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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

基本信息

批准号：
7920097
负责人：
JUN ZHANG
金额：
$ 53.75万
依托单位：
U.S. ARMY MEDICAL RESEARCH INST CHEM DEF
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7920097
关键词：
Active Sites Adenoviruses Advanced Development Biochemical Biological Assay Butyrylcholinesterase Chemical Warfare Chemical Warfare Agents Collaborations Development Drug Metabolic Detoxication Emergency Situation Enzymes Evolution Exposure to Expression Library Generations Genes Goals Human Hydrolysis In Vitro Libraries Life Liquid substance Medical Modeling Molecular Evolution Mutagenesis Mutation Organophosphates Organophosphorus Compounds Pesticides Pharmaceutical Preparations Phase Physiological Plasma Populations at Risk Preclinical Testing Process Production Proteins Recombinants Research Resistance Sarin Screening procedure Site Site-Directed Mutagenesis Solid Soman Specificity Structure Structure-Activity Relationship System Testing Toxic effect Validation Variant Work base bioscavenger catalyst combinatorial design efficacy testing immunogenicity improved in vivo mutant nerve agent next generation preclinical study prevent product development prophylactic recombinant virus research and development tabun tool

项目摘要

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基胆碱酯酶（huBuChE）变体