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) 是一组异质性常染色体隐性遗传疾病，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，但最近的观察强烈表明，患者的过氧化物酶体组装缺陷适合在细胞水平上进行干预。当具有轻度（NALD 和 IRD）表型的 PBD 患者的成纤维细胞在 30 摄氏度下生长时，过氧化物酶体组装可以恢复。这被认为反映了有缺陷的过氧化物酶在较低温度下构象的改善。当相互作用的过氧化物酶过度表达（反映有缺陷的过氧化物酶的功能冗余或稳定化）时，或者当细胞在过氧化物酶体增殖剂 4-苯基丁酸盐中培养时，也可以观察到拯救作用。在所有实验中，过氧化物酶体数量、基质蛋白输入和功能均得到改善。集体数据暗示了药物可以概括的几种机制。我们认为，筛选小分子文库是一种可靠的方法，可以识别可以在不偏向机制的情况下拯救过氧化物酶体组装的化合物。对于这种基于神经系统的疾病，药物渗透中枢神经系统至关重要，而小分子很可能会这样做。我们使用基于细胞的间接免疫荧光测定研究了不同等位基因在 30 摄氏度下拯救的普遍性，并能够通过肉眼对基质蛋白从细胞质到过氧化物酶体的重新分布进行评分。在当前的项目中，我们将调整我们的表型测定以实现高通量，并筛选一系列小药物分子，以确定可以挽救过氧化物酶体组装的化合物。我们将利用我们设计的 PEX1-G843D 等位基因纯合子细胞系（通过上述所有机制和 PBD 的最常见原因来拯救），并过表达在基线时保持胞质的 GFP 标记基质蛋白。我们将在每种化合物中培养细胞 5-10 天，并使用 Cellomics KSR 平台上的自动落射荧光显微镜对它们进行过氧化物酶体 GFP 评分。将使用优化的软件包分析图像，以区分细胞质和点状荧光。将通过评估过氧化物酶体酶活性的恢复来确认命中。鉴定出的化合物将在具有其他已知缺陷的患者的细胞系中进行测试，以确定其广泛的适用性。最终这些药物可以开发供患者使用。