Biochemical screen for protein ligation

描述（由申请人提供）：由于其参与广泛的细胞信号转导和其他机制，泛素途径现在被认为是发现治疗多种疾病的药物的新机会。2003年，蛋白酶体抑制剂Velcade被批准用于治疗难治性多发性骨髓瘤，取得了初步成功。人们相信，针对比蛋白酶体抑制更不普遍的机制，将产生比Velcade副作用更少的药物。E3连接酶是一种将泛素添加到特定靶蛋白或蛋白质群上的酶，在过去五年中得到了广泛的研究，被认为是选择性药物发现的有希望的目标。其中一些已经与特定疾病的遗传或生化联系起来，并且已经开发出通过高通量筛选找到抑制剂的检测方法。尽管做出了这样的努力，但还没有出现有希望的先导化合物。本提案的目标是开发一种简单的体外E3连接酶测定方法，该方法具有成本效益，可以检测聚泛素化，并且可以配置为高通量筛选。多泛素化将通过使用标记的，淬灭标记泛素相关结构域（UBA）来检测。这种检测方法将优于目前使用的方法，增加了发现选择性的、治疗上有用的抑制剂的可能性。最终的商业目标是开发一种可应用于各种E3酶和底物的分析格式，促进已知和新兴E3介导疾病过程的药物发现。酶（称为泛素E3连接酶）在关键细胞蛋白上添加泛素蛋白标签，与各种疾病有关；因此，抑制相关E3连接酶的化合物正在被寻找作为治疗疾病的潜在药物。该提案寻求资金开发生化分析来测量E3连接酶的活性；一旦该方法被开发出来，就可以通过高通量筛选来发现这种抑制剂。当E3连接酶将多种泛素添加到蛋白质上时，荧光信号会被破坏，当连接酶的作用被抑制时，荧光信号会被恢复。在第二阶段，化合物收集将使用这种方法进行筛选，目的是发现有可能导致药物的化合物。

DESCRIPTION (provided by applicant): Because of its involvement in a broad range of cellular signal transduction and other mechanisms, the ubiquitin pathway is now recognized as a new opportunity for discovering drugs to treat a variety of diseases. Initial success was achieved with the approval of the proteasome inhibitor Velcade in 2003 for refractory multiple myeloma. It is believed that targeting a less general mechanism than proteasome inhibition would yield drugs with fewer side effects than those seen with Velcade. E3 ligases, the enzymes that add ubiquitins to specific target proteins or groups of proteins, have been studied extensively in the last five years and are considered promising targets for selective drug discovery. Several of them have been linked genetically or biochemically with specific diseases, and assays have been developed to find inhibitors via high throughput screening. Despite this effort, no promising lead compounds have emerged. The goal of this proposal is to develop a simple in vitro E3 ligase assay that is cost effective, detects poly- ubiquitylation, and can be configured for high throughput screening. Poly-ubiquitylation will be detected by using a tagged, quencher-labeled ubiquitin-associated domain (UBA). Such an assay will be superior to those now in use, increasing the likelihood of discovering selective, therapeutically useful inhibitors. The ultimate commercial goal is to develop an assay format that can be applied to various E3 enzymes and substrates, facilitating drug discovery for known and emerging E3-mediated disease processes. Enzymes (called ubiquitin E3 ligases) that add a protein tag known as ubiquitin to key cellular proteins have been linked to various diseases; for this reason, compounds that inhibit relevant E3 ligases are being sought as potential drugs to treat the diseases. This proposal seeks funds to develop a biochemical assay to measure E3 ligase activity; once the assay is developed, it will be adapted to find such inhibitors through high throughput screening. The assay will be based on the destruction of a fluorescence signal when multiple ubiquitins are added to a protein by the E3 ligase and subsequent restoration of the signal when the ligase action is inhibited. In Phase II, compound collections will be screened using this assay, with the aim of finding compounds that have the potential to lead to drugs.