WO2007146003A2 - Methods for ester detoxication - Google Patents
Methods for ester detoxication Download PDFInfo
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- WO2007146003A2 WO2007146003A2 PCT/US2007/013333 US2007013333W WO2007146003A2 WO 2007146003 A2 WO2007146003 A2 WO 2007146003A2 US 2007013333 W US2007013333 W US 2007013333W WO 2007146003 A2 WO2007146003 A2 WO 2007146003A2
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- 0 C[C@@](*)[C@@]1C2=C[C@]2[C@]1C[C@@](C1)C1(CCCC1)[C@@]1[C@]1[C@]2[C@]1CCC*2 Chemical compound C[C@@](*)[C@@]1C2=C[C@]2[C@]1C[C@@](C1)C1(CCCC1)[C@@]1[C@]1[C@]2[C@]1CCC*2 0.000 description 2
- QADGHBJEXYHMPS-UHFFFAOYSA-N CC(C(c1ccccc1)O1)N(C)P1=CN(C)C Chemical compound CC(C(c1ccccc1)O1)N(C)P1=CN(C)C QADGHBJEXYHMPS-UHFFFAOYSA-N 0.000 description 1
- KWGRBVOPPLSCSI-WPRPVWTQSA-N C[C@@H]([C@@H](c1ccccc1)O)NC Chemical compound C[C@@H]([C@@H](c1ccccc1)O)NC KWGRBVOPPLSCSI-WPRPVWTQSA-N 0.000 description 1
- ZIKDDUAXXXHARZ-ZVXSNUFUSA-N C[C@@H]1N(C)P(C)O[C@H]1c1ccccc1 Chemical compound C[C@@H]1N(C)P(C)O[C@H]1c1ccccc1 ZIKDDUAXXXHARZ-ZVXSNUFUSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01008—Cholinesterase (3.1.1.8), i.e. butyrylcholine-esterase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10351—Methods of production or purification of viral material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
Definitions
- RhBchE monkey butyrylcholinesterase
- Human butyrylcholinesterase HuBchE
- HuBchE human butyrylcholinesterase
- HuBchE human butyrylcholinesterase
- HuBchE human butyrylcholinesterase
- a mutation library featuring HuBchE and RhBchE an expression vector containing DNA molecules of RhBchE and modified HuBchE
- organophosphate (OP) model compounds that mimic the structure of OP nerve agents
- use of OP model compounds to obtain antibodies for an array biosensor The invention provides a general method for detoxication of inorganic or organic esters including organophosphate nerve agents, and a diagnostic method for detecting OP agents in biological samples and environmental samples.
- Drug abuse is one of the major public health problems. More than 3 million Americans are heavy users and a like number of Americans are light abusers of cocaine. About 100,000 emergency room visits annually are cocaine-related. No effective treatment is available for the common complications of cocaine overdose on the cardiovascular and central nervous systems that produce cardiovascular distress and generalized seizures.
- Cocaine is an example of an organic ester that could be detoxicated in a therapeutic fashion by an esterase evolved utilizing the molecular evolution approach.
- organophosphate (OP) nerve agents or pesticides An example of inorganic ester that could be detoxicated in a therapeutic fashion are organophosphate (OP) nerve agents or pesticides.
- OP organophosphate
- the biological threat from nerve agents exposure has also become of great concern as these compounds are able to block numerous vital enzymes.
- serine esterases and proteases are rapidly and irreversibly inhibited by OP nerve agents.
- BchE is a soluble serum glycoprotein enzyme in the family of serine esterases. The physiological function of the enzyme is not clear. However, it has been known for decades that BchE scavenges low doses of OP and carbamate pesticides and protects people from the toxic effects of these poisons. BchE also serves as a marker for early detection of OP poisoning, because the exposure to OPs decreases the activity of the serum enzyme. BchE is also the primary enzyme for the metabolism and detoxication of cocaine and other esters in humans. BchE metabolizes cocaine to pharmacologically inactive compounds such as ecgonine methyl ester and benzoic acid. The enzyme poses great potential for use in cocaine detoxication and OP poisoning treatment.
- BchE represents an ideal enzyme for enzyme supplementation therapy: it doesn't require cofactors, it is soluble and highly functional at the pH of the plasma; and the products of hydrolysis are non-toxic.
- Purified BchE is stable for years and has a relatively long half-life after exogenous administration. Pegylation of HuBchE markedly improves its stability and half-life.
- purified HuBchE has been used safely for decades in Europe for treatment of humans with succinylcholine-induced apnea and OP poisoning. Regardless of its theoretical potential, HuBchE is not a very efficient enzyme toward either OPs or cocaine hydrolysis.
- the irreversible binding of the enzyme for OPs limits the ability of the enzyme to work only as a scavenger and not as a catalyst.
- the enzyme hydrolyzes natural (-)-cocaine 2000-times slower than (+)-cocaine.
- Rationally designed mutants generated by protein engineering showed that the enzyme can be converted to an OP hydrolase (Gl 17H and Gl 17H/E197Q mutants described by Millard et al., 1995; Millard et al., 1998) as well as to cocaine hydrolase
- An array format offers a number of advantages, such as the potential to analyze a sample for a large number of targets simultaneously. Furthermore, inclusion of positive and negative controls on each sensing surface is more reliable than such controls located on parallel but separate sensing surfaces.
- DNA array technology has led this effort in terms of laboratory devices and two notable systems employing optical waveguides include the systems marketed by Zeptosens (Pawlak et al., 2002) and Illumina (Epstein and Walt, 2003). These systems accommodate thousands of capture molecules and are highly sensitive. However, they are designed for use by highly trained laboratory personnel and have not been automated or adapted for on-site applications.
- the Array Biosensor described in this invention combines optical waveguide technology and the capacity to test multiple samples simultaneously for multiple targets with portability and automation.
- the biosensor is based on a planar waveguide with sufficient surface area to accommodate many small (mm 2 ) sensing regions.
- the waveguide a modified microscope slide, is illuminated using a 635 nm diode laser and a line generator, with the light launched into the proximal end.
- the first two-thirds of the slide provides a mode-mixing region so that the light is relatively uniform in the 2.4 cm 2 sensing region near the distal end (Feldstein et al., 1999). Under normal conditions, total internal reflection is achieved and an evanescent field is produced in the sensing region.
- the evanescent light excites fluorophores bound in the sensing region, and the emitted fluorescence is measured at 90° using a Peltier-cooled CCD camera (Wadkins et al., 1997; Golden et al, 2003).
- the location of the fluorescence within the array on the waveguide surface reveals the identity of the target detected. This system is commercially available.
- near-infrared fluorescence prevents interference from sample components, which may autofluoresce at shorter wavelengths, making separation of the target from complex samples unnecessary prior to analysis (Sapsford et al., 2001 ; Taitt et al., 2004).
- mass-sensitive sensors such as the surface plasmon resonance (SPR), resonant mirror, or interferometric systems (Homola et al., 2002; Kinning and Edwards, 2002; Campbell and McCloskey, 2002, Barzen et al., 2002)
- the fluorescence-based Array Biosensor requires a fluorophore-labeled molecule for signal generation. This makes the assay relatively immune to interference from nonspecific adsorption by sample components (Ligler et al., 2003; Rowe et ah, 1999; Sapsford et al, 2001 ; Taitt et al, 2004).
- the invention provides an isolated DNA molecule of monkey rhesus butyrylcholinesterase (RhBchE ) (Macaca mulatto).
- RhBchE monkey rhesus butyrylcholinesterase
- the invention provides an isolated DNA molecule of human butyrylcholinesterase (HuBchE) and mutants thereof.
- the invention provides a mutation library featuring HuBchE and
- RhBchE and a method for making a mutation library of BchE in a vector.
- the invention provides a method of infecting a cell with a vector by packaging of a mutation library of BchE in a vector particle [0017]
- the invention provides an expression vector containing DNA molecules of RhBchE and modified HuBchE; and a high-level adenovirus (AD)-based expression system of BchE.
- AD high-level adenovirus
- the invention provides racemic as well as enantiomerically pure
- OP model compounds that mimic the structure of OP nerve gases such as VX, tabun, GF, soman, and sarin.
- the invention provides means for use of the OP model compounds in a cell-based functional screening assay to identify OP resistant and/or catalytic
- the invention provides means for use of OP model compounds to obtain antibodies for the array biosensor.
- the invention provides a diagnostic method for detecting OP agents in biological samples.
- the invention provides a method for screening an OP model compound for BchE activity by incubating the BchE with the compound and further detecting the inhibition of BchE as indication of the activity of the compound.
- the invention provides a method for detecting BchE expression in culture medium of infected cells.
- the invention provides a method for detecting BchE by prokaryotic or eukaryotic cells expressing BchE in the presence of an OP compound or a nerve agent.
- the invention provides an array-based methodology to selectively detect nerve agents and other OPs.
- Figure IA shows RhBchE transfection.
- CHO cells were seeded overnight in 6- well plates. Plasmids containing background vector (N.C.), pRC-CMV-HuBchE (hBchE), pGS-RhBchE (GSl and GS2), and pRC-CMV-RhBchE (RCl and RC2) were transfected into to the cells with Lipofectamine as described by the manufacturer. The medium was collected at 24 hr and 48 hr post-transfection, and used for BchE activity assay using Ellman assay with 1 mM Bchl as substrate.
- Figure IB shows the results of western blot analysis of RhBchE.
- the BchE unit is defined as amount of enzyme that hydrolyzes 1 ⁇ mol substrate per min.
- Samples from 48 hr post-transfection were analyzed with western blot analysis using a polyclonal antibody developed against HuBchE (B).
- Figure 2A Purification of RhBchE. SDS-PAGE of purified RhBchE was detected with commassie blue.
- Figure 2B SDS-PAGE of purified RhBchE was detected with anti-
- FIG. 4B Inhibition of HuBchE by the specific concentrations of compound 2.
- Figure 4C Inhibition of HuBchE by the specific concentrations of compound 2.
- Figure 5 Diagram of eight randomly selected representative clones of gene shuffled recombinant BchEs.
- Figure 6A shows Butyrylthiocholine iodide (Bchl) hydrolysis activity.
- COS cells were pre-seeded in 6-well plates overnight. The cells were then infected with mock infection (Control) or 1x10 8 pfu and 4x10 8 pfu HuBchE- AD per well specifically at time 0.
- Bchl hydrolysis activity was examined for culture medium at the indicated time, post infection.
- a BchE unit was defined as enzyme needed to hydrolyze 1 ⁇ M/min Bchl substrate.
- Figure 6B shows the results of Western blot analysis of adenovirus (AD) expression vector meadiated BchE expression. Culture medium samples collected at indicated time post infection were analyzed on SDS-PAGE. 0, 1, and 2 represent cells infected by mock infection (Control) or 1x10 8 pfu and 4x10 8 pfu HuBchE- AD, respectively.
- AD adenovirus
- HuBchE in the medium was detected with western blot analysis.
- Figure 7 shows the BchE activity of 293 A cells transfected with Pad linearized indicated recombinant AD. BchE activity in the culture medium collected at indicated time post-transfection was analyzed with Ellman method.
- Figure 8A shows cell based HuBchE expression mediated Bchl hydrolysis in the solid-phase assay using different solid matrix between 0-3 hour .
- Figure 8B shows the Bchl hydrolysis in the same solid-phase assay as in 8A after 3.5-7.3 hr incubation.
- CHO-Kl cells stably expressing WT HuBchE were seeded in 24 well plates overnight. The cells were treated as described in the text. Absorbance at 405 nm in each well was recorded by a plate reader over time.
- the legend indicates the solid-phase material used for cell coating (1st layer) and substrate application (2nd layer).
- Figure 9 A shows the localization of the HuBchE expressing cells.
- the plates seeded with CHO-Kl cells stably expressed WT HuBchE were developed with an Ellman mixture with Bchl substrate as described in text.
- Figure 9B shows an inverted image of plate A.
- Figure 10 shows an illustration of specific plugs isolated during virus isolation from identified yellow spots.
- 293A cells in 6-well plate were infected with serial diluted HuBchE and stained for BchE activities as described.
- Indicated positive plugs, negative plugs, sample plugs, and background plugs were cored out from the plate and transferred to medium for virus extraction.
- the invention provides an isolated DNA molecule of monkey rhesus butyrylcholinesterase (RhBchE ) (Macaca mulatto).
- RhBchE monkey rhesus butyrylcholinesterase
- the RhBchE was cloned from RNA using the RACE kit and RT-PCR.
- the full length RhBchE was cloned into the Hindlll/Apal sites of pRC-CMV. Obtained plasmids were transfected into CHO cells and the expression of active BchE enzyme was monitored from the assay medium for Bchl hydrolysis.
- an isolated DNA molecule of HuBchE and mutants thereof are also provided.
- the invention provides a mutation library featuring HuBchE and RhBchE, a method for making a mutation library of BchE in a vector, and a method of infecting a cell with a vector by packaging of a mutation library of BchE in a vector particle.
- an expression vector containing DNA molecules of RhBchE and modified HuBchE and a high-level adenovirus (AD)-based expression system of butyrylcholinesterase may be any vector suitable for this purpose.
- the vector is a pENTRA vector or an adenovirus vector.
- the cell may be any cell, preferably a mammalian cell.
- a site-saturation mutagenesis technology was used.
- the mutagenesis can be performed at any chosen specific position, (e.g. position Gl 17 and El 97 for HuBchE library).
- the PCR product then can be cloned into the pENTRA 1 vector through Kpnl/Xhol sites.
- Plasmid DNA from pooled pENTRA-HuBchE clones can be used for recombination with an pAD/CMV/V5/DEST vector. Plasmids of the pAD-HuBchE mutation library pool can be digested with Pad enzyme and then transfected into 293 A cells using Lipofectamine 2000 for AD packing. A recombinant AD-HuBchE viral library can be collected from cell supernatant. The library is then screened with both a primary high-throughput solid phase functional screen and a secondary liquid based activity screen (Example 4).
- RhBchE or the mutants of the HuBchE having one or all of the RhBchE amino acid changes listed in Table 1 by site-directed mutagenesis or other means should provide an enzyme with greater hydrolytic activity. These residues could be responsible individually or in combination for the higher substrate binding affinity and velocity seen in the purified native enzyme. The different residues could alter the folding of the protein and/or the post-translational modification of the protein (i.e., N-glycosylation, phosphorylation), and therefore change the recognition and entry of cocaine or other esters to the binding site of the enzyme, the actual hydrolysis of cocaine, or the release of the hydrolysis products.
- the cell culture may be any cell culture suitable for the methods of the invention.
- the cell culture is a mammalian cell culture.
- the expression system for BchE may be any expression system suitable for such purpose.
- the expression system is a high level adenovirus(AD)-based expression system capable of incorporating mutation library expression and adapting to high throughput format functional screening. AD can infect a broad range of mammalian cells and permit expression of various proteins in different dividing and non-dividing cell lines (Example 10).
- the invention provides OP model compounds that mimic the structure of OP nerve agents such as VX, GF, tabun, soman, and sarin.
- the compounds are used in a concentration from 0.01 to 20 mM, preferrably in a concentration from 0.1 to 10 mM. In a specific embodiment, the concentration of a compound is 0.5 mM.
- the synthesis of the compounds is described in detail in Examples 12 & 13.
- the invention provides a method for screening an OP model compound for BchE activity by incubating the BchE with a compound and further detecting the inhibition of BchE as indication of the activity of the compound.
- the cells expressing BchE in the presence of an OP compound or a nerve agent may be prokaryotic or eukaryotic cells.
- the invention provides a diagnostic method for detecting OP agents in biological samples. As the acute toxic effects of OP compounds correlate well with their ability to inhibit AChE by reaction with an essential serine hydroxyl to form a relatively stable phosphoserine ester bond, the OP-ChE conjugates can serve as extraordinarly sensitive selective markers of OP exposure. Likewise, phosphorylated albumin (i.e., Tyr 411) can serve as a sensitive marker of OP or pesticide exposure.
- the invention further provides means for use of OP model compounds to obtain antibodies for the array biosensor by using an array-based methodology to selectively detect nerve agents and other OPs. This is a three-pronged approach.
- the final product is a fieldable Array Biosensor of use in detecting OPs in environmental samples or biological samples taken from animals exposed to low doses of nerve agents or other OPs (Example 14).
- the invention provides a clinically tested recombinant HuBchE (and/or a more active catalytic variant forms of this enzyme) as an efficient biological scavenger (and/or enzymatic catalyst) useful to remove nerve agents, cocaine, pesticides, other drugs of abuse in vivo before damage occurs.
- HuBchE and/or a more active catalytic variant forms of this enzyme
- the invention represents a promising approach to protect military personnel, first responders and civilians from the threat of OP exposure and provides an emergency treatment for cocaine or other agent overdose.
- This product can also be used to protect people potentially exposed to pesticides.
- the product can also be used as a detoxication device, such as nerve agent detoxication sponge, which absorbs and detoxifies nerve agents on skin or other surfaces; a detection device, such as testing strips to provide fast and sensitive detection of nerve agents; and a decontamination reagent for destruction and disposal of nerve agents.
- a detoxication device such as nerve agent detoxication sponge, which absorbs and detoxifies nerve agents on skin or other surfaces
- a detection device such as testing strips to provide fast and sensitive detection of nerve agents
- a decontamination reagent for destruction and disposal of nerve agents.
- the OP analogs and the OP hydrolysis assays provided herein can be readily applied to other enzymes such as paraoxonase, carboxylase, and catalytic antibodies; the diversified library can be used for screening for other commercially viable biotherapeutics (e.g., cocaine hydrolysis) and industrial enzymes.
- the invention is further described by the following non-limiting examples.
- Example 1 Cloning, expression and purification of RhBchE.
- RhBchE full length expression vector The 5' sequence of the RhBchE was cloned directly from rhesus monkey ⁇ Macaca mulatto) RNA using the RACE kit following the manufacture's procedure. Specifically, total RNA was prepared from a sample containing livers of three rhesus monkeys. After dephosphorylation of short mRNA, removing the cap structure of full length mRNA, an RNA RACE oligo was ligated onto the full length mRNA. Then through RT-PCR and cloning, the 5' sequence of the RhBchE was obtained.
- RhBchE sequence was confirmed through a recent input sequence (NCBI BV211040) and disclosed in the provisional application Serial No. 60/81 1,370, filed June 7, 2006, incorporated by reference in its entirety.
- the primer was designed based on the obtained sequence.
- the full length RhBchE, including the RhBchE signal peptide region and the mature BchE, was amplified through PCR and cloned into the Hindlll/Apal sites of pRC- CMV as well as the pGS vector.
- the pGS vector was essentially the same as the pRC-CMV vector, except that the selection marker of G418 in pRC-CMV was replaced with rat glutamine synthetase.
- Selection medium used is serum free Ultraculture medium without L-glutamine containing 25 ⁇ M methionine sulfoximine (MSX).
- MSX is a specific inhibitor of internal expressed glutamine synthetase. Therefore only cells carrying the pGS- RhBchE vector can replicate under this selective condition.
- the transfected cells were kept in the selective medium for 2 weeks to allow colony formation. Twenty four single colonies separated from the others were randomly selected. Culture medium was removed from the culture plates. Small filter paper soaked with trypsin was applied to selected colonies and incubated at room temperature for 2 minutes. The filter paper was then transferred to individual wells in 24-well plates provided with the selective medium indicated above. Cells were allowed to grow to confluence.
- the resin was then resuspended in 0.5 volume of 0.1 M l-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride containing procainamide at a concentration of 100 mol/ml of resin with the addition of 1 M HCl to maintain the pH 4.5 for 2.5 hr.
- the pH is adjusted every 5 min and the pH ranged about pH 4.45 to 4.65.
- the reaction was allowed to complete at room temperature overnight ( ⁇ 20 hr).
- the resin was then packed in a column and washed thoroughly with H 2 O with monitoring at UV280 for complete removal of unreacted procainamide. All flow- through was collected to measure un-coupled procainamide.
- RhBchE Purification of recombinant RhBchE from culture medium.
- Stimified recombinant protein stable cells expressing RhBchE were grown in Tl 80 flasks that allowed three layers of cell growth. Medium from the flasks was collected after two weeks of accumulation of secreted RhBchE. The medium was incubated with procainami de-resin overnight at 4 0 C with constant rotation. The resins were settled by 5 min centrifugation at 2000xg. The supernantant containing unbound materials was carefully removed and the resins were resuspended in 50 mM potassium phosphate pH 7.2, 1 mM EDTA (Buffer A) and packed into a column.
- RhBchE bound to the column was eluted with 0.05 to 0.5 M procainamide gradient solution in buffer A. Active fractions from the eluate were identified by the Ellman assay using Bchl substrate. The active fractions were pooled together and concentrated with Centricon (Millipore) with Molecular weight cut off of 30 kD.
- active fractions were pooled together and loaded on a DEAE sepharose column.
- the column was thoroughly washed with 20 mM Tris pH 8.0, 1 mM EDTA and protein bound to the column were eluted by step gradients of 0.1 M, 0.15 M, 0.2 M, 0.25 M, and 0.3 M NaCl in 20 mM Tris pH 8.0, 1 mM EDTA.
- the active enzyme was present in the 0.2 M NaCl eluant.
- the protein concentration was determined using the BCA method.
- the protein in each fraction was analyzed by SDS-PAGE followed by commassie blue staining and western blot analysis.
- the assays contained 25, 33.3, 50, 100, and 200 ⁇ M of Bchl, respectively, enzyme stock, and 50 mM potassium phosphate pH 7.2 buffer with 200 ⁇ M DTNB.
- the assay was carried out at 25 0 C.
- K n values were determined by Lineweaver-Burk analysis, and kau values were determined using the functional enzyme concentrations determined from echothiophate (ETP) titration.
- the competitive inhibition constant, K 1 of (+)-cocaine, (-)-cocaine and some of its metabolites was determined by measuring hydrolysis of Bchl in the presence of (+)-cocaine, (-)-cocaine, (-)-norcocaine, at a range of concentrations.
- Cocaine Hydrolysis Assay Cocaine hydrolysis was characterized by quantifying specific ecgonine methylester (EME) production by mass spectrometry (MS). Highly purified RhBchE or HuBchE was incubated with cocaine (1 , 2, 4, 10, and 40 ⁇ M final concentration in 10 mM potassium phosphate buffer pH 7.4) at 37°C. At intervals of 20, 40, and 60 minutes, aliquots were mixed with 6 N HCl to stop the reaction and stabilize the reaction products. Background reactions were performed simultaneously without added enzyme.
- EME ecgonine methylester
- MS mass spectrometry
- EME electrospray ionization
- the amount of EME produced at each time point was determined using flow injection of reaction aliquots directly into an Agilent MSD model MS, and ions ofm/z 199.9- 200.9 (EME) were quantified by selective ion monitoring.
- the amount of EME in the reaction mixtures was compared to a standard curve generated from EME solutions of known concentrations (125-1000 nM).
- the specific enzyme-catalyzed rate at each cocaine concentration was determined by subtracting the corresponding background rate.
- K n , and Vmax values were determined by Lineweaver-Burk analysis, and k cat was determined using the functional enzyme concentrations determined from echothiophate (ETP) titration.
- RhBchE and HuBchE by OP compound The kinetics of time-dependent inhibition of purified RhBchE and HuBchE by model OP compounds and ETP was studied in 50 mM potassium phosphate buffer pH 7.2 at 25 0 C. Inhibition of RhBchE and HuBchE was initiated by mixing highly purified HuBchE and RhBchE with various amounts of ETP. At defined times, the reaction mix containing 1 mM Bchl and 0.2 mM DTNB was added to the enzyme-compound mix and hydrolysis of Bchl was measured to determine residual BchE activity. Seven inhibitor concentrations were used for the assay with five time points for each inhibitor concentration.
- Gl 17H/E197Q HuBchE were individually incubated with 0.5 mM of compounds 1, 2, 3 (or 4-13, see Example 12) or ETP at 4°C for 48 hrs.
- Standard substrate Bchl (1 mM) was then used to measure percent of remaining enzyme activity using the Ellman method after 100- fold dilution of the original enzyme-compound incubation mixture.
- Inhibition rate constant determination for inhibition of HuBchE by model OP compounds The kinetics for time-dependent inhibition of purified HuBchE by the model OP compounds was studied in 50 mM potassium phosphate buffer pH 7.2 at 25 0 C. Inhibition of HuBchE was initiated by mixing 15 nM of highly purified HuBchE with various amounts of compounds 1, 2, 3, (or 4-13, see Example 12) or ETP. At defined times, the reaction mixture containing 1 mM Bchl and 0.2 mM DTNB was added to the enzyme- compound mixture and hydrolysis of Bchl was measured to determine residual HuBchE activity. Seven inhibitor concentrations were used for the assay and five time points of enzyme activity were determined for each inhibitor concentration. [0060] Example 4.
- Plasmid DNA from pooled pENTRA-huBchE clones were used for recombination with an pAD/CMV/V5/DEST vector. Plasmids of the pAD-huBchE mutation library pool were digested with Pad enzyme to expose the left and right viral ITRs and then transfected into 293 A cells using Lipofectamine 2000 for AD packing. A recombinant AD-huBuChE viral library was collected from cell supernatant 5 days post transfection. The library was then screened with both a primary high-throughput solid phase functional screen and a secondary liquid based activity screen.
- the functional screening platform was validated using the two position (Gl 17 and E 197) site-saturation mutagenesis-generated library above.
- the library recombinant viruses were used to infect 293 A cells, and the cells were coated with 1% agarose in MEM 24 hr post infection, then 0.4 mM of compound 4-13 (i.e., compound 5, sarin analog, see Example 13) was added to the coated cells to interact with expressed recombinant HuBchE variants.
- the coated cells were stained using Bchl as substrates in the presence of DTNB. The plates were monitored for 3 hrs for appearance of yellow spots, which indicate the presence of OP-hydrolyzing enzyme.
- the yellow agarose spot(s) were cored out from the plate and incubated in serum-free medium in individual tubes. Aliquots from the recombinant viruses released in the medium were added to 96- well plate pre-seeded with 293A cells. The 96-well culture plate was incubated for 3 days post-infection. This time allowed the multiplication of virus and expression of the encoded huBuChE variants. On day 3, culture medium from the 96-well plate was assayed for OP inhibition resistance/hydrolysis activity. Based on the results of all the above assays, the samples showing increased OP hydrolysis activity were selected for gene identification. The huBchE gene encoded in the recombinant virus was PCR amplified with viral vector specific primers from the culture medium. The PCR products were sequenced and mutations in the huBchE gene were identified.
- This double mutant variant was designed for improved cocaine hydrolysis.
- Two different concentrations (1x10 8 pfu and 4x10 8 pfu, (i.e. 200 multiplicity of infection (moi) and 800 moi respectively) of virus were used to infect CHO cells and COS cells pre-seeded overnight onto a 6-well plate.
- BchE activity in the culture medium was monitored at different time points post infection using 1 mM Bchl as substrate using standard Ellman reaction.
- AD expression vector for WT and G117H/E197Q HuBchE A ViralPower AD Expression system from Invitrogen was used to clone the HuBchE enzyme. The system involved the cloning of the target gene in an entry vector. The target gene was transferred to the pAD/CMV/V5/DEST through in vitro recombination using the Gateway technology as described by the manufacturer.
- PCR-amplified WT and Gl 17H/E197Q HuBchE from the original plasmid were made using Turbo Pfu to incorporate the restriction cloning sites Kpnl and Xhol.
- the PCR product was cloned in the Kpnl and Xhol sites of pENTRAl .
- the insert of selected clones was sequenced to verify that no mutations were introduced through the PCR step.
- the supercoiled plasmid was then incubated with the pAD/CMV/V5/DEST, the AD expression vector containing a CMV promoter for high level target protein expression and negative selection ccdB gene. After transformation, only recombined plasmids were able to grow on the selection plate. Plasmids were analyzed by restriction digestion and sequencing analysis to verify the correct insertion of HuBchE gene in pAD vector.
- the medium containing packaged recombinant virus was collected at day 12 post transfection, centrifuged to remove cell debris and stored in -80 0 C.
- recombinant virus was added into the medium of cultured COS cells. Bchl hydrolysis activity was determined for the culture medium at different time points post initial infection.
- Example 5 Development and Validation of functional screening assays.
- the Ellman reaction mixture containing substrate (1 mM Bchl or 1 mM ETP) and/or 0.1 mM DTNB were prepared in 1% agar (or 1% agarose) in colorless MEM.
- the reaction mixture was overlaid on cell culture plates prepared above and incubated at room temperature. The plates were visually monitored for appearance of yellow color and were also measured for OD405 absorption with a plate reader.
- CHO-Kl cells stably expressing WT HuBchE were serial diluted and seeded onto 10 cm culture dishes at different densities (i.e., 4, 20, and 100 cells/plate). The cells were allowed to grow for 1 week to form small colonies containing ⁇ 20 cells/colony. The cells were washed 2 times with serum free culture medium and then coated with 1% agarose in MEM. After overnight incubation, the plates were developed with an Ellman reagent mixture containing substrate (1 mM Bchl and 0.1 mM DTNB) prepared in 1% agarose in colorless MEM.
- the isolated plugs were transferred to tubes containing 0.5 ml of cultured medium and incubated at 4°C overnight. Medium incubated with plugs was then used to infect 293 A cells pre-seeded in 24- well plate. BchE activity in the medium was assayed at 24 and 48 hr post-infection. To recover the BchE gene from the isolated recombinant virus, 1 ⁇ l medium incubated with plugs was used as a template and amplified with AD vector specific primers T7 and pAD-V5R. Sequence of the PCR product was determined.
- RhBchE encoded a 574 amino acid polypeptide which had 95% sequence identity (96% sequence similarity) with HuBchE (gi:4557351) (SEQ ID NO:3), 91% identity (94% similarity) with rabbit BchE (gi:l 16354) (SEQ ID NO:4), 91% identity (95% similariTY) with horse BchE (gi:7381418) (SEQ ID NO:6), 88% identity (92% similarity) with cat BchE (gi:2981243) (SEQ ID NO:5), and 81% identity (89% similarity) with mouse BchE (gi:6857761) (SEQ ID NO:7).
- the polypeptide sequences of SEQ ID NOS:3-7 are shown below. [0073] The polypeptide sequence of HuBchE (SEQ ID NO:3):
- mouse BchE (SEQ ID NO:7): MQTQHTKVTQTHFLLWILLLCMPFGKSHTEEDFIITTKTGRVRGLSMPVLGGTVTAFLGI PYAQPPLGSLRFKKPQPLNKWPDIHNATQYANSCYQNIDQAFPGFQGSEMWNPNTNLSED CLYLNVWIRVPKPKNATVMVWIYGGGFQTGTSSLPVYDGKFLARVERVIWSMNYRVGAL GFLAFPGNPDAPGNMGLFDQQLALQWVQRNIAAFGGNPKSITIFGESAGAASVSLHLLCP QSYPLFTRAILESGSSNAPWAVKHPEEARNRTLTLAKFTGCSKENEMEMIKCLRSKDPQE ILRNERFVLPSDSILSINFGPTVDGDFLTDMPHTLLQLGKVKKAQILVGVNKDEGTAFLV YGAPGFSKDNDSLITRKEFQEGLNMYFPGVSRLGKEAVLFYYY
- RhBchE sequence of the invention SEQ ID NO:8 to M62777 (partial RhBchE) (SEQ ID NO:9) and HuBchE (NCBI NM 000055) (SEQ ID NO: 10).
- SEQ ID NO:8 The polynucleotide sequence encoding RhBchE (SEQ ID NO:8) is shown below.
- RhBchE residues that are of interest for comparison among different species are shown in Table 1 below.
- RhBchE Compared to the sequence of HuBchE, RhBchE contains 25 residues with different amino acids. Among these 25 different residues, 17 amino acids are conserved as similar amino acids. When compared to the BchE enzyme amino acid sequences from different animal species, including human, rabbit, cat, horse, and mouse, RhBchE has the highest similarity to HuBchE. There are six residues that are conserved between HuBchE and RhBchE but are not conserved with other species. However, seven out of the eight non- conserved amino acids (P215, S227, D342, D390, V454, G482, and K489) in RhBchE as compared to HuBchE are actually conserved with the other animal enzymes. The only amino acid residue that is specific for RhBchE is N348, whereas enzymes from all other species listed have Lys at this position (Table 1).
- Example 7 Expression of functional recombinant RhBchE. Expression of recombinant RhBchE was examined in both transient transfected and stably transfected CHO cells. Activity of expressed recombinant BchE enzyme was monitored from assay medium for Bchl hydrolysis using the Ellman reaction. The expression of BchE protein was also confirmed by western blot analysis, indicating that the recombinant RhBchE recognized by the anti-HuBchE polyclonal antibody and co-migrated with recombinant HuBchE ( Figure 1). [0086] Purification of RhBchE from M. mulatta serum.
- RhBchE was purified to approximately 70% purity based on SDS- PAGE and commassie blue staining ( Figure 2A).
- Western blot analysis with anti-HuBchE polyclonal antibody showed that the antibody recognized the RhBchE very efficiently and the immuno-reactive band co-migrated with purified HuBchE ( Figure 2B).
- Pegylation or other derivatizations of BchE can be done to improve the stability of any protein identified in the invention described herein.
- RhBchE Substrate specificity of RhBchE. Enzyme fractions purified, as described in Example 1, were analyzed for Bchl hydrolysis assay using the Ellman method (Table 2). Table 2 demonstrates kinetic constants for purified RhBchE and HuBchE determined in 50 mM potassium phosphate, pH 7.4, at 30 0 C for butyrylthiocholine and (-)-cocaine. [0088] Table 2. Kinetic constants for purified RhBchE and HuBchE.
- RhBchE showed a significant difference with HuBchE in substrate specificity. While the RhBchE had a 2.7-fold lower binding affinity to Bchl (Km
- RhBchE The substrate specificity of RhBchE was tested and cocaine hydrolytic products tested as inhibitors of the enzyme activity, the K 1 value for different compounds chemically related to cocaine were determined, as shown in Table 3 (Bchl hydrolysis inhibition in 50 mM potassium phosphate, pH 7.4, at 25 0 C).
- both (+)-cocaine and (-)-norcocaine has a ⁇ 2 fold higher K 1 for RhBchE than HuBchE, while (-)-cocaine has a ⁇ 3 fold lower K ⁇ for RhBchE than HuBchE, suggesting that the RhBchE is more structurally selective for (-)-cocaine than other substrates.
- (-)-Ecgonine methyl ester did not inhibit Bchl hydrolysis at the concentration tested (0-100 ⁇ M).
- Table 4 shows the kinetic constants for RhBchE and HuBchE for (-) cocaine (Xie et al., 1999; Mets et al., 1998) and shows that RhBchE has an improved k c JK m compared to previously reported cocaine hydrolysis catalysts and HuBchE mutants.
- RhBchE The catalytic efficiency (k ca /K m ) for cocaine hydrolysis for RhBchE is over 10- fold greater than the human counterpart.
- RhBchE was compared with HuBchE for its interaction with OP, the inhibition of RhBchE and HuBchE by ETP was first-order (Figure 3).
- Figure 3 A re-plot of the A aPP first order rate constant (for inhibition) versus the concentration of ETP resolved the inhibition constant for the reaction of RhBchE and HuBchE with ETP.
- the difference in sensitivity between RhBchE and HuBchE is shown in Table 5 (inhibition rate constants for ETP were obtained through double reciprocal plot of k app (min "1 ) using ETP concentrations presented in Figure 3).
- Example 8 OP analogue synthesis and inhibition of HuBchE.
- OP analogues mimicking the structures of sarin, soman, and VX respectively, were successfully synthesized as described in Examples 12 and 13.
- the OP analogues were tested for their interaction with HuBchE and the Gl 17H/E197Q HuBchE. The results are shown in Table 6 (HuBchE and Gl 17H/E197Q HuBchE were incubated with 0.5 mM of indicated compounds or control buffer for 48 hr at 4°C). [0098] Table 6. OP Inhibition of WT and G117H/E197Q HuBchE.
- Example 9 Gene shuffle to create RhBchE/HuBchE chimeras. We have shown that RhBchE has over 10-fold greater cocaine hydrolysis activity than HuBchE. There is a difference in 23 amino acids between RhBchE and HuBchE. The amino acid residues from eight randomly selected clones that are different between HuBchE and RhBchE are shown in Table 8 below.
- Example 10 AD-mediated expression of HuBchE.
- the AD system is another viral expression system that has been used extensively to express human proteins. AD can infect a broad range of mammalian cells and permit expression of recombinant proteins in many dividing and non-dividing mammalian cell lines. Medium from cells infected with various titers of the recombinant virus are analyzed for BchE activity, and show that BchE activity in the culture medium showed dose-dependent and time-dependent expression of HuBchE post-infection of COS cells. Similar results were obtained with CHO cells (data not shown). As shown in Figure 6, accumulated BchE activity in culture medium increased exponentially over time.
- the AD construct was replication-incompetent; no additional viral particles can be produced when COS cells are infected. Therefore the continuous increase of enzyme activity in the culture medium indicated the infected cells continuously produced the BchE enzyme over days.
- CPE cell round up
- Figure 6B showed that western blot analysis of the culture medium reflected the presence of HuBchE in a time-dependent and dose-depended manner and was in good agreement with results shown in Figure 6A.
- Figure 7 shows that transfection of pAD-WT-hBchE and p AD-Gl 17H/E197Q-hBchE resulted in increasing the HuBchE activity while transfection of the control vector pAD-lacZ only showed consistent background activity. This indicated that construction of the p AD- WT- hBchE and pAD-Gl 17H/E197Q-hBchE was successful.
- CHO-Kl cells stably expressing the HuBchE WT or Gl 17H/E197Q HuBchE were first used to establish the assay conditions. The preseeded cells were washed and then overlaid with 1% agar in colorless MEM. After the agar solidified, the cells were returned to the incubator (overnight) to allow the secretion of the BchE enzyme and diffusion of the enzyme into agar.
- Example 11 Application of the solid-phase BchE activity assay for detection of AD-BchE recombinant virus.
- Using the AD system usually involves a slow tittering process due to slow plaque formation.
- the BchE activity assay allowed development of an alternative method for estimating virus titer in a shorter time frame.
- Serial-diluted HuBchE- AD virus was used to infect 293 A cells pre-seeded in 6-well plate. An agarose-MEM mix was overlaid on cells at 1 hr post infection. Plates were stained for BchE activity in the following days after initial infection.
- Virus was isolated from identified yellow spots, and plugs were cored-out from the agarose culture plate.
- Figure 9 shows an illustration of specific plugs isolated during this experiment. Positive plugs were isolated from wells infected with high virus titers and showed yellow overall after BchE activity staining. Negative plugs were isolated from wells infected with highest virus dilutions with no yellow color showing after BchE activity staining. Sample plugs were isolated from wells with distinctive yellow spots developed after staining and the spots were cored as sample plugs. Background plugs were isolated from wells with distinctive yellow spots developed after staining and the non-stained area close to the spots were cored as background plugs.
- the isolated plugs were transferred to tubes containing 0.5 ml of cultured medium and incubated at 4°C overnight.
- the medium from the positive plug and sample plug were diluted 10-fold.
- Medium incubated with plugs (Nl to 3, Pl to 3, Sl to 3, and Bl to 3) and the diluted medium (P' 1 to 3 and S' 1 to 3) were then used to infect 293 A cells pre-seeded in 24-well plate.
- BchE activity in the medium was assayed at 24, and 48 hr post-infection and average activity was presented in Table 9. [00114] Table 9. BchE activity of various plugs in the medium.
- the coated cells were stained as described above using Bchl as substrates in the presence of DTNB.
- a similar number of yellow spots was identified in plates infected with same number of AD-Gl 17H/E197Q HuBchE virus mixed with increasing number of AD-WT HuBchE virus.
- the yellow agarose spots were cored out from the plate and incubated in serum-free medium in individual tubes.
- the recombinant viruses released in the medium were analyzed with PCR using AD specific primers that were across the gene insert. The PCR product was sequenced.
- OP analogs were used with hapten synthesis.
- the invention focused on the synthesis of enantiomerically pure OP analogs of five nerve agents they mimic.
- Scheme 13 OPs and the nerve agent they mimic required for hapten synthesis
- the mixture was diluted with 7.5 mL water and 12 mL ethanol then set up for hydrogenation using 10% PdVC (15 mg) and a balloon of H2.
- the system was covered, and the mixture was stirred overnight at room temperature.
- the catalyst was removed by filtration through celite, and the filtrate was concentrated in vacuo to remove the ethanol.
- the remaining aqueous layer was extracted extensively with ethyl ether, acidified with citric acid to pH 4, and extracted again with a mixture of isopropyl alcohol and chloroform (1 :4).
- the second set of extractions was dried over Na2SO4, filtered, and concentrated to yield a yellow oil.
- the crude material was purified via preparative TLC. Spectral data for each isomer is identical.
- the agents are significantly less potent inhibitors than echothiophate. It is notable that the Sp enantiomer of both the sarin analog and the GF analog is more potent an inhibitor of human BuChE than the Rp enantiomer. This is in agreement with the stereoselectivity of human BuChE inhibition by other organophosphates reported in the literature. This may be a useful feature of our overall strategy of obtaining agents that may have variable properties for in vitro and in vivo studies. Treatment of 1-13 (Table 10) with Che or albumin in vitro or in vivo will result in a phosphorylated protein or peptides that are used to procure antibodies to be used in the Array Biosensor.
- Example 14 Preparation and testing of antibodies to ChE- organophosphosphonates of nerve agents.
- the acute toxic effects of OP compounds correlate well with their ability to inhibit AChE by reaction with an essential serine hydroxyl to form a relatively stable phosphoserine ester bond.
- OP-ChE conjugates can serve as extraordinarily sensitive selective indicators of mechanism, information about adduct structure and potentially toxic outcomes.
- phosphorylated albumin i.e., Tyr 411
- Antibodies have been developed and used to distinguish phosphonylated ChE or albumin by denaturing the enzyme to expose the active site to antibodies. Previously, antibodies have been used to probe ChE structure and allostery over catalytic activity.
- the antibodies are used in a panel of ChE biomarkers including phosporylated albumin and other phosporylated proteins for the selective classification of chemical exposures in biological samples.
- the antibodies are placed in the Array Biosensor to make a fieldable and efficient nerve agent OP and other OP- like material biosensor.
- R -CH(CHj) 2 ; -CHCH 3 C(CH 3 ) 3 ; -cyclohexyl; -CH 2 CH 2 N[CH(CH3)2]2 ; and the corresponding tabun analog.
- FmocSer will also efficiently produce the desired OP- conjugated Fmoc serine or tyrosine. Incorporation of the OP-conjugated Fmoc serine or tyrosine into the normal octapeptide synthesis will provide the required OP-conjugated decapeptides used in the immunization studies.
- Anti-Sera to the active site orga ⁇ ophosphorylated serine of Che or Albumin.
- TLFGESAGAAS the five amino acids on either side of the serine active site are the same (i.e., TLFGESAGAAS) (SEQ ID NO:11).
- the decapeptide LFGES AGAAC (SEQ ID NO: 12) is used to develop OP-conjugate-selective anti-sera.
- Partial sequence of Human Albumin (YKFQNALLVRYTKKVPQV) (SEQ ID NO: 13), Rat Albumin (YGFQNAILVRYTQKAPQV) (SEQ ID NO: 14) and Mouse Albumin (YGFQNAILVRYTQKAPQV) (SEQ ID NO: 15) peptides are likewise be used for anti-sera procurement. Adducted protein can also be used directly. Of importance is the replacement of the terminal serine with a cysteine or a sulfur-containing linker. This will allow the necessary attachment chemistry to the biosensor (discussed below). The overall use of this decapeptide will conserve resources (as it will be of utility to both AChE and BuChE) and allow greater utility of the anti-sera.
- ChE will stand for both enzymes (and by analogy, albumin).
- the 1OS and 1OSP designation refers to the non-phosphonylated and phosphonylated decapeptide.
- Anti-ChElOS and anti-ChElOSP anti-sera is generated by immunizing rabbits with ChElOS and ChElOSP peptides conjugated to keyhole limpet hemocyanin as described before. It is important to have anti-sera from both the OP- conjugated peptide (ChElOSP) and the native or non-conjugated peptide (ChElOS) to serve as a control. Conjugation is done by standard procedures. Rabbits are immuninized using standard procedures by a commercial lab.
- the requisite peptides are synthesized by standard procedures described above with the exception that the ChElOSP decapeptide will contain the same OP-conjugates arising from nerve agents 1-13 (Example 13).
- Anti-sera is purified by chromatography over a DEAE Affi-gel Blue column.
- the OP-conjugated decapeptides is coupled to Affi-gel 15 beads thru the Cys and the anti-sera is purified with these beads.
- Less specific anti-sera is eluted with 1 M NaSCN and decapeptide-specific fractions (eluted with 1 M glycine-HCl and immediately buffered to pH 8) is analyzed by ELISA to ensure specific antibodies.
- Western blots are done as above.
- rat, primate and human AChE and BuChE or albumin is treated with 0.5 mM nerve agent analogs 1-13 (Example 13) or vehicle THF for 2 hr at 4°C or until enzyme activity is decreased to 1-2% based on the Ellman colorimetric method.
- Enzyme is affinity purified from animal sera or from recombinant sources. Recombinant HuChE and primate BuChE are available in our lab and rat BuChE are purified from rat sera.
- AChE are purified from animal blood cell membranes. The inhibited enzymes are analyzed by immunoblot with a denaturing gel.
- Densitometric analysis are used to quantify changes in the intensity of protein bands labeled with the anti- ChE 1 OSP anti-sera and control anti-sera within the linear range for each antisera.
- the antisera are used in an Array biosensor to detect OP-derivatized Chef as a selective probe of OP exposure in biological fluids or environmental samples.
Abstract
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WO2003054182A2 (en) * | 2001-12-21 | 2003-07-03 | Nexia Biotechnologies, Inc. | Production of butyrylcholinesterases in transgenic mammals |
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WO2003054182A2 (en) * | 2001-12-21 | 2003-07-03 | Nexia Biotechnologies, Inc. | Production of butyrylcholinesterases in transgenic mammals |
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Title |
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LYNCH T J ET AL: "Cocaine detoxification by human plasma butyrylcholinesterase." TOXICOLOGY AND APPLIED PHARMACOLOGY AUG 1997, vol. 145, no. 2, August 1997 (1997-08), pages 363-371, XP002476585 ISSN: 0041-008X * |
SUN HONG ET AL: "Cocaine metabolism accelerated by a re-engineered human butyrylcholinesterase." THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS AUG 2002, vol. 302, no. 2, August 2002 (2002-08), pages 710-716, XP002476584 ISSN: 0022-3565 * |
XIE WEIHUA ET AL: "An improved cocaine hydrolase: The A328Y mutant of human butyrylcholinesterase is 4-fold more efficient" MOLECULAR PHARMACOLOGY, BALTIMORE, MD, US, vol. 55, no. 1, January 1999 (1999-01), pages 83-91, XP002236142 ISSN: 0026-895X * |
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