Ubiquitin E3 ligase detection by fluorescence resonance transfer

描述(由申请人提供)：泛素-蛋白酶体途径可降解~80%-90%的细胞蛋白质，作为多种疾病的新疗法的来源非常有兴趣。该途径的蛋白酶体前阶段由泛素E1活化酶、E2结合酶和E3连接酶催化，预计将提供比蛋白酶体抑制剂更具选择性的药物，蛋白酶体抑制剂是从泛素蛋白酶体途径发展而来的第一类药物，因此比蛋白酶体抑制剂更不容易受到副作用的影响。有证据表明，E3酶与各种生理过程和疾病状态有关。出于这些原因，各大制药公司花了几年时间寻找新的、选择性的E3抑制剂用于药物开发，但到目前为止，结果一直令人失望，因为很少有E3抑制剂进入临床试验。由于需要改进的分析方法来发现筛选中的高质量分子，Progenra在该赠款项目的第一阶段开发了所选的E3连接酶的简便、均质分析方法，包括可用于底物泛素化和自动泛素化的基于筛选的分析方法。其生理底物(肌钙蛋白I)被配置用于底物分析的E3连接酶是简单的无名指E3MuRF1，它与肌肉萎缩或肌病有关，肌病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，以及缺乏运动和衰老的自然后果。肌肉萎缩是蛋白质分解速度增加和蛋白质合成速度降低的结果，肌肉重量的第二次净损失为10%-15%。MuRF1是在肌病中过度表达的几个基因之一，是治疗药物治疗肌肉萎缩的有效靶点。在第二阶段为期两年的项目中，建议利用第一阶段开发的MuRF1/肌钙蛋白I试验来筛选Progenra的220,000个成员小分子文库，以寻找该底物泛素化的抑制剂。将使用二次化验对这些抑制剂进行表征，以确定选择性和有效性。初步的临床前评估将使用体外和体内肌肉萎缩模型进行，并将使用药物化学进行铅优化。MuRF1特异性抑制剂的鉴定将是产生针对肌肉萎缩的新型靶向治疗的重要第一步。

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.