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-蛋白酶体途径可降解约 80-90% 的细胞蛋白质，作为 多种疾病新疗法的来源。该途径的前蛋白酶体阶段，由 泛素 E1 激活酶、E2 结合酶和 E3 连接酶预计可提供作用的药物 比蛋白酶体抑制剂更具选择性，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受到副作用的影响更小。有证据表明 E3 各种生理过程和疾病状态中的酶。由于这些原因，各大制药公司 公司花费了数年时间来寻找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。推理认为 Progenra 在第一阶段开发了筛选中需要改进的检测方法来发现高质量的分子 该资助项目的重点是对所选 E3 连接酶进行简便、均质的检测，包括基于筛选的检测 可用于底物泛素化以及自身泛素化。 E3连接酶的 为基于底物的测定配置的生理底物（肌钙蛋白 I）是简单的环指 E3 MuRF1，与肌肉萎缩或肌病有关，肌病是许多疾病的病理并发症 疾病，包括癌症、艾滋病毒/艾滋病和糖尿病，以及缺乏活动和衰老的自然后果。 肌肉萎缩是蛋白质分解率增加和蛋白质合成率降低的结果， 肌肉重量二次净损失 10-15%。 MuRF1 是在细胞中过度表达的几个基因之一 肌病，它是治疗肌肉萎缩的治疗药物的经过充分验证的靶点。在为期2年的第二阶段 项目中，建议利用第一阶段开发的 MuRF1/Troponin I 检测来筛选 Progenra 该底物泛素化抑制剂的 220,000 个成员小分子库。这些抑制剂将 使用二次测定来表征以确定选择性和功效。初步临床前评估将 使用肌肉萎缩的体外和体内模型进行，并将采用药物化学 领先优化。 MuRF1 特异性抑制剂的鉴定将是生产中重要的第一步 肌肉萎缩的新型靶向疗法。