛋白水平、总组蛋白与局部组蛋白乙酰化水平的作用持续时间。结果显示，在两个系统中，此类化合物的药效时长均超过了药物直接暴露的时间。
结论与意义 本研究结果支持以庚二酰二苯胺类HDACIs为基础的FRDA治疗策略推进至临床前开发阶段，并为此类药物未来的人体临床试验设计提供了关键参考，提示可采用间歇性给药方案。