Redesign of Butyrylcholinesterase for Cocaine Metabolism

项目描述（由申请人提供）：本项目主要研究人类丁基胆碱酯酶（BChE）的合理再设计，以加速人体可卡因的代谢。通过给药BChE来促进可卡因代谢已被认为是一种很有前途的治疗可卡因滥用的策略。然而，这种血浆酶对天然存在的(-)-可卡因的催化活性比对相对无生物活性的(+)-可卡因异构体的催化活性低三个数量级。在上一个资助周期中，该项目的主要目标是了解BChE催化(-)-可卡因和(+)-可卡因水解的机制差异，并测试计算方法是否适用于合理设计具有提高对(-)-可卡因催化效率的BChE突变体。该项目的进展揭示了bche催化(-)-可卡因和(+)-可卡因水解的基本催化途径。我们进一步开发了一种基于过渡状态模拟的新型计算设计策略，从而发现了几种BChE突变体，与文献中报道的所有BChE突变体相比，它们对(-)-可卡因的催化效率显著提高。利用这一有前途的设计策略和协议，在项目的下一阶段，我们提出了一个综合的计算-实验工作，以进一步提高BChE对(-)-可卡因的催化效率。提出的综合计算-实验方法将包括基于过渡状态建模和模拟的各种假设BChE突变体的大规模虚拟筛选，然后进行更复杂的计算评估和湿实验测试。具体目标包括：1。确定在上一个资助周期中发现的已知BChE高活性突变体催化(-)-可卡因水解的详细反应坐标和相应的自由能谱。2. 采用基于过渡状态建模和模拟的扩展计算设计方法，设计和发现新的BChE突变体，进一步提高对(-)-可卡因的催化效率，对大量假设的BChE突变体进行评估，然后进行湿实验测试，包括定点诱变，蛋白质表达和催化活性测定。这项研究的长期目标是最终利用高活性BChE突变体开发出一种有效的抗可卡因药物。

DESCRIPTION (provided by applicant): This project focuses on the rational redesign of human butyrylcholinesterase (BChE) in order to accelerate cocaine metabolism in human. Enhancing cocaine metabolism by administration of BChE has been recognized as a promising treatment strategy for cocaine abuse. However, the catalytic activity of this plasma enzyme is three orders-of-magnitude lower against the naturally occurring (-)-cocaine than that against the relatively biologically inactive (+)-cocaine isomer. The primary goal of this project in the previous funding cycle was to understand the mechanistic difference between BChE-catalyzed hydrolyses of (-)- cocaine and (+)-cocaine and to test whether a computational approach works or not for rational design of BChE mutants with an improved catalytic efficiency against (-)-cocaine. Progress on the project has revealed the fundamental catalytic pathways for BChE-catalyzed hydrolyses of (-)-cocaine and (+)-cocaine. We have further developed a novel computational design strategy based on transition state simulation, leading to discovery of several BChE mutants with significantly improved catalytic efficiency against (-)-cocaine compared to all BChE mutants reported in literature. Taking advantage of this promising design strategy and protocol, in the next phase of the project we propose an integrated computational-experimental effort to further improve the catalytic efficiency of BChE against (-)-cocaine. The proposed integrated computational- experimental approach will include a large-scale virtual screening of a variety of hypothetical BChE mutants based on the transition-state modeling and simulation, followed by more sophisticated computational evaluation and wet experimental tests. The Specific Aims include: 1. To determine the detailed reaction coordinates and the corresponding free energy profiles for (-)-cocaine hydrolysis catalyzed by the known high-activity mutants of BChE discovered in the previous funding cycle. 2. To design and discover new BChE mutants with further improved catalytic efficiency against (-)-cocaine by using an extended computational design approach based on the transition state modeling and simulation to evaluate a large number of hypothetical BChE mutants, followed by wet experimental tests including site-directed mutagenesis, protein expression, and catalytic activity assay. The long-term objective of this investigation will be to eventually develop an efficient anti-cocaine medication using a high-activity BChE mutant.