Redesign of Butyrylcholinesterase for Cocaine Metabolism

项目描述（由申请人提供）：本项目的重点是合理重新设计人丁酰胆碱酯酶（BChE），以加速可卡因在人体内的代谢。通过施用BChE来增强可卡因代谢已被认为是可卡因滥用的有希望的治疗策略。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生物学上无活性的（+）-可卡因异构体的催化活性低三个数量级。该项目在上一个供资周期的主要目标是了解BChE催化的（-）-可卡因和（+）-可卡因水解之间的机理差异，并测试计算方法是否适用于合理设计BChE突变体，提高对（-）-可卡因的催化效率。该项目的进展揭示了BChE催化水解（-）-可卡因和（+）-可卡因的基本催化途径。我们进一步开发了一种新的计算设计策略的基础上过渡态模拟，导致发现几个BChE突变体的催化效率显着提高对（-）-可卡因相比，文献中报道的所有BChE突变体。利用这个有前途的设计策略和协议，在该项目的下一阶段，我们提出了一个综合的计算实验的努力，以进一步提高催化效率的BChE对（-）-可卡因。所提出的综合计算-实验方法将包括基于过渡态建模和模拟的各种假设BChE突变体的大规模虚拟筛选，随后是更复杂的计算评估和湿实验测试。具体目标包括：1.确定前一个供资周期中发现的已知高活性BChE突变体催化的（-）-可卡因水解的详细反应坐标和相应的自由能分布。2.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，通过使用基于过渡态建模和模拟的扩展计算设计方法来评估大量的假设BChE突变体，随后进行湿实验测试，包括定点突变，蛋白表达和催化活性测定。这项研究的长期目标将是最终开发一种有效的抗可卡因药物，使用高活性的BChE突变体。

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.