HTS of Compounds Modulating PGC-1a Acetylation and Oxidative Metabolic Function

描述(申请人提供)：锻炼和卡路里限制对年龄相关的疾病有非常强大的保护作用，包括肥胖和糖尿病等新陈代谢障碍。在关键的靶组织中，骨骼肌被认为是这些保护作用的中介之一。这些效应的一个主要调节成分是转录辅助激活因子PGC-11，它的活性在运动或卡路里限制下上调。相反，PGC-11的活性在缺乏体力活动的情况下或在喂食高脂肪卡路里饮食后受到抑制。重要的是，骨骼肌中PGC-11活性的增加足以预防与年龄相关的疾病，延长寿命。PGC-11通过Sirt1去乙酰化是运动或营养缺乏条件下激活PGC-11的关键化学修饰之一。相比之下，PGC-11被GCN5乙酰化会导致一种在非运动或高脂肪卡路里饮食情况下发现的非活性蛋白质。这些结果导致了一种假设，即诱导PGC-11脱乙酰化的化合物可以模拟运动或营养缺乏的条件。为了解决这一假设，本提案旨在使用高通量筛选(HTS)定量测量PGC-11的乙酰化状态来鉴定这些化合物。我们有三个具体目标：1)与MLPCN合作实施有效的高通量筛选，以确定降低PGC-11乙酰化水平的小分子。我们已经开发并验证了一种基于生理相关细胞的检测方法，该方法使用ELISA法来筛选整个MLPCN文库。2)我们将使用先前验证的二次检测来评估识别的HITS与PGC-11靶基因激活的生物学相关性。我们将集中于(I)分析阳性化合物在抵消途径上的作用，以消除药物可能的副作用，(Ii)通过分析PGC-11靶标的基因表达变化，从初步的HTS检测中鉴定具有生物学意义的阳性化合物。3)与MLPCN合作，使用先前验证的第三级检测来开发和表征影响PGC-11乙酰化的化学探针，以测试特异性和作用机制。我们将着重于：(I)我们将证实PGC-11靶基因MCAD和CPT1b的表达增加依赖于PGC-11；(Ii)我们将分析阳性化学探针通过GCN5和SIRT1调节PGC-11乙酰化的特定靶点机制。这些研究的结果将提供化合物的鉴定以及控制PGC-11乙酰化的分子机制，从而将调节由该转录辅助激活因子调控的代谢活动。由于这些代谢活动至少在很大程度上模仿了运动和卡路里限制，因此这些化合物很有可能被用作治疗代谢紊乱和年龄相关疾病的潜在疗法。 公共卫生相关性：锻炼和限制卡路里摄入对包括代谢性疾病在内的年龄相关疾病具有强大的保护作用，因此，这项拨款提案中关于识别可能通过PGC-11去乙酰化模拟身体活动或营养剥夺的化合物的研究可能会转化为潜在的治疗方法。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.