Mutated organophosphorus acid anhydrolases and their uses thereof
Inventors
Harvey, Steven P. • Guelta, Mark A.
Assignees
United States Department of the Army
Publication Number
US-9617526-B1
Publication Date
2017-04-11
Expiration Date
2035-11-19
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Abstract
The invention is directed toward non-wild-type organophosphorus acid anhydrolases having three site mutations, method of production, and method of use to effectively degrade toxic chemical compounds such as (Ethyl({2-[bis(propan-2-yl)amino]ethyl}sulfanyl)(methyl)phosphinate (“VX”).
Core Innovation
The invention is directed toward non-wild-type organophosphorus acid anhydrolases (OPAAs) having three site mutations at sequence positions 212, 215, and 342 of SEQ ID NO: 1, methods of production, and methods of use to effectively degrade toxic chemical compounds such as the nerve agent VX (Ethyl({2-[bis(propan-2-yl)amino]ethyl}sulfanyl)(methyl)phosphinate). The non-wild-type OPAA proteins have specific amino acid substitutions at these positions, resulting in significantly enhanced catalytic activity degrading VX as compared to wild-type OPAA.
The problem being solved is the marginal or practically undetectable catalytic activity of wild-type OPAA enzymes against the highly toxic and persistent chemical warfare agent VX. Although wild-type OPAAs can degrade other organophosphorus compounds, their catalytic activity against VX is insufficient for practical use in detoxification or medical countermeasures. Previous efforts with single mutations showed only modest enhancement. Therefore, the invention provides mutated OPAAs with improved efficacy in VX degradation to address this critical detoxification need.
Claims Coverage
The patent includes four main inventive features claimed independently, focusing on the mutated OPAA enzyme, its encoding nucleic acid, methods to degrade VX using the mutant enzyme, and a kit for VX degradation.
Mutated OPAA with specific triple amino acid substitutions
An isolated organophosphorus acid anhydrolase (OPAA) comprising non-wild-type amino acids at positions 212, 215, and 342 of SEQ ID NO: 1, where tyrosine (Y) at position 212 is replaced by phenylalanine (F), isoleucine (I) at position 215 is replaced by phenylalanine (F), and valine (V) at position 342 is replaced by leucine (L).
Amino acid sequence of the mutated OPAA
The mutated OPAA comprises the amino acid sequence of SEQ ID NO: 2 embodying the triple substitutions Y212F, I215F, and V342L.
Nucleic acid encoding the mutated OPAA
An isolated nucleic acid sequence encoding the mutated OPAA with triple substitutions, specifically SEQ ID NO: 3.
Method of degrading VX using mutated OPAA
A method for degrading VX by contacting it with the mutated OPAA having the triple amino acid substitutions at positions 212, 215, and 342, with options for pharmaceutical administration to subjects, including specific dosage ranges and routes of administration.
The claims cover the mutated OPAA enzyme with specific triple amino acid substitutions, its nucleic acid encoding sequence, methods to degrade VX using this enzyme including pharmaceutical applications, and kits comprising the enzyme and pharmaceutically acceptable carriers, defining the core inventive enhancements over wild-type enzymes.
Stated Advantages
The mutated OPAA proteins exhibit at least ten to forty times greater catalytic activity in degrading VX compared to wild-type OPAA, enabling effective detoxification.
The triple mutation substantially improves binding and cleavage of VX, making the enzyme practically useful as a decontaminant and potential medical countermeasure.
Documented Applications
In vivo treatment of VX poisoning in subjects by administering pharmaceutical compositions containing the mutated OPAA.
Catalytic decontamination of surfaces or environments contaminated with VX by applying the mutated OPAA enzyme.
Pharmaceutical preparations and dosage regimens for administering the mutated OPAA by various routes including intravenous, subcutaneous, intramuscular, intranasal, and others.
Use in kits that contain the mutated OPAA and pharmaceutically acceptable carriers or adjuvants for VX degradation.
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