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

描述（由申请人提供）：运动和热量限制对年龄相关疾病（包括代谢紊乱，如肥胖和糖尿病）具有非常强大的保护作用。在关键的靶组织中，骨骼肌被认为是这些保护作用的介质之一。这些作用的主要调节成分是转录辅激活因子PGC-11，其活性在运动或热量限制下上调。相反，PGC-11的活性在身体不活动的条件下或在用高脂肪热量饮食喂养后被抑制。重要的是，骨骼肌中PGC-11活性的增加足以预防年龄相关疾病并延长寿命。通过Sirt 1对PGC-11进行脱乙酰化是在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，GCN 5对PGC-11的乙酰化导致在非运动或高脂肪热量饮食情况下发现的无活性蛋白质。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剥夺条件。为了解决这一假设，本提案旨在使用定量测量PGC-11的乙酰化状态的高通量筛选（HTS）来鉴定这些化合物。我们有三个具体目标：1）与MLPCN合作，实施经验证的高通量筛选，以识别降低PGC-11乙酰化水平的小分子。我们已经开发并验证了使用ELISA筛选整个MLPCN文库的生理相关的基于细胞的测定。2)我们将采用先前验证的二级试验来评估鉴定的命中对PGC-11靶基因激活的生物学相关性。我们将重点关注（i）分析阳性化合物对抵消途径的影响，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，从主要HTS试验中鉴定生物学相关的阳性化合物。3)与MLPCN合作开发和表征影响PGC-11乙酰化的化学探针，使用先前验证的三级测定法测试特异性和作用机制。我们将重点关注（i）我们将确认PGC-11靶基因MCAD和CPT 1b的表达增加依赖于PGC-11，（ii）我们将分析阳性化学探针通过GCN 5和SIRT 1调节PGC-11乙酰化的特定靶机制。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，并因此调节由该转录辅激活因子调节的代谢活动。由于这些代谢活动至少在很大程度上模仿运动和热量限制，因此这些化合物可能用作治疗代谢紊乱和年龄相关疾病的潜在疗法是非常合理的。 公共卫生相关性：运动和热量限制对年龄相关疾病（包括代谢疾病）具有有效的保护作用，因此这项研究旨在确定可能通过PGC-11脱乙酰化模拟身体活动或营养剥夺的化合物，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.