Substrate Analogues for the Enzyme-Catalyzed Detoxification of the Organophosphate Nerve Agents-Sarin, Soman, and Cyclosarin.

The G-type nerve agents, sarin (GB), soman (GD), and cyclosarin (GF), are among the most toxic compounds known. Much progress has been made in evolving the enzyme phosphotriesterase (PTE) from Pseudomonas diminuta for the decontamination of the G-agents; however, the extreme toxicity of the G-agents makes the use of substrate analogues necessary. Typical analogues utilize a chromogenic leaving group to facilitate high-throughput screening, and substitution of an O-methyl for the P-methyl group found in the G-agents, in an effort to reduce toxicity. Till date, there has been no systematic evaluation of the effects of these substitutions on catalytic activity, and the presumed reduction in toxicity has not been tested. A series of 21 G-agent analogues, including all combinations of O-methyl, p-nitrophenyl, and thiophosphate substitutions, have been synthesized and evaluated for their ability to unveil the stereoselectivity and catalytic activity of PTE variants against the authentic G-type nerve agents. The potential toxicity of these analogues was evaluated by measuring the rate of inactivation of acetylcholinesterase (AChE). All of the substitutions reduced inactivation of AChE by more than 100-fold, with the most effective being the thiophosphate analogues, which reduced the rate of inactivation by about 4-5 orders of magnitude. The analogues were found to reliably predict changes in catalytic activity and stereoselectivity of the PTE variants and led to the identification of the BHR-30 variant, which has no apparent stereoselectivity against GD and a kcat/Km of 1.4 × 106, making it the most efficient enzyme for GD decontamination reported till date.

Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of sarin and soman-butyrylcholinesterase adducts in human plasma.

Organophosphorus nerve agent (OPNA) adducts formed with human butyrylcholinesterase (HuBuChE) can be used as biomarker of OPNA exposure. Indeed, intoxication by OPNAs can be confirmed by the LC/MS2 analysis of a specific HuBuChE nonapeptide on which OPNAs covalently bind. A fast, selective, and highly sensitive online method was developed to detect sarin and soman adducts in plasma, including immunoextraction by anti-HuBuChE antibodies, pepsin digestion on immobilized enzyme reactors (IMER), and microLC/MS2 analysis of the OPNA adducts. The potential of three different monoclonal antibodies, covalently grafted on sepharose, was compared for the extraction of HuBuChE. The online method developed with the most promising antibodies allowed the extraction of up to 100% of HuBuChE contained in plasma and the digestion of 45% of it in less than 40 min. Moreover, OPNA-HuBuChE adducts, aged OPNA adducts, and unadducted HuBuChE could be detected (with S/N > 2000), even in plasma spiked with a low concentration of OPNA (10 ng mL-1). Finally, the potential of this method was compared to approaches involving other affinity sorbents, already described for HuBuChE extraction. Graphical abstract Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of organophosphorous nerve agent adducts formed with human butyrylcholinesterase.

Resolving pathways of interaction of mipafox and a sarin analog with human acetylcholinesterase by kinetics, mass spectrometry and molecular modeling approaches.

The hydroxyl oxygen of the catalytic triad serine in the active center of serine hydrolase acetylcholinesterase (AChE) attacks organophosphorus compounds (OPs) at the phosphorus atom to displace the primary leaving group and to form a covalent bond. Inhibited AChE can be reactivated by cleavage of the Ser-phosphorus bond either spontaneously or through a reaction with nucleophilic agents, such as oximes. At the same time, the inhibited AChE adduct can lose part of the molecule by progressive dealkylation over time in a process called aging. Reactivation of the aged enzyme has not yet been demonstrated. Here, our goal was to study oxime reactivation and aging reactions of human AChE inhibited by mipafox or a sarin analog (Flu-MPs, fluorescent methylphosphonate). Progressive reactivation was observed after Flu-MPs inhibition using oxime 2-PAM. However, no reactivation was observed after mipafox inhibition with 2-PAM or the more potent oximes used. A peptide fingerprinted mass spectrometry (MS) method, which clearly distinguished the peptide with the active serine (active center peptide, ACP) of the human AChE adducted with OPs, was developed by MALDI-TOF and MALDI-TOF/TOF. The ACP was detected with a diethyl-phosphorylated adduct after paraoxon inhibition, and with an isopropylmethyl-phosphonylated and a methyl-phosphonylated adduct after Flu-MPs inhibition and subsequent aging. Nevertheless, nonaged nonreactivated complexes were seen after mipafox inhibition and incubation with oximes, where MS data showed an ACP with an NN diisopropyl phosphoryl adduct. The kinetic experiments showed no reactivation of activity. The computational molecular model analysis of the mipafox-inhibited hAChE plots of energy versus distance between the atoms separated by dealkylation showed a high energy demand, thus little aging probability. However, with Flu-MPs and DFP, where aging was observed in our MS data and in previously published crystal structures, the energy demand calculated in modeling was lower and, consequently, aging appeared as a more likely reaction. We document here direct evidence for a phosphorylated hAChE refractory to oxime reactivation, although we observed no aging.

Synthesis and in vitro kinetic evaluation of N-thiazolylacetamido monoquaternary pyridinium oximes as reactivators of sarin, O-ethylsarin and VX inhibited human acetylcholinesterase (hAChE).

Presently available medications for treatment of organiphosphorus poisoning are not sufficiently effective due to various pharmacological and toxicological reasons. In this regard, herein we report the synthesis of a series of N-thiazolylacetamide monoquaternary pyridinium oximes and its analogs (1a-1b to 6a-6b) with diversely substituted thiazole ring and evaluation of their in vitro reactivation efficacies against nerve agent (sarin, O-ethylsarin and VX) inhibited human erythrocyte acetylcholinesterase (hAChE). Reactivation kinetics was performed to determine dissociation constant (KD), reactivity rate constant (kr) and the second order rate constant (kr2) for all the compounds and compared their efficacies with commercial antidotes viz. 2-PAM and obidoxime. All the newly synthesized oximes were evaluated for their physicochemical parameters (pKa) and correlated with their respective reactivation efficacies to assess the capability of the oxime reactivator. Three of these novel compounds showed promising reactivation efficacies toward OP inhibited hAChE. Molecular docking studies were performed in order to correlate the reactivation efficacies with their interactions in the active site of the AChE.

Novel nucleophiles enhance the human serum paraoxonase 1 (PON1)-mediated detoxication of organophosphates.

Paraoxonase 1 (PON1) is a calcium-dependent hydrolase associated with serum high-density lipoprotein particles. PON1 hydrolyzes some organophosphates (OPs), including some nerve agents, through nucleophilic attack of hydroxide ion (from water) in the active site. Most OPs are hydrolyzed inefficiently. This project seeks to identify nucleophiles that can enhance PON1-mediated OP degradation. A series of novel nucleophiles, substituted phenoxyalkyl pyridinium oximes, has been synthesized which enhance the degradation of surrogates of sarin (nitrophenyl isopropyl methylphosphonate; NIMP) and VX (nitrophenyl ethyl methylphosphonate; NEMP). Two types of in vitro assays have been conducted, a direct assay using millimolar concentrations of substrate with direct spectrophotometric quantitation of a hydrolysis product (4-nitrophenol) and an indirect assay using submicromolar concentrations of substrate with quantitation by the level of inhibition of an exogenous source of acetylcholinesterase from non-hydrolyzed substrate. Neither NIMP nor NEMP is hydrolyzed effectively by PON1 if one of these novel oximes is absent. However, in the presence of eight novel oximes, PON1-mediated degradation of both surrogates occurs. Computational modeling has created a model of PON1 embedded in phospholipid and has indicated general agreement of the binding enthalpies with the relative efficacy as PON1 enhancers. PON1 enhancement of degradation of OPs could be a unique and unprecedented mechanism of antidotal action.

Selective chromo-fluorogenic detection of DFP (a Sarin and Soman mimic) and DCNP (a Tabun mimic) with a unique probe based on a boron dipyrromethene (BODIPY) dye.

A novel colorimetric probe (P4) for the selective differential detection of DFP (a Sarin and Soman mimic) and DCNP (a Tabun mimic) was prepared. Probe P4 contains three reactive sites; i.e. (i) a nucleophilic phenol group able to undergo phosphorylation with nerve gases, (ii) a carbonyl group as a reactive site for cyanide; and (iii) a triisopropylsilyl (TIPS) protecting group that is known to react with fluoride. The reaction of P4 with DCNP in acetonitrile resulted in both the phosphorylation of the phenoxy group and the release of cyanide, which was able to react with the carbonyl group of P4 to produce a colour modulation from pink to orange. In contrast, phosphorylation of P4 with DFP in acetonitrile released fluoride that hydrolysed the TIPS group in P4 to yield a colour change from pink to blue. Probe P4 was able to discriminate between DFP and DCNP with remarkable sensitivity; limits of detection of 0.36 and 0.40 ppm for DCNP and DFP, respectively, were calculated. Besides, no interference from other organophosphorous derivatives or with presence of acid was observed. The sensing behaviour of P4 was also retained when incorporated into silica gel plates or onto polyethylene oxide membranes, which allowed the development of simple test strips for the colorimetric detection of DCNP and DFP in the vapour phase. P4 is the first probe capable of colorimetrically differentiating between a Tabun mimic (DCNP) and a Sarin and Soman mimic (DFP).

Fluorescent polymeric aggregates for selective response to sarin surrogates.

By combining the sensitivity of fluorescent units with the response of "smart" polymers to environmental changes, we propose a new approach for chemical detection applications. The system proved to be sensitive to 12 ppb of diethyl chlorophosphate (DCP), a Sarin surrogate, and to discriminate between the interfering molecules.

Characterization of an array of Love-wave gas sensors developed using electrospinning technique to deposit nanofibers as sensitive layers.

The electrospinning technique has allowed that very different materials are deposited as sensitive layers on Love-wave devices forming a low cost and successful sensor array. Their excellent sensitivity, good linearity and short response time are reported in this paper. Several materials have been used to produce the nanofibers: polymers as Polyvinyl alcohol (PVA), Polyvinylpyrrolidone (PVP) and Polystirene (PS); composites with polymers as PVA+SnCl4; combined polymers as PS+Poly(styrene-alt-maleic anhydride) (PS+PSMA) and metal oxides (SnO2). In order to test the array, well-known chemical warfare agent simulants (CWAs) have been chosen among the volatile organic compounds due to their importance in the security field. Very low concentrations of these compounds have been detected by the array, such as 0.2 ppm of DMMP, a simulant of sarin nerve gas, and 1 ppm of DPGME, a simulant of nitrogen mustard. Additionally, the CWA simulants used in the experiment have been discriminated and classified using pattern recognition techniques, such as principal component analysis and artificial neural networks.

Detoxification of alkyl methylphosphonofluoridates by an oxime-substituted ß-cyclodextrin--an in vitro structure-activity study.

Detoxification rates of a series of alkyl methylphosphonofluoridates by an oxime-substituted ß-cyclodextrin (ß-CD) were assessed quantitatively by using an AChE inhibition assay. The cyclodextrin (CD) derivative was identified in previous work as a highly active cyclosarin scavenger. Here, a structure-activity relationship was established by investigating the effect of this CD on the detoxification of sarin derivatives differing in the structure of the alkoxy residue. The results show that detoxification rates correlate with the steric bulk and chain length of the alkoxy group in the organophosphonate (OP). OPs with larger, more bulky residues are detoxified more rapidly, with the exception of soman, which is bearing a pinacolyloxy side chain. In addition, the substituted CD was in every case more active than unsubstituted, native ß-CD with up to a 400-fold difference. Comparing the kinetic results obtained with the known thermodynamic stabilities of related ß-CD complexes indicate that detoxification rates generally increase when the alkoxy residue on the OP is exchanged by a residue, which forms a more stable complex with ß-CD. This correlation lends support to the proposed mode of action of the substituted CD, involving initial complexation of the OP followed by reaction between the CD and the OP. The moderate to high efficacy on the detoxification of sarin derivatives suggests the potential applicability of this CD as a small molecule scavenger for G-type nerve agents.

Acute effects of a sarin-like organophosphorus agent, bis(isopropyl methyl)phosphonate, on cardiovascular parameters in anaesthetized, artificially ventilated rats.

The organophosphorus compound sarin irreversibly inhibits acetylcholinesterase. We examined the acute cardiovascular effects of a sarin-like organophosphorus agent, bis(isopropyl methyl)phosphonate (BIMP), in anaesthetized, artificially ventilated rats. Intravenous administration of BIMP (0.8mg/kg; the LD50 value) induced a long-lasting increase in blood pressure and tended to increase heart rate. In rats pretreated with the non-selective muscarinic-receptor antagonist atropine, BIMP significantly increased both heart rate and blood pressure. In atropine-treated rats, hexamethonium (antagonist of ganglionic nicotinic receptors) greatly attenuated the BIMP-induced increase in blood pressure without changing the BIMP-induced increase in heart rate. In rats treated with atropine plus hexamethonium, intravenous phentolamine (non-selective α-adrenergic receptor antagonist) plus propranolol (non-selective ß-adrenergic receptor antagonist) completely blocked the BIMP-induced increases in blood pressure and heart rate. In atropine-treated rats, the reversible acetylcholinesterase inhibitor neostigmine (1mg/kg) induced a transient increase in blood pressure, but had no effect on heart rate. These results suggest that in anaesthetized rats, BIMP induces powerful stimulation of sympathetic as well as parasympathetic nerves and thereby modulates heart rate and blood pressure. They may also indicate that an action independent of acetylcholinesterase inhibition contributes to the acute cardiovascular responses induced by BIMP.

Naltrexone prevents delayed encephalopathy in rats poisoned with the sarin analogue diisopropylflurophosphate.

BACKGROUND: Acute poisoning with organophosphate compounds can cause chronic neuropsychological disabilities not prevented by standard antidotes of atropine and pralidoxime. We determine the efficacy of naltrexone in preventing delayed encephalopathy after poisoning with the sarin analogue diisofluorophosphate (DFP) in rats. METHODS: A randomized controlled experiment was conducted. Rats were randomly assigned to receive a single intraperitoneal (IP) injection of 5 mg/kg DFP (n = 12) or vehicle control (isopropyl alcohol, n = 5). Rats were observed for cholinesterase toxicity and treated with IP atropine (2 mg/kg) and pralidoxime (25 mg/kg) as needed. After resolution of acute toxicity, rats injected with DFP were again randomized to receive daily injections of naltrexone (5 mg/kg per day) or saline (vehicle control). Control animals also received daily injections of saline. For 4 weeks after acute poisoning, rats underwent neurologic testing with the Morris Water Maze for assessment of spatial learning and reference memory. Comparisons on each test day were made across groups using analysis of variance followed by Fisher's least significant difference. Comparisons of changes in performance between first and last test day within each group were made using a paired t test. Significance was determined at P < .05. RESULTS: All rats receiving DFP developed toxicity requiring rescue. Spatial learning was significantly worse in the DFP-only group compared with the naltrexone-treated and control groups at day 10 (P = .0078), day 13 (P = .01), day 24 (P = .034), and day 31 (P = .03). No significant differences in reference memory were detected at any time point. CONCLUSION: Naltrexone protected against impairment of spatial learning from acute poisoning with DFP in rats.

A common mechanism for resistance to oxime reactivation of acetylcholinesterase inhibited by organophosphorus compounds.

Administration of oxime therapy is currently the standard approach used to reverse the acute toxicity of organophosphorus (OP) compounds, which is usually attributed to OP inhibition of acetylcholinesterase (AChE). Rate constants for reactivation of OP-inhibited AChE by even the best oximes, such as HI-6 and obidoxime, can vary >100-fold between OP-AChE conjugates that are easily reactivated and those that are difficult to reactivate. To gain a better understanding of this oxime specificity problem for future design of improved reactivators, we conducted a QSAR analysis for oxime reactivation of AChE inhibited by OP agents and their analogues. Our objective was to identify common mechanism(s) among OP-AChE conjugates of phosphates, phosphonates and phosphoramidates that result in resistance to oxime reactivation. Our evaluation of oxime reactivation of AChE inhibited by a sarin analogue, O-methyl isopropylphosphonofluoridate, or a cyclosarin analogue, O-methyl cyclohexylphosphonofluoridate, indicated that AChE inhibited by these analogues was at least 70-fold more difficult to reactivate than AChE inhibited by sarin or cyclosarin. In addition, AChE inhibited by an analogue of tabun (i.e., O-ethyl isopropylphosphonofluoridate) was nearly as resistant to reactivation as tabun-inhibited AChE. QSAR analysis of oxime reactivation of AChE inhibited by these OP compounds and others suggested that the presence of both a large substituent (i.e., ≥ the size of dimethylamine) and an alkoxy substituent in the structure of OP compounds is the common feature that results in resistance to oxime reactivation of OP-AChE conjugates whether the OP is a phosphate, phosphonate or phosphoramidate.

Testing of novel brain-penetrating oxime reactivators of acetylcholinesterase inhibited by nerve agent surrogates.

A critical need for combating the effects of organophosphate (OP) anticholinesterases, such as nerve agents, is the current lack of an effective oxime reactivator which can penetrate the blood-brain barrier (BBB), and therefore reactivate inhibited acetylcholinesterase (AChE) in the brain. Our laboratories have synthesized and have initiated testing of novel phenoxyalkyl pyridinium oximes (patent pending) that are more lipophilic than currently approved oximes. This is a preliminary report on these novel oximes which have been tested in vitro in rat brain homogenates with highly relevant surrogates for sarin (phthalimidyl isopropyl methylphosphonate; PIMP) and VX (nitrophenyl ethyl methylphosphonate; NEMP). The oximes demonstrated a range of 14-76% reactivation of rat brain AChE in vitro. An in vivo testing paradigm was developed in which the novel oxime was administered at the time of maximal brain AChE inhibition (about 80%) (1h) elicited by nitrophenyl isopropyl methylphosphonate (NIMP; sarin surrogate). This paradigm, with delayed administration of oxime to a time when brain AChE was starting to recover, was designed to minimize reactivation/reinhibition of peripheral AChE during the reactivation period which would decrease the availability of the surrogate for entry into the brain; this paradigm will allow proof of concept of BBB penetrability. The initial studies of these oximes in vivo with the sarin surrogate NIMP have indicated reactivation of up to about 25% at 30 min after oxime administration and substantial attenuation of seizure behavior from some of the oximes. Therefore these novel oximes have considerable potential as brain-protecting therapeutics for anticholinesterases.

Central respiratory effects on motor nerve activities after organophosphate exposure in a working heart brainstem preparation of the rat.

The impact of organophosphorus compound (OP) intoxication on the activity of central respiratory circuitry, causing acetylcholinesterase (AChE) inhibition and accumulation of acetylcholine in the respiratory brainstem circuits, is not understood. We investigated the central effect of the OP Crotylsarin (CRS) on respiratory network activity using the working heart brainstem preparation, which specifically allows for the analysis of central drug effects without changes in brainstem oxygenation possibly caused by drug effects on peripheral cardio-respiratory activity. Respiratory network activity was determined from phrenic and hypoglossal or vagal nerve activities (PNA, HNA, VNA). To investigate combined central and peripheral CRS effects hypo-perfusion was used mimicking additional peripheral cardiovascular collapse. Systemic CRS application induced a brief central apnea and complete AChE-inhibition in the brainstem. Subsequently, respiration was characterised by highly significant reduced PNA minute activity, while HNA showed expiratory related extra bursting indicative for activation of un-specified oro-pharyngeal behaviour. During hypo-perfusion CRS induced significantly prolonged apnoea. In all experiments respiratory activity fully recovered after 1h. We conclude that CRS mediated AChE inhibition causes only transient central breathing disturbance. Apparently intrinsic brainstem mechanisms can compensate for cholinergic over activation. Nevertheless, combination of hypo-perfusion and CRS exposure evoke the characteristic breathing arrests associated with OP poisoning.

Kinetic analysis of interactions of different sarin and tabun analogues with human acetylcholinesterase and oximes: is there a structure-activity relationship?

The repeated misuse of highly toxic organophosphorus compound (OP) based chemical warfare agents in military conflicts and terrorist attacks poses a continuous threat to the military and civilian sector. The toxic symptomatology of OP poisoning is mainly caused by inhibition of acetylcholinesterase (AChE, E.C. 3.1.1.7) resulting in generalized cholinergic crisis due to accumulation of the neurotransmitter acetylcholine (ACh) in synaptic clefts. Beside atropine as competitive antagonist of ACh at muscarinic ACh receptors oximes as reactivators of OP-inhibited AChE are a mainstay of standard antidotal treatment. However, human AChE inhibited by certain OP is rather resistant to oxime-induced reactivation. The development of more effective oxime-based reactivators may fill the gaps. To get more insight into a potential structure-activity relationship between human AChE, OPs and oximes in vitro studies were conducted to investigate interactions of different tabun and sarin analogues with human AChE and the oximes obidoxime and HI 6 by determination of various kinetic constants. Rate constants for the inhibition of human AChE by OPs, spontaneous dealkylation and reactivation as well as reactivation by obidoxime and HI 6 of OP-inhibited human AChE were determined. The recorded kinetic data did not allow a general statement concerning a structure-activity relationship between human AChE, OP and oximes.

Interaction of pentylsarin analogues with human acetylcholinesterase: a kinetic study.

Previous kinetic studies investigating the interactions between human acetylcholinesterase (AChE), structurally different organophosphorus compounds (OP) and oximes did not reveal a conclusive structure-activity relationship of the different reactions. The only exception was for a homologous series of methylphosphonofluoridates bearing C1-C4 O-n- or O-i-alkyl residues. Hence, it was tempting to investigate the kinetic interactions between different pentylsarin analogues, human AChE and two oximes, obidoxime and HI 6, in order to increase the understanding of structure-activity relationship between highly toxic OP and human AChE. The rate constants for the inhibition of human erythrocyte AChE by four pentylsarin compounds (k(i)), for the spontaneous dealkylation (aging, k(a)) and reactivation (k(s)) of inhibited AChE as well as for the oxime-induced reactivation of inhibited AChE by obidoxime and HI 6 reflected by the dissociation constant (K(D)) and the reactivity constant (k(r)) were determined. All pentylsarin analogues had a high inhibitory potency towards AChE. Inhibited AChE was subject to spontaneous reactivation which outweighed aging substantially. Pentylsarin-inhibited AChE could be reactivated by oximes, HI 6 being more potent than obidoxime. The determination of inhibition, reactivation and aging kinetics of pentylsarin analogues with human AChE extends the database on interactions between AChE and methylphosphonofluoridate homologues with C1-C4 n- and i-alkyl residues demonstrating a structure-activity relationship depending on the chain length with certain differences regarding inhibition and post-inhibitory reactions. Unfortunately, no structure-activity relationship could be observed for the oxime-induced reactivation of inhibited AChE. In view of previous results with numerous structurally different organophosphates, organophosphonates and phosphoramidates it has to be concluded that up to now kinetic studies did not provide decisive information for the development of more effective oxime-based reactivators.

Aging pathways for organophosphate-inhibited human butyrylcholinesterase, including novel pathways for isomalathion, resolved by mass spectrometry.

Some organophosphorus compounds are toxic because they inhibit acetylcholinesterase (AChE) by phosphylation of the active site serine, forming a stable conjugate: Ser-O-P(O)-(Y)-(XR) (where X can be O, N, or S and Y can be methyl, OR, or SR). The inhibited enzyme can undergo an aging process, during which the X-R moiety is dealkylated by breaking either the P-X or the X-R bond depending on the specific compound, leading to a nonreactivatable enzyme. Aging mechanisms have been studied primarily using AChE. However, some recent studies have indicated that organophosphate-inhibited butyrylcholinesterase (BChE) may age through an alternative pathway. Our work utilized matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry to study the aging mechanism of human BChE inhibited by dichlorvos, echothiophate, diisopropylfluorophosphate (DFP), isomalathion, soman, sarin, cyclohexyl sarin, VX, and VR. Inhibited BChE was aged in the presence of H2O18 to allow incorporation of (18)O, if cleavage was at the P-X bond. Tryptic-peptide organophosphate conjugates were identified through peptide mass mapping. Our results showed no aging of VX- and VR-treated BChE at 25 degrees C, pH 7.0. However, BChE inhibited by dichlorvos, echothiophate, DFP, soman, sarin, and cyclohexyl sarin aged exclusively through O-C bond cleavage, i.e., the classical X-R scission pathway. In contrast, isomalathion aged through both X-R and P-X pathways; the main aged product resulted from P-S bond cleavage and a minor product resulted from O-C and/or S-C bond cleavage.

Enzyme-kinetic investigation of different sarin analogues reacting with human acetylcholinesterase and butyrylcholinesterase.

The pertinent threat of using organophosphorus compound (OP)-type chemical warfare agents (nerve agents) during military conflicts and by non-state actors requires the continuous search for more effective medical countermeasures. OP inhibit acetylcholinesterase (AChE) and therefore standard treatment of respective poisoning includes AChE reactivators (oximes) in combination with antimuscarinic agents. Hereby, standard oximes, 2-PAM and obidoxime, are considered to be rather insufficient against various nerve agents. Numerous experimental oximes have been investigated in the last decades by in vitro and in vivo models. Recently, we studied the reactivating potency of several oximes with human AChE inhibited by structurally different OP and observed remarkable differences depending on the OP and oxime. In order to investigate structure-activity relationships we determined the various kinetic constants (inhibition, reactivation, aging) for a series of sarin analogues bearing a methyl, ethyl, n-propyl, n-butyl, i-propyl, i-butyl, cyclohexyl or pinacolyl group with human AChE and BChE. The rate constants for the inhibition of human erythrocyte AChE and plasma BChE by these OP (k(i)), for the spontaneous dealkylation (k(a)) and reactivation (k(s)) of OP-inhibited AChE and BChE as well as for the oxime-induced reactivation of OP-inhibited AChE and BChE by the oximes obidoxime, 2-PAM, HI 6, HLö 7 and MMB-4 were determined. With compounds bearing a n-alkyl group the inhibition rate constant increased with chain length. A relation between chain length and spontaneous reactivation velocity was also observed. In contrast, no structure-activity dependence could be observed for the oxime-induced reactivation of AChE and BChE inhibited by the compounds tested. In general, OP-inhibited AChE and BChE were susceptible towards reactivation by oximes. HLö 7 was the most potent reactivator followed by HI 6 and obidoxime while 2-PAM and MMB-4 were rather weak reactivators. These data indicate a potential structure-activity relationship concerning inhibition and spontaneous reactivation but not for oxime-induced reactivation.

Direct reaction of oximes with crotylsarin, cyclosarin, or VX in vitro.

The direct reaction of seven pyridinium oximes with the organophosphorus compounds (OPCs) crotylsarin, cyclosarin, and VX was studied by spectrophotometry. This method allows to quantify different parameters: (a) the half-life times (t (1/2)) of the oxime-OPC reactions on the basis of the changes in the absorption at the zwitterion (betaine) peak maximum, (b) the first- and second-order rate constants (k (1), k (2)), and (c) the maximum reaction velocities (v (max)). The results of the study show that the reaction velocity of the nerve agents with any of the oximes investigated decreased in the order crotylsarin > cyclosarin > VX. The comparison of the reaction rates of the three therapeutically used oximes (2-PAM, obidoxime, HI 6) with the respective OPC gave the highest rate for crotylsarin and cyclosarin with obidoxime and to a similar degree with HI 6, while in the case of VX the most reactive oxime was HI 6. The reaction velocity of the nerve agents with the monopyridinium oxime 2-PAM was lower as compared to the bispyridinium oximes (obidoxime, HI 6). The results obtained with the two sarin analogues indicate that the direct reaction with 2-PAM, obidoxime, or HI 6 could be used for non-corrosive decontamination purposes, especially, if sensitive biological surfaces like skin, mucous membranes, or wounds are considered. However, in view of the concentrations of nerve agents and oximes, which could be expected during OPC poisoning in man, the maximum reaction velocities would not be high enough to contribute markedly to the detoxication of nerve agents in vivo.

New safe method for preparation of sarin-exposed human erythrocytes acetylcholinesterase using non-toxic and stable sarin analogue isopropyl p-nitrophenyl methylphosphonate and its application to evaluation of nerve agent antidotes.

INTRODUCTION: A non-toxic and stable sarin analogue, isopropyl p-nitrophenyl methylphosphonate (INMP), was synthesized for safe preparation of sarin-exposed acetylcholinesterase (AChE). RESULTS AND DISCUSSION: This agent was stable for years, able to be handled in an ordinary laboratory without special care, and its 50% inhibitory concentration (IC50) on 0.04 U/ml human erythrocytes AChE was 15 nM. This reagent was thought to be especially useful since it enables experiments that require sarin-inhibited AChE, such as the development of antidotes for sarin, in a usual laboratory. To demonstrate the usefulness of this method, 40 known and novel pyridinealdoxime methiodide (PAM)-type oxime antidotes were synthesized, and their reactivation activities to INMP-exposed AChE and structure-activities correlation were studied. CONCLUSION: Among the antidotes tested in this experiment except for 2-PAM, the compound found to have the highest reactivation activity, was the novel hydrophobic 2-PAM-type compound, 2-[(hydroxyimino)methyl]-1-[4-(tert-butyl)benzyl] pyridinium bromide.