Mutant OPAA enzyme with increased catalytic efficiency on GP
Inventors
Harvey, Steven P. • Guelta, Mark A • McMahon, Leslie R
Assignees
United States Department of the Army
Publication Number
US-10363289-B1
Publication Date
2019-07-30
Expiration Date
2037-12-13
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Abstract
The invention is directed toward mutant, non-wild-type organophosphorus acid anhydrolase enzymes having three site mutations, methods of production, and methods of use to effectively degrade toxic organophosphorus compounds, most preferably GP (2, 2′-dimethylcyclopentyl methylphosphonofluoridate).
Core Innovation
The invention relates to novel mutant organophosphorus acid anhydrolase enzymes that effectively degrade toxic organophosphorus compounds, particularly the chemical nerve agent GP (2, 2′-dimethylcyclopentyl methylphosphonofluoridate). Specifically, the invention is directed to non-wild-type OPAA proteins having three site mutations at sequence positions 212, 342, and 215 of SEQ ID NO: 1, which greatly enhance catalytic efficiency on GP compared to the wild-type enzyme.
The problem addressed is the toxicity and environmental hazard of OP compounds such as nerve agents and pesticides, with GP being especially toxic and lacking an efficient and easily produced catalyst for degradation either in the environment or in vivo. While native wild-type OPAA exhibits catalytic activity against various nerve agents, its activity against GP is limited, making it marginally useful for decontamination or medical countermeasures for GP poisoning.
The invention solves this problem by providing OPAA mutants with substitutions at positions 212, 215, and 342, specifically including a preferred mutation combination Y212F, V342L, and I215L. This mutant OPAA (SEQ ID NO: 2) shows a catalytic efficiency on GP approximately 3.5 times greater than wild-type. This improved enzyme can be used for in vivo treatment of GP poisoning or for catalytic decontamination of GP from surfaces or environments. Methods of production, purification, and use (including pharmaceutical compositions and kits) for these mutant enzymes are also described.
Claims Coverage
The patent contains three independent claims covering a mutant OPAA enzyme, a method of degrading GP using the mutant enzyme, and a kit comprising the mutant enzyme with a carrier.
Mutant OPAA enzyme with specific amino acid substitutions
An organophosphorus acid anhydrolase enzyme comprising non-wild-type amino acids at sequence positions 212, 342, and 215 of SEQ ID NO: 1, specifically phenylalanine at position 212, leucine at position 342, and leucine at position 215.
Method of degrading GP using the mutant OPAA
A method of degrading the chemical nerve agent GP by contacting it with the mutant OPAA described above having the three specific amino acid substitutions.
Kit for degrading GP containing mutant OPAA and pharmaceutically acceptable carrier
A kit comprising the isolated mutant OPAA enzyme with the three specific amino acid mutations and a pharmaceutically acceptable carrier, optionally including pharmaceutically acceptable adjuvants or excipients.
The claims cover the mutant OPAA enzyme characterized by the three amino acid substitutions, a method of using this mutant enzyme to degrade GP, and a kit incorporating the mutant enzyme for such use.
Stated Advantages
The mutant OPAA enzyme has approximately 3.5 times greater catalytic efficiency on GP compared to the wild-type enzyme.
The improved catalytic efficiency is primarily due to a significantly lower Km, enhancing enzyme-substrate binding affinity.
The mutant enzyme provides an effective catalyst for the detoxification of GP in vivo or in the environment, useful as a medical countermeasure and for decontamination.
Documented Applications
In vivo treatment of GP poisoning in subjects.
Catalytic decontamination of GP from surfaces or environmental contamination.
Pharmaceutical compositions comprising the mutant OPAA for administration by various routes including intravenous, subcutaneous, intraperitoneal, oral, topical, aerosol, and inhalation.
Use of the mutant OPAA in kits containing the enzyme and carriers/adjuvants to facilitate administration or application for detoxification purposes.
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