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To obtain insight into the development of thermally stable insect acetylcholinesterases, 200 distinct, independent, unrestricted, unbiased, isobaric–isothermal, 316-ns molecular dynamics simulations of a substrate-bound mosquito acetylcholinesterase responsible for cholinergic functions (AP-agAChE)¹ were performed using forcefield FF12MC² and PMEMD of AMBER 11 with a periodic boundary condition at 1 atm and 340 K. In-depth conformational analysis of these simulations with an aggregated simulation time of 63.2 microseconds revealed partially unfolded regions of AP-agAChE that could be stabilized with mutations for developing thermally stable AP-agAChE variants and thereby enabling rigorous characterization³ of cysteine-targeting anticholinesterases as potential insecticides that are effective and environmentally safe and also spare beneficial insects¹.

The Glu197 of butyrylcholinesterase (BChE) has been long considered as deprotonated in various studies, e.g. discovering the dynamical characters, interpreting the binding properties of inhibitors, and proposing hypotheses for BChE-catalyzed reaction mechanism. By performing a series of 100 ns molecular dynamics simulations, we accidently discovered that Glu197 needed to be protonated to have the structures simulated appropriately, whereas the deprotonated Glu197 eventually caused the collapse of catalytic triad with long enough simulation time.[1] we found that a highly conserved water molecule required Glu197 to be protonated in order to form an important hydrogen bond network, which supported His438 to be preserved within the catalytic triad. Interestingly, catalytic triad and Glu197 have been long recognized for possibly deviating largely from their crystal structure positions, which could be catalytic deficient and is generally considered as the result from difference between crystal and aqueous environment. Here, our results suggest that the large deviations of catalytic triad and Glu197 from crystal structure are caused by inappropriate protonation state of Glu197. This finding of the unexpected protonation state of Glu197 shall provide an important clue that has been long missing for the better understanding of BChE related puzzles or even reconsideration of some BChE-catalyzed reaction mechanisms.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) hydrolyze the neurotransmitter acetylcholine and function thereby as regulators of cholinergic neurotransmission. Recently, interest has greatly increased in BChE. Firstly, BChE is a good broad spectrum bioscavenger of nerve agent and its efficiency could be significantly increased by the mean of specific reactivators. Secondly, BChE activity in the brain increases with the progression of Alzheimer’s disease, thus classifying BChE as a promising drug target in the advanced phase of the disease. AChE and BChE display specificities for substrates and ligands that only partially overlap. This disparity is largely due to differences in the number of aromatic residues lining the active site gorge, which leads to large differences in the shape of the gorge and potentially to distinct interactions with an individual ligand. Considerable structural information is available for the binding of a wide diversity of ligands to AChE. In contrast, structural data on the binding of reversible ligands to BChE was lacking. In the recent years, we solved the X-ray structures of multiple BChE-ligand complexes. Here we will present BChE structures with various ligands, some recently synthesized, to highlight the structural elements leading to their BChE affinity and specificity. These structural data will help to design specific reversible ligands that behave as inhibitors or reactivators.

The evolution of insecticide resistance is a model system for studying enzyme evolution. Three insect species have independently evolved catalytic organophosphate (OP) detoxification through a single active-site mutation in the αE7 carboxylesterase. To explore the evolutionary potential of αE7, we subjected αE7 from the sheep blowfly to nine rounds of mutation and screening. The final variant contained 11 mutations which increased the rate of OP-hydrolysis more than 1000-fold. Atomic resolution X-ray crystal structures of the evolutionary intermediates reveal the changes in structure and dynamics at each step in the evolutionary trajectory, and hint at the molecular basis for the increased rate of OP hydrolysis. This work explores the potential for the development of αE7 as an enzyme therapeutic for OP poisoning, and worryingly for insecticide resistance, this work suggests that more efficient OP detoxification could be readily acquired by insect pests.

To investigate how protein dynamics facilitates substrate entering and product exiting the phosphotriesterase active site, over 60 distinct, independent, unrestricted, unbiased, isobaric–isothermal, microsecond molecular dynamics simulations of zinc-containing phosphotriesterase in complex with a substrate analog¹ were performed using the second-generation cationic dummy atom model for the zinc divalent cation, forcefield FF12MC², and PMEMD of AMBER 16 with a periodic boundary condition at 1 atm and 277 K, 300 K, and 340 K. In-depth conformational analysis of these simulations with an aggregated simulation time of over 76 microseconds revealed atomic and dynamic details on the phosphtriesterase catalysis and its requirement of two cations, which offers insight into re-engineering of phosphotriesterase to develop an improved scavenger against phosphorous-containing inhibitors of acetylcholinesterase.

This talk will briefly discuss our newest progress in drug design, discovery and development involving cholinesterases, particularly in three major therapeutic areas. (1) On the basis of our previous design and discovery of cocaine hydrolases (CocHs) engineered from human butyrylcholinesterase (BChE), we have further developed a novel, long-acting CocH form, and demonstrated the promising clinical potential of CocHs for therapeutic treatment of cocaine overdose and addiction in clinically relevant animal models. One of the long-acting CocHs is currently in the large-scale protein drug manufacturing process development. (2) It has been demonstrated that a long-acting CocH (enzyme) is capable of both completely blocking cocaine-induced physiological effects and producing the desirable anti-obesity effects. Mice on a high-fat diet gained significantly less body weight when treated weekly with 1 mg/kg enzyme compared to control mice. (3) Most recently, we have also designed and tested a new therapeutic strategy for heroin detoxification based on a detailed analysis of the cholinesterases-involved chemical transformation and functional change of heroin in the body. It has been demonstrated in our animal models that a carefully selected cholinesterase inhibitor attenuated acute toxicity and physiological effects of heroin, whereas some other cholinesterase inhibitors may actually enhance the acute toxicity and physiological effects of heroin.

It is known that cholinesterases show homotropic pseudocooperative effects: their activity at millimolar substrate concentrations is higher than expected by simple saturation kinetics and they are strongly inhibited at the submolar concentrations. However, we have reported that the anionic site directed inhibitors tetramethylammonium and tetraethylamonium too, increase the activity of human butyrylcholinesterase. At that time, the same phenomenon could not be shown for the horse counterpart. Here, it was searched for other putative activators among often used compounds in cholinesterase research. Indeed, imidazole significantly  increase the activity of human enzyme, but also its atypical form and the horse enzyme. On the other hand, 2-PAM shows a certain degree of activation with both human enzymes, but inhibits the horse BChE in a classical competitive manner. To avoid substrate activation, the experiments were performed at around 50 micromolar starting substrate concentrations and were followed by its completion in the presence of different modulator(s) concentrations. Subsequently, the effect of 2-PAM on the phosphorylation by DFP was studied, since the bottom of the active site does not differ in these three enzymes. It seems that the distinctive action of activating agents on the wild type, the atypical human and horse BChE is a consequence of differences in the dynamics of the acylation loop at the active site entrance, rather then the composition of the enzyme’s peripheral anionic site.

Catalytic bioscavengers are second generation bioscavengers. These biopharmaceuticals can be used to degrade toxic organophosphorus agents (OPs) on the skin for decontamination or in the bloodstream for pre-treatment and post-exposure treatment of OP poisoning. Because degradation has to be fast, their catalytic efficiency has be as high as possible (k_cat/K_m>10⁶ M^-1min^-1). To be of interest, the catalytic activity of certain enzymes, in particular self-reactivating ChEs, has to be increased by several orders of magnitude. This can be reached by computer-redesign, directed evolution of existing enzymes, and combinational strategies. Rational design of novel ChE-based catalytic bioscavengers requires a better understanding of chemical mechanisms of inhibition, aging of conjugate, and spontaneous reactivation. Kinetic studies, X-ray crystallography and molecular modeling, in particular QM/MM calculations, present valuable insights into specific reaction routes, role of specific amino acids and obstacles against effective reactivation of phosphylated ChEs. Introducing new functional groups surrounding the phosphylated serine should create a stable H-bonded network susceptible to activate and orient water molecule, stabilize transition states, and intermediates. Direction of nucleophilic attack of water molecule on phosphorus atom may determine whether dephosphylation is favored over aging. Mutations of key residues surrounding human BChE active site, creating new reaction pathways, have been considered. QM/MM calculations suggest that introduction of a histidine, directing attack of water molecule from apical position competes with the aging reaction, while axial direction of water attack does not. Secondary mutations for stabilizing imidazolium upon activation of water molecule lead to lower energy barrier of reactivation reaction [1].

Tabun represents a phosphoramide class of organophosphosphates that are covalent inhibitors of acetylcholinesterase (AChE), an essential enzyme in neurotransmission. The currently used therapy in excessive cholinergic stimulation consists of the muscarinic antagonist of acetylcholine stimulation, an anti-seizure drug when indicated and an oxime as the reactivator of inhibited AChE. Since common oximes are particularly ineffective in tabun exposure, we probed the reactivation of phosphoramidate conjugates in more depth by using mutants of AChE and pyridinium oximes to reveal the structural subtleties and yield more information on the architecture of the active centre gorge needed for the reactivation of phosphoramidate agents used in terrorism and as pesticides. Our results indicated that the replacement of aromatic residues with aliphatic ones at the acyl pocket and choline binding site mostly interfered with the stabilization of the oxime’s pyridinium ring(s) in the proper orientation of the oxime group toward the phosphorylated active site serine. The peripheral binding site mutation resulted in a 2-5 fold increase in the reactivation rates by bis-pyridinium oximes when compared to the AChE wild type. In the case of mono-pyridinium oximes, we reported a 150-fold enhancement of the maximal reactivation rate for the choline binding site mutation, while the molecular recognition seemed to remain preserved. Therefore, our results emphasized the positive effect of several mutations on oxime embedding and orientation into a position for productive interactions with the tabun-phosphorylated active site serine indicating a future potential for further development of pseudo-catalytic bioscavengers based on AChE mutants.

The widespread deployment of insecticide-treated bednets (ITNs) in sub-Saharan Africa has led to a dramatic decline in malaria mortality. However, wide-spread and growing resistance of Anopheles gambiae mosquitoes to the pyrethroid class of voltage-gated Na⁺ channel modulators used on these nets jeopardizes this achievement, and has prompted the search for suitable insecticidal AChE inhibitors to replace pyrethroids. Such compounds would have three favorable characteristics:  excellent contact toxicity towards susceptible adult An. gambiae, good contact toxicity to those that bear the G119S resistance mutation of AChE, and very weak inhibition of human AChE.¹ We will review our work on the development of aromatic and heterocyclic core methyl and dimethylcarbamate AChE inhibitors,² and including both enzymatic inhibition potencies and mosquito contact toxicities.  Finally, the inhibition selectivities of particular compounds will be rationalized in the context of our recently obtained high resolution X-ray structures of G119S An. gambiae AChE.³

The toxicity of organophosphorus compounds (OPs) cannot be explained only by action on acetylcholinesterase or neuropathy target esterase (NTE). A fraction of the membrane bound phenylvalerate esterase activity (PVase) is associated to NTE, the key initiating molecular event in the OP-induced delayed neuropathy (OPIDN). An enzymatic fraction in chicken brain soluble PVase has been reported to be due to a butyrylcholinesterase protein, and we suggested that this enzymatic fraction could be related to the mode of action of the potentiation/promotion phenomenon of the OPIDN. We showed that human butyrylcholinesterase (hBuChE) shows PVase activity. Mipafox, iso-OMPA or PMSF inhibited both activities with similar kinetic constants for both activities. Moreover, the substrates acethylthiocholine and phenyl valerate showed competition in their activities. The results suggest that both activities are related to the same active center. This work studies in depth the kinetic interactions between phenyl valerate and acetylthiocholine in human butyrylcholinesterase, showing that the interactions are different to the competitive model of substrates according to the Michaelis-Menten reaction. The approach introduced in this work suggests that other site could be involved in the interaction with phenyl valerate. In addition, we have observed that human acetylcholinesterase has also phenyl valerate esterase activity, but with lower activity than human butyrylcholinesterase. The level of phenylvalerate esterase activity in cholinesterases depends on the species and the type of cholinesterase. Further evaluation of the molecular interactions is under study.

Carbamates are esters of substituted carbamic acids that react with acetylcholinesterase (AChE) in a two-step process, with initial transfer of the carbamoyl acyl group to a serine residue of AChE accompanied by loss of the carbamate leaving group followed by hydrolysis of the carbamoyl enzyme.  This hydrolysis, or decarbamoylation, is relatively slow, and half-lives of carbamoylated AChEs range from 4 min to more than 30 days.  Since carbamates are poor, slowly reversible AChE substrates, they are effective AChE inhibitors that have been developed as insecticides and therapeutic agents.  We show that decarbamoylation rates are independent of the leaving group for a series of carbamates with the same carbamoyl group.  For a given leaving group, when the alkyl substituents on the carbamoyl group increased in size from N-monomethyl- to N,N-dimethyl-, N-ethyl-N-methyl-, or N,N-diethyl-, the decarbamoylation rates decreased by 4-, 70-, and 1000-fold, respectively. Thus the larger the size of the alkyl groups, the slower the rate of decarbamoylation due to active site distortion.  Furthermore, solvent deuterium oxide isotope effects for decarbamoylation decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.3 for N,N-diethylcarbamoyl AChE, indicating a shift in the rate-limiting step from general acid-base catalysis to a likely conformational change.

Computer-aided drug design is based on molecular modelling which includes two steps; molecular docking accompanied by scoring docked poses. Molecular docking fits the right molecular “key” to a known receptor “lock” by optimizing the atomic coordinates of a ligand to adapt its 3D structure in such a way to accommodate the binding into the receptor. The second step is the determination of a good fit between the ligand “key” and receptor “lock” using a function that correctly prioritizes the docked ligand poses and predicts their binding affinities by taking into account molecular interactions between the ligand, protein and solvent. The 68 crystal structures of complexes between acetylcholinesterase (AChE, EC 3.1.1.7) and its ligands, deposited in PDB, were analysed by scoring the functions: LigScore1, LigScore2, PLP1, PLP2, Jain, PMF and PMF04. The scores derived from scoring functions were correlated with an inhibition constant for each ligand (Ki or IC₅₀) in a broad range 10^-3 – 10^-12 M. Scores were also correlated with other computational properties as the number of rotational bonds, number of H-bond donor or acceptor atoms, molecular complexity index and topological polar surface area. The linear correlation between the scores derived from the scoring function and matching pKi data resulted in the highest r value for the PLP2 function, r = 0.77, with 10% of the slope error. The LigScore1function resulted in the lowest r value of 0.47 with 23% of the slope error. The PLP2 scoring function is a good candidate in drug discovery related to AChE, although with a higher number of crystal structures of AChE complexes and reliable kinetic data, a better scoring function could be developed.

Background: Memantine is the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, used in the treatment of Alzheimer’s disease. Memantine pretreatment assured protection of skeletal muscles from poisoning with nerve agents and an interaction between memantine and AChE was proposed [1].  Aim: Memantine and its main metabolite (1-amino-3-hydroxymethyl-5-methyl adamantine, Mrz 2/373) were used to ascertain their interaction with erythrocyte acetylcholinesterase (AChE) in vitro. The effect of these two compounds on the kinetics of the soman-induced AChE inhibition and on the aging of the soman-AChE complex was also investigated.  Methods: Bovine AChE activity was measured titrimetrically and the effect on aging of the soman-AChE complex was studied [2].  Results: Memantine and Mrz 2/373 exerted concentration-dependent inhibition of AChE, with Mrz 2/373 being a more potent inhibitor than the parent compound. Addition of soman 2.5x10^-8 mol/l induced gradual AChE inhibition that became almost 100% after 20 min. Memantine (0.1, 0.5 and 1 mmol/l) and Mrz 2/373 (0.1 and 1 mmol/l) concentration-dependently slowed down the AChE inhibition. Neither memantine nor Mrz 2/373 prevented the aging of the soman-AChE complex. After 5 min incubation with AChE and soman, AChE activity was 11%, 36% and 30% in control medium and after adding of 1 mmol/l of memantine and Mrz 2/373, respectively. Conclusion: Since high micromolar and low millimolar concentrations of memantine can be achieved in rats [3], it is quite possible that memantine and Mrz 2/373 can prevent AChE from inhibition by soman, which could, along with known memantine’s neuroprotective activity, explain its potent antidotal effect in soman poisoning.

The series of bisquaternary oximes with ortho-positioned chlorine moiety was designed, prepared and evaluated. The novel compounds exhibited valuable pK_a properties [1] with improved in vitro reactivation ability of sarin, cyclosarin, VX, paraoxon- and dichlorvos-inhibited human AChE exceeding the standard monoquaternary or bisquaternary reactivators (pralidoxime, methoxime, trimedoxime, obidoxime and asoxime syn. HI-6). Additionally, some chlorinated compounds presented in vitro reactivation ability of tabun-inhibited human AChE similar to the efficiency of trimedoxime. The in vitro results were further explained by molecular docking study. The in vitro non-cytotoxic properties of novel compounds were determined with miscellaneous results. However, assessment of maximum tolerated dose highlighted that the selected chlorinated reactivator is well tolerated by mice on the level similar to the clinically or experimentally used oxime reactivators [2]. The in vivo reactivation study is in progress.

Acetylcholinesterase (AChE) is a key enzyme of the Central Nervous System (CNS), which hydrolyzes the neurotransmitter acetylcholine.¹ By targeting AChE, organophosphorus nerve agents (OPNA) and organophosphorus pesticides irreversibly inhibit the cholinergic transmission, which is leading to death if untreated.² Over several years, our group and colleagues have been concentrating on the development a new class of non-permanently charged bifunctional reactivators, that display higher affinity for AChE and high in vitro and in vivo efficiencies compared to 2-PAM and Hi6.³ By analogy, recently, we designed bifunctional reactivators that comprise a peripheral site ligand (PSL) connected to a fluorinated reactivator function using a covalent linker. On the basis of our previous work on the synthesis of central hybrid reactivators bearing 6-alkanyl-3-hydroxy-2-pyridinadoxime moiety, and with the goal to develop reactivator with greater lipophilicity and enhanced blood brain barrier (BBB)  permeability, we decided to substitute the 3-hydroxy group, initially designed to decrease the oxime pka, with a more electronegative and electron-withdrawing group such as fluorine. Fluorine is known to modulate the pka of the proximal oxime, the conformational bias and the binding properties via molecular interactions. This structural change, compared to the known 6-substituted 3-hydroxy-2-pyridinadoxime scaffold, appeared valuable for both practical and fundamental reasons, eventually providing reactivators with increased reactivation potency and better pharmacological profiles.

Despite extensive oxime research in the last 60 years pralidoxime is still the standard oxime in e.g. United States, British and French forces and obidoxime standard therapy for OP poisoning in several European countries. Oxime research focusses on highly potent oximes with activity against selected nerve agents, broad-spectrum oximes with activity against relevant nerve agents and centrally active (non-)oximes but virtually no compound brought significant improvements compared to the established obidoxime and pralidoxime. In the US MMB-4 is sought to replace pralidoxime and in Germany, France, UK, Canada and other European countries HI-6 is in advanced development for use as nerve agent antidote. Yet, both compounds are not considered as broad-spectrum antidotes and as a mid-term solution combinations of oximes in service with overlapping reactivation potency e.g. obidoxime and HI-6 have been proposed. We here set out to analyze the combination of obidoxime and HI-6 in both a static and dynamic model against poisoning with nerve agents and organophosphorus compound pesticides in vitro. In a cuvette based system the combination of HI-6 and obidoxime both 30 μM for sarin-, cyclosarin-, tabun-, VX- and paraoxon-inhibited human AChE did not result in an impaired reactivation compared to the sole use of both oximes but in a broadened spectrum. Similar results were gained with a dynamic model allowing simulation of nerve agent and pesticide toxicokinetics and oxime pharmacokinetics resembling in vivo conditions. Additional experiments in species closely related to humans e.g. swine are necessary to analyse a potential benefit in vivo.

The design of reactive molecules such as nerve agent antidotes is inherently challenging due to two intertwined processes imperative for their efficiency: The reversible binding of the initial non-covalent complex in a low energy conformation and the chemical reaction that proceeds via a transition state of high(er) energy. Furthermore, a structural and chemical diversity among different nerve agents and their corresponding complex with AChE complicates the design of broad-spectrum antidotes. The development of broad spectrum antidotes has proven challenging and although progress has been made, no new drugs with improved properties have been launched in several decades. Herein, we report a rational, structure-based approach for the development of broad-spectrum antidotes. Based on a hit molecule identified in a high throughput screening targeting the non-inhibited species of AChE, 18 new analogous molecules were designed and synthesized. This resulted in a set of compounds with a diversity in their potency, as desired for subsequent (quantitative) structure-activity relationship ((Q)SAR) modeling. The 18 compounds were investigated for their ability to bind to four different phosphonylated forms of AChE (i.e. human AChE inhibited by the nerve agents VX, VR, and tabun, or the substance DFP). The QSAR model was subsequently used to guide the development of a novel set of pyridinium-oxime based broad spectrum antidotes. The mechanism of reactivation of the developed antidotes has been investigated using a combination of X-ray crystallography and molecular modelling.

Organophosphorus (OP) compounds are potent acetylcholinesterase (AChE) inhibitors that have found use as both chemical warfare agents (CWAs) and as pesticides. Following inhibition of AChE by OP compounds, a competitive dealkylation reaction of the phosphylated serine residue occurs – a process referred to as aging. Current therapeutic reactivators of OP-inhibited AChE, mainly oximes, are not effective once aging has occurred. For the first time, we have demonstrated in vitro conversion of the aged AChE to the native form using small drug-like molecular therapeutics.  As part of this effort, a diverse library of small molecule therapeutics have been developed to both recover the activity of aged-AChE, termed resurrection, as well as the activity of inhibited-AChE, referred to as reactivation. The structure of such therapeutics is derived from pyridyl-based quinone methide precursors (QMPs), sharing structural similarities to known therapeutic oximes. A structure-activity relationship study of synthesized QMP therapeutics was conducted to determine the effect electron-donating and electron-withdrawing groups have on the efficiency of both processes and to design optimized small molecule therapeutics for in vivo biological efficacy. Our successes will be presented.

Nanotechnological "two-in-one" approach using nanoparticles for packaging two oximes in single carriers and nose-to-brain delivery for brain protection against poisoning by organophosphorus agents have been developed. Strategies for designing nanocarriers for drug delivery to the CNS and crossing the BBB showed that nanoparticles based on natural and biodegradable materials are promising. Solid lipid nanoparticles (SLNs) are biocompatible, biodegradable and have very low toxicity, thereby fulfilling the requirements of preclinical safety [1]. 2-PAM and a novel reactivator of VX-, paraoxon-, and tabun-phosphylated AChE [2] a poorly water soluble 6-(5-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentyl)-3-hydroxypicolinaldehyde oxime (3-HPA), were loaded in SLNs to offer distinct release profile and half-life for both oximes. To increase the therapeutic time window of both oximes, SLNs with two different compartments were designed. Oxime-loaded SLNs of hydrodynamic diameter 100-160 nm and zeta potential (from -30 to -25 mV) were stable for a period of 10 months at 4ºC. SLNs displayed longer circulation time in the bloodstream compared to free 3-HPA and free 2-PAM. Oxime-loaded SLNs were suitable for intravenous administration. Paraoxon-poisoned rats (0.8×LD50) were treated with 5mg/kg of 3-HPA-loaded SLNs and 2-PAM+3-HPA-loaded SLNs. Brain AChE reactivation up to 30% was slowly achieved in 5 h after administration of 3-HPA-SLNs. Synergistic effect and increased reactivation up to 35% was observed with combination of both oximes. In addition, new cationic liposomes based on L-α-phosphatidylcholine and cationic surfactant were administered via the intranasal route. These liposomes were found to reach directly central AChEs. This last approach provides evidence that reactivation of central AChEs can be achieved by a non-invasive approach that bypasses the BBB.

Organophosphorus nerve agents are highly toxic compounds which pose a threat worldwide. These compounds induce toxicity by covalently binding to the active site serine of acetylcholinesterase, which results in inhibition of the enzyme. Without functional acetylcholinesterase, the levels of the neurotransmitter acetylcholine in neuromuscular junctions rise quickly, causing overstimulation of the nervous system, which will culminate in death if not treated. Current treatments rely on small molecules to interact with inhibited enzyme to disrupt the covalently bound phosphorus moiety at the active site. The most effective molecules incorporate a pyridinium oxime which acts via direct nucleophilic attack on the phosphorus to achieve reactivation of the enzyme. These compounds have limited effectiveness because the charged portion of the molecule does not allow them to cross into the central nervous system where acetylcholinesterase inhibition is most harmful. The results of studies that characterized a small molecule reactivator (4-amino-2-((diethylamino)methyl)phenol [ADOC]) that does not incorporate an oxime but is capable of reactivating nerve agent-inhibited enzyme as well as or better than current treatments have been used to inform the design of additional novel compounds. This study describes the in vitro characterization of these novel compounds as reactivators of phosphonylated human acetylcholinesterase.

Organophosphate (OP) compounds inhibit the enzyme acetylcholinesterase (AChE) resulting in severe symptoms and ultimately death. OP intoxications are currently treated by administration of atropine and certain oxime compounds (Obidoxime, HI-6 or 2-PAM). The latter compounds contain nucleophilic oximes that reactivate OP-inhibited AChE by liberating the phosphylated serine. However, these oximes have several drawbacks such as their intrinsic toxicity, their permanent charge which thwarts penetration of brain tissues and their inability to effectively reactivate all types of nerve agent inhibited AChEs. Therefore, the search for new (non-ionic) antidotes of nerve agent poisoning is of great importance. Recently, several papers reported on the discovery of non-oxime compounds as a result of the in vitro or in silico screening of libraries of bioactive compounds and approved drugs. For instance, Katz et al reported¹ a novel class of compounds in which the 4-amino-2-(diethylamino)phenol (ADOC) appeared to be a key motif responsible for reactivation of OP-inhibited AChE.² In addition, several structural derivatives of ADOC were synthesized and evaluated for OP-AChE reactivation by Cadieux et al.³ That study provided valuable information on key structural features of ADOC with respect to reactivation potency and enzyme inhibition, but unfortunately, none of the reported derivatives performed equal or better than the ADOC parent. We here report the design and synthesis of a new series of ADOC derivatives. We report that one of the compounds synthesized so far showed a remarkably improved in vitro performance compared to ADOC towards VX-, sarin-, cyclosarin- and paraoxon-inhibited human AChE.

The chemicals known as the organophosphates (OPs) are found in hundreds of useful agricultural, industrial, and commercial products; however, they have also been associated with a variety of adverse health effects in humans and other non-target organisms.  The acute toxicity of OPs is attributed to the inhibition of the enzyme acetylcholinesterase; however, this mechanism is inadequate to explain all of the long-term adverse effects of OPs.   In both live imaging studies in primary neuronal culture as well as in manganese-enhanced magnetic resonance imaging (MEMRI) studies of the brains of living rats, we have observed impairments in axonal transport (AXT) associated with both the insecticide OP chlorpyrifos and the nerve agent OP diisopropylfluorophosphate.  These observations may be important since AXT is an essential process that is responsible for the movement of a variety of important macromolecules to and from a neuron's cell body.   In this presentation, a brief overview of the results of these neuronal culture (trafficking) and MEMRI experiments will be provided.  In addition, the results of experiments conducted to date to identify specific molecular targets of OPs that might negatively influence axonal transport will be summarized.  These targets include post-translational modifications of structural proteins that affect AXT through the regulation of microtubule dynamics and stability (e.g., Tau phosphorylation, Tubulin Acetylation), and specific signaling kinases (e.g., ERK GSKIIIβ) that are known to regulate various components of the AXT process.  These experiments are expected to help us begin to develop novel therapeutic strategies to improve the neuronal deficits associated with OPs.

The choice of biomarkers for establishment of exposure to organophosphorus compounds (OPs) is made based on the results of assessment of the real situation with account for such factors as the required timeframe for providing the results of expert examination, nature and volume of biosamples, available equipment, and the degree of confidence of information on the influencing factor (substance, dose, way of entry, use of antidote). We estimated the efficiency of express methods of diagnosis of exposure to OPs, specifically, Ellman’s cholinesterase activity assay, as well as GC-MS/MS and HPLC/MS/MS determination of OPs fluoride-regenerated from protein adducts and low-molecular hydrolytic metabolites of OPs, respectively. The objects of study were blood and urine samples of rats exposed to VR in a dose of 2×0.4LD50 under conditions of antidotal therapy with Carboxim {5-[[[2-[benzyl(diemthyl)ammonio]ethyl]amino]carbonyl]-2-[(hydroxyimino)methyl]-1-methylpy-ridinium dichloride}. Carboxim therapy led to AChE reactivation 3 h after exposure to VR, while in the absence of the therapy the AChE activity recovered within 3 days. Fluoride regeneration of VR from its blood plasma protein adducts was possible within 7 days after poisoning irrespective of whether the therapy was applied or not О-isobutyl methylphosphonate was detected in urine 24 h after exposure in the urine samples of animals both subjected and not subjected to antidotal therapy, whereas after 3 days it was detected exclusively in the urine samples of animals not given the antidote. It was also found that blood plasma levels of free and esterified fatty acids can serve as an additional toxicodynamic parameter of VR poisoning.

Trichloronate is a racemic organophosphatioate insecticide. It induced delayed neuropathic in hens and human. The avian are species with greater susceptibility to organophosphorus poisoning due to their low levels of A-esterases. However, a copper-dependent hydrolyzing activity of hexyl dichlorophenyl phosphoramidate (HDCP), known as “antogonistic stereoselectivity” was recently identified in chicken serum. This study shows the activating effect of copper on the hydrolysis of trichloronate enantiomers by turkey serum and albumin (TSA) using chiral chromatography with CHIRALCEL OD column and heptane HPLC as mobile phase. The trichloronate hydrolysis levels (µM remaining concentration of each isomer) quantified at 37 °C, pH 7.4 and 60 minutes of turkey serum (10 μL) incubated with 300 μM of copper were statistically higher p˂0.05) for (-)-trichloronate (65 %) than (+)-trichloronate (32%). This estereoselective hydrolysis observed in turkey serum was confirmed by the incubation of 200 μg of turkey serum albumin (amount of this protein estimated in the 10 mL of turkey serum) with 400 μM of racemic trichloronate and 300 μM of copper at physiological condition during 60 minutes; hydrolysis values of 90% and 72% were obtained for (-)-trichloronate and (+)-tricholoronate. In conclusion, the present study evidences the hydrolysis of an organophosphatioate racemic for an A-esterase activity in turkey serum and identifies albumin as the cuproprotein responsible of this Cu²⁺-dependent stereoselective hydrolysis of this chiral insecticide in the turkey serum.

Chronic illness from exposure to organophosphorus toxicants is hypothesized to involve modification of unknown proteins.  Tyrosine readily reacts with organophosphorus toxicants in proteins that have no active site serine.  We developed a monoclonal antibody, depY, that specifically recognizes diethoxyphospho-tyrosine in proteins and peptides, independent of the surrounding amino acid sequence ¹. Our goal was to identify diethoxyphosphorylated proteins in human HEK293 cell lysate treated with chlorpyrifos oxon.  Cell lysates treated with chlorpyrifos oxon were examined by ELISA and capillary electrophoresis Western blot.  Tryptic peptides were analyzed by liquid chromatography-tandem mass spectrometry.  The depY antibody recognized diethoxyphospho-tyrosine containing proteins by ELISA and Western blotting.  Mass spectrometry identified 40 diethoxyphospho-tyrosine peptides from 24 proteins in immunopurified samples, but found only 9 diethoxyphospho-tyrosine peptides from 6 proteins when the same sample was not immunopurified on depY. The most abundant proteins in the cell lysate, Histone H4, Heat shock 70 kDa protein 1A/1B, Heat shock protein HSP 90 beta, and Alpha-enolase, were represented by several diethoxyphospho-tyrosine peptides.  It was concluded that use of immobilized depY improved the number of diethoxyphospho-tyrosine peptides identified in a complex mixture.  The mass spectrometry results confirmed the specificity of depY for diethoxyphospho-tyrosine peptides independent of the context of the modified tyrosine, which means depY could be used to analyze modified proteins in any species.

Pesticides and warfare nerve agents are frequently organophosphates (OPs) or related compounds. Their acute toxicity highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and/or detoxification of these compounds. Herein, we report the use of two different thermostable enzyme families capable to detect and inactivate OPs. In particular, mutants of carboxylesterase-2 from Alicyclobacillus acidocaldarius and of phosphotriesterase-like lactonases from Sulfolobus solfataricus and Sulfolobus acidocaldarius, have been selected and assembled in an optimized format for the development of an electrochemical biosensor and a decontamination formulation, respectively. The features of the developed tools have been tested in an ad-hoc fabricated chamber, to mimic an alarming situation of exposure to a nerve agent. Choosing ethyl-paraoxon as nerve agent simulant, a limit of detection (LOD) of 0.4 nM, after 5 s of exposure time was obtained. Furthermore, an optimized enzymatic formulation was used for a fast and efficient environmental detoxification (>99%) of the nebulized nerve agent simulants in the air and on surfaces. Crucial, large-scale experiments have been possible thanks to production of grams amounts of pure (>90%) enzymes.

Small ionizable, zwitterionic oximes of limited toxicity show successful outcomes in non-human primates upon intramuscular post-treatment of exposures to OP’s, that enter via the respiratory tract.  Along with their inherent limitations, we consider the bases for success in post-exposure treatment of OP toxicity and reversal of OP-induced sequelae of symptoms¹.  (1) High vapor pressure OPs carry the largest acute exposure risk in mass terrorism.  Toxic OPs released from explosive devices or into controlled ventilation environments are governed by partial pressure and Fick’s Second Law of Diffusion (inverse square of the distance);  (2) Low molecular weight, zwitterionic oximes confer optimal nucleophile orientation and activity within the confines of the OP-impacted, active center gorge of human acetylcholinesterase (AChE).  (3) We emphasize features of ionizable neutral oximes of low toxicity that allow facile passage of membranes to peripheral and central AChE targets and optimal attack angles in the AChE active center.  Hence, for volatile OP’s, antidotes must rapidly enter the circulation, post-exposure, to chase the offending OP.  Following entry, antidotes should then hastily equilibrate between tissue compartments and cross the blood-brain barrier.  Accordingly, we examine the ionization states of zwitterionic oximes and other cationic and anionic (F^-) nucleophiles in relation to their kinetic parameters of reactivation².  Toxicities, both realized and potential, of nucleophilic antidotes in different ionization states, and pharmacokinetics in mice and macaques, under control and exposure conditions, emerge as critical factors for determining in vivo antidote efficacy.  Data will be presented on multiple OP’s and their enzyme conjugates^1-3, comparator oximes and in three animal species/strains.

DDeliberate sarin releases in Syria with large numbers of fatalities emphasize the need for OP countermeasures for both military and civilian populations. Therapeutic countermeasures  involve several strategies: (i) preventing OP poisoning through administering pre-exposure treatments that scavenge OPs before they inhibit their physiological AChE targets in the brain and in the periphery (ii) post-exposure oxime that can rapidly reactivate OP-inhibited AChE or (iii) a combination of both. In terms of a pretreatment, our recent studies have demonstrated that administration of an aerosolized (aer)-rHuBChE employing a user friendly nebulizer, forms a protective pulmonary bioshield in the lungs of macaques which to date remains intact for at least 4 days. Thus 8 mg/kg of aer-rHuBChE deposited in the lung can prevent symptoms and inhibition of RBC-AChE and plasma BChE following a high (55ug/kg) inhaled dose of aer-paraoxon (Px) 4 days later; an amount known to inhibit circulating ChEs by >95% and cause tremors.  In terms of oxime efficacy, macaque studies have demonstrated that a single IM post-exposure injection of the zwitterionic, centrally acting oxime RS194B (62-80ug/kg) plus low-dose atropine rapidly reactivates OP-inhibited RBC-AChE and circulating BChE and dramatically reverse both early and advanced clinical OP symptoms following lethal inhalation exposure to both sarin vapor (49.6ug/kg) and lethal aerosolized paraoxon (100ug/kg).            The increased efficacy of nebulizers in humans and the known synergy between aer-rHuBChE pretreatment with IM RS194B post exposure bodes well for a prophylactic or combination treatment which can protect against potent inhaled OP agents for >6 days without multiple injections.

Organophosphorus compounds (OP) pose a significant threat. Administration of human butyrylcholinesterase (Hu BChE) may reduce or prevent OP toxicity. Thus, we evaluated the safety and efficacy of Hu BChE in monkeys using sensitive neurobehavioral tests while concurrently characterizing absorption and elimination in the presence and absence of high-dose soman exposure to predict time course and degree of protection. Eight young adult male cynomolgus macaques were trained on two distinct automated tests of neurobehavioral functioning. Hu BChE purified under current-Good-Manufacturing Practices (CGMP) was injected intramuscularly at 13.1 mg/kg, producing an average peak plasma value (C_max) of 28 Units/ml. The apparent time to maximum concentration (T_max) approximated 12 hours and the elimination half-life approximated 80 hours, returning to pre-administration (baseline) levels by 14 days. No behavioral disruptions following Hu BChE administration were observed on either neurobehavioral test, even in monkeys injected 24 hours later with an otherwise lethal dose of soman. Thus, Hu BChE provided complete neurobehavioral protection from soman challenge. These data replicate and extend previous results that used a different route of administration (intravenous), a different species (rhesus macaque), and a different BChE product (non-CGMP material). The addition of two sensitive neurobehavioral tests coupled with the PK/PD results convincingly demonstrates the neurobehavioral safety of plasma-derived Hu BChE at therapeutic levels. Protection against an otherwise-lethal dose of soman by a pre-exposure treatment dose that is devoid of side effects establishes a foundation for additional testing using other exposure routes and treatment times, other challenge agents/routes, or other classes of organophosphate scavengers.