Screening Small Molecules for Rescue of Peroxisome Assembly Defects

描述（由申请人提供）： 过氧化物酶体生物发生障碍（PBD）是一组具有高发病率和死亡率的异质性常染色体隐性遗传疾病，其发病率约为1/50，000。虽然没有令人满意的治疗方法，最近的观察强烈表明，患者的过氧化物酶体组装缺陷是在细胞水平上进行干预。当细胞在30 ℃下生长时，过氧化物酶体组装可以在来自具有轻度（NALD和IRD）表型的PBD患者的成纤维细胞中恢复。这被认为反映了在较低温度下有缺陷的过氧化物酶的构象改善。救援时也观察到相互作用的过氧化物酶过表达，反映功能冗余或稳定的缺陷过氧化物酶，或当细胞培养在4-苯基丁酸，过氧化物酶体增殖。在所有实验中，过氧化物酶体数量，基质蛋白的输入和功能的改善。收集的数据暗示了药物可以概括的几种机制。我们建议，筛选小分子库是一个强大的方法，以确定化合物，可以拯救过氧化物酶体组装没有偏向机制。对于这种基于神经系统的疾病，药物穿透CNS是至关重要的，小分子可能会这样做。我们调查了救援的泛化在30摄氏度之间的不同等位基因使用基于细胞的间接免疫荧光测定，并能够评分，通过眼睛，从细胞质的过氧化物酶体的基质蛋白的再分配。在目前的项目中，我们将调整我们的高通量表型检测和筛选一系列小的药物样分子，以确定化合物，可以拯救过氧化物酶体组装。我们将利用我们已经工程化的PEX 1-G843 D等位基因纯合的细胞系（通过上述所有机制和PBD的单一最常见原因拯救），并过表达GFP标记的基质蛋白，这些基质蛋白在基线时保持在胞质中。我们将在每种化合物中培养细胞5-10天，并在Cellomics KSR平台上使用自动落射荧光显微镜对其进行过氧化物酶体GFP评分。将使用优化的软件包分析图像，以区分胞质荧光和点状荧光。通过评价过氧化物酶体酶活性的回收率来确认命中。将在患有其他已知缺陷的患者的细胞系中测试所鉴定的化合物，以确定广泛的适用性。最终，这些药物可以被开发用于患者。

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.