Atrogin-1 inhibitors for Muscle Wasting

描述(申请人提供)：肌肉萎缩(消瘦)是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，已在大鼠中发现了在萎缩性饥饿条件下表达增强的各种蛋白质。特别是，在禁食生物的骨骼肌中，一组名为萎缩素(萎缩特异性基因)的新基因的表达显著增加，当恢复进食时，表达迅速下降。其中一个基因-阿托金-1的产物是F-box蛋白泛素E3连接酶，它是泛素-蛋白酶体降解途径的关键酶。这一途径与肌肉萎缩有关，因为在模型系统中，蛋白酶体抑制剂可以防止肌肉萎缩。使用阿托金-1的抑制剂可以选择性地减弱与肌肉萎缩相关的蛋白酶体活性。在第一阶段，在酵母中异位表达的阿托金-1的E3泛素化活性将与已知底物钙调神经磷酸酶结合。然后，将建立和验证一种基于酵母的阿托金-1检测方法，为高通量筛选化合物和天然产物集合以发现阿托金-1的第二阶段抑制剂做准备。在该检测中，以下内容将被克隆并在酿酒酵母中表达：阿托金-1的底物，与连接到β-半乳糖苷酶报告基因的P53融合；阿托金-1 E3连接酶复合体；以及适合作为选择性控制的E3连接酶。验证这一模块化实验的初步实验结果表明，在酵母中异位表达的人E3连接酶、p-53激活的报告质粒和P53融合连接酶底物给出零信号，但当E3连接酶缺失时产生强烈的报告信号，与E3泛素化/降解融合P53一致。第一阶段的成功完成将导致一项有效的筛选试验，可用于第二阶段。在第二阶段，活性成分将从最好的提取物引线中分离出来，目标是确定新的、有效的和选择性的阿托金-1抑制剂，用于开发用于治疗糖尿病相关肌肉萎缩的辅助疗法。

DESCRIPTION (provided by applicant): Muscle atrophy (wasting) is a serious clinical complication of diabetes and other chronic pathoses, 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 (atrophy-specific genes) increases significantly in skeletal muscles of fasting organisms and decreases rapidly when feeding is resumed. The product of one of these genes - atrogin-1 - is an F-box protein ubiquitin E3 ligase, a critical enzyme of the ubiquitin-proteasomal degradation pathway. This pathway is implicated in muscle atrophy, since proteasome inhibitors protect against muscle wasting in model systems. Selective attenuation of proteasomal activity associated with muscle wasting may be achievable using inhibitors of atrogin-1. In Phase 1, E3 ubiquitination activity of atrogin-1 ectopically expressed in yeast will be demonstrated against a known substrate, calcineurin. Then, a yeast-based assay for atrogin-1 will be developed and validated, in preparation for high throughput screening of compound and natural products collections to discover inhibitors of atrogin-1 in Phase 2. For the assay, the following will be cloned and expressed in yeast S. cerevisiae: the substrate of atrogin-1, fused to p53 linked to a beta-galactosidase reporter; atrogin-1 E3 ligase complex; and an E3 ligase suitable as a selectivity control. Results of pilot experiments to validate this modular assay demonstrate that human E3 ligases, p-53 activated reporter plasmid, and p53-fused ligase substrate ectopically expressed in yeast give a null signal, but produce a robust reporter signal when E3 ligase is left out, consistent with ubiquitination/degradation of fused p53 by E3. Successful completion of Phase 1 should result in a validated screening assay that can be utilized in Phase 2. In Phase 2, active principles will be isolated from the best extract leads, with the goal of identifying novel, potent, and selective inhibitors of atrogin-1 for development as adjuvant therapy of muscle wasting associated with diabetes.