Thermostabilized CocE Mutants for Cocaine Detoxification
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Solution Overview
Problem
Current methods for treating cocaine overdose and addiction lack effective antagonists and thermostable cocaine esterase (CocE) formulations, which are crucial for rapid and efficient detoxification and dependence reduction.
Innovation Solution
Development of thermostabilized cocaine esterase (CocE) compositions and mutants, such as L169K/G173Q, that maintain enzymatic activity and stability at 37°C, combined with specific compounds like phenylboronic acid, to enhance the half-life and efficacy of CocE in reducing cocaine toxicity and dependence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type CocE is used for cocaine detoxification, then rapid cocaine breakdown is achieved, but the enzyme has short half-life and low thermostability at 37°C
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions (L169K, G173Q, and combinations thereof) into the CocE protein sequence. These mutations alter the physical-chemical parameters of the enzyme, specifically increasing its thermostability and half-life at 37°C while preserving or enhancing its catalytic activity toward cocaine hydrolysis.
Solution Approach 2:
The patent creates composite enzyme formulations by combining mutated CocE variants with stabilizing compounds such as phenylboronic acid and its derivatives. This composite approach synergistically enhances both the thermostability and catalytic efficiency of the enzyme system for cocaine detoxification.
2Duration of action of stationary object
If CocE is engineered for increased thermostability, then half-life at 37°C is extended, but enzymatic activity may be reduced
Solution Approach 1:
The patent optimizes multiple amino acid positions simultaneously (including L169K, G173Q, and their combinations) to achieve a balance where thermostability improvements do not come at the cost of catalytic activity. The specific mutation combinations were selected and optimized to maintain productive enzyme-substrate interaction while enhancing structural stability.
Solution Approach 2:
The patent combines thermostable CocE mutants with small molecule stabilizers like phenylboronic acid that can enhance both stability and activity. This composite system allows the enzyme to maintain high catalytic efficiency while achieving the desired thermostability profile for therapeutic application.
3Duration of action of stationary object
If stabilizing compounds like phenylboronic acid are added to CocE, then thermostability and half-life are significantly extended, but formulation complexity increases
Solution Approach 1:
The patent uses small molecules like phenylboronic acid and its derivatives as intermediary stabilizing agents that bind to the enzyme and enhance its thermostability. These intermediaries act as molecular chaperones or protective agents that simplify the overall formulation by providing stability through well-defined molecular interactions rather than complex polymeric or lipid-based systems.
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 thermostabilized CocE compositions significantly extend the enzyme's half-life and activity, providing rapid and effective detoxification and reducing cocaine-induced toxicity and dependence, offering a more stable and potent therapeutic option compared to wild-type CocE.
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 bacterium expresses a cocaine esterase (CocE) that acts similarly to BChE to hydrolyze the benzoyl ester of cocaine
Implementation Method 3
The inventors have discovered that certain compounds thermostabilize wild-type CocE, and further thermostabilize CocE mutants that were already more thermostable than wild-type CocE.
Data Source
AI summary
Provided are compositions comprising a cocaine esterase (CocE) and a compound that thermostabilizes the CocE. Also provided are methods of thermostabilizing a cocaine esterase. Additionally provided are methods of treating a mammal undergoing a cocaine-induced condition. Methods of determining whether a compound is a thermostabilizing agent for a protein are also provided. Uses of the above-described compositions for the treatment of a cocaine-induced condition is additionally provided. Additionally provided is an isolated nucleic acid encoding a CocE polypeptide having the substitutions L169K and G173Q, and the CocE polypeptide encoded by that nucleic acid, and pharmaceutical compositions thereof. Further provided is the use of that composition for the manufacture of a medicament for the treatment of a cocaine-induced condition and for the treatment of a cocaine-induced condition.