Engineered organophosphorus acid anhydrolases and methods of use thereof

Patent metadata

Field	Value
Publication number	US-2016355792-A1
Application number	US-201615174719-A
Country	US
Kind code	A1
Filing date	Jun 6, 2016
Priority date	Jun 5, 2015
Publication date	Dec 8, 2016
Grant date	—

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.

First claim

We claim:

1 . A genetically engineered OPAA polypeptide, wherein the genetically engineered OPPA polypeptide has broadened substrate specificity relative to an unmodified OPPA polypeptide by replacing at least one basic amino acid within the small pocket of the OPAA with an acidic amino acid or its amide under physiological conditions.

2 . The genetically engineered OPAA polypeptide of claim 1 , wherein the OPPA comprises an amino acid sequence at least 90% identical to the region from amino acid residue 212 to residue 343 of SEQ ID NO:12, and wherein the amino acid at the residue corresponding to residue 343 is not a histidine.

3 . The genetically engineered OPAA polypeptide of claim 1 , wherein the OPPA comprises the amino acid sequence of the region from amino acid residue 212 to residue 343 of SEQ ID NO:12, and wherein the amino acid at the residue corresponding to residue 343 is not a histidine.

4 . The genetically engineered OPAA polypeptide of claim 1 , wherein the OPAA comprises the amino acid sequence of the region from amino acid residue 212 to residue 343 of SEQ ID NO:6, 14, or 15.

5 . The genetically engineered OPAA polypeptide of claim 1 , wherein the OPAA comprises the amino acid sequence of the region from amino acid residue 212 to residue 343 of SEQ ID NO:7.

6 . The genetically engineered OPAA polypeptide of claim 1 , wherein the OPAA comprises the amino acid sequence of SEQ ID NO:12, with or without the N-terminal methionine.

7 . The genetically engineered OPAA polypeptide of claim 1 , wherein the OPAA comprises the amino acid sequence of SEQ ID NO:6, 14, or 15 with or without the N-terminal methionine.

8 . The genetically engineered OPAA polypeptide of claim 1 , wherein the OPAA comprises the amino acid sequence of SEQ ID NO:7, with or without the N-terminal methionine.

9 . The genetically engineered OPAA polypeptide of claim 1 , wherein the OPAA has catalytic activity for both enantiomers a V-type nerve agent; has increased OPAA activity towards the Sp/P(−) enantiomer of a V-type nerve agent; or the combination thereof.

10 . The genetically engineered OPAA polypeptide of claim 9 , wherein the V-type nerve agent is VR or VX.

11 . A method of treating or inhibiting organophosphate exposure or poisoning in a subject comprising administering the subject an effective amount the engineered OPAA of claim 1 to reduce the level or activity of the organophosphate.

12 . The method of claim 9 , wherein the subject was exposed to or is at risk of being exposed to a V-type nerve agent.

13 . The method of claim 12 , wherein the V-type nerve agent is VX or VR.

14 . The method of claim 11 , wherein the subject has one or more symptoms of organophosphorus exposure.

15 . The method of claim 14 , wherein the one or more symptoms comprise peripheral or central nervous system damage, myopathy, psychosis, general paralysis, twitching, trembling, paralyzed breathing, convulsions, miosis, blurred vision, dark vision, headache, nausea, dizziness, vomiting, hypersecretion, sweating, salivation, lacrimation, rhinorrhea, abdominal cramps, diarrhea, urinary incontinence, muscle twitching/fasciculations, paralysis, pallor, muscle weakness, tremors, convulsions, incoordination, diaphoresis, bronchospasm, bronchorrhea, tightness in chest, wheezing, productive cough, pulmonary edema, bradycardia, sinus arrest, tachycardia, hypertension, toxic myocardiopathy, mydriasis, ataxia, anxiety, restlessness, choreliform movement, loss of consciousness, respiratory depression, fatigue, seizures, depression, memory loss, confusion, or a combination thereof.

16 . The method of claim 11 , further comprising administering the subject atropine, 2-PAM, a benzodiazepine, or a combination thereof.

17 . The method of claim 11 , wherein the OPAA is administered in an effective amount to reduce the LD50 of the organophosphate.

18 . The method of claim 11 , wherein the OPAA is present in a pharmaceutical composition that is administered into the bloodstream of the subject via injection, infusion, pulmonary administration, or intranasal administration.

19 . A method of decontaminating a surface or liquid exposed to an organophosphate comprising contacting the surface or liquid with an effective amount of the OPAA of claim 1 to reduce the level or activity of the agent.

20 . The method of claim 19 , wherein the OPAA is a liquid, powder, or foam formulation.

Assignees

• Univ Georgia
• Government Of The United States As Represented By The Secretary Of The Army

Inventors

• Pegan Scott
• Harvey Steven P

Classifications

• C12Y301/08002

  Diisopropyl-fluorophosphatase (3.1.8.2) · CPC title

• C12N9/16 Primary

  acting on ester bonds (3.1) · CPC title

• A61K31/5513

  1,4-Benzodiazepines, e.g. diazepam {or clozapine} · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• A61K31/439

  the ring forming part of a bridged ring system, e.g. quinuclidine (8-azabicyclo [3.2.1] octanes A61K31/46) · CPC title