Stereo-specific synthesis of analogs of nerve agents and their utilization for selection and characterization of paraoxonase (PON1) catalytic scavengers.

Fluorogenic organophosphate inhibitors of acetylcholinesterase (AChE) homologous in structure to nerve agents provide useful probes for high throughput screening of mammalian paraoxonase (PON1) libraries generated by directed evolution of an engineered PON1 variant with wild-type like specificity (rePON1). Wt PON1 and rePON1 hydrolyze preferentially the less-toxic R(P) enantiomers of nerve agents and of their fluorogenic surrogates containing the fluorescent leaving group, 3-cyano-7-hydroxy-4-methylcoumarin (CHMC). To increase the sensitivity and reliability of the screening protocol so as to directly select rePON1 clones displaying stereo-preference towards the toxic S(P) enantiomer, and to determine accurately K(m) and k(cat) values for the individual isomers, two approaches were used to obtain the corresponding S(P) and R(P) isomers: (a) stereo-specific synthesis of the O-ethyl, O-n-propyl, and O-i-propyl analogs and (b) enzymic resolution of a racemic mixture of O-cyclohexyl methylphosphonylated CHMC. The configurational assignments of the S(P) and R(P) isomers, as well as their optical purity, were established by X-ray diffraction, reaction with sodium fluoride, hydrolysis by selected rePON1 variants, and inhibition of AChE. The S(P) configuration of the tested surrogates was established for the enantiomer with the more potent anti-AChE activity, with S(P)/R(P) inhibition ratios of 10-100, whereas the R(P) isomers of the O-ethyl and O-n-propyl were hydrolyzed by wt rePON1 about 600- and 70-fold faster, respectively, than the S(P) counterpart. Wt rePON1-induced R(P)/S(P) hydrolysis ratios for the O-cyclohexyl and O-i-propyl analogs are estimated to be >>1000. The various S(P) enantiomers of O-alkyl-methylphosphonyl esters of CHMC provide suitable ligands for screening rePON1 libraries, and can expedite identification of variants with enhanced catalytic proficiency towards the toxic nerve agents.

Laboratory setup for long-term monitoring of the volatilization of hazardous materials: preliminary tests of O-ethyl S-2-(N,N-diisopropylamino)ethyl methylphosphonothiolate on asphalt.

Contrary to commonly used pesticides, the rate of volatilization of extremely toxic chemicals such as the nerve agent O-ethyl S-2-(N,N-diisopropylamino)ethyl methylphosphonothiolate (VX) cannot be readily obtained under environmental conditions due to its high mammalian toxicity that would require extraordinary precautions. An alternative is a laboratory setup that would be used to obtain environmentally relevant data required for risk assessment studies. In this paper we describe a newly designed climatic hood that enables control of temperature, humidity, and air velocity within less than +/- 0.5% fluctuations during continuous operation. The performance of the evaporation system togetherwith the sampling and analytical procedures produced a meaningful concentration profile of vapors obtained from a 15 mg sample of VX dispersed as small droplets over a 10 x 16 cm piece of asphalt road. The released vapors amounted to approximately 30% of the applied mass, and its time course was best fitted to a triexponential curve with rate constants changing over time from 2.2 to 0.03 h(-1). The asphalt enhanced a specific degradation pathway of VX that is relatively minor in aqueous solutions. Results provide the first data on the volatilization of VX from samples of asphalt road, and offer an insight into VX behavior in the environment.

Role of edrophonium in prevention of the re-inhibition of acetylcholinesterase by phosphorylated oxime.

We examined the role of edrophonium in the acceleration phenomenon using mouse wild-type and mutant D74N AChE inhibited with 7-(O,O-diethyl-phosphinyloxy)-1-methylquinolinium methylsulfate (DEPQ). With DEPQ-inhibited wild-type mouse acetylcholinesterase (AChE), the reactivation kinetic profile demonstrated one-phase exponential association only when 2-[hydroxyimino methyl]-1-methylpyridinium chloride (2-PAM) and 1-(2-hydroxy-iminomethyl-1-pyridinium)-1-(4-carboxy-aminopyridi nium)-dimethyl ether hydrochloride (HI-6) were used as reactivators. When 1,1[oxybis-methylene)bis[4-(hydroxyimino)methyl] pyridinium dichloride (LüH6) and 1,1-trimethylene bis(4-hydroxyimino methyl) pyridinium dichloride (TMB4) were used, the reactivation kinetic profile was biphasic in nature. Edrophonium had no effect on reactivation by 2-PAM and HI-6, but significantly accelerated LüH6- and TMB4-induced reactivation of DEPQ-inhibited wild-type mouse AChE. Comparison of the initial and overall reactivation rate constants with five oximes indicated that acceleration by edrophonium may be due to the prevention of re-inhibition of the reactivated enzyme by the phosphorylated oxime (POX) produced during the reactivation. With LüH6 and TMB4, about 2.5-fold increase in the reactivation rate constants was observed in the presence of edrophonium, but little or no effect was observed with the other three oximes. The initial reactivation rate constants were 5.4- and 4.2-fold of the overall rate constants with LüH6 and TMB4 as reactivators respectively, however, very little change was found between the initial and overall rate constants with the other three oximes. In experiments with D74N AChE, for which the inhibition potency of charged organophosphate (OP) was two to three orders less than wild-type enzyme, edrophonium had no effect on the reactivation by LüH6 and TMB4 and the time courses of reactivation were monophasic. The data from mutant enzyme substantiate the involvement of edrophonium in protecting POX re-inhibition of reactivated enzyme formed during the reactivation of OP-inhibited AChE.

Characterization of O,O-diethylphosphoryl oximes as inhibitors of cholinesterases and substrates of phosphotriesterases.

Reactivators of organophosphate (OP)-inhibited cholinesterases (ChEs) are believed to give rise to phosphorylated oximes (POX) that reinhibit the enzyme. Diethylphosphoryl oximes (DEP-OX) that were generated in situ were demonstrated in the past to be unstable, yet were more potent inhibitors of acetylcholinesterase (AChE) than the parent OPs. In view of the inconsistencies among reported results, and the potential toxicity of POXs, it seemed important to characterize authentic DEP-OXs, and to evaluate their interference with reactivation of diethylphosphoryl-ChE (DEP-ChE) conjugates. To this end, the diethylphosphoric acid esters of 1-methyl-2-pyridinium carboxaldehyde oxime (DEP-2PAM) and 1-methyl-4 pyridinium carboxaldehyde oxime (DEP-4PAM) were synthesized and chemically defined. The half-lives of DEP-2PAM and DEP-4PAM in 10 mM Tris buffer, pH 7.8, at 29 degrees were found to be 10 and 980 sec, respectively. The two DEP-OXs inhibited ChEs with the following ranking order: for DEP-2PAM, human butyrylcholinesterase (HuBChE, k(i) = 2.03 x 10(9) M(-1) min(-1)) > mouse AChE (MoAChE) approximately equal to fetal bovine serum AChE (FBS-AChE) approximately equal to equine BChE (EqBChE); for DEP-4PAM, HuBChE (k(i) = 0.71 x 10(9) M(-1) min(-1)) > EqBChE > MoAChE > FBS-AChE. A dialkylarylphosphate hydrolase (phosphotriesterase; PTE) from Pseudomonas sp. catalyzed the hydrolysis of DEP-4PAM with k(cat)/Km = 3.56 x 10(7) M(-1) min(-1) and Km = 0.78 mM. Reactivation of DEP-ChEs was enhanced by PTE when 4-PAM-based oximes were used as reactivators, whereas reactivation with 2-PAM-based oximes was not affected by PTE. This observation is attributed primarily to the short half-life of DEP-OXs derived from the latter oximes. Relatively low doses of PTE can detoxify large quantities of DEP-OXs rapidly, and thereby augment the efficacy of antidotes that contain the oxime function in position 4 of the pyridine ring.

Prophylaxis against soman inhalation toxicity in guinea pigs by pretreatment alone with human serum butyrylcholinesterase.

Human butyrylcholinesterase (HuBChE) has previously been shown to protect mice, rats, and monkeys against multiple lethal toxic doses of organophosphorus (OP) anticholinesterases that were challenged by i.v. bolus injections. This study examines the concept of using a cholinesterase scavenger as a prophylactic measure against inhalation toxicity, which is the more realistic simulation of exposure to volatile OPs. HuBChE-treated awake guinea pigs were exposed to controlled concentration of soman vapors ranging from 417 to 430 micrograms/liter, for 45 to 70 s. The correlation between the inhibition of circulating HuBChE and the dose of soman administered by sequential i.v. injections and by respiratory exposure indicated that the fraction of the inhaled dose of soman that reached the blood was 0.29. HuBChE to soman molar ratio of 0.11 was sufficient to prevent the manifestation of toxic signs in guinea pigs following exposure to 2.17x the inhaled LD50 dose of soman (ILD50, 101 micrograms/kg). A slight increase in HuBChE:soman ratio (0.15) produced sign-free animals after two sequential respiratory exposures with a cumulative dose of 4.5x ILD50. Protection was exceptionally high and far superior to the currently used traditional approach that consisted of pretreatment with pyridostigmine and postexposure combined administration of atropine, benactyzine, and an oxime reactivator. Quantitative analysis of the results suggests that in vivo sequestration of soman, and presumably other OPs, by exogenously administered HuBChE, is independent of the species used or the route of challenge entry. This assuring conclusion significantly expands the database of the bioscavenger strategy that now offers a dependable extrapolation from animals to human.

Acceleration of oxime-induced reactivation of organophosphate-inhibited fetal bovine serum acetylcholinesterase by monoquaternary and bisquaternary ligands.

Reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) by oximes is the primary reason for their effectiveness in the treatment of OP poisoning. Reactivation is reported to accelerate by quaternary ligands such as decamethonium, which is devoid of nucleophilicity. The mechanism of this enhancement is not known. To better understand the acceleration phenomenon, we examined ligand modulations of oxime-induced reactivation of methylphosphonylated AChE using 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide and fetal bovine serum AChE. Edrophonium, decamethonium, and propidium, three quaternary AChE ligands of different types, were tested as potential accelerators. Experiments were carried out with both soluble enzyme preparation and AChE conjugated to polyurethane. Kinetic measurements with oximes 2-[hydroxyiminomethyl]-1-methylpyridinium chloride, 1,1'-trimethylene bis-(4-hydroxyimino methyl)-pyridinium dibromide, and 1, 1'-[oxybis-methylene)bis[4-(hydroxyimino)methyl]pyridiniu um dichloride showed that in the presence of 50 microM edrophonium, the reactivation rate constants increased 3.3-12.0-fold; 200 microM decamethonium produced a 1.6-3.0-fold enhancement of reactivation rate constants by the same oximes. Reactivation of the inhibited enzyme by 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(4-carboxy-aminopyridinium )-d imethyl ether hydrochloride, 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(3-carboxy-aminopyridinium )-d imethyl ether hydrochloride, and 1-[[[4-(aminocarbonyl)pyridino]methoxy]methyl]-2, 4, -bis(hydroxyimino)methyl pyridinium dichloride was not affected by either ligand. Propidium slowed the reactivation of 7-(methylethoxyphosphinyloxy)-1- methylquinolinium iodide-inhibited AChE by all oximes. Results suggest that the accelerator site may reside inside the catalytic gorge rather than at its entrance and acceleration may be due to the prevention of reinhibition of the regenerated enzyme by the putative product, the phosphonylated oxime. In addition to the nucleophilic property of the oximate anion, some of the reactivators may carry an accelerating determinant, as characterized with respect to edrophonium and decamethonium. Results offer possible explanations for the superiority of 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(4-carboxy-aminopyridinium )-d imethyl ether hydrochloride over other oximes in the reactivation of specific AChE-OP conjugates.

Combined effect of organophosphorus hydrolase and oxime on the reactivation rate of diethylphosphoryl-acetylcholinesterase conjugates.

Reactivation of inhibited acetylcholinesterase (AChE) is essential for rapid recovery after organophosphate (OP) poisoning. However, following administration of an oxime reactivator, such as pralidoxime mesylate (P2S), in patients poisoned with certain diethylphosphorothioate pesticides, no reactivation is observed, presumably due to reinhibition by circulating anti-cholinesterase OPs. Pretreatment alone with organophosphorus hydrolases (OPH) that are capable of rapidly hydrolyzing OPs was demonstrated, in animals, to confer significant protection against OP toxicity. One strategy to augment the potentially therapeutic scope of OPHs is a combined post-exposure treatment consisting of a drug(s) commonly used against OP toxicity and a suitable hydrolase. In this study, we examined the in vitro ability of OPH from Pseudomonas sp. (OPHps) to prevent reinhibition of P2S-reactivated AChE by excess OPs. The kinetic parameters of the reactivation of a series of diethylphosphoryl-AChE (DEP--AChE) conjugates, obtained by the use of various diethylphosphates, were determined and compared with the rates of reactivation in the presence of OPHps, with and without the OP inhibitors in the reactivation medium. Extrapolation of the in vitro results to in vivo conditions suggests that an OPHps concentration as low as 1 microgram/mL blood would result in a 100-fold decrease in the concentration of circulating anti-AChE pesticides within less than one blood-circulation time, thereby minimizing reinhibition of the reactivated enzyme. Thus, for DEP-based pesticides, the combination of P2S-OPH treatment can significantly improve clinical recovery after OP intoxication. In addition, it is shown here for the first time that an OPH can effectively hydrolyze quaternary ammonium-containing OPs. This indicates that hydrolysis of phosphorylated oximes, toxic side products of oxime treatment, may also be accelerated by OPHs.

Role of oligosaccharides in the pharmacokinetics of tissue-derived and genetically engineered cholinesterases.

To understand the role of glycosylation in the circulation of cholinesterases, we compared the mean residence time of five tissue-derived and two recombinant cholinesterases (injected intravenously in mice) with their oligosaccharide profiles. Monosaccharide composition analysis revealed differences in the total carbohydrate, galactose, and sialic acid contents. The molar ratio of sialic acid to galactose residues on tetrameric human serum butyrylcholinesterase, recombinant human butyrylcholinesterase, and recombinant mouse acetylcholinesterase was found to be approximately 1.0. For Torpedo californica acetylcholinesterase, monomeric and tetrameric fetal bovine serum acetylcholinesterase, and equine serum butyrylcholinesterase, this ratio was approximately 0.5. However, the circulatory stability of cholinesterases could not be correlated with the sialic acid-to-galactose ratio. Fractionation of the total pool of oligosaccharides obtained after neuraminidase digestion revealed one major oligosaccharide for human serum butyrylcholinesterase and three or four major oligosaccharides in other cholinesterases. The glycans of tetrameric forms of plasma cholinesterases (human serum butyrylcholinesterase, fetal bovine serum acetylcholinesterase, and equine serum butyrylcholinesterase) clearly demonstrated a reduced heterogeneity and higher maturity compared with glycans of monomeric fetal bovine serum acetylcholinesterase, dimeric tissue-derived T. californica acetylcholinesterase, and recombinant cholinesterases. T. californica acetylcholinesterase, recombinant cholinesterases, and monomeric fetal bovine serum acetylcholinesterase showed a distinctive shorter mean residence time (44-304 min) compared with tetrameric forms of plasma cholinesterases (1902-3206 min). Differences in the pharmacokinetic parameters of cholinesterases seem to be due to the combined effect of the molecular weight and charge- and size-based heterogeneity in glycans.

The stoichiometry of protection against soman and VX toxicity in monkeys pretreated with human butyrylcholinesterase.

Bioscavengers of organophophates (OP) have been examined as potential substitutes for the currently approved drug treatment against OP toxicity. The present work was designed to assess the ability of butyrylcholinesterase, purified from human serum (HuBChE), to prevent the toxicity induced by soman and VX in rhesus monkeys. The consistency of the data across species was then evaluated as the basis for the extrapolation of the data to humans. The average mean residence time of the enzyme in the circulation of monkeys following an intravenous loading was 34 hr. High bioavailability of HuBChE in blood (>80%) was demonstrated after intramuscular injection. A molar ratio of HuBChE:OP approximately 1.2 protected against an i.v. bolus injection of 2.1 x LD50 VX, while a ratio of 0.62 was sufficient to protect monkeys against an i.v. dose of 3.3 x LD50 of soman, with no additional postexposure therapy. A remarkable protection was also seen against soman-induced behavioral deficits detected in the performance of a spatial discrimination task. The consistency of the results across several species offers a reliable prediction of both the stoichiometry of the scavenging and the extent of prophylaxis with HuBChE against nerve agent toxicity in humans.

Structure of glycan moieties responsible for the extended circulatory life time of fetal bovine serum acetylcholinesterase and equine serum butyrylcholinesterase.

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

Large-scale purification and long-term stability of human butyrylcholinesterase: a potential bioscavenger drug.

Butyrylcholinesterase from human plasma (HuBChE) is a potential drug candidate for detoxification of certain harmful chemicals that contain carboxylic or phosphoric acid ester bonds. Large quantities of purified HuBChE, displaying a high stability upon long-term storage, are required for the evaluation of its therapeutic capacity and its pharmaceutical properties. Several modifications of a previously reported procedure enabled us to purify the enzyme > 15,000-fold from pools of up to 100 1 of human plasma. The three-step procedure is based on precipitation of plasma proteins by ammonium sulfate (step I) and batch adsorption of HuBChE on procainamide-Sepharose 4B gel (step II). Ammonium sulfate was also employed in the third stage to fractionate the final product from procainamide-containing HuBChE solution. The overall yield (63%) of electrophoretically pure enzyme was significantly higher than that previously reported (34%) for the purification of HuBChE from 12.5 1 of plasma or from 5 kg of Cohn fraction IV-4. Purified HuBChE was stored at 5 degrees C in 10 mM phosphate buffer (pH 7.4) containing 1 mM EDTA and 0.02% NaN3. The specific activity, protein migration on gel electrophoresis, thermostability at 54 degrees C and the mean residence time in the circulation of mice remained essentially constant for at least 46 months. The modifications introduced can provide large quantities of purified enzyme that maintains its activity and bioavailability properties for several years.

Amino acid residues controlling reactivation of organophosphonyl conjugates of acetylcholinesterase by mono- and bisquaternary oximes.

Single and multiple site mutants of recombinant mouse acetylcholinesterase (rMoAChE) were inhibited with racemic 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide (MEPQ) and the resulting mixture of two enantiomers, CH3PR,S(O)(OC2H5)-AChE(EMPR,S-AChE), were subjected to reactivation with 2-(hydroxyiminomethyl)-1-methylpyridinium methanesulfonate (P2S) and 1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4"-carbamoyl-1"- pyridinium)-2-oxapropane dichloride (HI-6). Kinetic analysis of the reactivation profiles revealed biphasic behavior with an approximate 1:1 ratio of two presumed reactivatable enantiomeric components. Equilibrium dissociation and kinetic rate constants for reactivation of site-specific mutant enzymes were compared with those obtained for wild-type rMoAChE, tissue-derived Torpedo AChE and human plasma butyrylcholinesterase. Substitution of key amino acid residues at the entrance to the active-site gorge (Trp-286, Tyr-124, Tyr-72, and Asp-74) had a greater influence on the reactivation kinetics of the bisquaternary reactivator HI-6 compared with the monoquaternary reactivator P2S. Replacement of Phe-295 by Leu enhanced reactivation by HI-6 but not by P2S. Of residues forming the choline-binding subsite, the E202Q mutation had a dominant influence where reactivation by both oximes was decreased 16- to 33-fold. Residues Trp-86 and Tyr-337 in this subsite showed little involvement. These kinetic findings, together with energy minimization of the oxime complex with the phosphonylated enzyme, provide a model for differences in the reactivation potencies of P2S and HI-6. The two kinetic components of oxime reactivation of MEPQ-inhibited AChEs arise from the chirality of O-ethyl methylphosphonyl moieties conjugated with Ser-203 and may be attributable to the relative stability of the phosphonyl oxygen of the two enantiomers in the oxyanion hole.

Characterization of monoclonal antibodies that inhibit the catalytic activity of acetylcholinesterases.

Monoclonal antibodies were generated against fetal bovine serum acetylcholinesterase and fetal bovine serum acetylcholinesterase inhibited by diisopropyl fluorophosphate or 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide. Six monoclonal antibodies inhibited 70 to > 98% of the catalytic activity of fetal bovine serum acetylcholinesterase. Inhibition of serum acetylcholinesterase from several mammalia by four monoclonal antibodies showed broad cross-reactivity. In all cases, monoclonal antibodies bound to the native form of acetylcholinesterases. None reacted with serum butyrylcholinesterases from various species. Although all monoclonal antibodies inhibited catalytic activity of acetylcholinesterases, the site of interaction with acetylcholinesterase appeared to differ for several antibodies. Two types of acetylcholinesterase:monoclonal antibody complexes were formed: one between tetrameric forms and another between catalytic subunits within the tetramer. Monoclonal antibodies that inhibited acetylcholinesterase activity at > 98% also considerably slowed binding of diisopropyl fluorophosphate and other organophosphorus compounds to the acetylcholinesterase:monoclonal antibody complex. Binding of these monoclonal antibodies to acetylcholinesterase influenced function of the enzyme's peripheral anionic site. None of the antibodies bound to the esteratic site of acetylcholinesterase. Monoclonal antibodies caused changes in catalytic activity of acetylcholinesterase by interaction at a site remote from the catalytic site, presumably at the entrance to the active site gorge.

Role of tyrosine 337 in the binding of huperzine A to the active site of human acetylcholinesterase.

Huperzine A (HUP), a natural, potent, 'slow,' reversible inhibitor of antiacetylcholinesterase (AChE), has been suggested to be superior to antiacetylcholinesterase drugs now being used for management of Alzheimer's disease. To delineate the binding site of human AChE (HuAChE) for HUP, the biochemical constants kon, koff, and Ki were determined for complexes formed between HUP and single-site (Y337F, Y337A, F295A, W286A, and E202Q) or double-site (F295L/F297V) mutants of recombinant HuAChE (rHuAChE). The kinetic and dissociation constants were compared with those obtained for wild-type rHuAChE and AChE from Torpedo californica. Results demonstrate that the inhibition of AChE by HUP occurs through association with residues located inside the active site 'gorge,' rather than at the rim of the gorge. Tyrosine at position 337 (Y337) is essential for inhibition of rHuAChE by HUP (Ki = 26 nM). An aromatic array constituted from residues Y337, F295, and probably W86 is likely to offer a multicontact subsite that interacts with the ammonium group and with both the exo-and endocyclic double bond moieties of HUP. Lack of the aromatic side chain in the position homologous to Y337 explains the poor inhibitory potency of HUP toward human butyrylcholinesterase (Ki > 20,000 nM). Replacement of the carboxylate-containing E202 by glutamine had only marginal effect on the stability of the complex formed between HUP and rHuAChE. The pH-rate profiles suggest that destabilization of the complex after proton gain cannot be attributed solely to protonation of E202. These findings are expected to establish HUP as a lead compound for the design of new anti-AChE drugs.

Huperzine A as a pretreatment candidate drug against nerve agent toxicity.

Huperzine A (HUP) is a naturally-occurring, potent, reversible inhibitor of acetylcholinesterase (AChE) that crosses the blood-brain barrier. To examine its ability to protect against nerve agent poisoning, HUP was administered i.p. to mice, and the s.c. LD50 of soman was determined at various time intervals after pretreatment. Results were compared to those obtained for animals treated with physostigmine. A protective ratio of approximately 2 was maintained for at least 6 hr after a single injection of HUP, without the need for any post-challenge drug therapy. By contrast, pretreatment with physostigmine increased the LD50 of soman by 1.4- to 1.5-fold for only up to 90 min. The long-lasting antidotal efficacy displayed by HUP correlated with the time course of the blood-AChE inhibition. The results suggest that the protection of animals by HUP from soman poisoning was achieved by temporarily sequestering the active site region of the physiologically important AChE.

Amplification of the effectiveness of acetylcholinesterase for detoxification of organophosphorus compounds by bis-quaternary oximes.

Pretreatment of rhesus monkeys with fetal bovine serum acetylcholinesterase (FBS AChE) provides complete protection against 5 LD50 of organophosphate (OP) without any signs of toxicity or performance decrements as measured by serial probe recognition tests or primate equilibrium platform performance (Maxwell et al., Toxicol Appl Pharmacol 115: 44-49, 1992; Wolfe et al., Toxicol Appl Pharmacol 117: 189-193, 1992). Although such use of enzyme as a single pretreatment drug for OP toxicity is sufficient to provide complete protection, a relatively large (stoichiometric) amount of enzyme was required in vivo to neutralize OP. To improve the efficacy of cholinesterases as pretreatment drugs, we have developed an approach in which the catalytic activity of OP-inhibited FBS AChE was rapidly and continuously restored, thus detoxifying the OP and minimizing enzyme aging by having sufficient amounts of appropriate oxime present. The efficacy of FBS AChE to detoxify several OPs was amplified by addition of bis-quaternary oximes, particularly 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(4-carboxyaminopyridinium) -dimethyl ether hydrochloride (HI-6). When mice were pretreated with sufficient amounts of FBS AChE and HI-6 and challenged with repeated doses of O-isopropyl methylphosphonofluridate (sarin), the OP was continuously detoxified so long as the molar concentration of the sarin dose was less than the molar concentration of AChE in circulation. The in vitro experiments showed that the stoichiometry of sarin:FBS AChE was higher than 3200:1 and in vivo stoichiometry with mice was as high as 57:1. Addition of HI-6 to FBS AChE as a pretreatment drug amplified the efficacy of enzyme as a scavenger of nerve agents.

Direct observation and elucidation of the structures of aged and nonaged phosphorylated cholinesterases by 31P NMR spectroscopy.

31P NMR spectroscopy of butyrylcholinesterase (BChE), acetylcholinesterase (AChE), and chymotrypsin (Cht) inhibited by pinacolyl methylphosphonofluoridate (soman), methylphosphonodifluoridate (MPDF), and diisopropyl phosphorofluoridate (DFP) allowed direct observation of the OP-linked moiety of aged (nonreactivatable) and nonaged organophosphorus (OP)-ChE conjugates. The 31P NMR chemical shifts of OP-ChE conjugates clearly demonstrated insertion of a P-O- bond into the active site of aged OP-ChE adducts. The OP moiety of nonaged OP-ChEs was shown to be uncharged. The OP-bound pinacolyl moiety of soman-inhibited and aged AChE was detached completely, whereas only partial dealkylation of the pinacolyl group was observed for soman-inhibited BChEs. This suggests that the latter enzyme reacted with the less active stereoisomer(s) of soman. In the case of soman-inhibited Cht, no dealkylation could be experimentally detected for any of the four stereoisomers of OP-Cht adducts. Results are consistent with the contention that the phenomenon of enzyme-catalyzed dealkylation of OP adducts of serine hydrolases strongly depends on the orientation of both the catalytic His and the carboxyl side chain of either Glu or Asp positioned next to the catalytic Ser. The denatured protein of aged OP-ChE or OP-Cht is a convenient leaving group in nucleophilic displacements of tetrahedral OP compounds despite the presence of a P-O- bond. This indicates that the unusual resistance to reactivation of the aged enzyme cannot be ascribed to simple electrostatic repulsion of an approaching nucleophile. The broadening of the 31P NMR signal of native OP-ChEs relative to that of OP-Cht is in agreement with the crystal structure of AChE, showing that the active site region of ChEs in solution resides in a deep, narrow gorge.

Prevention of soman-induced cognitive deficits by pretreatment with human butyrylcholinesterase in rats.

This study examined the ability of pretreatment with human serum butyrylcholinesterase (HuBChE) to prevent soman-induced cognitive impairments. Behavioral testing was carried out using the Morris water maze task evaluating learning, memory, and reversal learning processes. Pretreatment with HuBChE significantly prevented the memory and reversal learning impairments induced by soman. A small deficiency in performance was observed only during part of the learning period in HuBChE-treated rats after administration of soman. Results support the contention that pretreatment alone with HuBChE is sufficient to increase survival and to prevent impairment in cognitive functioning following exposure to soman.

Human butyrylcholinesterase as a general prophylactic antidote for nerve agent toxicity. In vitro and in vivo quantitative characterization.

Butyrylcholinesterase purified from human plasma (HuBChE) was evaluated both in vitro and in vivo in mice and rats as a single prophylactic antidote against the lethal effects of highly toxic organophosphates (OP). The variation among the bimolecular rate constants for the inhibition of HuBChE by tabun, VX, sarin, and soman was 10-fold (0.47 to 5.12 x 10(7) M-1 min-1; pH 8.0, 26 degrees). The half-life of HuBChE in blood after its i.v. administration in mice and rats was 21 and 46 hr, respectively. The peak blood-enzyme level was obtained in both species approximately 9-13 hr following i.m. injection of HuBChE, and the fraction of the enzyme activity absorbed into the blood was 0.9 and 0.54 for rats and mice, respectively. The stoichiometry of the in vivo sequestration of the anti-cholinesterase toxicants was consistent with the HuBChE/OP ratio of the molar concentration required to inhibit 100% enzyme activity in vitro. Linear correlation was demonstrated between the blood level of HuBChE and the extent of protection conferred against the toxicity of nerve agents. Pretreatment with HuBChE alone was sufficient not only to increase survivability following exposure to multiple median lethal doses of a wide range of potent OPs, but also to alleviate manifestation of toxic symptoms in mice and rats without the need for additional post-exposure therapy. It appeared that in order to confer protection against lethality nerve agents had to be scavenged to a level below their median lethal dose LD50 within less than one blood circulation time. Since the high rate of sequestration of nerve agents by HuBChE is expected to underlie the activity of the scavenger in other species as well, a reliable extrapolation of its efficacy from experimental animals to humans can be made.

Immunochemical characterization of anti-acetylcholinesterase inhibitory monoclonal antibodies.

Monoclonal antibodies (mAbs) were prepared against native or DFP-inhibited Torpedo californica acetylcholinesterase and native or DFP-, MEPQ-, and soman-inhibited fetal bovine serum acetylcholinesterase. The cross reactivity of these antibodies with acetylcholinesterases from various species and their ability to inhibit catalytic activity were determined. Eight antibodies were found to inhibit catalytic activity of either Torpedo or fetal bovine serum enzyme. In all cases the antibodies bound to the native form of the enzymes and in some cases even to the denatured form. None of the antibodies recognized human or horse serum butyrylcholinesterase. Sucrose density gradient centrifugation of enzyme-antibody complexes provided two types of profiles, one with multiple peaks, indicating numerous complexes between tetrameric forms of the enzyme, and the other with single peaks, demonstrating complex formation within the tetrameric form. Different antibodies appeared to interact with slightly different regions, but in all cases the binding encompassed the peripheral anionic site. Decrease in catalytic activity of the enzyme was most likely caused by conformational changes in the enzyme molecule resulting from interaction with these mAbs.