Biochemical screen for protein ligation

蛋白质连接的生化筛选

• 批准号: 7271822
• 负责人: Michael R Mattern
• 金额: $ 25.31万
• 依托单位: PROGENRA, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-07 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7271822
• 关键词: Adverse effects; Anti-Inflammatory Agents; Anti-inflammatory; Antineoplastic Agents; Area; Attention; Award; Binding; Biochemical; Biological Assay; Biological Factors; Cell Nucleus; Cells; Chemistry; Clinical Trials; Collection; Condition; Cytosol; Degradation Pathway; Detection; Development; Disease; Effectiveness; Endoplasmic Reticulum; Enzymes; Family; Fluorescence; Funding; Goals; Human Biology; Hydrolase; In Vitro; Inflammatory; Label; Lead; Libraries; Ligase; Ligation; Link; Malignant Neoplasms; Measures; Mediating; Methods; Molecular Conformation; Multiple Myeloma; Muscle; Nobel Prize; Noise; Numbers; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phase II Clinical Trials; Physiological Processes; Polyubiquitin; Process; Proteasome Inhibition; Proteasome Inhibitor; Proteins; Range; Reaction; Refractory; Relapse; Screening procedure; Signal Transduction; Testing; Toxic effect; Troponin; UBA Domain; Ubiquitin; Ubiquitin-Activating Enzymes; Ubiquitination; United States Food and Drug Administration; Velcade; Work; base; cost; cost effective; drug discovery; enzyme substrate; fluorophore; high throughput screening; improved; in vitro Assay; inhibitor/antagonist; interest; isopeptidase; multicatalytic endopeptidase complex; novel; novel therapeutics; protein degradation; restoration; small molecule; success; therapeutic target; tumorigenesis; ubiquitin isopeptidase; ubiquitin-protein ligase; wasting

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.

描述（由申请人提供）：由于泛素途径涉及广泛的细胞信号转导和其他机制，它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 获批用于治疗难治性多发性骨髓瘤，取得了初步成功。据信，针对比蛋白酶体抑制更不通用的机制，将产生比万珂副作用更少的药物。 E3 连接酶是将泛素添加到特定目标蛋白或蛋白组上的酶，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。其中一些已在遗传或生化方面与特定疾病相关，并且已开发出通过高通量筛选来寻找抑制剂的测定方法。尽管做出了这些努力，但尚未出现有前景的先导化合物。该提案的目标是开发一种简单的体外 E3 连接酶测定，该测定具有成本效益、检测多聚泛素化并可配置用于高通量筛选。多聚泛素化将通过使用标记的、淬灭剂标记的泛素相关结构域 (UBA) 进行检测。这种测定法将优于目前使用的测定法，增加发现选择性的、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 酶和底物的检测形式，促进已知和新兴 E3 介导的疾病过程的药物发现。将称为泛素的蛋白质标签添加到关键细胞蛋白质上的酶（称为泛素 E3 连接酶）已与多种疾病相关。因此，人们正在寻找抑制相关 E3 连接酶的化合物作为治疗这些疾病的潜在药物。该提案寻求资金来开发一种生化检测方法来测量 E3 连接酶活性；一旦开发出检测方法，它将适用于通过高通量筛选来寻找此类抑制剂。该测定将基于当 E3 连接酶将多个泛素添加到蛋白质时荧光信号的破坏以及随后当连接酶作用被抑制时信号的恢复。在第二阶段，将使用这种测定法筛选化合物集合，目的是寻找有可能产生药物的化合物。