Exercise and caloric restriction exert very powerful protective effects against age-associated diseases including metabolic disorders such as obesity and diabetes. Among the key target tissues skeletal muscle is thought to be one of the mediators of these protective effects. A main regulatory component of these effects is the transcriptional coactivator PGC-11 which activity is upregulated upon exercise or under caloric restriction. Conversely, the activity of PGC-11 is suppressed in conditions of physical inactivity or after feeding with high fat calorie diets. Importantly, increases of PGC-11 activity in skeletal muscle are sufficient to prevent age- associated diseases and prolong life span. Deacetylation of PGC-11 through Sirt1 is one of the key chemical modifications that activate PGC-11 under exercise or nutrient deprivation conditions. In contrast, acetylation of PGC-11 by GCN5 results in an inactive protein found in non-exercise or high fat calorie diets situations. These results lead to the hypothesis that chemical compounds that induce PGC-11 deacetylation could mimic exercise or nutrient deprivation conditions. To address this hypothesis, this present proposal is aimed to identify these chemical compounds using a high throughput screening (HTS) that quantitatively measures the acetylation state of PGC-11. We have three Specific Aims: 1) Collaborate with MLPCN to implement a validated high-throughput screen to identify small molecules that decrease the acetylation level of PGC-11. We have developed and validated a physiological relevant cell-based assay using ELISA to screen the entire MLPCN library. 2) We will employ previously validated secondary assays to assess the biological relevance of identified hits on activation of PGC-11 target genes. We will focus on (i) to analyze the effects of the positive chemical compounds on counteracting pathways to eliminate possible side-effects of the drugs and, (ii) to identify the biologically relevant positive chemical compounds from the primary HTS assay by analyzing gene expression changes of PGC-11 targets. 3) Collaborate with MLPCN to develop and characterize chemical probes that affect PGC-11 acetylation using previously validated tertiary assays to test specificity and mechanism of action. We will focus on (i) we will confirm that increased expression of the PGC-11 target genes MCAD and CPT1b is dependent upon PGC-11 and, (ii) we will analyze the specific target mechanism by which positive chemical probes modulate PGC-11 acetylation through GCN5 and SIRT1. The outcomes of these studies will provide the identification of chemical compounds as well as the molecular mechanisms that control PGC-11 acetylation and as a consequence will modulate the metabolic activities regulated by this transcriptional coactivator. Since these metabolic activities mimic, at least to a large extend, exercise and calorie restriction it is very plausible that these chemical compounds might be used as potential therapies to treat metabolic disorders and age-associated diseases.
Exercise and calorie restriction have potent protective effects against age-associated diseases including metabolic diseases, thus studies in this grant proposal to identify chemical compounds that might mimic physical activity or nutrient deprivation through PGC-11 deacetylation might translate into potential therapies.
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