Discovery of inhibitors against ubiquitin specific protease in human DNA damage r

人类 DNA 损伤中泛素特异性蛋白酶抑制剂的发现

• 批准号: 8063542
• 负责人: Zhihao Zhuang
• 金额: $ 3.71万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8063542
• 关键词: Adverse effects; Animal Model; Biological Assay; Cancerous; Catalytic Domain; Cell model; Cell physiology; Cells; Chemicals; Chemistry; Childhood Acute Lymphocytic Leukemia; Chromatin; Collaborations; Colon Carcinoma; Complex; DNA Crosslinking; DNA Damage; DNA biosynthesis; Detection; Deubiquitinating Enzyme; Disease; Dot Immunoblotting; Enzymes; Fanconi' s Anemia; Fluorescence; Fluorogenic Substrate; Future; Genomics; Goals; Human; Human Ubiquitin; Individual; Intervention; Lead; Libraries; Malignant Neoplasms; Malignant neoplasm of prostate; Membrane Protein Traffic; Molecular Bank; Natural Immunity; Neurodegenerative Disorders; Pathway interactions; Pharmaceutical Preparations; Phase; Phosphotransferases; Platinum; Play; Proteasome Inhibitor; Protein Deregulation; Proteins; Proteome; Reporter; Role; Screening procedure; Signal Transduction; Specificity; Staging; Structure-Activity Relationship; Testing; Therapeutic; Therapeutic Index; United States National Institutes of Health; Validation; Yeasts; base; cancer cell; high throughput screening; human DNA damage; human disease; in vivo; inhibitor/antagonist; malignant breast neoplasm; novel therapeutics; protein degradation; public health relevance; repository; response; small molecule; ubiquitin C-terminal 7-amido-4-methylcoumarin; ubiquitin-specific protease

DESCRIPTION (provided by applicant): The overall goal of this project is to identify potent and specific inhibitors for human deubiquitylating enzyme through a quantitative high-throughput screening (qHTS). Besides the well-known roles in protein degradation, protein ubiquitylation also plays essential roles in many other cellular processes, including membrane trafficking, innate immunity, kinase signaling, chromatin dynamics and DNA damage response. Deregulation of protein ubiquitylation often leads to devastating human diseases, including cancer and neurodegenerative disease. Given the large number of deubiquitylating enzymes (DUBs) existing in the human proteome and the implication of many DUBs in human diseases, it is imperative to identify potent and specific inhibitors for the individual deubiquitylating enzymes. Human ubiquitin specific protease 1, a prototypical USP, forms a stable complex with a WD40 repeat-containing protein. It plays important roles in the human DNA damage response pathways, i.e. the Fanconi anemia pathway and the translesion DNA synthesis pathway. Inhibitors of ubiquitin specific protease 1 will serve as useful chemical probes for investigating the role of deubiquitylation in both pathways. The inhibitors will also have therapeutic potential for the recalcitrant diseases cancer. Together with Anton Simeonov's group at the NIH Chemical Genomics Center (NCGC), we have developed a fluorescence- based primary assay at a low 4 ¿L volume in 1536-well format. Secondary assay orthogonal to the fluorescence assay was developed to eliminate the false positive hits obtained in the primary screening. The specificity and selectivity of the small molecules' effect on USP1 will be determined by screening the compound library on other human and yeast USPs. The optimized lead compounds will be used to probe the in vivo deubiquitylation in DNA damage response. Moreover, we will test the compounds as sensitizers for cancer cells to DNA-damaging drugs in a cellular model. The inhibitors of deubiquitinating enzymes hold promise as the next-generation therapeutics that can overcome the narrow therapeutic index and toxic side effects facing the proteasome inhibitors. PUBLIC HEALTH RELEVANCE: Ubiquitin specific proteases (USPs) are emerging as promising targets for pharmacological intervention because of their connection to many human diseases, including prostate, colon and breast cancer, pediatric acute lymphoblastic leukemia, and familial cylindromatosis. So far few small molecule inhibitors have been developed for inhibiting the human USPs. The goal of this project is to develop lead compounds that can specifically inhibit a USP in DNA damage response and thus sensitize cancer cells to DNA crosslinking drugs.)

描述（由申请人提供）：本项目的总体目标是通过定量高通量筛选（qHTS）鉴定人去泛素化酶的有效和特异性抑制剂。除了在蛋白质降解中的众所周知的作用之外，蛋白质泛素化还在许多其他细胞过程中发挥重要作用，包括膜运输、先天免疫、激酶信号传导、染色质动力学和DNA损伤反应。蛋白质泛素化的失调常常导致毁灭性的人类疾病，包括癌症和神经退行性疾病。鉴于人类蛋白质组中存在大量的去泛素化酶（DUB）以及许多DUB在人类疾病中的意义，鉴定针对个体去泛素化酶的有效且特异性的抑制剂是必要的。人泛素特异性蛋白酶1，一种典型的USP，与含有WD 40重复序列的蛋白质形成稳定的复合物。它在人类DNA损伤反应途径，即范可尼贫血途径和跨损伤DNA合成途径中起重要作用。泛素特异性蛋白酶1的抑制剂将作为有用的化学探针，用于研究去泛素化在这两种途径中的作用。这些抑制剂也将具有治疗恶性肿瘤的潜力。我们与NIH化学基因组学中心（NCGC）的Anton Simeonov小组一起，在1536孔格式中以低4L体积开发了基于荧光的初步测定。开发了与荧光试验正交的二级试验，以消除初步筛选中获得的假阳性命中。将通过筛选其他人和酵母USP上的化合物文库来确定小分子对USP 1影响的特异性和选择性。优化的先导化合物将用于探测体内DNA损伤反应中的去泛素化。此外，我们将在细胞模型中测试这些化合物作为癌细胞对DNA损伤药物的敏化剂。去泛素化酶抑制剂有望成为下一代治疗药物，可以克服蛋白酶体抑制剂所面临的狭窄治疗指数和毒副作用。 公共卫生相关性：泛素特异性蛋白酶（USP）由于与许多人类疾病（包括前列腺癌、结肠癌和乳腺癌、小儿急性淋巴细胞白血病和家族性圆柱瘤病）的联系而成为药物干预的有希望的靶点。到目前为止，已经开发了很少的小分子抑制剂用于抑制人USP。该项目的目标是开发可以特异性抑制DNA损伤反应中的USP的先导化合物，从而使癌细胞对DNA交联药物敏感。

项目成果

Selective and cell-active inhibitors of the USP1/ UAF1 deubiquitinase complex reverse cisplatin resistance in non-small cell lung cancer cells.

USP1/ UAF1去泛素酶复合物的选择性和细胞活性抑制剂在非小细胞肺癌细胞中反向顺铂抗性。

• DOI: 10.1016/j.chembiol.2011.08.014
• 发表时间: 2011-11-23
• 期刊: Chemistry & biology
• 作者: Chen J;Dexheimer TS;Ai Y;Liang Q;Villamil MA;Inglese J;Maloney DJ;Jadhav A;Simeonov A;Zhuang Z
• 通讯作者: Zhuang Z