Histone Deacetylase Inhibitors as Therapeutics for Friedreich's ataxia

描述（由申请人提供）：弗里德赖希共济失调（FRDA）是一种遗传性神经退行性疾病，由核编码线粒体蛋白frataxin缺乏引起。目前尚无有效的治疗方法。在98%的FRDA患者中发现的DNA异常是在frataxin基因的第一个内含子中GAA三联体的不稳定超扩增，这干扰了基因转录，无论是在DNA水平还是在染色质结构水平。基于核心组蛋白的乙酰化状态可能导致含有扩增GAA重复序列的frataxin基因沉默的假设，我们筛选了具有代表性的商业组蛋白去乙酰化酶（HDAC）抑制剂，并鉴定了一种化合物（来自BioMol的BML-210），该化合物部分缓解了FRDA患者淋巴样细胞中frataxin基因的抑制。基于这一结果，我们设计了一个合成方案来获得该先导化合物的衍生物库。HDAC抑制剂的icso将在FRDA患者淋巴细胞衍生的细胞系中测定。Real-time PCR将用于确定BML-210和/或其衍生物是否增加frataxin mRNA在人FRDA细胞系、从FRDA供血中分离的淋巴样细胞以及gaa扩增的frataxin敲入小鼠建立的神经细胞系中的表达。抑制剂对细胞frataxin蛋白的影响将通过western blotting来确定，最活跃的抑制剂的全基因组效应将通过DNA微阵列分析来评估。动物研究将在正常小鼠中进行，以确定生物利用度、组织分布、药代动力学、化合物在血清中的半衰期、毒性和最大耐受剂量。我们将确定HDAC抑制剂是否穿过血脑屏障并在体内发挥作用。将利用含有扩展GAA等位基因的frataxin基因敲入小鼠来确定HDAC抑制剂是否激活体内frataxin基因表达，并缓解这些动物的神经功能缺陷。这一提议旨在开发新药来治疗遗传性神经疾病弗里德赖希共济失调（FRDA），这是由人类重要基因中简单序列GAA的重复扩增引起的。这些重复可能通过将DNA包装在不活跃的染色体环境中使基因沉默。我们正在开发小分子来逆转这种效应，希望这些分子可以治疗FRDA。

DESCRIPTION (provided by applicant): Friedreich's ataxia (FRDA) is an inherited neurodegenerative disease caused by a deficiency in the nuclear- encoded mitochondrial protein frataxin. At present there is no effective cure or treatment for FRDA. The DNA abnormality found in 98% of FRDA patients is the unstable hyper-expansion of a GAA triplet in the first intron of the frataxin gene that interferes with gene transcription, either at the level of DNA or chromatin structure. Based on the hypothesis that the acetylation state of the core histones might be responsible for silencing the frataxin gene harboring expanded GAA repeats, we screened a representative collection of commercial histone deacetylase (HDAC) inhibitors, and identified one compound (BML-210 from BioMol) that partially relieves repression of the frataxin gene in lymphoid cells derived from FRDA patients. Based on this result, we have devised a synthetic scheme to obtain a library of derivatives of this lead compound. ICSOs for the HDAC inhibitors will be determined in cell lines derived from FRDA patient lymphocytes. Real-time PCR will be used to determine whether BML-210 and/or its derivatives increase frataxin mRNA expression in human FRDA cell lines, lymphoid cells isolated from FRDA donor blood, and in neuronal cell lines established from GAA-expanded frataxin knock-in mice. The effects of the inhibitors on cellular frataxin protein will be determined by western blotting, and the genome-wide effects of the most active inhibitors will be assessed by DNA microarray analysis. Animal studies will be performed in normal mice to determine the bioavailability, tissue distribution, pharmacokinetics, half-lives of the compounds in serum, toxicity, and maximum tolerated dosage. We will determine whether the HDAC inhibitors cross the blood brain barrier and function in vivo. Knock-in mice harboring frataxin genes with expanded GAA alleles will be utilized to determine whether HDAC inhibitors activate frataxin gene expression in vivo, and relieve neurological deficits in these animals. This proposal is aimed at the development of new drugs to treat the inherited neurological disease Friedreich's ataxia (FRDA), which is caused by expansion of repeats of the simple sequence GAA in an essential human gene. These repeats silence the gene, possibly by packaging the DNA in an inactive chromosomal environment. We are developing small molecules to reverse this effect, with the hope that these molecules may be therapeutic for FRDA.