Redesign of Butyrylcholinesterase for Cocaine Metabolism

This project focuses on the rational redesign of human butyryl cholinesterase (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 computationalexperimental 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.