Mutant organophosphorus acid anhydrolase enzymes having increased catalytic efficiency on V-agents
Inventors
Harvey, Steven P • Guelta, Mark A
Assignees
United States Department of the Army • Government of the United States of America
Publication Number
US-10421952-B1
Publication Date
2019-09-24
Expiration Date
2038-01-30
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Abstract
The invention is directed toward mutant, non-wild-type organophosphorus acid anhydrolases (OPPAs) having three or more site mutations, methods of production, kits and methods of use to effectively degrade toxic V-agent type chemical compounds such as VX, VR, CVX, and VM.
Core Innovation
The invention provides mutant, non-wild-type organophosphorus acid anhydrolase (OPAA) enzymes with mutations at amino acid sequence positions 212, 215, 342, and 343 of SEQ ID NO: 1, and optionally at position 332. These mutants include substitutions such as Y212F, I215Y, V342L, and variations at 343 (A, G, or D), and at 332 (Q). Specific mutant enzymes named FLYA, FLYG, and FLYD-Q exhibit substantially increased catalytic efficiency in degrading toxic V-type agents including VX, VR, CVX, and VM.
The problem addressed by the invention arises from the limited catalytic activity of the native OPAA enzyme on particularly toxic and persistent V-agents such as VX, VR, CVX, and VM. Native OPAA shows only marginally detectable activity towards these agents, rendering it ineffective as a decontaminant or medical countermeasure. Thus, there is a need for new OPAA variants that can effectively degrade these V-agents with higher catalytic efficiency.
The invention also discloses methods for producing these mutant OPAAs by recombinant expression, chemical synthesis, and purification techniques. Kits and methods are provided for catalytic degradation of V-agents by contacting the agents with these mutant enzymes. The mutants have demonstrated fold-increases in catalytic efficiency compared to wild-type enzymes, enabling practical applications in detoxification and treatment scenarios.
Claims Coverage
The patent contains multiple independent claims covering mutant OPAA enzymes with specific mutations, methods of degrading V-agents using these enzymes, and kits containing the mutant enzymes and carriers.
Mutant OPAA enzyme with mutations at specific positions
An OPAA enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, with non-wild-type amino acids at each of positions 212, 215, 342, and 343.
Mutant OPAA enzymes with specified amino acid substitutions
Mutant enzymes including specific amino acid substitutions such as phenylalanine at 212, leucine at 342, tyrosine at 215, and alanine, glycine, or aspartic acid at 343, with optional glutamine at position 332.
Mutant OPAA enzymes having sequences SEQ ID NO: 2, 3, or 4
Mutant enzymes comprising the amino acid sequences of SEQ ID NO: 2 (FLYA), SEQ ID NO: 3 (FLYG), or SEQ ID NO: 4 (FLYD-Q) encompassing the stated mutations.
Methods of degrading V-agents using mutant OPAA enzymes
Methods of degrading VX and/or VR using SEQ ID NO: 2 enzyme, degrading VX, VR, and/or CVX using SEQ ID NO: 3 enzyme, and degrading VM and/or CVX using SEQ ID NO: 4 enzyme by contacting the agents with the respective mutant enzyme.
Kits for degrading V-agents containing mutant OPAA and carrier
Kits comprising a mutant organophosphorus acid anhydrolase enzyme with sequences SEQ ID NO: 2, 3, or 4, combined with a pharmaceutically acceptable carrier for degrading VX, VR, CVX, and/or VM.
The independent claims cover mutant OPAA enzymes with defined mutations at key positions improving catalytic activity on V-agents, methods for using these enzymes to degrade specific V-agents, and kits comprising these enzymes with carriers for practical applications.
Stated Advantages
Significantly increased catalytic efficiency of mutant OPAA enzymes on toxic V-agents such as VX, VR, CVX, and VM, surpassing wild-type enzyme activity by up to 33-fold in some cases.
Enhanced ability to effectively degrade persistent and highly toxic chemical warfare agents, enabling practical use as medical countermeasures or decontaminants.
Capability to produce mutant enzymes by recombinant DNA technology and other methods allows scalable production for various use cases.
Mutant enzymes enable pharmaceutical formulations and delivery in multiple administration routes for treatment and environmental detoxification.
Documented Applications
In vivo treatment of VX poisoning by administering mutant OPAA enzymes.
Catalytic decontamination of surfaces or environment contaminated with V-agents including VX, VR, CVX, and VM.
Formulation of pharmaceutical compositions and kits for controlled delivery of mutant OPAA enzymes for detoxifying chemical warfare agents.
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