Screening Small Molecules for Rescue of Peroxisome Assembly Defects

描述（由申请人提供）：

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.