Molecular evolution of human butrylcholinesterase for nerve agent detoxification

我们的长期目标是开发临床安全的人类丁酰胆碱酯酶(HuBuChE)变体，如 用于保护易受化学战剂或药物危害的人群的水解剂 有机磷(OP)农药。将huBuChE转化为OP水解催化剂的挑战是 鉴定抗灭活的huBuChE变异体，高效快速地催化OP水解，并 自发脱磷。我们工作的基本假设是，HuBuChE的组合 需要在靠近和/或远离活性部位的多个残基上发生突变，才能显著改善 OP的水解活性最终可作为临床上有用的水解催化剂。的主要目标是 目前的工作是应用强大的分子进化策略来产生huBuChE变体 显著提高了OP神经毒剂的水解性。本研究的目的包括：1) 不同类型对映体纯OP模型底物的立体选择性合成 筛选；2)点饱和突变功能筛选体系的优化与验证 具有催化活性的huBuChE变体的进化；使用两个模型化合物表示 Tabun和Soman；4)具有广泛特异性的huBuChE变体的进化；5)体外和体内 与美国医学会合作，使用正宗神经毒剂对进化的huBuChE候选药物进行疗效测试 中心。这项工作提供了鉴定OP催化剂所需的研究和开发(R&D)工作 胡布切特变种通过包括分子进化、理性设计在内的组合方法 诱变、体外和体内药效功能筛选。这项研究完成后，我们将拥有 第三代虎步车产品准备进入高级开发阶段，包括在 良好的生产规范(GMP)条件、先进的药理筛选和临床前测试， 成功建立了两代huBuChE蛋白的过渡过程 用于高级产品的药物，人血浆来源的huBuChE和重组野生型huBuChE 发展。

Our long-term goals are to develop clinically safe human butyrylcholinesterase (huBuChE) variants as hydrolytic catalysts to protect populations at risk from exposure to chemical warfare agents or organophosphate (OP) pesticides. The challenge to convert huBuChE into an OP hydrolytic catalyst is to identify huBuChE variants that are resistant to inactivation, catalyze OP hydrolysis efficiently and rapidly and spontaneously dephosporylate. The underlying hypothesis for our work is that combinations of huBuChE mutations at multiple residues, close to and/or far from the active site, are needed to markedly improve the OP hydrolysis activity to ultimately serve as a clinically useful hydrolytic catalyst. The primary goal of the current work is to apply powerful molecular evolution strategies to produce huBuChE variants with significantly improved hydrolytic activity for OP nerve agents. The Aims for this research include: 1) Stereoselective synthesis of different classes of enantiomerically pure OP model substrates for functional screening; 2) Optimization and validation of the functional screening system with a site-saturation mutation library; 3) Evolution of huBuChE variants with catalytic activity using two model compounds representing tabun and soman; 4) Evolution of huBuChE variants with broad specificity; and 5) In vitro and in vivo efficacy testing of evolved huBuChE candidates using authentic nerve agents in collaboration with the Center. This work provides the research and development (R&D) effort required to identify OP catalytic huBuChE variants through a combinatorial approaches including molecular evolution, rational designed mutagenesis, in vitro and in vivo efficacy functional screening. Upon completion of this study, we will have the third generation huBuChE products ready to enter advanced development including production under good manufacturing practice (GMP) conditions, advanced pharmacological screening, and preclinical testing, a process that has been successfully established to transition two generations of huBuChE based protein drugs, human plasma derived huBuChE and recombinant wild-type huBuChE, for advanced product development.