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

• 批准号: 7487881
• 负责人: Christopher M. Hadad
• 金额: $ 35.51万
• 依托单位: U.S. ARMY MEDICAL RESEARCH INST CHEM DEF
• 依托单位国家: 美国
• 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/7487881
• 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; Methods; Military Personnel; Modeling; Mutate; Photoaffinity Labels; Poisoning; Production; Proteins; Proteomics; Pseudocholinesterase; Reaction; Recombinant Proteins; Recombinants; Scanning; Screening procedure; Selenocysteine; Specificity; Structural Models; Structure; Therapeutic; Thermodynamics; Time; Tryptophan; Validation; absorption; aryldialkylphosphatase; base; computer studies; design; desire; enzyme activity; esterase; expectation; improved; in vivo; insight; molecular dynamics; mutant; nerve agent; novel; novel therapeutics; prophylactic; tool

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

酶作为催化人体治疗药物对抗有机磷(OP)神经的关键障碍 这些药物的活性和特异性较差。使用计算的、机械的、光谱的和蛋白质 利用诱变方法，我们提出了人酶突变体的产生和优化。 (对氧磷酶I、丁酰胆碱酯酶和乙酰胆碱酯酶)催化化学物质的水解。 战争神经毒剂比迄今发现的具有更大的活性和特异性。自变异以来 BUCHE、AChE和HuPONI是基于人类蛋白质的，预计这些蛋白质将具有 很少或根本没有免疫和行为副作用，使这些合理的人类治疗 候选人。我们将阐明催化所需的潜在化学作用机制 化学战神经毒剂的水解，设计活性增强的人类蛋白质突变体 神经毒剂，并适当设计人类来源的重组蛋白，然后可以 以足够的数量表达，以供随后的体内疗效验证。总体预期是 发展足够的科学数据，以允许选择一到两种蛋白质产品 作为新一代预防性生物制剂向高级开发的过渡 有可能被授予NDA地位，这些生物制剂将提供增强的保护，防止 军事或民用环境中的神经毒剂中毒。计算的，机械的，光谱的， 光亲和标记、质谱学、蛋白质组学和生化工具将用于野生型和 胆碱酯酶和酯酶的重组突变形式用于开发这些新的 治疗有机磷(OP)神经毒剂。 这些新形式的胆碱酯酶的开发将提供一种生物治疗 反对在军事和民用环境中使用有机磷神经毒剂。这些疗法， 作为人类起源的，将具有所需的化学特异性，也很少或根本没有免疫学和 行为副作用