Ubiquitin E3 ligase detection by fluorescence resonance transfer

通过荧光共振转移检测泛素 E3 连接酶

基本信息

批准号：
8848079
负责人：
David E Sterner
金额：
$ 61.12万
依托单位：
PROGENRA, INC.
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-02-06 至 2017-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8848079
关键词：
AIDS/HIV problem Acquired Immunodeficiency Syndrome Adverse effects Affect Aging Animal Model Anti-Inflammatory Agents Anti-inflammatory Antineoplastic Agents Applications Grants Atrophic Biological Assay Cachexia Cells Chemicals Clinical Clinical Trials Complex Complication Degradation Pathway Denervation Detection Diabetes Mellitus Disease Dose-Limiting Enzymes Exhibits FDA approved Fasting Fingers Fluorescence Fluorescence Resonance Energy Transfer Genes Glucocorticoids Goals Grant Health Homeostasis Housing Human Genome In Vitro Inflammation Knockout Mice Lead Ligase Malignant Neoplasms Methods Modeling Monitor Multiple Myeloma Muscle Muscle Fibers Muscular Atrophy Myopathy Myosin Heavy Chains Organism Pathology Pathway interactions Performance Pharmaceutical Chemistry Pharmaceutical Preparations Pharmacologic Substance Phase Physiological Physiological Processes Process Property Proteasome Inhibitor Protein Biosynthesis Proteins Proteolysis Refractory Relapse Reporting Resistance Rodent Secondary to Side Skeletal Muscle Small Business Innovation Research Grant Source Specificity Starvation Testing Therapeutic Agents Toxic effect Troponin I Ubiquitin Ubiquitin-Activating Enzymes Weight Work base design drug development drug discovery enzyme pathway feeding high throughput screening improved in vivo Model inhibitor/antagonist interest meetings member multicatalytic endopeptidase complex new therapeutic target novel novel therapeutics overexpression preclinical evaluation prevent protein degradation protein metabolism screening small molecule libraries targeted treatment ubiquitin-protein ligase wasting

项目摘要

DESCRIPTION (provided by applicant): The ubiquitin-proteasome pathway degrades ~80-90% of cellular proteins and is of great interest as a source of new therapeutics for multiple diseases. The pre-proteasomal phase of the pathway, catalyzed by the ubiquitin E1 activating enzyme, E2 conjugating enzyme, and E3 ligase, is predicted to afford drugs that act more selectively than proteasome inhibitors, the first class of drug developed from the ubiquitin proteasome pathway, and thus be less compromised than proteasome inhibitors by side effects. Evidence implicates E3 enzymes in various physiological processes and disease states. For these reasons, major pharmaceutical companies have spent several years identifying novel, selective E3 inhibitors for drug development, but to date the results have been disappointing, as very few E3 inhibitors have entered clinical trials. Reasoning that improved assays were needed to discover high quality molecules in screening, Progenra developed, in Phase I of this grant project, facile, homogeneous assays for E3 ligases of choice, including an -screen based assay that can be employed for substrate ubiquitylation as well as auto-ubiquitylation. The E3 ligase whose physiological substrate (Troponin I) was configured for the substrate based assay is the simple RING finger E3 MuRF1, which is associated with muscle wasting, or myopathy, a pathological complication of numerous diseases, including cancer, HIV/AIDS, and diabetes, as well as a natural consequence of inactivity and aging. Muscle wasting is a result of increased rates of protein breakdown and decreased rates of protein synthesis, with a secondary net loss in muscle weight of 10-15%. MuRF1 is one of several genes overexpressed in myopathy and it is a well-validated target for therapeutic agents to treat muscle wasting. In the phase II 2-year project, it is proposed to utilize the MuRF1/Troponin I assay developed in Phase I to screen Progenra's 220,000 member small molecule library for inhibitors of this substrate ubiquitylation. These inhibitors will be characterized using secondary assays to establish selectivity and efficacy. Initial preclinical evaluation will be performed using in vitro and in vivo models of muscle wasting, and medicinal chemistry will be employed for lead optimization. The identification of specific inhibitors of MuRF1 will be an important first step in producing novel targeted therapies for muscle atrophy.

描述（由申请人提供）：泛素 - 蛋白酶体途径降解了约80-90％的细胞蛋白，并且是多种疾病的新疗法的引起人们的极大兴趣。途径的前膜前阶段是由泛素E1激活酶，E2共轭酶和E3连接酶催化的，预计将提供比蛋白酶体抑制剂更有选择性地起作用的药物，蛋白酶体抑制剂是由泛素蛋白酶体pathway和蛋白酶蛋白酶的构成效应的，而不是蛋白酶蛋白酶蛋白酶的效应，而不是蛋白酶蛋白酶蛋白酶蛋白酶蛋白酶的效应，并且是蛋白酶的效应。证据暗示在各种生理过程和疾病状态下E3酶。由于这些原因，主要的制药公司花了几年的时间来识别新颖的，选择性的E3药物开发抑制剂，但迄今为止，结果令人失望，因为很少有E3抑制剂进入了临床试验。需要改进的测定方法来发现筛查中的高质量分子，后代在该赠款项目的第一阶段开发了E3连接酶的第一阶段，均匀的均质测定法，包括可用于基于底物的基板泛素化的基于屏幕的测定法，并且是自动启动性的。简单的环手指E3 MURF1与肌肉浪费或肌病有关的E3连接酶的配置为基于底物测定的E3连接酶是癌症，癌症，HIV/AIDS和糖尿病，以及无性和无效的自然疾病。肌肉浪费是蛋白质分解率增加和蛋白质合成率降低的结果，肌肉体重的继发净损失为10-15％。 MURF1是肌病中过表达的几种基因之一，它是治疗肌肉浪费的治疗剂的验证靶标。在II期2年期间，建议利用在I期中开发的MURF1/Troponin I测定法，以筛选Gepenra的220,000个成员小分子库来抑制此底物泛素化的抑制剂。这些抑制剂将使用次要测定来确定选择性和功效。最初的临床前评估将使用体外和体内肌肉浪费模型进行，并将采用药物化学进行铅优化。 MURF1的特定抑制剂的鉴定将是产生新型肌肉萎缩靶向疗法的重要第一步。