Screening Small Molecules for Rescue of Peroxisome Assembly Defects

筛选小分子以挽救过氧化物酶体组装缺陷

• 批准号: 7136980
• 负责人: Nancy Elise Braverman
• 金额: $ 22.38万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-18 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7136980
• 关键词: peroxisome; proteins; small molecule

DESCRIPTION (provided by applicant): The Peroxisome Biogenesis Disorders (PBD), are a heterogeneous group of autosomal recessive disorders with high morbidity and mortality, and have an incidence of around 1/50,000. Although there is no satisfactory therapy available, recent observations strongly suggest that patient defects in peroxisome assembly are amenable to intervention at the cellular level. Peroxisome assembly can be restored in fibroblasts from PBD patients with mild (NALD and IRD) phenotypes when their cells are grown at 30 degrees C. This is thought to reflect improved conformation of the defective peroxin at lower temperature. Rescue is also observed when interacting peroxins are overexpressed, reflecting functional redundancy or stabilization of the defective peroxin, or when cells are cultured in 4-phenylbutyrate, a peroxisome proliferator. In all experiments, peroxisome number, matrix protein import and functions improve. The collective data implicate several mechanisms drugs could recapitulate. We propose that screening small molecule libraries is a robust method to identify compounds that can rescue peroxisome assembly without bias towards mechanism. For this neurologically based disorder, it is critical that drugs penetrate the CNS and small molecules are likely to do so. We investigated the generalization of rescue at 30 degrees C amongst diverse alleles using a cell-based indirect immunofluorescence assay and were able to score, by eye, redistribution of matrix proteins from the cytosol to the peroxisome. In the current project, we will adapt our phenotype assay for high throughput and screen a collection of small drug-like molecules to identify compounds that can rescue peroxisome assembly. We will utilize a cell line we have engineered homozygous for the PEX1-G843D allele (rescued by all the mechanisms discussed above and the single most common cause of PBD) and overexpressing GFPtagged matrix proteins that remain cytosolic at baseline. We will culture the cells in each compound for 5-10 days and score them for peroxisomal GFP using automated epifluorescent microscopy on the Cellomics KSR platform. Images will be analyzed using a software package optimized to discriminate cytoslic from punctate fluorescence. Hits will be confirmed by evaluating recovery of peroxisomal enzymatic activities. Identified compounds will be tested in cell lines from patients with other known defects to determine broad applicability. Ultimately these drugs could be developed for patient use.

描述（由申请人提供）： 过氧化物酶体生物发生障碍（PBD）是一组异类的常染色体隐性疾病，具有高发病率和死亡率，发病率约为1/50,000。尽管没有令人满意的治疗方法，但最近的观察结果强烈表明，过氧化物酶体组装中的患者缺陷可以在细胞水平上进行干预。当其细胞在30摄氏度下生长时，可以在PBD患者的成纤维细胞中恢复过氧化物酶体组装。这被认为反映了在较低温度下有缺陷的过氧蛋白的改善构象。当相互作用过表达相互作用时，还可以观察到救援，反映了有缺陷的过氧蛋白的功能冗余或稳定，或者在过氧化物酶体增殖剂4-苯基丁酸中培养细胞时。在所有实验中，过氧化物酶体数，基质蛋白导入和功能有所改善。集体数据暗示药物可能会概括。我们建议筛选小分子库是一种可靠的方法，可以识别可以挽救过氧化物酶体组装而不会偏向机制的化合物。对于这种基于神经学的疾病，至关重要的是，药物渗透到中枢神经系统和小分子可能会这样做。我们使用基于细胞的间接免疫荧光测定法研究了30摄氏度的拯救在30度C中的概括，并能够通过眼睛将基质蛋白从细胞质溶胶重新分布到过氧化物酶体进行得分。在当前项目中，我们将适应表型测定法，以进行高吞吐量，并筛选出小型药物样分子的集合，以鉴定可以挽救过氧化物酶体组装的化合物。 We will utilize a cell line we have engineered homozygous for the PEX1-G843D allele (rescued by all the mechanisms discussed above and the single most common cause of PBD) and overexpressing GFPtagged matrix proteins that remain cytosolic at baseline.我们将使用自动化的表体显微镜在Cellomics KSR平台上培养每种化合物中的细胞5-10天，并为其对过氧化物酶体GFP进行评分。将使用优化的软件包对图像进行分析，以区分点状荧光。通过评估过氧化物酶体酶活性的恢复，将确认命中。确定的化合物将在来自其他已知缺陷患者的细胞系中进行测试，以确定广泛的适用性。最终，这些药物可以用于患者使用。