Computational Design of a Human Butyrylcholinesterase Mutant for Accelerating Cocaine Hydrolysis Based on the Transition‐State Simulation

Abstract

Cocaine is recognized as the most reinforcing of all drugs of abuse. 1,2,3 There is no available anti-cocaine medication. The disastrous medical and social consequences of cocaine addiction have made the development of an effective pharmacological treatment a high priority. 4,5,6 An ideal anti-cocaine medication would be to accelerate cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. cocaine hydrolysis catalyzed by plasma enzyme butyrylcholinesterase (BChE). 5, 7 , 8 , 9 , 10 ,11 However, the native BChE has a low catalytic efficiency against naturally occurring (−)-cocaine.12 , 13 , 14 , 15 (−)-cocaine has a plasma half-life of ~ 45 -90 min, long enough for manifestation of the central nervous system (CNS) effects which peak in minutes. 13 , 16 Here we report an unconventional computational design leading to discovery of a human BChE mutant with a ~151-fold improved catalytic efficiency, which can be used as an exogenous enzyme in human to prevent (−)-cocaine from reaching CNS. The encouraging outcome not only provides a hopeful anti-cocaine medication, but also demonstrates that a novel general approach of studying enzymatic mechanism and computational drug design is promising.For rational design of a mutant enzyme with a higher catalytic activity for a given substrate, in general, one needs to design a mutation that can accelerate the rate-determining step of the entire catalytic reaction process while the other steps are not slowed down by the mutation. Reported computational modeling and experimental data indicated that the formation of the prereactive BChE-(−)-cocaine complex (ES) is the rate-determining step of (−)-cocaine hydrolysis catalyzed by wild-type BChE, 17, 18 , 19 , 20 , 21 , 22 ,23 whereas the rate-determining step for the faster hydrolysis of the biologically inactive (+)-cocaine enantiomer is the chemical reaction process consisting of four individual reaction steps (see Scheme 1).18 This mechanistic understanding is consistent with the experimental observation 17 that the catalytic rate constant of wild-type BChE against (+)-cocaine is pH-dependent, whereas that of the same enzyme against (−)-cocaine is independent of the pH. The pH-dependence of the rate constant for (+)-cocaine hydrolysis is clearly associated with the protonation of H438 residue in the NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript catalytic triad (S198, H438, and E325). For the first and third steps of the reaction process, when H438 is protonated, the catalytic triad cannot function and, therefore, the enzyme becomes inactive. The lower the pH of the reaction solution is, the higher the concentration of the protonated H438 is, and the lower the concentration of the active enzyme is. Hence, the rate constant was found to decrease with decreasing the pH of the reaction solution for the enzymatic hydrolysis of (+)-cocaine. 17 Based on the above mechanistic understanding, the previously reported efforts for ratio...

Free Energy Perturbation (FEP) Simulation on the Transition States of Cocaine Hydrolysis Catalyzed by Human Butyrylcholinesterase and Its Mutants

“…[24][25][26][27][28][29] For free enzyme structures, the +1 charge of the enzyme was neutralized by adding one chloride counterion. For TS1 structure, the +2 charge of the system was neutralized by adding two chloride counterions.…”

First-Principles Calculation of pKa for Cocaine, Nicotine, Neurotransmitters, and Anilines in Aqueous Solution

“…1,2,3 The disastrous medical and social consequences of cocaine addiction have made the development of an anti-cocaine medication a high priority. 4,5,6,7,8,9,10,11,12,13,14 The alkaloid nicotine (12), initially found in tobacco leaves, is the addictive amine compound that maintains tobacco smoking behavior. 15,16,17 More than any other abused psychostimulant, nicotine addiction is the number one cause of preventable mortality and is responsible for over 4 million smoking-related deaths each year.…”

Structure-and-mechanism-based design and discovery of therapeutics for cocaine overdose and addiction

“…The possible effects of some mutations on the hydrogen bonding were examined by performing MD simulations on the TS1 structures for (−)-cocaine hydrolysis catalyzed by wild-type BChE and its various mutants. 54,55 Based on extensive MD simulations54 ,55 on various TS1 structures associated with wild-type BChE and its mutants, some mutants were predicted to have stronger overall hydrogen bonding between the carbonyl oxygen of (−)-cocaine and the protein environment. For example, the carbonyl oxygen of (−)-cocaine in the simulated TS1 structure associated with the wild-type has two N-H ⋯ O type of hydrogen bonds with the peptidic NH of G117 and A199 residues (Fig.…”