Lenz DE

General

Full name : Lenz David E

First name : David E

Mail : USAMRICD\; 3100 Ricketts Point Rd\; Aberdeen Proving Ground\; MD 21010

Zip Code :

City :

Country : USA

Email : lenz@asia.apgea.army.mil

Phone : 410-436-2372

Fax : 410-436-1960

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References (43)

Title : Butyrylcholinesterase, a stereospecific in vivo bioscavenger against nerve agent intoxication - Cerasoli_2019_Biochem.Pharmacol__113670
Author(s) : Cerasoli DM , Armstrong SJ , Reeves TE , Hodgins SM , Kasten SA , Lee-Stubbs RB , Cadieux CL , Otto TC , Capacio BR , Lenz DE
Ref : Biochemical Pharmacology , :113670 , 2019
Abstract : Human butyrylcholinesterase (E.C. 3.1.1.8) purified from blood plasma has previously been shown to provide protection against up to five and a half times the median lethal dose of an organophosphorus nerve agent in several animal models. In this study the stoichiometric nature of the protection afforded by human butyrylcholinesterase against organophosphorus nerve agents was investigated in guinea pigs. Animals were administered human butyrylcholinesterase (26.15mg/kg identical with 308nmol/kg) by the intravascular or intramuscular route. Animals were subsequently dosed with either soman or VX in accordance with a stage-wise adaptive dose design to estimate the modified median lethal dose in treated animals. Human butyrylcholinesterase (308nmol/kg) increased the median lethal dose of soman from 154nmol/kg to 770nmol/kg. Comparing the molar ratio of agent molecules to enzyme active sites yielded a stoichiometric protective ratio of 2:1 for soman, likely related to the similar stereoselectivity the enzyme has compared to the toxic target, acetylcholinesterase. In contrast, human butyrylcholinesterase (308nmol/kg) increased the median lethal dose of VX from 30nmol/kg to 312nmol/kg, resulting in a stoichiometric protective ratio of only 1:1, suggesting a lack of stereoselectivity for this agent.
ESTHER : Cerasoli_2019_Biochem.Pharmacol__113670
PubMedSearch : Cerasoli_2019_Biochem.Pharmacol__113670
PubMedID: 31628910

Title : Radiolabelled soman binding to sera from Rats, Guinea Pigs and Monkeys - Lenz_2017_Toxicol.Lett_283_86
Author(s) : Lenz DE , Cerasoli DM , Maxwell DM
Ref : Toxicol Lett , 283 :86 , 2017
Abstract : Soman is a highly toxic organophosphorus chemical warfare compound that binds rapidly and irreversibility to a variety of serine active enzymes, i.e., butyryl- and acetyl-cholinesterases and carboxylesterase. The in vivo toxicity of soman has been reported to vary significantly in different animal species, such as rats and guinea pigs or non-human primates. This species variation makes it difficult to identify appropriate animal models for therapeutic drug development under the US Food and Drug Administration (FDA) Animal Rule. Since species variation in soman toxicity has been correlated with species variation in serum carboxylesterase, we undertook to determine if serum from guinea pigs, rats and non-human primates bound different levels of soman in vitro in the presence of equimolar concentrations of soman. Our results demonstrated that the amount of soman bound in the serum of rats was 4 uM, but essentially null in guinea pigs or non-human primates. The results strongly correlate with the presence or absence of carboxylesterase in the serum of animals and the difference in the toxic dose of soman in various species. Our results support prior suggestions that guinea pigs and non-human primates may be better animal models for the development of antidotes under the FDA Animal Rule.
ESTHER : Lenz_2017_Toxicol.Lett_283_86
PubMedSearch : Lenz_2017_Toxicol.Lett_283_86
PubMedID: 29155040

Title : Human butyrylcholinesterase efficacy against nerve agent exposure - Reed_2017_J.Biochem.Mol.Toxicol_31_
Author(s) : Reed BA , Sabourin CL , Lenz DE
Ref : J Biochem Mol Toxicol , 31 : , 2017
Abstract : Acetylcholinesterase is vital for normal operation of many processes in the body. Following exposure to organophosphorus (OP) nerve agents, death can ensue without immediate medical intervention. Current therapies mitigate the cholinergic crisis caused by nerve agents but do not fully prevent long-term health concerns, for example, brain damage following seizures. Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger being investigated as an antidote for OP nerve agent poisoning. HuBChE sequesters OP nerve agent in the bloodstream preventing the nerve agent from reaching critical target organ systems. HuBChE was effective when used as both a pre-treatment and as a post-exposure therapy. HuBChE has potential for use in both military settings and to protect civilian first responders in situations where nerve agent usage is suspected. We reviewed various animal models studies evaluating the efficacy of HuBChE against nerve agent exposure, pursuant to its submission for approval under the FDA Animal Rule.
ESTHER : Reed_2017_J.Biochem.Mol.Toxicol_31_
PubMedSearch : Reed_2017_J.Biochem.Mol.Toxicol_31_
PubMedID: 28225154

Title : Solubilization and humanization of paraoxonase-1 - Sarkar_2012_J.Lipids_2012_610937
Author(s) : Sarkar M , Harsch CK , Matic GT , Hoffman K , Norris JR, 3rd , Otto TC , Lenz DE , Cerasoli DM , Magliery TJ
Ref : J Lipids , 2012 :610937 , 2012
Abstract : Paraoxonase-1 (PON1) is a serum protein, the activity of which is related to susceptibility to cardiovascular disease and intoxication by organophosphorus (OP) compounds. It may also be involved in innate immunity, and it is a possible lead molecule in the development of a catalytic bioscavenger of OP pesticides and nerve agents. Human PON1 expressed in E. coli is mostly found in the insoluble fraction, which motivated the engineering of soluble variants, such as G2E6, with more than 50 mutations from huPON1. We examined the effect on the solubility, activity, and stability of three sets of mutations designed to solubilize huPON1 with fewer overall changes: deletion of the N-terminal leader, polar mutations in the putative HDL binding site, and selection of the subset of residues that became more polar in going from huPON1 to G2E6. All three sets of mutations increase the solubility of huPON1; the HDL-binding mutant has the largest effect on solubility, but it also decreases the activity and stability the most. Based on the G2E6 polar mutations, we "humanized" an engineered variant of PON1 with high activity against cyclosarin (GF) and found that it was still very active against GF with much greater similarity to the human sequence.
ESTHER : Sarkar_2012_J.Lipids_2012_610937
PubMedSearch : Sarkar_2012_J.Lipids_2012_610937
PubMedID: 22720164

Title : Post-exposure therapy with human butyrylcholinesterase following percutaneous VX challenge in guinea pigs - Mumford_2011_Clin.Toxicol.(Phila)_49_287
Author(s) : Rice H , M EP , Lenz DE , Cerasoli DM
Ref : Clinical Toxicology (Phila) , 49 :287 , 2011
Abstract : CONTEXT: Human butyrylcholinesterase (huBuChE) has potential utility as a post-exposure therapy following percutaneous nerve agent poisoning as there is a slower absorption of agent by this route and hence a later onset of poisoning. METHODS. We used surgically implanted radiotelemetry devices to monitor heart rate, EEG, body temperature and locomotor activity in guinea pigs challenged with VX via the percutaneous route. RESULTS. Treatment with huBuChE (24.2 mg/kg, i.m.) at 30 or 120 min following percutaneous VX (~2.5 x LD(50)) protected 9 out of 10 animals from lethality. When i.m. huBuChE administration was delayed until the onset of observable signs of systemic cholinergic poisoning, only one out of six animals survived to 7 days. Survival increased to 50% when the same dose of huBuChE was given intravenously at the onset of signs of poisoning. This dose represents approximately 1/10th the stoichiometric equivalent of the dose of VX administered (0.74 mg/kg). Intramuscular administration of huBuChE (24.2 mg/kg) alone did not produce any changes in heart rate, brain electrical activity, temperature or locomotion compared to saline control. Survival following VX and huBuChE treatment was associated with minimal incapacitation and observable signs of poisoning, and the mitigation or prevention of detrimental physiological changes (e.g. seizure, bradycardia and hypothermia) observed in VX + saline-treated animals. At 7 days, cholinesterase activity in the erythrocytes and most brain areas of guinea pigs that received huBuChE at either 18 h prior to or 30 min following VX was not significantly different from that of naive, weight-matched control animals. CONCLUSION. Percutaneous VX poisoning was successfully treated using post-exposure therapy with huBuChE bioscavenger. The opportunity for post-exposure treatment may have particular relevance in civilian settings, and this is a promising indication for the use of huBuChE.
ESTHER : Mumford_2011_Clin.Toxicol.(Phila)_49_287
PubMedSearch : Mumford_2011_Clin.Toxicol.(Phila)_49_287
PubMedID: 21563904

Title : Comparison of human and guinea pig acetylcholinesterase sequences and rates of oxime-assisted reactivation - Cadieux_2010_Chem.Biol.Interact_187_229
Author(s) : Cadieux CL , Broomfield CA , Kirkpatrick MG , Kazanski ME , Lenz DE , Cerasoli DM
Ref : Chemico-Biological Interactions , 187 :229 , 2010
Abstract : Poisoning via organophosphorus (OP) nerve agents occurs when the OP binds and inhibits the enzyme acetylcholinesterase (AChE). This enzyme is responsible for the metabolism of the neurotransmitter acetylcholine (ACh) which transmits signals between nerves and several key somatic regions. When AChE is inhibited, the signal initiated by ACh is not properly terminated. Excessive levels of ACh result in a cholinergic crisis, and in severe cases can lead to death. Current treatments for OP poisoning involve the administration of atropine, which blocks ACh receptors, and oximes, which reactivate AChE after inhibition. Efforts to improve the safety, efficacy, and broad spectrum utility of these treatments are ongoing and usually require the use of appropriate animal model systems. For OP poisoning, the guinea pig (Cavia porcellus) is a commonly used animal model because guinea pigs more closely mirror primate susceptibility to OP poisoning than do other animals such as rats and mice. This is most likely because among rodents and other small mammals, guinea pigs have a very low relative concentration of serum carboxylesterase, an enzyme known to bind OPs in vitro and to act as an endogenous bioscavenger in vivo. Although guinea pigs historically have been used to test OP poisoning therapies, it has been found recently that guinea pig AChE is substantially more resistant to oxime-mediated reactivation than human AChE. To examine the molecular basis for this difference, we reverse transcribed mRNA encoding guinea pig AChE, amplified the resulting cDNA, and sequenced this product. The nucleotide and deduced amino acid sequences of guinea pig AChE were then compared to the human version. Several amino acid differences were noted, and the predicted locations of these differences were mapped onto a structural model of human AChE. To examine directly how these differences affect oxime-mediated reactivation of AChE after inhibition by OPs, human and guinea pig red blood cell ghosts were prepared and used as sources of AChE, and the relative capacity of several different oximes to reactivate each OP-inhibited AChE were determined. The differences we report between human and guinea pig AChE raise additional concerns about the suitability of the guinea pig as an appropriate small animal model to approximate human responses to OP poisoning and therapies.
ESTHER : Cadieux_2010_Chem.Biol.Interact_187_229
PubMedSearch : Cadieux_2010_Chem.Biol.Interact_187_229
PubMedID: 20433814
Gene_locus related to this paper: cavpo-d3ye96

Title : Engineering human PON1 in an E. coli expression system - Suzuki_2010_Adv.Exp.Med.Biol_660_37
Author(s) : Suzuki SM , Stevens RC , Richter RJ , Cole TB , Park S , Otto TC , Cerasoli DM , Lenz DE , Furlong CE
Ref : Advances in Experimental Medicine & Biology , 660 :37 , 2010
Abstract : Expression and purification of recombinant human paraoxonase-1 (rHuPON1) from bacterial systems have proven elusive. Most systems for successful production of recombinant PON1 have relied on either eukaryotic expression in baculovirus or prokaryotic expression of synthetic, gene-shuffled rabbit-mouse-human PON1 hybrid molecules. We review here methods and protocols for the production of pure, native rHuPON1 using an E. coli expression system followed by conventional column chromatographic purification. The resulting rHuPON1 is stable, active, and capable of protecting PON1 knockout mice (PON1(-/-)) from exposure to high levels of the organophosphorus (OP) compound diazoxon. Bacterially-derived rHuPON1 can be produced in large quantities and lacks the glycosylation of eukaryotic systems that produces immunogenic complications when used as a therapeutic. The rHuPON1 should be useful for treating insecticide OP exposures and reducing risks of other diseases resulting from low PON1 status. The ease of mutagenesis in bacterial systems will also allow for the generation and screening of rHuPON1 variants with enhanced catalytic efficiencies against nerve agents and other OP compounds.
ESTHER : Suzuki_2010_Adv.Exp.Med.Biol_660_37
PubMedSearch : Suzuki_2010_Adv.Exp.Med.Biol_660_37
PubMedID: 20221869

Title : Efficacy and physiological effects of human butyrylcholinesterase as a post-exposure therapy against percutaneous poisoning by VX in the guinea-pig - Mumford_2010_Chem.Biol.Interact_187_304
Author(s) : Mumford H , Price ME , Cerasoli DM , Teschner W , Ehrlich H , Schwarz HP , Lenz DE
Ref : Chemico-Biological Interactions , 187 :304 , 2010
Abstract : The physiological effects of human plasma-derived butyrylcholinesterase (huBuChE) administration and its modulation of the effects of percutaneous VX challenge are poorly understood. Percutaneously administered nerve agents are more slowly absorbed than inhaled agents; consequently, signs of poisoning occur later, with a longer duration. Telemetry was used to monitor heart rate, EEG, temperature and activity in guinea-pigs. Treatment with huBuChE at 30 or 120 min following percutaneous VX challenge ( approximately 2.5 x LD(50)) provided 100% protection from lethality. When huBuChE administration was delayed until the onset of observable signs of poisoning only 1 out of 6 animals survived to the end of the experiment at 7 days. This study adds to the body of evidence demonstrating the efficacy of huBuChE in animals by describing the successful therapeutic use of a protein bioscavenger as a post-exposure treatment against dermal exposure to VX up to 2h post-exposure. This study simultaneously used telemetric methods to show that the efficacy of huBuChE is linked to the prevention of detrimental physiological changes observed in control VX-treated animals. Post-exposure therapy is a promising additional indication for the concept of use of this material, and one that has particular relevance in a civilian exposure scenario.
ESTHER : Mumford_2010_Chem.Biol.Interact_187_304
PubMedSearch : Mumford_2010_Chem.Biol.Interact_187_304
PubMedID: 20176007

Title : Butyrylcholinesterase as a Therapeutic Drug for Protection against Percutaneous VX - Lenz_2010_Chem.Biol.Interact_187_249
Author(s) : Lenz DE , Clarkson ED , Schulz SM , Cerasoli DM
Ref : Chemico-Biological Interactions , 187 :249 , 2010
Abstract : The administration of purified human plasma-derived butyrylcholinesterase (HuBCHE) as a pretreatment has been demonstrated to enhance survival and protect against decreased cognitive function after exposure to organophosphorus poisons (OPs). Based on efficacy data obtained with guinea pigs and non-human primates and the lack of behavioral side effects, plasma-derived HuBCHE has been granted investigational new drug status by the US Food and Drug Administration. The recent availability of a recombinant form of HuBCHE (rHuBCHE) from the milk of transgenic goats has now allowed us to determine the pharmacokinetics of that material in guinea pigs and use it as a therapy following exposure to the VX. The rHuBCHE was expressed as a dimer and following intramuscular (i.m.) administration had more a rapid adsorption and clearance profile in guinea pigs than the plasma-derived material. Based on those data, we administered rHuBCHE i.m. 1h after a percutaneous exposure of guinea pigs to either 2xLD(50) or 5xLD(50) of VX. Post-exposure therapy with rHuBCHE provided improved survival at both challenge levels, 90% and 33% respectively versus 20% or 0% respectively for animals that did not receive therapy. These studies showed that BCHE can be efficacious as a therapy against percutaneous exposure to VX.
ESTHER : Lenz_2010_Chem.Biol.Interact_187_249
PubMedSearch : Lenz_2010_Chem.Biol.Interact_187_249
PubMedID: 20513442

Title : Plant-derived human butyrylcholinesterase, but not an organophosphorous-compound hydrolyzing variant thereof, protects rodents against nerve agents - Geyer_2010_Proc.Natl.Acad.Sci.U.S.A_107_20251
Author(s) : Geyer BC , Kannan L , Garnaud PE , Broomfield CA , Cadieux CL , Cherni I , Hodgins SM , Kasten SA , Kelley K , Kilbourne J , Oliver ZP , Otto TC , Puffenberger I , Reeves TE , Robbins N, 2nd , Woods RR , Soreq H , Lenz DE , Cerasoli DM , Mor TS
Ref : Proc Natl Acad Sci U S A , 107 :20251 , 2010
Abstract : The concept of using cholinesterase bioscavengers for prophylaxis against organophosphorous nerve agents and pesticides has progressed from the bench to clinical trial. However, the supply of the native human proteins is either limited (e.g., plasma-derived butyrylcholinesterase and erythrocytic acetylcholinesterase) or nonexisting (synaptic acetylcholinesterase). Here we identify a unique form of recombinant human butyrylcholinesterase that mimics the native enzyme assembly into tetramers; this form provides extended effective pharmacokinetics that is significantly enhanced by polyethylene glycol conjugation. We further demonstrate that this enzyme (but not a G117H/E197Q organophosphorus acid anhydride hydrolase catalytic variant) can prevent morbidity and mortality associated with organophosphorous nerve agent and pesticide exposure of animal subjects of two model species.
ESTHER : Geyer_2010_Proc.Natl.Acad.Sci.U.S.A_107_20251
PubMedSearch : Geyer_2010_Proc.Natl.Acad.Sci.U.S.A_107_20251
PubMedID: 21059932

Title : Purification and characterization of functional human paraoxonase-1 expressed in Trichoplusia ni larvae - Otto_2010_Chem.Biol.Interact_187_388
Author(s) : Otto TC , Kasten SA , Kovaleva E , Liu Z , Buchman G , Tolosa M , Davis D , Smith JR , Balcerzak R , Lenz DE , Cerasoli DM
Ref : Chemico-Biological Interactions , 187 :388 , 2010
Abstract : Human serum paraoxonase-1 (HuPON1) is difficult to either purify from plasma or functionally express in high yield from recombinant sources. Here, we describe the characterization of functional HuPON1 expressed and purified from Trichoplusia ni (T. ni) larvae infected with an orally active form of baculovirus. SDS-PAGE and anti-HuPON1 Western blot analyses yielded only three bands of approximately 41, 42, and 44 kDa. MALDI-TOF confirmed the identity of each of these bands as HuPON1 with greater than 95% confidence. These isoforms result from differential glycosylation of the enzyme as indicated by peptide mapping, mass analysis, and PNGase F deglycosylation experiments. Recombinant insect-produced HuPON1 hydrolyzed phenyl acetate, paraoxon, and the nerve agents GF, VX, and VR. The enzyme had dramatic stereoselectivity for the P+ isomers of VX and VR. T. ni larvae expressing HuPON1 were remarkably resistant to the pesticide chlorpyrifos. Together, these results demonstrate that the caterpillar of the T. ni moth can be used as an expression system to produce large quantities of functional recombinant HuPON1. Insect production of HuPON1 may provide a source for both in vitro enzymatic and crystallographic studies and in vivo stability and anti-nerve agent efficacy testing.
ESTHER : Otto_2010_Chem.Biol.Interact_187_388
PubMedSearch : Otto_2010_Chem.Biol.Interact_187_388
PubMedID: 20176005

Title : Protection by pyridostigmine bromide of marmoset hemi-diaphragm acetylcholinesterase activity after soman exposure - Haigh_2010_Chem.Biol.Interact_187_416
Author(s) : Haigh JR , Adler M , Apland JP , Deshpande SS , Barham CB , Desmond P , Koplovitz I , Lenz DE , Gordon RK
Ref : Chemico-Biological Interactions , 187 :416 , 2010
Abstract : Pyridostigmine bromide (PB) was approved by the U.S. Food and Drug Administration (FDA) in 2003 as a pretreatment in humans against the lethal effects of the irreversible nerve agent soman (GD). Organophosphate (OP) chemical warfare agents such as GD exert their toxic effects by inhibiting acetylcholinesterase (AChE) from terminating the action of acetylcholine at postsynaptic sites in cholinergic nerve terminals (including crucial peripheral muscle such as diaphragm). As part of the post-marketing approval of PB, the FDA required (under 21CFR314, the "two animal rule") the study of a non-human primate model (the common marmoset Callithrix jacchus jacchus) to demonstrate increased survival against lethal GD poisoning, and protection of physiological hemi-diaphragm function after PB pretreatment and subsequent GD exposure. Marmosets (male and female) were placed in the following experimental groups: (i) control (saline pretreatment only), (ii) low dose PB (12.5 microg/kg), or (iii) high dose (39.5 microg/kg) PB. Thirty minutes after the PB dose, animals were challenged with either saline (control) or soman (GD, 45 microg/kg), followed 1 min later by atropine (2mg/kg) and 2-PAM (25mg/kg). After a further 16 min, animals were euthanized and the complete diaphragm removed; the right hemi-diaphragm was frozen immediately at -80 degrees C, and the left hemi-diaphragm was placed in a tissue bath for 4h (to allow for decarbamylation to occur), then frozen. AChE activities were determined using the automated WRAIR cholinesterase assay. Blood samples were collected for AChE activities prior to PB, before GD challenge, and after sacrifice. RBC-AChE was inhibited by approximately 18% and 50% at the low and high doses of PB, respectively, compared to control (baseline) activity. In the absence of PB pretreatment, the inhibition of RBC-AChE by GD was 98%. The recovery of hemi-diaphragm AChE activity after the 4h wash period (decarbamylation) was approximately 8% and 17%, at the low and high PB doses, respectively, compared with the baseline (control) AChE activity prior to PB pretreatment or soman exposure. The results suggest that PB pretreatment protects a critical fraction of AChE activity in the marmoset diaphragm, which is sufficient to allow the animal to breathe despite exposure to a dose of soman that is lethal in unprotected animals.
ESTHER : Haigh_2010_Chem.Biol.Interact_187_416
PubMedSearch : Haigh_2010_Chem.Biol.Interact_187_416
PubMedID: 20144889

Title : Computational Modeling of Human Paraoxonase 1: Preparation of Protein Models, Binding Studies, and Mechanistic Insights - Sanan_2010_J.Phys.Org.Chem_23_357
Author(s) : Sanan TT , Muthukrishnan S , Beck JM , Tao P , Hayes CJ , Otto TC , Cerasoli DM , Lenz DE , Hadad CM
Ref : J Phys Org Chem , 23 :357 , 2010
Abstract : The enzyme human paraoxonase 1 (huPON1) has demonstrated significant potential for use as a bioscavenger for treatment of exposure to organophosphorus (OP) nerve agents. Herein we report the development of protein models for the human isoform derived from a crystal structure of a chimeric version of the protein (pdb ID: 1V04) and a homology model derived from the related enzyme diisopropylfluorophosphatase (pdb ID: 1XHR). From these structural models, binding modes for OP substrates are predicted, and these poses are found to orient substrates in proximity to residues known to modulate specificity of the enzyme. Predictions are made with regard to the role that residues play in altering substrate binding and turnover, in particular with regard to the stereoselectivity of the enzyme, and the known differences in activity related to a natural polymorphism in the enzyme. Potential mechanisms of action of the protein for catalytic hydrolysis of OP substrates are also evaluated in light of the proposed binding modes.
ESTHER : Sanan_2010_J.Phys.Org.Chem_23_357
PubMedSearch : Sanan_2010_J.Phys.Org.Chem_23_357
PubMedID: 24077808

Title : DNA aptamers developed against a soman derivative cross-react with the methylphosphonic acid core but not with flanking hydrophobic groups - Bruno_2009_J.Mol.Recognit_22_197
Author(s) : Bruno JG , Carrillo MP , Cadieux CL , Lenz DE , Cerasoli DM , Phillips T
Ref : J Mol Recognit , 22 :197 , 2009
Abstract : Twelve rounds of systematic evolution of ligands by exponential enrichment (SELEX) were conducted against a magnetic bead conjugate of the para-aminophenylpinacolylmethylphosphonate (PAPMP) derivative of the organophosphorus (OP) nerve agent soman (GD). The goal was to develop DNA aptamers that could scavenge GD in vivo, thereby reducing or eliminating the toxic effects of this dangerous compound. Aptamers were sequenced and screened in peroxidase-based colorimetric plate assays after rounds 8 and 12 of SELEX. The aptamer candidate sequences exhibiting the highest affinity for the GD derivative from round 8 also reappeared in several clones from round 12. Each of the highest affinity PAPMP-binding aptamers also bound methylphosphonic acid (MPA). In addition, the aptamer with the highest overall affinity for PAPMP carried a sequence motif (TTTAGT) thought to bind MPA based on previously published data (J. Fluoresc 18: 867-876, 2008). This sequence motif was found in several other relatively high affinity PAPMP aptamer candidates as well. In studies with the nerve agent GD, pre-incubation of a large molar excess of aptamer candidates failed to protect human butyrylcholinesterase (BuChE) from inhibition. With the aid of three-dimensional molecular modeling of the GD derivative it appears that a hydrophilic cleft sandwiched between the pinacolyl group and the p-aminophenyl ring might channel nucleotide interactions to the phosphonate portion of the immobilized GD derivative. However, bona fide GD free in solution may be repulsed by the negative phosphate backbone of aptamers and rotate its phosphonate and fluorine moieties away from the aptamer to avoid being bound. Future attempts to develop aptamers to GD might benefit from immobilizing the pinacolyl group of bona fide GD to enhance exposure of the phosphonate and fluorine to the random DNA library.
ESTHER : Bruno_2009_J.Mol.Recognit_22_197
PubMedSearch : Bruno_2009_J.Mol.Recognit_22_197
PubMedID: 19051203

Title : Dramatic differences in organophosphorus hydrolase activity between human and chimeric recombinant mammalian paraoxonase-1 enzymes - Otto_2009_Biochemistry_48_10416
Author(s) : Otto TC , Harsch CK , Yeung DT , Magliery TJ , Cerasoli DM , Lenz DE
Ref : Biochemistry , 48 :10416 , 2009
Abstract : Human serum paraoxonase-1 (HuPON1) has the capacity to hydrolyze aryl esters, lactones, oxidized phospholipids, and organophosphorus (OP) compounds. HuPON1 and bacterially expressed chimeric recombinant PON1s (G2E6 and G3C9) differ by multiple amino acids, none of which are in the putative enzyme active site. To address the importance of these amino acid differences, the abilities of HuPON1, G2E6, G3C9, and several variants to hydrolyze phenyl acetate, paraoxon, and V-type OP nerve agents were examined. HuPON1 and G2E6 have a 10-fold greater catalytic efficiency toward phenyl acetate than G3C9. In contrast, bacterial PON1s are better able to promote hydrolysis of paraoxon, whereas HuPON1 is considerably better at catalyzing the hydrolysis of nerve agents VX and VR. These studies demonstrate that mutations distant from the active site of PON1 have large and unpredictable effects on the substrate specificities and possibly the hydrolytic mechanisms of HuPON1, G2E6, and G3C9. The replacement of residue H115 in the putative active site with tryptophan (H115W) has highly disparate effects on HuPON1 and G2E6. In HuPON1, variant H115W loses the ability to hydrolyze VR but has improved activity toward paraoxon and VX. The H115W variant of G2E6 has paraoxonase activity similar to that of wild-type G2E6, modest activity with phenyl acetate and VR, and enhanced VX hydrolysis. VR inhibits H115W HuPON1 competitively when paraoxon is the substrate and noncompetitively when VX is the substrate. We have identified the first variant of HuPON1, H115W, that displays significantly enhanced catalytic activity against an authentic V-type nerve agent.
ESTHER : Otto_2009_Biochemistry_48_10416
PubMedSearch : Otto_2009_Biochemistry_48_10416
PubMedID: 19764813

Title : In silico analyses of substrate interactions with human serum paraoxonase 1 - Hu_2009_Proteins_75_486
Author(s) : Hu X , Jiang X , Lenz DE , Cerasoli DM , Wallqvist A
Ref : Proteins , 75 :486 , 2009
Abstract : Human paraoxonase (HuPON1) is a serum enzyme that exhibits a broad spectrum of hydrolytic activities, including the hydrolysis of various organophosphates, esters, and recently identified lactone substrates. Despite intensive site-directed mutagenesis and other biological studies, the structural basis for the specificity of substrate interactions of HuPON1 remains elusive. In this study, we apply homology modeling, docking, and molecular dynamic (MD) simulations to probe the binding interactions of HuPON1 with representative substrates. The results suggest that the active site of HuPON1 is characterized by two distinct binding regions: the hydrophobic binding site for arylesters/lactones, and the paraoxon binding site for phosphotriesters. The unique binding modes proposed for each type of substrate reveal a number of key residues governing substrate specificity. The polymorphic residue R/Q192 interacts with the leaving group of paraoxon, suggesting it plays an important role in the proper positioning of this substrate in the active site. MD simulations of the optimal binding complexes show that residue Y71 undergoes an "open-closed" conformational change upon ligand binding, and forms strong interactions with substrates. Further binding free energy calculations and residual decomposition give a more refined molecular view of the energetics and origin of HuPON1/substrate interactions. These studies provide a theoretical model of substrate binding and specificity associated with wild type and mutant forms of HuPON1, which can be applied in the rational design of HuPON1 variants as bioscavengers with enhanced catalytic activity.
ESTHER : Hu_2009_Proteins_75_486
PubMedSearch : Hu_2009_Proteins_75_486
PubMedID: 18951406

Title : A collaborative endeavor to design cholinesterase-based catalytic scavengers against toxic organophosphorus esters - Masson_2008_Chem.Biol.Interact_175_273
Author(s) : Masson P , Nachon F , Broomfield CA , Lenz DE , Verdier L , Schopfer LM , Lockridge O
Ref : Chemico-Biological Interactions , 175 :273 , 2008
Abstract : Wild-type human butyrylcholinesterase (BuChE) has proven to be an efficient bioscavenger for protection against nerve agent toxicity. Human acetylcholinesterase (AChE) has a similar potential. A limitation to their usefulness is that both cholinesterases (ChEs) react stoichiometrically with organophosphosphorus (OP) esters. Because OPs can be regarded as pseudo-substrates for which the dephosphylation rate constant is almost zero, several strategies have been attempted to promote the dephosphylation reaction. Oxime-mediated reactivation of phosphylated ChEs generates a turnover, but it is too slow to make pseudo-catalytic scavengers of pharmacological interest. Alternatively, it was hypothesized that ChEs could be converted into OP hydrolases by using rational site-directed mutagenesis based upon the crystal structure of ChEs. The idea was to introduce a nucleophile into the oxyanion hole, at an appropriate position to promote hydrolysis of the phospho-serine bond via a base catalysis mechanism. Such mutants, if they showed the desired catalytic and pharmacokinetic properties, could be used as catalytic scavengers. The first mutant of human BuChE that was capable of hydrolyzing OPs was G117H. It had a slow rate. Crystallographic study of the G117H mutant showed that hydrolysis likely occurs by activation of a water molecule rather than direct nucleophilic attack by H117. Numerous BuChE mutants were made later, but none of them was better than the G117H mutant at hydrolyzing OPs, with the exception of soman. Soman aged too rapidly to be hydrolyzed by G117H. Hydrolysis was however accomplished with the double mutant G117H/E197Q, which did not age after phosphonylation with soman. Multiple mutations in the active center of human and Bungarus AChE led to enzymes displaying low catalytic activity towards OPs and unwanted kinetic complexities. A new generation of human AChE mutants has been designed with the assistance of molecular modelling and computational methods. According to the putative water-activation mechanism of G117H BChE, a new histidine/aspartate dyad was introduced into the active center of human AChE at the optimum location for hydrolysis of the OP adduct. Additional mutations were made for optimizing activity of the new dyad. It is anticipated that these new mutants will have OP hydrolase activity.
ESTHER : Masson_2008_Chem.Biol.Interact_175_273
PubMedSearch : Masson_2008_Chem.Biol.Interact_175_273
PubMedID: 18508040

Title : Substantially improved pharmacokinetics of recombinant human butyrylcholinesterase by fusion to human serum albumin - Huang_2008_BMC.Biotechnol_8_50
Author(s) : Huang YJ , Lundy PM , Lazaris A , Huang Y , Baldassarre H , Wang B , Turcotte C , Cote M , Bellemare A , Bilodeau AS , Brouillard S , Touati M , Herskovits P , Begin I , Neveu N , Brochu E , Pierson J , Hockley DK , Cerasoli DM , Lenz DE , Wilgus H , Karatzas CN , Langermann S
Ref : BMC Biotechnol , 8 :50 , 2008
Abstract : BACKGROUND: Human butyrylcholinesterase (huBChE) has been shown to be an effective antidote against multiple LD50 of organophosphorus compounds. A prerequisite for such use of huBChE is a prolonged circulatory half-life. This study was undertaken to produce recombinant huBChE fused to human serum albumin (hSA) and characterize the fusion protein.
RESULTS: Secretion level of the fusion protein produced in vitro in BHK cells was approximately 30 mg/liter. Transgenic mice and goats generated with the fusion constructs expressed in their milk a bioactive protein at concentrations of 0.04-1.1 g/liter. BChE activity gel staining and a size exclusion chromatography (SEC)-HPLC revealed that the fusion protein consisted of predominant dimers and some monomers. The protein was confirmed to have expected molecular mass of approximately 150 kDa by Western blot. The purified fusion protein produced in vitro was injected intravenously into juvenile pigs for pharmacokinetic study. Analysis of a series of blood samples using the Ellman assay revealed a substantial enhancement of the plasma half-life of the fusion protein (approximately 32 h) when compared with a transgenically produced huBChE preparation containing >70% tetramer (approximately 3 h). In vitro nerve agent binding and inhibition experiments indicated that the fusion protein in the milk of transgenic mice had similar inhibition characteristics compared to human plasma BChE against the nerve agents tested. CONCLUSION: Both the pharmacokinetic study and the in vitro nerve agent binding and inhibition assay suggested that a fusion protein retaining both properties of huBChE and hSA is produced in vitro and in vivo. The production of the fusion protein in the milk of transgenic goats provided further evidence that sufficient quantities of BChE/hSA can be produced to serve as a cost-effective and reliable source of BChE for prophylaxis and post-exposure treatment.
ESTHER : Huang_2008_BMC.Biotechnol_8_50
PubMedSearch : Huang_2008_BMC.Biotechnol_8_50
PubMedID: 18485214

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 : A gas chromatographic-mass spectrometric approach to examining stereoselective interaction of human plasma proteins with soman - Yeung_2008_J.Anal.Toxicol_32_86
Author(s) : Yeung DT , Smith JR , Sweeney RE , Lenz DE , Cerasoli DM
Ref : J Anal Toxicol , 32 :86 , 2008
Abstract : The organophosphorus (OP) nerve agent soman (GD) contains two chiral centers (a carbon and a phosphorus atom), resulting in four stereoisomers (C+P+, C-P+, C+P-, and C-P-); the P- isomers exhibit a mammalian toxicity that is approximately 1000-fold greater than that of the P+ isomers. The capacity to assess the binding or hydrolysis of each of the four stereoisomers is an important tool in the development of enzymes with the potential to protect against GD intoxication. Using a gas chromatography-mass spectrometry-based approach, we have examined the capacity of plasma-derived human serum albumin, plasma-purified human butyrylcholinesterase, goat milk-derived recombinant human butyrylcholinesterase, and recombinant human paraoxonase 1 to interact with each of the four stereoisomers of GD in vitro at pH 7.4 and 25 degrees C. Under these experimental conditions, the butyrylcholinesterase samples were found to bind GD with a relative preference for the more toxic stereoisomers (C-P- > C+P- > C-P+ > C+P+), while human serum albumin and paraoxonase 1 interacted with GD with a relative preference for the less toxic isomers (C-P+/C+P+ > C+P-/C-P-). The results indicate that these human proteins exhibit distinct stereoselective interactions with GD. The approach described presents a means to rapidly assess substrate stereospecificity, supporting future efforts to develop more effective OP bioscavenger proteins.
ESTHER : Yeung_2008_J.Anal.Toxicol_32_86
PubMedSearch : Yeung_2008_J.Anal.Toxicol_32_86
PubMedID: 18269799

Title : Recombinant human butyrylcholinesterase from milk of transgenic animals to protect against organophosphate poisoning - Huang_2007_Proc.Natl.Acad.Sci.U.S.A_104_13603
Author(s) : Huang YJ , Huang Y , Baldassarre H , Wang B , Lazaris A , Leduc M , Bilodeau AS , Bellemare A , Cote M , Herskovits P , Touati M , Turcotte C , Valeanu L , Lemee N , Wilgus H , Begin I , Bhatia B , Rao K , Neveu N , Brochu E , Pierson J , Hockley DK , Cerasoli DM , Lenz DE , Karatzas CN , Langermann S
Ref : Proc Natl Acad Sci U S A , 104 :13603 , 2007
Abstract : Dangerous organophosphorus (OP) compounds have been used as insecticides in agriculture and in chemical warfare. Because exposure to OP could create a danger for humans in the future, butyrylcholinesterase (BChE) has been developed for prophylaxis to these chemicals. Because it is impractical to obtain sufficient quantities of plasma BChE to treat humans exposed to OP agents, the production of recombinant BChE (rBChE) in milk of transgenic animals was investigated. Transgenic mice and goats were generated with human BChE cDNA under control of the goat beta-casein promoter. Milk from transgenic animals contained 0.1-5 g/liter of active rBChE. The plasma half-life of PEGylated, goat-derived, purified rBChE in guinea pigs was 7-fold longer than non-PEGylated dimers. The rBChE from transgenic mice was inhibited by nerve agents at a 1:1 molar ratio. Transgenic goats produced active rBChE in milk sufficient for prophylaxis of humans at risk for exposure to OP agents.
ESTHER : Huang_2007_Proc.Natl.Acad.Sci.U.S.A_104_13603
PubMedSearch : Huang_2007_Proc.Natl.Acad.Sci.U.S.A_104_13603
PubMedID: 17660298

Title : Stoichiometric and catalytic scavengers as protection against nerve agent toxicity: a mini review - Lenz_2007_Toxicology_233_31
Author(s) : Lenz DE , Yeung D , Smith JR , Sweeney RE , Lumley LA , Cerasoli DM
Ref : Toxicology , 233 :31 , 2007
Abstract : Currently fielded treatments for nerve agent intoxication promote survival, but do not afford complete protection against either nerve agent-induced motor and cognitive deficits or neuronal pathology. The use of human plasma-derived butyrylcholinesterase (HuBuChE) to neutralize the toxic effects of nerve agents in vivo has been shown to both aid survival and protect against decreased cognitive function after nerve agent exposure. Recently, a commercially produced recombinant form of human butyrylcholinesterase (r-HuBuChE; PharmAthene Inc.) expressed in the milk of transgenic goats has become available. This material is biochemically similar to plasma-derived HuBuChE in in vitro assays. The pharmacokinetic characteristics of a polyethylene glycol coated (pegylated) form of r-HuBuChE were determined in guinea pigs; the enzyme was rapidly bioavailable with a half-life (t(1/2)) and pharmacokinetic profile that resembled that of plasma-derived huBuChE. Guinea pigs were injected with 140mg/kg (i.m.) of pegylated r-HuBuChE 18h prior to exposure (sc) to 5.5xLD(50) VX or soman. VX and soman were administered in a series of three injections of 1.5xLD(50), 2.0xLD(50), and 2.0xLD(50), respectively, with injections separated by 2h. Pretreatment with pegylated r-HuBuChE provided 100% survival against multiple lethal doses of VX and soman. Guinea pigs displayed no signs of nerve agent toxicity following exposure. Assessments of motor activity, coordination, and acquisition of spatial memory were performed for 2 weeks following nerve agent exposure. There were no measurable decreases in motor or cognitive function during this period. In contrast, animals receiving 1.5xLD(50) challenges of soman or VX and treated with standard atropine, 2-PAM, and diazepam therapy showed 50 and 100% survival, respectively, but exhibited marked decrements in motor function and, in the case of GD, impaired spatial memory acquisition. The advances in this field have resulted in the decision to select both the plasma-derived and the recombinant form of BuChE for advanced development and transition to clinical trials. Efforts have now been expanded to identify a catalytic protein capable of not only binding, but also rapidly hydrolyzing the standard threat nerve agents. Recent work has focused on paraoxonase-1 (PON1), a naturally occurring human serum enzyme with the capacity to catalyze the hydrolysis of nerve agents, albeit too slowly to afford dramatic protection. Using rational design, several amino acids involved in substrate binding have been identified and site-directed mutations have revealed that residue H115 plays an important role in binding. In addition, the stereospecificity of PON1 for the catalytic hydrolysis of soman has been examined. The enzyme exhibits a slight stereospecificity for the C+P+ isomer of soman, which is due more to preferential binding than to selective hydrolysis of this isomer. The results suggest that it may be possible to engineer a mutant form of PON1 with enhanced activity and stereospecificity for the most toxic nerve agent isoforms.
ESTHER : Lenz_2007_Toxicology_233_31
PubMedSearch : Lenz_2007_Toxicology_233_31
PubMedID: 17188793

Title : Direct detection of stereospecific soman hydrolysis by wild-type human serum paraoxonase - Yeung_2007_FEBS.J_274_1183
Author(s) : Yeung DT , Smith JR , Sweeney RE , Lenz DE , Cerasoli DM
Ref : Febs J , 274 :1183 , 2007
Abstract : Human serum paraoxonase 1 (HuPON1; EC 3.1.8.1) is a calcium-dependent six-fold beta-propeller enzyme that has been shown to hydrolyze an array of substrates, including organophosphorus (OP) chemical warfare nerve agents. Although recent efforts utilizing site-directed mutagenesis have demonstrated specific residues (such as Phe222 and His115) to be important in determining the specificity of OP substrate binding and hydrolysis, little effort has focused on the substrate stereospecificity of the enzyme; different stereoisomers of OPs can differ in their toxicity by several orders of magnitude. For example, the C+/-P- isomers of the chemical warfare agent soman (GD) are known to be more toxic by three orders of magnitude. In this study, the catalytic activity of HuPON1 towards each of the four chiral isomers of GD was measured simultaneously via chiral GC/MS. The catalytic efficiency (k(cat)/K(m)) of the wild-type enzyme for the various stereoisomers was determined by a simultaneous solution of hydrolysis kinetics for each isomer. Derived k(cat)/K(m) values ranged from 625 to 4130 mm(-1).min(-1), with isomers being hydrolyzed in the order of preference C+P+ > C-P+ > C+P- > C-P-. The results indicate that HuPON1 hydrolysis of GD is stereoselective; substrate stereospecificity should be considered in future efforts to enhance the OPase activity of this and other candidate bioscavenger enzymes.
ESTHER : Yeung_2007_FEBS.J_274_1183
PubMedSearch : Yeung_2007_FEBS.J_274_1183
PubMedID: 17286579

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 : 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 : Role of immunogen design in induction of soman-specific monoclonal antibodies - Johnson_2005_Immunol.Lett_96_121
Author(s) : Johnson JK , Cerasoli DM , Lenz DE
Ref : Immunol Lett , 96 :121 , 2005
Abstract : The study of monoclonal antibodies raised against defined hapten epitopes has been a useful approach to understanding antibody repertoire. The situation in which antibodies are raised against different epitopes of the same hapten but have some common recognition or binding features has been less frequently examined. To explore the latter situation, we have characterized three monoclonal antibodies previously raised against two structurally different epitopes of the same organophosphorus nerve agent hapten, pinacolymethyl phosphonofluoridate (soman). Two antibodies, BE2-IA10 (BE2) and CC1-IIA4 (CC1), raised against the hydrophobic pinacolyl motif of soman, bind exclusively to soman and not to any other organophosphorus nerve agents. We determined that these antibodies have the same heavy chain sequence, which they share with the unrelated antibodies MOPC 21 and H17-L19. While all these antibodies share the same heavy chain sequence, they each possess different light chain sequences. Binding studies revealed that each of these antibodies has a unique reactivity with a panel of structurally related ligands, suggesting that the light chains are critically important in determining specificity in these antibodies. The third antibody, #2.ID8.2, raised against the methyl phosphoryl portion of soman, has unique heavy and light chain sequences. This antibody binds to all the currently identified chemical warfare agents. Given that the presenting epitope used to induce #2.ID8.2 is common to sarin, soman, tabun and VX, the ability of this antibody to recognize each of these haptens versus the inability of BE2 or CC1 to do so demonstrates the important role that immunogen design can play in the specificity of an antibody response.
ESTHER : Johnson_2005_Immunol.Lett_96_121
PubMedSearch : Johnson_2005_Immunol.Lett_96_121
PubMedID: 15585315

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 : Analysis of active-site amino-acid residues of human serum paraoxonase using competitive substrates - Yeung_2005_FEBS.J_272_2225
Author(s) : Yeung DT , Lenz DE , Cerasoli DM
Ref : Febs J , 272 :2225 , 2005
Abstract : Serum paraoxonase (PON1) is a calcium-dependent six-fold beta-propeller protein structurally similar to the di-isopropylfluorophosphatase (DFPase) found in the squid Loligo vulgaris. Human serum paraoxonase (HuPON1) has been shown to hydrolyze an array of substrates even though relatively little is known about its physiological role(s) or its catalytic mechanism. Through site-directed mutagenesis studies, designed from a DFPase-like homology model, and from a crystal structure of a hybrid PON1 molecule, amino-acid residues essential for enzyme function, including H115 and F222, have been identified. It was shown previously that, when H115 is replaced with tryptophan, the resulting enzyme hydrolyzes paraoxon but not phenyl acetate. This study shows that, when present simultaneously, phenyl acetate competitively inhibits paraoxon hydrolysis by H115W. Conversely, when F222 is replaced with tyrosine, mutant F222Y can hydrolyze phenyl acetate but not paraoxon. The presence of DFP, an inhibitor of both arylesterase and paraoxonase activities of wild-type HuPON1 (mean Ki=0.48+/-0.15 mM), has no effect on the ability of F222Y to catalyze the hydrolysis of phenyl acetate, suggesting that the F222Y mutant is unable to bind DFP. Together, the results suggest that, in wild-type HuPON1, H115 and F222 are important in determining substrate binding and specificity, but are not likely to be directly involved in substrate hydrolysis.
ESTHER : Yeung_2005_FEBS.J_272_2225
PubMedSearch : Yeung_2005_FEBS.J_272_2225
PubMedID: 15853807

Title : Oral administration of pyridostigmine bromide and huperzine A protects human whole blood cholinesterases from ex vivo exposure to soman - Gordon_2005_Chem.Biol.Interact_157-158_239
Author(s) : Gordon RK , Haigh JR , Garcia GE , Feaster SR , Riel MA , Lenz DE , Aisen PS , Doctor BP
Ref : Chemico-Biological Interactions , 157-158 :239 , 2005
Abstract : Cholinesterases (ChEs) are classified as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) according to their substrate specificity and sensitivity to selected inhibitors. The activities of AChE in red blood cells (RBC-AChE) and BChE in serum can be used as potential biomarkers of suppressed and/or heightened activity in the central and peripheral nervous systems. Exposure to organophosphate (OP) chemical warfare agents (CWAs), pesticides, anesthetics, and a variety of drugs such as cocaine, as well as some neurodegenerative and liver disease states, selectively reduces AChE or BChE activity. In humans, the toxicity of pesticides is well documented. Therefore, blood cholinesterase activity can be exploited as a tool for confirming exposure to these agents and possible treatments. Current assays for measurement of RBC-AChE and serum BChE require several labor-intensive processing steps, suffer from wide statistical variation, and there is no inter-laboratory conversion between methods. These methods, which determine only the serum BChE or RBC-AChE but not both, include the Ellman, radiometric, and deltapH (modified Michel) methods. In contrast, the Walter Reed Army Institute of Research Whole Blood (WRAIR WB, US Patent #6,746,850) cholinesterase assay rapidly determines the activity of both AChE and BChE in unprocessed (uncentrifuged) whole blood, uses a minimally invasive blood sampling technique (e.g., blood from a finger prick), and is semi-automated for high-throughput using the Biomek 2000 robotic system. To date, the WRAIR whole blood assay was used to measure AChE and BChE activities in human blood from volunteers in FDA clinical trials. In the first FDA study, 24 human subjects were given either 30 mg PB orally (n = 19) or placebo (n = 5). Blood samples were obtained pre-dosing and 2.5, 5, 8, and 24 h post-dosing. The samples were analyzed for AChE and BChE activity using the WRAIR WB robotic system, and for PB concentration by HPLC. We found that maximal inhibition of AChE (26.2%) and concentration of PB (17.1 ng/mL) occurred at 2.5 h post-PB dosing. AChE activity returned to almost 100% of pre-dose values by 6 h. A dose-dependent linear correlation was found between the amount of PB measured in the blood and the inhibition of AChE. Following soman (GD) exposure, recovered AChE activity was similar to levels that were reversibly protected by the PB administration. Therefore, the WRAIR ChE WB data clearly supports the conclusion that PB is an effective pre-treatment drug for nerve agent exposure (GD). In the second FDA human study for the treatment of Alzheimer's disease, the WRAIR ChE WB assay was used to determine the RBC-AChE and serum BChE profile of healthy elderly volunteers receiving Huperzine A. Huperzine A is a plant-derived reversible and selective AChE inhibitor compared to BChE, and is a more potent inhibitor of AChE than PB. Huperzine A is available as a nutraceutical, a natural supplement reported to improve memory, and has a variety of neuroprotective effects. Individuals received an increasing dose regimen of huperzine A (final dose 200 microg after 4 weeks), which produced more than 50% inhibition of RBC-AChE. Huperzine A was well tolerated by these patients at doses that sequestered more RBC-AChE than PB, and thus warrants further study as a prophylaxis for OP poisoning in addition to Alzheimer's therapy. Due to the documented use of OPs by terrorists and in warfare around the globe, Federal, State, and local authorities need a reliable, fast, inexpensive, and standard method for confirming such an assault in order to initiate appropriate containment, decontamination, and treatment measures. This assay is ideal for prescreening military personnel for atypical ChE activities that would preclude their deployment to areas of potential CWA exposure. The WRAIR WB ChE assay will fulfill the requirement for rapid and reliable monitoring of such exposure in military and civilian populations.
ESTHER : Gordon_2005_Chem.Biol.Interact_157-158_239
PubMedSearch : Gordon_2005_Chem.Biol.Interact_157-158_239
PubMedID: 16256090

Title : Structure\/function analyses of human serum paraoxonase (HuPON1) mutants designed from a DFPase-like homology model - Yeung_2004_Biochim.Biophys.Acta_1702_67
Author(s) : Yeung DT , Josse D , Nicholson JD , Khanal A , McAndrew CW , Bahnson BJ , Lenz DE , Cerasoli DM
Ref : Biochimica & Biophysica Acta , 1702 :67 , 2004
Abstract : Human serum paraoxonase (HuPON1) is a calcium-dependent enzyme that hydrolyzes esters, including organophosphates and lactones, and exhibits anti-atherogenic properties. A few amino acids have been shown to be essential for the enzyme's arylesterase and organophosphatase activities. Until very recently, a three-dimensional model was not available for HuPON1, so functional roles have not been assigned to those residues. Based on sequence-structure alignment studies, we have folded the amino acid sequence of HuPON1 onto the sixfold beta-propeller structure of squid diisopropylfluorophosphatase (DFPase). We tested the validity of this homology model by circular dichroism (CD) spectroscopy and site-directed mutagenesis. Consistent with predictions from the homology model, CD data indicated that the structural composition of purified HuPON1 consists mainly of beta-sheets. Mutants of HuPON1 were assayed for enzymatic activity against phenyl acetate and paraoxon. Substitution of residues predicted to be important for substrate binding (L69, H134, F222, and C284), calcium ion coordination (D54, N168, N224, and D269), and catalytic mechanism of HuPON1 (H285) led to enzyme inactivation. Mutants F222Y and H115W exhibited substrate-binding selectivity towards phenyl acetate and paraoxon, respectively. The homology model presented here is very similar to the recently obtained PON1 crystal structure, and has allowed identification of several residues within the enzyme active site.
ESTHER : Yeung_2004_Biochim.Biophys.Acta_1702_67
PubMedSearch : Yeung_2004_Biochim.Biophys.Acta_1702_67
PubMedID: 15450851

Title : Development of an immunoassay for diagnosis of exposure to toxic organophosphorus compounds - Miller_2001_J.Appl.Toxicol_21 Suppl 1_S23
Author(s) : Miller JK , Lenz DE
Ref : J Appl Toxicol , 21 Suppl 1 :S23 , 2001
Abstract : Currently, diagnosis of exposure to toxic low-molecular-weight compounds is effected by the use of chromatographic techniques. Such an approach is limited by the need for expensive equipment and sample clean-up before carrying out the analysis. To overcome those drawbacks, we have been involved in the development of an immunoassay for diagnosis of exposure to toxic organophosphorus compounds such as pinacolylmethyl phosphonofluoridate (soman), which is a chemical warfare agent. Prior estimates suggested that it is necessary to be able to detect soman at a concentration below 2.5 x 10(-7) M. Using four previously developed monoclonal antibodies, an enzyme-linked immunosorbant assay (ELISA) was used to optimize assay conditions and identify the antibody with the highest apparent affinity. The minimum required assay time was 2.0-2.5 h with no loss in sensitivity. To determine the specificity of the highest affinity antibody, a competitive inhibition enzyme immunoassay (CIEIA) was performed with six structural analogs of soman. The IC50 values for these analogues were 5 x 10(-7) M for 4-nitrophenylpinacolylmethylphosphonate, 8 x 10(-7) M for dipinacolylmethylphosphonate, 2 x 10(-6) M for diisopropylmethylphosphonate, 3 x 10(-5) M for 4-nitrophenylmethyl(phenylphosphinate) and 6.5 x 10(-5) M for 4-nitrophenylethyl(phenyl)phosphinate. 4-Nitrophenyl-di(n-butyl)phosphinate did not inhibit binding. Those inhibitors with branched alkyl side-chains, similar to the soman molecule, were effective inhibitors. Compounds, which contained predominately aromatic groups, were poor inhibitors. We are continuing to probe the binding specificity of the monoclonal antibody to determine its utility in further assay development. Our present results suggest that the antibody chosen may have the appropriate specificity and affinity for immunodiagnosis of exposure to soman.
ESTHER : Miller_2001_J.Appl.Toxicol_21 Suppl 1_S23
PubMedSearch : Miller_2001_J.Appl.Toxicol_21 Suppl 1_S23
PubMedID: 11920916

Title : Protective action of the serine protease inhibitor N-tosyl-L-lysine chloromethyl ketone (TLCK) against acute soman poisoning - Cowan_2001_J.Appl.Toxicol_21_293
Author(s) : Cowan FM , Broomfield CA , Lenz DE , Shih TM
Ref : J Appl Toxicol , 21 :293 , 2001
Abstract : Soman-poisoned rats display cholinergic crisis, a systemic mast cell degranulation characteristic of anaphylactic reactions and an excitotoxin-like sequential seizure and neuronal degeneration. The protection of guinea pigs from soman lethality by prophylactic administration of the serine protease inhibitor suramin suggests a possible proteolytic component in soman poisoning. The present study tested the effect of N-tosyl-L-lysine chloromethyl ketone (TLCK), an inhibitor of trypsin-like serine proteases, on soman-induced toxic signs (convulsions, righting reflex) and survival time. Nine control guinea pigs receiving 2 x LD(50) (56 microg kg(-1), s.c.) of soman immediately followed by a therapeutic dose of atropine sulfate (17.4 mg kg(-1) i.m.) experienced severe convulsions, and 8/9 lost the righting reflex. Six of these nine animals expired within 65 min; the three remaining animals survived 24 h to termination of the experiment. When a second group of animals were given TLCK (12 mg kg(-1), i.p.) 30 min prior to a 2 x LD(50) soman challenge and atropine-sulfate therapy, 5/9 experienced convulsions and only 3/9 lost the righting reflex. All nine animals survived beyond 4 h, with six surviving to 24 h. Compared with soman controls, prophylaxis with TLCK significantly prevented the loss of righting reflex (P = 0.05) and enhanced 4-h survival (P = 0.005). Although, convulsions were reduced and 24-h survival was improved in TLCK-treated animals, these results were not statistically significant. The protection from soman toxicity by chemically distinct protease inhibitors such as suramin and TLCK suggests a role for pathological proteolytic pathways in soman intoxication.
ESTHER : Cowan_2001_J.Appl.Toxicol_21_293
PubMedSearch : Cowan_2001_J.Appl.Toxicol_21_293
PubMedID: 11481662

Title : Organophosphate skin decontamination using immobilized enzymes - Gordon_1999_Chem.Biol.Interact_119-120_463
Author(s) : Gordon RK , Feaster SR , Russell AJ , LeJeune KE , Maxwell DM , Lenz DE , Ross M , Doctor BP
Ref : Chemico-Biological Interactions , 119-120 :463 , 1999
Abstract : We previously demonstrated that a combination of cholinesterase (ChE) pre-treatment with an oxime is an effective measure against soman and sarin. We describe here a novel approach for the preparation of covalently linked ChEs which are immobilized to a polyurethane matrix. Such preparation of ChE-sponges enhances the stability and usefulness of the enzymes in non-physiological environments. The ChE-sponges, which can be molded to any form, can effectively be used to remove and decontaminate organophosphates (OPs) from surfaces, biological (skin or wounds) or otherwise (clothing or sensitive medical equipment), or the environment. The ChE-sponges retained their catalytic activity under conditions of temperature, time, and drying where the native soluble enzyme would rapidly denature, and can be reused in conjunction with oximes many times. The ChE-sponge in the presence of oxime repeatedly detoxified OPs such as DFP or MEPQ. These developments in ChE technology have extended the applicability of OP scavengers from in vivo protection, to a variety of external detoxification and decontamination schemes. In addition to treatment of OP-contaminated soldiers, the ChE-sponge could protect medical personnel from secondary contamination while attending chemical casualties, and civilians exposed to pesticides or highly toxic nerve agents such as sarin.
ESTHER : Gordon_1999_Chem.Biol.Interact_119-120_463
PubMedSearch : Gordon_1999_Chem.Biol.Interact_119-120_463
PubMedID: 10421484

Title : Characterization of ChEs Immobilized on Polyurethane Foams -
Author(s) : Gordon RK , Feaster SR , Herron PC II , Lowe ER , LeJeune KE , Russell AJ , Lenz DE , Ross M , 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. :307 , 1998
PubMedID:

Title : Behavioral decrements persist in rhesus monkeys trained on a serial probe recognition task despite protection against soman lethality by butyrylcholinesterase - Castro_1994_Neurotoxicol.Teratol_16_145
Author(s) : Castro CA , Gresham VC , Finger AV , Maxwell DM , Solana RP , Lenz DE , Broomfield CA
Ref : Neurotoxicology & Teratology , 16 :145 , 1994
Abstract : Recently, it has been demonstrated that an exogenously administered enzyme such as butyrylcholinesterase (BCHE) can prevent death in rhesus monkeys exposed to multiple-lethal doses of the acetylcholinesterase inhibitor soman when the enzyme is given prior to soman exposure (3). We report that despite BCHE protecting against soman-induced lethality, behavioral effects are seen in these monkeys which last for at least 6 days as measured by performance on a serial probe recognition (SPR) task. Analyses of the serial position curves showed that performance was lower on the probe trials when the probe items were from the middle of the list than when the probe items were from the beginning or end of the list which were unaffected. BCHE given alone also produced behavioral effects, causing all animals not to respond on the probe trials until 8 h following BCHE administration. Taken together, these findings suggest that the BCHE is not completely binding all of the soman and that a concentration of soman which is capable of causing behavioral effects may be entering the CNS.
ESTHER : Castro_1994_Neurotoxicol.Teratol_16_145
PubMedSearch : Castro_1994_Neurotoxicol.Teratol_16_145
PubMedID: 8052188

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 : Catalytic antibodies hydrolysing organophosphorus esters - Brimfield_1993_Chem.Biol.Interact_87_95
Author(s) : Brimfield AA , Lenz DE , Maxwell DM , Broomfield CA
Ref : Chemico-Biological Interactions , 87 :95 , 1993
Abstract : Transition state stabilization is considered one means by which enzymes reduce free energy of activation. The transition state of phosphonic acid anhydrides acted on by OPA hydrolase is postulated to be pentacoordinate, which ordains either a square pyramid or a trigonal bipyramid structure. The advent of catalytic monoclonal antibodies has provided a system in which these assumptions can be tested. By immunizing mice with the protein conjugate of a trigonal bipyramid transition state analog, we have produced hybridomas secreting monoclonal antibodies which hydrolyze phosphonates. To date, activity has been shown toward pinacolyl methylphosphonofluoridic acid (soman). Preliminary results suggest that the antibody is an IgG2a with kappa light chain character. Our results support the trigonal bipyramidal transition state for this group of enzymes.
ESTHER : Brimfield_1993_Chem.Biol.Interact_87_95
PubMedSearch : Brimfield_1993_Chem.Biol.Interact_87_95
PubMedID: 8344006

Title : Protection by butyrylcholinesterase against organophosphorus poisoning in nonhuman primates - Broomfield_1991_J.Pharmacol.Exp.Ther_259_633
Author(s) : Broomfield CA , Maxwell DM , Solana RP , Castro CA , Finger AV , Lenz DE
Ref : Journal of Pharmacology & Experimental Therapeutics , 259 :633 , 1991
Abstract : Butyrylcholinesterase (BCHE) was examined as an in vivo exogenous scavenger for highly toxic organophosphorus (OP) poisons. Protection studies with equine BCHE were carried out in rhesus monkeys trained to perform a Serial Probe Recognition task. The pharmacokinetics of equine BCHE administered i.v. in rhesus monkeys revealed an elimination T1/2 of approximately 620 hr. Animals given 503 nmol of BCHE i.v. and then challenged with 220 to 260 nmol of soman (two LD50; a lethal dose in untreated animals) all survived with no clinical signs of OP poisoning. Serial Probe Recognition performance was depressed after enzyme administration and at 1 hr postsoman. However, all monkeys performed the task at base-line levels at 8 hr after soman and throughout the remainder of the experimental period. Two different monkeys each were given two doses of sarin, 183 nmol/dose (one LD50) after 460 nmol of BCHE. No signs were observed. A third group of monkeys given 253 or 340 nmol (three and four LD50, respectively) of soman after 460 nmol of BCHE required 1 mg/kg of atropine i.v. 10 min postsoman, but recovered completely within 24 hr. Our results indicate that BCHE has the required properties to function as a biological scavenger to protect against the pharmacological and behavioral toxicity of OP poisons.
ESTHER : Broomfield_1991_J.Pharmacol.Exp.Ther_259_633
PubMedSearch : Broomfield_1991_J.Pharmacol.Exp.Ther_259_633
PubMedID: 1941611

Title : Poster: Protection by butyry1cholinesterase against soman poisoning -
Author(s) : Lenz DE , Broomfield CA , Maxwell CA , Solana RP , Finger AV , Woodard CL
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :302 , 1991
PubMedID:

Title : Hepatic subcellular localization of cresylbenzodioxaphosphorin oxide (CBDP)-sensitive soman binding sites - Little_1990_Biochem.Pharmacol_40_1733
Author(s) : Little JS , Maxwell DM , Fox-Talbot MK , Brecht K , Lenz DE
Ref : Biochemical Pharmacology , 40 :1733 , 1990
Abstract : The toxicity of the organophosphorus poison soman (pinacolylmethylphosphonofluoridate) is attributable to its irreversible inhibition of the enzyme acetylcholinesterase. In addition, soman binds irreversibly to a number of noncholinesterase tissue binding sites which appear to be its major means of in vivo detoxification. This study was conducted to determine the hepatic subcellular localization of these sites. Subcellular fractions of liver from male Sprague-Dawley rats (200-250 g) were prepared by differential and isopycnic density gradient centrifugation. The binding of [14C]soman to these subcellular fractions was determined in the presence and absence of cresylbenzodioxaphosphorin oxide (CBDP), a compound that binds irreversibly to the noncholinesterase soman binding sites. Crude fractionation of liver homogenates into nuclear, mitochondrial, microsomal, and soluble fractions revealed that 78% of the total CBDP-sensitive binding activity was localized in the nuclear and microsomal fractions. Further purification of these fractions indicated that all of the homogenate binding activity could be accounted for in the purified microsomal fraction. When purified liver microsomes were solubilized and fractionated on linear sucrose gradients, 90% of the CBDP-sensitive soman binding activity cosedimented with carboxylesterase activity which suggests that these binding sites are carboxylesterase.
ESTHER : Little_1990_Biochem.Pharmacol_40_1733
PubMedSearch : Little_1990_Biochem.Pharmacol_40_1733
PubMedID: 2242010

Title : Effect of carboxylesterase inhibition on carbamate protection against soman toxicity - Maxwell_1988_J.Pharmacol.Exp.Ther_246_986
Author(s) : Maxwell DM , Brecht KM , Lenz DE , O'Neill BL
Ref : Journal of Pharmacology & Experimental Therapeutics , 246 :986 , 1988
Abstract : The ability of the carbamates pyridostigmine and physostigmine to protect against the lethal effects of soman, an extremely toxic anticholinesterase agent, was measured in rats, guinea pigs and rabbits. Pharmacologically equivalent doses of these carbamates that inhibited 70% of the blood acetylcholinesterase in each species were injected i.m. 25 min before s.c. injection with soman. Pretreatment with either carbamate, in combination with 17.4 mg/kg of atropine, produced protection against soman toxicity in all species. When protection was expressed as the ratio between the soman LD50 values in carbamate-protected animals and control animals, this protective ratio varied 3-fold between species (2.1-6.1 for pyridostigmine; 2.2-6.6 for physostigmine). When protection was expressed as the difference in the soman LD50 values between carbamate-protected animals and control animals, this protective difference was consistent among species (126 +/- 19 micrograms/kg). Species variation in protective ratios was observed largely because the control LD50 values defining soman toxicity in unprotected animals varied among species (20 micrograms/kg in rabbits, 28 micrograms/kg in guinea pigs and 126 micrograms/kg in rats). The species variation of the soman LD50 values in control animals was eliminated by pretreating animals with cresylbenzodioxaphosphorin oxide, which reduced the species variation in soman detoxification. The LD50 values for soman in cresylbenzodioxaphosphorin oxide-treated animals (9.8-15.6 micrograms/kg) did not differ significantly between species. Similarly, protective ratios for carbamates against soman in cresylbenzodioxaphosphorin oxide-treated animals were also clustered in a narrow range (8.5-11.4 for pyridostigmine; 9.0-13.4 for physostigmine) that did not differ significantly, regardless of species or carbamate.(ABSTRACT TRUNCATED AT 250 WORDS)
ESTHER : Maxwell_1988_J.Pharmacol.Exp.Ther_246_986
PubMedSearch : Maxwell_1988_J.Pharmacol.Exp.Ther_246_986
PubMedID: 3418520

Title : Structural and stereochemical specificity of mouse monoclonal antibodies to the organophosphorous cholinesterase inhibitor soman - Brimfield_1985_Mol.Pharmacol_28_32
Author(s) : Brimfield AA , Hunter KW, Jr. , Lenz DE , Benschop HP , van Dijk C , De Jong LP
Ref : Molecular Pharmacology , 28 :32 , 1985
Abstract : To test the usefulness of immunotherapy in organophosphate poisoning, two mouse monoclonal antibodies were prepared to the chemical warfare agent soman. The antibodies bound reversibly to soman and afforded considerable protection to acetylcholinesterase in vitro. However, they were only marginally effective in preventing the consequences of soman poisoning in mice (these data have been published elsewhere). Since potential for immunotherapeutic usefulness resides in antibody affinity and specificity, we conducted experiments to define these parameters to enable us to maximize them in the production of later antibodies. Interaction of the antibodies (CC1 and BE2) in affinity-purified form with a series of soman analogs in a competitive inhibition enzyme immunoassay was used to assess the contribution to binding affinity of each functional group on the soman molecule. Neither antibody interacted with the -P = S analog of soman or methylphosphonic acid. A decrease in the number of methyl groups on the pinacolyl side chain reduced or eliminated binding with both antibodies while increasing the size of this group had a mixed result. The major metabolite of soman, its basic hydrolysis product, interacted weakly with BE2 and failed to interact with CC1. Alkyl ester group substitution at the fluorine position increased antibody binding up to the symmetrical dipinacolyl analog. Stereochemical specificity was determined by measuring the apparent decrease in the rate of inhibition of cholinesterases (acetylcholine acetylhydrolase, EC 3.1.1.7, or acylcholine acylhydrolase, EC 3.1.1.8) by pure soman stereoisomers in the presence of increasing concentrations of each antibody. CC1 demonstrated specificity that varied as C(+)P(+) less than C(-)P(+) less than C(-)P(-) less than C(+)P(-). Although affinities were much lower, BE2 also showed a preference for the more toxic P(-) isomers.
ESTHER : Brimfield_1985_Mol.Pharmacol_28_32
PubMedSearch : Brimfield_1985_Mol.Pharmacol_28_32
PubMedID: 4021995

Title : Inhibition of rat cerebrum acetylcholinesterase isoenzymes after acute administration of soman -
Author(s) : Lenz DE , Maxwell DM
Ref : Biochemical Pharmacology , 30 :1369 , 1981
PubMedID: 7271832