Cerasoli DM

General

Full name : Cerasoli Douglas M

First name : Douglas Mark

Mail : US Army Medical Research Instititute of Chemical Defense, Research Division - USAMRICD, 2900 Ricketts Point Road, 21010-5400 Aberdeen Proving Grounds

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Country : USA

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Website : \/\/www.tributes.com\/obituary\/show\/Douglas-Mark-Cerasoli-104901103

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References (64)

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 : Adeno-associated virus-mediated expression of human butyrylcholinesterase to treat organophosphate poisoning - Gupta_2019_PLoS.One_14_e0225188
Author(s) : Gupta V , Cadieux CL , McMenamin D , Medina-Jaszek CA , Arif M , Ahonkhai O , Wielechowski E , Taheri M , Che Y , Goode T , Limberis MP , Li M , Cerasoli DM , Tretiakova AP , Wilson JM
Ref : PLoS ONE , 14 :e0225188 , 2019
Abstract : Rare diseases defined by genetic mutations are classic targets for gene therapy. More recently, researchers expanded the use of gene therapy in non-clinical studies to infectious diseases through the delivery of vectorized antibodies to well-defined antigens. Here, we further extend the utility of gene therapy beyond the "accepted" indications to include organophosphate poisoning. There are no approved preventives for the multi-organ damage resulting from acute or chronic exposure to organophosphates. We show that a single intramuscular injection of adeno-associated virus vector produces peak expression (~0.5 mg/ml) of active human butyrylcholinesterase (hBChE) in mice serum within 3-4 weeks post-treatment. This expression is sustained for up to 140 days post-injection with no silencing. Sustained expression of hBChE provided dose-dependent protection against VX in male and female mice despite detectable antibodies to hBChE in some mice, thereby demonstrating that expression of hBChE in vivo in mouse muscle is an effective prophylactic against organophosphate poisoning.
ESTHER : Gupta_2019_PLoS.One_14_e0225188
PubMedSearch : Gupta_2019_PLoS.One_14_e0225188
PubMedID: 31765413

Title : Assessment of mouse strain differences in baseline esterase activities and toxic response to sarin - Matson_2018_Toxicology_410_10
Author(s) : Matson LM , Lee-Stubbs RB , Cadieux CL , Koenig JA , Ardinger CE , Chandler J , Johnson EA , Hoard-Fruchey HM , Shih TA , Cerasoli DM , McDonough JH
Ref : Toxicology , 410 :10 , 2018
Abstract : Genetics likely play a role in various responses to nerve agent (NA) exposure, as genetic background plays an important role in behavioral, neurological, and physiological responses. This study uses different mouse strains to identify if mouse strain differences in sarin exposure exist. In Experiment 1, basal levels of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CE) were measured in different strains of naive mice to account for potential pharmacokinetic determinants of individual differences. In Experiment 2, median lethal dose (MLD) levels were estimated in 8 inbred mouse strains following subcutaneous (s.c.) administration of sarin. Few strain or sex differences in esterase activity levels were observed, with the exception of erythrocyte AChE activity in the C57BL/6J strain. Both sex and strain differences in toxicity were observed, with the most resistant strains being the BALB/cByJ and FVB/NJ strains and the most sensitive strain being the DBA/2J strain. These findings can be expanded to explore pathways involved in NA response, which may provide an avenue to develop therapeutics for preventing and treating the damaging effects of NA exposure.
ESTHER : Matson_2018_Toxicology_410_10
PubMedSearch : Matson_2018_Toxicology_410_10
PubMedID: 30172647

Title : Purification, characterization, and N-glycosylation of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures - Corbin_2018_Biotechnol.Bioeng_115_1301
Author(s) : Corbin JM , Kailemia MJ , Cadieux CL , Alkanaimsh S , Karuppanan K , Rodriguez RL , Lebrilla CB , Cerasoli DM , McDonald KA , Nandi S
Ref : Biotechnol Bioeng , 115 :1301 , 2018
Abstract : Recombinant butyrylcholinesterase produced in a metabolically regulated transgenic rice cell culture (rrBChE) was purified to produce a highly pure (95%), active form of enzyme. The developed downstream process uses common manufacturing friendly operations including tangential flow filtration, anion-exchange chromatography, and affinity chromatography to obtain a process recovery of 42% active rrBChE. The purified rrBChE was then characterized to confirm its comparability to the native human form of the molecule (hBChE). The recombinant and native enzyme demonstrated comparable enzymatic behavior and had an identical amino acid sequence. However, rrBChE differs in that it contains plant-type complex N-glycans, including an alpha-1,3 linked core fucose, and a beta-1,2 xylose, and lacking a terminal sialic acid. Despite this difference, rrBChE is demonstrated to be an effective stoichiometric bioscavenger for five different organophosphorous nerve agents in vitro. Together, the efficient downstream processing scheme and functionality of rrBChE confirm its promise as a cost-effective alternative to hBChE for prophylactic and therapeutic use.
ESTHER : Corbin_2018_Biotechnol.Bioeng_115_1301
PubMedSearch : Corbin_2018_Biotechnol.Bioeng_115_1301
PubMedID: 29411865

Title : Evaluating Mice lacking Serum Carboxylesterase as a Behavioral Model for Nerve Agent Intoxication - Dunn_2018_Toxicol.Mech.Methods__1
Author(s) : Dunn EN , Ferrara-Bowens TM , Chachich ME , Honnold CL , Rothwell CC , Hoard-Fruchey HM , Lesyna CA , Johnson EA , Cerasoli DM , McDonough JH , Cadieux CL
Ref : Toxicol Mech Methods , :1 , 2018
Abstract : Mice and other rodents are typically utilized for chemical warfare nerve agent research. Rodents have large amounts of carboxylesterase in their blood, while humans do not. Carboxylesterase non-specifically binds to and detoxifies nerve agent. The presence of this natural bioscavenger makes mice and other rodents poor models for studies identifying therapeutics to treat humans exposed to nerve agents. To obviate this problem, a serum carboxylesterase knockout (Es1 KO) mouse was created. In this study, Es1 KO and wild type (WT) mice were assessed for differences in gene expression, nerve agent (soman; GD) median lethal dose (MLD) values, and behavior prior to and following nerve agent exposure. No expression differences were detected between Es1 KO and WT mice in more than 34,000 mouse genes tested. There was a significant difference between Es1 KO and WT mice in MLD values, as the MLD for GD-exposed WT mice was significantly higher than the MLD for GD-exposed Es1 KO mice. Behavioral assessments of Es1 KO and WT mice included an open field test, a zero maze, a Barnes maze, and a sucrose preference test (SPT). While sex differences were observed in various measures of these tests, overall, Es1 KO mice behaved similarly to WT mice. The two genotypes also showed virtually identical neuropathological changes following GD exposure. Es1 KO mice appear to have an enhanced susceptibility to GD toxicity while retaining all other behavioral and physiological responses to this nerve agent, making the Es1 KO mouse a more human-like model for nerve agent research.
ESTHER : Dunn_2018_Toxicol.Mech.Methods__1
PubMedSearch : Dunn_2018_Toxicol.Mech.Methods__1
PubMedID: 29768075
Gene_locus related to this paper: mouse-Ces1g

Title : The role of genetic background in susceptibility to chemical warfare nerve agents across rodent and non-human primate models - Matson_2017_Toxicology_393_51
Author(s) : Matson LM , McCarren HS , Cadieux CL , Cerasoli DM , McDonough JH
Ref : Toxicology , 393 :51 , 2017
Abstract : Genetics likely play a role in various responses to nerve agent exposure, as genetic background plays an important role in behavioral, neurological, and physiological responses to environmental stimuli. Mouse strains or selected lines can be used to identify susceptibility based on background genetic features to nerve agent exposure. Additional genetic techniques can then be used to identify mechanisms underlying resistance and sensitivity, with the ultimate goal of developing more effective and targeted therapies. Here, we discuss the available literature on strain and selected line differences in cholinesterase activity levels and response to nerve agent-induced toxicity and seizures. We also discuss the available cholinesterase and toxicity literature across different non-human primate species. The available data suggest that robust genetic differences exist in cholinesterase activity, nerve agent-induced toxicity, and chemical-induced seizures. Available cholinesterase data suggest that acetylcholinesterase activity differs across strains, but are limited by the paucity of carboxylesterase data in strains and selected lines. Toxicity and seizures, two outcomes of nerve agent exposure, have not been fully evaluated for genetic differences, and thus further studies are required to understand baseline strain and selected line differences.
ESTHER : Matson_2017_Toxicology_393_51
PubMedSearch : Matson_2017_Toxicology_393_51
PubMedID: 29113833

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 : Targeting of organophosphorus compound bioscavengers to the surface of red blood cells - McCranor_2016_Chem.Biol.Interact_259_205
Author(s) : McCranor BJ , Hofstetter CA , Olert MA , Moorad-Doctor D , Cerasoli DM , Garcia GE
Ref : Chemico-Biological Interactions , 259 :205 , 2016
Abstract : To develop a prophylactic for organophosphorus (OP) poisoning utilizing catalytic bioscavengers, the circulatory stability of the enzymes needs to be increased. One strategy for increasing the bioavailability of OP bioscavengers is to target them to the surface of red blood cells (RBCs). Given the circulatory lifespan of 120 days for human RBCs, this strategy has the potential for creating a persistent pool of bioscavenger. Here we report the development of fusion proteins with a single chain variable fragment (scFv) of Ter119, a molecule that associates with glycophorin A on the surface of RBCs, and the VIID11 variant of paraoxonase 1 (scFv-PON1). We show that scFv-PON1 variants expressed by Trichoplusia ni larvae are catalytically active and that one variant in particular can successfully bind to the surface of murine RBCs both in vitro and in vivo. This study represents a proof of concept for targeting catalytic bioscavengers to the surface of RBCs and is an early step in developing catalytic bioscavengers that can remain in circulation for an extended period of time.
ESTHER : McCranor_2016_Chem.Biol.Interact_259_205
PubMedSearch : McCranor_2016_Chem.Biol.Interact_259_205
PubMedID: 27163849

Title : Probing the Activity of a Non-Oxime Reactivator for Acetylcholinesterase Inhibited by Organophosphorus Nerve Agents - Cadieux_2016_Chem.Biol.Interact_259_133
Author(s) : Cadieux CL , Wang H , Zhang Y , Koenig JA , Shih TM , McDonough J , Koh J , Cerasoli DM
Ref : Chemico-Biological Interactions , 259 :133 , 2016
Abstract : Currently fielded treatments for nerve agent intoxication include atropine, an acetylcholine receptor antagonist, and pralidoxime (2PAM), a small molecule reactivator of acetylcholinesterase (AChE). 2PAM reactivates nerve agent-inhibited AChE via direct nucleophilic attack by the oxime moiety on the phosphorus center of the bound nerve agent. Due to a permanently charged pyridinium motif, 2PAM is not thought to cross the blood brain barrier and therefore cannot act directly in the neuronal junctions of the brain. In this study, ADOC, a non-permanently charged, non-oxime molecule initially identified using pesticide-inhibited AChE, was characterized in vitro against nerve agent-inhibited recombinant human AChE. The inhibitory and reactivation potentials of ADOC were determined with native AChE and AChE inhibited with tabun, sarin, soman, cyclosarin, VX, or VR and then compared to those of 2PAM. Several structural analogs of ADOC were used to probe the reactivation mechanism of the molecule. Finally, guinea pigs were used to examine the protective efficacy of the compound after exposure to sarin. The results of both in vitro and in vivo testing will be useful in the design of future small molecule reactivators.
ESTHER : Cadieux_2016_Chem.Biol.Interact_259_133
PubMedSearch : Cadieux_2016_Chem.Biol.Interact_259_133
PubMedID: 27062893

Title : A rationally designed mutant of plasma platelet-activating factor acetylhydrolase hydrolyzes the organophosphorus nerve agent soman - Kirby_2015_Biochim.Biophys.Acta_1854_1809
Author(s) : Kirby SD , Norris J , Sweeney R , Bahnson BJ , Cerasoli DM
Ref : Biochimica & Biophysica Acta , 1854 :1809 , 2015
Abstract : Organophosphorus compounds (OPs) such as sarin and soman are some of the most toxic chemicals synthesized by man. They exert toxic effects by inactivating acetylcholinesterase (AChE) and bind secondary target protein. Organophosphorus compounds are hemi-substrates for enzymes of the serine hydrolase superfamily. Enzymes can be engineered by amino acid substitution into OP-hydrolyzing variants (bioscavengers) and used as therapeutics. Some enzymes associated with lipoproteins, such as human plasma platelet-activating factor acetylhydrolase (pPAF-AH), are also inhibited by OPs; these proteins have largely been ignored for engineering purposes because of complex interfacial kinetics and a lack of structural data. We have expressed active human pPAF-AH in bacteria and previously solved the crystal structure of this enzyme with OP adducts. Using these structures as a guide, we created histidine mutations near the active site of pPAF-AH (F322H, W298H, L153H) in an attempt to generate novel OP-hydrolase activity. Wild-type pPAF-AH, L153H, and F322H have essentially no hydrolytic activity against the nerve agents tested. In contrast, the W298H mutant displayed novel somanase activity with a kcat of 5min-1 and a KM of 590muM at pH7.5. There was no selective preference for hydrolysis of any of the four soman stereoisomers.
ESTHER : Kirby_2015_Biochim.Biophys.Acta_1854_1809
PubMedSearch : Kirby_2015_Biochim.Biophys.Acta_1854_1809
PubMedID: 26343853
Gene_locus related to this paper: human-PLA2G7

Title : An in vitro and in vivo evaluation of the efficacy of recombinant human liver prolidase as a catalytic bioscavenger of chemical warfare nerve agents - Rezk_2015_Drug.Chem.Toxicol_38_37
Author(s) : Rezk PE , Zdenka P , Sabnekar P , Kajih T , Mata DG , Wrobel C , Cerasoli DM , Chilukuri N
Ref : Drug & Chemical Toxicology , 38 :37 , 2015
Abstract : In this study, we determined the ability of recombinant human liver prolidase to hydrolyze nerve agents in vitro and its ability to afford protection in vivo in mice. Using adenovirus containing the human liver prolidase gene, the enzyme was over expressed by 200- to 300-fold in mouse liver and purified to homogeneity by affinity and gel filtration chromatography. The purified enzyme hydrolyzed sarin, cyclosarin and soman with varying rates of hydrolysis. The most efficient hydrolysis was with sarin, followed by soman and by cyclosarin {apparent kcat/Km [(1.9 +/- 0.3), (1.7 +/- 0.2), and (0.45 +/- 0.04)] x 10(5 )M(-1 )min(-1), respectively}; VX and tabun were not hydrolyzed by the recombinant enzyme. The enzyme hydrolyzed P (+) isomers faster than the P (-) isomers. The ability of recombinant human liver prolidase to afford 24 hour survival against a cumulative dose of 2 x LD50 of each nerve agent was investigated in mice. Compared to mice injected with a control virus, mice injected with the prolidase expressing virus contained (29 +/- 7)-fold higher levels of the enzyme in their blood on day 5. Challenging these mice with two consecutive 1 x LD50 doses of sarin, cyclosarin, and soman resulted in the death of all animals within 5 to 8 min from nerve agent toxicity. In contrast, mice injected with the adenovirus expressing mouse butyrylcholinesterase, an enzyme which is known to afford protection in vivo, survived multiple 1 x LD50 challenges of these nerve agents and displayed no signs of toxicity. These results suggest that, while prolidase can hydrolyze certain G-type nerve agents in vitro, the enzyme does not offer 24 hour protection against a cumulative dose of 2 x LD50 of G-agents in mice in vivo.
ESTHER : Rezk_2015_Drug.Chem.Toxicol_38_37
PubMedSearch : Rezk_2015_Drug.Chem.Toxicol_38_37
PubMedID: 24641262

Title : Investigation of evolved paraoxonase-1 variants for prevention of organophosphorous pesticide compound intoxication - Mata_2014_J.Pharmacol.Exp.Ther_349_549
Author(s) : Mata DG , Rezk PE , Sabnekar P , Cerasoli DM , Chilukuri N
Ref : Journal of Pharmacology & Experimental Therapeutics , 349 :549 , 2014
Abstract : We investigated the ability of the engineered paraoxonase-1 variants G3C9, VII-D11, I-F11, and VII-D2 to afford protection against paraoxon intoxication. Paraoxon is the toxic metabolite of parathion, a common pesticide still in use in many developing countries. An in vitro investigation showed that VII-D11 is the most efficient variant at hydrolyzing paraoxon with a kcat/Km of 2.1 x 10(6) M(-1) min(-1) and 1.6 x 10(6) M(-1) min(-1) for the enzyme expressed via adenovirus infection of 293A cells and mice, respectively. Compared with the G3C9 parent scaffold, VII-D11 is 15- to 20-fold more efficacious at hydrolyzing paraoxon. Coinciding with these results, mice expressing VII-D11 in their blood survived and showed no symptoms against a cumulative 6.3 x LD50 dose of paraoxon, whereas mice expressing G3C9 experienced tremors and only 50% survival. We then determined whether VII-D11 can offer protection against paraoxon when present at substoichiometric concentrations. Mice containing varying concentrations of VII-D11 in their blood (0.2-4.1 mg/ml) were challenged with doses of paraoxon at fixed stoichiometric ratios that constitute up to a 10-fold molar excess of paraoxon to enzyme (1.4-27 x LD50 doses) and were assessed for tremors and mortality. Mice were afforded complete asymptomatic protection below a paraoxon-to-enzyme ratio of 8:1, whereas higher ratios produced tremors and/or mortality. VII-D11 in mouse blood coeluted with high-density lipoprotein, suggesting an association between the two entities. Collectively, these results demonstrate that VII-D11 is a promising candidate for development as a prophylactic catalytic bioscavenger against organophosphorous pesticide toxicity.
ESTHER : Mata_2014_J.Pharmacol.Exp.Ther_349_549
PubMedSearch : Mata_2014_J.Pharmacol.Exp.Ther_349_549
PubMedID: 24706983

Title : Chiral separation of G-type chemical warfare nerve agents via analytical supercritical fluid chromatography - Kasten_2014_Chirality_26_817
Author(s) : Kasten SA , Zulli S , Jones JL , Dephillipo T , Cerasoli DM
Ref : Chirality , 26 :817 , 2014
Abstract : Chemical warfare nerve agents (CWNAs) are extremely toxic organophosphorus compounds that contain a chiral phosphorus center. Undirected synthesis of G-type CWNAs produces stereoisomers of tabun, sarin, soman, and cyclosarin (GA, GB, GD, and GF, respectively). Analytical-scale methods were developed using a supercritical fluid chromatography (SFC) system in tandem with a mass spectrometer for the separation, quantitation, and isolation of individual stereoisomers of GA, GB, GD, and GF. Screening various chiral stationary phases (CSPs) for the capacity to provide full baseline separation of the CWNAs revealed that a Regis WhelkO1 (SS) column was capable of separating the enantiomers of GA, GB, and GF, with elution of the P(+) enantiomer preceding elution of the corresponding P(-) enantiomer; two WhelkO1 (SS) columns had to be connected in series to achieve complete baseline resolution. The four diastereomers of GD were also resolved using two tandem WhelkO1 (SS) columns, with complete baseline separation of the two P(+) epimers. A single WhelkO1 (RR) column with inverse stereochemistry resulted in baseline separation of the GD P(-) epimers. The analytical methods described can be scaled to allow isolation of individual stereoisomers to assist in screening and development of countermeasures to organophosphorus nerve agents.
ESTHER : Kasten_2014_Chirality_26_817
PubMedSearch : Kasten_2014_Chirality_26_817
PubMedID: 25298066

Title : Human paraoxonase double mutants hydrolyze V and G class organophosphorus nerve agents - Kirby_2013_Chem.Biol.Interact_203_181
Author(s) : Kirby SD , Norris JR , Richard Smith J , Bahnson BJ , Cerasoli DM
Ref : Chemico-Biological Interactions , 203 :181 , 2013
Abstract : Variants of human paraoxonase 1 (PON1) are being developed as catalytic bioscavengers for the organophosphorus chemical warfare agents (OP). It is preferable that the new PON1 variants have broad spectrum hydrolase activities to hydrolyze both G- and V-class OPs. H115W PON1 has shown improvements over wild type PON1 in its capacity to hydrolyze some OP compounds. We improved upon these activities either by substituting a tryptophan (F347W) near the putative active site residues for enhanced substrate binding or by reducing a bulky group (Y71A) at the periphery of the putative enzyme active site. When compared to H115W alone, we found that H115W/Y71A and H115W/F347W maintained VX catalytic efficiency but showed mixed results for the capacity to hydrolyze paraoxon. Testing our double mutants against racemic sarin, we observed reduced values of KM for H115W/F347W that modestly improved catalytic efficiency over wild type and H115W. Contrary to previous reports, we show that H115W can hydrolyze soman, and the double mutant H115W/Y71A is nearly 4-fold more efficient than H115W for paraoxon hydrolysis. We also observed modest stereoselectivity for hydrolysis of the P(-) stereoisomer of tabun by H115W/F347W. These data demonstrate enhancements made in PON1 for the purpose of developing an improved catalytic bioscavenger to protect cholinesterase against chemical warfare agents.
ESTHER : Kirby_2013_Chem.Biol.Interact_203_181
PubMedSearch : Kirby_2013_Chem.Biol.Interact_203_181
PubMedID: 23159884

Title : Assessing protection against OP pesticides and nerve agents provided by wild-type HuPON1 purified from Trichoplusia ni larvae or induced via adenoviral infection - Hodgins_2013_Chem.Biol.Interact_203_177
Author(s) : Hodgins SM , Kasten SA , Harrison J , Otto TC , Oliver ZP , Rezk P , Reeves TE , Chilukuri N , Cerasoli DM
Ref : Chemico-Biological Interactions , 203 :177 , 2013
Abstract : Human paraoxonase-1 (HuPON1) has been proposed as a catalytic bioscavenger of organophosphorus (OP) pesticides and nerve agents. We assessed the potential of this enzyme to protect against OP poisoning using two different paradigms. First, recombinant HuPON1 purified from cabbage loopers (iPON1; Trichoplusia ni) was administered to guinea pigs, followed by exposure to at least 2times the median lethal dose (LD50) of the OP nerve agents tabun (GA), sarin (GB), soman (GD), and cyclosarin (GF), or chlorpyrifos oxon, the toxic metabolite of the OP pesticide chlorpyrifos. In the second model, mice were infected with an adenovirus that induced expression of HuPON1 and then exposed to sequential doses of GD, VX, or (as reported previously) diazoxon, the toxic metabolite of the OP pesticide diazinon. In both animal models, the exogenously added HuPON1 protected animals against otherwise lethal doses of the OP pesticides but not against the nerve agents. Together, the results support prior modeling and in vitro activity data which suggest that wild-type HuPON1 does not have sufficient catalytic activity to provide in vivo protection against nerve agents.
ESTHER : Hodgins_2013_Chem.Biol.Interact_203_177
PubMedSearch : Hodgins_2013_Chem.Biol.Interact_203_177
PubMedID: 23123254

Title : Identification and characterization of novel catalytic bioscavengers of organophosphorus nerve agents - Otto_2013_Chem.Biol.Interact_203_186
Author(s) : Otto TC , Scott JR , Kauffman MA , Hodgins SM , diTargiani RC , Hughes JH , Sarricks EP , Saturday GA , Hamilton TA , Cerasoli DM
Ref : Chemico-Biological Interactions , 203 :186 , 2013
Abstract : In an effort to discover novel catalytic bioscavengers of organophosphorus (OP) nerve agents, cell lysates from a diverse set of bacterial strains were screened for their capacity to hydrolyze the OP nerve agents VX, VR, and soman (GD). The library of bacterial strains was identified using both random and rational approaches. Specifically, two representative strains from eight categories of extremophiles were chosen at random. For the rational approach, the protein sequence of organophosphorus hydrolase (OPH) from Brevundimonas diminuta was searched against a non-redundant protein database using the Basic Local Alignment Search Tool to find regions of local similarity between sequences. Over 15 protein sequences with significant sequence similarity to OPH were identified from a variety of bacterial strains. Some of these matches were based on predicted protein structures derived from bacterial genome sequences rather than from bona fide proteins isolated from bacteria. Of the 25 strains selected for nerve agent testing, three bacterial strains had measurable levels of OP hydrolase activity. These strains are Ammoniphilus oxalaticus, Haloarcula sp., and Micromonospora aurantiaca. Lysates from A. oxalaticus had detectable hydrolysis of VR; Haloarcula sp. had appreciable hydrolysis of VX and VR, whereas lysates from M. aurantiaca had detectable hydrolysis of VR and GD.
ESTHER : Otto_2013_Chem.Biol.Interact_203_186
PubMedSearch : Otto_2013_Chem.Biol.Interact_203_186
PubMedID: 23041042

Title : Repeated exposure to sublethal doses of the organophosphorus compound VX activates BDNF expression in mouse brain - Pizarro_2012_Toxicol.Sci_126_497
Author(s) : Pizarro JM , Chang WE , Bah MJ , Wright LK , Saviolakis GA , Alagappan A , Robison CL , Shah JD , Meyerhoff JL , Cerasoli DM , Midboe EG , Lumley LA
Ref : Toxicol Sci , 126 :497 , 2012
Abstract : The highly toxic organophosphorus compound VX [O-ethyl S-[2-(diisopropylamino)ethyl]methylphosphonate] is an irreversible inhibitor of the enzyme acetylcholinesterase (AChE). Prolonged inhibition of AChE increases endogenous levels of acetylcholine and is toxic at nerve synapses and neuromuscular junctions. We hypothesized that repeated exposure to sublethal doses of VX would affect genes associated with cell survival, neuronal plasticity, and neuronal remodeling, including brain-derived neurotrophic factor (BDNF). We examined the time course of BDNF expression in C57BL/6 mouse brain following repeated exposure (1/day x 5 days/week x 2 weeks) to sublethal doses of VX (0.2 LD(50) and 0.4 LD(50)). BDNF messenger RNA expression was significantly (p < 0.05) elevated in multiple brain regions, including the dentate gyrus, CA3, and CA1 regions of the hippocampal formation, as well as the piriform cortex, hypothalamus, amygdala, and thalamus, 72 h after the last 0.4 LD(50) VX exposure. BDNF protein expression, however, was only increased in the CA3 region of the hippocampus. Whether increased BDNF in response to sublethal doses of VX exposure is an adaptive response to prevent cellular damage or a precursor to impending brain damage remains to be determined. If elevated BDNF is an adaptive response, exogenous BDNF may be a potential therapeutic target to reduce the toxic effects of nerve agent exposure.
ESTHER : Pizarro_2012_Toxicol.Sci_126_497
PubMedSearch : Pizarro_2012_Toxicol.Sci_126_497
PubMedID: 22240983

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 : Structure of Recombinant Human Carboxylesterase 1 Isolated from Whole Cabbage Looper Larvae - Greenblatt_2012_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_F68_269
Author(s) : Greenblatt HM , Otto TC , Kirkpatrick MG , Kovaleva E , Brown S , Buchman G , Cerasoli DM , Sussman JL
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , F68 :269 , 2012
Abstract : The use of whole insect larvae as a source of recombinant proteins offers a more cost-effective method of producing large quantities of human proteins than conventional cell-culture approaches. Human carboxylesterase 1 has been produced in and isolated from whole Trichoplusia ni larvae. The recombinant protein was crystallized and its structure was solved to 2.2 resolution. The results indicate that the larvae-produced enzyme is essentially identical to that isolated from cultured Sf21 cells, supporting the use of this expression system to produce recombinant enzymes for crystallization studies.
ESTHER : Greenblatt_2012_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_F68_269
PubMedSearch : Greenblatt_2012_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_F68_269
PubMedID: 22442219
Gene_locus related to this paper: human-CES1

Title : Production of ES1 plasma carboxylesterase knockout mice for toxicity studies - Duysen_2011_Chem.Res.Toxicol_24_1891
Author(s) : Duysen EG , Koentgen F , Williams GR , Timperley CM , Schopfer LM , Cerasoli DM , Lockridge O
Ref : Chemical Research in Toxicology , 24 :1891 , 2011
Abstract : The LD(50) for soman is 10-20-fold higher for a mouse than a human. The difference in susceptibility is attributed to the presence of carboxylesterase in mouse but not in human plasma. Our goal was to make a mouse lacking plasma carboxylesterase. We used homologous recombination to inactivate the carboxylesterase ES1 gene on mouse chromosome 8 by deleting exon 5 and by introducing a frame shift for amino acids translated from exons 6 to 13. ES1-/- mice have no detectable carboxylesterase activity in plasma but have normal carboxylesterase activity in tissues. Homozygous ES1-/- mice and wild-type littermates were tested for response to a nerve agent model compound (soman coumarin) at 3 mg/kg sc. This dose intoxicated both genotypes but was lethal only to ES1-/- mice. This demonstrated that plasma carboxylesterase protects against a relatively high toxicity organophosphorus compound. The ES1-/- mouse should be an appropriate model for testing highly toxic nerve agents and for evaluating protection strategies against the toxicity of nerve agents.
ESTHER : Duysen_2011_Chem.Res.Toxicol_24_1891
PubMedSearch : Duysen_2011_Chem.Res.Toxicol_24_1891
PubMedID: 21875074
Gene_locus related to this paper: mouse-Ces1c

Title : Computational characterization of how the VX nerve agent binds human serum paraoxonase 1 - Fairchild_2011_J.Mol.Model_17_97
Author(s) : Fairchild SZ , Peterson MW , Hamza A , Zhan CG , Cerasoli DM , Chang WE
Ref : J Mol Model , 17 :97 , 2011
Abstract : Human serum paraoxonase 1 (HuPON1) is an enzyme that can hydrolyze various chemical warfare nerve agents including VX. A previous study has suggested that increasing HuPON1's VX hydrolysis activity one to two orders of magnitude would make the enzyme an effective countermeasure for in vivo use against VX. This study helps facilitate further engineering of HuPON1 for enhanced VX-hydrolase activity by computationally characterizing HuPON1's tertiary structure and how HuPON1 binds VX. HuPON1's structure is first predicted through two homology modeling procedures. Docking is then performed using four separate methods, and the stability of each bound conformation is analyzed through molecular dynamics and solvated interaction energy calculations. The results show that VX's lone oxygen atom has a strong preference for forming a direct electrostatic interaction with HuPON1's active site calcium ion. Various HuPON1 residues are also detected that are in close proximity to VX and are therefore potential targets for future mutagenesis studies. These include E53, H115, N168, F222, N224, L240, D269, I291, F292, and V346. Additionally, D183 was found to have a predicted pKa near physiological pH. Given D183's location in HuPON1's active site, this residue could potentially act as a proton donor or accepter during hydrolysis. The results from the binding simulations also indicate that steered molecular dynamics can potentially be used to obtain accurate binding predictions even when starting with a closed conformation of a protein's binding or active site.
ESTHER : Fairchild_2011_J.Mol.Model_17_97
PubMedSearch : Fairchild_2011_J.Mol.Model_17_97
PubMedID: 20379755

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 : VX hydrolysis by human serum paraoxonase 1: a comparison of experimental and computational results - Peterson_2011_PLoS.One_6_e20335
Author(s) : Peterson MW , Fairchild SZ , Otto TC , Mohtashemi M , Cerasoli DM , Chang WE
Ref : PLoS ONE , 6 :e20335 , 2011
Abstract : Human Serum paraoxonase 1 (HuPON1) is an enzyme that has been shown to hydrolyze a variety of chemicals including the nerve agent VX. While wildtype HuPON1 does not exhibit sufficient activity against VX to be used as an in vivo countermeasure, it has been suggested that increasing HuPON1's organophosphorous hydrolase activity by one or two orders of magnitude would make the enzyme suitable for this purpose. The binding interaction between HuPON1 and VX has recently been modeled, but the mechanism for VX hydrolysis is still unknown. In this study, we created a transition state model for VX hydrolysis (VX(ts)) in water using quantum mechanical/molecular mechanical simulations, and docked the transition state model to 22 experimentally characterized HuPON1 variants using AutoDock Vina. The HuPON1-VX(ts) complexes were grouped by reaction mechanism using a novel clustering procedure. The average Vina interaction energies for different clusters were compared to the experimentally determined activities of HuPON1 variants to determine which computational procedures best predict how well HuPON1 variants will hydrolyze VX. The analysis showed that only conformations which have the attacking hydroxyl group of VX(ts) coordinated by the sidechain oxygen of D269 have a significant correlation with experimental results. The results from this study can be used for further characterization of how HuPON1 hydrolyzes VX and design of HuPON1 variants with increased activity against VX.
ESTHER : Peterson_2011_PLoS.One_6_e20335
PubMedSearch : Peterson_2011_PLoS.One_6_e20335
PubMedID: 21655255

Title : Nerve agent hydrolysis activity designed into a human drug metabolism enzyme - Hemmert_2011_PLoS.One_6_e17441
Author(s) : Hemmert AC , Otto TC , Chica RA , Wierdl M , Edwards JS , Lewis SM , Edwards CC , Tsurkan L , Cadieux CL , Kasten SA , Cashman JR , Mayo SL , Potter PM , Cerasoli DM , Redinbo MR
Ref : PLoS ONE , 6 :e17441 , 2011
Abstract : Organophosphorus (OP) nerve agents are potent suicide inhibitors of the essential neurotransmitter-regulating enzyme acetylcholinesterase. Due to their acute toxicity, there is significant interest in developing effective countermeasures to OP poisoning. Here we impart nerve agent hydrolysis activity into the human drug metabolism enzyme carboxylesterase 1. Using crystal structures of the target enzyme in complex with nerve agent as a guide, a pair of histidine and glutamic acid residues were designed proximal to the enzyme's native catalytic triad. The resultant variant protein demonstrated significantly increased rates of reactivation following exposure to sarin, soman, and cyclosarin. Importantly, the addition of these residues did not alter the high affinity binding of nerve agents to this protein. Thus, using two amino acid substitutions, a novel enzyme was created that efficiently converted a group of hemisubstrates, compounds that can start but not complete a reaction cycle, into bona fide substrates. Such approaches may lead to novel countermeasures for nerve agent poisoning.
ESTHER : Hemmert_2011_PLoS.One_6_e17441
PubMedSearch : Hemmert_2011_PLoS.One_6_e17441
PubMedID: 21445272
Gene_locus related to this paper: human-CES1

Title : Bisquaternary pyridinium oximes: Comparison of in vitro reactivation potency of compounds bearing aliphatic linkers and heteroaromatic linkers for paraoxon-inhibited electric eel and recombinant human acetylcholinesterase - Bharate_2010_Bioorg.Med.Chem_18_787
Author(s) : Bharate SB , Guo L , Reeves TE , Cerasoli DM , Thompson CM
Ref : Bioorganic & Medicinal Chemistry , 18 :787 , 2010
Abstract : Oxime reactivators are the drugs of choice for the post-treatment of OP (organophosphorus) intoxication and used widely for mechanistic and kinetic studies of OP-inhibited cholinesterases. The purpose of the present study was to evaluate new oxime compounds to reactivate acetylcholinesterase (AChE) inhibited by the OP paraoxon. Several new bisquaternary pyridinium oximes with heterocyclic linkers along with some known bisquaternary pyridinium oximes bearing aliphatic linkers were synthesized and evaluated for their in vitro reactivation potency against paraoxon-inhibited electric eel acetylcholinesterase (EeAChE) and recombinant human acetylcholinesterase (rHuAChE). Results herein indicate that most of the compounds are better reactivators of EeAChE than of rHuAChE. The reactivation potency of two different classes of compounds with varying linker chains was compared and observed that the structure of the connecting chain is an important factor for the activity of the reactivators. At a higher concentration (10(-3)M), compounds bearing aliphatic linker showed better reactivation than compounds with heterocyclic linkers. Interestingly, oximes with a heterocyclic linker inhibited AChE at higher concentration (10(-3)M), whereas their ability to reactivate was increased at lower concentrations (10(-4)M and 10(-5)M). Compounds bearing either a thiophene linker 26, 46 or a furan linker 31 showed 59%, 49% and 52% reactivation of EeAChE, respectively, at 10(-5)M. These compounds showed 14%, 6% and 15% reactivation of rHuAChE at 10(-4)M. Amongst newly synthesized analogs with heterocyclic linkers (26-35 and 45-46), compound 31, bearing furan linker chain, was found to be the most effective reactivator with a k(r) 0.042min(-1), which is better than obidoxime (3) for paraoxon-inhibited EeAChE. Compound 31 showed a k(r) 0.0041min(-1) that is near equal to pralidoxime (1) for paraoxon-inhibited rHuAChE.
ESTHER : Bharate_2010_Bioorg.Med.Chem_18_787
PubMedSearch : Bharate_2010_Bioorg.Med.Chem_18_787
PubMedID: 20005727

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 : 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 : Human carboxylesterase 1 stereoselectively binds the nerve agent cyclosarin and spontaneously hydrolyzes the nerve agent sarin - Hemmert_2010_Mol.Pharmacol_77_508
Author(s) : Hemmert AC , Otto TC , Wierdl M , Edwards CC , Fleming CD , MacDonald M , Cashman JR , Potter PM , Cerasoli DM , Redinbo MR
Ref : Molecular Pharmacology , 77 :508 , 2010
Abstract : Organophosphorus (OP) nerve agents are potent toxins that inhibit cholinesterases and produce a rapid and lethal cholinergic crisis. Development of protein-based therapeutics is being pursued with the goal of preventing nerve agent toxicity and protecting against the long-term side effects of these agents. The drug-metabolizing enzyme human carboxylesterase 1 (hCE1) is a candidate protein-based therapeutic because of its similarity in structure and function to the cholinesterase targets of nerve agent poisoning. However, the ability of wild-type hCE1 to process the G-type nerve agents sarin and cyclosarin has not been determined. We report the crystal structure of hCE1 in complex with the nerve agent cyclosarin. We further use stereoselective nerve agent analogs to establish that hCE1 exhibits a 1700- and 2900-fold preference for the P(R) enantiomers of analogs of soman and cyclosarin, respectively, and a 5-fold preference for the P(S) isomer of a sarin analog. Finally, we show that for enzyme inhibited by racemic mixtures of bona fide nerve agents, hCE1 spontaneously reactivates in the presence of sarin but not soman or cyclosarin. The addition of the neutral oxime 2,3-butanedione monoxime increases the rate of reactivation of hCE1 from sarin inhibition by more than 60-fold but has no effect on reactivation with the other agents examined. Taken together, these data demonstrate that hCE1 is only reactivated after inhibition with the more toxic P(S) isomer of sarin. These results provide important insights toward the long-term goal of designing novel forms of hCE1 to act as protein-based therapeutics for nerve agent detoxification.
ESTHER : Hemmert_2010_Mol.Pharmacol_77_508
PubMedSearch : Hemmert_2010_Mol.Pharmacol_77_508
PubMedID: 20051531
Gene_locus related to this paper: human-CES1

Title : Stereoselective hydrolysis of organophosphate nerve agents by the bacterial phosphotriesterase - Tsai_2010_Biochemistry_49_7978
Author(s) : Tsai PC , Bigley A , Li Y , Ghanem E , Cadieux CL , Kasten SA , Reeves TE , Cerasoli DM , Raushel FM
Ref : Biochemistry , 49 :7978 , 2010
Abstract : Organophosphorus compounds include many synthetic, neurotoxic substances that are commonly used as insecticides. The toxicity of these compounds is due to their ability to inhibit the enzyme acetylcholine esterase. Some of the most toxic organophosphates have been adapted for use as chemical warfare agents; the most well-known are GA, GB, GD, GF, VX, and VR. All of these compounds contain a chiral phosphorus center, with the S(P) enantiomers being significantly more toxic than the R(P) enantiomers. Phosphotriesterase (PTE) is an enzyme capable of detoxifying these agents, but the stereochemical preference of the wild-type enzyme is for the R(P) enantiomers. A series of enantiomerically pure chiral nerve agent analogues containing the relevant phosphoryl centers found in GB, GD, GF, VX, and VR has been developed. Wild-type and mutant forms of PTE have been tested for their ability to hydrolyze this series of compounds. Mutant forms of PTE with significantly enhanced, as well as relaxed or reversed, stereoselectivity have been identified. A number of variants exhibited dramatically improved kinetic constants for the catalytic hydrolysis of the more toxic S(P) enantiomers. Improvements of up to 3 orders of magnitude relative to the value of the wild-type enzyme were observed. Some of these mutants were tested against racemic mixtures of GB and GD. The kinetic constants obtained with the chiral nerve agent analogues accurately predict the improved activity and stereoselectivity against the authentic nerve agents used in this study.
ESTHER : Tsai_2010_Biochemistry_49_7978
PubMedSearch : Tsai_2010_Biochemistry_49_7978
PubMedID: 20701311

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 : Crystal structures of human group-VIIA phospholipase A2 inhibited by organophosphorus nerve agents exhibit non-aged complexes - Samanta_2009_Biochem.Pharmacol_78_420
Author(s) : Samanta U , Kirby SD , Srinivasan P , Cerasoli DM , Bahnson BJ
Ref : Biochemical Pharmacology , 78 :420 , 2009
Abstract : The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun. The sarin and soman complexes displayed a racemic mix of P(R) and P(S) stereoisomers at the P-chiral center. The tabun complex displayed only the P(R) stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix-assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme. The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long-term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents.
ESTHER : Samanta_2009_Biochem.Pharmacol_78_420
PubMedSearch : Samanta_2009_Biochem.Pharmacol_78_420
PubMedID: 19394314
Gene_locus related to this paper: human-PLA2G7

Title : New series of monoquaternary pyridinium oximes: Synthesis and reactivation potency for paraoxon-inhibited electric eel and recombinant human acetylcholinesterase - Bharate_2009_Bioorg.Med.Chem.Lett_19_5101
Author(s) : Bharate SB , Guo L , Reeves TE , Cerasoli DM , Thompson CM
Ref : Bioorganic & Medicinal Chemistry Lett , 19 :5101 , 2009
Abstract : The preparation of a series of monoquaternary pyridinium oximes bearing either a heterocyclic side chain or a functionalized aliphatic side chain and the corresponding in vitro evaluation for reactivation of paraoxon-inhibited electric eel acetylcholinesterase (EeAChE) and recombinant human acetylcholinesterase (rHuAChE) are reported. Several newly synthesized compounds efficiently reactivated inhibited EeAChE, but were poor reactivators of inhibited rHuAChE. Compounds bearing a thiophene ring in the side chain (20, 23, 26 and 29) showed better reactivation (24-37% for EeAChE and 5-9% for rHuAChE) compared to compounds with furan and isoxazole heterocycles (0-8% for EeAChE and 2-3% for rHuAChE) at 10(-5)M. The N-pyridyl-CH(2)COOH analog 8 reactivated EeAChE (36%) and rHuAChE (15%) at 10(-4)M with a k(r) value better than 2-pyridine aldoxime methiodide (2-PAM) for rHuAChE.
ESTHER : Bharate_2009_Bioorg.Med.Chem.Lett_19_5101
PubMedSearch : Bharate_2009_Bioorg.Med.Chem.Lett_19_5101
PubMedID: 19640713

Title : Crystal structures of brain group-VIII phospholipase A2 in nonaged complexes with the organophosphorus nerve agents soman and sarin - Epstein_2009_Biochemistry_48_3425
Author(s) : Epstein TM , Samanta U , Kirby SD , Cerasoli DM , Bahnson BJ
Ref : Biochemistry , 48 :3425 , 2009
Abstract : Insecticide and nerve agent organophosphorus (OP) compounds are potent inhibitors of the serine hydrolase superfamily of enzymes. Nerve agents, such as sarin, soman, tabun, and VX exert their toxicity by inhibiting human acetycholinesterase at nerve synapses. Following the initial phosphonylation of the active site serine, the enzyme may reactivate spontaneously or through reaction with an appropriate nucleophilic oxime. Alternatively, the enzyme-nerve agent complex can undergo a secondary process, called "aging", which dealkylates the nerve agent adduct and results in a product that is highly resistant to reactivation by any known means. Here we report the structures of paraoxon, soman, and sarin complexes of group-VIII phospholipase A2 from bovine brain. In each case, the crystal structures indicate a nonaged adduct; a stereoselective preference for binding of the P(S)C(S) isomer of soman and the P(S) isomer of sarin was also noted. The stability of the nonaged complexes was corroborated by trypsin digest and electrospray ionization mass spectrometry, which indicates nonaged complexes are formed with diisopropylfluorophosphate, soman, and sarin. The P(S) stereoselectivity for reaction with sarin was confirmed by reaction of racemic sarin, followed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate each stereoisomer. The P(S) stereoisomers of soman and sarin are known to be the more toxic stereoisomers, as they react preferentially to inhibit human acetylcholinesterase. The results obtained for nonaged complexes of group-VIII phospholipase A2 are compared to those obtained for other serine hydrolases and discussed to partly explain determinants of OP aging. Furthermore, structural insights can now be exploited to engineer variant versions of this enzyme with enhanced nerve agent binding and hydrolysis functions.
ESTHER : Epstein_2009_Biochemistry_48_3425
PubMedSearch : Epstein_2009_Biochemistry_48_3425
PubMedID: 19271773

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 : 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 : Balancing the stability and the catalytic specificities of OP hydrolases with enhanced V-agent activities - Reeves_2008_Protein.Eng.Des.Sel_21_405
Author(s) : Reeves TE , Wales ME , Grimsley JK , Li P , Cerasoli DM , Wild JR
Ref : Protein Engineering Des Sel , 21 :405 , 2008
Abstract : Rational site-directed mutagenesis and biophysical analyses have been used to explore the thermodynamic stability and catalytic capabilities of organophosphorus hydrolase (OPH) and its genetically modified variants. There are clear trade-offs in the stability of modifications that enhance catalytic activities. For example, the H254R/H257L variant has higher turnover numbers for the chemical warfare agents VX (144 versus 14 s(-1) for the native enzyme (wild type) and VR (Russian VX, 465 versus 12 s(-1) for wild type). These increases are accompanied by a loss in stability in which the total Gibb's free energy for unfolding is 19.6 kcal/mol, which is 5.7 kcal/mol less than that of the wild-type enzyme. X-ray crystallographic studies support biophysical data that suggest amino acid residues near the active site contribute to the chemical and thermal stability through hydrophobic and cation-pi interactions. The cation-pi interactions appear to contribute an additional 7 kcal/mol to the overall global stability of the enzyme. Using rational design, it has been possible to make amino acid changes in this region that restored the stability, yet maintained effective V-agent activities, with turnover numbers of 68 and 36 s(-1) for VX and VR, respectively. This study describes the first rationally designed, stability/activity balance for an OPH enzyme with a legitimate V-agent activity, and its crystal structure.
ESTHER : Reeves_2008_Protein.Eng.Des.Sel_21_405
PubMedSearch : Reeves_2008_Protein.Eng.Des.Sel_21_405
PubMedID: 18434422

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 : Crystal structures of human carboxylesterase 1 in covalent complexes with the chemical warfare agents soman and tabun - Fleming_2007_Biochemistry_46_5063
Author(s) : Fleming CD , Edwards CC , Kirby SD , Maxwell DM , Potter PM , Cerasoli DM , Redinbo MR
Ref : Biochemistry , 46 :5063 , 2007
Abstract : The organophosphorus nerve agents sarin, soman, tabun, and VX exert their toxic effects by inhibiting the action of human acetylcholinesterase, a member of the serine hydrolase superfamily of enzymes. The current treatments for nerve agent exposure must be administered quickly to be effective, and they often do not eliminate long-term toxic side effects associated with organophosphate poisoning. Thus, there is significant need for effective prophylactic methods to protect at-risk personnel from nerve agent exposure, and protein-based approaches have emerged as promising candidates. We present the 2.7 A resolution crystal structures of the serine hydrolase human carboxylesterase 1 (hCE1), a broad-spectrum drug metabolism enzyme, in covalent acyl-enzyme intermediate complexes with the chemical weapons soman and tabun. The structures reveal that hCE1 binds stereoselectively to these nerve agents; for example, hCE1 appears to react preferentially with the 10(4)-fold more lethal PS stereoisomer of soman relative to the PR form. In addition, structural features of the hCE1 active site indicate that the enzyme may be resistant to dead-end organophosphate aging reactions that permanently inactivate other serine hydrolases. Taken together, these data provide important structural details toward the goal of engineering hCE1 into an organophosphate hydrolase and protein-based therapeutic for nerve agent exposure.
ESTHER : Fleming_2007_Biochemistry_46_5063
PubMedSearch : Fleming_2007_Biochemistry_46_5063
PubMedID: 17407327
Gene_locus related to this paper: human-CES1

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 : 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 : 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 : CD4+Thy1- thymocytes with a Th-type 2 cytokine response - Cerasoli_2001_Int.Immunol_13_75
Author(s) : Cerasoli DM , Kelsoe G , Sarzotti M
Ref : Int Immunol , 13 :75 , 2001
Abstract : We have identified a small subset of CD3(+), CD4(+), CD8(-) thymocytes that do not express Thy1 (CD90). This Thy1(-) subset represents 1-3.7% of the total number of thymocytes in a naive mouse. CD4(+)Thy1(-) thymocytes express high levels of CD3, intermediate to high levels of heat-stable antigen (HSA), and low levels of CD25, CD45RB, CD69, CD44 and CD62L. They produce high titers of IL-4 and no IFN-gamma upon stimulation in vitro, a response characteristic of T(h)2 cells. In the thymi of mice infected neonatally with a high dose of the retrovirus Cas-Br-E MuLV, the frequency of CD4(+)Thy1(-) cells increased approximately 10-fold. High-dose virus infection resulted in decreased HSA and increased CD44 expression on CD4(+)Thy1(-) cells relative to cells from naive mice. CD4(+)Thy1(-) cells from high-dose infected mice also secreted IL-4 and not IFN-gamma upon in vitro stimulation. We previously reported that infection of newborn mice with a high dose of murine retrovirus results in the induction of a non-protective anti-viral T(h)2 T cell response; CD4(+)Thy1(-) thymocytes with a T(h)2-like cytokine profile may play a role in determining the cytokine bias of this anti-viral response.
ESTHER : Cerasoli_2001_Int.Immunol_13_75
PubMedSearch : Cerasoli_2001_Int.Immunol_13_75
PubMedID: 11133836

Title : CD4+ T cells that evade deletion by a self peptide display Th1-biased differentiation - Riley_2001_Eur.J.Immunol_31_311
Author(s) : Riley MP , Shih FF , Jordan MS , Petrone AL , Cerasoli DM , Scott P , Caton AJ
Ref : European Journal of Immunology , 31 :311 , 2001
Abstract : We have examined factors governing the differentiation of autoreactive CD4+ T cells that have evaded deletion by a self peptide. Two lineages of transgenic mice (HA12 and HA104) expressing the influenza virus hemagglutinin (HA) were mated with TS1 mice that express a clonotypic T cell receptor (TCR) specific for the I-Ed-restricted determinant site 1 (S1) of HA. Thymocytes expressing high levels of the clonotypic TCR were deleted in both TS1xHA transgenic lineages. However, through allelic inclusion, thymocytes expressing low levels of the clonotypic TCR and high levels of endogenous TCR alpha-chains evaded deletion in TS1xHA12 and TS1xHA104 mice to graded degrees. When stimulated with S1 peptide in vitro, the non-autoreactive TS1 T cells were biased toward differentiation into Th2 effectors. By contrast, CD4+ T cells that evaded deletion in TS1xHA12 and TS1xHA104 mice were progressively biased toward Th1-like differentiation. Moreover, the effector cells from TS1xHA12 and TS1xHA104 mice secreted higher levels of IFN-gamma , on a per cell basis, than were secreted by their non-autoreactive counterparts. Thus, CD4+ T cells that evade deletion by a self peptide can exhibit an intrinsic bias toward differentiation into Th1 effector cells.
ESTHER : Riley_2001_Eur.J.Immunol_31_311
PubMedSearch : Riley_2001_Eur.J.Immunol_31_311
PubMedID: 11265648

Title : Graded deletion and virus-induced activation of autoreactive CD4+ T cells - Riley_2000_J.Immunol_165_4870
Author(s) : Riley MP , Cerasoli DM , Jordan MS , Petrone AL , Shih FF , Caton AJ
Ref : J Immunol , 165 :4870 , 2000
Abstract : We have examined factors governing the negative selection of autoreactive CD4(+) T cells in transgenic mice expressing low (HA12 mice) vs. high (HA104 mice) amounts of the influenza virus hemagglutinin (HA). When mated with TS1 mice that express a transgenic TCR specific for the I-Ed-restricted determinant site 1 (S1) of HA, thymocytes expressing high levels of the clonotypic TCR were deleted in both HA-transgenic lineages. However, through allelic inclusion, thymocytes with lower levels of the clonotypic TCR evaded deletion in TS1 x HA12 and TS1 x HA104 mice to graded degrees. Moreover, in both lineages, peripheral CD4(+) T cells could be activated by the S1 peptide in vitro, and by influenza virus in vivo. These findings indicate that allelic inclusion can allow autoreactive CD4(+) thymocytes to evade thymic deletion to varying extents reflecting variation in the expression of the self peptide, and can provide a basis for the activation of autoreactive peripheral T cells by viruses bearing homologues of self peptides ("molecular mimicry").
ESTHER : Riley_2000_J.Immunol_165_4870
PubMedSearch : Riley_2000_J.Immunol_165_4870
PubMedID: 11046011

Title : Relaxed negative selection in germinal centers and impaired affinity maturation in bcl-xL transgenic mice - Takahashi_1999_J.Exp.Med_190_399
Author(s) : Takahashi Y , Cerasoli DM , Dal Porto JM , Shimoda M , Freund R , Fang W , Telander DG , Malvey EN , Mueller DL , Behrens TW , Kelsoe G
Ref : J Exp Med , 190 :399 , 1999
Abstract : The role of apoptosis in affinity maturation was investigated by determining the affinity of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific antibody-forming cells (AFCs) and serum antibody in transgenic mice that overexpress a suppressor of apoptosis, Bcl-xL, in the B cell compartment. Although transgenic animals briefly expressed higher numbers of splenic AFCs after immunization, the bcl-xL transgene did not increase the number or size of germinal centers (GCs), alter the levels of serum antibody, or change the frequency of NP-specific, long-lived AFCs. Nonetheless, the bcl-xL transgene product, in addition to endogenous Bcl-xL, reduced apoptosis in GC B cells and resulted in the expansion of B lymphocytes bearing VDJ rearrangements that are usually rare in primary anti-NP responses. Long-lived AFCs bearing these noncanonical rearrangements were frequent in the bone marrow and secreted immunoglobulin G(1) antibodies with low affinity for NP. The abundance of noncanonical cells lowered the average affinity of long-lived AFCs and serum antibody, demonstrating that Bcl-xL and apoptosis influence clonal selection/maintenance for affinity maturation.
ESTHER : Takahashi_1999_J.Exp.Med_190_399
PubMedSearch : Takahashi_1999_J.Exp.Med_190_399
PubMedID: 10430628

Title : Immune recognition of influenza hemagglutinin as a viral and a neo-self-antigen - Caton_1998_Immunol.Res_17_23
Author(s) : Caton AJ , Cerasoli DM , Shih FF
Ref : Immunol Res , 17 :23 , 1998
Abstract : To analyze mechanisms governing tolerance and autoimmunity to self-antigens, we have generated lineages of transgenic mice that express the influenza virus PR8 hemagglutinin (HA) as a neo-self-antigen. By comparing the HA-specific T and B cell responses that can be induced in HA Tg mice with those that are induced in non-Tg (BALB/c) mice, the specificity and genetic basis with which tolerance is induced to the HA has been examined. This article summarizes studies using lineages of HA Tg mice that express different forms and amounts of the HA under the control of the SV40 promoter/enhancer. Our studies have revealed that specific subsets of HA-specific T and B cells are negatively selected from the primary repertoires of HA Tg mice. However, substantial populations of HA-specific T and B cells evade negative selection and can be activated by virus immunization. Understanding the capacity of these autoreactive lymphocytes to differentiate and participate in antigen-specific immune responses will provide important insights into mechanisms by which autoimmunity might be induced by viruses bearing structural similarities with self-antigens.
ESTHER : Caton_1998_Immunol.Res_17_23
PubMedSearch : Caton_1998_Immunol.Res_17_23
PubMedID: 9479564

Title : In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. V. Affinity maturation develops in two stages of clonal selection - Takahashi_1998_J.Exp.Med_187_885
Author(s) : Takahashi Y , Dutta PR , Cerasoli DM , Kelsoe G
Ref : J Exp Med , 187 :885 , 1998
Abstract : To examine the role of germinal centers (GCs) in the generation and selection of high affinity antibody-forming cells (AFCs), we have analyzed the average affinity of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific AFCs and serum antibodies both during and after the GC phase of the immune response. In addition, the genetics of NP-binding AFCs were followed to monitor the generation and selection of high affinity AFCs at the clonal level. NP-binding AFCs gradually accumulate in bone marrow (BM) after immunization and BM becomes the predominant locale of specific AFCs in the late primary response. Although the average affinity of NP-specific BM AFCs rapidly increased while GCs were present (GC phase), the affinity of both BM AFCs and serum antibodies continued to increase even after GCs waned (post-GC phase). Affinity maturation in the post-GC phase was also reflected in a shift in the distribution of somatic mutations as well as in the CDR3 sequences of BM AFC antibody heavy chain genes. Disruption of GCs by injection of antibody specific for CD154 (CD40 ligand) decreased the average affinity of subsequent BM AFCs, suggesting that GCs generate the precursors of high affinity BM AFCs; inhibition of CD154-dependent cellular interactions after the GC reaction was complete had no effect on high affinity BM AFCs. Interestingly, limited affinity maturation in the BM AFC compartment still occurs during the late primary response even after treatment with anti-CD154 antibody. Thus, GCs are necessary for the generation of high affinity AFC precursors but are not the only sites for the affinity-driven clonal selection responsible for the maturation of humoral immune responses.
ESTHER : Takahashi_1998_J.Exp.Med_187_885
PubMedSearch : Takahashi_1998_J.Exp.Med_187_885
PubMedID: 9500791

Title : A major T cell determinant from the influenza virus hemagglutinin (HA) can be a cryptic self peptide in HA transgenic mice - Shih_1997_Int.Immunol_9_249
Author(s) : Shih FF , Cerasoli DM , Caton AJ
Ref : Int Immunol , 9 :249 , 1997
Abstract : Transgenic (Tg) mice expressing the influenza virus PR8 hemagglutinin (PR8 HA) were infected with PR8 virus and analyzed for their ability to generate T cell responses to individual MHC class II-restricted T cell determinants from the HA. HAmemb and HAtrunc mice each express HA transgene mRNA in many tissues (including the thymus), but differ in the form and amount of the HA that is expressed: HAmemb mice express the entire viral HA as a membrane-bound neo-self antigen, whereas HAtrunc mice express lower levels of a truncated HA polypeptide. HAmemb mice were found to mediate efficient negative selection of autoreactive T cells directed to the major I-Ed-restricted and I-Ad-restricted determinants from the HA (designated S1 and S2 respectively). S1-specific T cell responses were similarly undetectable in PR8-infected HAtrunc mice. However, S2-specific T cells were only partially eliminated in HAtrunc mice; indeed, even though their frequency was reduced relative to non-Tg mice, S2-specific T cells still constituted a sizable population in PR8-infected HAtrunc mice. Moreover, the S2-specific T cells from HAtrunc and non-Tg mice appeared to be equally sensitive to stimulation with S2 peptide, and in each case utilized highly diverse T cell receptors to recognize S2-I-Ad. The findings demonstrate that an individual class II-restricted T cell determinant can be recognized as a cryptic self peptide during T cell repertoire formation and as an immunodominant peptide in the context of an anti-viral T cell response, providing a basis for the induction of autoreactive T cells by viruses containing homologs of self antigens.
ESTHER : Shih_1997_Int.Immunol_9_249
PubMedSearch : Shih_1997_Int.Immunol_9_249
PubMedID: 9040007

Title : Transgenic mice that express different forms of the influenza virus hemagglutinin as a neo-self-antigen - Caton_1995_J.Clin.Immunol_15_106S
Author(s) : Caton AJ , Stark SE , Shih FF , Cerasoli DM
Ref : J Clin Immunol , 15 :106S , 1995
Abstract : We have generated transgenic mouse lineages that express the influenza virus hemagglutinin in different physical forms. One kind expresses the full-length hemagglutinin molecule as a cell surface glycoprotein and can be recognized by hemagglutinin-specific B and T cells. The other expresses a truncated polypeptide corresponding to the N-terminal third of the hemagglutinin molecule. This polypeptide encodes known hemagglutinin-specific T-cell determinants; however, it contains no native B-cell epitopes, since these depend on the conformation of the fully folded protein. In each case, the hemagglutinin transgenic mice display ubiquitous expression of transgenic messenger RNA and induce T-cell tolerance to the transgene-encoded T-cell determinant site 1. Thus, the hemagglutinin is a neo-self-antigen in both kinds of hemagglutinin transgenic mice and should provide a useful system for understanding the factors and mechanisms that govern tolerance and autoimmunity to self-antigens.
ESTHER : Caton_1995_J.Clin.Immunol_15_106S
PubMedSearch : Caton_1995_J.Clin.Immunol_15_106S
PubMedID: 8613482

Title : Low avidity recognition of a class II-restricted neo-self peptide by virus-specific T cells - Cerasoli_1995_Int.Immunol_7_935
Author(s) : Cerasoli DM , McGrath J , Carding SR , Shih FF , Knowles BB , Caton AJ
Ref : Int Immunol , 7 :935 , 1995
Abstract : The specificity with which CD4+ T cells recognize self peptides in vivo was examined in transgenic mice that express an influenza virus PR8 hemagglutinin (HA) polypeptide in many tissues, including the thymus (HA Tg mice). HA Tg and non-Tg mice were analyzed for their T cell responses to the major PR8 HA I-E(d)-restricted CD4+ T cell determinant S1. Negative selection eliminated S1-specific T cells from HA Tg mice. Nevertheless, HA Tg mice retained the ability to mount a T cell response to a closely related analog of the S1 determinant [S1(K113)], and some S1(K113)-specific TCRs displayed a partial reactivity with S1 as indicated by their ability to transmit signals for IL-3 but not IL-2 secretion in response to the neo-self peptide. Moreover, the neo-self S1 peptide antagonized the ability of these TCRs to signal IL-2 secretion in response to the foreign S1(K113) determinant. Thus, TCRs that exhibit a partial reactivity with a self peptide are present in the peripheral T cell repertoire and can be activated by a virus containing an analog of the self peptide. These findings provide a model for the induction of autoimmunity by viruses that are close homologs of self peptides, and suggest a way in which TCRs could react with self peptides during positive selection of developing thymocytes.
ESTHER : Cerasoli_1995_Int.Immunol_7_935
PubMedSearch : Cerasoli_1995_Int.Immunol_7_935
PubMedID: 7577802

Title : Genetic basis for T cell recognition of a major histocompatibility complex class II-restricted neo-self peptide - Cerasoli_1995_J.Exp.Med_182_1327
Author(s) : Cerasoli DM , Riley MP , Shih FF , Caton AJ
Ref : J Exp Med , 182 :1327 , 1995
Abstract : We have analyzed the genetic basis for T cell recognition of an endogenous major histocompatibility complex class II-restricted self peptide. Transgenic mice expressing the influenza virus PR8 hemagglutinin I-Ed-restricted determinant S1 (HA Tg mice) mediate negative selection of PR8 S1-specific T cells, but respond to immunization with a virus containing a closely related analogue, S1(K113). Sequence analysis of S1(K113)-specific T cell receptors (TCR) from nontransgenic mice revealed a dominant TCR clonotype that cross-reacts with PR8 S1. This clonotype is eliminated by negative selection in HA Tg mice; nonetheless, modified versions of this TCR that used altered junctional sequences and a novel V alpha/V beta pairing to evade negative selection by the S1 self peptide were identified. The remaining S1(K113)-specific TCRs from HA Tg mice were highly diverse; 13 of 15 S1(K113)-specific TCRs from HA Tg mice used unique V alpha/V beta pairings. Thus, tolerance to PR8 S1 as a self peptide does not limit the diversity of the T cell response to S1(K113).
ESTHER : Cerasoli_1995_J.Exp.Med_182_1327
PubMedSearch : Cerasoli_1995_J.Exp.Med_182_1327
PubMedID: 7595203

Title : T cells from unprimed mice respond to the self antigen heme, in a class II restricted manner, at a frequency similar to alloresponses - Sutherland_1995_Int.Immunol_7_771
Author(s) : Sutherland RM , Pan ZK , Caton AJ , Tang XX , Cerasoli DM , Paterson Y
Ref : Int Immunol , 7 :771 , 1995
Abstract : Heme is a non-protein autoantigen which stimulates potent proliferative responses by T cells from unprimed mice of some strains. These studies show that T cells responding to heme in primary responses are predominantly CD4+, classically I-A restricted, and use diverse TCR characterized by the expression of distinct V, D and J gene segments. These characteristics distinguish heme from superantigens and mitogens which exhibit degenerate MHC restriction and, in the case of superantigens, restricted V gene usage. Using limiting dilution analysis these studies also show that the potent primary response of H-2s mice reflects a high frequency (0.26-0.45%) of heme responsive T cells in the periphery, comparable to the frequency of alloresponsive T cells reported by others in primary mixed lymphocyte reactions. In contrast, heme responsive T cells occur at -10-fold lower frequency in unprimed H-2d mice (0.03%). To determine the antigen recognized by heme reactive T cells, the mass spectra of peptides eluted from the high responder haplotype, I-A(s), were examined. These indicated a markedly different molecular weight distribution of peptides isolated from cells grown in the presence of heme, compared with those from cells grown in its absence. This suggests that heme mediates the expansion of diverse T cells in the peripheral repertoire by a mechanism similar to that for allogeneic responses in which the profile of naturally processed peptides bound to the MHC class II molecule is changed.
ESTHER : Sutherland_1995_Int.Immunol_7_771
PubMedSearch : Sutherland_1995_Int.Immunol_7_771
PubMedID: 7547704

Title : Effect of antibody binding on protein motions studied by hydrogen-exchange labeling and two-dimensional NMR - Mayne_1992_Biochemistry_31_10678
Author(s) : Mayne L , Paterson Y , Cerasoli DM , Englander SW
Ref : Biochemistry , 31 :10678 , 1992
Abstract : We have used hydrogen-exchange labeling detected by 2D NMR to study antibody-protein interactions for two monoclonal antibodies raised against horse cytochrome c. The data show that these antibodies bind mainly to the large 37-59 omega-loop of the cytochrome c molecule. In addition, the results provide some suggestive evidence concerning units of local structural flexibility in cytochrome c.
ESTHER : Mayne_1992_Biochemistry_31_10678
PubMedSearch : Mayne_1992_Biochemistry_31_10678
PubMedID: 1384698