Small molecule therapeutics for Friedreich's ataxia

弗里德赖希共济失调的小分子疗法

• 批准号: 6807511
• 负责人: JOEL M. GOTTESFELD
• 金额: $ 21.7万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-15 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6807511
• 关键词: DNA; DNA footprinting; Friedreich' s ataxia; amides; brain disorder chemotherapy; cell line; chemical binding; drug design /synthesis /production; fluorescent dye /probe; gene expression; imidazole; microarray technology; microscopy; nucleic acid sequence; nucleic acid structure; pharmacokinetics; phosphotransferases; plasmids; polymerase chain reaction; polymers; pyrroles; small molecule; tissue /cell culture; transfection; western blottings

DESCRIPTION (provided by applicant): Friedreich's ataxia (FRDA) is an inherited neurodegenerative disease caused by deficiency of the nuclear-encoded mitochondrial protein frataxin. At present there is no effective cure or treatment for FRDA. The DNA abnormality found in 98 percent of FRDA patients is the unstable hyperexpansion of a GAA triplet repeat in the first intron of the frataxin gene, which adopts unusual DNA structures that interfere with gene transcription. We will explore whether synthetic DNA ligands, pyrrole-imidizole (Py-lm) polyamides, can be designed to bind GAA repeats and whether such molecules will relieve transcription inhibition by stabilizing canonical Watson-Crick B-type DNA structure in the frataxin gene. Py-lm polyamides bind predetermined DNA sequences with subnanomolar affinities, comparable to the binding affinities of natural transcriptional regulatory proteins; moreover, these molecules have been shown to access target sites in the nucleus of cultured cells. A series of Py-lm polyamides will be synthesized to target the GAA repeat sequence in the frataxin gene, and DNase I footprinting will be used to measure polyamide binding affinities and specificities for their target sequences. The effect of polyamides on GAA repeat DNA structure in plasmid DNAs will be determined. Polyamides that bind GAA repeat DNA with high affinity are expected to stabilize B-type DNA, and prevent transitions to triplex or other unusual DNA structures. The effects of polyamides on transcription of the frataxin gene will be monitored both in vitro and in cell culture experiments. Deconvolution microscopy will be used to monitor the subcellular localization and kinetics of uptake of fluorescent dye-polyamide conjugates in cultured cells. Real-time PCR will be used to determine the effects of polyamides on frataxin mRNA expression in appropriate human FRDA cell lines. The effects of polyamides on cellular frataxin protein will be determined by western blotting, and the genome-wide effects of polyamide treatment will be assessed by DNA microarray analysis.

描述（由申请人提供）：Friedreich的共济失调（FRDA）是由核编码的线粒体蛋白Frataxin缺乏引起的遗传神经退行性疾病。目前，FRDA尚无有效的治疗或治疗方法。在98％的FRDA患者中发现的DNA异常是Frataxin基因的第一个内含子中GAA三重序重复的不稳定过度脱位，该基因采用了干扰基因转录的异常DNA结构。我们将探索合成DNA配体，吡咯米 - 咪唑（PY-LM）聚酰胺是否可以设计用于结合GAA重复序列，以及该分子是否会通过稳定Frataxin基因中的稳定范围的沃森 - 克里克B-type DNA结构来缓解转录抑制作用。 PY-LM聚酰胺与预定的DNA序列与亚洋摩尔亲和力结合，与天然转录调节蛋白的结合亲和力相当。此外，这些分子已被证明可以访问培养细胞核中的目标位点。将合成一系列PY-LM聚酰胺，以靶向Frataxin基因中的GAA重复序列，DNase I足迹将用于测量其目标序列的聚酰胺结合亲和力和特异性。将确定聚酰胺对质粒DNA中GAA重复DNA结构的影响。与高亲和力结合GAA重复DNA的聚酰胺有望稳定B型DNA，并防止过渡到三型或其他异常的DNA结构。聚酰胺对Frataxin基因转录的影响将在体外和细胞培养实验中受到监测。反卷积显微镜将用于监测培养细胞中荧光染料 - 聚解酰胺偶联物的摄取的亚细胞定位和动力学。实时PCR将用于确定聚酰胺对适当的人FRDA细胞系中FRATAXIN mRNA表达的影响。聚酰胺对细胞Frataxin蛋白的影响将通过蛋白质印迹确定，并且将通过DNA微阵列分析评估聚酰胺治疗的全基因组效应。