Thermostable Cocaine Esterase Mutants for Toxicity Reduction
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Solution Overview
Problem
Current treatments for cocaine abuse and overdose lack effective antagonists, with existing therapies such as endogenous esterases and antibodies showing limited efficacy due to rapid cocaine metabolism and pleiotropic effects, necessitating a more stable and efficient cocaine degradation solution.
Innovation Solution
Development of highly efficient, thermostable, and long-lasting cocaine esterase mutants with increased thermostability and catalytic efficiency, which can hydrolyze cocaine and its metabolites, such as the T172R and G173Q mutants, for use in pharmaceutical compositions and therapeutic applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild-type cocaine esterase (BChE) is used to hydrolyze cocaine, then cocaine breakdown occurs, but the enzyme shows limited stability and short plasma half-life at physiological temperatures
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of cocaine esterase through site-directed mutagenesis. Specific residues (e.g., Ser140, Ser159, Trp220) are mutated to alter the enzyme's thermal stability parameters while preserving catalytic activity, thereby extending plasma half-life at physiological temperatures
Solution Approach 2:
The patent creates stable variants by copying the wild-type BChE structure and introducing targeted mutations. The mutant enzymes replicate the catalytic function of wild-type BChE while incorporating stabilizing amino acid substitutions that enhance thermal resistance and prolong circulation time
2Productivity
If naturally occurring esterases are used for cocaine hydrolysis, then cocaine metabolism occurs, but the enzymes lack sufficient catalytic efficiency and stability for effective therapy
Solution Approach 1:
The patent simultaneously optimizes multiple parameters including catalytic efficiency (kcat/Km) and thermal stability. Mutations are designed to enhance substrate binding affinity and turnover rate while introducing stabilizing interactions that maintain structural integrity at 37°C, achieving both high productivity and reliability
3Reliability
If cocaine antibodies or catalytic antibodies are administered to prevent cocaine from reaching sites of action, then cocaine blockade occurs, but the therapies show limited efficacy compared to enzymatic degradation
Solution Approach 1:
The patent extracts and utilizes the catalytic function of esterases to directly degrade cocaine into inactive metabolites. This enzymatic approach is more efficient than antibody-based blockade because it actively destroys the substrate rather than merely preventing its action, simplifying the therapeutic mechanism while enhancing efficacy
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The thermostable cocaine esterase mutants provide prolonged protection against cocaine toxicity and addiction by maintaining high catalytic activity at physiological temperatures, significantly extending the plasma half-life and reducing cocaine-induced lethality, offering a more effective treatment option compared to wild-type esterases.
Implementation Method 1
Naturally occurring cocaine is hydrolyzed at the benzoyl ester by serum butyrylcholinesterase (BChE) to nontoxic ecgonine methyl ester and benzoic acid
Implementation Method 2
The purified enzyme (MW ∼65kDa) catalyzes cocaine very efficiently with Michaelis-Menten kinetics kcat =7.2 s-1
Data Source
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AI summary
Embodiments of the invention disclosed herein generally relate to anti-cocaine therapeutics. Specifically, some embodiments of the invention relate to highly efficient, thermostable, and long-lasting cocaine esterase (CocE) mutants that can protect against the toxic and reinforcing effects of cocaine in subjects. Provided herein are mutant CocE polypeptides displaying thermostable esterase activity. Also provided are methods of treating cocaine-induced conditions in a subject in need via administration of mutant CocE as well as methods for high-throughput screening of candidate esterase polypeptides.