Proteolytic silencing of cancer targets using engineered ubiquitin ligases

使用工程泛素连接酶对癌症靶标进行蛋白水解沉默

• 批准号: 8584010
• 负责人: MATTHEW P DELISA
• 金额: $ 20.9万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-16 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8584010
• 关键词: 26S proteasome; Ablation; Acetylation; Affinity; Antibodies; Antigens; Antineoplastic Agents; Apoptotic; Area; Binding; Cancer Etiology; Catalytic RNA; Cell Death; Cell Line; Cell Proliferation; Cells; Chimera organism; Consensus; Disease; Dissection; Drug Targeting; Ectopic Expression; Engineering; Enzymes; Eukaryotic Cell; Evaluation; Event; Family; Gene Expression; Gene Targeting; Genes; Genetic Engineering; Goals; Growth; Human; Inflammation; Knock-out; Life; Lysine; MAPK1 gene; Malignant Neoplasms; Mammalian Cell; Measures; Mediating; Methods; Methylation; Mitogen-Activated Protein Kinases; Modification; Molecular; Neoplasm Metastasis; Oncogenes; Oncogenic; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Physiological; Play; Polyubiquitin; Post-Translational Protein Processing; Property; Protein Conformation; Protein Isoforms; Proteins; Proteomics; RNA; Recruitment Activity; Reporting; Research; Role; Route; Signal Pathway; Signal Transduction; Site; Somatic Cell; Specificity; Substrate Specificity; System; Technology; Testing; Therapeutic; Therapeutic Intervention; Time; Tumor Cell Invasion; Ubiquitin; Ubiquitination; United States National Institutes of Health; base; cellular targeting; drug discovery; glycosylation; inhibitor/antagonist; innovation; intercellular communication; interest; member; multicatalytic endopeptidase complex; new technology; novel; novel therapeutics; protein degradation; public health relevance; tool; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): A variety of technologies such as gene targeting, ribozymes, antisense, or RNA-mediated interference (RNAi) have been developed for silencing of specific genes and assessing the physiological function of their products. Together, these approaches are enabling the discovery of novel drug targets in cancer and even the therapeutic knockdown of cancer-causing or cancer-promoting genes. However, for many applications it may be desirable to target specific protein conformations or post-translational modifications. In particular, protein phosphorylation plays a major role in numerous intracellular signaling pathways and represents a very active area of research in the quest for new therapeutic cancer targets. Unfortunately, modification-specific silencing is not possible using the above methods, all of which target gene expression. Therefore, this proposal seeks to develop a new functional proteomics technology based on antibody-like E3 ubiquitin ligase enzymes called "ubiquibodies" that are capable of silencing target proteins and their post-translationally modified isoforms. The innovativeness of this method lies in the creation of the ubiquibodies themselves, which are antibody-enzyme chimeras that combine the robust proteolytic activity of E3 ubiquitin ligases (E3s) with the affinity, specificity and modularity of intracellular single-chain Fv (scFv) antibodies. The resulting ubiquibodies are capable of recruiting the cell's robust protein degradation system to virtually any protein of interest. Since the scFv domain of a ubiquibody can be created to bind specific protein conformations or post-translational modifications such as glycosylation or phosphorylation, the technology has the potential for silencing of certain protein isoforms while sparing others. The long-term goal is to develop ubiquibodies as an innovative new protein silencing technology for functional analysis of cancer-associated proteins as well as for identification and pharmacological manipulation of cancer drug targets. Towards this goal, the following specific aims are proposed: first, under Aim 1, ubiquibodies will be engineered against the nonphosphorylated (inactive) and phosphorylated (active) isoforms of a well-studied target, ERK2, a member of the MAPK family whose members are well known to regulate several physiological and pathological phenomena including inflammation, apoptotic cell death, oncogenic transformation, tumor cell invasion, and metastasis. Second, under Aim 2, the consequences of ectopic expression of anti-ERK2 ubiquibodies will be examined with respect to ERK2 signaling as well as growth and proliferation of mammalian cells. It is anticipated that isoform-specific silencing will help to uncover valuable information about ERK's role in signaling and cell proliferation. More broadly, this new protein knockout system will provide a simple and efficient tool for creating artificial E3 ligases that enable: (i) dissection of diverse functional properties of cellular proteins in somatic cells; (ii) evaluation of whether specific cellular protins are valid targets for therapeutic intervention; and (iii) ablation of intracellular protein targetsas an effective therapeutic strategy for certain human malignancies such as cancer.

描述（由申请人提供）：已经开发了多种技术，例如基因靶向，核酶，反义或RNA介导的干扰（RNAi），用于沉默特定基因并评估其产物的生理功能。总之，这些方法可以在癌症中发现新的药物靶标，甚至可以发现造成癌症或癌症的基因的治疗性敲低。但是，对于许多应用，可能需要针对特定​​的蛋白质构象或翻译后修饰。特别是，蛋白质磷酸化在许多细胞内信号通路中起着重要作用，并且代表了寻求新的治疗癌症靶标的非常活跃的研究领域。不幸的是，使用上述方法不可能进行特异性的沉默，所有方法靶向基因表达。因此，该提案旨在开发一种基于抗体样E3泛素连接酶酶的新功能蛋白质组学技术，称为“泛质”，能够使靶蛋白及其后翻译后修饰的同工型沉降。这 这种方法的创新性在于泛质本身的创造，它们是抗体 - 酶嵌合体，结合了E3泛素连接酶（E3S）的鲁棒蛋白水解活性与亲和力，特异性和细胞内单链FV（SCFV）抗体的亲和力，特异性和模态。所得的泛质能够募集细胞的稳健蛋白质降解系统几乎任何感兴趣的蛋白质。由于可以创建泛质的SCFV结构域来结合特定的蛋白质构象或翻译后修饰，例如糖基化或磷酸化，因此该技术具有沉默某些蛋白质的潜力 在保留他人的同时。长期的目标是开发泛质作为创新的新蛋白质沉默技术，用于对癌症相关蛋白的功能分析以及对癌症药物靶标的鉴定和药理操纵。为了实现这一目标，提出了以下具体目标：首先，在目标1下，将针对非磷酸化（不活跃）和磷酸化和磷酸化的（主动）同工型ERK2，MAPK家族成员ERK2的成员ERK2的成员众所周知，众所周知的几个物理学和病理元件的apoptoct thul thy thul thy thul thy thy thy thy，转移。其次，在AIM 2下，将检查抗ERK2泛质的异位表达的后果，相对于ERK2信号传导以及哺乳动物细胞的生长和增殖。可以预料，同工型特异性的沉默将有助于发现有关ERK在信号传导和细胞增殖中的作用的有价值的信息。更广泛地说，这种新的蛋白质敲除系统将为创建人工E3连接酶提供一个简单有效的工具，以启用：（i）解剖多种功能 细胞中细胞蛋白的特性； （ii）评估特定的细胞蛋白质是否是治疗干预的有效靶标； （iii）消融细胞内蛋白靶标的一种有效的治疗策略，例如某些人类恶性肿瘤，例如癌症。