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: 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
• 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

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-蛋白酶体途径降解约80-90%的细胞蛋白，是多种疾病新疗法的重要来源。该途径的前蛋白酶体阶段，由泛素E1激活酶、E2结合酶和E3连接酶催化，预计可以提供比蛋白酶体抑制剂更有选择性作用的药物，蛋白酶体抑制剂是由泛素蛋白酶体途径开发的第一类药物，因此比蛋白酶体抑制剂更少受到副作用的影响。证据表明E3酶参与多种生理过程和疾病状态。由于这些原因，大型制药公司花了数年时间寻找新的、选择性的E3抑制剂用于药物开发，但迄今为止的结果令人失望，因为很少有E3抑制剂进入临床试验。考虑到在筛选过程中需要改进检测方法来发现高质量的分子，Progenra在该资助项目的第一阶段开发了简便、均匀的E3连接酶检测方法，包括一种基于筛选的检测方法，可用于底物泛素化和自泛素化。E3连接酶的生理底物（肌钙蛋白I）被配置为基于底物的测定是简单的无名指E3 MuRF1，它与肌肉萎缩或肌病有关，这是许多疾病的病理并发症，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结果。肌肉萎缩是蛋白质分解率增加和蛋白质合成率下降的结果，肌肉重量的二次净损失为10-15%。MuRF1是肌病中过度表达的几个基因之一，它是治疗肌肉萎缩的药物的一个经过充分验证的靶点。在为期2年的II期项目中，建议利用I期开发的MuRF1/Troponin I检测来筛选Progenra的22万个成员小分子文库，以寻找这种底物泛素化的抑制剂。这些抑制剂将使用二级分析来确定选择性和有效性。初步的临床前评估将通过体外和体内肌肉萎缩模型进行，药物化学将用于先导优化。鉴定特异性的MuRF1抑制剂将是开发针对肌肉萎缩的新型靶向疗法的重要的第一步。