Engineered organophosphorus acid anhydrolases and methods of use thereof
Inventors
Pegan, Scott • Harvey, Steven P.
Assignees
University of Georgia Research Foundation Inc UGARF • United States Department of the Army • Government of the United States of America
Publication Number
US-10260054-B2
Publication Date
2019-04-16
Expiration Date
2036-06-06
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
Genetically engineered organophosphorus acid anhydrolases (OPAA) with improved catalytic efficiency and relaxed stereospecificity are provided. The variants typically include a mutation at the residue corresponding to H343 of wildtype Alteromonas sp. OPAA. The mutation allows the OPAA enzyme to effectively process both VR enantiomers. The OPAA optionally include one or more mutations selected the residues corresponding to Y212, V342, and I215 of wildtype Alteromonas sp. OPAA which improve the enzyme's catalytic efficiency for VX and VR. A particularly preferred OPAA includes mutations at the residues corresponding Y212F, V342L, I215Y, and H343D relative to wildtype Alteromonas sp. OPAA. Compositions including an effective amount of OPAA to increase hydrolysis of an organophosphate, and methods of use thereof for treating subjects exposed to an organophosphate, or a surface or liquid contaminated with an organophosphate are also provided.
Core Innovation
The invention provides genetically engineered organophosphorus acid anhydrolases (OPAAs) with improved catalytic efficiency and relaxed stereospecificity. These engineered OPAAs typically include a mutation at the residue corresponding to H343 of wildtype Alteromonas sp. OPAA, enabling the enzyme to effectively process both enantiomers of the V-type nerve agent VR. Additional optional mutations at residues corresponding to Y212, V342, and I215 further improve catalytic efficiency for nerve agents VX and VR.
The problem addressed is the inadequate catalytic efficiency and stereospecificity of wild-type OPAA enzymes against V-type nerve agents such as VX and VR. Wild-type OPAA shows low activity for VR and a strong preference for the less toxic enantiomers of soman and related agents, limiting their effectiveness as broad-spectrum enzymatic antidotes. Existing treatments for organophosphate poisoning are limited, and enzymatic detoxification offers advantages by degrading poisons before they interact with acetylcholinesterases.
Claims Coverage
The claims focus on genetically engineered OPAA polypeptides with mutations enhancing substrate specificity and catalytic activity, methods of treatment, and decontamination uses. There are multiple inventive features covering polypeptide sequences, functional mutations, therapeutic methods, and compositions.
Broadened substrate specificity by mutation of basic amino acids in small pocket
The OPAA polypeptides have broadened substrate specificity relative to SEQ ID NO:5 by replacing at least one basic amino acid within the small pocket with an acidic amino acid or its amide, particularly mutation of the residue corresponding to histidine at position 343 to non-histidine residues (e.g., aspartate). The polypeptides have at least 90% sequence identity from residues 212 to 343 of SEQ ID NO:12, with non-histidine at position 343.
Specific engineered OPAA sequences with improved activity
The inventive OPAA includes sequences corresponding to SEQ ID NOs:6, 7, 12, 14, or 15, with mutations such as Y212F, V342L, I215Y, and H343D. These engineered sequences exhibit catalytic activity for both enantiomers of V-type nerve agents VX or VR, with increased activity particularly towards the toxic P(−) enantiomer.
Therapeutic methods using engineered OPAAs
Methods of treating or inhibiting organophosphate poisoning by administering an effective amount of engineered OPAA polypeptides to subjects exposed or at risk of exposure to organophosphates, especially V-type nerve agents VX or VR, to reduce their level or activity and alleviate symptoms associated with poisoning.
Decontamination methods using engineered OPAAs
Methods for decontaminating surfaces or liquids exposed to organophosphates by contacting with effective amounts of engineered OPAAs in liquid, powder, or foam formulations to reduce organophosphate levels or activity.
The claims cover genetically engineered OPAA polypeptides with specific mutations that broaden substrate specificity and improve catalysis against V-type nerve agents, pharmaceutical and prophylactic methods of their use for organophosphate poisoning, and environmental decontamination methods applying these engineered enzymes.
Stated Advantages
Improved catalytic efficiency allowing effective processing of both enantiomers of V-type nerve agents such as VX and VR.
Broadened stereospecificity relaxing the substrate specificity to include the more toxic enantiomers of organophosphates.
High levels of expression and stability suitable for therapeutic and industrial applications.
Compatible with combination therapies using atropine, pralidoxime, and benzodiazepines to improve survival rates in organophosphate poisoning.
Documented Applications
Treatment and prophylaxis of organophosphate poisoning in subjects exposed or at risk, including hydrolysis of VX, VR, or related agents in blood or tissues.
Decontamination of surfaces and liquids contaminated with organophosphates by application of OPAA compositions in foams, sprays, powders, or liquids for environmental and industrial detoxification.
Interested in licensing this patent?