Ubiquitin E3 ligase detection by fluorescence resonance transfer

通过荧光共振转移检测泛素 E3 连接酶

• 批准号: 8648089
• 负责人: David E Sterner
• 金额: $ 72.33万
• 依托单位: PROGENRA, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-06 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8648089
• 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; 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; public health relevance; screening; small molecule libraries; ubiquitin-protein ligase; wasting

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.

泛素-蛋白酶体途径降解约80-90%的细胞蛋白质，并且作为一种生物降解途径而引起极大兴趣。 多种疾病的新疗法的来源。蛋白酶体前阶段的途径，催化的 泛素E1激活酶、E2结合酶和E3连接酶被预测可以提供作用于 比蛋白酶体抑制剂更有选择性，这是从泛素蛋白酶体开发的第一类药物 途径，因此比蛋白酶体抑制剂更少受到副作用的损害。证据显示E3 酶在各种生理过程和疾病状态。因此，大型制药公司 许多公司已经花了数年时间来确定用于药物开发的新型选择性E3抑制剂，但迄今为止， 结果令人失望，因为很少有E3抑制剂进入临床试验。推理 Progenra开发的第一阶段，需要改进的检测方法来发现高质量的筛选分子 在这个资助项目中，我们选择了一种简便、均质的E3连接酶测定方法，包括一种基于筛选的测定方法 其可用于底物泛素化以及自身泛素化。E3连接酶， 用于基于底物的测定的生理底物（肌钙蛋白I）是简单的RING指E3 MuRF 1与肌肉萎缩或肌病有关，肌肉萎缩或肌病是许多肌肉萎缩的病理并发症。 这些疾病包括癌症、艾滋病毒/艾滋病和糖尿病，以及不活动和衰老的自然后果。 肌肉萎缩是蛋白质分解速率增加和蛋白质合成速率降低的结果， 肌肉重量的二次净损失为10- 15%。MuRF 1是在大肠杆菌中过表达的几个基因之一。 并且它是治疗肌肉萎缩的治疗剂的充分验证的靶标。第二阶段，2年 项目中，建议利用I期开发的MuRF 1/肌钙蛋白I检测来筛选Progenra的 220，000个成员的小分子文库，用于抑制该底物泛素化。这些抑制剂将 使用二次测定来表征以确定选择性和功效。初始临床前评价将 使用体外和体内肌肉萎缩模型进行，药物化学将用于 铅优化。鉴定MuRF 1的特异性抑制剂将是生产MuRF 1的重要的第一步。 肌肉萎缩的新靶向疗法。