Author Report for: Ashani Y

Anthropogenic organophosphorus compounds (AOPCs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents, and pesticides. To date, only a handful of soil bacteria bearing a phosphotriesterase (PTE), the key enzyme in the AOPC degradation pathway, have been identified. Therefore, the extent to which bacteria are capable of utilizing AOPCs as a phosphorus source, and how widespread this adaptation may be, remains unclear. Marine environments with phosphorus limitation and increasing levels of pollution by AOPCs may drive the emergence of PTE activity. Here, we report the utilization of diverse AOPCs by four model marine bacteria and 17 bacterial isolates from the Mediterranean Sea and the Red Sea. To unravel the details of AOPC utilization, two PTEs from marine bacteria were isolated and characterized, with one of the enzymes belonging to a protein family that, to our knowledge, has never before been associated with PTE activity. When expressed in Escherichia coli with a phosphodiesterase, a PTE isolated from a marine bacterium enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPCs may provide bacteria a source of phosphorus in depleted environments and offers a prospect for the bioremediation of a pervasive class of anthropogenic pollutants.

Anthropogenic organophosphates (AOPs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents and pesticides. Soil bacteria bearing a phosphotriesterase (PTE) can degrade AOPs, but whether bacteria are capable of utilizing AOPs as a phosphorus source, and how widespread PTEs are in nature, remains unclear. Here, we report the utilization of diverse AOPs by four model marine bacteria and seventeen bacterial isolates from seawater samples. To unravel the details of AOP utilization, two novel PTEs from marine bacteria were isolated and characterized. When expressed in E. coli, these PTEs enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPs provides bacteria with a source of phosphorus in depleted environments and offers a new prospect for the bioremediation of a pervasive class of anthropogenic pollutants.

Stabilization of Torpedo californica acetylcholinesterase by the divalent cations Ca(+2) , Mg(+2) and Mn(+2) was investigated. All three substantially protect the enzyme from thermal inactivation. Electron paramagnetic resonance revealed one high-affinity binding site for Mn(+2) and several much weaker sites. Differential scanning calorimetry showed a single irreversible thermal transition. All three cations raise both the temperature of the transition and the activation energy, with the transition becoming more cooperative. The crystal structures of the Ca(+2) and Mg(+2) complexes with Torpedo acetylcholinesterase were solved. A principal binding site was identified. In both cases, it consists of four aspartates (a 4D motif), within which the divalent ion is embedded, together with several waters molecules. It makes direct contact with two of the aspartates, and indirect contact, via waters, with the other two. The 4D motif has been identified in 31 acetylcholinesterase sequences and 28 butyrylcholinesterase sequences. Zebrafish acetylcholinesterase also contains the 4D motif; it, too, is stabilized by divalent metal ions. The ASSAM server retrieved 200 other proteins that display the 4D motif, in many of which it is occupied by a divalent cation. It is a very versatile motif, since, even though tightly conserved in terms of rmsd values, it can contain from one to as many as three divalent metal ions, together with a variable number of waters. This novel motif, which binds primarily divalent metal ions, is shared by a broad repertoire of proteins. This article is protected by copyright. All rights reserved.

Nerve agents are organophosphates (OPs) that potently inhibit acetylcholinesterase, and their enzymatic detoxification has been a long-standing goal. Nerve agents vary widely in size, charge, hydrophobicity and the cleavable ester bond. A single enzyme is therefore unlikely to efficiently hydrolyze all agents. Here, we describe a mixture of three previously developed variants of the bacterial phosphotriesterase (Bd-PTE) that are highly stable and nearly sequence identical. This mixture enables effective detoxification of a broad spectrum of known threat agents-GA (tabun), GB (sarin), GD (soman), GF (cyclosarin), VX and Russian-VX. The potential for dimer dissociation and exchange that could inactivate Bd-PTE has minimal impact, and the three enzyme variants are as active in a mixture as they are individually. To our knowledge, this engineered enzyme 'cocktail' comprises the first solution for enzymatic detoxification of the entire range of threat nerve agents.

Recent years have seen an increasing number of incidence, in which organophosphate nerve agents (OPNAs) have been used against civilians with devastating outcomes. Current medical countermeasures against OPNA intoxications are aimed at mitigating their symptoms, but are unable to effectively prevent them. In addition, they may fail to prevent the onset of a cholinergic crisis in the brain and its secondary toxic manifestations. The need for improved medical countermeasures has led to the development of bioscavengers; proteins and enzymes that may prevent intoxication by binding and inactivating OPNAs before they can reach their target organs. Non-catalytic bioscavengers such as butyrylcholinesterase, can rapidly bind OPNA molecules in a stoichiometric and irreversible manner, but require the administration of large protein doses to prevent intoxication. Thus, many efforts have been made to develop catalytic bioscavengers that could rapidly detoxify OPNAs without being inactivated in the process. Such enzymes may provide effective prophylactic protection and improve post-exposure treatments using much lower protein doses. Here we review attempts to develop catalytic bioscavengers using molecular biology, directed evolution and enzyme engineering techniques; and natural or computationally designed enzymes. These include both stoichiometric scavengers and enzymes that can hydrolyze OPNAs with low catalytic efficiencies. We discuss the catalytic parameters of evolved and engineered enzymes and the results of in-vivo protection and post-exposure experiments performed using OPNAs and bioscavengers. Finally, we briefly address some of the challenges that need to be met in order to transition these enzymes into clinically approved drugs.

Catalytic scavengers of organophosphates (OPs) are considered very promising antidote candidates for preventing the adverse effects of OP intoxication as stand alone treatments. This study aimed at correlating the in-vivo catalytic efficiency ((kcat/KM)[Enzyme]pl), established prior to the OP challenge, with the severity of symptoms and survival rates of intoxicated animals. The major objective was to apply a theoretical approach to estimate a lower limit for (kcat/KM)[Enzyme]pl that will be adequate for establishing the desired kcat/KM value and plasma concentration of efficacious catalytic bioscavengers. Published data sets by our group and others, from in vivo protection experiments executed in the absence of any supportive medicine, were analyzed. The kcat/KM values of eight OP hydrolyzing enzymes and their plasma concentrations in four species exposed to OPs via s.c., i.m. and oral gavage, were analyzed. Our results show that regardless of the OP type and the animal species employed, sign-free animals were observed following bioscavenger treatment provided the theoretically estimated time period required to detoxify 96% of the OP (t96%) in-vivo was </=10 s. This, for example, can be achieved by an enzyme with kcat/KM = 5 x 107 M-1 min-1 and a plasma concentration of 0.4 muM ((kcat/KM)[Enzyme]pl = 20 min-1). Experiments in which animals were intoxicated by i.v. OP injections did not always conform to this rule, and in some cases resulted in high mortality rates. We suggest that in vivo evaluation of catalytic scavengers should avoid the unrealistic bolus i.v. route of OP exposure.

Structure-based drug design utilizes apo-protein or complex structures retrieved from the PDB. >57% of crystallographic PDB entries were obtained with polyethyleneglycols (PEGs) as precipitant and/or as cryoprotectant, but >6% of these report presence of individual ethyleneglycol oligomers. We report a case in which ethyleneglycol oligomers' presence in a crystal structure markedly affected the bound ligand's position. Specifically, we compared the positions of methylene blue and decamethonium in acetylcholinesterase complexes obtained using isomorphous crystals precipitated with PEG200 or ammonium sulfate. The ligands' positions within the active-site gorge in complexes obtained using PEG200 are influenced by presence of ethyleneglycol oligomers in both cases bound to W84 at the gorge's bottom, preventing interaction of the ligand's proximal quaternary group with its indole. Consequently, both ligands are approximately 3.0A further up the gorge than in complexes obtained using crystals precipitated with ammonium sulfate, in which the quaternary groups make direct pi-cation interactions with the indole. These findings have implications for structure-based drug design, since data for ligand-protein complexes with polyethyleneglycol as precipitant may not reflect the ligand's position in its absence, and could result in selecting incorrect drug discovery leads. Docking methylene blue into the structure obtained with PEG200, but omitting the ethyleneglycols, yields results agreeing poorly with the crystal structure; excellent agreement is obtained if they are included. Many proteins display features in which precipitants might lodge. It will be important to investigate presence of precipitants in published crystal structures, and whether it has resulted in misinterpreting electron density maps, adversely affecting drug design. This article is protected by copyright. All rights reserved.

Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at approximately 2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20 degrees C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.

Organophosphate (OP) based pesticides are highly toxic compounds that are still widely used in agriculture around the world. According to World Health Organization (WHO) data, it is estimated that between 250,000 and 370,000 deaths occur yearly around the globe as a result of acute intoxications by pesticides. Currently available antidotal drug treatments of severe OP intoxications are symptomatic, do not reduce the level of intoxicating OP in the body and have limited ability to prevent long-term brain damage. Pesticide poisonings present a special therapeutic challenge since in many cases, such as with parathion, their toxicity stems from their metabolites that inhibit the essential enzyme acetylcholinesterase. Our goal is to develop a new treatment strategy for parathion intoxication by combining a catalytic bioscavenger that rapidly degrades the intoxicating parathion-metabolite (paraoxon) in the blood, with a glutamate bioscavenger that reduces the elevated concentration of extracellular glutamate in the brain following OP intoxication. We report on the development of a novel catalytic bioscavenger by directed evolution of serum paraoxonase 1 (PON1) that effectively detoxifies paraoxon in-vivo. We also report preliminary results regarding the utilization of this PON1 variant together with a recombinant human enzyme glutamate oxaloacetate transaminase 1 (rGOT1), suggesting that a dual PON-GOT treatment may increase survival and recovery from parathion and paraoxon intoxications.

The nearly 200,000 fatalities following exposure to organophosphorus (OP) pesticides each year and the omnipresent danger of a terroristic attack with OP nerve agents emphasize the demand for the development of effective OP antidotes. Standard treatments for intoxicated patients with a combination of atropine and an oxime are limited in their efficacy. Thus, research focuses on developing catalytic bioscavengers as an alternative approach using OP-hydrolyzing enzymes such as Brevundimonas diminuta phosphotriesterase (PTE). Recently, a PTE mutant dubbed C23 was engineered, exhibiting reversed stereoselectivity and high catalytic efficiency (k cat/K M) for the hydrolysis of the toxic enantiomers of VX, CVX, and VR. Additionally, C23's ability to prevent systemic toxicity of VX using a low protein dose has been shown in vivo. In this study, the catalytic efficiencies of V-agent hydrolysis by two newly selected PTE variants were determined. Moreover, in order to establish trends in sequence-activity relationships along the pathway of PTE's laboratory evolution, we examined k cat/K M values of several variants with a number of V-type and G-type nerve agents as well as with different OP pesticides. Although none of the new PTE variants exhibited k cat/K M values >107 M-1 min-1 with V-type nerve agents, which is required for effective prophylaxis, they were improved with VR relative to previously evolved variants. The new variants detoxify a broad spectrum of OPs and provide insight into OP hydrolysis and sequence-activity relationships.

The recent attacks with the nerve agent sarin in Syria reveal the necessity of effective countermeasures against highly toxic organophosphorus compounds. Multiple studies provide evidence that a rapid onset of antidotal therapy might be life-saving but current standard antidotal protocols comprising reactivators and competitive muscarinic antagonists show a limited efficacy for several nerve agents. We here set out to test the newly developed phosphotriesterase (PTE) mutant C23AL by intravenous (i.v.), intramuscular (i.m.; model for autoinjector) and intraosseous (i.o.; model for intraosseous insertion device) application in an in vivo guinea pig model after VX challenge ( approximately 2LD50). C23AL showed a Cmax of 0.63mumolL(-1) after i.o. and i.v. administration of 2mgkg(-1) providing a stable plasma profile up to 180min experimental duration with 0.41 and 0.37mumolL(-1) respectively. The i.m. application of C23AL did not result in detectable plasma levels. All animals challenged with VX and subsequent i.o. or i.v. C23AL therapy survived although an in part substantial inhibition of erythrocyte, brain and diaphragm AChE was detected. Theoretical calculation of the time required to hydrolyze in vivo 96.75% of the toxic VX enantiomer is consistent with previous studies wherein similar activity of plasma containing catalytic scavengers of OPs resulted in non-lethal protection although accompanied with a variable severity of cholinergic symptoms. The relatively low C23AL plasma level observed immediately after its i.v. or i.o load, point at a possible volume of distribution greater than the guinea pig plasma content, and thus underlines the necessity of in vivo experiments in antidote research. In conclusion the i.o. application of PTE is efficient and resulted in comparable plasma levels to the i.v. application at a given time. Thus, i.o. vascular access systems could improve the post-exposure PTE therapy of nerve agent poisoning.

The potent human toxicity of organophosphorus (OP) nerve agents calls for the development of effective antidotes. Standard treatment for nerve agent poisoning with atropine and an oxime has a limited efficacy. An alternative approach is the development of catalytic bioscavengers using OP-hydrolyzing enzymes such as paraoxonases (PON1). Recently, a chimeric PON1 mutant, IIG1, was engineered toward the hydrolysis of the toxic isomers of soman and cyclosarin with high in vitro catalytic efficiency. In order to investigate the suitability of IIG1 as a catalytic bioscavenger, an in vivo guinea pig model was established to determine the protective effect of IIG1 against the highly toxic nerve agent cyclosarin. Prophylactic i.v. injection of IIG1 (1 mg/kg) prevented systemic toxicity in cyclosarin (~2LD50)-poisoned guinea pigs, preserved brain acetylcholinesterase (AChE) activity, and protected erythrocyte AChE activity partially. A lower IIG1 dose (0.2 mg/kg) already prevented mortality and reduced systemic toxicity. IIG1 exhibited a high catalytic efficiency with a homologous series of alkylmethylfluorophosphonates but had low efficiency with the phosphoramidate tabun and was virtually ineffective with the nerve agent VX. This quantitative analysis validated the model for predicting in vivo protection by catalytic bioscavengers based on their catalytic efficiency, the level of circulating enzyme, and the dose of the intoxicating nerve agent. The in vitro and in vivo results indicate that IIG1 may be considered as a promising candidate bioscavenger to protect against the toxic effects of a range of highly toxic nerve agents.

The highly toxic organophosphorus (OP) nerve agent VX is characterized by a remarkable biological persistence which limits the effectiveness of standard treatment with atropine and oximes. Existing OP hydrolyzing enzymes show low activity against VX and hydrolyze preferentially the less toxic P(+)-VX enantiomer. Recently, a phosphotriesterase (PTE) mutant, C23, was engineered towards the hydrolysis of the toxic P(-) isomers of VX and other V-type agents with relatively high in vitro catalytic efficiency (kcat/KM=5x106M-1min-1). To investigate the suitability of the PTE mutant C23 as a catalytic scavenger, an in vivo guinea pig model was established to determine the efficacy of post-exposure treatment with C23 alone against VX intoxication. Injection of C23 (5mgkg-1 i.v.) 5min after s.c. challenge with VX ( approximately 2LD50) prevented systemic toxicity. A lower C23 dose (2mgkg-1) reduced systemic toxicity and prevented mortality. Delayed treatment (i.e., 15min post VX) with 5mgkg-1 C23 resulted in survival of all animals and only in moderate systemic toxicity. Although C23 did not prevent inhibition of erythrocyte acetylcholinesterase (AChE) activity, it partially preserved brain AChE activity. C23 therapy resulted in a rapid decrease of racemic VX blood concentration which was mainly due to the rate of degradation of the toxic P(-)-VX enantiomer that correlates with the C23 blood levels and its kcat/KM value. Although performed under anesthesia, this proof-of-concept study demonstrated for the first time the ability of a catalytic bioscavenger to prevent systemic VX toxicity when given alone as a single post-exposure treatment, and enables an initial assessment of a time window for this approach. In conclusion, the PTE mutant C23 may be considered as a promising starting point for the development of highly effective catalytic bioscavengers for post-exposure treatment of V-agents intoxication.

VX and its Russian (RVX) and Chinese (CVX) analogues rapidly inactivate acetylcholinesterase and are the most toxic stockpile nerve agents. These organophosphates have a thiol leaving group with a choline-like moiety and are hydrolyzed very slowly by natural enzymes. We used an integrated computational and experimental approach to increase Brevundimonas diminuta phosphotriesterase's (PTE) detoxification rate of V-agents by 5000-fold. Computational models were built of the complex between PTE and V-agents. On the basis of these models, the active site was redesigned to be complementary in shape to VX and RVX and to include favorable electrostatic interactions with their choline-like leaving group. Small libraries based on designed sequences were constructed. The libraries were screened by a direct assay for V-agent detoxification, as our initial studies showed that colorimetric surrogates fail to report the detoxification rates of the actual agents. The experimental results were fed back to improve the computational models. Overall, five rounds of iterating between experiment and model refinement led to variants that hydrolyze the toxic SP isomers of all three V-agents with kcat/KM values of up to 5 x 10(6) M(-1) min(-1) and also efficiently detoxify G-agents. These new catalysts provide the basis for broad spectrum nerve agent detoxification.

The photosensitizer, methylene blue (MB), generates singlet oxygen ((1)O2) that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark MB inhibits reversibly, binding being accompanied by a bathochromic shift that can be used to show its displacement by other reversible inhibitors binding to the catalytic 'anionic' subsite (CAS), the peripheral 'anionic' subsite (PAS), or bridging them. Data concerning both reversible and irreversible inhibition are here reviewed. MB protects TcAChE from thermal denaturation, and differential scanning calorimetry reveals a approximately 8 degrees C increase in the denaturation temperature. The crystal structure of the MB/TcAChE complex reveals a single MB stacked against W279 in the PAS, pointing down the gorge towards the CAS. The intrinsic fluorescence of the irreversibly inhibited enzyme displays new emission bands that can be ascribed to N'-formylkynurenine (NFK); this was indeed confirmed using anti-NFK antibodies. Mass spectroscopy revealed that two Trp residues, Trp84 in the CAS, and Trp279 in the PAS, were the only Trp residues, out of a total of 14, significantly modified by photo-oxidation, both being converted to NFK. In the presence of competitive inhibitors that displace MB from the gorge, their modification is completely prevented. Thus, photo-oxidative damage caused by MB involves targeted release of (1)O2 by the bound photosensitizer within the aqueous milieu of the active-site gorge.

A preferred strategy for preventing nerve agents intoxication is catalytic scavenging by enzymes that hydrolyze them before they reach their targets. Using directed evolution, we simultaneously enhanced the activity of a previously described serum paraoxonase 1 (PON1) variant for hydrolysis of the toxic S(P) isomers of the most threatening G-type nerve agents. The evolved variants show <=340-fold increased rates and catalytic efficiencies of 0.2-5 x 10(7) M(-1) min(-1). Our selection for prevention of acetylcholinesterase inhibition also resulted in the complete reversion of PON1's stereospecificity, from an enantiomeric ratio (E) < 6.3 x 10(-4) in favor of the R(P) isomer of a cyclosarin analog in wild-type PON1, to E > 2,500 for the S(P) isomer in an evolved variant. Given their ability to hydrolyze G-agents, these evolved variants may serve as broad-range G-agent prophylactics.

The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (k(cat)/K(m)) of ~10(4) M(-1) s(-1) after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the R(P) isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.

The photosensitizer methylene blue MB generates singlet oxygen that irreversibly inhibits Torpedo californica acetylcholinesterase TcAChE In the dark it inhibits reversibly Binding is accompanied by a bathochromic absorption shift used to demonstrate displacement by other acetylcholinesterase inhibitors interacting with the catalytic anionic subsite CAS the peripheral anionic subsite PAS or bridging them MB is a noncompetitive inhibitor of TcAChE competing with reversible inhibitors directed at both anionic subsites but a single site is involved in inhibition MB also quenches TcAChE's intrinsic fluorescence It binds to TcAChE covalently inhibited by a small organophosphate OP but not an OP containing a bulky pyrene Differential scanning calorimetry shows an 8 degrees increase in the denaturation temperature of the MB/TcAChE complex relative to native TcAChE and a less than twofold increase in cooperativity of the transition The crystal structure reveals a single MB stacked against Trp279 in the PAS oriented down the gorge toward the CAS it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330 midway down the gorge in the MB/TcAChE crystal structure precludes simultaneous binding of a second MB at the CAS Conversely binding of ligands at the CAS dislodges MB from its preferred locus at the PAS The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage.

The principal role of AChE (acetylcholinesterase) is termination of impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine. The active site of AChE is near the bottom of a long and narrow gorge lined with aromatic residues. It contains a CAS (catalytic 'anionic' subsite) and a second PAS (peripheral 'anionic' site), the gorge mouth, both of which bind acetylcholine via pi-cation interactions, primarily with two conserved tryptophan residues. It was shown previously that generation of 1O2 by illumination of MB (Methylene Blue) causes irreversible inactivation of TcAChE (Torpedo californica AChE), and suggested that photo-oxidation of tryptophan residues might be responsible. In the present study, structural modification of the TcAChE tryptophan residues induced by MB-sensitized oxidation was investigated using anti-N-formylkynurenine antibodies and MS. From these analyses, we determined that N-formylkynurenine derivatives were specifically produced from Trp84 and Trp279, present at the CAS and PAS respectively. Peptides containing these two oxidized tryptophan residues were not detected when the competitive inhibitors, edrophonium and propidium (which should displace MB from the gorge) were present during illumination, in agreement with their efficient protection against the MB-induced photo-inactivation. Thus the bound MB elicited selective action of 1O2 on the tryptophan residues facing on to the water-filled active-site gorge. The findings of the present study thus demonstrate the localized action and high specificity of MB-sensitized photo-oxidation of TcAChE, as well as the value of this enzyme as a model system for studying the mechanism of action and specificity of photosensitizing agents.

An ex vivo protocol was developed to assay the antidotal capacity of rePON1 variants to protect endogenous acetylcholinesterase and butyrylcholinesterase in human whole blood against OP nerve agents. This protocol permitted us to address the relationship between blood rePON1 concentrations, their kinetic parameters, and the level of protection conferred by rePON1 on the cholinesterases in human blood, following a challenge with cyclosarin (GF). The experimental data thus obtained were in good agreement with the predicted percent residual activities of blood cholinesterases calculated on the basis of the rate constants for inhibition of human acetylcholinesterase and butyrylcholinesterase by GF, the concentration of the particular rePON1 variant, and its k(cat)/K(m) value for GF. This protocol thus provides a rapid and reliable ex vivo screening tool for identification of rePON1 bioscavenger candidates suitable for protection of humans against organophosphorus-based toxicants. The results also permitted the refinement of a mathematical model for estimating the efficacious dose of rePON1s variants required for prophylaxis in humans.

Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates. We improved PON1's catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1's activity toward the coumarin analog of S(p)-cyclosarin by approximately 10(5)-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with k(cat)/K(M) approximately 10(7) M(-1) min(-1). We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.

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.

Organophosphate compounds (OP) are potent inhibitors of acetylcholinesterases (AChEs) and can cause lethal poisoning in humans. Inhibition of AChEs by the OP soman involves phosphonylation of the catalytic serine, and subsequent dealkylation produces a form known as the "aged" enzyme. The nonaged form can be reactivated to a certain extent by nucleophiles, such as pralidoxime (2-PAM), whereas aged forms of OP-inhibited AChEs are totally resistant to reactivation. Here, we solved the X-ray crystal structures of AChE from Torpedo californica (TcAChE) conjugated with soman before and after aging. The absolute configuration of the soman stereoisomer adduct in the nonaged conjugate is P(S)C(R). A structural reorientation of the catalytic His440 side chain was observed during the aging process. Furthermore, the crystal structure of the ternary complex of the aged conjugate with 2-PAM revealed that the orientation of the oxime function does not permit nucleophilic attack on the phosphorus atom, thus providing a plausible explanation for its failure to reactivate the aged soman/AChE conjugate. Together, these three crystal structures provide an experimental basis for the design of new reactivators.

The dimeric form of Torpedo californica acetylcholinesterase provides a valuable experimental system for studying transitions between native, partially unfolded, and unfolded states since long-lived partially unfolded states can be generated by chemical modification of a nonconserved buried cysteine residue, Cys 231, by denaturing agents, by oxidative stress, and by thermal inactivation. Elucidation of the 3D structures of complexes of Torpedo californica acetylcholinesterase with a repertoire of reversible inhibitors permits their classification into three categories: (a) active-site directed inhibitors, which interact with the catalytic anionic subsite, at the bottom of the active-site gorge, such as edrophonium and tacrine; (b) peripheral anionic site inhibitors, which interact with a site at the entrance to the gorge, such as propidium and d-tubocurarine; and (c) elongated gorge-spanning inhibitors, which bridge the two sites, such as BW284c51 and decamethonium. The effects of these three categories of reversible inhibitors on the stability of Torpedo californica acetylcholinesterase were investigated using spectroscopic techniques and differential scanning calorimetry. Thermodynamic parameters obtained calorimetrically permitted quantitative comparison of the effects of the inhibitors on the enzyme's thermal stability. Peripheral site inhibitors had a relatively small effect, while gorge-spanning ligands and those binding at the catalytic anionic site, had a much larger stabilizing effect. The strongest effect was, however, observed with the polypeptide toxin, fasciculin II (FasII), even though, in terms of its binding site, it belongs to the category of peripheral site ligands. The stabilizing effect of the ligands binding at the anionic subsite of the active site, like that of the gorge-spanning ligands, may be ascribed to their capacity to stabilize the interaction between the two subdomains of the enzyme. The effect of fasciculin II may be ascribed to the large surface area of interaction (>2000 A(2)) between the two proteins. Stabilization of Torpedo californica acetylcholinesterase by both divalent cations and chemical chaperones was earlier shown to be due to a shift in equilibrium between the native state and a partially unfolded state ( Millard et al. ( 2003 ) Protein Sci. 12 , 2337 - 2347 ). The low molecular weight inhibitors used in the present study may act similarly and can thus be considered as pharmacological chaperones for stabilizing the fully folded native form of the enzyme.

Controlled inhibition of brain acetyl- and butyrylcholinesterases (AChE and BChE, respectively) and of monoamine oxidase-B (MAO-B) may slow neurodegeneration in Alzheimer's and Parkinson's diseases. It was postulated that certain carbamate esters would inhibit AChE and BChE with the concomitant release in the brain of the OH-derivatives of rasagiline or selegiline that can serve as inhibitors of MAO-B and as antioxidants. We conducted a detailed in vitro kinetic study on two series of novel N-methyl, N-alkyl carbamates and compared them with rivastigmine, a known anti-Alzheimer drug. The rates of carbamylation (k(i)) and decarbamylation (k(r)) of recombinant human AChE were mainly determined by the size of the N-alkyl substituent and to a lesser extent by the nature of the leaving group. k(i) was highest when the alkyl was methyl, hexyl, cyclohexyl, or an aromatic substituent and lowest when it was ethyl. This suggested that k(i) depends on a delicate balance between the length of the residue and its degree of freedom of rotation. By contrast, presumably because of its wider gorge, inhibition of human BChE was less influenced by the size of the alkyl group and more dependent on the structure of the leaving group. The data show how the degree of enzyme inhibition can be manipulated by structural changes in the N-methyl, N-alkyl carbamates and the corresponding leaving group to achieve therapeutic levels of brain AChE, BChE, and MAO-B inhibition.

In the present study, we applied for the first time (31)P diffusion NMR to resolve different species obtained by the addition of organophosphorus compounds (OP) such as diisopropyl phosphorofluoridate (DFP) or 1-pyrenebutyl phosphorodichloridate (PBPDC) to alpha-chymotrypsin (Cht). (31)P diffusion NMR was used since the products of these reactions constitute a mixture of OP-covalent conjugates of the enzyme and OP-containing hydrolysis products that have noninformative (1)H NMR spectra. It was shown that the peak, attributed to the covalent native diisopropylphosphoryl-Cht (DIP-Cht) conjugate by chemical shift considerations, has a greater diffusion coefficient (D = (0.65 +/- 0.01) x 10(-5) cm(2) s(-1)) than expected from its molecular mass (approximately 25 kDa). This peak was therefore suggested to consist of at least two superimposed signals of diisopropyl phosphoryl (DIP) pools of high and low molecular weights that happen to have the same chemical shift. This conclusion was substantiated by the use of DMSO-d(6) that separated the overlapping signals. Diffusion measurements performed on the extensively dialyzed and unfolded DIP-Cht conjugate still resulted in a high diffusion coefficient ((0.30 +/- 0.05) x 10(-5) cm(2) s(-1)) relative to the assumed molecular mass. This observation was attributed to a dynamic dealkylation at the OP moiety (i.e., aging) that occurred during the relatively long diffusion measurements, where DIP-Cht was converted to the corresponding monoisopropyl phosphoryl Cht (MIP-Cht) conjugate. Homogeneous aged forms of OP-Cht were obtained by use of DFP and heat-induced dealkylation of DIP-Cht, and by PBPDC that provided the aged form via the hydrolysis of a P-Cl bond (PBP-Cht). The thermally stable aged conjugates enabled a reliable determination of the diffusion coefficients over several days of data acquisition, and the values found were (0.052 +/- 0.002) x 10(-5) cm(2) s(-1) and (0.054 +/-0.004) x 10(-5) cm(2) s(-1) for the MIP-Cht and the PBP-Cht adducts, respectively, values in the range expected for a species with a molecular weight of 25 kDa. The advantages and limitations of (31)P diffusion NMR in corroborating the type of species that prevail in such systems are briefly discussed.

Human butyrylcholinesterase (HuBChE) is a drug candidate for protection against organophosphates (OP) intoxication. A mathematically based model was validated and employed to better understand the role of the endogenous HuBChE in detoxification of OPs and to estimate the dose of exogenous HuBChE required for enhancing protection of humans from lethal exposure to OPs. The model addresses the relationship between the HuBChE dose needed to maintain a certain residual activity of human acetylcholinesterase (HuAChE) and the following parameters: (1) level and duration of exposure, (2) bimolecular rate constants of inhibition of HuAChE (kA) and HuBChE (kB) by OPs, and (3) time elapsed from enzyme load. The equation derived for the calculation of HuBChE dose requires the knowledge of kA/kB in human blood and the rate constant of HuBChE elimination. Predictions of HuBChE doses were validated by in vitro experiments and data of published human studies. These predictions highlight two parameters that are likely to decrease the calculated dose: (1) the rapid consumption of the less toxic isomers of OPs in human plasma, and (2) the volume of distribution of HuBChE that appears significantly greater than the volume of plasma. The first part of the analysis of the proposed model was focused on acute bolus exposures and suggests that upper limit doses of 134, 115, and 249 mg/70 kg are sufficient to protect RBC AChE above 30% of baseline activity following a challenge with 1 LD(50) VX, soman, and sarin, respectively. The principles of the validated model should be applicable for advanced predictions of HuBChE dose for protection against continuous exposures to OPs.

Fluoride ion is a reversible inhibitor of human butyrylcholinesterase (HuBChE) that is a viable drug candidate against organophosphates (OPs) toxicity. Since large numbers of communities in many countries are occasionally exposed to relatively high amount of fluoride, its effect on the kinetics of inhibition of HuBChE by OPs was investigated. In saline phosphate, pH 7.4, fluoride in the lower millimolar range significantly slowed the inhibition of HuBChE by paraoxon, DFP, echothiophate, soman, sarin, and VX. The kinetics of the inhibition was found consistent with the formation of a reversible fluoride-HuBChE complex that is at least 25-fold less active towards phosphorylation or phosphonylation than the free enzyme. Heat inactivation experiments indicate that the binding of fluoride to HuBChE probably involves enhanced cross-domain interaction via hydrogen bonds formation that may decrease enzyme activity. In spite of distinct structural differences among the OP used, the dissociation constants of the fluoride-HuBChE reversible complex varied over a narrow range (KF, 0.31-0.70 mM); however, KF in human plasma increased to 2.75-3.40 mM. 19F-NMR spectroscopy revealed that fluoride ion is complexed to plasma components, an observation that explains in part the apparent increase in KF. Results suggest that an estimate of the relative decrease in the rate of OPs sequestration in presence of fluoride can be obtained from the fraction of the free HuBChE (1 + [F]/K(F))(-1). Considering KF values in human plasma, it is concluded that the scavenging efficacy of OPs by HuBChE is not compromised by the normal concentration range of circulating fluoride ions.

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

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

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Huperzine A, an alkaloid isolated from Huperzia serrata was found to reversibly inhibit acetylcholinesterases (EC 3.1.1.7) and butyrylcholinesterases (EC 3.1.1.8) with on- and off-rates that depend on both the type and the source of enzyme. Long-term incubation of high concentrations of purified cholinesterases (1-8 microM) with huperzine A did not show any chemical modification of huperzine A. A low dissociation constant KI was obtained for mammalian acetylcholinesterase-huperzine (20-40 nM) compared to mammalian butyrylcholinesterase-huperzine (20-40 microM). This indicates that the thermodynamic stability of huperzine-cholinesterase complex may depend on the number and type of aromatic amino acid residues in the catalytic pocket region of the cholinesterase molecule.

We demonstrate here the correlation between protection afforded by pretreatment alone with parathion hydrolase purified from Pseudomonas sp. against tabun toxicity in mice and the kinetic parameters which are assumed to determine the in vivo detoxification of tabun by the same enzyme. Results show that 15 and 22 micrograms of parathion hydrolase per animal conferred a protective ratio of 3.94 and 5.65 respectively, against tabun toxicity, without post-exposure treatment.

Parathion hydrolase purified from Pseudomonas sp. was injected i.v. into mice to demonstrate the feasibility of using organophosphorus acid anhydride (OPA) hydrolases as pretreatment against organophosphates (OP) poisoning. Results show that exogenous administration of as low as 7 to 26 micrograms of parathion hydrolase conferred protection against challenge with multiple median lethal doses (LD50) of diethyl p-nitrophenyl phosphate (paraoxon; 3.8-7.3 x LD50) and diethylfluorophosphate (DEFP; 2.9 x LD50) without administration of supportive drugs. The extent of protection observed was consistent with blood-parathion hydrolase levels and the kinetic constants of the enzymatic hydrolysis of paraoxon and DEFP by parathion hydrolase. OPA hydrolases not only appear to be potential prophylactic drugs capable of increasing survival ratio following OP intoxication but also to alleviate post-exposure symptoms.

Human butyrylcholinesterase (BChE, EC 3.1.1.8) or acetylcholinesterase (AChE, EC 3.1.1.7) from fetal bovine serum (FBS), administered i.v. in mice, sequestered at approximately 1:1 stoichiometry the highly toxic anti-ChE organophosphate, 1,2,2-trimethylpropyl methyl-fluorophosphonate (soman). A quantitative linear correlation was demonstrated between blood-ChE levels and the protection conferred by exogeneously administered ChE. Results presented here demonstrate that either human BChE or FBS-AChE is an effective prophylactic measure sufficient to protect mice from multiple LD50S of soman without the administration of post-treatment supportive drugs.

We have successfully demonstrated that exogenously administered acetyl- or butyrylcholinesterase (AChE, BChE respectively) will sequester organophosphates (OPs) before they reach their physiological targets. In addition, a third enzyme, endogenous carboxylesterase is known to be capable of scavenging OPs. In these studies, we have administered AChE and BChE to three different species of animals (mice, marmosets and monkeys) which were challenged with three different OPs (VX, MEPQ and soman). Results obtained from these systematic studies demonstrate that: (a) a quantitative linear correlation exists between blood AChE levels and the protection afforded by exogenously administered ChEs in animals challenged with OP, (b) approximately one mole of either AChE or BChE sequesters one mole of OP, (c) such prophylactic measures are sufficient to protect animals against OPs without the administration of any supportive drugs. Thus the OP dose, the blood-level of esterase, the ratio of the circulating enzyme to OP challenge, and the rate of reaction between them determine the overall efficacy of an enzyme as a pretreatment drug. The biochemical mechanism underlying the sequestration of various OPs by the use of exogenously administered scavenging esterases is the same in all species of animals studied. Therefore, the extrapolation of the results obtained by the use of ChE prophylaxis in animals to humans should be more reliable and effective than extrapolating the results from currently used multidrug antidotal modalities.

"Aged" organophosphoryl conjugates of serine hydrolases differ from the corresponding "non-aged" conjugates in their striking resistance to nucleophilic reactivation. The refined X-ray structures of "aged" and "non-aged" organophosphoryl conjugates of gamma-chymotrypsin were compared in order to understand the molecular basis for this resistance of "aged" conjugates. "Aged" and "non-aged" crystalline organophosphoryl-gamma-chymotrypsin conjugates were obtained by prolonged soaking of native gamma-chymotrypsin crystals with appropriate organophosphates. Thus, a representative "non-aged" conjugate, diethylphosphoryl-gamma-chymotrypsin, was obtained by soaking native crystals with paraoxon (diethyl-p-nitrophenyl phosphate), and a closely related "aged" conjugate, monoisopropyl-gamma-chymotrypsin, was obtained by soaking with diisopropylphosphorofluoridate. In both crystalline conjugates, the refined structures clearly reveal a high occupancy of the active site by the appropriate organophosphoryl moiety within covalent bonding distance of Ser195 O gamma. Whereas in the "non-aged" conjugate both ethyl groups can be visualized clearly, in the putative "aged" conjugate, as expected, only one isopropyl group is present. There is virtually no difference between the "aged" and "non-aged" conjugates either with respect to the conformation of the polypeptide backbone as a whole or with respect to the positioning of the side-chains within the active site. In the "aged" conjugate, however, close proximity (2.6 A) of the negatively charged phosphate oxygen atom of the dealkylated organophosphoryl group to His57 N epsilon 2 indicates the presence of a salt bridge between these two moieties. In contrast, in the "non-aged" conjugate the DEP moiety retains its two alkyl groups; thus, lacking a negative oxygen atom, it does not enter into such a charge-charge interaction and its nearest oxygen atom is 3.6 A away from His57 N epsilon 2. It is suggested that steric constraints imposed by the salt bridge in the "aged" conjugate lie at the basis of its resistance to reactivation.

Monoclonal antibody 25B1 generated against diisopropyl phosphorofluoridate inhibited fetal bovine serum acetylcholinesterase has been extensively characterized with respect to its anticholinesterase properties. This antibody demonstrated considerably different properties from previously reported inhibitory antibodies raised against acetylcholinesterase in terms of the degree of inhibition (greater than 98%), the high degree of specificity, and the stability of the antigen-antibody complex. Monoclonal antibody 25B1 appears to be directed against a conformational epitope located in close proximity to the catalytic center of the enzyme and was found to be most suitable for studying the stabilization of the active site of acetylcholinesterase against denaturation by heat or guanidine following phosphorylation by organophosphorus anticholinesterase compounds. This approach allowed the determination of stability rank order of various phosphorylated acetylcholinesterases. Among all the organophosphates tested, the combination of a methyl group and a negatively charged oxygen attached to the P atom, CH3P(O)(O-)-AChE, conferred the greatest protection to the active site of aged or nonaged organophosphoryl conjugates of acetylcholinesterase.

Fetal bovine serum acetylcholinesterase (FBS-AChE, EC 3.1.1.7) was titrated, both in vitro and in vivo, with a highly toxic anti-ChE organophosphate, 7-(methylethoxyphosphinyloxy)-1-methyl-quinolinium iodie (MEPQ). Approximately 1:1 stoichiometry was obtained for the sequestration of MEPQ by FBS-AChE in mice. A quantitative, linear correlation was demonstrated between blood-AChE levels and the protection afforded by exogenously administered AChE in mice when challenged with anti-ChE MEPQ. The results presented in this report demonstrate that such prophylactic measures are indeed sufficient to protect animals against poisoning by as high as an 8 x LD50 dose of organophosphate without the administration of any supportive drug. Despite the relatively large toxic dose, most of the mice that survived the challenge did not show any classical clinical signs of severe anti-ChE poisoning. MEPQ may be considered a suitable model compound for studying the quantitative aspects of the scavenger prophylactic approach described here.

Homologous aged and nonaged fluorescent organophosphorus conjugates of alpha-chymotrypsin (Cht) were used in a comparative spectroscopic study of the conformation of their active sites, employing the pyrene group as the fluorescent probe. Steady-state fluorescence measurements showed that the quantum yield of the pyrene probe which is stoichiometrically attached to the active site is ca. 20% lower in the aged conjugate, pyrenebutyl-O-P(O)(O-)-Cht (PBP-Cht), than in the nonaged conjugate, pyrenebutyl-O-P-(O)(OC2H5)-Cht (PBEP-Cht). Furthermore, fluorescence decay data indicate that quenching is dynamic and is not caused by oxygen. These data, together with collisional quenching data, imply that quenching originates in an internal interaction of the fluorophore with a group within the protein. Thus, interaction of the pyrene moiety with the polypeptide chain is significantly stronger in the aged than in the nonaged conjugate, implying a different orientation of the fluorophore with respect to the protein. Circular dichroism measurements, which reflect the asymmetry of the bound pyrene in the ground state, as well as circularly polarized luminescence studies, which reflect its asymmetry in the excited state, also show that the relative configuration of the pyrene moiety and the polypeptide chain is significantly altered upon aging. Aged conjugates obtained by use of various fluorescenct organophosphates [pyrenebutyl-O-P(O)Cl2, pyrenebutyl-O-P(O)(p-nitrophenoxy)Cl, pyrenebutyl-O-P(O)(p-nitrophenoxy)2] exhibit similar spectroscopic features, thus substantiating the hypothesis that instantaneous aging, by use of pyrenebutyl-O-P(O)Cl2, and dynamic aging, by gradual removal of an aryloxy group, yield a similar product. This finding provides strong support for the formation of a P-O- moiety in the aged conjugates, since the only expected common product of the two processes is PB-O-P(O)(O-)-Cht. Formation of excimers of the pyrene-containing organophosphorylchymotrypsin conjugates at concentrations above 3 X 10(-6) M is also reported.

7-(Methylethoxy phosphinyloxy)-1-methyl-quinolinium iodide (MEPQ), a powerful anti-cholinesterase methylphosphonate ester, was labeled with tritium (9 Ci/mmol) at the methylphosphonyl moiety (TCH2P(O)(OR)X) by an iodine-tritium replacement reaction. Kinetic measurements of the rate of inhibition of acetylcholinesterase (AChE) by [3H]MEPQ and its rate of hydrolysis in alkaline solution confirmed the identity of [3H]MEPQ with authentic MEPQ, which was prepared by the same reaction sequences. Gel-filtration experiments verified the radiospecificity of [3H]MEPQ. In vitro radiolabeling of both AChE and butyrylcholinesterase along with the whole-body autoradiography of [3H]MEPQ-treated mice suggests that [3H]MEPQ is a convenient marker for studying biological systems containing these esterases.

To substantiate reported data and improve the properties of anticholinesterase drugs in blood-brain barrier (B-BB) research, 7-(methylethoxyphosphinyloxy) 1-methyl-quinolinium iodide (MEPQ) was prepared and evaluated as an inhibitor of both acetyl- and butyrylcholinesterase (AChE and BuChE, respectively) from various sources. The second-order rate constants for the inhibition of cholinesterase from eel, mice brain and horse serum at 25 degrees were found to be 5.3 X 10(8), 1.3 X 10(8) and 5.4 X 10(7) M-1 min-1 respectively. The inhibited enzyme could be reactivated by 1-methyl-2-hydroxy iminomethylpyridinium iodide (2-PAM). The two enantiomers of the racemic mixture MEPQ inhibited AChE at similar rates. Low concentrations of AChE could be determined by the residual enzyme activity and by fluorescence measurements of the leaving group, thus suggesting the application of MEPQ as a sensitive titrant of cholinesterase, as well as a potential tool in studying B-BB permeability changes.

Covalent molecular combinations of a cyclic phosphate (dioxaphosphorinane) and a potential leaving group, such as 3-(trimethylammonio)phenol iodide (TMPH), suggested the synthesis of O-[3-(trimethylammonio)phenyl]-1,3,2-dioxaphosphorinane 2-oxide iodide (TDPI). TDPI inhibited acetylcholinesterase (AChE) (ki = 8.4 x 10(3) M-1 min-1) via the formation of an unstable covalent intermediate. TDPI-inhibited AChE hydrolyzed spontaneously with t1/2 approximately equal to 10 min. Butyrylcholinesterase (BuChE) was also inhibited by TDPI (ki = 1.8 x 10(4) M-1 min-1), but the inhibited BuChE was more stable (greater than 10 times) than the corresponding AchE-TDPI conjugate. Pretreatment of mice with TDPI conferred protection against 22 LD50's of paraoxon and 5 LD50's of soman, provided that treatment with anticholinergics and an oxime followed administration of these anticholinesterase poisons. Correlation between in vitro and in vivo observations suggests that the main protection of AChE conferred by TDPI results from temporary masking of the active site of the enzyme. The acute toxicity of TDPI was found to be 444 mg/kg (sc, mice), whereas analogous carbamates and a noncyclic phosphate also displaying antidotal properties are greater than 170 times more toxic.

Aging of organophosphoryl-acetylcholinesterase (AChE) conjugates, involving dealkylation of the bound organophosphoryl group, renders AChE resistant to reactivation by 2-pyridinealdoxime methiodide (2-PAM). The fluorescent organophosphates 1-pyrenebutyl ethylphosphorochloridate (PBEPC) and 1-pyrenebutylphosphorodichloridate (PBPDC) react stoichiometrically with purified electric eel AChE. PBEPC forms a non-aged AChE conjugate readily reactivated by 2-PAM; PBPDC forms an aged conjugate which cannot be reactivated. There is no difference in the wavelengths of excitation and emission maxima between the aged and non-aged AChE conjugates. However, the fluorescence quantum yield of pyrene in the non-aged conjugate is reduced by ca. 50% compared to the aged conjugate and from the shortening of the fluorescence decay time in the non-aged conjugate, it is concluded that the quenching is primarily dynamic. It is suggested that in the aged conjugate the organophosphoryl moiety is less accessible to the external medium than in the non-aged conjugate.

Aging of acetylcholinesterase (AChE) inhibited by certain organophosphates such as diisopropylfluorophosphate apparently involves dealkylation of the bound organophosphoryl moiety; this renders the inactive enzyme resistant to reactivation by quaternary oximes such as 2-pyridinealdoxime methiodide (2-PAM) which are used in therapy of organophosphate intoxication. The fluorescent pyrenyl organophosphates synthesized in this study were designed to detect putative conformational changes which might explain this resistance. The following inhibitors: 1-pyrenebutyl phosphorodichloride (PBPDC), 1-pyrenebutyl ethylphosphorochloridate (PBEPC), and 1-pyrenebutyl ethylphosphorofluoridate (PBEPF), react specifically with purified electric eel AChE (ki = 10(6)-10(7) M-1 min-1). AChE inhibited by PBEPC and PBEPF was readily reactivated by 2-PAM, while enzyme inhibited PBPDC could not be reactivated. Conjugates were prepared of both PBEPC and PBPDC with AChE, each containing one molecule of florophore per catalytic subunit. Thus two stoichiometric conjugates, PBEP-AChE (non-aged) and POBP-AChE (aged), were obtained. The two complexes exhibited identical absorption spectra, but differed in their steady-state fluorescence spectra. Although the wave-lenths of the excitation and emission spectra were similar, the pyrene fluorescence of the non-aged conjugate was ca. 50% quenched relative to the aged conjugate. Nanosecond fluorescence decay studies revealed two principal lifetime components of pyrene fluorescence. Both were longer for the aged (PBP-AChE) than for the non-aged (PBEP-AChE) conjugate and revealed a ca. 50% lower quantum yield for the non-aged as compared to the aged conjugate. A possible interpretation for these results is that in the aged conjugate the organophosphoryl moiety is less acessible to the external medium. Measurement of quenching of pyrene fluorescence in the aged and non-aged conjugates by the peripheral anionic site ligand propidium also indicated marked conformational differences between the two conjugates, and circular polarization of luminescence measurements revealed that propidium itself induced a substantial conformational change in both conjugates. Fluorescence lifetime measurements revealed that whereas propidium had little effect on the decay parameters for the non-aged conjugate it caused a decrease in lifetime and in relative quantum yield for the aged conjugate. PBEPF virtually eliminated cholinesterase activity in dissociated cord and brain cultures. Fluorescence microscopy reveals fine green fluorescent grains distinctly located throughout many neurons and glia. Labelling is much more pronounced in larger and older neurons. No specific fluorescence could be detected in cultures preincubated with nonfluorescent organophosphates.

In attempt to improve distribution and transport qualities of antidotes against organophosphorus poisoning, a new series of pyridine aldoximes linked to glucose moiety were synthesized and studied both in vivo and in vitro. Preliminary results describing the biological activity of the new compounds are presented and discussed in this report.