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：
8117141
负责人：
DAVID E LENZ
金额：
$ 8.83万
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/8117141
关键词：
Acetylcholinesterase Active Sites Address Adoption Affinity Agreement Amino Acid Substitution Amino Acids Antidotes Binding Blood Blood Circulation 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 Medical Research 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 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 human PON1 protein improved kidney cell liquid chromatography mass spectrometry mutant nerve agent neuroregulation novel programs prophylactic response tabun toxicant weapons

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.

化学神经剂暴露期间观察到的毒性作用主要是对乙酰胆碱酯酶的不可逆转抑制（ACHES）。这些酶在调节整个身体的神经信号传导。疼痛的抑制总是导致放松管制后突触后靶标（即心脏，肺等），可能导致死亡。人抛物酶（Huponi）是血清蛋白能够水解这些致命的毒素，但是，必须优化其催化能力在成功地用作治疗神经毒剂中毒的可行解毒剂之前。这个目的应用是能够在神经剂之前对神经剂进行排毒的设计和合成可以达到其生理靶标并发挥致命作用。为此，我们将定义氨基酸残基（在酶的活跃部位或附近）可能在化学神经剂的分解中起作用。经过使用合理设计，我们将生成本机Huponi酶的变体，并测试其能力细胞基水解神经剂，例如Tabun（GA），Sarin（GB），Soman，（GD）和VX。这将是通过检查Huponi对神经药物和 /或实际增强其机械以分解这些毒素。目标1和2将解决最多的问题成功表达各种突变蛋白的功能表达方法。根据获得的结果，目标 3和4试图完善我们对该酶底物特异性的了解。我们估计如果 Huponi的催化潜力至少可以增强10倍，它确实可以是可行的抗神经药物药品。