Enzymatic Protein Labeling

酶蛋白标记

• 批准号: 7887726
• 负责人: MARK D DISTEFANO
• 金额: $ 27.48万
• 依托单位: UNIVERSITY OF MINNESOTA
• 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 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7887726
• 关键词: Alkenes; Alkynes; Alzheimer' s Disease; Antibodies; Autoimmune Diseases; Azides; Binding Sites; Biological; Brain-Derived Neurotrophic Factor; Cells; DNA; Development; Diagnostic; Diphosphates; Disadvantaged; Disease; Enzymes; Erythropoietin; Gray unit of radiation dose; Hypoxia; In Vitro; Ketones; Label; Lead; Malignant Neoplasms; Methodology; Methods; Modeling; Molecular; N-terminal; Nerve Degeneration; Peptides; Pharmaceutical Preparations; Polyethylene Glycols; Post-Translational Protein Processing; Preparation; Property; Proteins; Rattus; Reaction; Research Personnel; Site; Small Interfering RNA; Specificity; Stroke; Substrate Specificity; System; T-Cell Leukemia; T-Lymphocyte; Therapeutic; Therapeutic Agents; Transferase; Translating; Work; antibody conjugate; antigen binding; base; biological systems; design; directed evolution; enzyme substrate; functional group; improved; in vivo; isoprenoid; mutant; novel therapeutics; protein aminoacid sequence; protein farnesyltransferase; public health relevance; success

DESCRIPTION (provided by applicant): Proteins perform a particularly diverse range of tasks in biological systems. To study how these molecules function in vitro and in vivo and to create new molecular entities with therapeutic and diagnostic capabilities, researchers have developed a broad range of methods to chemically modify proteins. While all of these methods are useful, they also all have disadvantages that limit their utility. Recently, we developed a method for enzymatically modifying proteins using the enzyme protein farnesyltransferase (PFTase). This method allows azide and alkyne-containing substrates to be transferred to proteins containing a small, four residue, and sequence at their C-terminus. Subsequent bio- orthogonal reaction of the azide or alkyne-functionalized protein can be used to prepare a wide variety of protein conjugates. What makes this approach unique is the fact that it allows selective covalent protein modification to be achieved with a minimalist tag. In this application we propose to capitalize on the utility of this protein modification strategy by applying it to several important problems in therapeutics while we continue to improve and refine it. The specific aims of this project are: (1) Design and synthesize simplified azide-, alkyne-, and other functional group-containing substrates for PFTase (2) Develop a general directed evolution system to produce useful new mutants of PFTase including enzymes with relaxed and/or alternative isoprenoid and peptide/protein substrate specificity. (3) Use the PFTase catalyzed enzymatic protein modification method to prepare PEGylated forms of erythropoietin (EPO) and brain derived neurotrophic factor (BDNF) with increased stability and evaluate their neuroprotective efficacy following intranasal application in a rat hypoxia model. (4) Use a similar approach to prepare antibody-RNA conjugates that can be used to target siRNAs to T cells. If successful, the work described in this application could lead to improved protein- based drugs that could be used for the treatment of stroke, Alzheimer's and other neurodegenerative conditions as well as new, more selective agents for the treatment of autoimmune diseases and T cell leukemias. Moreover, the protein modification methodology developed here should be useful for the preparation of a wide variety of other protein conjugates that could be employed for a plethora of therapeutic and diagnostic applications. PUBLIC HEALTH RELEVANCE: If successful, the work described in this application could lead to improved protein-based drugs that could be used for the treatment of stroke, Alzheimer's and other neurodegenerative conditions as well as autoimmune diseases and cancer. New methods will also be developed that could be translated into the development of novel therapeutic agents for other diseases as well.

描述（由申请人提供）：蛋白质在生物系统中执行特别多样化的任务。为了研究这些分子如何在体外和体内发挥作用，并创造具有治疗和诊断能力的新分子实体，研究人员开发了广泛的方法来化学修饰蛋白质。虽然所有这些方法都很有用，但它们也都有限制其实用性的缺点。最近，我们开发了一种使用酶蛋白法尼基转移酶（PFTase）酶促修饰蛋白质的方法。该方法允许将含叠氮化物和炔的底物转移到在其C-末端含有小的四个残基和序列的蛋白质。叠氮化物或炔官能化蛋白质的后续生物正交反应可用于制备多种蛋白质缀合物。这种方法的独特之处在于，它允许用极简标签实现选择性共价蛋白质修饰。在本申请中，我们建议通过将其应用于治疗学中的几个重要问题来利用这种蛋白质修饰策略的效用，同时我们继续改进和完善它。本项目的具体目标是：（1）设计并合成了简化的叠氮基、炔基、和其它含官能团的PFTase底物（2）开发一个通用的定向进化系统，以产生PFT酶的有用的新突变体，包括具有松弛和/或替代类异戊二烯和肽/蛋白质底物特异性的酶。(3)使用PFTase催化的酶促蛋白质修饰方法制备稳定性增加的聚乙二醇化形式的促红细胞生成素（EPO）和脑源性神经营养因子（BDNF），并在大鼠缺氧模型中鼻内应用后评价其神经保护功效。(4)使用类似的方法制备可用于将siRNA靶向T细胞的抗体-RNA缀合物。如果成功的话，本申请中描述的工作可以导致改进的基于蛋白质的药物，其可以用于治疗中风、阿尔茨海默氏症和其他神经退行性病症，以及用于治疗自身免疫性疾病和T细胞白血病的新的更具选择性的药剂。此外，这里开发的蛋白质修饰方法应该可用于制备各种各样的其他蛋白质缀合物，这些缀合物可用于过多的治疗和诊断应用。 公共卫生相关性：如果成功，本申请中描述的工作可能会导致改进的基于蛋白质的药物，可用于治疗中风，阿尔茨海默氏症和其他神经退行性疾病以及自身免疫性疾病和癌症。还将开发新的方法，这些方法也可以转化为其他疾病的新型治疗药物的开发。