Butyrylcholinesterase variants and methods of use

a technology of butyrylcholinesterase and variants, applied in the field of butyrylcholinesterase variants, can solve the problems of cocaine abuse, life-threatening overdoses, long-term dependency, etc., and achieve the effect of enhancing cocaine hydrolysis activity and increasing cocaine hydrolysis activity

Inactive Publication Date: 2006-03-23
LOCKRIDGE OKSANA +1
View PDF5 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011] The invention provides four butyrylcholinesterase variants having increased cocaine hydrolysis activity as well as the corresponding encoding nucleic acids. The invention also provides libraries comprising butyrylcholinesterase variants having at least one amino acid alteration in one or more regions of butyrylcholinesterase and further having at least one butyrylcholinesterase variant exhibiting enhanced cocaine hydrolysis activity compared to butyrylcholinesterase. The invention further provides methods of hydrolyzing a cocaine-based butyrylcholinesterase substrate as well as methods of treating a cocaine-induced condition.

Problems solved by technology

Cocaine abuse is a significant social and medical problem in the United States as evidenced by the estimated 3.6 million chronic users.
Cocaine abuse often, leads to long-term dependency as well as life-threatening overdoses.
However, no effective antagonist is currently available that combats the reinforcing and toxic effects of cocaine.
One difficulty in identifying an antagonist to treat cocaine abuse arises largely from the narcotic's mechanism of action.
Specifically, cocaine inhibits the re-uptake of neurotransmitters resulting in overstimulation of the reward pathway.
In addition, at higher concentrations, cocaine interacts with multiple receptors in both the central nervous and cardiovascular systems, leading to toxicities associated with overdose.
Because of this multifarious mechanism of action of cocaine, it is difficult to identify selective antagonists to treat both the reinforcing and toxic effects of cocaine.
Additionally, antagonists that block cocaine's binding to its receptors tend to display many of the same deleterious effects as cocaine.
In addition, these dopamine antagonists produce profound decreases in other behaviors when the doses are increased only slightly above the levels that display an effect on cocaine self-administration behavior.
Therefore, it has been suggested that individuals with defective versions of the butyrylcholinesterase gene are at higher risk for life-threatening reactions to cocaine.
Recently, administration of butyrylcholinesterase has been demonstrated to confer limited protection against cocaine overdose in mice and rats.
Although administration of butyrylcholinesterase provides some effect against cocaine toxicity in vivo, it is not an efficient catalyst of cocaine hydrolysis.
The low cocaine hydrolysis activity of wild-type butyrylcholinesterase requires the use of prohibitively large quantities of purified enzyme for therapy.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Butyrylcholinesterase variants and methods of use
  • Butyrylcholinesterase variants and methods of use
  • Butyrylcholinesterase variants and methods of use

Examples

Experimental program
Comparison scheme
Effect test

example i

A Butyrylcholinesterase Variant with Increased Cocaine Hydrolysis Activity

[0105] This example describes the discovery and characterization of the butyrylcholinesterase variant designated SEQ ID NO: 2, in which Alanine (A) at amino acid position 328 of human butyrylcholinesterase is replaced with Tryptophan (W). The A328W butyrylcholinesterase variant designated SEQ ID NO: 2 exhibits a 15-fold increase in cocaine hydrolysis activity compared to human butyrylcholinesterase.

Structural Modeling of Cocaine in the Active Site of Human Butyrylcholinesterase

[0106] In order to determine amino acid residues important for cocaine hydrolysis activity, cocaine was docked into the active site of butyrylcholinesterase with the FlexiDock program (Tripos Inc., St. Louis, Mo.) in Sybyl 6.4 software on a Silicone Graphics Octane computer. Flexidock allows docking of ligands into protein active sites, allowing the user to define bonds which are flexible during the docking process. The user must ide...

example ii

Development of a Cocaine Hydrolysis Assay

[0118] This example describes the development of a cocaine hydrolysis assay that permits the efficient analysis of hundreds of butyrylcholinesterase variants simultaneously.

Development of an Isotope Tracer Cocaine Hydrolysis Assay.

[0119] For the purpose of validating new cocaine hydrolysis assays, butyrylcholinesterase hydrolysis of cocaine was first measured as described previously (Xie et al., Mol. Pharmacol. 55:83-91 (1999)), using high-performance liquid chromatography (HPLC). Briefly, reactions containing 100 μM cocaine in 10 mM Tris, pH 7.4 were initiated by the addition of horse butyrylcholinesterase (ICN Pharmaceuticals, Inc., Costa Mesa, Calif.) and incubated 2-4 hours at 37° C. Following the incubation, the pH was adjusted to 3, and the sample was filtered. Subsequently, the sample was applied to a Hypersil ODS-C 18 reversed phase column (Hewlett Packard, Wilmington, Del.) previously equilibrated with an 80:20 mixture of 0.05 M ...

example iii

Synthesis and Characterization of Butyrylcholinesterase Variant Libraries

[0126] This example describes the synthesis and characterization of butyrylcholinesterase variant libraries expressed in mammalian cells.

[0127] In order to facilitate the synthesis of libraries of butyrylcholinesterase variants, DNA encoding wild-type human butyrylcholinesterase, a truncated, enzymatically active, monomeric version of human butyrylcholinesterase, and the A328Y mutant that displays a four-fold increased cocaine hydrolysis activity are cloned into a modified doublelox targeting vector, using unique restriction sites. In preliminary assays the wild-type human butyrylcholinesterase was captured more efficiently and, therefore, serves as the initial DNA template for the synthesis of libraries of butyrylcholinesterase variants. Synthesis of focused libraries of butyrylcholinesterase variants by codon-based mutagenesis.

[0128] A variety of information can be used to focus the synthesis of the initia...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
pHaaaaaaaaaa
volumesaaaaaaaaaa
pHaaaaaaaaaa
Login to view more

Abstract

The invention provides four butyrylcholinesterase variants having increased cocaine hydrolysis activity as well as the corresponding encoding nucleic acids. The invention also provides libraries comprising butyrylcholinesterase variants having at least one amino acid alteration in one or more regions of butyrylcholinesterase and further having at least one butyrylcholinesterase variant exhibiting enhanced cocaine hydrolysis activity compared to butyrylcholinesterase. The invention further provides methods of hydrolyzing a cocaine-based butyrylcholinesterase substrate as well as methods of treating a cocaine-induced condition.

Description

[0001] This invention was made with government support under grant number 1R01 DA011707 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.BACKGROUND OF THE INVENTION [0002] This invention relates to butyrylcholinesterase variants and, more specifically to the production and therapeutic use thereof. [0003] Cocaine abuse is a significant social and medical problem in the United States as evidenced by the estimated 3.6 million chronic users. Cocaine abuse often, leads to long-term dependency as well as life-threatening overdoses. However, no effective antagonist is currently available that combats the reinforcing and toxic effects of cocaine. [0004] One difficulty in identifying an antagonist to treat cocaine abuse arises largely from the narcotic's mechanism of action. Specifically, cocaine inhibits the re-uptake of neurotransmitters resulting in overstimulation of the reward pathway. It is this overstimulation that is prop...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/18C07H21/04A61K38/00A61K38/46C12Q1/46
CPCC12N9/18A61K38/00C12Y301/01008C12Q1/46
Inventor LOCKRIDGE, OKSANAWATKINS, JEFFRY
Owner LOCKRIDGE OKSANA
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap