Screen for MURF-1 inhibitors to treat myopathy

描述（由申请人提供）：肌病（肌肉萎缩或肌肉萎缩）是癌症、HIV/AIDS、糖尿病和其他慢性疾病的严重临床并发症，导致发病率增加和预期寿命缩短。虽然已经通过一些干预措施解决了症状，但尚未开发出成功的治疗方法。最近，已经在大鼠中鉴定了在萎缩诱导饥饿条件下表达增强的各种蛋白质。特别是，一组新的基因称为atrogins（atrophysicgenes）的表达显着增加，在骨骼肌的禁食生物体和迅速下降时，恢复喂养。这些基因之一- MURF-1 -的产物是环指结构域泛素E3连接酶，泛素-蛋白酶体降解途径的关键酶。基因敲除和药理学研究表明，这一途径与肌肉萎缩有关。因此，使用MURF-1的抑制剂可以实现与肌肉萎缩相关的蛋白酶体活性的衰减。在第一阶段，将针对适当的底物证明MURF-1的E3泛素化活性。然后，将开发和验证基于酵母的MURF-1抑制剂测定，并筛选天然产物提取物的集合。对于该试验，将克隆以下基因并在酵母S.酿酒酵母：MURF-1的底物，与连接β-半乳糖苷酶报告基因的p53融合;游离MURF-1（E3连接酶）;适合作为选择性对照的E3连接酶。在使测定适应多孔板和高通量筛选可接受的参数后，将筛选来自NCI的天然产物提取物和一组筛选化合物的集合中的MURF-1抑制剂。阶段I的成功完成应导致至少一种具有合理效价（IC 50 1-5 μ g/ml）的提取物先导化合物。在第二阶段，将从最佳提取物先导化合物中分离出活性成分，并从小分子筛选中选择最佳命中物用于先导化合物优化，目的是鉴定新型、有效和选择性的MURF-1抑制剂，以开发用于治疗与各种病理状态相关的肌肉萎缩。

DESCRIPTION (provided by applicant): Myopathy (muscle atrophy, or muscle wasting) is a serious clinical complication of cancer, HIV/AIDS, diabetes, and other chronic diseases, leading to increased morbidity and reduced life expectancy. While symptoms have been addressed by a number of interventions, no successful therapy has been developed. Recently, various proteins whose expression is enhanced under conditions of atrophy-inducing starvation have been identified in rats. In particular, the expression of a set of novel genes called atrogins (atrophyspecific genes) increases significantly in skeletal muscles of fasting organisms and decreases rapidly when feeding is resumed. The product of one of these genes - MURF-1 - is RING-finger domain ubiquitin E3 ligase, a critical enzyme of the ubiquitin-proteasomal degradation pathway. Genetic knockout and pharmacological studies have implicated this pathway in muscle atrophy. Thus, attenuation of proteasomal activity associated with muscle wasting may be achievable using inhibitors of MURF-1. In Phase I, E3 ubiquitination activity of MURF-1 will be demonstrated against an appropriate substrate. Then, a yeastbased assay for inhibitors of MURF-1 will be developed and validated, and a collection of natural products extracts will be screened. For the assay, the following will be cloned and expressed in yeast S. cerevisiae: the substrate of MURF-1, fused to p53 linked to a (3-galactosidase reporter; free MURF-1 (E3 ligase); an E3 ligase suitable as a selectivity control. After the assay is adapted to multiwell plates and to parameters acceptable for high throughput screening, a collection of natural product extracts and a set of screening compounds from the NCI will be screened for inhibitors of MURF-1. Successful completion of Phase I should result in at least one extract lead with reasonable potency (IC50 1-5 ug/ml). In Phase II, active principles will be isolated from the best extract leads and best hits from the small molecule screen will be selected for lead optimization, with the goal of identifying novel, potent, and selective inhibitors of MURF-1 for development as therapies for muscle wasting associated with various pathological states.