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

发现导致肥胖的新型代谢途径的小分子抑制剂的检测方法开发

基本信息

批准号：
10320035
负责人：
PAUL L FOX
金额：
$ 40.25万
依托单位：
CLEVELAND CLINIC LERNER COM-CWRU
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-03-01 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10320035
关键词：
19 year old Adipocytes Adipose tissue Adult Affect Affinity Automobile Driving Binding Biological Assay Body mass index Cardiovascular Diseases Cell physiology Cells Cellular Assay Chemicals Child Complex Cyclin-Dependent Kinase 5 Detergents Development Diabetes Mellitus Diabetic Diet Dietary Fats Disease Dyslipidemias Endocrinology Enzyme-Linked Immunosorbent Assay Epidemic Event Exhibits FRAP1 gene Fatty acid glycerol esters Flowcharts Funding Funding Opportunities Future Genetic Goals Health High Fat Diet Human Hyperinsulinism Hypertension Immunoglobulin Joining Region Immunosuppression In Vitro Insulin Kinetics Laboratories Libraries Ligase Lipids Longevity Malignant Neoplasms Mediating Mental Depression Metabolic Metabolic Pathway Miniaturization Modification Molecular Molecular Weight Mus Mutation National Institute of Diabetes and Digestive and Kidney Diseases Obesity Outcome Pathologic Pathology Pathway interactions Peptides Phosphorylation Phosphotransferases Population Prevention Procedures Protein Biosynthesis Proteins Reagent Recombinants Reporting Research Ribosomal Protein S6 Ribosomal Protein S6 Kinase Risk Factors Signal Pathway Sirolimus Site Solvents Structure-Activity Relationship Surface Plasmon Resonance Technology Testing Therapeutic Therapeutic Agents Therapeutic Uses Thinness Time Toxic effect Triage Triglycerides Validation Weight analog assay development base candidate validation diet-induced obesity dietary experimental study fatty acid-transport protein glucose tolerance glutamyl-prolyl-tRNA synthetase high throughput screening imaging agent improved inhibitor interest lipid metabolism long chain fatty acid mimetics mouse model novel pre-clinical prevent programs prototype response side effect small molecule inhibitor small molecule libraries therapeutic candidate uptake

项目摘要

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.

项目摘要/摘要在美国，肥胖是一个流行级问题，影响了约35％的成年人口，其中20％ 19岁以下的儿童。升高的体重指数与高血压，血脂异常和高胰岛素血症 - 多种病理的所有危险因素，包括糖尿病，心血管疾病，癌症，寿命缩短，甚至抑郁。我们的研究计划的长期目标是开发低 - 新发现的肥胖驱动代谢途径的分子量抑制剂（LMWI）实验室。本申请的直接目标是制定评估和必要的试剂以允许飞行员屏幕并准备小分子抑制剂的未来高通量屏幕（HTS）发现预防或减少肥胖及其病理后果的治疗剂。我们最近报告了一个新的 MTORC1-S6K1轴的靶标，即谷氨酸 - 丙酰tRNA合成酶（EPRS）。 S6K1直接在连接催化合成酶结构域的接头域中的Ser999磷酸化EPRS。值得注意的是具有磷酸化的SER999-TO-ALA突变的遗传改性小鼠暴露了重量的降低和白色脂肪组织。由于葡萄糖耐量的提高，它们在代谢上具有健康状态喂养高脂饮食时，寿命延长，小鼠保持苗条。这些结果极大地暗示了EPRS MTORC1-S6K1的临界下游靶标决定肥胖。我们建议使用alphascreen 寻求S6K1介导的EPR磷酸化LMWI的技术。我们将利用最近的优势我们实验室中的发现表明S6K1和EPR之间具有强大的约束力。抑制这种结合特异性阻断EPRS磷酸化而不抑制S6K1的催化活性或磷酸化的磷酸化或它的规范靶标，例如核糖体蛋白S6。这，我们预计我们的方法将揭示小分子防止脂肪积累的抑制剂，而不会破坏MTORC1-S6K1轴的主要功能，例如全球蛋白质合成。我们进一步期望这样的LMWIS会显着减少广告方面与已知的MTORC1抑制剂（如雷帕霉素）相比。作为一个特定的假设，我们提出了 S6K1*与EPR的相互作用的有效LMWI可以安全有效地减少脂肪的积累在脂肪细胞和全身肥胖中。在这里，我们将开发体外和细胞分析以促进发现并验证这种抑制剂。在AIM 1中，我们将开发基于字母的测定法以询问 S6K1*/EPRS相互作用及其抑制作用。在AIM 2中，我们将制定正交评估以进行验证，确定选择性和结构活性关系，以及细胞功能和毒性的评估。 AIM 3我们将使用这些新开发的测定法进行试验屏幕，并验证和分类候选人。这些研究的完成将为未来的大型潜水复合库，候选物的验证和优先级，结构相关化合物的测试以及化学修饰以最大化效率，以允许在饮食肥胖的小鼠模型中进行随后的测试。