Molecular evolution of human butrylcholinesterase for nerve agent detoxification

人丁酰胆碱酯酶用于神经毒剂解毒的分子进化

• 批准号: 7235227
• 负责人: JUN ZHANG
• 金额: $ 72.98万
• 依托单位: U.S. ARMY MEDICAL RESEARCH INST CHEM DEF
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-30 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7235227
• 关键词: active sites; biochemical evolution; bioterrorism /chemical warfare; catalyst; conditioning; detoxification; evolution; gene mutation; human; hydrolysis; library; model; pesticides; plasma; proteins

Our long-term goals are to develop clinically safe human butyrylcholinesterase (huBuChE) variants as hydrolytic catalysts to protect populations at risk from exposure to chemical warfare agents or organophosphate (OP) pesticides. The challenge to convert huBuChE into an OP hydrolytic catalyst is to identify huBuChE variants that are resistant to inactivation, catalyze OP hydrolysis efficiently and rapidly and spontaneously dephosporylate. The underlying hypothesis for our work is that combinations of huBuChE mutations at multiple residues, close to and/or far from the active site, are needed to markedly improve the OP hydrolysis activity to ultimately serve as a clinically useful hydrolytic catalyst. The primary goal of the current work is to apply powerful molecular evolution strategies to produce huBuChE variants with significantly improved hydrolytic activity for OP nerve agents. The Aims for this research include: 1) Stereoselective synthesis of different classes of enantiomerically pure OP model substrates for functional screening; 2) Optimization and validation of the functional screening system with a site-saturation mutation library; 3) Evolution of huBuChE variants with catalytic activity using two model compounds representing tabun and soman; 4) Evolution of huBuChE variants with broad specificity; and 5) In vitro and in vivo efficacy testing of evolved huBuChE candidates using authentic nerve agents in collaboration with the Center. This work provides the research and development (R&D) effort required to identify OP catalytic huBuChE variants through a combinatorial approaches including molecular evolution, rational designed mutagenesis, in vitro and in vivo efficacy functional screening. Upon completion of this study, we will have the third generation huBuChE products ready to enter advanced development including production under good manufacturing practice (GMP) conditions, advanced pharmacological screening, and preclinical testing, a process that has been successfully established to transition two generations of huBuChE based protein drugs, human plasma derived huBuChE and recombinant wild-type huBuChE, for advanced product development.

我们的长期目标是开发临床安全的人丁酰胆碱酯酶（huBuChE）变体， 水解催化剂，以保护处于危险中的人口免受化学战剂的危害， 有机磷农药将huBuChE转化为OP水解催化剂的挑战是 鉴定huBuChE变体，所述huBuChE变体对失活具有抗性，有效且快速地催化OP水解， 自发脱磷酸化。我们工作的基本假设是， 需要在接近和/或远离活性位点的多个残基处的突变，以显著改善生物活性。 OP水解活性，最终用作临床上有用的水解催化剂。的首要目标 目前的工作是应用强大的分子进化策略来产生huBuChE变体， 显著提高了对OP神经毒剂的水解活性。本研究的目的包括：1） 不同类型的对映体纯OP模型底物的立体选择性合成用于功能化的 筛选; 2）位点饱和突变的功能筛选系统的优化和验证 3）使用两种模型化合物进化具有催化活性的huBuChE变体，所述两种模型化合物代表 tabun和梭曼; 4）具有广泛特异性的huBuChE变体的进化;和5）体外和体内 使用真实的神经毒剂，与 中心这项工作提供了研究和开发（R&D）的努力，需要确定OP催化 huBuChE变体通过组合方法包括分子进化、合理设计 诱变、体外和体内功效功能筛选。在完成这项研究后，我们将 第三代huBuChE产品准备进入高级开发，包括生产下 良好生产规范（GMP）条件、先进的药理学筛选和临床前试验， 已经成功建立了一种将两代基于huBuChE的蛋白质 药物，人血浆衍生的huBuChE和重组野生型huBuChE，用于高级产品 发展