Saxena A

General

Full name : Saxena Ashima

First name : Ashima

Mail : Division of Bacterial & Rickettsial Diseases\; Walter Reed Army Institute of Research\; 503 Robert Grant Ave.\; Silver Spring\; MD 20910-7500

Zip Code :

City :

Country : USA

Email : ashima.saxena.civ@mail.mil

Phone : +13013199406

Fax : 202-782-6304

Website :

Directory :

References (100)

Title : Low levels of endogenous cholinesterases support the choice of cows, sheep and goats for the transgenic expression of human butyrylcholinesterase in milk - Belinskaya_2023_Chem.Biol.Interact_14ChEPon_110691
Author(s) : Belinskaya T , Saxena A
Ref : Chemico-Biological Interactions , :110691 , 2023
Abstract : Butyrylcholinesterase purified from human plasma (Hu BChE) as well as recombinant (r) Hu BChE are candidate enzymes that can protect humans from toxicity of organophosphorus compounds (OPs). Domestic animals such as cows, pigs, sheep, and goats have been used for the transgenic expression of a variety of valuable therapeutic proteins. Indeed, rHu BChE was successfully expressed in the milk of transgenic goats, but the presence of any endogenous cholinesterases (ChE) in milk would interfere with the isolation of expressed rHu BChE. The aim of this study was to determine the presence of endogenous ChEs in bovine, ovine, caprine, and porcine milk to determine the suitability of these species for the production of rHu BChE. Using acetyl- and butyryl- thiocholine as substrates, ChE activity (2-4 U/mL) was detected in pig milk only. ChE activities in milk from other animals were <0.01 U/mL and could only be detected following enrichment on procainamide-Sepharose gel. Two different methods based on measuring activity in the presence of acetylcholinesterase (AChE)- or BChE- specific inhibitors were used to estimate the proportions of AChE and BChE activities in enriched milk. Monoclonal antibodies (MAbs), against fetal bovine serum AChE that recognize AChEs from ruminants only, were also used to confirm the identity of AChEs. While bovine and ovine milk contained both AChE and BChE activities, caprine and porcine milk contained predominantly BChE activity. The presence of very low ChE activity supports the choice of cows, sheep, and goats for the transgenic expression of rHu BChE in milk.
ESTHER : Belinskaya_2023_Chem.Biol.Interact_14ChEPon_110691
PubMedSearch : Belinskaya_2023_Chem.Biol.Interact_14ChEPon_110691
PubMedID: 37659623

Title : Conjugates of Human Serum Butyrylcholinesterase and Nerve Agents are Behaviorally Safe in Rhesus Macaques - Saxena_2021_Chem.Biol.Interact__109499
Author(s) : Saxena A , Myers TM , Sipos ML
Ref : Chemico-Biological Interactions , :109499 , 2021
Abstract : Exogenously administered human serum butyrylcholinesterase (Hu BChE) affords protection by binding to organophosphorus (OP) nerve agents and pesticides in circulation. The resulting Hu BChE-OP conjugate undergoes 'aging' and the conjugate circulates until cleared from the body. Thus, we evaluated the effects of Hu BChE-OP conjugates on the general health and operant behavior of macaques. Rhesus macaques trained to perform a six-item serial probe recognition (SPR) task were administered 30 mg/kg of Hu BChE-soman conjugate (n=4) or Hu BChE-VX conjugate (n=4) by intramuscular injection. Performance on the SPR task was evaluated at 60-90 min after conjugate administration and daily thereafter for the next 4 weeks. Diazepam (3.2 mg/kg), a positive control, was administered 5 weeks after conjugate administration and performance on the SPR task was evaluated as before. Blood collected throughout the study was analyzed for acetylcholinesterase (AChE) and BChE activities. Residual BChE activity of conjugates displayed a similar pharmacokinetic profile as free Hu BChE. Neither of the Hu BChE-OP conjugates produced clear or pronounced degradations in performance on the SPR task. In contrast, diazepam clearly impaired performance on the SPR task on the day of administration in 7 of 8 macaques (and sometimes longer). Taken together, these results suggest that Hu BChE-OP conjugates are safe and provide further support for the development of Hu BChE as a bioscavenger for use in humans.
ESTHER : Saxena_2021_Chem.Biol.Interact__109499
PubMedSearch : Saxena_2021_Chem.Biol.Interact__109499
PubMedID: 33961835

Title : Acetylcholinesterase inhibition resulting from exposure to inhaled OP can be prevented by pretreatment with BChE in both macaques and minipigs - Rosenberg_2020_Neuropharmacol__108150
Author(s) : Rosenberg YJ , Saxena A
Ref : Neuropharmacology , :108150 , 2020
Abstract : More frequent and widespread nerve agent attacks highlight the need for efficacious pre- and post-exposure organophosphate (OP) counter-measures to protect military and civilian populations. Because of critical targeting of acetylcholinesterase (AChE) in the CNS by OPs, a pre-treatment candidate for preventing/reducing poisoning will be broadly acting molecules that scavenges OPs in blood before they reach their physiological targets. Prophylactic human butyrylcholinesterase (HuBChE), the leading pretreatment candidate, has been shown to protect against multiple LD50's of nerve agents in rodents, macaques, and minipigs. This review describes the development of a HuBChE bioscavenger pretreatment from early proof-of-concept studies to pre-clinical studies with the native injectable enzyme and the development of aerosolized forms of recombinant enzyme, which can be delivered by inhalation nebulizer devices, to effect protection against inhaled OP nerve agents and insecticides. Early animal studies utilized parenteral exposure. However, lungs are the portal of entry for most volatile OP vapors and represent the major means of OP intoxication. In this regard, pretreat-ment with 7.5mg/kg of HuBChE by IM injection protected minipigs against lethal sarin vapor and preventing AChE inhibition in the blood. This is similar to the five-day protection in macaques by an aerosolized rHuBChE using a nebulizer against aerosolized paraoxon (estimated to be an 8mg/kg estimated human dose). Importantly, lethal inhaled doses of OP may be smaller relative to the same dose delivered by injection, thus reducing the protective HuBChE dose while a combination of HuBChE and post-exposure oxime may prolong protection.
ESTHER : Rosenberg_2020_Neuropharmacol__108150
PubMedSearch : Rosenberg_2020_Neuropharmacol__108150
PubMedID: 32442543

Title : Proline 285 is integral for the reactivation of organophosphate-inhibited human butyrylcholinesterase by 2-PAM - diTargiani_2020_Chem.Biol.Interact__109092
Author(s) : diTargiani RC , Belinskaya T , Tipparaju P , Lockridge O , Saxena A
Ref : Chemico-Biological Interactions , :109092 , 2020
Abstract : Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger that protects from the toxicity of nerve agents. Non-human primates are suitable models for toxicity studies that cannot be performed in humans. We evaluated the biochemical properties of native macaque (MaBChE) tetramers, compared to recombinant MaBChE monomers, PEGylated recombinant MaBChE tetramers and monomers, and native HuBChE tetramers. Km and kcat values for butyrylthiocholine were independent of subunit assembly status. The Km for all forms of MaBChE was about 70muM, compared to 13muM for HuBChE. The kcat was about 100,000 min(-1) for MaBChE and 30,000 min(-1) for HuBChE. The reversible inhibitor ethopropazine had similar Ki values of 0.05muM for all MaBChE forms and HuBChE. The bimolecular rate constant, ki, for inhibition by diisopropylfluorophosphate (DFP), an analog of sarin, was 2.2 to 2.5x10(7)M(-1)min(-1) for all MaBChE forms and for HuBChE. A major difference between MaBChE and HuBChE was the rate of reactivation by 2-PAM. The second order rate constant for reactivation of DFP-inhibited MaBChE by 2-PAM was 1.4M(-1)min(-1), but was 380 fold faster for DFP-inhibited HuBChE (kr 531M(-1)min(-1)). The acyl pocket of MaBChE has Leu285 in place of Pro285 in HuBChE. The reactivation rate of DFP-inhibited HuBChE mutant P285L by 2-PAM was reduced 5.8% (kr 92M(-1)min(-1)) indicating that P285 determines whether 2-PAM binds in an orientation that favors release of diisopropylphosphate. DFP-inhibited MaBChE treated with 0.2M 2-PAM recovered 10% of its original activity, whereas DFP-inhibited HuBChE recovered 80% activity. It was concluded that the biochemical properties of MaBChE are similar to those of HuBChE except for the reactivation of DFP-inhibited BChE.
ESTHER : diTargiani_2020_Chem.Biol.Interact__109092
PubMedSearch : diTargiani_2020_Chem.Biol.Interact__109092
PubMedID: 32278739

Title : Monoclonal antibodies to fetal bovine serum acetylcholinesterase distinguish between acetylcholinesterases from ruminant and non-ruminant species - Naik_2020_Chem.Biol.Interact__109225
Author(s) : Naik RS , Belinskaya T , Vinayaka CR , Saxena A
Ref : Chemico-Biological Interactions , :109225 , 2020
Abstract : Two types of cholinesterases (ChEs) are present in mammalian blood and tissues: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). While AChE regulates neurotransmission by hydrolyzing acetylcholine at the postsynaptic membranes and neuromuscular junctions, BChE in plasma has been suggested to be involved in detoxifying toxic compounds. This study was undertaken to establish the identity of circulating ChE activity in plasmas from domestic animals (bovine, ovine, caprine, porcine and equine) by assessing sensitivity to AChE-specific inhibitors (BW284c51 and edrophonium) and BChE-specific inhibitors (dibucaine, ethopropazine and Iso-OMPA) as well as binding to anti-FBS AChE monoclonal antibodies (MAbs). Based on the inhibition of ChE activity by ChE-specific inhibitors, it was determined that bovine, ovine and caprine plasma predominantly contain AChE, while porcine and equine plasma contain BChE. Three of the anti-FBS AChE MAbs, 4E5, 5E8 and 6H9, inhibited 85-98% of enzyme activity in bovine, ovine and caprine plasma, confirming that the esterase in these plasmas was AChE. These MAbs did not bind to purified recombinant human or mouse AChE, demonstrating that these MAbs were specific for AChEs from ruminant species. These MAbs did not inhibit the activity of purified human BChE, or ChE activity in porcine and equine plasma, confirming that the ChE in these plasmas was BChE. Taken together, these results demonstrate that anti-FBS AChE MAbs can serve as useful tools for distinguishing between AChEs from ruminant and non-ruminant species and BChEs.
ESTHER : Naik_2020_Chem.Biol.Interact__109225
PubMedSearch : Naik_2020_Chem.Biol.Interact__109225
PubMedID: 32795450

Title : Identification of Carboxylesterase, Butyrylcholinesterase, Acetylcholinesterase, Paraoxonase, and Albumin Pseudoesterase in Guinea Pig Plasma through Nondenaturing Gel Electrophoresis - Napon_2018_Comp.Med_68_367
Author(s) : Napon G , Dafferner AJ , Saxena A , Lockridge O
Ref : Comp Med , 68 :367 , 2018
Abstract : Drugs to protect against nerve agent toxicity are tested in animals. The current preferred small animal model is guinea pigs because their plasma bioscavenging capacity resembles that of NHP. We stained nondenaturing polyacrylamide slab gels with a variety of substrates, inhibitors, and antibodies to identify the esterases in heparinized guinea pig plasma. An intense band of carboxylesterase activity migrated behind albumin. Minor carboxylesterase bands were revealed after background activity from paraoxonase was inhibited by using EDTA. The major butyrylcholinesterase band was a disulfide-linked dimer. Incubation with the antihuman butyrylcholinesterase antibody B2 18-5 shifted the butyrylcholinesterase dimer band to slower migrating complexes. Carboxylesterases were distinguished from butyrylcholinesterase by their sensitivity to inhibition by bis-p-nitrophenyl phosphate. Acetylcholinesterase tetramers formed a complex with the antihuman acetylcholinesterase antibody HR2. Organophosphorus toxicants including cresyl saligenin phosphate, dichlorvos, and chlorpyrifos oxon irreversibly inhibited the serine esterases but not paraoxonase. Albumin pseudoesterase activity was seen in gels stained with alpha- or beta-naphthyl acetate and fast blue RR. We conclude that guinea pig plasma has 2 types of carboxylesterase, butyrylcholinesterase dimers and 5 minor butyrylcholinesterase forms, a small amount of acetylcholinesterase tetramers, paraoxonase, and albumin pseudoesterase activity. A knockout mouse with no carboxylesterase activity in plasma is available and may prove to be a better model for studies of nerve agent toxicology than guinea pigs.
ESTHER : Napon_2018_Comp.Med_68_367
PubMedSearch : Napon_2018_Comp.Med_68_367
PubMedID: 30278860

Title : Tetramer organizing polyproline-rich peptides identified by mass spectrometry after release of the peptides from Hupresin-purified butyrylcholinesterase tetramers isolated from milk of domestic pig (Sus scrofa) - Saxena_2018_Data.Brief_20_1607
Author(s) : Saxena A , Belinskaya T , Schopfer LM , Lockridge O
Ref : Data Brief , 20 :1607 , 2018
Abstract : Milk of the domestic pig has 10 times more butyrylcholinesterase (BChE) per mL than porcine serum. We purified BChE from porcine milk by affinity chromatography on Hupresin-Sepharose. The pure porcine BChE (PoBChE) was a tetramer with a molecular weight of 340,000, similar to that of human BChE tetramers. The C-terminal 40 residues of PoBChE constitute the tetramerization domain. The glue that holds the 4 BChE subunits together is a polyproline-rich peptide. Mass spectrometry analysis of trypsin-digested PoBChE identified a variety of polyproline-rich peptides originating from 12 different proteins. The donor proteins exist in the nucleus or cytoplasm of cells and contribute their polyproline-rich peptides after a cell is degraded. The secreted PoBChE scavenges the polyproline-rich peptides and incorporates one polyproline peptide per PoBChE tetramer, where the polyproline peptide is bound noncovalently but very tightly with an estimated dissociation constant of 10(-12) M. The most abundant polyproline-rich peptides were derived from acrosin, homeobox protein HoxB4, lysine-specific demethylase 6B, proline-rich protein 12, and proline-rich membrane anchor 1 (PRiMA). The research article associated with the data in this report can be found in Saxena et al. (2018). The Data in Brief report lists all the polyproline-rich peptides identified in PoBChE tetramers.
ESTHER : Saxena_2018_Data.Brief_20_1607
PubMedSearch : Saxena_2018_Data.Brief_20_1607
PubMedID: 30263913

Title : Characterization of butyrylcholinesterase from porcine milk - Saxena_2018_Arch.Biochem.Biophys_652_38
Author(s) : Saxena A , Belinskaya T , Schopfer LM , Lockridge O
Ref : Archives of Biochemistry & Biophysics , 652 :38 , 2018
Abstract : Human butyrylcholinesterase (HuBChE) is under development for use as a pretreatment antidote against nerve agent toxicity. Animals are used to evaluate the efficacy of HuBChE for protection against organophosphorus nerve agents. Pharmacokinetic studies of HuBChE in minipigs showed a mean residence time of 267h, similar to the half-life of HuBChE in humans, suggesting a high degree of similarity between BChE from 2 sources. Our aim was to compare the biochemical properties of PoBChE purified from porcine milk to HuBChE purified from human plasma. PoBChE hydrolyzed acetylthiocholine slightly faster than butyrylthiocholine, but was sensitive to BChE-specific inhibitors. PoBChE was 50-fold less sensitive to inhibition by DFP than HuBChE and 5-fold slower to reactivate in the presence of 2-PAM. The amino acid sequence of PoBChE determined by liquid chromatography tandem mass spectrometry was 91% identical to HuBChE. Monoclonal antibodies 11D8, mAb2, and 3E8 (HAH 002) recognized both PoBChE and HuBChE. Assembly of 4 identical subunits into tetramers occurred by noncovalent interaction with polyproline-rich peptides in PoBChE as well as in HuBChE, though the set of polyproline-rich peptides in milk-derived PoBChE was different from the set in plasma-derived HuBChE tetramers. It was concluded that the esterase isolated from porcine milk is PoBChE.
ESTHER : Saxena_2018_Arch.Biochem.Biophys_652_38
PubMedSearch : Saxena_2018_Arch.Biochem.Biophys_652_38
PubMedID: 29908755
Gene_locus related to this paper: pig-BCHE

Title : Probing the role of amino acids in oxime-mediated reactivation of nerve agent-inhibited human acetylcholinesterase - Chambers_2015_Toxicol.In.Vitro_29_408
Author(s) : Chambers C , Luo C , Tong M , Yang Y , Saxena A
Ref : Toxicol In Vitro , 29 :408 , 2015
Abstract : In this study, we employed site-directed mutagenesis to understand the role of amino acids in the gorge in oxime-induced reactivation of nerve agent-inhibited human (Hu) acetylcholinesterase (AChE). The organophosphorus (OP) nerve agents studied included GA (tabun), GB (sarin), GF (cyclosarin), VX, and VR. The kinetics of reactivation were examined using both the mono-pyridinium oxime 2-PAM and bis-pyridinium oximes MMB4, HI-6, and HLo-7. The second-order reactivation rate constants were used to compare reactivation of nerve agent-inhibited wild-type (WT) and mutant enzymes. Residues including Y72, Y124 and W286 were found to play important roles in reactivation by bis-pyridinium, but not by mono-pyridinium oximes. Residue Y124 also was found to play a key role in reactivation by HI-6 and HLo-7, while E202 was important for reactivation by all oximes. Residue substitutions of F295 by Leu and Y337 by Ala showed enhanced reactivation by bis-pyridinium oximes MMB4, HI-6, and HLo-7, possibly by providing more accessibility of the OP moiety associated at the active-site serine to the oxime. These results are similar to those observed previously with bovine AChE and demonstrate that there is significant similarity between human and bovine AChEs with regard to oxime reactivation.
ESTHER : Chambers_2015_Toxicol.In.Vitro_29_408
PubMedSearch : Chambers_2015_Toxicol.In.Vitro_29_408
PubMedID: 25451328

Title : IL-17-producing CD4(+) T cells contribute to the loss of B-cell tolerance in experimental autoimmune myasthenia gravis - Schaffert_2015_Eur.J.Immunol_45_1339
Author(s) : Schaffert H , Pelz A , Saxena A , Losen M , Meisel A , Thiel A , Kohler S
Ref : European Journal of Immunology , 45 :1339 , 2015
Abstract : The role of Th17 cells in the pathogenesis of autoantibody-mediated diseases is unclear. Here, we assessed the contribution of Th17 cells to the pathogenesis of experimental autoimmune myasthenia gravis (EAMG), which is induced by repetitive immunizations with Torpedo californica acetylcholine receptor (tAChR). We show that a significant fraction of tAChR-specific CD4(+) T cells is producing IL-17. IL-17(ko) mice developed fewer or no EAMG symptoms, although the frequencies of tAChR-specific CD4(+) T cells secreting IL-2, IFN-gamma, or IL-21, and the percentage of FoxP3(+) Treg cells were similar to WT mice. Even though the total anti-tAChR antibody levels were equal, the complement fixating IgG2b subtype was reduced in IL-17(ko) as compared to WT mice. Most importantly, pathogenic anti-murine AChR antibodies were significantly lower in IL-17(ko) mice. Furthermore, we confirmed the role of Th17 cells in EAMG pathogenesis by the reconstitution of TCR beta/delta(ko) mice with WT or IL-17(ko) CD4(+) T cells. In conclusion, we show that the level of IgG2b and the loss of B-cell tolerance, which results in pathogenic anti-murine AChR-specific antibodies, are dependent on IL-17 production by CD4(+) T cells. Thus, we describe here for the first time how Th17 cells are involved in the induction of classical antibody-mediated autoimmunity.
ESTHER : Schaffert_2015_Eur.J.Immunol_45_1339
PubMedSearch : Schaffert_2015_Eur.J.Immunol_45_1339
PubMedID: 25676041

Title : Prophylaxis with human serum butyrylcholinesterase protects Gottingen minipigs exposed to a lethal high-dose of sarin vapor - Saxena_2015_Chem.Biol.Interact_238_161
Author(s) : Saxena A , Hastings NB , Sun W , Dabisch PA , Hulet SW , Jakubowski EM , Mioduszewski RJ , Doctor BP
Ref : Chemico-Biological Interactions , 238 :161 , 2015
Abstract : Serum-derived human butyrylcholinesterase (Hu BChE) is a stoichiometric bioscavenger that is being developed as a potential prophylactic nerve agent countermeasure. Previously, we reported the prophylactic efficacy of Hu BChE in Gottingen minipigs against a whole-body exposure to 4.1mg/m3 of sarin (GB) vapor, which produced lethality over 60min. Since the toxicity of nerve agent is concentration-dependent, in the present study, we investigated the toxic effects of an almost 3-fold higher rate of GB vapor exposure and the ability of Hu BChE to protect minipigs against this exposure. Male minipigs were subjected to: (1) air exposure; (2) GB vapor exposure; or (3) pretreatment with 7.5mg/kg of Hu BChE by i.m. injection, 24h prior to whole-body exposure to 11.4mg/m3 of GB vapor for 10min. Electrocardiogram, electroencephalogram, and pupil size were monitored throughout exposure. Blood drawn before and throughout exposure was analyzed for blood gases, electrolytes, metabolites, acetylcholinesterase and BChE activities, and amount of GB bound to red blood cells and plasma. A novel finding was that saline-treated animals exposed to GB vapor did not develop any seizures, but manifested a variety of cardiac and whole blood toxic signs and rapidly died due to respiratory failure. Strikingly, pre-treatment with 7.5mg/kg of Hu BChE not only prevented lethality, but also avoided all cardiac toxic signs manifested in the non-treated cohort. Thus, Hu BChE alone can serve as an effective prophylactic countermeasure versus a lethal high-dose exposure to GB vapor.
ESTHER : Saxena_2015_Chem.Biol.Interact_238_161
PubMedSearch : Saxena_2015_Chem.Biol.Interact_238_161
PubMedID: 26145887

Title : Genome Sequence of Novosphingobium lindaniclasticum LE124T, Isolated from a Hexachlorocyclohexane Dumpsite - Saxena_2013_Genome.Announc_1_e00715
Author(s) : Saxena A , Nayyar N , Sangwan N , Kumari R , Khurana JP , Lal R
Ref : Genome Announc , 1 : , 2013
Abstract : Novosphingobium lindaniclasticum LE124(T) is a hexachlorocyclohexane (HCH)-degrading bacterium isolated from a high-dosage-point HCH dumpsite (450 mg HCH/g soil) located in Lucknow, India (27 degrees 00'N and 81 degrees 09'E). Here, we present the annotated draft genome sequence of strain LE124(T), which has an estimated size of 4.86 Mb and is comprised of 4,566 coding sequences.
ESTHER : Saxena_2013_Genome.Announc_1_e00715
PubMedSearch : Saxena_2013_Genome.Announc_1_e00715
PubMedID: 24029761
Gene_locus related to this paper: 9sphn-t0hqb1 , 9sphn-t0hmn5 , 9sphn-t0iya6 , 9sphn-t0gy64 , 9sphn-t0hhu1 , 9sphn-t0i5p8 , 9sphn-t0hp61

Title : Multifunctional cholinesterase and amyloid Beta fibrillization modulators. Synthesis and biological investigation - Butini_2013_ACS.Med.Chem.Lett_4_1178
Author(s) : Butini S , Brindisi M , Brogi S , Maramai S , Guarino E , Panico A , Saxena A , Chauhan V , Colombo R , Verga L , De Lorenzi E , Bartolini M , Andrisano V , Novellino E , Campiani G , Gemma S
Ref : ACS Med Chem Lett , 4 :1178 , 2013
Abstract : In order to identify novel Alzheimer's modifying pharmacological tools, we developed bis-tacrines bearing a peptide moiety for specific interference with surface sites of human acetylcholinesterase (hAChE) binding amyloid-beta (Abeta). Accordingly, compounds 2a-c proved to be inhibitors of hAChE catalytic and noncatalytic functions, binding the catalytic and peripheral sites, interfering with Abeta aggregation and with the Abeta self-oligomerization process (2a). Compounds 2a-c in complex with TcAChE span the gorge with the bis-tacrine system, and the peptide moieties bulge outside the gorge in proximity of the peripheral site. These moieties are likely responsible for the observed reduction of hAChE-induced Abeta aggregation since they physically hamper Abeta binding to the enzyme surface. Moreover, 2a was able to significantly interfere with Abeta self-oligomerization, while 2b,c showed improved inhibition of hAChE-induced Abeta aggregation.
ESTHER : Butini_2013_ACS.Med.Chem.Lett_4_1178
PubMedSearch : Butini_2013_ACS.Med.Chem.Lett_4_1178
PubMedID: 24900626

Title : Effect of polyethylene glycol conjugation on the circulatory stability of plasma-derived human butyrylcholinesterase in mice - Sun_2013_Chem.Biol.Interact_203_172
Author(s) : Sun W , Luo C , Tipparaju P , Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 203 :172 , 2013
Abstract : Exogenously administered human serum butyrylcholinesterase (Hu BChE) was demonstrated to function as a bioscavenger of highly toxic organophosphorus (OP) compounds in several animal species. Since the enzyme is isolated from human serum, it is currently the most suitable pretreatment for human use. A dose of 200-300mg/70kg human adult is projected to provide protection from 2 X LD50 of soman. Due to the limited supply of Hu BChE, strategies aimed at reducing the dose of enzyme are being explored. In this study, we investigated the effect of modification with polyethylene glycol (PEG) on the in vivo stability of Hu BChE. Mice were given two injections of either Hu BChE or Hu BChE modified with PEG-5K or PEG-20K, six weeks apart. Pharmacokinetic parameters, such as mean residence time (MRT), maximal concentration (Cmax), elimination half-life (T1/2), and area under the plasma concentration time curve extrapolated to infinity (AUC), were determined. For the first injection, values for MRT, T1/2, Cmax, and AUC for PEG-5K-Hu BChE and PEG-20K-Hu BChE were similar to those for Hu BChE. These values for the second injection of Hu BChE as well as PEG-Hu BChEs were lower as compared to those for the first injections, likely due to antibody-mediated clearance.
ESTHER : Sun_2013_Chem.Biol.Interact_203_172
PubMedSearch : Sun_2013_Chem.Biol.Interact_203_172
PubMedID: 23220586

Title : Development and validation of a simple assay for the determination of cholinesterase activity in whole blood of laboratory animals - Naik_2013_J.Appl.Toxicol_33_290
Author(s) : Naik RS , Liu W , Saxena A
Ref : Journal of Applied Toxicology , 33 :290 , 2013
Abstract : Current methods for measuring acetylcholinesterase (AChE) activities in whole blood use butyrylcholinesterase (BChE)-selective inhibitors. However, the poor selectivity of these inhibitors results in the inhibition of AChE activity to some degree, leading to errors in reported values. The goal of this study was to develop and validate a simple assay for measuring AChE and BChE activities in whole blood from humans as well as experimental animals. Blood was fractionated into plasma and erythrocytes, and cholinesterase activities were titrated against ethopropazine and (-)-huperzine A to determine the lowest concentration of ethopropazine that inhibited BChE completely without affecting AChE activity and the lowest concentration of (-)-huperzine A that inhibited AChE completely without interfering with BChE activity. Results indicate that 20 microm ethopropazine can be successfully used for the accurate measurement of AChE activity in blood from humans as well as animals. Use of (-)-huperzine A is not required for measuring BChE activity in normal or 'exposed' blood samples. The method was validated for blood from several animal species, including mice, rats, guinea pigs, dogs, minipigs, and African green, cynomolgus and rhesus monkeys. This method is superior to all reported methods, does not require the separation of erythrocyte and plasma fractions, and is suitable for measuring cholinesterase activities in fresh or frozen blood from animals that were exposed to nerve agents or those that were administered high doses of BChE. The method is simple, direct, reproducible, and reliable and can easily be adapted for high-throughput screening of blood samples. Published 2012. This article is a US Government work and is in the public domain in the USA.
ESTHER : Naik_2013_J.Appl.Toxicol_33_290
PubMedSearch : Naik_2013_J.Appl.Toxicol_33_290
PubMedID: 22407886

Title : In vitro characterization of organophosphorus compound hydrolysis by native and recombinant human prolidase - Chandrasekaran_2013_Toxicol.In.Vitro_27_499
Author(s) : Chandrasekaran L , Belinskaya T , Saxena A
Ref : Toxicol In Vitro , 27 :499 , 2013
Abstract : Human prolidase is a binuclear metalloenzyme, which can potentially function as a catalytic bioscavenger for organophosphorus (OP) nerve agents. Although the biochemical properties of native prolidase purified from human erythrocytes, liver, kidney, and fibroblast cells are well known, it is very poorly characterized with regard to its OP hydrolyzing activity. Also, the high cost of purification of large quantities of native enzyme limits its use as a bioscavenger. Thus, recombinant human prolidase with similar biochemical properties to those of native enzyme would be more suitable as a catalytic bioscavenger. In this study, we established an Escherichia coli expression system, which produced a large amount of tagged human liver prolidase that was purified to over 95% purity from the soluble fraction of cell lysate by affinity chromatography on Streptavidin-agarose resin. The catalytic properties of the recombinant enzyme were compared in vitro with those of highly purified prolidase I isolated from human erythrocytes. The catalytic properties of recombinant prolidase overlap with those of the erythrocyte-derived native enzyme. Both enzymes efficiently hydrolyzed diisopropylfluorophosphate, sarin, soman, tabun and cyclosarin, but were much less efficient at hydrolyzing paraoxon and methyl paraoxon. These results suggest that human prolidase expressed in E. coli is suitable for further development as a catalytic bioscavenger for OP nerve agents.
ESTHER : Chandrasekaran_2013_Toxicol.In.Vitro_27_499
PubMedSearch : Chandrasekaran_2013_Toxicol.In.Vitro_27_499
PubMedID: 22677456

Title : Polyproline tetramer organizing peptides in fetal bovine serum acetylcholinesterase - Biberoglu_2013_Biochim.Biophys.Acta_1834_745
Author(s) : Biberoglu K , Schopfer LM , Saxena A , Tacal O , Lockridge O
Ref : Biochimica & Biophysica Acta , 1834 :745 , 2013
Abstract : Acetylcholinesterase (AChE) in the serum of fetal cow is a tetramer. The related enzyme, butyrylcholinesterase (BChE), in the sera of humans and horse requires polyproline peptides for assembly into tetramers. Our goal was to determine whether soluble tetrameric AChE includes tetramer organizing peptides in its structure. Fetal bovine serum AChE was denatured by boiling to release non-covalently bound peptides. Bulk protein was separated from peptides by filtration and by high performance liquid chromatography. Peptide mass and amino acid sequence of the released peptides were determined by MALDI-TOF-TOF and LTQ-Orbitrap mass spectrometry. Twenty polyproline peptides, divided into 5 families, were identified. The longest peptide contained 25 consecutive prolines and no other amino acid. Other polyproline peptides included one non-proline amino acid, for example serine at the C-terminus of 20 prolines. A search of the mammalian proteome database suggested that this assortment of polyproline peptides originated from at least 5 different precursor proteins, none of which were the ColQ or PRiMA of membrane-anchored AChE. To date, AChE and BChE are the only proteins known that include polyproline tetramer organizing peptides in their tetrameric structure.
ESTHER : Biberoglu_2013_Biochim.Biophys.Acta_1834_745
PubMedSearch : Biberoglu_2013_Biochim.Biophys.Acta_1834_745
PubMedID: 23352838

Title : Amino acid residues at the N- and C-termini are essential for the folding of active human butyrylcholinesterase polypeptide - Naik_2013_Chem.Biol.Interact_203_24
Author(s) : Naik RS , Pattabiraman N , Patel KA , Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 203 :24 , 2013
Abstract : Human serum butyrylcholinesterase (HuBChE) is currently the most suitable bioscavenger for the prophylaxis of highly toxic organophosphate (OP) nerve agents. A dose of 200mg of HuBChE is envisioned as a prophylactic treatment that can protect humans from an exposure of up to 2xLD50 of soman. The limited availability and administration of multiple doses of this stoichiometric bioscavenger make this pretreatment difficult. Thus, the goal of this study was to produce a smaller enzymatically active HuBChE polypeptide (HBP) that could bind to nerve agents with high affinity thereby reducing the dose of enzyme. Studies have indicated that the three-dimensional structure and the domains of HuBChE (acyl pocket, lip of the active center gorge, and the anionic substrate-binding domain) that are critical for the binding of substrate are also essential for the selectivity and binding of inhibitors including OPs. Therefore, we designed three HBPs by deleting some N- and C-terminal residues of HuBChE by maintaining the folds of the active site core that includes the three active site residues (S198, E325, and H438). HBP-4 that lacks 45 residues from C-terminus but known to have BChE activity was used as a control. The cDNAs for the HBPs containing signal sequences were synthesized, cloned into different mammalian expression vectors, and recombinant polypeptides were transiently expressed in different cell lines. No BChE activity was detected in the culture media of cells transfected with any of the newly designed HBPs, and the inactive polypeptides remained inside the cells. Only enzymatically active HBP-4 was secreted into the culture medium. These results suggest that residues at the N- and C-termini are required for the folding and/or maintenance of HBP into an active stable, conformation.
ESTHER : Naik_2013_Chem.Biol.Interact_203_24
PubMedSearch : Naik_2013_Chem.Biol.Interact_203_24
PubMedID: 23044488

Title : Draft Genome Sequence of Agrobacterium sp. Strain UHFBA-218, Isolated from Rhizosphere Soil of Crown Gall-Infected Cherry Rootstock Colt - Dua_2013_Genome.Announc_1_E00302
Author(s) : Dua A , Sangwan N , Kaur J , Saxena A , Kohli P , Gupta AK , Lal R
Ref : Genome Announc , 1 : , 2013
Abstract : We report here the draft genome sequence of the alphaproteobacterium Agrobacterium sp. strain UHFBA-218, which was isolated from rhizosphere soil of crown gall-infected cherry rootstock Colt. The draft genome of strain UHFBA-218 consists of 112 contigs (5,425,303 bp) and 5,063 coding sequences with a G+C content of 59.8%.
ESTHER : Dua_2013_Genome.Announc_1_E00302
PubMedSearch : Dua_2013_Genome.Announc_1_E00302
PubMedID: 23723402
Gene_locus related to this paper: rhird-m8b858 , rhird-m8b836 , rhird-a0a067u386 , rhird-m8b397

Title : Characterization of human serum butyrylcholinesterase in rhesus monkeys: behavioral and physiological effects - Myers_2012_Neurotoxicol.Teratol_34_323
Author(s) : Myers TM , Sun W , Naik RS , Clark MG , Doctor BP , Saxena A
Ref : Neurotoxicology & Teratology , 34 :323 , 2012
Abstract : The effects of a large dose of human serum butyrylcholinesterase (HuBChE) were evaluated in rhesus monkeys using a serial-probe recognition (SPR) task designed to assess attention and short-term memory. Each monkey received an intravenous injection of 150 mg (105,000 U or 30 mg/kg) of HuBChE 60 min prior to testing on the SPR task. Concurrent with the cognitive-behavioral assessment, blood was collected at various time points throughout the study and was analyzed for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities, anti-BChE antibody production and gross clinical pathology (i.e., complete blood count and clinical chemistry panel). HuBChE revealed a peak blood activity of 227 U/ml at 5 min after intravenous injection and a mean residence time of approximately 72 h. No cognitive-behavioral decrements of any kind in SPR performance and no toxic signs in clinical pathology were detected in any of the blood assays during the 5 weeks of observation. Anti-HuBChE antibodies peaked at about 14 days after injection, with no concomitant behavioral changes. These results demonstrate the behavioral and physiological safety of HuBChE in rhesus monkeys and support its development as a bioscavenger for the prophylaxis of chemical warfare agent toxicity in humans.
ESTHER : Myers_2012_Neurotoxicol.Teratol_34_323
PubMedSearch : Myers_2012_Neurotoxicol.Teratol_34_323
PubMedID: 22402122

Title : Genome sequence of Acinetobacter sp. strain HA, isolated from the gut of the polyphagous insect pest Helicoverpa armigera - Malhotra_2012_J.Bacteriol_194_5156
Author(s) : Malhotra J , Dua A , Saxena A , Sangwan N , Mukherjee U , Pandey N , Rajagopal R , Khurana P , Khurana JP , Lal R
Ref : Journal of Bacteriology , 194 :5156 , 2012
Abstract : In this study, Acinetobacter sp. strain HA was isolated from the midgut of a fifth-instar larva of Helicoverpa armigera. Here, we report the draft genome sequence (3,125,085 bp) of this strain that consists of 102 contigs, 2,911 predicted coding sequences, and a G+C content of 41%.
ESTHER : Malhotra_2012_J.Bacteriol_194_5156
PubMedSearch : Malhotra_2012_J.Bacteriol_194_5156
PubMedID: 22933775
Gene_locus related to this paper: 9gamm-i4zrl8 , 9gamm-i4zpd5

Title : Differences in amino acid residues in the binding pockets dictate substrate specificities of mouse senescence marker protein-30, human paraoxonase1, and squid diisopropylfluorophosphatase - Belinskaya_2012_Biochim.Biophys.Acta_1824_701
Author(s) : Belinskaya T , Pattabiraman N , diTargiani RC , Choi M , Saxena A
Ref : Biochimica & Biophysica Acta , 1824 :701 , 2012
Abstract : Senescence marker protein-30 (SMP-30) is a candidate enzyme that can function as a catalytic bioscavenger of organophosphorus (OP) nerve agents. We purified SMP-30 from mouse (Mo) liver and compared its hydrolytic activity towards various esters, lactones, and G-type nerve agents with that of human paraoxonase1 (Hu PON1) and squid diisopropylfluorophosphatase (DFPase). All three enzymes contain one or two metal ions in their active sites and fold into six-bladed beta-propeller structures. While Hu PON1 hydrolyzed a variety of lactones, the only lactone that was a substrate for Mo SMP-30 was d-(+)-gluconic acid delta-lactone. Squid DFPase was much more efficient at hydrolyzing DFP and G-type nerve agents as compared to Mo SMP-30 or Hu PON1. The K(m) values for DFP were in the following order: Mo SMP-30>Hu PON1>squid DFPase, suggesting that the efficiency of DFP hydrolysis may be related to its binding in the active sites of these enzymes. Thus, homology modeling and docking were used to simulate the binding of DFP and selected delta-lactones in the active sites of Hu SMP-30, Hu PON1, and squid DFPase. Results from molecular modeling studies suggest that differences in metal-ligand coordinations, the hydrophobicity of the binding pockets, and limited space in the binding pocket due to the presence of a loop, are responsible for substrate specificities of these enzymes.
ESTHER : Belinskaya_2012_Biochim.Biophys.Acta_1824_701
PubMedSearch : Belinskaya_2012_Biochim.Biophys.Acta_1824_701
PubMedID: 22401958

Title : Pretreatment with human serum butyrylcholinesterase alone prevents cardiac abnormalities, seizures, and death in Gottingen minipigs exposed to sarin vapor - Saxena_2011_Biochem.Pharmacol_82_1984
Author(s) : Saxena A , Sun W , Dabisch PA , Hulet SW , Hastings NB , Jakubowski EM , Mioduszewski RJ , Doctor BP
Ref : Biochemical Pharmacology , 82 :1984 , 2011
Abstract : Human serum butyrylcholinesterase (Hu BChE) is a stoichiometric bioscavenger that is being developed as a prophylactic countermeasure against organophosphorus nerve agents. This study was designed to evaluate the efficacy of Hu BChE against whole-body inhalation exposure to a lethal dose of sarin (GB) vapor. Male Gottingen minipigs were subjected to: air exposure, GB vapor exposure, or pretreatment with Hu BChE followed by GB vapor exposure. Hu BChE was administered by i.m. injection 24 h prior to exposure to 4.1 mg/m(3) of GB vapor for 60 min. Electrocardiograms (ECG), electroencephalograms (EEG), and pupil size were recorded throughout exposure. Blood drawn before and throughout exposure was analyzed for blood gases, electrolytes, metabolites, acetylcholinesterase and BChE activities, and amount of GB present. Untreated animals exposed to GB vapor exhibited cardiac abnormalities and generalized seizures, ultimately succumbing to respiratory failure. Pretreatment with 3.0 or 6.5 mg/kg of Hu BChE delayed blood gas and acid-base disturbances and the onset of cardiac and neural toxic signs, but failed to increase survivability. Pretreatment with 7.5 mg/kg of Hu BChE, however, completely prevented toxic signs, with blood chemistry and ECG and EEG parameters indistinguishable from control during and after GB exposure. GB bound in plasma was 200-fold higher than plasma from pigs that did not receive Hu BChE, suggesting that Hu BChE scavenged GB in blood and prevented it from reaching other tissues. Thus, prophylaxis with Hu BChE alone not only increased survivability, but also prevented cardiac abnormalities and neural toxicity in minipigs exposed to a lethal dose of GB vapor.
ESTHER : Saxena_2011_Biochem.Pharmacol_82_1984
PubMedSearch : Saxena_2011_Biochem.Pharmacol_82_1984
PubMedID: 21968035

Title : Whole genome sequence of the rifamycin B-producing strain Amycolatopsis mediterranei S699 - Verma_2011_J.Bacteriol_193_5562
Author(s) : Verma M , Kaur J , Kumar M , Kumari K , Saxena A , Anand S , Nigam A , Ravi V , Raghuvanshi S , Khurana P , Tyagi AK , Khurana JP , Lal R
Ref : Journal of Bacteriology , 193 :5562 , 2011
Abstract : Amycolatopsis mediterranei S699 is an actinomycete that produces an important antibiotic, rifamycin B. Semisynthetic derivatives of rifamycin B are used for the treatment of tuberculosis, leprosy, and AIDS-related mycobacterial infections. Here, we report the complete genome sequence (10.2 Mb) of A. mediterranei S699, with 9,575 predicted coding sequences.
ESTHER : Verma_2011_J.Bacteriol_193_5562
PubMedSearch : Verma_2011_J.Bacteriol_193_5562
PubMedID: 21914879
Gene_locus related to this paper: amymu-d8hka5 , amymu-d8hpp2 , amymu-d8hu68 , amymu-d8hy73 , amymu-d8i2j5 , amymu-d8i8i8 , amyms-g0fkj6 , amyms-g0g7f0 , amyms-g0fps5

Title : Prophylaxis with human serum butyrylcholinesterase protects guinea pigs exposed to multiple lethal doses of soman or VX - Saxena_2011_Biochem.Pharmacol_81_164
Author(s) : Saxena A , Sun W , Fedorko JM , Koplovitz I , Doctor BP
Ref : Biochemical Pharmacology , 81 :164 , 2011
Abstract : Human serum butyrylcholinesterase (Hu BChE) is currently under advanced development as a bioscavenger for the prophylaxis of organophosphorus (OP) nerve agent toxicity in humans. It is estimated that a dose of 200mg will be required to protect a human against 2xLD(50) of soman. To provide data for initiating an investigational new drug application for the use of this enzyme as a bioscavenger in humans, we purified enzyme from Cohn fraction IV-4 paste and initiated safety and efficacy evaluations in mice, guinea pigs, and non-human primates. In mice, we demonstrated that a single dose of enzyme that is 30 times the therapeutic dose circulated in blood for at least four days and did not cause any clinical pathology in these animals. In this study, we report the results of safety and efficacy evaluations conducted in guinea pigs. Various doses of Hu BChE delivered by i.m. injections peaked at approximately 24h and had a mean residence time of 78-103h. Hu BChE did not exhibit any toxicity in guinea pigs as measured by general observation, serum chemistry, hematology, and gross and histological tissue changes. Efficacy evaluations showed that Hu BChE protected guinea pigs from an exposure of 5.5xLD(50) of soman or 8xLD(50) of VX. These results provide convincing data for the development of Hu BChE as a bioscavenger that can protect humans against all OP nerve agents.
ESTHER : Saxena_2011_Biochem.Pharmacol_81_164
PubMedSearch : Saxena_2011_Biochem.Pharmacol_81_164
PubMedID: 20846507

Title : Systemic administration of the potential countermeasure huperzine reversibly inhibits central and peripheral acetylcholinesterase activity without adverse cognitive-behavioral effects - Myers_2010_Pharmacol.Biochem.Behav_94_477
Author(s) : Myers TM , Sun W , Saxena A , Doctor BP , Bonvillain AJ , Clark MG
Ref : Pharmacol Biochem Behav , 94 :477 , 2010
Abstract : Huperzine A is potentially superior to pyridostigmine bromide as a pretreatment for nerve agent intoxication because it inhibits acetylcholinesterase both peripherally and centrally, unlike pyridostigmine, which acts only peripherally. Using rhesus monkeys, we evaluated the time course of acetylcholinesterase and butyrylcholinesterase inhibition following four different doses of -(-)huperzine A: 5, 10, 20, and 40 microg/kg. Acetylcholinesterase inhibition peaked 30 min after intramuscular injection and varied dose dependently, ranging from about 30% to 75%. Subsequently, cognitive-behavioral functioning was also evaluated at each dose of huperzine A using a six-item serial-probe recognition task that assessed attention, motivation, and working memory. Huperzine did not impair performance, but physostigmine did. The results demonstrate that huperzine A can selectively and reversibly inhibit acetylcholinesterase without cognitive-behavioral side effects, thus warranting further study.
ESTHER : Myers_2010_Pharmacol.Biochem.Behav_94_477
PubMedSearch : Myers_2010_Pharmacol.Biochem.Behav_94_477
PubMedID: 19909771

Title : In search of a catalytic bioscavenger for the prophylaxis of nerve agent toxicity - diTargiani_2010_Chem.Biol.Interact_187_349
Author(s) : diTargiani RC , Chandrasekaran L , Belinskaya T , Saxena A
Ref : Chemico-Biological Interactions , 187 :349 , 2010
Abstract : A novel approach for treating organophosphorus (OP) poisoning is the use of enzymes, both stoichiometric and catalytic, as bioscavengers to sequester these compounds in circulation before they reach their physiological targets. Human serum butyrylcholinesterase and a recombinant form of this enzyme produced in the milk of transgenic goats have completed Phase I clinical trials as stoichiometric bioscavengers for the protection of humans against OP nerve agents. However, a major limitation of the first generation bioscavenger is the 1:1 stoichiometry between the enzyme and the OP. Therefore, efforts are underway to develop the second generation catalytic bioscavenger, which will neutralize/hydrolyze multiple OP molecules. To avoid any complications related to adverse immune reactions, three enzymes from human (Hu) sources are being considered for development as catalytic bioscavengers: (1) prolidase; (2) paraoxonase 1 (PON1); (3) senescence marker protein-30 (SMP-30). Towards this effort, native or recombinant (r) forms of candidate catalytic bioscavengers were isolated and characterized for their ability to hydrolyze G-type nerve agents at concentrations of 10muM and 1mM. Results show that mammalian enzymes were significantly less efficient at hydrolyzing nerve agents as compared to bacterial organophosphorus hydrolase (OPH) and organophosphorus acid anhydrolase (OPAA). Recombinant Hu prolidase was the most efficient and the only mammalian enzyme that hydrolyzed all four G-type nerve agents. On the other hand, both rHu PON1 and Mo SMP-30 showed 10-fold lower activity towards sarin compared to rHu prolidase and did not hydrolyze tabun. Based on these results, Hu prolidase appears to be the most promising candidate for further development: (1) it can be easily expressed in E. coli; (2) of the three candidate enzymes, it is the only enzyme that hydrolyzes all four G-type agents. Efforts to improve the catalytic efficiency of this enzyme towards OP nerve agents are underway.
ESTHER : diTargiani_2010_Chem.Biol.Interact_187_349
PubMedSearch : diTargiani_2010_Chem.Biol.Interact_187_349
PubMedID: 20176006

Title : Safety of administration of human butyrylcholinesterase and its conjugates with soman or VX in rats - Genovese_2010_Basic.Clin.Pharmacol.Toxicol_106_428
Author(s) : Genovese RF , Sun W , Johnson CC , diTargiani RC , Doctor BP , Saxena A
Ref : Basic Clin Pharmacol Toxicol , 106 :428 , 2010
Abstract : We evaluated the effects of conjugated enzyme-nerve agent product resulting from the inhibition of bioscavenger human serum butyrylcholinesterase (Hu BChE) by nerve agents soman or VX. Rats were trained on a multiple Fixed-Ratio 32, Extinction 30 sec. (FR32, Ext30) schedule of food reinforcement and then injected (i.m.) with Hu BChE (30 mg/kg), equivalent amounts of Hu BChE-soman conjugate (GDC), Hu BChE-VX conjugate, oxotremorine (OXO) (0.316 mg/kg) or vehicle (n = 8, each group). On the day of injection and on 10 subsequent daily sessions, performance was evaluated on the FR32, Ext30 schedule. Neither conjugates nor Hu BChE produced a performance deficit under the schedule. OXO produced a substantial decrease in responding on the day of administration, with complete recovery observed on subsequent sessions. None of the treatments affected circulating acetylcholinesterase (AChE) activity when evaluated 24-72 hr after injection. The dose of Hu BChE produced a 20,000-fold increase above baseline in circulating BChE activity. Pathological evaluation of organ systems approximately 2 weeks following administration of conjugates or Hu BChE alone did not show toxicity. Taken together, these results suggest that Hu BChE - nerve agent conjugates produced following bioscavenger protection against nerve agents soman and VX do not appear to be particularly toxic. These results add to the safety assessment of Hu BChE as a bioscavenger countermeasure against nerve agent exposure.
ESTHER : Genovese_2010_Basic.Clin.Pharmacol.Toxicol_106_428
PubMedSearch : Genovese_2010_Basic.Clin.Pharmacol.Toxicol_106_428
PubMedID: 20050840

Title : Mechanism for potent reactivation ability of H oximes analyzed by reactivation kinetic studies with cholinesterases from different species - Luo_2010_Chem.Biol.Interact_187_185
Author(s) : Luo C , Chambers C , Yang Y , Saxena A
Ref : Chemico-Biological Interactions , 187 :185 , 2010
Abstract : Oxime-induced reactivation of organophosphorus (OP) nerve agent-inhibited acetylcholinesterase (AChE) is a very important step for the treatment of nerve agent toxicity. Therefore, extensive efforts are being made to develop more efficient and broad-spectrum oximes to replace the currently used oximes 2-PAM or obidoxime. In the 1970s and 1980s, several H oximes (such as HI-6 and HLo-7) were found to be very potent reactivators of non-aged soman-inhibited AChE. Later these oximes were shown to rapidly reactivate GF- and VR-inhibited AChE as well. However, the mechanism for the high potency of these H oximes is still unknown. In this study, the relationship between the reactivation rate constant of nerve agent-inhibited rhesus monkey AChE, human AChE and guinea pig AChE and the size of the alkoxyl (OR) group of nerve agents was analyzed. Results demonstrate that for nerve agent-inhibited rhesus monkey and human AChEs, reactivation by H oximes accelerated as the size of the OR group was increased. But with guinea pig AChE, reactivation by H oximes declined as the size of the OR group was increased. Reactivation kinetic study using GF- and VR-inhibited wild-type and mutant bovine AChEs has shown that mutations of Y124Q and W286A particularly reduced reactivation by these H oximes. Since these 2 amino acid residues are highly conserved in all AChEs sequenced to date, it is unlikely that the remarkable reduction observed in H oxime reactivation with guinea pig AChE is caused by a change in these two amino acid residues.
ESTHER : Luo_2010_Chem.Biol.Interact_187_185
PubMedSearch : Luo_2010_Chem.Biol.Interact_187_185
PubMedID: 20096273

Title : Y124 at the peripheral anionic site is important for the reactivation of nerve agent-inhibited acetylcholinesterase by H oximes - Luo_2010_Biochem.Pharmacol_80_1427
Author(s) : Luo C , Chambers C , Pattabiraman N , Tong M , Tipparaju P , Saxena A
Ref : Biochemical Pharmacology , 80 :1427 , 2010
Abstract : The toxicity of organophosphorus (OP) nerve agents is manifested through irreversible inhibition of acetylcholinesterase (AChE) at the cholinergic synapses, which stops nerve signal transmission, resulting in a cholinergic crisis and eventually death of the poisoned person. Oxime compounds used in nerve agent antidote regimen reactivate nerve agent-inhibited AChE and halt the development of this cholinergic crisis. Due to diversity in structures of OP nerve agents, none of the currently available oximes is able to reactivate AChE inhibited by different nerve agents. To understand the mechanism for the differential activities of oximes toward AChE inhibited by diverse nerve agents in order to aid the design of new broad-spectrum AChE reactivators, we undertook site-directed mutagenesis and molecular modeling studies. Recombinant wild-type and mutant bovine (Bo) AChEs were inhibited by two bulky side-chain nerve agents, GF and VR, and used for conducting reactivation kinetics with five oximes. A homology model for wild-type Bo AChE was built using the recently published crystal structure of human AChE and used to generate models of 2-PAM and HI-6 bound to the active-sites of GF- and VR-inhibited Bo AChEs before nucleophilic attack. Results revealed that the peripheral anionic site (PAS) of AChE as a whole plays a critical role in the reactivation of nerve agent-inhibited AChE by all 4 bis-pyridinium oximes examined, but not by the mono-pyridinium oxime 2-PAM. Of all the residues at the PAS, Y124 appears to be critical for the enhanced reactivation potency of H oximes.
ESTHER : Luo_2010_Biochem.Pharmacol_80_1427
PubMedSearch : Luo_2010_Biochem.Pharmacol_80_1427
PubMedID: 20655881

Title : Demonstration of in vivo stability and lack of immunogenicity of a polyethyleneglycol-conjugated recombinant CHO-derived butyrylcholinesterase bioscavenger using a homologous macaque model - Rosenberg_2010_Chem.Biol.Interact_187_279
Author(s) : Rosenberg YJ , Saxena A , Sun W , Jiang X , Chilukuri N , Luo C , Doctor BP , Lee KD
Ref : Chemico-Biological Interactions , 187 :279 , 2010
Abstract : Human serum and recombinant butyrylcholinesterase (rHuBChE) are the most advanced prophylactics against organophosphate (OP) toxicity due to nerve agent or insecticide exposure. For ethical reasons, such potential multi-use treatments cannot be tested in humans and will require extensive testing in animal models and the "Animal Rule" 21 (21 CFR 601.90) for regulatory approval. This will involve multiple injections of rHuBChE into heterologous animals, e.g. macaques, rodents with inevitable immunogenicity and subsequent elimination of the enzyme on repeat injections. In order to accurately assess pharmacokinetics, efficacy and safety of a candidate rBChE in an "antibody free" system, a homologous macaque (Ma) model has been developed. In these studies, macaques received single or multiple intravenous injections of native MaBChE as well as unmodified or PEG-conjugated forms of rMaBChE produced in CHO cells. Compared to the poor plasma retention of unmodified rBChE (MRT: <10h), three injections of 1.5-2.3mg/kg of PEG-conjugated tetrameric rBChE resulted in high circulatory stability (MRT: >134h) and lack of immunogenicity similar to native MaBChE. PEG-conjugation of the monomeric rMaBChE form also exhibited pharmacokinetic profiles comparable to the tetrameric form (MRT: >113h). However, despite the increased bioavailability of PEG-rBChE, antigenicity studies using sandwich ELISA showed that while macaque BChE was not immunogenic in macaques, PEGylation of rMaBChE did not prevent binding to anti-BChE antibodies, suggesting PEGylation may not be sufficient to mask non-human epitopes on rBChE. This homologous model can provide necessary preclinical protection data for the use of PEG-rHuBChE in humans and bodes well for a safe and efficacious CHO-derived rHuBChE therapeutic.
ESTHER : Rosenberg_2010_Chem.Biol.Interact_187_279
PubMedSearch : Rosenberg_2010_Chem.Biol.Interact_187_279
PubMedID: 20211615

Title : Pharmacokinetics and immunologic consequences of repeated administrations of purified heterologous and homologous butyrylcholinesterase in mice - Sun_2009_Life.Sci_85_657
Author(s) : Sun W , Luo C , Naik RS , Doctor BP , Saxena A
Ref : Life Sciences , 85 :657 , 2009
Abstract : AIM: To assess the consequences of repeated administrations of purified human serum butyrylcholinesterase (Hu BChE) and mouse serum (Mo) BChE into mice. MAIN METHODS: Purified Hu BChE and Mo BChE isolated from the sera of CD-1 mice were administered into Balb/c or CD-1 mice. The enzymes were delivered by i.m. injections of approximately 100U (0.15mg) on day 1 and on day 28, respectively. The effects of two injections were monitored by following blood BChE and anti-BChE IgG levels. KEY FINDINGS: Hu BChE displayed a mean residence time (MRT) of 50h, and an area under the curve (AUC) of 1220U/ml.h in Balb/c or CD-1 mice. Mo BChE exhibited an MRT of 78h and an AUC of 1815U/ml.h in Balb/c mice; the AUC increased to 2504U/ml.h in CD-1 mice. A second injection of Hu BChE in both strains exhibited a marked reduction in circulatory stability. The circulatory stability of the second injection of Mo BChE was reduced in Balb/c mice, but was almost identical to the first injection in CD-1 mice. Consistent with these observations, circulating anti-BChE IgGs were observed in mice injected with Hu BChE; low levels of anti-BChE IgGs were observed only in Balb/c mice injected with Mo BChE. No antibody response was detected in CD-1 mice following either injection of homologous Mo BChE. SIGNIFICANCE: The identical pharmacokinetic profiles and the absence of an immunologic response following a second administration of homologous BChE support the development of Hu BChE as a detoxifying drug in humans.
ESTHER : Sun_2009_Life.Sci_85_657
PubMedSearch : Sun_2009_Life.Sci_85_657
PubMedID: 19772863

Title : Direct correlation between molecular dynamics and enzymatic stability: a comparative neutron scattering study of native human butyrylcholinesterase and its aged soman conjugate - Gabel_2009_Biophys.J_96_1489
Author(s) : Gabel F , Masson P , Froment MT , Doctor BP , Saxena A , Silman I , Zaccai G , Weik M
Ref : Biophysical Journal , 96 :1489 , 2009
Abstract : An incoherent elastic neutron scattering study of the molecular dynamics of native human butyrylcholinesterase and its "aged" soman-inhibited conjugate revealed a significant change in molecular flexibility on an angstrom-nanosecond scale as a function of temperature. The results were related to the stability of each state as established previously by differential scanning calorimetry. A striking relationship was found between the denaturation behavior and the molecular flexibility of the native and inhibited enzymes as a function of temperature. This was reflected in a quantitative correlation between the atomic mean-square displacements on an angstrom-nanosecond scale determined by neutron spectroscopy and the calorimetric specific heat. By the application of a simple two-state model that describes the transition from a folded to a denatured state, the denaturation temperatures of the native and the inhibited enzyme were correctly extracted from the atomic mean-square displacements. Furthermore, the transition entropy and enthalpy extracted from the model fit of the neutron data were, within the experimental accuracy, compatible with the values determined by differential scanning calorimetry.
ESTHER : Gabel_2009_Biophys.J_96_1489
PubMedSearch : Gabel_2009_Biophys.J_96_1489
PubMedID: 19217865

Title : Adenovirus-transduced human butyrylcholinesterase in mouse blood functions as a bioscavenger of chemical warfare nerve agents - Chilukuri_2009_Mol.Pharmacol_76_612
Author(s) : Chilukuri N , Duysen EG , Parikh K , diTargiani RC , Doctor BP , Lockridge O , Saxena A
Ref : Molecular Pharmacology , 76 :612 , 2009
Abstract : Human serum butyrylcholinesterase (Hu BChE) is a promising therapeutic against the toxicity of chemical warfare nerve agents. We have showed previously that recombinant (r) Hu BChE can be expressed at very high levels, 400 to 600 U/ml in mouse blood, by delivering the Hu BChE gene using adenovirus (Ad). Here, we report the biochemical properties of the Ad-expressed full-length and truncated rHu BChE in mouse blood. The molecular sizes of the full-length rHu BChE subunit and its oligomers were similar to those of native Hu BChE, although only a small portion of the full-length rHu BChE subunit underwent assembly into dimers and tetramers. As expected, Ad containing the truncated Hu BChE gene transduced the expression of monomeric rHu BChE only. Compared with 415 U of rHu BChE per milliliter in blood, tissues including liver, lung, heart, brain, kidney, muscle, intestine, diaphragm, salivary gland, and fat expressed <10 U/g of rHu BChE activity. Ad-expressed rHu BChE in mouse blood neutralized soman and O-ethyl S-2-N,N-diisopropylaminoethyl methylphosphonothiolate at rates similar to those of native Hu BChE and rHu BChE expressed in vitro. Because the expression of rHu BChE rapidly decreased 6 days after virus administration, sera were assayed for the presence of anti-Hu BChE antibodies. Anti-Hu BChE antibodies were detected on day 7 and in increased amounts thereafter, which coincided with the loss of Hu BChE expression in sera. In conclusion, the delivery of Hu BChE gene using Ad can be a promising strategy that can provide protection against multiple lethal doses of chemical warfare nerve agents in vivo.
ESTHER : Chilukuri_2009_Mol.Pharmacol_76_612
PubMedSearch : Chilukuri_2009_Mol.Pharmacol_76_612
PubMedID: 19542320

Title : Adenovirus-mediated gene transfer of human butyrylcholinesterase results in persistent high-level transgene expression in vivo - Chilukuri_2008_Chem.Biol.Interact_175_327
Author(s) : Chilukuri N , Duysen EG , Parikh K , Sun W , Doctor BP , Lockridge O , Saxena A
Ref : Chemico-Biological Interactions , 175 :327 , 2008
Abstract : Human serum butyrylcholinesterase (Hu BChE) is a promising therapeutic against the toxicity of chemical warfare nerve agents, pesticide intoxication, and cocaine overdose. However, its widespread application is hampered by difficulties in large-scale production of the native protein from human plasma and/or availability as a recombinant protein suitable for use in vivo. This limitation may be resolved by in vivo delivery and expression of the Hu BChE gene. In this study, recombinant (r) adenoviruses (Ads) encoding full-length and truncated rHu BChEs were tested for in vivo expression in mice. Mice injected with these rAds intraperitoneally failed to express rHu BChE. However, a single tail vein injection of both rAds resulted in persistent high serum levels of rHu BChE in BChE knockout mice, which peaked on days 4/5 at 377+/-162U/ml for full-length rHu BChE and 574+/-143U/ml for truncated rHu BChE. These activity levels are orders of magnitude higher than 1.9U/ml of mouse BChE present in wild-type mouse serum. Thereafter, rHu BChE levels dropped rapidly and very little or no activity was detected in the serum 10 days post-virus administration. In conclusion, the present study demonstrates the potential of rAd-mediated Hu BChE gene therapy to counteract multiple lethal doses of chemical warfare nerve agent toxicity.
ESTHER : Chilukuri_2008_Chem.Biol.Interact_175_327
PubMedSearch : Chilukuri_2008_Chem.Biol.Interact_175_327
PubMedID: 18499092

Title : Effect of polyethylene glycol modification on the circulatory stability and immunogenicity of recombinant human butyrylcholinesterase - Chilukuri_2008_Chem.Biol.Interact_175_255
Author(s) : Chilukuri N , Sun W , Naik RS , Parikh K , Tang L , Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 175 :255 , 2008
Abstract : The therapeutic value of human serum butyrylcholinesterase (Hu BChE) as a bioscavenger of chemical warfare agents is due to its high reactivity with organophosphorus compounds and prolonged circulatory stability. Native Hu BChE is mostly tetrameric in form while the enzyme produced using molecular cloning technology is a mixture of tetramers, dimers, and monomers. Previous studies revealed that monomers and dimers of recombinant human (rHu) BChE cleared rapidly from the circulation of mice compared to tetrameric rHu BChE and native Hu BChE, which have mean residence times (MRTs) of 18h and 45h, respectively. It was also shown that polyethylene glycol-20K (PEG) modification of tetrameric rHu BChE prolonged its circulatory stability and bioavailability in vivo. The goal of this study was to determine if modification with PEG could prolong the circulatory stability and eliminate the immunogenicity of monomeric rHu BChE. Monomeric rHu BChE was expressed in human 293A cells using a cDNA lacking the 45 amino acid tetramerization domain from the carboxyl terminus and the adenovirus expression system. The catalytic and inhibitory properties of purified monomeric rHu BChE were similar to those for native Hu BChE and were not affected by PEG modification. As expected, monomeric rHu BChE rapidly cleared from the circulation of mice (MRT=3.2+/-0.3h) while monomeric PEG-rHu BChE demonstrated significant improvement in its bioavailability and circulatory stability in blood (MRT=31.4+/-5.4h). However, a second injection of monomeric PEG-rHu BChE, 28 days after the first, displayed a much shorter MRT=11.6+/-0.4h, and circulating anti-monomeric PEG-rHu BChE antibodies were detected in the blood of mice. These results suggest that PEG modification increased the circulatory stability of monomeric rHu BChE but failed to reduce or eliminate its immunogenicity.
ESTHER : Chilukuri_2008_Chem.Biol.Interact_175_255
PubMedSearch : Chilukuri_2008_Chem.Biol.Interact_175_255
PubMedID: 18603232

Title : Comparison of methods used for the determination of cholinesterase activity in whole blood - Naik_2008_Chem.Biol.Interact_175_298
Author(s) : Naik RS , Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 175 :298 , 2008
Abstract : Cholinesterases (ChEs) are classified as either acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) based on their substrate and inhibitor specificity. Organophosphate and carbamate compounds commonly represented by herbicides, pesticides, and nerve gases irreversibly inhibit ChEs. Therefore, exposure to organophosphates and carbamates is normally assessed by measuring ChE activity in blood. There are two approaches for measuring AChE and BChE activity present in whole blood: (1) separating blood into erythrocytes, which contain only AChE, and plasma which contains only BChE, to measure their activity individually, or (2) use a BChE-specific inhibitor to measure the activity of AChE in whole blood. A number of studies have reported the use of different inhibitors for the simultaneous measurement of AChE and BChE activities. However, the inhibitors used for completely inhibiting BChE activity also inhibited AChE activity leading to errors in reported values. The goal of this study was to find the most accurate and simple method for the simultaneous determination of AChE and BChE activity in animal whole blood. Solutions containing human AChE and BChE in various proportions were prepared and AChE and BChE activities were measured using three reported methods. Results demonstrate that ethopropazine and (-) huperzine A appear to be the most specific ChE inhibitors. Preliminary results with human and animal whole blood suggest that 20 microM ethopropazine and 500 nM (-) huperzine A can be used for measuring AChE and BChE activities across species.
ESTHER : Naik_2008_Chem.Biol.Interact_175_298
PubMedSearch : Naik_2008_Chem.Biol.Interact_175_298
PubMedID: 18555980

Title : Efficacy of human serum butyrylcholinesterase against sarin vapor - Saxena_2008_Chem.Biol.Interact_175_267
Author(s) : Saxena A , Sun W , Dabisch PA , Hulet SW , Hastings NB , Jakubowski EM , Mioduszewski RJ , Doctor BP
Ref : Chemico-Biological Interactions , 175 :267 , 2008
Abstract : Human serum butyrylcholinesterase (Hu BChE) is currently under advanced development as a pretreatment drug for organophosphate (OP) poisoning in humans. It was shown to protect mice, rats, guinea pigs, and monkeys against multiple LD(50) challenges of OP nerve agents by i.v. or s.c. bolus injections. Since inhalation is the most likely route of exposure to OP nerve agents on the battlefield or in public places, the aim of this study was to evaluate the efficacy of Hu BChE against whole-body inhalation exposure to sarin (GB) vapor. Male Gottingen minipigs were subjected to one of the following treatments: (1) air exposure; (2) GB vapor exposure; (3) pretreatment with 3 mg/kg of Hu BChE followed by GB vapor exposure; (4) pretreatment with 6.5 mg/kg of Hu BChE followed by GB vapor exposure; (5) pretreatment with 7.5 mg/kg of Hu BChE followed by GB vapor exposure. Hu BChE was administered by i.m. injection, 24h prior to whole-body exposure to GB vapor at a concentration of 4.1 mg/m(3) for 60 min, a dose lethal to 99% of untreated exposed pigs (LCt99). EEG, ECG, and pupil size were monitored throughout exposure, and blood drawn from a surgically implanted jugular catheter before and throughout the exposure period, was analyzed for acetylcholinesterase (AChE) and BChE activities, and the amount of GB present in plasma. All animals exposed to GB vapor alone or pretreated with 3 or 6.5 mg/kg of Hu BChE, died following exposure to GB vapor. All five animals pretreated with 7.5 mg/kg of Hu BChE survived the GB exposure. The amount of GB bound in plasma was 200-fold higher compared to that from plasma of pigs that did not receive Hu BChE, suggesting that Hu BChE was effective in scavenging GB in blood. Additionally, pretreatment with 7.5 mg/kg of Hu BChE prevented cardiac abnormalities and seizure activity observed in untreated animals and those treated with lower doses of Hu BChE.
ESTHER : Saxena_2008_Chem.Biol.Interact_175_267
PubMedSearch : Saxena_2008_Chem.Biol.Interact_175_267
PubMedID: 18597747

Title : A repeated injection of polyethyleneglycol-conjugated recombinant human butyrylcholinesterase elicits immune response in mice - Chilukuri_2008_Toxicol.Appl.Pharmacol_231_423
Author(s) : Chilukuri N , Sun W , Parikh K , Naik RS , Tang L , Doctor BP , Saxena A
Ref : Toxicol Appl Pharmacol , 231 :423 , 2008
Abstract : Human serum butyrylcholinesterase (Hu BChE) serves as an efficacious bioscavenger of highly toxic organophosphorus (OP) compounds. Since there is a concern that the supply of native Hu BChE may be limited, monomeric and tetrameric forms of recombinant Hu BChE (rHu BChE) were evaluated as replacements and found that they lacked sufficient stability in vivo. However, their in vivo stability could be significantly prolonged by conjugation with polyethyleneglycol-20K (PEG) suggesting that monomeric and tetrameric PEG-rHu BChE could function as bioscavengers. Here, the immunogenicity of PEG-rHu BChE was evaluated in mice following two injections given four weeks apart. In addition to pharmacokinetic parameters, such as mean residence time, maximal concentration, time to reach the maximal concentration, elimination half-life and area under the plasma concentration-time curve extrapolated to infinity, the presence of circulating anti-rHu BChE antibodies was also determined. Although the pharmacokinetic parameters were significantly improved for the first injection of monomeric and tetrameric PEG-rHu BChEs, they were much lower for the second injection. Anti-rHu BChE antibodies were detected in the blood of mice following the first and second enzyme injections and their levels were approximately higher by 5-fold and 2-fold in mice injected with monomeric and tetrameric PEG-rHu BChEs as compared to mice injected with unconjugated enzymes. The findings that the rapid clearance of a repeat injection of PEG-rHu BChEs in mice which coincides with the presence of circulating anti-rHu BChE antibodies suggest that PEG conjugation prolonged the circulatory stability of rHu BChE but failed to eliminate its immunogenicity in mice.
ESTHER : Chilukuri_2008_Toxicol.Appl.Pharmacol_231_423
PubMedSearch : Chilukuri_2008_Toxicol.Appl.Pharmacol_231_423
PubMedID: 18586293

Title : Exploiting protein fluctuations at the active-site gorge of human cholinesterases: further optimization of the design strategy to develop extremely potent inhibitors - Butini_2008_J.Med.Chem_51_3154
Author(s) : Butini S , Campiani G , Borriello M , Gemma S , Panico A , Persico M , Catalanotti B , Ros S , Brindisi M , Agnusdei M , Fiorini I , Nacci V , Novellino E , Belinskaya T , Saxena A , Fattorusso C
Ref : Journal of Medicinal Chemistry , 51 :3154 , 2008
Abstract : Protein conformational fluctuations are critical for biological functions, although the relationship between protein motion and function has yet to be fully explored. By a thorough bioinformatics analysis of cholinesterases (ChEs), we identified specific hot spots, responsible for protein fluctuations and functions, and those active-site residues that play a role in modulating the cooperative network among the key substructures. This drew the optimization of our design strategy to discover potent and reversible inhibitors of human acetylcholinesterase and butyrylcholinesterase (hAChE and hBuChE) that selectively interact with specific protein substructures. Accordingly, two tricyclic moieties differently spaced by functionalized linkers were investigated as molecular yardsticks to probe the finest interactions with specific hot spots in the hChE gorge. A number of SAR trends were identified, and the multisite inhibitors 3a and 3d were found to be the most potent inhibitors of hBuChE and hAChE known to date.
ESTHER : Butini_2008_J.Med.Chem_51_3154
PubMedSearch : Butini_2008_J.Med.Chem_51_3154
PubMedID: 18479118

Title : Tacrine based human cholinesterase inhibitors: synthesis of peptidic-tethered derivatives and their effect on potency and selectivity - Butini_2008_Bioorg.Med.Chem.Lett_18_5213
Author(s) : Butini S , Guarino E , Campiani G , Brindisi M , Coccone SS , Fiorini I , Novellino E , Belinskaya T , Saxena A , Gemma S
Ref : Bioorganic & Medicinal Chemistry Lett , 18 :5213 , 2008
Abstract : Tacrine based reversible inhibitors of cholinesterases (ChEIs) containing peptidic tethers were synthesized to interact with specific regions at the gorge level, and their potency was determined with human (h) acetylcholinesterase and butyrylcholinesterase. Analogues 3i,j and 3l,m were identified as promising hits and may pave the way for the development of a new series of tacrine based enzyme selective hChEIs.
ESTHER : Butini_2008_Bioorg.Med.Chem.Lett_18_5213
PubMedSearch : Butini_2008_Bioorg.Med.Chem.Lett_18_5213
PubMedID: 18786825

Title : Developing procedures for the large-scale purification of human serum butyrylcholinesterase - Saxena_2008_Protein.Expr.Purif_61_191
Author(s) : Saxena A , Luo C , Doctor BP
Ref : Protein Expr Purif , 61 :191 , 2008
Abstract : Human serum butyrylcholinesterase (Hu BChE) is the most viable candidate for the prophylactic treatment of organophosphate poisoning. A dose of 200 mg/70 kg is predicted to protect humans against 2x LD(50) of soman. Therefore, the aim of this study was to develop procedures for the purification of gram quantities of this enzyme from outdated human plasma or Cohn Fraction IV-4. The purification of Hu BChE was accomplished by batch adsorption on procainamide-Sepharose-CL-4B affinity gel followed by ion-exchange chromatography on a DEAE-Sepharose column. For the purification of enzyme from Cohn Fraction IV-4, it was resuspended in 25 mM sodium phosphate buffer, pH 8.0, and fat was removed by decantation, prior to batch adsorption on procainamide-Sepharose gel. In both cases, the procainamide gel was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0, containing 0.05 M NaCl, and the enzyme was eluted with the same buffer containing 0.1 M procainamide. The enzyme was dialyzed and the pH was adjusted to 4.0 before loading on the DEAE column equilibrated in sodium acetate buffer, pH 4.0. The column was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0 containing 0.05 M NaCl before elution with a gradient of 0.05-0.2M NaCl in the same buffer. The purity of the enzyme following these steps ranged from 20% to 40%. The purity of the enzyme increased to >90% by chromatography on an analytical procainamide affinity column. Results show that Cohn Fraction IV-4 is a much better source than plasma for the large-scale isolation of purified Hu BChE.
ESTHER : Saxena_2008_Protein.Expr.Purif_61_191
PubMedSearch : Saxena_2008_Protein.Expr.Purif_61_191
PubMedID: 18602477

Title : Comparison of oxime reactivation and aging of nerve agent-inhibited monkey and human acetylcholinesterases - Luo_2008_Chem.Biol.Interact_175_261
Author(s) : Luo C , Tong M , Maxwell DM , Saxena A
Ref : Chemico-Biological Interactions , 175 :261 , 2008
Abstract : Non-human primates are valuable animal models that are used for the evaluation of nerve agent toxicity as well as antidotes and results from animal experiments are extrapolated to humans. It has been demonstrated that the efficacy of an oxime primarily depends on its ability to reactivate nerve agent-inhibited acetylcholinesterase (AChE). If the in vitro oxime reactivation of nerve agent-inhibited animal AChE is similar to that of human AChE, it is likely that the results of an in vivo animal study will reliably extrapolate to humans. Therefore, the goal of this study was to compare the aging and reactivation of human and different monkey (Rhesus, Cynomolgus, and African Green) AChEs inhibited by GF, GD, and VR. The oximes examined include the traditional oxime 2-PAM, two H-oximes HI-6 and HLo-7, and the new candidate oxime MMB4. Results indicate that oxime reactivation of all three monkey AChEs was very similar to human AChE. The maximum difference in the second-order reactivation rate constant between human and three monkey AChEs or between AChEs from different monkey species was 5-fold. Aging rate constants of GF-, GD-, and VR-inhibited monkey AChEs were very similar to human AChE except for GF-inhibited monkey AChEs, which aged 2-3 times faster than the human enzyme. The results of this study suggest that all three monkey species are suitable animal models for nerve agent antidote evaluation since monkey AChEs possess similar biochemical/pharmacological properties to human AChE.
ESTHER : Luo_2008_Chem.Biol.Interact_175_261
PubMedSearch : Luo_2008_Chem.Biol.Interact_175_261
PubMedID: 18555982

Title : Long-term effects of human butyrylcholinesterase pretreatment followed by acute soman challenge in cynomolgus monkeys - Sun_2008_Chem.Biol.Interact_175_428
Author(s) : Sun W , Doctor BP , Lenz DE , Saxena A
Ref : Chemico-Biological Interactions , 175 :428 , 2008
Abstract : Human serum butyrylcholinesterase (Hu BChE) was demonstrated previously to be an effective prophylaxis that can protect animals from organophosphate nerve agents. However, in most of those studies, the maximum dose used to challenge animals was low (<2x LD(50)), and the health of these animals was monitored for only up to 2 weeks. In this study, six cynomolgus monkeys received 75 mg of Hu BChE followed by sequential doses (1.5, 2.0, 2.0 x LD(50)) of soman 10h later for a total challenge of 5.5x LD(50). Four surviving animals that did not show any signs of soman intoxication were transferred to WRAIR for the continuous evaluation of long-term health effects for 14 months. Each month, blood was drawn from these monkeys and analyzed for serum chemistry and hematology parameters, blood acetylcholinesterase (AChE) and BChE levels. Based on the serum chemistry and hematology parameters measured, no toxic effects or any organ malfunctions were observed up to 14 months following Hu BuChE protection against exposure to 5.5x LD(50) of soman. In conclusion, Hu BChE pretreatment not only effectively protects monkeys from soman-induced toxicity of the immediate acute phase but also for a long-term outcome.
ESTHER : Sun_2008_Chem.Biol.Interact_175_428
PubMedSearch : Sun_2008_Chem.Biol.Interact_175_428
PubMedID: 18674756

Title : An in vitro comparative study on the reactivation of nerve agent-inhibited guinea pig and human acetylcholinesterases by oximes - Luo_2007_Biochemistry_46_11771
Author(s) : Luo C , Tong M , Chilukuri N , Brecht K , Maxwell DM , Saxena A
Ref : Biochemistry , 46 :11771 , 2007
Abstract : The reactivation of nerve agent-inhibited acetylcholinesterase (AChE) by oxime is the most important step in the treatment of nerve agent poisoning. Since the evaluation of nerve agent antidotes cannot be conducted in humans, results from animal experiments are extrapolated to humans. Guinea pig is one of the animal models that is frequently used for conducting nerve agent antidote evaluations. Several investigations have demonstrated that the efficacy of an oxime primarily depends on its ability to reactivate nerve agent-inhibited AChE. If the in vitro oxime reactivation of nerve agent-inhibited animal AChE is similar to that of human AChE, it is likely that the results of an in vivo animal study will reliably extrapolate to humans. Therefore, the goal of this study was to compare the reactivation of guinea pig and human AChEs inhibited by six different G and V type nerve agents. Reactivation kinetic studies with five mono- and bis-pyridinium oximes showed that oxime reactivation of nerve agent-inhibited human AChE in most cases was faster than guinea pig AChE. The most significant enhancement was observed in the reactivation of human AChE inhibited by nerve agents containing bulky side chains GF, GD, and VR, by H-series oximes HLo-7, HI-6, and ICD-585. In these cases, species-related differences observed between the two AChEs, based on the second-order reactivation rate constants, were 90- to over 400-fold. On the other hand, less than 3-fold differences were observed in the rates of aging of nerve agent-inhibited guinea pig and human AChEs. These results suggest that the remarkable species-related differences observed in the reactivation of nerve agent-inhibited guinea pig and human AChEs were not due to differences in the rates of aging. These results also suggest that guinea pig may not be an appropriate animal model for the in vivo evaluation of oxime therapy.
ESTHER : Luo_2007_Biochemistry_46_11771
PubMedSearch : Luo_2007_Biochemistry_46_11771
PubMedID: 17900152

Title : Evaluation of cognitive and biochemical effects of low-level exposure to sarin in rhesus and African green monkeys - Genovese_2007_Toxicology_231_11
Author(s) : Genovese RF , Oubre JL , Jakubowski EM , Fleming PJ , Saxena A , Rockwood GA , Tipparaju P , Willmore CB
Ref : Toxicology , 231 :11 , 2007
Abstract : We investigated the potential of low-level exposures to the chemical warfare nerve agent, sarin, to produce adverse effects. Rhesus (Macaca mulatta) and African green monkeys (Chlorocebus acthiops) were trained on a serial probe recognition (SPR) task before IM administration of a low-level concentration (5.87 microg/kg or 2.93 microg/kg) of sarin. Blood was sampled before agent administration and at various times following administration. Sarin administration did not disrupt performance on the SPR task in either species. Major dependent measures characterizing performance (accuracy, number of completed trials per session, average choice response time) were largely unaffected on the day sarin was administered as well as on subsequent testing sessions occurring over several weeks following administration. Analyses of red blood cell (RBC) and plasma samples revealed that sarin administration produced a substantial degree of inhibition of circulating acetylcholinesterase (AChE) in RBC fractions and butyrylcholinesterase (BChE) in plasma fractions, which only slowly recovered. In this regard, AChE activity was inhibited to a greater extent than BChE activity. Blood samples were also evaluated for regenerated sarin, which was found in RBC and plasma fractions in both species and showed orderly elimination functions. More sarin was regenerated from RBC fractions than from plasma fractions. Elimination of regenerated sarin was much slower in RBC than plasma and exceeded the expected time of AChE aging, suggesting the presence of additional sarin binding sites. In general, effects were similar in both species. Taken together, our results show that while the concentrations of sarin administered were clearly biochemically active, they were below those that are required to produce a disruption of behavioral performance.
ESTHER : Genovese_2007_Toxicology_231_11
PubMedSearch : Genovese_2007_Toxicology_231_11
PubMedID: 17126468

Title : Bioscavenger for protection from toxicity of organophosphorus compounds - Saxena_2006_J.Mol.Neurosci_30_145
Author(s) : Saxena A , Sun W , Luo C , Myers TM , Koplovitz I , Lenz DE , Doctor BP
Ref : Journal of Molecular Neuroscience , 30 :145 , 2006
Abstract : Current antidotal regimens for organophosphorus compound (OP) poisoning consist of a combination of pretreatment with a spontaneously reactivating AChE inhibitor such as pyridostigmine bromide, and postexposure therapy with anticholinergic drugs such as atropine sulfate and oximes such as 2-PAM chloride (Gray, 1984). Although these antidotal regimens are effective in preventing lethality of animals from OP poisoning, they do not prevent postexposure incapacitation, convulsions, performance deficits, or, in many cases, permanent brain damage (Dunn and Sidell, 1989). These problems stimulated the development of enzyme bioscavengers as a pretreatment to sequester highly toxic OPs before they reach their physiological targets. Several studies over the last two decades have demonstrated that exogenously administered human serum butyrylcholinesterase (Hu BChE) can be used successfully as a safe, efficacious, and single prophylactic treatment to counteract the toxicity of OPs. It also has potential use for first responders (civilians) reacting to terrorist nerve gas release, pesticide overexposure, or succinylcholine-induced apnea. A dose of 200 mg of Hu BChE in humans is envisioned as a prophylactic treatment that can protect from exposure of 2-5 x LD50 of nerve agents (Ashani, 2000).
ESTHER : Saxena_2006_J.Mol.Neurosci_30_145
PubMedSearch : Saxena_2006_J.Mol.Neurosci_30_145
PubMedID: 17192662

Title : Discovery of huperzine A-tacrine hybrids as potent inhibitors of human cholinesterases targeting their midgorge recognition sites - Gemma_2006_J.Med.Chem_49_3421
Author(s) : Gemma S , Gabellieri E , Huleatt P , Fattorusso C , Borriello M , Catalanotti B , Butini S , De Angelis M , Novellino E , Nacci V , Belinskaya T , Saxena A , Campiani G
Ref : Journal of Medicinal Chemistry , 49 :3421 , 2006
Abstract : We describe herein the development of novel huperzine A-tacrine hybrids characterized by 3-methylbicyclo[3.3.1]non-3-ene scaffolds. These compounds were specifically designed to establish tight interactions, through different binding modes, with the midgorge recognition sites of human acetylcholinesterase (hAChE: Y72, D74) and human butyrylcholinesterase (hBuChE: N68, D70) and their catalytic or peripheral sites. Compounds 5a-c show a markedly improved biological profile relative to tacrine and huperzine A.
ESTHER : Gemma_2006_J.Med.Chem_49_3421
PubMedSearch : Gemma_2006_J.Med.Chem_49_3421
PubMedID: 16722663

Title : Effects of soman inhibition and of structural differences on cholinesterase molecular dynamics: a neutron scattering study - Gabel_2005_Biophys.J_89_3303
Author(s) : Gabel F , Weik M , Masson P , Renault F , Fournier D , Brochier L , Doctor BP , Saxena A , Silman I , Zaccai G
Ref : Biophysical Journal , 89 :3303 , 2005
Abstract : Incoherent elastic neutron scattering experiments on members of the cholinesterase family were carried out to investigate how molecular dynamics is affected by covalent inhibitor binding and by differences in primary and quaternary structure. Tetrameric native and soman-inhibited human butyrylcholinesterase (HuBChE) as well as native dimeric Drosophila melanogaster acetylcholinesterase (DmAChE) hydrated protein powders were examined. Atomic mean-square displacements (MSDs) were found to be identical for native HuBChE and for DmAChE in the whole temperature range examined, leading to the conclusion that differences in activity and substrate specificity are not reflected by a global modification of subnanosecond molecular dynamics. MSDs of native and soman-inhibited HuBChE were identical below the thermal denaturation temperature of the native enzyme, indicating a common mean free-energy surface. Denaturation of the native enzyme is reflected by a relative increase of MSDs consistent with entropic stabilization of the unfolded state. The results suggest that the stabilization of HuBChE phosphorylated by soman is due to an increase in free energy of the unfolded state due to a decrease in entropy.
ESTHER : Gabel_2005_Biophys.J_89_3303
PubMedSearch : Gabel_2005_Biophys.J_89_3303
PubMedID: 16100272

Title : Human serum butyrylcholinesterase: in vitro and in vivo stability, pharmacokinetics, and safety in mice - Saxena_2005_Chem.Biol.Interact_157-158_199
Author(s) : Saxena A , Sun W , Luo C , Doctor BP
Ref : Chemico-Biological Interactions , 157-158 :199 , 2005
Abstract : The use of exogenously administered cholinesterases (ChEs) as bioscavengers of highly toxic organophosphate (OP) nerve agents is now sufficiently well documented to make them a highly viable prophylactic treatment against this potential threat. Of the ChEs evaluated so far, human serum butyrylcholinesterase (HuBChE) is most suitable for human use. A dose of 200 mg (3 mg/kg) of HuBChE is envisioned as a prophylactic treatment in humans that can protect from an exposure of up to 2 x LD50 of soman. In addition to its use as a prophylactic for a variety of wartime scenarios, including covert actions, it also has potential use for first responders (civilians) reacting to terrorist nerve gas release. We recently, developed a procedure for the large-scale purification of HuBChE, which yielded approximately 6 g of highly purified enzyme from 120 kg of Cohn fraction IV-4. The enzyme had a specific activity of 700-750 U/mg and migrated as a single band on SDS-PAGE. To provide data for initiating an investigational new drug (IND) application for the use of this enzyme as a bioscavenger in humans, we established its pharmacokinetic properties, examined its safety in mice, and evaluated its shelf life at various temperatures. In mice administered various doses up to 90 mg/kg, enzyme activity reached peak levels in circulation at 10 and 24 h following i.p. and i.m. injections, respectively. The enzyme displayed a mean residence time (MRT) of 40-50 h, regardless of the route of administration or dose of injected enzyme. Mice were euthanized 2 weeks following enzyme administration and tissues were examined grossly or microscopically for possible toxic effects. Results suggest that HuBChE does not exhibit any toxicity in mice as measured by general observation, serum chemistry, hematology, gross or histologic tissue changes. The shelf life of this enzyme stored at 4, 25, 37, and 45 degrees C was determined in lyophilized form. The enzyme was found to be stable when stored in lyophilized form at -20, 4, 25, or 37 degrees C to date (2 years), as measured by specific activity and SDS polyacrylamide gel electrophoresis. The effect of storage on circulatory stability was determined by measuring MRT in mice; there was no change in the MRT of lyophilized enzyme stored at -20 degrees C to date (2 years). These results provide convincing data that HuBChE is a safe bioscavenger that can provide protection against all OP nerve agents. Efforts are now underway to prepare the required documentation for submission of an IND application to the United States Food and Drug Administration (USFDA).
ESTHER : Saxena_2005_Chem.Biol.Interact_157-158_199
PubMedSearch : Saxena_2005_Chem.Biol.Interact_157-158_199
PubMedID: 16263104

Title : Bioscavengers for the protection of humans against organophosphate toxicity - Doctor_2005_Chem.Biol.Interact_157-158_167
Author(s) : Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 157-158 :167 , 2005
Abstract : Current antidotes for organophosphorus compounds (OP) poisoning consist of a combination of pretreatment with carbamates (pyridostigmine bromide), to protect acetylcholinesterase (AChE) from irreversible inhibition by OP compounds, and post-exposure therapy with anti-cholinergic drugs (atropine sulfate) to counteract the effects of excess acetylcholine and oximes (e.g., 2-PAM chloride) to reactivate OP-inhibited AChE. These antidotes are effective in preventing lethality from OP poisoning, but they do not prevent post-exposure incapacitation, convulsions, seizures, performance decrements, or in many cases permanent brain damage. These symptoms are commonly observed in experimental animals and are likely to occur in humans. The problems intrinsic to these antidotes stimulated attempts to develop a single protective drug, itself devoid of pharmacological effects, which would provide protection against the lethality of OP compounds and prevent post-exposure incapacitation. One approach is the use of enzymes such as cholinesterases (ChEs), beta-esterases in general, as single pretreatment drugs to sequester highly toxic OP anti-ChEs before they reach their physiological targets. This approach turns the irreversible nature of the OP: ChE interaction from disadvantage to an advantage; instead of focusing on OP as an anti-ChE, one can use ChE as an anti-OP. Using this approach, it was shown that administration of fetal bovine serum AChE (FBSAChE) or equine serum butyrylcholinesterase (EqBChE) or human serum BChE (HuBChE) protected the animals from multiple LD50s of a variety of highly toxic OPs without any toxic effects or performance decrements. The bioscavengers that have been explored to date for the detoxification of OPs fall into three categories: (A) those that can catalytically hydrolyze OPs and thus render them non-toxic, such as OP hydrolase and OP anhydrase; (B) those that stoichiometrically bind to OPs, that is, 1 mol of enzyme neutralizes one or 2 mol of OP inactivating both, such as ChEs and related enzymes; and (C) and those generally termed as "pseudo catalytic", e.g., a combination of ChE and an oxime pre-treatment such that the catalytic activity of OP-inhibited ChE can rapidly and continuously be restored in the presence of an oxime. Since the biochemical mechanism underlying prophylaxis by exogenous esterases such as ChEs is established and tested in several animal species, including non-human primates, this concept should allow a reliable extrapolation of results from animal experiments to human application. Having being extensively investigated by several groups, plasma derived HuBChE is judged to be the most suitable bioscavenger for its advancement for human use. The program is being developed at the present time for conducting a safety clinical trial in human volunteers. Several other candidate bioscavengers will follow; e.g., recombinant HuBChE expressed in the milk of transgenic goats, pseudo catalytic scavenger(s), e.g., a combination of ChE and oxime, and possibly PON 1 as a catalytic scavenger in the future.
ESTHER : Doctor_2005_Chem.Biol.Interact_157-158_167
PubMedSearch : Doctor_2005_Chem.Biol.Interact_157-158_167
PubMedID: 16293236

Title : Development of molecular probes for the identification of extra interaction sites in the mid-gorge and peripheral sites of butyrylcholinesterase (BuChE). Rational design of novel, selective, and highly potent BuChE inhibitors - Campiani_2005_J.Med.Chem_48_1919
Author(s) : Campiani G , Fattorusso C , Butini S , Gaeta A , Agnusdei M , Gemma S , Persico M , Catalanotti B , Savini L , Nacci V , Novellino E , Holloway HW , Greig NH , Belinskaya T , Fedorko JM , Saxena A
Ref : Journal of Medicinal Chemistry , 48 :1919 , 2005
Abstract : Tacrine heterobivalent ligands were designed as novel and reversible inhibitors of cholinesterases. On the basis of the investigation of the active site gorge topology of butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) and by using flexible docking procedures, molecular modeling studies formulated the hypothesis of extra interaction sites in the active gorge of hBuChE, namely, a mid-gorge interaction site and a peripheral interaction site. The design strategy led to novel BuChE inhibitors, balancing potency and selectivity. Among the compounds identified, the heterobivalent ligand 4m, containing an amide nitrogen and a sulfur atom at the 8-membered tether level, is one of the most potent and selective BuChE inhibitors described to date. The novel inhibitors, bearing postulated key features, validated the hypothesis of the presence of extra interaction sites within the hBuChE active site gorge.
ESTHER : Campiani_2005_J.Med.Chem_48_1919
PubMedSearch : Campiani_2005_J.Med.Chem_48_1919
PubMedID: 15771436

Title : Polyethylene glycosylation prolongs the circulatory stability of recombinant human butyrylcholinesterase - Chilukuri_2005_Chem.Biol.Interact_157-158_115
Author(s) : Chilukuri N , Parikh K , Sun W , Naik R , Tipparaju P , Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 157-158 :115 , 2005
Abstract : Previous studies in rodents and non-human primates have demonstrated that pretreatment of animals with cholinesterases could provide significant protection against organophosphate (OP) nerve agent toxicity. Gene delivery/therapy is emerging as an approach to achieve high-level expression of proteins in vivo that are very similar to their native counterparts. Recently, adenoviral (Ad) vectors have proven to be excellent vehicles for delivering genes to cells in vitro and in vivo. In this study, we explored the use of the newly designed AdenoVATOR system for the expression of recombinant human butyrylcholinesterase (rHu BChE) in human embryonic kidney 293A (HEK-293A) cells. In these cells, rHu BChE was expressed as mostly tetrameric form by the simultaneous expression of proline-rich attachment domain. By optimizing the culture conditions, 1.5-2.0 U/ml of rHu BChE could be expressed in HEK-293A cells. Recombinant Hu BChE was purified to homogeneity by ammonium sulfate fractionation followed by affinity column chromatography using procainamide Sepharose and cobalt Sepharose gels. The enzymatic and physico-chemical properties of purified rHu BChE were similar to those of native serum-derived Hu BChE. To determine the suitability of this preparation for use as an antidote against highly toxic nerve agents, its pharmacokinetics were evaluated in mice. Recombinant Hu BChE exhibited a mean residence time of 18.3 h which was 2.5-fold shorter than that observed for native Hu BChE in mice. However, rHu BChE chemically modified with polyethyleneglycol (PEG) displayed a mean residence time of 36.2 h suggesting that PEG-modification can prolong the circulatory stability of rHu BChE. The efficacy of Ad-Hu BChE to induce the production of therapeutic levels of bioscavenger in vivo is under evaluation.
ESTHER : Chilukuri_2005_Chem.Biol.Interact_157-158_115
PubMedSearch : Chilukuri_2005_Chem.Biol.Interact_157-158_115
PubMedID: 16253215

Title : Safety and pharmacokinetics of human serum butyrylcholinesterase in guinea pigs - Sun_2005_Chem.Biol.Interact_157-158_428
Author(s) : Sun W , Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 157-158 :428 , 2005
Abstract : Human serum butyrylcholinesterase (Hu BChE) has been demonstrated to be a highly effective detoxifying enzyme for counteracting the acute toxicity of organophosphorus (OP) nerve agents. In order to initiate an investigational new drug (IND) application for human use, the safety and pharmacokinetic properties of the enzyme were assessed in guinea pigs. Sixty milligrams per kilogram of Hu BChE was administered to guinea pigs by either i.p. or i.m. injection. Blood was drawn at various time points for up to 2 weeks following enzyme injection for the measurement of blood BChE activity. Hu BChE displayed a mean residence time of 110 h, regardless of the route of administration and the enzyme activity remained almost 10-fold above baseline level even after 2 weeks post enzyme injection. Fourteen days post Hu BChE administration, all animals were subjected to 20 panel serum chemistry, hematology, and complete gross/histopathology examination. Results showed no toxic effects as measured by general observation, serum chemistry, hematology, and gross and histological tissue changes. In conclusion, Hu BChE displays a long-lasting stability in the circulation of guinea pigs, and is devoid of any toxic side effects. These results provide convincing data for the safe and effective use of Hu BChE as a bioscavenger to protect humans against all OP nerve agents.
ESTHER : Sun_2005_Chem.Biol.Interact_157-158_428
PubMedSearch : Sun_2005_Chem.Biol.Interact_157-158_428
PubMedID: 16429577

Title : In vitro and in vivo characterization of recombinant human butyrylcholinesterase (Protexia) as a potential nerve agent bioscavenger - Cerasoli_2005_Chem.Biol.Interact_157-158_363
Author(s) : Cerasoli DM , Griffiths EM , Doctor BP , Saxena A , Fedorko JM , Greig NH , Yu QS , Huang Y , Wilgus H , Karatzas CN , Koplovitz I , Lenz DE
Ref : Chemico-Biological Interactions , 157-158 :363 , 2005
Abstract : Previous studies in rodents and nonhuman primates have demonstrated that pretreatment with cholinesterases can provide significant protection against behavioral and lethal effects of nerve agent intoxication. Human butyrylcholinesterase (HuBuChE) purified from plasma has been shown to protect against up to 5 x LD50s of nerve agents in guinea pigs and non-human primates, and is currently being explored as a bioscavenger pretreatment for human use. A recombinant form of HuBuChE has been expressed in the milk of transgenic goats as a product called Protexia. Protexia was supplied by Nexia Biotechnologies (Que., Canada) as a purified solution with a specific activity of 600 U/mg. Initial in vitro studies using radiolabeled 3H-soman or 3H-DFP (diisopropyl fluorophosphate) demonstrated that these inhibitors specifically bind to Protexia. When Protexia was mixed with soman, sarin, tabun or VX using varying molar ratios of enzyme to nerve agent (8:1, 4:1, 1:1 and 1:4, respectively), the data indicated that 50% inhibition of enzyme activity occurs around the 1:1 molar ratio for each of the nerve agents. Protexia was further characterized for its interaction with pyridostigmine bromide and six unique carbamate inhibitors of cholinesterase. IC50 and Ki values for Protexia were determined to be very similar to those of HuBuChE purified from human plasma. These data suggest that Protexia has biochemical properties very similar to those HuBuChE when compared in vitro. Together these data the continued development of the goat milk-derived recombinant HuBuChE Protexia as a potential bioscavenger of organophosphorus nerve agents.
ESTHER : Cerasoli_2005_Chem.Biol.Interact_157-158_363
PubMedSearch : Cerasoli_2005_Chem.Biol.Interact_157-158_363
PubMedID: 16429486

Title : Effects of physostigmine and human butyrylcholinesterase on acoustic startle reflex and prepulse inhibition in C57BL\/6J mice - Clark_2005_Pharmacol.Biochem.Behav_81_497
Author(s) : Clark MG , Sun W , Myers TM , Bansal R , Doctor BP , Saxena A
Ref : Pharmacol Biochem Behav , 81 :497 , 2005
Abstract : The use of exogenously administered cholinesterases as bioscavengers of highly toxic organophosphorus nerve agents is a viable prophylactic against this threat. To use this strategy, cholinesterases must provide protection without disrupting behavior when administered alone. To assess behavioral safety, the acoustic startle reflex and prepulse inhibition (PPI) of C57BL/6J mice were investigated following administration of human plasma-derived butyrylcholinesterase (HuBChE). Two hours before testing, four groups of mice (n=10 per group) were pretreated with saline or HuBChE (2000 U, ip). Fifteen minutes before testing, subjects received either saline or the carbamate physostigmine (0.4 mg/kg, sc). Mice exposed to physostigmine exhibited a significant attenuation of the startle reflex, an increased time to peak startle amplitude, and significantly increased PPI. This effect was partially mitigated in mice pretreated with HuBChE. HuBChE alone did not change startle behavior or PPI significantly compared to saline controls. The circulatory time-course of butyrylcholinesterase was assessed in a separate group of mice and revealed levels approximately 600 times the physiological norm 2-4 h post administration. Thus, HuBChE does not appear to significantly alter startle or PPI behavior at a dose 30-fold higher than that estimated to be necessary for protection against 2LD50 of soman in humans.
ESTHER : Clark_2005_Pharmacol.Biochem.Behav_81_497
PubMedSearch : Clark_2005_Pharmacol.Biochem.Behav_81_497
PubMedID: 15913750

Title : Protection against soman or VX poisoning by human butyrylcholinesterase in guinea pigs and cynomolgus monkeys - Lenz_2005_Chem.Biol.Interact_157-158_205
Author(s) : Lenz DE , Maxwell DM , Koplovitz I , Clark CR , Capacio BR , Cerasoli DM , Federko JM , Luo C , Saxena A , Doctor BP , Olson C
Ref : Chemico-Biological Interactions , 157-158 :205 , 2005
Abstract : Human butyrylcholinesterase (HuBuChE), purified from outdated human plasma, is being evaluated for efficacy against nerve agents in guinea pigs and cynomolgus monkeys. Previous studies in rodents and nonhuman primates demonstrated that pretreatment of animals with enzymes that can scavenge nerve agents could provide significant protection against behavioral and lethal effects of nerve agent intoxication. In preparation for evaluation of efficacy of HuBuChE prior to initiating an investigational new drug (IND) application, the pharmacokinetics of HuBuChE were evaluated in guinea pigs and in cynomolgus monkeys. HuBuChE was injected intramuscularly (i.m.) at two doses, and blood samples were taken to follow the time-course of HuBuChE in blood for up to 168 h after administration. In guinea pigs, the two doses of HuBuChE, 19.9 and 32.5 mg/kg, produced similar times of maximal blood concentration (T(max) of 26.0 and 26.8 h, respectively) and similar elimination half-times (t(1/2) of 64.6 and 75.5 h, respectively). Enzyme levels were still 10-fold over baseline at 72 h. Based on these data, guinea pigs were administered 150 mg/kg of enzyme i.m. and challenged at T(max). Soman or VX doses were approximately 1.5, 2.0 and 2.0 x LD50 administered subcutaneously (s.c.) in sequence at 90-120 min apart. None of the animals displayed signs of organophosphorus (OP) anticholinesterase intoxication at any of the challenge levels, and all survived for the 14-day duration of the experiment. Similar experiments were carried out with cynomolgus monkeys to determine the pharmacokinetics of HuBuChE and its efficacy against soman. The complete survival of nearly all animals tested to date, coupled with the maximal blood concentration and half-life elimination profile obtained for HuBuChE after i.m. injection, provides strong support for the continued development of HuBuChE as a product to protect against nerve agents.
ESTHER : Lenz_2005_Chem.Biol.Interact_157-158_205
PubMedSearch : Lenz_2005_Chem.Biol.Interact_157-158_205
PubMedID: 16289064

Title : An ex vivo approach for the evaluation of reversible inhibitors as potential pretreatments against organophosphate toxicity - Tonduli_2005_Chem.Biol.Interact_157-158_426
Author(s) : Tonduli LS , Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 157-158 :426 , 2005
Abstract : Several studies demonstrated that pretreatment with reversible acetylcholinesterase (AChE) inhibitor, such as (pyridostigmine) PYR, improved the survival of animals intoxicated by organophosphate nerve agents (OP). These compounds temporarily inhibited a fraction of the enzyme and protected it from inactivation by nerve agents. An important criterion for effective pretreatment is that it must ensure the recovery of the protected fraction of the enzyme. We thus designed a simple ex vivo method to investigate the recovery of AChE activity, that was protected by PYR, prior to irreversible inhibition by an OP, using a modified Ellman assay. Results show that our approach is suitable for routine use and can successfully predict the potential use of various AChE inhibitors as pretreatment drugs against OP nerve agent intoxication.
ESTHER : Tonduli_2005_Chem.Biol.Interact_157-158_426
PubMedSearch : Tonduli_2005_Chem.Biol.Interact_157-158_426
PubMedID: 16429574

Title : Species-related differences in the oxime-induced reactivation of organophosphate-inhibited acetylcholinesterases -
Author(s) : Luo C , Dawson M , Chambers C , Chilukuri N , Radic Z , Taylor P , Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 157-158 :393 , 2005
PubMedID: 16429528

Title : Poster (32) Bioscavengers: antidotes for organophosphate chemical warfare agent toxicity. -
Author(s) : Doctor BP , Saxena A , Ashani Y , Ross M
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :337 , 2004
PubMedID:

Title : Poster (33) Strategy for the reactivation of organophosphate-inhibited human butyrylcholinesterase -
Author(s) : Luo C , McKissic D , Doctor BP , Saxena A
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :338 , 2004
PubMedID:

Title : Poster (36) Human serum butyrylcholinesterase: a future generation antidote for organophosphate chemical warfare agent toxicity -
Author(s) : Saxena A , Luo C , Bansal R , Sun W , Clark M , Ashani Y , Ross M , Doctor BP
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :339 , 2004
PubMedID:

Title : Protective effect of equine butyrylcholinesterase in inhalation intoxication of rats with sarin: determination of blood and brain cholinesterase activities - Sevelova_2004_Inhal.Toxicol_16_531
Author(s) : Sevelova L , Bajgar J , Saxena A , Doctor BP
Ref : Inhal Toxicol , 16 :531 , 2004
Abstract : The effect of pretreatment with equine butyrylcholinesterase (EqBuChE) on cholinesterase inhibition in the blood and brain of rats following inhalation intoxication with low concentrations (1.25 microg/L for 60 min) of sarin were studied. Animals pretreated with different doses of equine butyrylcholinesterase showed significant increases in plasma butyrylcholinesterase activity. However, erythrocyte acetylcholinesterase activity was unchanged. The decrease in acetylcholinesterase and butyrylcholinesterase activity after inhalation intoxication was dependent on the dose of equine butyrylcholinesterase used for pretreatment and was always greater for erythrocyte acetylcholinesterase. Acetylcholinesterase activity in different brain regions was unchanged following pretreatment with equine butyrylcholinesterase. After inhalation exposure to sarin, acetylcholinesterase activity was diminished markedly in the pontomedullar area (51.5% of normal activity) and frontal cortex (72.0% of normal activity), and slightly in basal ganglia (91.4% of normal activity). Plasma levels of sarin were determined using fluoride-induced reactivation of inhibited enzyme. As expected, the amounts of sarin in plasma were almost identical in rats pretreated with EqBuChE as well as in untreated rats. In pretreated animals, the plasma amount of sarin did not depend on the dose of equine butyrylcholinesterase used for pretreatment. Our results demonstrate that equine butyrylcholinesterase pretreatment can be considered as an effective prophylaxis against nerve agents (at least with sarin) and seems to be an alternative or superior to prophylaxis provided by reversible cholinesterase inhibitors.
ESTHER : Sevelova_2004_Inhal.Toxicol_16_531
PubMedSearch : Sevelova_2004_Inhal.Toxicol_16_531
PubMedID: 15204744

Title : Two possible orientations of the HI-6 molecule in the reactivation of organophosphate-inhibited acetylcholinesterase. -
Author(s) : Luo C , Leader H , Radic Z , Maxwell DM , Taylor P , Doctor BP , Saxena A
Ref : Cholinergic Mechanisms, CRC Press :627 , 2004
PubMedID:

Title : The influence of solvent composition on global dynamics of human butyrylcholinesterase powders: a neutron-scattering study - Gabel_2004_Biophys.J_86_3152
Author(s) : Gabel F , Weik M , Doctor BP , Saxena A , Fournier D , Brochier L , Renault F , Masson P , Silman I , Zaccai G
Ref : Biophysical Journal , 86 :3152 , 2004
Abstract : A major result of incoherent elastic neutron-scattering experiments on protein powders is the strong dependence of the intramolecular dynamics on the sample environment. We performed a series of incoherent elastic neutron-scattering experiments on lyophilized human butyrylcholinesterase (HuBChE) powders under different conditions (solvent composition and hydration degree) in the temperature range from 20 to 285 K to elucidate the effect of the environment on the enzyme atomic mean-square displacements. Comparing D(2)O- with H(2)O-hydrated samples, we were able to investigate protein as well as hydration water molecular dynamics. HuBChE lyophilized from three distinct buffers showed completely different atomic mean-square displacements at temperatures above approximately 200 K: a salt-free sample and a sample containing Tris-HCl showed identical small-amplitude motions. A third sample, containing sodium phosphate, displayed highly reduced mean-square displacements at ambient temperature with respect to the other two samples. Below 200 K, all samples displayed similar mean-square displacements. We draw the conclusion that the reduction of intramolecular protein mean-square displacements on an Angstrom-nanosecond scale by the solvent depends not only on the presence of salt ions but also on their type.
ESTHER : Gabel_2004_Biophys.J_86_3152
PubMedSearch : Gabel_2004_Biophys.J_86_3152
PubMedID: 15111428

Title : Poster (99) Rational design, synthesis and pharmacological evaluation of novel, selective and highly potent cholinesterase inhibitors -
Author(s) : Gaeta A , Savini L , Fattorusso C , Catalanotti B , Campiani G , Chiasserini L , Pellerano C , McKissic D , Saxena A
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :372 , 2004
PubMedID:

Title : Strategy for reactivation of organophosphate-inhibited human butyrylcholinesterase -
Author(s) : Luo C , Dawson M , Maxwell DM , Doctor BP , Saxena A
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :251 , 2004
PubMedID:

Title : Human serum butyrylcholinesterase: A future generation antidote for organophosphate chemical warfare agent toxicity . -
Author(s) : Saxena A , Luo C , Bansal R , Sun W , Clark M , Ashani Y , Ross M , Doctor BP
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :269 , 2004
PubMedID:

Title : Two possible orientations of the HI-6 molecule in the reactivation of organophosphate-inhibited acetylcholinesterase - Luo_2003_Biochem.Pharmacol_66_387
Author(s) : Luo C , Leader H , Radic Z , Maxwell DM , Taylor P , Doctor BP , Saxena A
Ref : Biochemical Pharmacology , 66 :387 , 2003
Abstract : The inhibition of acetylcholinesterase (AChE) by organophosphorus compounds (OPs) causes acute toxicity or death of the intoxicated individual. One group of these compounds, the OP nerve agents, pose an increasing threat in the world due to their possible use in the battlefield or terrorist acts. Antidotes containing oxime compounds to reactivate the inhibited enzyme are highly valued for treatment against OP poisoning. One of these reactivators, HI-6, was shown to be significantly more effective in treating soman toxicity than other oximes, such as 2-PAM, TMB4, and obidoxime. However, HI-6 was less effective in reactivating AChE inhibited by the OP pesticide, paraoxon. In this study, the mechanism for HI-6-induced reactivation of OP-AChE conjugates was investigated using mouse mutant AChEs inhibited with different OPs including organophosphate paraoxon, and several methylphosphonates. Results indicate that the HI-6 molecule may assume two different orientations in the reactivation of AChE inhibited by organophosphate and Sp methylphosphonates. These conclusions were further corroborated by reactivation studies using an analog of HI-6 in which the bispyridinium moieties are linked by a methylene bridge rather than an ether oxygen.
ESTHER : Luo_2003_Biochem.Pharmacol_66_387
PubMedSearch : Luo_2003_Biochem.Pharmacol_66_387
PubMedID: 12907237

Title : Aromatic amino-acid residues at the active and peripheral anionic sites control the binding of E2020 (Aricept) to cholinesterases - Saxena_2003_Eur.J.Biochem_270_4447
Author(s) : Saxena A , Fedorko JM , Vinayaka CR , Medhekar R , Radic Z , Taylor P , Lockridge O , Doctor BP
Ref : European Journal of Biochemistry , 270 :4447 , 2003
Abstract : E2020 (R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]methyl)piperidine hydrochloride is a piperidine-based acetylcholinesterase (AChE) inhibitor that was approved for the treatment of Alzheimer's disease in the United States. Structure-activity studies of this class of inhibitors have indicated that both the benzoyl containing functionality and the N-benzylpiperidine moiety are the key features for binding and inhibition of AChE. In the present study, the interaction of E2020 with cholinesterases (ChEs) with known sequence differences, was examined in more detail by measuring the inhibition constants with Torpedo AChE, fetal bovine serum AChE, human butyrylcholinesterase (BChE), and equine BChE. The basis for particular residues conferring selectivity was then confirmed by using site-specific mutants of the implicated residue in two template enzymes. Differences in the reactivity of E2020 toward AChE and BChE (200- to 400-fold) show that residues at the peripheral anionic site such as Asp74(72), Tyr72(70), Tyr124(121), and Trp286(279) in mammalian AChE may be important in the binding of E2020 to AChE. Site-directed mutagenesis studies using mouse AChE showed that these residues contribute to the stabilization energy for the AChE-E2020 complex. However, replacement of Ala277(Trp279) with Trp in human BChE does not affect the binding of E2020 to BChE. Molecular modeling studies suggest that E2020 interacts with the active-site and the peripheral anionic site in AChE, but in the case of BChE, as the gorge is larger, E2020 cannot simultaneously interact at both sites. The observation that the KI value for mutant AChE in which Ala replaced Trp286 is similar to that for wild-type BChE, further confirms our hypothesis.
ESTHER : Saxena_2003_Eur.J.Biochem_270_4447
PubMedSearch : Saxena_2003_Eur.J.Biochem_270_4447
PubMedID: 14622273

Title : Natural monomeric form of fetal bovine serum acetylcholinesterase lacks the C-terminal tetramerization domain - Saxena_2003_Biochemistry_42_15292
Author(s) : Saxena A , Hur RS , Luo C , Doctor BP
Ref : Biochemistry , 42 :15292 , 2003
Abstract : Acetylcholinesterase isolated from fetal bovine serum (FBS AChE) was previously characterized as a globular tetrameric form. Analysis of purified preparations of FBS AChE by gel permeation chromatography revealed the presence of a stable, catalytically active, monomeric form of this enzyme. The two forms could be distinguished from each other based on their molecular weight, hydrodynamic properties, kinetic properties, thermal stability, and the type of glycans they carry. No differences between the two forms were observed for the binding of classical inhibitors such as edrophonium and propidium or inhibitors that are current or potential drugs for the treatment of Alzheimer's disease such as (-) huperzine A and E2020; tacrine inhibited the monomeric form 2-3-fold more potently than the tetrameric form. Sequencing of peptides obtained from an in-gel tryptic digest of the monomer and tetramer by tandem mass spectrometry indicated that the tetramer consists of 583 amino acid residues corresponding to the mature form of the enzyme, whereas the monomer consists of 543-547 amino acid residues. The subunit molecular weight of the protein component of the monomer (major species) was determined to be 59 414 Da and that of the tetramer as 64 239 Da. The N-terminal of the monomer and the tetramer was Glu, suggesting that the monomer is not a result of truncation at the N-terminal. The only differences detected were at the C-terminus. The tetramer yielded the expected C-terminus, CSDL, whereas the C-terminus of the monomer yielded a mixture of peptides, of which LLSATDTLD was the most abundant. These results suggest that monomeric FBS AChE is trimmed at the C-terminus, and the results are consistent with the involvement of C-terminal amino acids in the assembly of monomers into tetramers.
ESTHER : Saxena_2003_Biochemistry_42_15292
PubMedSearch : Saxena_2003_Biochemistry_42_15292
PubMedID: 14690439

Title : Specific targeting of acetylcholinesterase and butyrylcholinesterase recognition sites. Rational design of novel, selective, and highly potent cholinesterase inhibitors - Savini_2003_J.Med.Chem_46_1
Author(s) : Savini L , Gaeta A , Fattorusso C , Catalanotti B , Campiani G , Chiasserini L , Pellerano C , Novellino E , McKissic D , Saxena A
Ref : Journal of Medicinal Chemistry , 46 :1 , 2003
Abstract : Tacrine-based AChE and BCHE inhibitors were designed by investigating the topology of the active site gorge of the two enzymes. The homobivalent ligands characterized by a nitrogen-bridged atom at the tether level could be considered among the most potent and selective cholinesterase inhibitors described to date. The nitrogen-containing homobivalent ligands 3e,g and the sulfur-containing 3h validated the hypothesis of extra sites of interaction in the AChE and BCHE active site gorges.
ESTHER : Savini_2003_J.Med.Chem_46_1
PubMedSearch : Savini_2003_J.Med.Chem_46_1
PubMedID: 12502352

Title : Inhibition of cholinesterases with cationic phosphonyl oximes highlights distinctive properties of the charged pyridine groups of quaternary oxime reactivators - Ashani_2003_Biochem.Pharmacol_66_191
Author(s) : Ashani Y , Bhattacharjee AK , Leader H , Saxena A , Doctor BP
Ref : Biochemical Pharmacology , 66 :191 , 2003
Abstract : Oxime-induced reactivation of phosphonylated cholinesterases (ChEs) produces charged phosphonyl pyridine oxime intermediates (POXs) that are most potent organophosphate (OP) inhibitors of ChEs. To understand the role of cationic pyridine oxime leaving groups in the enhanced anti-ChE activity of POXs, the bimolecular rate constants for the inhibition (k(i)) of acetylcholinesterases (AChE) and butyrylcholinesterases (BChE), and the rate of decomposition (k(d)) of authentic O-alkyl methylphosphonyl pyridine oximes (AlkMeP-POXs) and N,N-dimethylamidophosphoryl pyridine oximes (EDMP-POXs), were studied. Stability ranking order in aqueous solutions correlated well with the electronic features and optimized geometries that were obtained by ab initio calculations at 6-31G(**) basis set level. AlkMeP-POXs of the 2-pyridine oxime series were found to be 4- to 8-fold more stable (t(1/2)=0.7 to 1.5 min) than the homologous O,O-diethylphosphoryl (DEP) oxime. Results suggest that re-inhibition of enzyme activity by POX is less likely during the reactivation of DEP-ChEs (obtained by use of DEP-containing pesticides) by certain oximes, compared to nerve agent-inhibited ChEs. The greatest inhibition was observed for the O-cyclohexyl methylphosphonyl-2PAM derivative (4.0 x 10(9)M(-1)min(-1); mouse AChE) and is 10-fold higher than the k(i) of cyclosarin. Increasing the size of the O-alkyl substituent of AlkMeP-POXs had only a small to moderate effect on the k(i) of ChEs, signifying a major role for the cationic pyridine oxime leaving group in the inhibition reaction. The shape of plots of logk(i) vs. pK(a) of the leaving groups for AlkMeP-PAMs and DEP-PAMs, could be used as a diagnostic tool to highlight and rationalize the unique properties of the cationic moiety of pyridine oxime reactivators.
ESTHER : Ashani_2003_Biochem.Pharmacol_66_191
PubMedSearch : Ashani_2003_Biochem.Pharmacol_66_191
PubMedID: 12826262

Title : Pharmacokinetics and immunologic consequences of exposing macaques to purified homologous butyrylcholinesterase - Rosenberg_2002_Life.Sci_72_125
Author(s) : Rosenberg YJ , Luo C , Ashani Y , Doctor BP , Fischer R , Wolfe G , Saxena A
Ref : Life Sciences , 72 :125 , 2002
Abstract : Exposure to organophosphorus compounds (OPs), in the form of nerve agents and pesticides poses an ever increasing military and civilian threat. In recent years, attention has focused on the use of exogenously administered cholinesterases as an effective prophylactic treatment for protection against OPs. Clearly, a critical prerequisite for any potential bioscavenger is a prolonged circulatory residence time, which is influenced by the size of protein, the microheterogeneity of carbohydrate structures, and the induction (if any) of anti-enzyme antibodies following repeated injections of the enzyme. Previously, it was demonstrated that multiple injections of equine butyrylcholinesterase (BChE) into rabbits, rats, or rhesus monkeys, resulted in a mean residence time spanning several days, and variable immune responses. The present study sought to assess the pharmacokinetics and immunological consequences of administration of purified macaque BChE into macaques of the same species at a dose similar to that required for preventing OP toxicity. An i.v. injection of 7,000 U of homologous enzyme in monkeys demonstrated much longer mean residence times in plasma (MRT = 225 +/- 19 h) compared to those reported for heterologous Hu BChE (33.7 +/- 2.9 h). A smaller second injection of 3,000 U given four weeks later, attained predicted peak plasma levels of enzyme activity, but surprisingly, the MRT in the four macaques showed wide variation and ranged from 54 to 357 h. No antibody response was detected in macaques following either injection of enzyme. These results bode well for the potential use of human BChE as a detoxifying drug in humans.
ESTHER : Rosenberg_2002_Life.Sci_72_125
PubMedSearch : Rosenberg_2002_Life.Sci_72_125
PubMedID: 12417246

Title : Synthesis of more potent analogues of the acetylcholinesterase inhibitor, huperzine B - Rajendran_2002_Bioorg.Med.Chem.Lett_12_1521
Author(s) : Rajendran V , Saxena A , Doctor BP , Kozikowski AP
Ref : Bioorganic & Medicinal Chemistry Lett , 12 :1521 , 2002
Abstract : The synthesis and acetylcholinesterase inhibition activity of analogues of huperzine B are reported. These new racemic analogues show a better AChE inhibitory activity than the natural product huperzine B.
ESTHER : Rajendran_2002_Bioorg.Med.Chem.Lett_12_1521
PubMedSearch : Rajendran_2002_Bioorg.Med.Chem.Lett_12_1521
PubMedID: 12031333

Title : Novel and potent tacrine-related hetero- and homobivalent ligands for acetylcholinesterase and butyrylcholinesterase - Savini_2001_Bioorg.Med.Chem.Lett_11_1779
Author(s) : Savini L , Campiani G , Gaeta A , Pellerano C , Fattorusso C , Chiasserini L , Fedorko JM , Saxena A
Ref : Bioorganic & Medicinal Chemistry Lett , 11 :1779 , 2001
Abstract : Based upon synthetic and biochemical results, a novel and potent tacrine analogue and heterobivalent analogues of tacrine, were designed. The role played by the amino groups of homo- and heterobivalent ligands in the interaction with the peripheral and catalytic sites of AChE and BuChE were investigated. The syntheses of these materials together with the results of AChE/BuChE inhibition assays are detailed.
ESTHER : Savini_2001_Bioorg.Med.Chem.Lett_11_1779
PubMedSearch : Savini_2001_Bioorg.Med.Chem.Lett_11_1779
PubMedID: 11425559

Title : Allosteric control of acetylcholinesterase activity by monoclonal antibodies -
Author(s) : Saxena A , Hur RS , Doctor BP
Ref : Biochemistry , 38 :15688 , 1999
PubMedID: 10569956

Title : Role of edrophonium in prevention of the re-inhibition of acetylcholinesterase by phosphorylated oxime - Luo_1999_Chem.Biol.Interact_119-120_129
Author(s) : Luo C , Saxena A , Ashani Y , Leader H , Radic Z , Taylor P , Doctor BP
Ref : Chemico-Biological Interactions , 119-120 :129 , 1999
Abstract : We examined the role of edrophonium in the acceleration phenomenon using mouse wild-type and mutant D74N AChE inhibited with 7-(O,O-diethyl-phosphinyloxy)-1-methylquinolinium methylsulfate (DEPQ). With DEPQ-inhibited wild-type mouse acetylcholinesterase (AChE), the reactivation kinetic profile demonstrated one-phase exponential association only when 2-[hydroxyimino methyl]-1-methylpyridinium chloride (2-PAM) and 1-(2-hydroxy-iminomethyl-1-pyridinium)-1-(4-carboxy-aminopyridi nium)-dimethyl ether hydrochloride (HI-6) were used as reactivators. When 1,1[oxybis-methylene)bis[4-(hydroxyimino)methyl] pyridinium dichloride (LuH6) and 1,1-trimethylene bis(4-hydroxyimino methyl) pyridinium dichloride (TMB4) were used, the reactivation kinetic profile was biphasic in nature. Edrophonium had no effect on reactivation by 2-PAM and HI-6, but significantly accelerated LuH6- and TMB4-induced reactivation of DEPQ-inhibited wild-type mouse AChE. Comparison of the initial and overall reactivation rate constants with five oximes indicated that acceleration by edrophonium may be due to the prevention of re-inhibition of the reactivated enzyme by the phosphorylated oxime (POX) produced during the reactivation. With LuH6 and TMB4, about 2.5-fold increase in the reactivation rate constants was observed in the presence of edrophonium, but little or no effect was observed with the other three oximes. The initial reactivation rate constants were 5.4- and 4.2-fold of the overall rate constants with LuH6 and TMB4 as reactivators respectively, however, very little change was found between the initial and overall rate constants with the other three oximes. In experiments with D74N AChE, for which the inhibition potency of charged organophosphate (OP) was two to three orders less than wild-type enzyme, edrophonium had no effect on the reactivation by LuH6 and TMB4 and the time courses of reactivation were monophasic. The data from mutant enzyme substantiate the involvement of edrophonium in protecting POX re-inhibition of reactivated enzyme formed during the reactivation of OP-inhibited AChE.
ESTHER : Luo_1999_Chem.Biol.Interact_119-120_129
PubMedSearch : Luo_1999_Chem.Biol.Interact_119-120_129
PubMedID: 10421446

Title : Differences in active-site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase - Saxena_1999_Chem.Biol.Interact_119-120_61
Author(s) : Saxena A , Redman AM , Jiang X , Lockridge O , Doctor BP
Ref : Chemico-Biological Interactions , 119-120 :61 , 1999
Abstract : We examined the role of A328(F330) in the binding of various inhibitors to cholinesterases (ChEs) using human butyrylcholinesterase (BChE) mutants to determine if the conclusions drawn from studies with acetylcholinesterase (AChE) mutants could be extended to BChE. For huperzine A and edrophonium, the results obtained with AChE mutants could be directly correlated with those obtained with native ChEs and site-specific mutants of human BChE. Inhibition studies of ethopropazine with BChE mutants, where A328 was modified to either F or Y, suggested that A328 was not solely responsible for the selectivity of ethopropazine. Volume calculations for the active-site gorge showed that the poor inhibitory activity of ethopropazine towards AChE was due to the smaller dimension of the active-site gorge. The volume of the BChE active-site gorge is approximately 200 A3 larger than that of the AChE gorge, which allows the accommodation of ethopropazine in two different orientations as demonstrated by rigid-body refinement and molecular dynamics calculations. These results suggest that, although the overall scaffolding of the two enzymes may be highly similar, the dimensions and the micro-environment of the gorge play a significant role in determining the selectivity of substrate and inhibitors for ChEs.
ESTHER : Saxena_1999_Chem.Biol.Interact_119-120_61
PubMedSearch : Saxena_1999_Chem.Biol.Interact_119-120_61
PubMedID: 10421439

Title : Improvements in scavenger protection against organophosphorus agents by modification of cholinesterases - Maxwell_1999_Chem.Biol.Interact_119-120_419
Author(s) : Maxwell DM , Saxena A , Gordon RK , Doctor BP
Ref : Chemico-Biological Interactions , 119-120 :419 , 1999
Abstract : The ability of stoichiometric scavengers, such as ChEs, to protect against a variety of OP agents has been demonstrated in several in vivo models. To improve the detoxification of OP agents by ChEs, several approaches have been recently used to increase the stoichiometry, stability, and in vivo effectiveness of ChEs as OP scavengers. For example, the in vitro stoichiometric neutralization of sarin by AChE was increased from 1:1 to 3200:1 by the addition of the oxime HI-6, while the in vivo stoichiometry was increased to 57:1 in mice by HI-6. The aging rate of soman-inhibited mouse AChE was reduced 12-fold in a mutant AChE (E202Q) which resulted in a two-fold increase in oxime-assisted detoxification of soman. To improve the duration of scavenger protection provided by ChEs, the mean residence times of five tissue-derived and two recombinant ChEs injected i.v. in mice were compared with their oligosaccharide profiles. The mean residence times of these ChEs were found to increase with molecular weight and with the levels of oligosaccharide sialylation. The stability of AChE in non-physiological environments was improved by immobilizing it in a polyurethane foam matrix that allowed AChE to retain enzymatic activity at high temperature (75 degrees C) where soluble enzyme denatured. These developments in scavenger technology have improved the in vivo protection provided by OP scavengers and extended their applicability to provide external decontamination of chemical agents and pesticides.
ESTHER : Maxwell_1999_Chem.Biol.Interact_119-120_419
PubMedSearch : Maxwell_1999_Chem.Biol.Interact_119-120_419
PubMedID: 10421479

Title : Phosphoryl oxime inhibition of acetylcholinesterase during oxime reactivation is prevented by edrophonium - Luo_1999_Biochemistry_38_9937
Author(s) : Luo C , Saxena A , Smith M , Garcia GE , Radic Z , Taylor P , Doctor BP
Ref : Biochemistry , 38 :9937 , 1999
Abstract : Reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) is a key objective in the treatment of OP poisoning. This study with native, wild-type, and mutant recombinant DNA-expressed AChEs, each inhibited by representative OP compounds, establishes a relationship between edrophonium acceleration of oxime-induced reactivation of OP-AChE conjugates and phosphoryl oxime inhibition of the reactivated enzyme that occurs during reactivation by pyridinium oximes LH6 and TMB4. No such recurring inhibition could be observed with HI-6 as the reactivator due to the extreme lability of the phosphoryl oximes formed by this oxime. Phosphoryl oximes formed during reactivation of the ethoxy methylphosphonyl-AChE conjugate by LH6 and TMB4 were isolated for the first time and their structures confirmed by (31)P NMR. However, phosphoryl oximes formed during the reactivation of the diethylphosphoryl-AChE conjugate were not sufficiently stable to be detected by (31)P NMR. The purified ethoxy methylphosphonyl oximes formed during the reactivation of ethoxy methylphosphonyl-AChE conjugate with LH6 and TMB4 are 10- to 22-fold more potent than MEPQ as inhibitors of AChE and stable for several hours at pH 7.2 in HEPES buffer. Reactivation of both ethoxy methylphosphonyl- and diethylphosphoryl-AChE by these two oximes was accelerated in the presence of rabbit serum paraoxonase, suggesting that organophosphorus hydrolase can hydrolyze phosphoryl oxime formed during the reactivation. Our results emphasize that certain oximes, such as LH6 and TMB4, if used in the treatment of OP pesticide poisoning may cause prolonged inhibition of AChE due to formation of phosphoryl oximes.
ESTHER : Luo_1999_Biochemistry_38_9937
PubMedSearch : Luo_1999_Biochemistry_38_9937
PubMedID: 10433700

Title : The Role of Oligosaccharides in the Pharmacokinetics of Cholinesterases -
Author(s) : Saxena A , Ashani Y , Raveh L , Doctor BP
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :283 , 1998
PubMedID:

Title : Allosteric control of acetylcholinesterase activity by monoclonal antibodies - Saxena_1998_Biochemistry_37_145
Author(s) : Saxena A , Hur RS , Doctor BP
Ref : Biochemistry , 37 :145 , 1998
Abstract : Previous studies showed that monoclonal antibodies raised against phosphorylated fetal bovine serum acetylcholinesterase appeared to modulate the catalytic activity of the enzyme by binding to a conformational epitope located at or near the region of the peripheral anionic site. The mechanism of inhibition of acetylcholinesterase by these monoclonal antibodies was further investigated by determining their effect on (i) substrate inhibition due to the binding of excess substrate to the peripheral anionic site and (ii) binding of peripheral anionic site ligands, such as propidium and fasciculin. Results of these experiments demonstrate that the accessibility of substrate to the peripheral anionic site in these complexes was restricted but not completely blocked, as none of the monoclonal antibodies eliminated the phenomenon of excess substrate inhibition. The results also show that propidium clearly slowed the inhibition of fetal bovine serum acetylcholinesterase by all six inhibitory monoclonal antibodies but to different levels. Complexation of fetal bovine serum acetylcholinesterase with monoclonal antibodies 25B1, 4E5, 6H9, and 5E8 interfered with the binding of fasciculin to the complexed enzyme, suggesting that part of their epitope overlapped with the fasciculin binding site. These monoclonal antibodies bind, in part, at the peripheral anionic site, since polyclonal anti-idiotypic antibodies generated against two monoclonal antibodies, 25B1 and 6H9, bound stoichiometric amounts of propidium. Like fasciculin, binding of these monoclonal antibodies in the vicinity of the peripheral anionic site at the rim of the active site gorge allosterically affects the orientation of W86 located at the base of the gorge, resulting in inhibition of enzyme activity.
ESTHER : Saxena_1998_Biochemistry_37_145
PubMedSearch : Saxena_1998_Biochemistry_37_145
PubMedID: 9425034

Title : Role of oligosaccharides in the pharmacokinetics of tissue-derived and genetically engineered cholinesterases - Saxena_1998_Mol.Pharmacol_53_112
Author(s) : Saxena A , Ashani Y , Raveh L , Stevenson D , Patel T , Doctor BP
Ref : Molecular Pharmacology , 53 :112 , 1998
Abstract : To understand the role of glycosylation in the circulation of cholinesterases, we compared the mean residence time of five tissue-derived and two recombinant cholinesterases (injected intravenously in mice) with their oligosaccharide profiles. Monosaccharide composition analysis revealed differences in the total carbohydrate, galactose, and sialic acid contents. The molar ratio of sialic acid to galactose residues on tetrameric human serum butyrylcholinesterase, recombinant human butyrylcholinesterase, and recombinant mouse acetylcholinesterase was found to be approximately 1.0. For Torpedo californica acetylcholinesterase, monomeric and tetrameric fetal bovine serum acetylcholinesterase, and equine serum butyrylcholinesterase, this ratio was approximately 0.5. However, the circulatory stability of cholinesterases could not be correlated with the sialic acid-to-galactose ratio. Fractionation of the total pool of oligosaccharides obtained after neuraminidase digestion revealed one major oligosaccharide for human serum butyrylcholinesterase and three or four major oligosaccharides in other cholinesterases. The glycans of tetrameric forms of plasma cholinesterases (human serum butyrylcholinesterase, fetal bovine serum acetylcholinesterase, and equine serum butyrylcholinesterase) clearly demonstrated a reduced heterogeneity and higher maturity compared with glycans of monomeric fetal bovine serum acetylcholinesterase, dimeric tissue-derived T. californica acetylcholinesterase, and recombinant cholinesterases. T. californica acetylcholinesterase, recombinant cholinesterases, and monomeric fetal bovine serum acetylcholinesterase showed a distinctive shorter mean residence time (44-304 min) compared with tetrameric forms of plasma cholinesterases (1902-3206 min). Differences in the pharmacokinetic parameters of cholinesterases seem to be due to the combined effect of the molecular weight and charge- and size-based heterogeneity in glycans.
ESTHER : Saxena_1998_Mol.Pharmacol_53_112
PubMedSearch : Saxena_1998_Mol.Pharmacol_53_112
PubMedID: 9443938

Title : Comparison of Cholinesterases and Carboxylesterase as Bioscavengers for Organophosphorus Compounds -
Author(s) : Maxwell DM , Brecht KM , Saxena A , Feaster SR , Doctor BP
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :387 , 1998
PubMedID:

Title : Synthesis and anticholinesterase activity of huperzine A analogues containing phenol and catechol replacements for the pyridone ring - Campiani_1998_Bioorg.Med.Chem.Lett_8_1413
Author(s) : Campiani G , Kozikowski AP , Wang S , Ming L , Nacci V , Saxena A , Doctor BP
Ref : Bioorganic & Medicinal Chemistry Lett , 8 :1413 , 1998
Abstract : Based upon modeling results obtained using the crystal structure of huperzine A in complex with acetylcholinesterase (AChE), two novel analogues of this potent AChE inhibitor were designed with phenol or catechol rings replacing the pyridone ring. From the modeling studies, the catechol analogue appeared capable of replacing one of the crystallographic waters bridging huperzine with Tyr 130 and Glu 199 of AChE. The synthesis of these materials by use of a palladium catalyzed bicycloannulation strategy is detailed together with the results of AChE inhibition assays.
ESTHER : Campiani_1998_Bioorg.Med.Chem.Lett_8_1413
PubMedSearch : Campiani_1998_Bioorg.Med.Chem.Lett_8_1413
PubMedID: 9871776

Title : Acceleration of Oxime-Induced Reactivation of Organophosphate-Inhibited Acetylcholinesterase by Quaternary Ligands -
Author(s) : Luo C , Ashani Y , Saxena A , Leader H , Maxwell DM , Taylor P , Doctor BP
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :215 , 1998
PubMedID:

Title : Amino Acid Sequence of Horse Serum Butyrylcholinesterase -
Author(s) : Moorad DR , Luo C , Saxena A , Doctor BP , Garcia GE
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :145 , 1998
PubMedID:
Gene_locus related to this paper: horse-BCHE

Title : The pH Dependence of Dealkylation in Soman-Inhibited Cholinesterases and Their Mutants: Further Evidence for a Push-Pull Mechanism - Saxena_1998_Biochemistry_37_15086
Author(s) : Saxena A , Viragh C , Frazier DS , Kovach IM , Maxwell DM , Lockridge O , Doctor BP
Ref : Biochemistry , 37 :15086 , 1998
Abstract : Bimolecular rate constants for the inactivation of recombinant (r) human (Hu) butyrylcholinesterase (BChE) with P(S)C(S)- and P(S)C(R)-2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman) are (92 +/- 7) x 10(6) M-1 min-1 and (13.7 +/- 0.8) x 10(6) M-1 min-1 at pH 7.4, mu = 0.1 M and 25 degreesC. Mutations of E197(199) to D or Q and W82(84) to A result in reductions in the rate constants for inactivation with P(S)C(S)-soman 4.3-, 11.8-, and 263-fold and with P(S)C(R)-soman by 6.5-, 47.3-, and 685-fold, respectively. The pH dependence of dealkylation (aging) in r mouse (Mo) acetylcholinesterase (AChE) and rHu BChE and their mutants inactivated with P(S)C(S)- and P(S)C(R)-soman was compared. Best-fit parameters for the asymmetric bell curves for the adducts of wild-type Mo AChE are pK1 = pK2 = 4.0-4.9 and pK3 = 5.2-6.6. These pKs are consistent with the involvement of two carboxylic acids, possibly E202(199) and either E334(327) or E450(443), and H447(440)H+ in the dealkylation of AChE. E202Q MoAChE inactivated with the soman diastereomers yielded pK3 = 5.5-5.8. Nearly symmetric pH curves for soman-inhibited wild-type and E197D Hu BChE gave pK2 = 3.7-4.6 and pK3 = 7.3-8.0, but much lower, pK3 approximately 5, for the corresponding adduct of the E197Q mutant. Dealkylation in soman-inhibited BChE is consistent with the participation of one carboxylic acid side chain and H438(440)H+. Maximal rate constants for dealkylation (kmax) are 1-6 min-1 for AChE and 2 min-1 for BChE at 25 degreesC. The W82 to A mutation in BChE results in the largest reduction, 2500-6000-fold, in the rate constant for dealkylation. The reduction in the rate constants for dealkylation in the E197 mutants is highly pH dependent. The solvent isotope effects at the pH maxima are 1.3-1.4, indicating unlikely preprotonation or proton in "flight" at the enzymic transition states. The new results support the push-pull mechanism of dealkylation in soman-inhibited cholinesterases proposed previously.
ESTHER : Saxena_1998_Biochemistry_37_15086
PubMedSearch : Saxena_1998_Biochemistry_37_15086
PubMedID: 9790671

Title : pH Dependence of Dealkylation in Soman-Inhibited Cholinesterases and Their Mutants -
Author(s) : Viragh C , Saxena A , Frazier DS , Kovach IM , Lockridge O , Doctor BP
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :247 , 1998
PubMedID:

Title : Differences in active site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase - Saxena_1997_Biochemistry_36_14642
Author(s) : Saxena A , Redman AM , Jiang X , Lockridge O , Doctor BP
Ref : Biochemistry , 36 :14642 , 1997
Abstract : Amino acid sequence alignments of cholinesterases revealed that 6 of 14 aromatic amino acid residues lining the active center gorge of acetylcholinesterase are replaced by aliphatic amino acid residues in butyrylcholinesterase. The Y337 (F330) in mammalian acetylcholinesterase, which is replaced by A328 in human butyrylcholinesterase, is implicated in the binding of ligands such as huperzine A, edrophonium, and acridines and one end of bisquaternary compounds such as BW284C51 and decamethonium. Y337 may sterically hinder the binding of phenothiazines such as ethopropazine, which contains a bulky exocyclic substitution. Inhibition studies of (-)-huperzine A with human butyrylcholinesterase mutants, where A328 (KI = 194.6 microM) was modified to either F (KI = 0.6 microM, as in Torpedo acetylcholinesterase) or Y (KI = 0.032 microM, as in mammalian acetylcholinesterase), confirmed previous observations made with acetylcholinesterase mutants that this residue is important for binding huperzine A. Inhibition studies of ethopropazine with butyrylcholinesterase mutants, where A328 (KI = 0.18 microM) was modified to either F (KI = 0.82 microM) or Y (KI = 0.28 microM), suggested that A328 was not solely responsible for the selectivity of ethopropazine. Volume calculations for the active site gorge showed that the poor inhibitory activity of ethopropazine toward acetylcholinesterase was due to the smaller dimension of the active site gorge which was unable to accommodate the bulky inhibitor molecule. The volume of the butyrylcholinesterase active site gorge is approximately 200 A3 larger than that of the acetylcholinesterase gorge, which allows the accommodation of ethopropazine in two different orientations as demonstrated by rigid-body refinement and molecular dynamics calculations.
ESTHER : Saxena_1997_Biochemistry_36_14642
PubMedSearch : Saxena_1997_Biochemistry_36_14642
PubMedID: 9398183

Title : Structure of glycan moieties responsible for the extended circulatory life time of fetal bovine serum acetylcholinesterase and equine serum butyrylcholinesterase - Saxena_1997_Biochemistry_36_7481
Author(s) : Saxena A , Raveh L , Ashani Y , Doctor BP
Ref : Biochemistry , 36 :7481 , 1997
Abstract : Cholinesterases are serine hydrolases that can potentially be used as pretreatment drugs for organophosphate toxicity, as drugs to alleviate succinylcholine-induced apnea, and as detoxification agents for environmental toxins such as heroin and cocaine. The successful application of serum-derived cholinesterases as bioscavengers stems from their relatively long residence time in the circulation. To better understand the relationship between carbohydrate structure and the stability of cholinesterases in circulation, we determined the monosaccharide composition, the distribution of various oligosaccharides, and the structure of the major asparagine-linked oligosaccharides units present in fetal bovine serum acetylcholinesterase and equine serum butyrylcholinesterase. Our findings indicate that 70-80% of the oligosaccharides in both enzymes are negatively charged. This finding together with the molar ratio of galactose to sialic acid clearly suggests that the beta-galactose residues are only partially capped with sialic acid, yet they displayed a long duration in circulation. The structures of the two major oligosaccharides from fetal bovine serum acetylcholinesterase and one major oligosaccharide from equine serum butyrylcholinesterase were determined. The three carbohydrate structures were of the biantennary complex type, but only the ones from fetal bovine serum acetylcholinesterase were fucosylated on the innermost N-acetylglucosamine residue of the core. Pharmacokinetic studies with native, desialylated, and deglycosylated forms of both enzymes indicate that the microheterogeneity in carbohydrate structure may be responsible, in part, for the multiphasic clearance of cholinesterases from the circulation of mice.
ESTHER : Saxena_1997_Biochemistry_36_7481
PubMedSearch : Saxena_1997_Biochemistry_36_7481
PubMedID: 9200697

Title : Mutant acetylcholinesterases as potential detoxification agents for organophosphate poisoning - Saxena_1997_Biochem.Pharmacol_54_269
Author(s) : Saxena A , Maxwell DM , Quinn DM , Radic Z , Taylor P , Doctor BP
Ref : Biochemical Pharmacology , 54 :269 , 1997
Abstract : It has been demonstrated that cholinesterases (ChEs) are an effective mode of pretreatment to prevent organophosphate (OP) toxicity in mice and rhesus monkeys. The efficacy of ChE as a bioscavenger of OP can be enhanced by combining enzyme pretreatment with oxime reactivation, since the scavenging capacity extends beyond a stoichiometric ratio of ChE to OP. Aging has proven to be a major barrier to achieving oxime reactivation of acetylcholinesterase (AChE) inhibited by the more potent OPs. To further increase the stoichiometry of OP to ChE required, we have sought AChE mutants that are more easily reactivated than wild-type enzyme. Substitution of glutamine for glutamate (E199) located at the amino-terminal to the active-site serine (S200) in Torpedo AChE generated an enzyme largely resistant to aging. Here we report the effect of the corresponding mutation on the rate of inhibition, reactivation by 1-(2-hydroxyiminomethyl-1-pyridinium)-1(4-carboxyaminopyridinium)- dimethyl ether hydrochloride (HI-6), and aging of mouse AChE inhibited by C(+)P(-)- and C(-)P(-)-epimers of soman. The E202 to Q mutation decreased the affinity of soman for AChE, slowed the reactivation of soman-inhibited AChE by HI-6, and decreased the aging of mutant AChE. These effects were more pronounced with C(-)P(-)-soman than with C(+)P(-)-soman. In vitro detoxification of soman and sarin by wild-type and E202Q AChE in the presence of 2 mM HI-6 showed that, E202Q AChE was 2-3 times more effective in detoxifying soman and sarin than wild-type AChE. These studies show that these recombinant DNA-derived AChEs are a great improvement over wild-type AChE as bioscavengers. They can be used to develop effective methods for the safe disposal of stored OP nerve agents and potential candidates for pre- or post-exposure treatment for OP toxicity.
ESTHER : Saxena_1997_Biochem.Pharmacol_54_269
PubMedSearch : Saxena_1997_Biochem.Pharmacol_54_269
PubMedID: 9271331

Title : Comparison of Acetylcholinesterase, Pyridostigmine, and HI-6 as Antidotes against Organophosphorus Compounds -
Author(s) : Maxwell DM , Brecht KM , Saxena A , Taylor P , Doctor BP
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :353 , 1995
PubMedID:

Title : Structural Analysis of the Asparagine-Linked Oligosaccharides of Cholinesterases -
Author(s) : Saxena A , Doctor BP
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :105 , 1995
PubMedID:

Title : Modulation of Catalysis and Inhibition of Fetal Bovine Serum Acetylcholinesterase by Monoclonal Antibodies -
Author(s) : Doctor BP , Gentry MK , Saxena A , Ashani Y
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :141 , 1995
PubMedID:

Title : Characterization of monoclonal antibodies that inhibit the catalytic activity of acetylcholinesterases - Gentry_1995_J.Neurochem_64_842
Author(s) : Gentry MK , Moorad DR , Hur RS , Saxena A , Ashani Y , Doctor BP
Ref : Journal of Neurochemistry , 64 :842 , 1995
Abstract : Monoclonal antibodies were generated against fetal bovine serum acetylcholinesterase and fetal bovine serum acetylcholinesterase inhibited by diisopropyl fluorophosphate or 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide. Six monoclonal antibodies inhibited 70 to > 98% of the catalytic activity of fetal bovine serum acetylcholinesterase. Inhibition of serum acetylcholinesterase from several mammalia by four monoclonal antibodies showed broad cross-reactivity. In all cases, monoclonal antibodies bound to the native form of acetylcholinesterases. None reacted with serum butyrylcholinesterases from various species. Although all monoclonal antibodies inhibited catalytic activity of acetylcholinesterases, the site of interaction with acetylcholinesterase appeared to differ for several antibodies. Two types of acetylcholinesterase:monoclonal antibody complexes were formed: one between tetrameric forms and another between catalytic subunits within the tetramer. Monoclonal antibodies that inhibited acetylcholinesterase activity at > 98% also considerably slowed binding of diisopropyl fluorophosphate and other organophosphorus compounds to the acetylcholinesterase:monoclonal antibody complex. Binding of these monoclonal antibodies to acetylcholinesterase influenced function of the enzyme's peripheral anionic site. None of the antibodies bound to the esteratic site of acetylcholinesterase. Monoclonal antibodies caused changes in catalytic activity of acetylcholinesterase by interaction at a site remote from the catalytic site, presumably at the entrance to the active site gorge.
ESTHER : Gentry_1995_J.Neurochem_64_842
PubMedSearch : Gentry_1995_J.Neurochem_64_842
PubMedID: 7830078

Title : Identification of amino acid residues involved in the binding of Huperzine A to cholinesterases - Saxena_1994_Protein.Sci_3_1770
Author(s) : Saxena A , Qian N , Kovach IM , Kozikowski AP , Pang YP , Vellom DC , Radic Z , Quinn DM , Taylor P , Doctor BP
Ref : Protein Science , 3 :1770 , 1994
Abstract : Huperzine A, a potential agent for therapy in Alzheimer's disease and for prophylaxis of organophosphate toxicity, has recently been characterized as a reversible inhibitor of cholinesterases. To examine the specificity of this novel compound in more detail, we have examined the interaction of the 2 stereoisomers of Huperzine A with cholinesterases and site-specific mutants that detail the involvement of specific amino acid residues. Inhibition of fetal bovine serum acetylcholinesterase by (-)-Huperzine A was 35-fold more potent than (+)-Huperzine A, with KI values of 6.2 nM and 210 nM, respectively. In addition, (-)-Huperzine A was 88-fold more potent in inhibiting Torpedo acetylcholinesterase than (+)-Huperzine A, with KI values of 0.25 microM and 22 microM, respectively. Far larger KI values that did not differ between the 2 stereoisomers were observed with horse and human serum butyrylcholinesterases. Mammalian acetylcholinesterase, Torpedo acetylcholinesterase, and mammalian butyrylcholinesterase can be distinguished by the amino acid Tyr, Phe, or Ala in the 330 position, respectively. Studies with mouse acetylcholinesterase mutants, Tyr 337 (330) Phe and Tyr 337 (330) Ala yielded a difference in reactivity that closely mimicked the native enzymes. In contrast, mutation of the conserved Glu 199 residue to Gln in Torpedo acetylcholinesterase produced only a 3-fold increase in KI value for the binding of Huperzine A.
ESTHER : Saxena_1994_Protein.Sci_3_1770
PubMedSearch : Saxena_1994_Protein.Sci_3_1770
PubMedID: 7849595

Title : The role of glutamate-199 in the aging of cholinesterase - Saxena_1993_Biochem.Biophys.Res.Commun_197_343
Author(s) : Saxena A , Doctor BP , Maxwell DM , Lenz DE , Radic Z , Taylor P
Ref : Biochemical & Biophysical Research Communications , 197 :343 , 1993
Abstract : Aging of organophosphate-conjugated acetylcholinesterase results from the loss of an alkoxy group with concomitant stabilization of the conjugate to spontaneous or nucleophile-induced deacylation. We have examined the kinetics of aging in a pinacolylmethylphosphonofluoridate (soman)-inhibited mutant enzyme in which the glutamate (E199) located at the amino-terminal to the active-site serine (S200) was converted to glutamine (Q). For wild type enzyme, the soman-acetylcholinesterase conjugate aged immediately, giving rise to a form of enzyme resistant to reactivation by oximes. In contrast, the E199Q mutant enzyme was largely resistant to aging and could be reactivated by oximes. Since the pH dependence for aging was not altered appreciably, the primary influence of the loss of charge appears to be on the intrinsic rate of aging. The negative charge on E199 likely imparts an inductive effect on the conjugated organophosphate to facilitate removal of the alkoxy group.
ESTHER : Saxena_1993_Biochem.Biophys.Res.Commun_197_343
PubMedSearch : Saxena_1993_Biochem.Biophys.Res.Commun_197_343
PubMedID: 7902714

Title : Immunochemical characterization of anti-acetylcholinesterase inhibitory monoclonal antibodies - Gentry_1993_Chem.Biol.Interact_87_227
Author(s) : Gentry MK , Saxena A , Ashani Y , Doctor BP
Ref : Chemico-Biological Interactions , 87 :227 , 1993
Abstract : Monoclonal antibodies (mAbs) were prepared against native or DFP-inhibited Torpedo californica acetylcholinesterase and native or DFP-, MEPQ-, and soman-inhibited fetal bovine serum acetylcholinesterase. The cross reactivity of these antibodies with acetylcholinesterases from various species and their ability to inhibit catalytic activity were determined. Eight antibodies were found to inhibit catalytic activity of either Torpedo or fetal bovine serum enzyme. In all cases the antibodies bound to the native form of the enzymes and in some cases even to the denatured form. None of the antibodies recognized human or horse serum butyrylcholinesterase. Sucrose density gradient centrifugation of enzyme-antibody complexes provided two types of profiles, one with multiple peaks, indicating numerous complexes between tetrameric forms of the enzyme, and the other with single peaks, demonstrating complex formation within the tetrameric form. Different antibodies appeared to interact with slightly different regions, but in all cases the binding encompassed the peripheral anionic site. Decrease in catalytic activity of the enzyme was most likely caused by conformational changes in the enzyme molecule resulting from interaction with these mAbs.
ESTHER : Gentry_1993_Chem.Biol.Interact_87_227
PubMedSearch : Gentry_1993_Chem.Biol.Interact_87_227
PubMedID: 7688272