Cocaine is a widely abused and addictive drug, resulting in serious medical and social problems in modern society. Currently, there is no FDA-approved medication specific for cocaine abuse treatment. The disastrous medical and social consequences of cocaine abuse have made the development of an anti-cocaine medication a high priority. However, despite decades of efforts, traditional pharmacodynamic approach has failed to yield a truly useful small-molecule drug due to the difficulties inherent in blocking a blocker like cocaine without affecting the normal functions of the transporters or receptors. An alternative approach, i.e. pharmacokinetic approach, is to interfere with the delivery of cocaine to its receptors/transporters and/or accelerate its metabolism in the body. It would be an ideal anti-cocaine medication to accelerate cocaine metabolism producing biologically inactive metabolites. Two natural enzymes may catalyze hydrolysis of cocaine: human butyrylcholinesterase (BChE) and bacterial cocaine esterase (CocE). However, the wild-type enzymes are not suitable as anti-cocaine therapeutics, due to the low catalytic activity, thermoinstability, or short biological half-life. In this investigation, we performed integrated computational-experimental studies to rationally design and discover mutants of these enzymes in order to improve the catalytic activity, thermostability, and/or biological half-life. To rationally design desirable mutants of the enzymes, we have successfully developed computational models, including those for BChE gating, glycosylated BChE structure, BChE-substrate complex structures, BChE dimer/tetramer structures, CocE monomer/dimer structures, and CocE-substrate complex structures. Development of the computational models enabled us to rationally design new amino-acid mutations that may improve the catalytic activity, thermostability, and/or prolonged biological half-life. The computational design was followed by wet experimental tests, including both in vitro and in vivo experiments, leading to discovery of new enzyme forms with not only a high catalytic efficiency against cocaine, but also an improved thermostability and/or prolonged biological half-life. The identified new mutants of BChE and CocE are expected to be valuable candidates for development of a more efficient enzyme therapy for cocaine abuse. The encouraging outcomes of the present study also suggest that the structure-and-mechanism-based design and integrated computational-experimental approach is promising for rational drug design and discovery.

Cocaine is one of the most reinforcing drugs of abuse and has caused serious medical and social problems. There is no FDA-approved medication specific for cocaine. It is of a high priority to develop an effective therapeutic treatment for cocaine abuse. Human butyrylcholinesterase (BChE) has been recognized as a promising candidate of enzyme therapy to metabolize cocaine into biologically inactive metabolites and prevent it from reaching central nervous system (CNS). However, the catalytic activity of wide-type human BChE against cocaine is not sufficiently high for treatment of cocaine abuse. Dr. Zhan’s lab has successfully designed and discovered a series of high-activity mutants of human BChE specific for cocaine metabolism. This dissertation is mainly focused to address the possible concerns in further development of promising human BChE mutants for cocaine detoxification, including whether the administration of this exogenous enzyme will affect the cholinergic system, whether it can efficiently hydrolyze cocaine’s toxic metabolites, and whether the commonly used therapeutic agents will significantly affect the catalytic activity of the BChE mutants against cocaine when they are co-administered. According to the results obtained, all of the examined BChE mutants have a considerably improved catalytic efficiency against (-)-cocaine, without significantly improving the catalytic efficiency against any of the other examined substrates, including neurotransmitter acetylcholine. Two representative mutants (including E12-7) also have a considerably improved catalytic activity against cocaethylene (formed from combined use of cocaine and alcohol) compared to wild-type BChE, and E12-7 can rapidly metabolize cocaethylene, in addition to cocaine, in rats. Further evaluation of possible drug-drug interactions between E12-7 and some other commonly used therapeutic agents revealed that all of the examined agents, except some tricyclic antidepressants, do not significantly inhibit E12-7. In addition, an effort to discover new mutants with further improved activity against cocaine led to the discovery of a new BChE mutant, denoted as E20-7, according to both the in vitro and in vivo assays. The encouraging outcomes of the present investigation suggest that it is possible to develop a more effective enzyme therapy for cocaine abuse treatment using one of the most promising BChE mutants, such as E12-7 or E20-7.