Enhancement of organophosphinate hydrolase activity therough mechanistic evaluati

通过机理评估增强有机次膦酸酯水解酶活性

基本信息

批准号：
7920099
负责人：
Christopher M. Hadad
金额：
$ 24.72万
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/7920099
关键词：
Acetylcholinesterase Ache Active Sites Advanced Development Adverse effects Affinity Amino Acids Behavioral Binding Biochemical Biological Biological Products Catalysis Chemical Warfare Chemicals Cholinesterases Chromogenic Substrates Collaborations Computer Analysis Cysteine Data Development Engineering Enzymes Escherichia coli Evaluation Experimental Designs Free Energy Generations Grant Human Hybrids Hydrolase Hydrolysis Libraries Maps Medical Methods Military Personnel Modeling Mutate Photoaffinity Labels Poisoning Production Proteins Proteomics Pseudocholinesterase Reaction Recombinant Proteins Recombinants Research Scanning Screening procedure Selenocysteine Specificity Structural Models Structure Therapeutic Thermodynamics Time Tryptophan Validation absorption aryldialkylphosphatase base bioscavenger computer studies design enzyme activity esterase expectation improved in vivo insight molecular dynamics mutant nerve agent novel novel therapeutics prophylactic tool

项目摘要

A critical barrier to using enzymes as catalytic human therapeutics against organophosphorous (OP) nerve agents is their poor activity and specificity. Using computational, mechanistic, spectroscopic, and protein mutagenic approaches, we propose the production and optimization of mutants of human enzymes (paraoxonase I, butyrylcholine esterase, and acetylcholinesterase) to catalyze the hydrolysis of chemical warfare nerve agents with greater activity and specificity than those identified to date. Since mutated BuChE, AChE and HuPONI are based on human proteins, the expectation is that these proteins would have few or no immunological and behavioral side effects, making these reasonable human therapeutic candidates. We will elucidate the underlying chemical mechanisms of action necessary for catalytic hydrolysis of chemical warfare nerve agents, design mutants of human proteins with enhanced activity toward nerve agents, and appropriately design recombinant proteins of human origin which can then be expressed in sufficient quantities for subsequent in vivo validation of efficacy. The overall expectation is to develop a sufficient body of scientific data to allow for a selection to be made of one or two protein products for the transition to advanced development as a new generation of prophylactic biological agents with the potential to be granted NDA status and that these biological agents will provide enhanced protection against nerve agent poisoning in a military or civilian setting. Computational, mechanistic, spectroscopic, photoaffinity labeling, mass spectrometric, proteomic, and biochemical tools will be used with wild-type and recombinant mutant forms of cholinesterase and esterase enzymes for the development of these novel therapeutics against organophosphorous (OP) nerve agents. The development of these novel forms of cholinesterase enzymes will provide a biological therapeutic against the use of organophosphorous nerve agents in military and civilian settings. These therapeutics, being of human origin, will have the desired chemical specificity and also few or no immunological and behavioral side effects

使用酶作为有机磷（OP）神经的催化人类疗法的关键障碍代理是他们的活动和特异性不佳。使用计算，机械，光谱和蛋白质诱变方法，我们提出了人类酶突变体的生产和优化（二氧蛋白酶I，丁甘氨酸酯酶和乙酰胆碱酯酶）催化化学水解与迄今为止所确定的活动相比，活动和特异性具有更大的活动和特异性。自突变以来 Buche，Ache和Huponi是基于人类蛋白质的，期望这些蛋白质将具有很少或没有免疫学和行为副作用，使这些合理的人类治疗候选人。我们将阐明催化所必需的基本化学机制化学战神经剂的水解，人类蛋白质的设计突变体，具有增强的活性朝向神经剂，并适当设计人类来源的重组蛋白以足够的数量表示疗效的体内验证。总体期望是开发足够的科学数据，以允许选择一种或两种蛋白质产品作为新一代预防生物学剂的过渡到高级发展有可能被授予NDA地位的潜力，这些生物代理将提供增强的保护在军事或平民环境中中毒。计算，机械，光谱，光性标记，质谱，蛋白质组学和生化工具将与野生型和重组突变形式的胆碱酯酶和酯酶酶的发展，用于开发这些新型针对有机磷（OP）神经剂的治疗剂。这些新型胆碱酯酶酶的开发将提供生物学治疗反对在军事和平民环境中使用有机磷神经。这些治疗学，作为人类来源，将具有所需的化学特异性，也很少或没有免疫学和行为副作用