We will optimize a high-content assay to identify compounds that promote peroxisome biogenesis in human cells. Compounds we identify can be valuable chemical probes for modeling peroxisome biogenesis disorders and evaluating the contribution of peroxisomes to normal metabolism and physiology in animal models. In addition, compounds may have therapeutic potential for the treatment of metabolic syndrome (MetS) and type 2 diabetes (T2D). The initial assay development will be performed in a high-content/image based screening format, with the intent of translating the assay to the Molecular Libraries Production Centers Network (MLPCN). Peroxisomes represent a major site of fatty acid -oxidation and the only site of very long-chain fatty acid (VLCFA) -oxidation in the cell. Compounds that increase peroxisome biogenesis in human cells might provide a novel therapeutic avenue for type-II diabetes and metabolic syndrome. Increased plasma fatty acids contribute to insulin resistance and hyperglycemia, which are risk factors for both metabolic syndrome and T2D. The lipotoxicity hypothesis suggests that elevated plasma fatty acids may lead to ectopic lipid accumulation in non- adipose tissue, particularly in the liver ("fatty liver"), which would impair its function. Therefore, preventing lipid accumulation in non-adipose tissue and decreasing fatty acids in plasma by increasing fatty acid oxidation could provide treatments for metabolic disorders. Structurally unrelated compounds that increase peroxisome biogenesis do improve metabolic syndrome and diabetic symptoms in rodents, validating this phenotypic target. We will use a high- content screening assay to identify compounds that promote peroxisome biogenesis in human cells using mechanisms independent of direct PPAR activation (non-classical peroxisomal biogenesis). Compounds we identify will be counter-screened with multiple orthogonal secondary assays to confirm increased peroxisomal functionality in cell culture. Gene expression profiling will be performed with compounds stimulating peroxisome biogenesis to identify distinct mechanisms. Chemical probes identified in this study can be used for modeling peroxisome biogenesis disorders and evaluating the contribution of peroxisomes to normal metabolism and physiology in animal models. The assay development will be performed in a high-content/image based screening format, with the intent of transferring the assay to the Molecular Libraries Production Centers Network (MLPCN) high-content specialized screening center (Burnham Institute).
This proposal utilizes a novel high throughput screening (HTS) assay to identify novel regulators of peroxisome biogenesis and function. Due to peroxisomes function in fatty acid oxidation and their known roles in alleviating symptoms of metabolic syndrome/Type 2 diabetes in mice, compounds that could increase peroxisome biogenesis/functionality in human cells could provide a first step towards developing novel therapeutics for metabolic syndrome and Type 2 diabetes. We have validated a known therapeutic compound in mice as a potent activator in our assay to use for compound screening and secondary/orthogonal assay development.
|Law, Courtney J; Ashcroft, Harriet A; Zheng, Weifan et al. (2014) Assay development and multivariate scoring for high-content discovery of chemoprotectants of endoplasmic-reticulum-stress-mediated amylin-induced cytotoxicity in pancreatic beta cells. Assay Drug Dev Technol 12:375-84|