Novel Protein-Based Therapeutics for Nerve Agent Detoxification

描述（由申请人提供）：有机磷神经毒剂塔本（GA），沙林（GB），索曼（GD），环沙林（GF）， VX和俄罗斯VX （R-VX）是已知最致命的化合物之一。目前针对化学武器接触的治疗提供的保护有限，必须立即实施，并可能产生长期的毒副作用。人羧酸酯酶1 （hCE1）是一种混杂的药物代谢酶，有望作为一种广谱的基于蛋白质的神经毒剂解毒治疗药物。我们已经确定了几种hCE1与活神经毒剂复合物的晶体结构。这些初步结果表明，该酶具有立体选择性和抗衰老性，并提供了两个假定的神经毒剂结合位点：活性位点和混杂表面结合位点。该应用的重点是利用一系列靶向结构、生化和体内研究将hCE1转化为高效的神经毒剂水解酶。北卡罗来纳大学教堂山分校、圣裘德儿童研究医院和美国陆军化学防御医学研究所的研究小组已经组建起来，将追求四个具体目标：1。阐明hCE1与多种活性神经毒剂配合物的晶体结构。2. 引入旨在改善神经毒剂结合和水解的靶向突变。3. 检测定点突变体对体外hCETs神经毒剂水解活性的影响。4. 评估工程形式的hCE1在体内保护血清酯酶缺陷小鼠免受神经毒剂暴露的能力。这些正在进行的研究的总体目标是开发一种新的、有效的、广谱的基于蛋白质的治疗方法，以预防神经毒剂暴露。显然，迫切需要有效的催化对策，以安全保护军事人员和平民第一反应者免受化学武器袭击。

DESCRIPTION (provided by applicant): The organophosphate nerve agents tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), VX, and Russian VX (R-VX) are among the deadliest compounds known. Current treatments for chemical weapons exposure offer limited protection, must be administered immediately, and can generate long-term toxic side effects. Human carboxylesterase 1 (hCE1), a promiscuous drug metabolism enzyme, offers promise as a broad- spectrum protein-based therapeutic for nerve agent detoxification. We have determined several crystal structures of hCE1 in complex with live nerve agents. These preliminary results have indicated that the enzyme is both stereoselective and resistant to aging, and provides two putative nerve agent binding sites: the active site and a promiscuous surface binding site. The focus of this application is to convert hCE1 into a highly efficient nerve agent hydrolase using a range of targeted structural, biochemical and in vivo studies. A team of investigators at the University of North Carolina at Chapel Hill, St. Jude Children's Research Hospital, and the US Army Medical Research Institute of Chemical Defense has been assembled, and four specific aims will be pursued: 1. Elucidate crystal structures of hCE1 in complex with a wide variety of live nerve agents. 2. Introduce targeted mutations designed to improve nerve agent binding and hydrolysis. 3. Examine the impact site-directed mutants have on hCETs nerve agent hydrolysis activity in vitro. 4. Assess the in vivo ability of engineered forms of hCE1 to protect serum esterase-deficient mice from exposure to nerve agents. The overall goal of these on-going investigations is to develop a novel, efficient, broad-spectrum protein- based therapeutic for prophylactic protection against nerve agent exposure. It is clear that effective catalytic countermeasures are urgently needed to safely protect military personnel and civilian first-responders from attacks involving chemical weapons.