PEGylated Butyrylcholinesterase used as a Bioscavenger

聚乙二醇化丁酰胆碱酯酶用作生物清除剂

• 批准号: 6990389
• 负责人: MARY S. ROSENDAHL
• 金额: $ 12.18万
• 依托单位: BOLDER BIOTECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-26 至 2006-09-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6990389
• 关键词: biotechnology; chemical conjugate; cholinesterases; cysteine; laboratory mouse; laboratory rat; neuroprotectants; polyethylene glycols; protein engineering; site directed mutagenesis; spectrometry

DESCRIPTION (provided by applicant): A novel concept for dealing with nerve agent toxicity involves taking advantage of a naturally occurring enzyme, butyrylcholinesterase (BuChE) that is able to sequester or catalyze the hydrolysis of nerve agents. This macromolecule has the potential of providing protection against a number of nerve agents with minimal side effects, since it can stoichiometrically bind and/or metabolize a nerve agent before its distribution to the site of toxic effect. The main obstacle to development of this type of therapeutic for use in humans is the need for frequent administration of a relatively short-lived product. We propose to create a novel BuChE protein that can be administered less frequently, but with greater potency, than the unmodified BuChE. We will achieve this goal using the published structural information for butylcholinesterase to rationally design polyethylene glycol (PEG)-BuChE conjugates using cysteine-reactive PEG. We will introduce a new "free" cysteine using she-directed mutagenesis in regions of BuChE that are believed to be non-essential for biological activity. The "free" cysteine residue will serve as the site for the covalent modification of the protein using a thiol-reactive PEG. This technology allows for the creation of novel, fully active PEG-Cys-BuChE analogues of defined structure and overcomes the problems of reduced bioactivity and heterogeneity when a protein is modified using a standard amine-reactive PEG. During Phase I we will identify sites in BuChE that can be modified without affecting the protein's in vitro bioactivity and further, demonstrate that PEGylation enhances BuChE's circulating half-life and efficacy in vivo. During Phase II, we develop a cost effective manufacturing process and produce sufficient quantities of the PEGylated recombinant BuChE for testing in animal toxicity models and biochemical characterization.

描述（由申请人提供）：处理神经毒剂毒性的新概念涉及利用天然存在的酶丁酰胆碱酯酶（BuChE），该酶能够螯合或催化神经毒剂的水解。这种大分子有可能提供对许多神经毒剂的保护，副作用最小，因为它可以化学计量地结合和/或代谢神经毒剂，然后再分布到毒性作用部位。开发用于人类的这种类型的治疗剂的主要障碍是需要频繁施用相对短寿命的产品。我们建议创建一种新的BuChE蛋白，可以不太频繁地给药，但具有更大的效力，比未修饰的BuChE。我们将实现这一目标，使用已发表的结构信息的丁基胆碱酯酶，合理地设计聚乙二醇（PEG）-BuChE共轭物，使用半胱氨酸反应性PEG。我们将引入一个新的“自由”半胱氨酸使用她定向诱变的BuChE的区域，被认为是非必需的生物活性。“游离”半胱氨酸残基将用作使用硫醇反应性PEG共价修饰蛋白质的位点。该技术允许产生具有确定结构的新型、完全活性的PEG-Cys-BuChE类似物，并且克服了当使用标准胺反应性PEG修饰蛋白质时生物活性降低和异质性降低的问题。在第一阶段，我们将确定在BuChE的网站，可以被修改，而不影响蛋白质的体外生物活性，并进一步证明，聚乙二醇化增强BuChE的循环半衰期和疗效在体内。在第二阶段，我们开发了一种具有成本效益的生产工艺，并生产了足够数量的聚乙二醇化重组BuChE，用于动物毒性模型和生化表征的测试。