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

描述(由申请人提供)：该项目的总体目标是通过定量高通量筛选(QHTS)确定有效和特定的人类去泛素化酶抑制剂。蛋白质泛素化除了在蛋白质降解中起重要作用外，还在许多其他细胞过程中发挥重要作用，包括膜转运、先天免疫、激酶信号、染色质动力学和DNA损伤反应。解除对蛋白质泛素化的调控往往会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脱泛素化酶(DUB)，以及许多DUB在人类疾病中的意义，寻找针对单个脱泛素化酶的有效和特异的抑制剂是当务之急。人泛素特异性蛋白水解酶1是一种典型的USP，它与WD40重复蛋白形成稳定的复合体。它在人类DNA损伤反应途径，即Fanconi贫血途径和跨损伤DNA合成途径中发挥重要作用。泛素特异性蛋白水解酶1的抑制剂将作为有用的化学探针来研究去泛素化在这两个途径中的作用。这些抑制剂还将对顽固性癌症具有治疗潜力。与美国国立卫生研究院化学基因组学中心的Anton Simeonov团队一起，我们开发了一种基于荧光的初级分析方法，在1536孔格式下，L体积较小。建立了与荧光测定法正交的二次测定法，以消除初筛中的假阳性。小分子对USP1作用的特异性和选择性将通过筛选其他人和酵母USP1上的化合物文库来确定。优化后的先导化合物将用于探索体内DNA损伤反应中的去泛素化反应。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破坏药物的敏感剂。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面临的治疗指数狭窄和毒副作用。 公共卫生相关性：泛素特异性蛋白水解酶(USP)由于与许多人类疾病有关，包括前列腺癌、结肠癌和乳腺癌、儿童急性淋巴细胞白血病和家族性圆柱状细胞瘤病，正成为药物干预的有希望的靶点。到目前为止，用于抑制人USPS的小分子抑制剂还很少。该项目的目标是开发能够在DNA损伤反应中特异性抑制USP的先导化合物，从而使癌细胞对DNA交联剂敏感。)

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.)