Osteoporosis Screen for Praja 1 E3 Ligase Inhibitors

描述（由申请人提供）：骨质疏松症是老年人群维持健康和生活质量的主要挑战。其中最有前途和最少利用的治疗干预措施是那些旨在增加骨密度的治疗。选择和验证可以被操纵以实现这种合成代谢作用的靶点取决于与骨形成相关的信息量的增加。药物发现的一个新领域-通过泛素途径酶的蛋白质稳态调节-最近已被证明与合成代谢性骨质疏松症药物的研究有关。Prajal是一种环指E3泛素连接酶，已发现其催化泛素化，随后诱导Dlxin-1的蛋白酶体降解，Dlxin-1是转录激活因子DIx 5的共激活因子。DIx 5与程序性骨形成相关。因此，假设E3连接酶的选择性抑制剂可增加骨密度。在第一阶段，建议建立一个基于酵母的Prajal抑制剂筛选试验。E3系统（Prajal）的基本组成部分将在S.酿酒酵母，沿着与p53连接的人Dlxin-1，和监测p53活性（β-半乳糖苷酶活化）的报道构建体。然后将重构的E3连接酶功能和报告系统配置并验证为Prajal介导的Dlxin-1泛素化抑制剂的高通量筛选。植物和海洋生物提取物的集合，沿着一个小的化合物集合，将被筛选Prajal E3泛素连接酶活性的有效抑制剂。在第二阶段，活性提取物的分馏将由测定指导，以鉴定活性成分。从这一努力中产生的新的纯化合物将被认为是骨质疏松症的开发候选者。模块化测定构建格式将允许评价与各种疾病相关的其他E3。

DESCRIPTION (provided by applicant): 0steoporosis represents a major challenge to the maintenance of health and quality of life in an aging population. Among the most promising and least exploited therapeutic interventions are those treatments designed to increase bone density. The selection and validation of targets that can be manipulated to achieve this anabolic effect depends on an increasing amount of information relating to bone formation. A novel area for drug discovery - protein homeostatic regulation via ubiquitin pathway enzymes -- has recently been demonstrated to have relevance to the search for anabolic osteoporosis drugs. Prajal, a RING-finger E3 ubiquitin ligase, has been found to catalyse ubiquitination and, subsequently, induce proteasomal degradation of Dlxin-1, a coactivator of the transcriptional activator DIx5. DIx5 is associated with programmed bone formation. Thus, selective inhibitors of this E3 ligase are hypothesized to increase bone density. In Phase I, it is proposed to establish a yeast-based screening assay for inhibitors of Prajal. Essential components of the E3 system (Prajal) will be cloned and expressed in S. cerevisiae, along with human Dlxin-1 linked to p53, and a reporter construct that monitors p53 activity (beta-galactosidase activation). The reconstructed E3 ligase function and reporter system will then be configured and validated as a high throughput screen for inhibitors of Prajal-mediated Dlxin-1 ubiquitination. Collections of plant and marine organism extracts, along with a small compound collection, will be screened for potent inhibitors of Prajal E3 ubiquitin ligase activity. In Phase II, fractionation of active extracts will be guided by the assay, to identify active principles. Novel pure compounds arising from this effort will be considered as development candidates for osteoporosis. The modular assay construction format will permit evaluation of other E3s that are associated with a variety of diseases.