Redesign of Butyrylcholinesterase for Cocaine Metabolism

描述(申请人提供)：该项目致力于合理重新设计人丁酰胆碱酯酶(BChE)，以加速人类可卡因的代谢。通过给予BChE促进可卡因代谢已被认为是治疗可卡因滥用的一种有前途的策略。然而，这种血浆酶对自然产生的(-)-可卡因的催化活性比对相对生物活性不高的(+)-可卡因异构体低三个数量级。该项目在上一个资金周期的主要目标是了解BChE催化的(-)-可卡因和(+)-可卡因的水解物之间的机理差异，并测试计算方法是否适用于合理设计BChE突变体，并提高对(-)-可卡因的催化效率。该项目的进展揭示了BChE催化的(-)-可卡因和(+)-可卡因的基本催化途径。我们进一步开发了一种基于过渡态模拟的新的计算设计策略，导致发现了几个BChE突变体，与文献报道的所有BChE突变体相比，它们对(-)-可卡因的催化效率显著提高。利用这一有希望的设计策略和协议，在该项目的下一阶段，我们提出了一种整合的计算-实验工作，以进一步提高BChE对(-)-可卡因的催化效率。提出的计算-实验一体化方法将包括基于过渡态建模和模拟的各种假设的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.确定在上一个资助周期中发现的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分布。2.设计和发现对(-)-可卡因的催化效率进一步提高的新的BChE突变体，采用基于过渡态建模和模拟的扩展计算设计方法对大量假想的BChE突变体进行评估，然后进行定点突变、蛋白质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使用高活性BChE突变体的高效抗可卡因药物。

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.