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.

项目概要/摘要在美国，肥胖是一个普遍存在的问题，影响着约 35% 的成年人口和 20% 的成年人口。 19岁以下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、我们研究计划的长期目标是开发一种低寿命的方法。我们的研究人员阐明了新发现的肥胖驱动代谢途径的分子量抑制剂（LMWI）该应用程序的直接目标是开发分析方法和必要的试剂以进行试点。筛选并为未来的小分子抑制剂高通量筛选 (HTS) 做准备我们最近报道了一种新的治疗药物来预防或减少肥胖及其病理后果。 mTORC1-S6K1 轴的靶点，即谷氨酰脯氨酰 tRNA 合成酶 (EPRS) 直接。磷酸化连接催化合成酶结构域的接头结构域中 Ser999 处的 EPRS。具有磷酸缺陷 Ser999 至 Ala 突变的转基因小鼠表现出体重显着减轻葡萄糖耐量改善表明它们代谢健康。延长寿命，并且在喂食高脂肪饮食时小鼠仍保持苗条。这些结果强烈表明 EPRS 是一种有效的治疗方法。 mTORC1-S6K1 决定肥胖的关键下游靶标我们建议使用 AlphaScreen。我们将利用最近的技术来寻找 S6K1 介导的 EPRS 磷酸化的 LMWI。我们实验室的研究结果表明 S6K1 和 EPRS 之间有很强的结合，这种结合受到抑制。特异性阻断 EPRS 磷酸化，而不抑制 S6K1 的催化活性或因此，我们预计我们的方法将揭示小分子。防止脂肪堆积而不破坏 mTORC1-S6K1 轴主要功能的抑制剂，例如整体蛋白质合成，我们进一步预计此类 LMWI 将表现出显着减少的不利副作用。与已知的 mTORC1 抑制剂（例如雷帕霉素）相比，我们提出了一个具体的假设。 S6K1* 与 EPRS 相互作用的有效 LMWI 将安全有效地减少脂肪堆积在这里，我们将开发体外和细胞测定法以促进发现。在目标 1 中，我们将开发一种基于 AlphaScreen 的检测方法来询问。 S6K1*/EPRS 相互作用及其抑制在目标 2 中，我们将开发正交测定法进行验证，选择性和构效关系的测定，以及细胞功能和毒性的评估。目标 3 我们将使用这些新开发的检测方法进行试点筛选，并对候选药物进行验证和分类。这些研究的完成将为未来大规模、多样化的高温超导提供必要的试剂和分析方法。化合物库、候选化合物的验证和优先排序、结构相关化合物的测试，以及进行化学修饰以最大限度地提高功效，以便随后在饮食肥胖小鼠模型中进行测试。