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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：
10115720
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 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10115720
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 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/antagonist 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.

项目总结/文摘