Enzymatic Protein Labeling

酶蛋白标记

• 批准号: 8067036
• 负责人: MARK D DISTEFANO
• 金额: $ 27.21万
• 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/8067036
• 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; RNA; 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，研究人员开发了多种化学修饰蛋白质的方法。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呢基转移酶（PFTase）对蛋白质进行酶促修饰的方法。该方法允许将含有叠氮化物和炔烃的底物转移到含有小的四个残基和 C 末端序列的蛋白质上。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使用极简标签实现选择性共价蛋白质修饰。在此应用中，我们建议利用这种蛋白质修饰策略的实用性，将其应用于治疗学中的几个重要问题，同时我们继续改进和完善它。该项目的具体目标是：（1）设计和合成简化的含叠氮化物、炔烃和其他官能团的 PFTase 底物（2）开发通用定向进化系统以产生有用的新 PFTase 突变体，包括具有松弛的酶和/或替代类异戊二烯和肽/蛋白质底物特异性。 (3)采用PFTase催化酶蛋白修饰方法制备稳定性更高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估其神经保护功效。 (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，本申请中描述的工作可能会带来改进的基于蛋白质的药物，可用于治疗中风、阿尔茨海默病和其他神经退行性疾病，以及用于治疗自身免疫性疾病和 T 细胞白血病的新的、更具选择性的药物。此外，这里开发的蛋白质修饰方法应该可用于制备多种其他蛋白质缀合物，这些蛋白质缀合物可用于多种治疗和诊断应用。 公共健康相关性：如果成功，本申请中描述的工作可能会改进基于蛋白质的药物，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。还将开发新方法，这些方法也可以转化为其他疾病的新型治疗剂的开发。