Ubiquitin ligase as a therapeutic target for arthritis

描述（由申请人提供）：类风湿性关节炎（RA）与慢性炎症和滑膜细胞过度生长有关，参与这些过程的因子是发现治疗药物的潜在靶点。Hrd 1，也被称为滑液蛋白，是一种E3泛素连接酶，最近发现在关节炎患者的类风湿性滑膜细胞中存在高水平，进一步的小鼠体内研究将其鉴定为关节病的新致病因子。HrdVs泛素连接酶活性的抑制剂，其引起某些蛋白质的泛素化和最终蛋白酶体降解，因此预期是针对RA或其他炎性病症的活性的优异候选物。与识别这样的抑制剂的目标，该提案描述了一种基于酵母的检测方法，其中人Hrd 1和荧光GFP融合的版本，其底物CD 3-d在酿酒酵母中表达的发展。当这些成分起作用时，人Hrd 1与内源性酵母泛素-蛋白酶体途径元件（E1，E2，蛋白酶体）合作，将CD 3-d-GFP的水平保持在最低水平，导致没有荧光信号。Hrd 1的抑制允许CD 3-d-GFP融合保持完整，产生阳性信号（荧光）。在克隆必要组分并在酵母中表达后，将建立试验。在调整测定条件以产生可接受的信噪比和高通量筛选的其他参数后，天然产物提取物和化合物的集合将通过筛选以鉴定确认的命中物，以作为鉴定合适的先导物的基础。拟议工作的最终商业目标是发现一种有效治疗RA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.