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Rational design of human paraoxonase to design enhanced catalytic efficiency

合理设计人对氧磷酶以提高催化效率

基本信息

批准号：
7487878
负责人：
DAVID E LENZ
金额：
$ 11.31万
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/7487878
关键词：
Acetylcholinesterase Active Sites Address Adoption Affinity Agreement Amino Acid Substitution Amino Acids Antidotes Binding Blood Blood Circulation Catalytic Domain Cell Line Cessation of life Chemical Agents Chemical Weapons Chemicals China Cholinesterase Inhibitors Collection Computer Simulation Conflict (Psychology)Detection Development Embryo Enzyme Tests Enzymes Ethiopia Fluorides Germany Goals Heart Human Human Resources Hydrolysis Individual Insecticides Institution International Investigation Iran Iraq Isomerism Japan Kinetics Knowledge Lead Lung Medical Military Personnel Monitor Mutation Nature O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphate Office of Administrative Management Paraoxon Pesticides Pharmaceutical Preparations Phosphorescent Assays Physiological Play Poison Poisoning Population Proteins Rate Relative (related person)Research Research Infrastructure Research Institute Research Project Grants Resources Role Sampling Sarin Serum Proteins Signal Transduction Site Soman Specificity Stereoisomer Stream Substrate Specificity Synapses Testing Therapeutic Toxic Actions Toxic effect Toxin Transfection United States National Institutes of Health VX nerve gas Variant Vertebrates Vulnerable Populations Western Blotting World War II analog antinerve agent aryldialkylphosphatase base bioscavenger design drug discovery drug production emergency service responder gallium alloy GF human PON1 protein improved kidney cell liquid chromatography mass spectrometry mutant nerve agent neuroregulation novel programs prophylactic response tabun toxicant

项目摘要

The toxic effects observed during episodes of chemical nerve agent exposure are primarily the result of the irreversible inhibition of acetylcholinesterases (AChEs). These enzymes play an important role in the regulation of neural signalling throughout the body. Inhibition of AChEs invariably leads to the deregulation of post-synaptic targets (i.e. heart, lungs, etc) and may lead to death. Human paraoxonase (HuPONI) is a serum protein capable of hydrolyzing these deadly toxins, however, its catalytic capacity must be optimized before it can be successfully used as a viable antidote to treat nerve agent poisoning. The objective of this application is the design and synthesis of HuPONI variants capable of detoxifying nerve agents before they can reach their physiological targets and exert a lethal effect. To do this we will define amino acid residues (in or near the active site of the enzyme) that may play a role in the breakdown of chemical nerve agents. By using rational design we will generate variants of the native HuPONI enzyme and test their capacity to catalyltically hydrolyze nerve agents such as tabun (GA), sarin (GB), soman, (GD), and VX. This will be accomplished by the examination of the alteration in the affinity of HuPONI for the nerve agents and / or actual enhancement of its machinery to break down these toxins. Aims 1 and 2 will address the most successful approach to functional expression of various mutant proteins. Based on the results obtained, aims 3 and 4 seek to refine our knowledge of the substrate specificity of this enzyme. We estimate that if the catalytic potential of HuPONI can be enhanced by at least 10-fold, it truly could be a viable anti-nerve agent drug.

在接触化学神经毒剂期间观察到的毒性效应主要是由于乙酰胆碱酯酶（AChE）的不可逆抑制。这些酶在生物体内起着重要的作用。调节全身神经信号。乙酰胆碱酯酶的抑制总是导致放松管制，突触后靶点（即心脏、肺等），并可能导致死亡。人对氧磷酶（HuPONI）是一种血清蛋白能够水解这些致命的毒素，但是，它的催化能力必须优化才能成功地用作治疗神经毒剂中毒的有效解毒剂。的目的应用是HuPONI变体的设计和合成，其能够在神经毒剂之前解毒。可以达到它们的生理目标并产生致命的效果。为此，我们将定义氨基酸残基 (in或在酶的活性位点附近），其可能在化学神经毒剂的分解中起作用。通过使用合理的设计，我们将产生天然HuPONI酶的变体，并测试它们的能力，催化水解神经毒剂，如塔崩（GA）、沙林（GB）、梭曼（GD）和VX。这将是通过检查HuPONI对神经毒剂的亲和力的改变来完成，和/或实际上增强了它的机制来分解这些毒素。目标1和2将解决最多的问题，各种突变蛋白功能性表达的成功方法。根据取得的成果，目标图3和图4试图完善我们对这种酶的底物特异性的认识。我们估计，如果 HuPONI的催化潜力可以提高至少10倍，它确实可以成为一种可行的抗神经毒剂药