Ubiquitin ligase as a therapeutic target for arthritis

描述(申请人提供)：类风湿性关节炎(RA)与慢性炎症和滑膜细胞过度生长有关，参与这些过程的因素是发现治疗药物的潜在靶点。Hrd1，又称滑膜小提素，是一种E3泛素连接酶，最近发现在关节炎患者的类风湿滑膜细胞中存在高水平，在小鼠体内的进一步研究发现，它是关节病的一个新的致病因素。Hrdvs泛素连接酶活性的抑制剂可导致泛素化，最终导致某些蛋白质的蛋白酶体降解，因此有望成为治疗RA或其他炎症性疾病的优秀候选药物。为了识别这些抑制物，本提案描述了一种基于酵母的检测方法的发展，在该方法中，人Hrd1及其底物CD3-d的荧光GFP融合版本在酿酒酵母中表达。当这些成分发挥作用时，人类Hrd1与内源酵母泛素-蛋白酶体途径元件(E1、E2、蛋白酶体)合作，将CD3-d-GFP的水平保持在最低水平，导致没有荧光信号。抑制Hrd1使CD3-d-GFP融合保持完整，产生积极的信号(荧光)。在克隆必要的成分并在酵母中表达后，将建立检测方法。在调整分析条件以产生可接受的高通量筛选的信噪比和其他参数后，将通过筛查运行天然产品提取物和化合物的集合，以确定确认的命中，作为确定合适线索的基础。这项拟议工作的最终商业目标是发现一种有效治疗类风湿关节炎或其他自身免疫性疾病的药物。

DESCRIPTION (provided by applicant): Rheumatoid arthritis (RA) is associated with chronic inflammation and overgrowth of synovial cells, and factors involved in these processes are potential targets for discovery of therapeutic drugs. Hrd1, also known as synoviolin, is an E3 ubiquitin ligase enzyme recently found to be present at high levels in rheumatoid synovial cells from arthritis patients, and further in vivo studies in mice identified it as a novel causative factor in arthropathy. Inhibitors of HrdVs ubiquitin ligase activity, which causes ubiquitination and ultimately proteasomal degradation of certain proteins, would thus be expected to be excellent candidates for activity against RA or other inflammatory disorders. With the goal of identifying such inhibitors, this proposal describes the development of a yeast-based assay in which human Hrd1 and a fluorescent GFP-fused version of its substrate CD3-d are expressed in Saccharomyces cerevisiae. When these components are functional, human Hrd1, in collaboration with endogenous yeast ubiquitin-proteasomal pathway elements (E1, E2, proteasomes), keeps the level of CD3-d-GFP at a minimum, resulting in no fluorescent signal. Inhibition of Hrd1 allows the CD3-d-GFP fusion to remain intact, producing a positive signal (fluorescence). The assay will be set up following cloning of the necessary components and expressing them in yeast. After adjusting assay conditions to produce an acceptable signal:noise ratio and other parameters of high throughput screening, collections of natural product extracts and compounds will be run through the screen to identify confirmed hits, to serve as a basis for identifying suitable leads. The ultimate commercial goal of the proposed work is to discover a drug with efficacy as a therapy against RA or other autoimmune diseases.