Assay Development for Discovery of a Small Molecule Inhibitor of a Novel Metabolic Pathway that Drives Obesity

项目摘要/摘要 在美国，肥胖是一个流行的问题，影响着大约35%的成年人口和20%的 19岁以下的儿童。体重指数升高与高血压、血脂异常和 高胰岛素血症-多种疾病的所有危险因素，包括糖尿病、心血管疾病、癌症、 寿命缩短，甚至抑郁。我们研究计划的长期目标是开发一种低成本的 我们新发现的肥胖代谢途径的分子量抑制物(LMWI) 实验室。这个应用程序的直接目标是开发分析和必要的试剂，以允许进行试验 筛选并为未来高通量筛选(HTS)发现小分子抑制剂做准备 预防或减少肥胖及其病理后果的治疗剂。我们最近报道了一起新的 MTORC1-S6K1轴的靶标，即谷氨酰脯氨基tRNA合成酶(Eprs)。S6K1直接 在连接催化合成酶结构域的连接器域中，使Ser999处的EPR磷酸化。值得注意的是， 携带磷酸缺失Ser999-to-Ala突变的转基因小鼠体重显著减轻 和白色脂肪组织。他们的代谢是健康的，这表明糖耐量的改善和 延长寿命，当喂食高脂肪食物时，小鼠保持苗条。这些结果强烈地表明EPRS是一种 决定肥胖的mTORC1-S6K1的关键下游靶点。我们建议使用AlphaScreen 寻找S6K1介导的EPRS磷酸化的低相对分子质量的技术。我们将利用最近的 我们实验室的研究结果表明，S6K1与Eprs之间有很强的结合。抑制这种结合 在不抑制S6K1的催化活性或S6K1的磷酸化的情况下，特异性地阻断EPRS的磷酸化 它的典型靶点是核糖体蛋白S6。因此，我们预计我们的方法将揭示小分子 在不破坏mTORC1-S6K1轴主要功能的情况下防止脂肪堆积的抑制剂， 比如全球蛋白质合成。我们进一步预计，这种低体重指数的不利因素将显著减少。 与已知的mTORC1抑制剂(如雷帕霉素)相比的效果。作为一个具体的假设，我们提出 S6K1*与Eprs相互作用的有效LMWI将安全有效地减少脂肪堆积 脂肪细胞和全身肥胖症。在这里，我们将开发体外和细胞测试，以促进发现 以及此类抑制剂的有效性。在目标1中，我们将开发一种基于AlphaScreen的测试来审问 S6K1*/Eprs相互作用及其抑制作用。在目标2中，我们将开发用于验证的正交分析， 选择性和构效关系的测定，细胞功能和毒性的评估。在……里面 目标3我们将使用这些新开发的化验方法进行试点筛选，并对候选人进行验证和分类。 这些研究的完成将为未来大规模、多样化的HTS提供必要的试剂和分析方法 化合物文库，候选化合物的验证和优先顺序，结构相关化合物的测试，以及 化学修饰以最大限度地提高疗效，以允许随后在小鼠饮食肥胖模型中进行测试。

Project Summary/Abstract Obesity is an epidemic-scale problem in the U.S. affecting about 35% of the adult population and 20% of children under 19 years of age. Elevated body mass index is associated with hypertension, dyslipidemia, and hyperinsulinemia – all risk factors for multiple pathologies including diabetes, cardiovascular disease, cancer, shortened lifespan, and even depression. The long-term goal of our research program is to develop a low- molecular weight inhibitor (LMWI) of a newly discovered adiposity-driving metabolic pathway elucidated by our laboratory. The immediate goal of this application is to develop assays and necessary reagents to permit pilot screens and prepare for a future high-throughput screen (HTS) of small-molecule inhibitors to discover therapeutic agents to prevent or reduce obesity and its pathological consequences. We recently reported a new target of the mTORC1-S6K1 axis, namely, glutamyl-prolyl tRNA synthetase (EPRS). S6K1 directly phosphorylates EPRS at Ser999 in the linker domain that joins the catalytic synthetase domains. Remarkably, genetically-modified mice with a phospho-deficient Ser999-to-Ala mutation exhibit marked reduction in weight and white adipose tissue. They are metabolically healthy as indicated by improved glucose tolerance and extended lifespan, and mice remain lean when fed a high-fat diet. These results strongly implicate EPRS as a critical downstream target of mTORC1-S6K1 that determines adiposity. We propose to use AlphaScreen technology to seek LMWIs of S6K1-mediated phosphorylation of EPRS. We will take advantage of recent findings in our laboratory that show strong binding between S6K1 and EPRS. Inhibition of this binding specifically blocks EPRS phosphorylation without inhibiting the catalytic activity of S6K1 or phosphorylation of its canonical targets such as ribosomal protein S6. Thus, we anticipate our approach will reveal small-molecule inhibitors that prevent fat accumulation without disrupting the principal functions of the mTORC1-S6K1 axis, such as global protein synthesis. We further expect that such LMWIs will exhibit markedly reduced adverse side effects compared to known inhibitors of mTORC1, such as rapamycin. As a specific hypothesis, we propose that an effective LMWI of the interaction of S6K1* with EPRS will safely and efficiently reduce fat accumulation in adipocytes and whole body adiposity. Here we will develop in vitro and cellular assays to facilitate discovery and validation of such inhibitors. In Aim 1 we will develop an AlphaScreen-based assay to interrogate S6K1*/EPRS interaction, and its inhibition. In Aim 2 we will develop orthogonal assays for validation, determination of selectivity and structure-activity relationship, and assessment of cell function and toxicity. In Aim 3 we will use these newly-developed assays to conduct pilot screens, and validate and triage candidates. Completion of these studies will provide the reagents and assays necessary for a future HTS of large, diverse compound libraries, validation and prioritization of candidates, testing of structurally-related compounds, and chemical modification to maximize efficacy to permit subsequent testing in mouse models of dietary obesity.