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）：各种各样的技术，如基因靶向、核酶、反义或rna介导干扰（RNAi），已被开发用于沉默特定基因和评估其产物的生理功能。总之，这些方法使得在癌症中发现新的药物靶点，甚至治疗性地敲除致癌或促癌基因成为可能。然而，对于许多应用来说，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，并且在寻求新的治疗癌症靶点的研究中代表了一个非常活跃的领域。不幸的是，使用上述所有针对基因表达的方法，修饰特异性沉默是不可能的。因此，本提案寻求开发一种新的功能蛋白质组学技术，该技术基于抗体样E3泛素连接酶，称为“泛素体”，能够沉默靶蛋白及其翻译后修饰的同种异构体。的