Histone Deacetylase Inhibitors as Therapeutics for Friedreich's ataxia

描述(申请人提供)：Friedreich‘s共济失调(FRDA)是一种遗传性神经退行性疾病，由核编码的线粒体蛋白Frataxin缺乏引起。目前，FRDA尚无有效的治愈或治疗方法。在98%的FRDA患者中发现的DNA异常是Frataxin基因第一内含子中GAA三联体的不稳定过度扩张，这在DNA或染色质结构水平上干扰了基因转录。基于核心组蛋白的乙酰化状态可能导致含有扩展的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组蛋白去乙酰化酶(HDAC)抑制剂集合，并从生物分子中鉴定出一种化合物(BML-210)，它部分缓解了FRDA患者淋巴细胞中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将在来自FRDA患者淋巴细胞的细胞系中确定。实时定量聚合酶链式反应将被用来确定BML-210和/或其衍生物是否增加了人FRDA细胞系、从FRDA供体血中分离的淋巴细胞以及从GAA扩增的Frataxin基因敲除小鼠建立的神经细胞系中Frataxin的mRNA表达。这些抑制剂对细胞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。将在正常小鼠身上进行动物研究，以确定这些化合物的生物利用度、组织分布、药代动力学、血清半衰期、毒性和最大耐受量。我们将确定HDAC抑制剂是否通过血脑屏障并在体内发挥作用。携带Frataxin基因的转基因小鼠具有扩展的GAA等位基因，将被用来确定HDAC抑制剂是否在体内激活Frataxin基因的表达，并缓解这些动物的神经缺陷。这项建议旨在开发治疗遗传性神经疾病Friedreich‘s ataxia(FRDA)的新药，FRDA是由人类基本基因中简单序列GAA重复序列的扩展引起的。这些重复序列可能通过将DNA包装在不活跃的染色体环境中而使基因沉默。我们正在开发小分子来逆转这种效应，希望这些分子可能对FRDA具有治疗作用。

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.