Nerve agents: what they are, how they work, how to counter them

Review Cite This: ACS Chem. Neurosci. 2018, 9, 873−885

pubs.acs.org/chemneuro

Nerve Agents: What They Are, How They Work, How to Counter Them Stefano Costanzi,*,†,‡ John-Hanson Machado,§,∥ and Moriah Mitchell† †

Department of Chemistry and ‡Center for Behavioral Neuroscience, American University, 4400 Massachusetts Avenue, NW, Washington, DC 20016, United States § Department of Chemistry, The George Washington University, 800 22nd Street NW, Washington, DC 20052, United States ∥ Computational Biology Institute, The George Washington University, 45085 University Drive Suite 305, Ashburn, Virginia 20147, United States ABSTRACT: Nerve agents are organophosphorus chemical warfare agents that exert their action through the irreversible inhibition of acetylcholinesterase, with a consequent overstimulation of cholinergic transmission followed by its shutdown. Beyond warfare, they have notoriously been employed in acts of terrorism as well as high profile assassinations. After a brief historical introduction on the development and deployment of nerve agents, this review provides a survey of their chemistry, the way they affect cholinergic transmission, the available treatment options, and the current directions for their improvement. As the review illustrates, despite their merits, the currently available treatment options present several shortcomings. Current research directions involve the search for improved antidotes, antagonists of the nicotinic receptors, small-molecule pretreatment options, as well as bioscavengers as macromolecular pretreatment options. These efforts are making good progress in many different directions and, hopefully, will lead to a lower target susceptibility, thus reducing the appeal of nerve agents as chemical weapons. KEYWORDS: Nerve agents, development and deployment, acetylcholinesterase, cholinergic transmission, treatment options, drug development

■

INTRODUCTION Nerve agents are highly toxic organophosphorus compounds discovered in Germany in the 1930s within the scope of industrial research for the development of pesticides and subsequently developed as chemical warfare agents. They exert their toxicity on both the central and the peripheral branches of the nervous system by interfering with the breakdown of the neurotransmitter acetylcholine in the synaptic cleft. In particular, nerve agents act as irreversible inhibitors of acetylcholinesterase, an enzyme responsible for acetylcholine breakdown. As a result, they cause an excessive accumulation of the neurotransmitter, leading to an overstimulation of cholinergic neurotransmission (i.e., the neurotransmission mediated by acetylcholine) followed by desensitization with toxic and fatal consequences. Poisoning symptoms and signs include vomiting, wheezing, twitching, pupil constriction (miosis), and paralysis. Death typically occurs from respiratory failure or seizures.1,2 In this paper, after a brief historical introduction on the development and deployment of nerve agents (Sections 1 and 2), we provide a survey of their chemistry (Section 3), the physiological aspects of cholinergic transmission (Section 4), the biochemistry of acetylcholinesterase and its inhibition by nerve agents (Sections 5 and 6), the available treatment options, and the current directions for their improvement (Sections 7 and © 2018 American Chemical Society

8). All bidimensional chemical structures were sketched with ChemDraw Professional 15, ChemDraw 150.0.106, PerkinElmer Informatics, Inc. (www.cambridgesoft.com). All three-dimensional structures were represented with Maestro, version 10.4, Schrödinger Suite, Release 2015-4, Schrödinger, LLC, New York, NY, 2015 www.schrodinger.com).

1. BRIEF HISTORICAL NOTES ON THE DEVELOPMENT OF NERVE AGENTS The first nerve agent, known as tabun or GA (Figure 1, tabun), was synthesized as a potential pesticide in 1936 by Gerhard Schrader at the German chemical corporation IG Farben. The high human toxicity of the compound became evident when, after accidental exposure to a small quantity of it, Schrader experienced persistent and severe symptoms, including shortness of breath and miosis.2 The mechanism of action of tabun and its analogues, however, became known to German scientists only in 1943, when Nobel Laureate Richard Kuhn and coworkers discovered that they acted as irreversible inhibitors of the enzyme acetylcholinesterase. Received: March 30, 2018 Accepted: April 17, 2018 Published: April 17, 2018 873

DOI: 10.1021/acschemneuro.8b00148 ACS Chem. Neurosci. 2018, 9, 873−885

Review

ACS Chemical Neuroscience

Figure 1. Chemical structures of common nerve agents. For all the compounds, the more toxic P(−) isomers are presented. In terms of absolute stereochemistry, for all the shown compounds with the exception of tabun, the P(−) isomer corresponds to the S configuration. Conversely, due to the different priority order of its substituents, for tabun, the P(−) isomer corresponds to the R isomer. The chiral carbon of soman, the configuration of which is less important for its toxicity, is indicated with a star.20−25

Figure 2. Chemical structures of the A232 and A234 Novichok agents, also known as Novichok 5 and Novichok 7, respectively. For both compounds, the structures suggested by Mirzayanov are presented on the top row, while alternative structures proposed by Hoenig and Ellison are presented on the bottom row.6,9,11

by Mirzayanov, some Novichok agents were synthesized on a microscale for analytical purposes by Hosseini and coworkers, with the aim of enriching the Central Analytical Database (OCAD) maintained by the Organization for the Prohibition of Chemical Weapons (OPCW).10 Despite the fact that a scientist directly involved in the Foliant program exposed the structures of the Novichok agents, some uncertainty remains, as Hoenig and Ellison have suggested alternative structures for the same compounds.9,11,12 As evident from Figure 2, the alternative structures proposed for compounds A232 and A234 are completely different from those indicated by Mirzayanov. Although, by his account, Mirzayanov was directly involved in the Novichok program, there is not sufficient published information to rule out the structures provided by Hoenig and Ellison. As the Novichok series illustrates, toward the end of the Cold War, both blocks focused on the development of known or new nerve agents into binary agents, i.e. agents that could be stored in the form of two precursors, intended to be combined only at the time of deployment, yielding in situ synthesis of the nerve agent. For instance, the precursors could be stored in two separate compartments of a chemical ammunition, designed to bring them into contact only upon deployment. For example, a binary ammunition that leads to the production of sarin might contain the precursors methylphosphonyl difluoride and isopropanol, spatially separated by a physical barrier. When the precursors come into contact, sarin is synthesized in situ (Figure 3). Hydrofluoric acid is also produced as a byproduct of the reaction and is typically neutralized with a basic amine. The main advantage of binary nerve agents is that the chemical precursors are less toxic than the final product, reducing the risks for storage, transportation, and accidental dispersion.7,13

In accordance with the Reich Ordinance of 1935, IG Farben reported discovery of tabun to the German Ministry of War, leading to the beginning of a chemical weapons program intended to develop further nerve agents.2,3 In 1938, the program led to the synthesis of sarin, or GB (Figure 1, sarin).2,3 Subsequently, in 1944, Richard Kuhn along with Konrad Henkel synthesized and tested soman, or GD (Figure 1, soman), an agent far more potent than sarin, especially with respect to skin exposure (see below, Section 6).2 Collectively, the nerve agents synthesized in Germany during World War II were named “G” agents. Following the Second World War, further nerve agents were synthesized during the Cold War both in the Western block and the Soviet Union. The agents synthesized in the West were dubbed “V” agents, as homage to the victory of the Allied forces in WWII. The first of these agents, known as VX (Figure 1, VX), was synthesized in 1952 in the United Kingdom by British chemist Ranajit Ghosh. The agent, significantly more potent than sarin and soman and endowed with stronger percutaneous effects, was then passed on to the United States and Canada for further development.3,4 Subsequently, the Soviet Union began development of compounds similar to the V agents, such as VR, which is also known as Russian VX or R-33 (Figure 1, VR).5 Meanwhile, the United States developed a new generation of compounds known as intermediate volatility agents (IVA), an example of which is GV (Figure 1, GV).6,7 A final class of nerve agents, known as Novichok agents, was developed by the Soviet Union toward the end of the cold war, within the context of a chemical weapons program known as Foliant, which was exposed in the early 1990s by the Soviet defector Vil Mirzayanov. Although there are many uncertainties surrounding the Foliant program, it appears that the Novichok agents were developed as binary agents, with the lethal compounds synthesized from less toxic precursors only at the time of delivery (see below for more information on binary agents). Moreover, the agents are reportedly endowed with very high potencies, which is some cases exceed that of VX by more than five times.8 Chemical structures for several members of the series were revealed by Mirzayanov. The top row of Figure 2 show the structures of two of the compounds revealed by the Russian scientist, namely A232 and A234, also known as Novichok 5 and Novichok 7.9 Based on the structures disclosed

Figure 3. Chemical reaction for the production of sarin in a binary chemical weapon from methylphosphonyl difluoride and isopropanol. Hydrofluoric acid is also produced as a byproduct of the reaction and is typically neutralized with a basic amine. In the binary weapon, the precursors are separated by a physical barrier that breaks as the device is deployed. 874


Review


2. DEPLOYMENT OF NERVE AGENTS IN CHEMICAL WARFARE, TERRORISM, AND ASSASSINATIONS Although their discovery and development dates back to the 1930s, nerve agents were not used in warfare until Iraqi military deployed tabun against Iranian troops on Majnoon Island February of 1984, resulting in more than 300 deaths and thousands of casualties.14 Sarin, VX, and tabun were used against Iran and the Kurds later in the Iran−Iraq war.14 Moreover, between 2013 and 2017, sarin was used in a series of chemical attacks in the Syrian civil war. In particular, such attacks occurred in several regions of the country, among which Damascus and the surrounding East Ghouta region, Aleppo and the nearby Idlib region, and possibly the Hama region.15 Of note, the identification of basic amines in environmental samples by the United Nations Mission in the Ghouta area indicates that nerve agents might have been deployed in binary form, possibly through cruder versions of binary weapons, where the components were loaded into the ammunitions prior to launching without a separating barrier.13 At the time of this writing, the Fact-Finding Mission (FFM) of the OPCW was deployed to Syria to investigate the allegations of another chemical weapons attack, possibly involving nerve agents, that occurred in Douma in April 2018.16 Beyond warfare, nerve agents have been used in terrorism. The religious sect Aum Shrinrikyo employed sarin on several occasions in the 1990s.2,3 On June 27, 1994 the doomsday cult sprayed the agent from a converted refrigerator truck in a residential neighborhood in Matsumoto, Japan.17 The intended target of this act of terror was a dormitory housing judges who had ruled against Aum Shrinrikyo in court; however, all three judges survived with injuries, while seven civilians died and hundreds were injured.3 Subsequently, on March 20, 1995, cult members used umbrella tips to simultaneously puncture plastic bags containing sarin on 5 subway trains in Tokyo, Japan, resulting in 12 deaths and nearly 3800 injuries.3,17 Nerve agents have also been used in recent high-profile assassinations and assassination attempts. In particular, VX was used to assassinate Kim Jong-nam, the half-brother of Kim Jongun, at the Kuala Lumpur airport in February 2017.18 Moreover, Novichok agents have been implicated in the March 2018 assassination attempt perpetrated in Salisbury, UK, against Sergei Skripal, a former Russian intelligence officer who acted as an agent for the British MI6, and his daughter Yulia.19 As these historical notes illustrate, nerve agents maintain a high degree of appeal for states and terrorists. Thus, the development of improved treatment options is essential to reduce their effectiveness and protect armed forces and civilians alike. As we will see in Sections 7 and 8, the scientific community is putting forth a significant amount of efforts in this direction.

configuration are associated with higher toxicity with respect to the corresponding isomer that feature the phosphorus atom in the R configuration. The fact that tabun is an exception is purely a nomenclature issue: as a result of the different priority order of its substituent, the special arrangement that results in the S configuration for the phosphorus atom of the other compounds shown in Figure 1 yields the R configuration for tabun. For soman, which as we mentioned presents a second chiral center, the stereochemical configuration of the chiral carbon appears less significant than that of the phosphorus atom.20−25 Due to the different nature of the substituents on the phosphorus atoms, nerve agents present a range of physicochemical properties (Table 1), which in turn affect their Table 1. Physicochemical Properties of Selected Nerve Agents.6,27,49,89,90 property

tabun

sarin

soman

cyclosarin

VX

VR

molecular weight density at 25 °C (g/cm3) boiling point (°C) melting point (°C) vapor pressure at 25 °C (mm Hg) volatility at 25 °C (mg/m3) water solubility at 25 °C (%)

162.1

140.1

182.2

180.2

267.4

267.4

1.073

1.089

1.022

1.120

1.008

1.008

247

147

167

92

300

323

−50

−56

−42

< −30

−39

NAa

0.07

2.9

0.3

0.06

0.0007

0.0003

610

22 000

3,900

600

10

9

10

∞

2

∼2

3

NAa

a

NA: data not available.

characteristics as chemical warfare agents. For instance, lipophilic compounds such as VX are absorbed more readily through the skin than hydrophilic compounds. Conversely, compounds of a greater hydrophilic character such as sarin penetrate more deeply into the lungs. Notably, vapor pressure and volatility are key properties to determine the environmental persistency of the compounds, as less volatile compounds such as VX are endowed with a greater environmental persistency with respect to more volatile compounds such as sarin.26,27

4. PHYSIOLOGICAL ASPECTS OF CHOLINERGIC TRANSMISSION We mentioned earlier that nerve agents exert their toxicity by inhibiting the breakdown of acetylcholine. As we will see in this section, acetylcholine is a key neurotransmitter endowed with a wide range of physiological functions, which has activity on multiple branches of the nervous system.28 Molecularly, acetylcholine triggers signaling by binding to and activating two different classes of receptors: (a) a family of voltage-gated ion channels know as nicotinic receptors, which mediate the faster ionotropic component of cholinergic signaling, and (b) a family of G protein-coupled receptors (GPCRs) known as muscarinic receptors, which mediate the slower metabotropic component of cholinergic signaling. Nicotinic receptors are oligomeric channels resulting from the combination of five subunits with four different subunit families (α, β, δ, ε), each of which comprises multiple subtypes. Conversely, muscarinic receptors exist in five different subtypes, some coupling preferentially to Gq/11 proteins (M1, M3, M5) and some coupling preferentially to Gi proteins (M2 and M4).28 The

3. THE CHEMISTRY OF NERVE AGENTS Chemically, nerve agents are phosphonic or phosphoric acid derivatives featuring a chiral phosphorus atom linked to four different substituents (Figure 1). As a result, nerve agents exist in the form of two enantiomers endowed with different affinities for the enzyme acetylcholinesterase and, consequently, different toxicity levels. In addition to the chiral phosphorus atom, soman also features a chiral carbon atom, leading to the existence of four stereoisomers. Structural evidence and deductive stereochemistry suggest that, for all the agents shown in Figure 1 with the exception of tabun, the isomers that present the phosphorus atom in the S 875


Review


Figure 4. Efferent pathways of the peripheral nervous system, showing the parasympathetic, sympathetic, and somatic branches. The neurotransmitter acetylcholine is represented by green dots; norepinephrine is represented by orange dots, and epinephrine is represented by purple dots. Receptors are indicated with the following letters: M = muscarinic receptors; N = nicotinic receptors; α = α-adrenergic receptors; β = β-adrenergic receptors. By way of example, a nonexhaustive list of the physiological effects of the stimulation of the various branches is provided; see McCorry for a comprehensive table of the effects.31

wide range of nicotinic and muscarinic receptors is consistent with the plethora of functions mediated by acetylcholine. Of note, nicotinic signaling is traditionally regarded as significantly faster than the muscarinic signaling, requiring only milliseconds as opposed to seconds or minutes. Albeit, more recent data indicate that muscarinic signaling can also occur at high speed, especially in the case of presynaptic muscarinic receptors that control the release of the neurotransmitter.29 Within both the parasympathetic and the sympathetic branch of the autonomic nervous system, acetylcholine is a key neurotransmitter for the transmission of the nervous impulse from preganglionic to postganglionic efferent neurons. In particular, stimulated preganglionic efferent neurons release acetylcholine at the ganglionic synapse, with a consequent activation of postsynaptic nicotinic receptors that results in the transmission of the nervous impulse to the postganglionic nerve (Figure 4). Similarly, acetylcholine is also released at the synapse between the efferent sympathetic neurons and cells of the adrenal medulla, once again activating postsynaptic nicotinic receptors (Figure 4). The adrenal medulla is essentially a modified sympathetic ganglion that, upon stimulation, secretes epinephrine and norepinephrine into the bloodstream. Beyond its central role as a ganglionic neurotransmitter, acetylcholine is also released by all axons of the postganglionic nerves of the parasympathetic system, leading to the activation of postsynaptic muscarinic receptors in the target organs and tissues (Figure 4). Moreover, while most postganglionic axons in the sympathetic system secrete norepinephrine and activate postsynaptic adrenergic receptors, some of them, e.g. those that innervate sweat glands, release acetylcholine and lead to the activation of

postsynaptic muscarinic receptors (Figure 4). Furthermore, in all acetylcholine-secreting synapses of the parasympathetic and the sympathetic nervous system, presynaptic muscarinic receptors are deputed to the control of further release of the neurotransmitter.30,31 As a result, acetylcholine is essential for both the rest-and-digest functions mediated by the parasympathetic system as well as the opposite fight-or-flight reaction of the sympathetic system, which results in opposite effects (see Figure 4 for some examples of these effects).28 Beyond the autonomic nervous system, acetylcholine is a key neurotransmitter at the neuromuscular junction, where it is released by presynaptic vesicles of neuronal terminals and binds to postsynaptic nicotinic receptors on muscle cells as well as presynaptic muscarinic receptors that control further release of the neurotransmitter (Figure 4).32,33 Finally, within the central nervous system (CNS), acetylcholine contributes to the function of the amygdala, hippocampus, cortex, hypothalamus, and striatum, through the activation of muscarinic and nicotinic receptors. Often these effects are mediated by the modulation of the release of other neurotransmitters such as glutamate and γaminobutyric acid.34,35 Some of the functions of acetylcholine in the CNS include the regulation of wakefulness and rapid eye movement (REM), sleep, as well as memory and learning.36,37

5. STRUCTURE OF ACETYLCHOLINESTERASE AND MOLECULAR MECHANISM OF ACETYLCHOLINE BREAKDOWN After exerting its action, acetylcholine is hydrolyzed within the synaptic cleft by acetylcholinesterase into choline and acetate. In particular, as illustrated in Figure 5, the degradation of 876


Review


Figure 5. Acetylcholine degradation by acetylcholinesterase.

acetylcholine proceeds through an acylation step, in which the acetyl group of the substrate is cleaved from the choline moiety and forms a covalent bond to a serine residue of the enzyme, followed by a deacylation step, in which the acetyl group is hydrolyzed from the serine residue. Acetylcholinesterase is an extremely efficient enzyme, with a catalytic efficiency (Kcat/Km) of the order of 108, which approximates its diffusion control limit. For instance, for the recombinant human enzyme, Ordentlich et al. measured a Km and Kcat for the substrate thioacetylcholine of 0.14 mM and 370 × 103 min−1, respectively, resulting in a catalytic efficiency of 26.4 × 108 M−1 × min−1.38 Near instant degradation of acetylcholine following its secretion terminates the signal, thus posing a strict temporal limit to the neurotransmitter’s cellular response.39 Following hydrolysis, the resulting choline is up-taken within the presynaptic terminal and employed as a substrate for the resynthesis of the neurotransmitter.40 Structures for acetylcholinesterase from several organisms were solved via X-ray crystallography, providing snapshots of the enzyme in complex with a variety of molecules, including acetylcholine, acetylcholine analogues, inhibitors, and antidotes: by way of example, an overview of the backbone structure of the murine enzyme in complex with an analogue of the substrate (PDB ID: 2HA0) is shown in Figure 6.41 Macroscopically, the enzyme belongs to the superfamily of the α/β hydrolase fold enzymes, being composed of α-helical and β-sheet domains. The β-sheet domain, which is highly conserved within the α/β hydrolases, provides the catalytic machinery of the enzyme. Conversely, the α-helical domains, mostly located on the surface of the protein, are highly enzyme-dependent and are major players in providing the basis to ensure substrate exclusivity. The enzyme’s active site is located at the bottom of a gorge. The entrance of acetylcholine into the active site is thought to be guided by 14 conserved aromatic amino acid residues located along the gorge’s surface due to a weak affinity. Moreover, acetylcholinesterase has a large dipole moment oriented along the axis of the active site at the gorge’s bottom produced by seven acidic amino acid residues located near the entrance of the gorge. This large dipole moment, at low substrate concentrations, is thought to contribute to guiding acetylcholine to the active site.42,43 To explain how choline exits the enzyme when it experiences the same attractive forces that also push the parent species toward the active site, the existence of a backdoor was postulated by Ripoll et al. in 1993.44 After several years of controversy, this hypothesis has been finally validated through X-ray crystallography and molecular dynamics experiments, which evidenced how the disruption of a hydrogen bond between Trp84 and Tyr442 (Torpedo californica numbering, corresponding to Trp86 and Tyr449 in the human enzyme) indeed opens a backdoor channel.45,46

Figure 6. Ribbon structure for mutant murine acetylcholinesterase bound to the acetylcholine analogue 4k-TMA (PDB ID: 2HA0).41 The backbone of the enzyme is schematically represented as a ribbon, colored by secondary structure (red: α-helices; cyan: β-strands; white: connecting loops). Two 4k-TMA molecules, both shown as space-filling models, can be seen in the structure: one, shown with green carbon atoms, is in the catalytic site; the other, shown with pink carbon atoms, is in the gorge. The two amino acid residues that have been proposed to form the backdoor exit are shown as ball-and-stick models with gray carbons.

Within the superfamily of α/β hydrolases, acetylcholinesterase belongs to the family of serine hydrolases because its active site features a catalytic serine residue that performs a nucleophilic attack to the substrate, which eventually leads to its hydrolysis.42 Panel A of Figure 7 shows the catalytic site for the murine enzyme in complex with a nonhydrolyzable acetylcholine analogue dubbed 4k-TMA, in which the oxygen ester of the neurotransmitter is replaced with a carbon atom.41 As the figure illustrates, the active serine residue (Ser203 in the murine enzyme) is in close proximity to the carbonyl carbon of acetylcholine, at a distance consistent with covalent bonding (of note, residue numbering for murine and human enzymes are identical). At the opposite end of the molecule, the ammonium group of acetylcholine forms a cation-π interaction with the aromatic ring of Trp46. Two additional residues located in the catalytic site, namely His447 and Glu334, together with Ser203, form a catalytic triad that support Ser203 in its catalytic role. An NMR study on the human enzyme revealed how the carboxylate of the side chain of Glu334 forms a short, strong hydrogen bond with the NH group of His447. In turn, this confers a stronger negative partial charge to the other nitrogen of the imidazole ring of His447, which facilitates the deprotonation of Ser203 in the acylation phase of the reaction and the deprotonation of a water molecule in the deacylation phase of the reaction.47 Catalytic triads are common in serine hydrolases, but they are usually composed of a serine, a histidine, and an aspartate residue, not a glutamate. The triad’s acidic residue, glutamate, was discovered in a 1991 crystallographic study of acetylcholinesterase from Torpedo californica.42 877


Review


Figure 7. Catalytic site of the murine acetylcholinesterase as shown in four crystallograpically solved structures. (A) Acetylcholinesterase bound to the acetylcholine analogue 4k-TMA (PDB ID: 2HA0).41 (B) Nonaged acetylcholinesterase inhibited by sarin (PDB ID: 2Y2V).54 (C) Nonaged acetylcholinesterase inhibited by sarin and bound to the oxime antidote HI-6 (PDB ID: 5FPP).55 (D) Aged acetylcholinesterase inhibited by sarin and bound to the oxime antidote HI-6 (PDB ID 2WHQ);51 notably, only parts of the antidote are visible in this structure. The ligands and the catalytic triad residues, namely Ser203, His447, and Glu334, are shown as balls and sticks, while the other amino acid residues are shown as thin tubes. Carbon atoms are colored in green for the ligands and in gray for the acetylcholinesterase residues. Nitrogen atoms are colored in blue, carbon atoms in green, and oxygen atoms in red. The 2D structure of the antidote HI-6 is provided as an inset in panel C.

Table 2. Toxicological Properties5,48,49 and Aging Half Times56 of Selected Nerve Agents property

tabun

sarin

soman

cyclosarin

VX

VR

humana inhalation LCt50 (mg min/m3) humana percutaneous LD50 (mg/kg) aging half time (h)b

150−400 14.3 19.2

70−100 24.3 3.04

35−50 5 0.105

35 5 7.00

10−30 0.14 36.5

40 0.1−0.01 139

a Human toxicity values are estimates extrapolated from animal data. bIn vitro data for human erythrocyte cholinesterase. Half times calculated from the rate constants for spontaneous dealkylation presented in the reference article, assuming a first order reaction.

6. INHIBITION OF ACETYLCHOLINESTERASE BY NERVE AGENTS: PATHOLOGICAL CONSEQUENCES AND MOLECULAR MECHANISM OF ACTION

consequences. Toxicity depends on the nature of the nerve agent as well as the route of exposure. As shown in Table 2, estimated LCt50 for inhalation in human can vary from 150−400 mg min/m3 for tabun to 10−30 mg min/m3 for VX.48,49 Likewise, the estimated LD50 for percutaneous exposure in human ranges from 24.3 mg/kg for sarin to 0.1 mg/kg for Russian VX.5,49 The array of symptoms associated with nerve agent poisoning is complex and varied due to the many physiological implications of muscarinic and nicotinic signaling and the above-mentioned acetylcholine-mediated modulation of the release of other neurotransmitters. To complicate the picture, overstimulation of muscarinic and nicotinic receptors eventually leads to their desensitization. Hence, further symptoms of cholinergic poison-

As mentioned, acetylcholinesterase inhibitors enhance the concentration of acetylcholine in the synaptic cleft, yielding a sustained stimulation of the cholinergic system. Reversible inhibitors, such as donezepil and galantamine, and hydrolyzable carbamate inhibitors, such as rivastigmine, are important therapeutic agents used for the treatment of a series of conditions such as Alzheimer’s disease, autism, Parkinson’s disease, and glaucoma.40 However, the irreversible covalent inhibition of acetylcholinesterase by nerve agents causes an excessive accumulation of the neurotransmitter with severe toxic 878


Review

ACS Chemical Neuroscience ing result from a lack of cholinergic transmission. Symptoms such as vomiting, miosis, wheezing, increased nasal and submucosal mucus secretion, and blood pressure drop are attributed to alteration of nicotinic signaling in the parasympathetic ganglia and, ultimately, muscarinic signaling at the synapse of the postganglionic parasympathetic nerves. Elevated blood pressure is attributed to alteration of nicotinic signaling in the sympathetic ganglia. Fasciculation and paralysis are attributed to alteration of the nicotinic signaling at the neuromuscular junction. Death typically results from respiratory drive loss or seizure.1 Mechanistically, the reaction of acetylcholinesterase with nerve agents closely follows its reaction with the neurotransmitter acetylcholine, with the acylation step that takes place in acetylcholine breakdown replaced by a phosphonylation step (Figure 8). Just as the nucleophilic Ser203 residue attacks the

Figure 9. Adducts formed by tabun, sarin, and VX with acetylcholinesterase, before and after aging.

7. AVAILABLE TREATMENT OPTIONS In the 1930s, physiologists at the Army Proving Ground Raubkammer in Münster, Germany, began developing medications to treat tabun poisoning. After testing small doses of tabun on volunteers, physiologists observed that the chemical agent resulted in slowed heartbeat, miosis, and salivary gland stimulation. Physicians reasoned that administering a dose of the fast acting atropine (Figure 10, atropine), or the more slowly

Figure 8. Covalent inhibition of acetylcholinesterase by a general nerve agent and subsequent aging of the adduct.

electrophilic carbon of the carbonyl group of acetylcholine, leading to a covalent bond between the enzyme and the acetyl group and the cleavage of the choline moiety (Figure 5), the same serine residue attacks the electrophilic phosphorus of the nerve agent, leading to a covalent bond between the enzyme and the cleavage of the electron withdrawing R1 group (Figure 8). However, while the acetylation of Ser203 by acetylcholine is readily hydrolyzable, the phosophonylation by a nerve agent is not.50 Following phosphonylation, after a period of time dependent on the specific nerve agent, a subsequent elimination reaction occurs that results in dealkylation of the phosphoester group. Thus, the acetylcholinesterase inhibited by an organophosphorus compound can be in one of two successive phases: prior to the dealkylation process, the inhibited enzyme is in the nonaged state; after the dealkylation process, it is in the aged state (Figure 8). By way of example, Figure 9 shows the agents tabun, sarin, and VX in their free form and in the nonaged and aged adducts that they form with AChE, as deduced from X-ray crystallography.51−53 The aging process is also illustrated by the crystallographic structures provided in Figure 7, with the enzyme-sarin adduct in its nonaged form shown in panels B and C (PDB IDs: 2Y2V and 5FPP, respectively)54,55 and in its aged form in panel D (PDB ID: 2WHQ).51 Aging half times range from a few minutes, as in the case of soman adducts, to 48 h, as in the case of VX adducts (Table 2).56 As we will see in the next section, the currently known antidotes are unable to reactivate acetylcholinesterase after the aging of the adducts. Hence, nerve agents that give rise to fast-aging adducts are particularly difficult to treat.

Figure 10. First row: atropine, pralidoxime (2-PAM), and diazepam are the three components of the antidote treatment nerve agent autoinjector ATNAA kit, while pyridostigmine is an FDA approved pretreatment option. Second row: RS194B is a candidate for the development of antidotes permeable to the blood brain barrier, while galantamine and huperzine A are being explored as novel pretreatment options.

acting scopolamine, would be effective because those drugs had opposing physiological effects.2 To this day, atropine remains one of the primary medical countermeasures for nerve agent poisoning.1 Although not understood at the time of introduction, atropine is a competitive antagonist of the muscarinic receptors. It does not restore acetylcholinesterase functionality; thus, it is a treatment but not an antidote for nerve agent poisoning. Moreover, it only counteracts the effects of nerve agent poisoning that involve postsynaptic muscarinic receptors. For instance, it does not treat those that arise from the stimulation of somatic motor neurons, which is based on nicotinic postsynaptic receptors, or those that arise from the stimulation of adrenergic 879


Review


not permeable to the blood−brain barrier; thus, its action is confined to outside the CNS.62

component of the sympathetic nervous system, which involve nicotinic and adrenergic postsynaptic receptors.1,4 Irwin B. Wilson and Sara Ginsburg developed the first clinically relevant acetylcholinesterase reactivator at Columbia University in the early 1950s.57 Building on Schlomo Hestrin’s discovery of hydroxylamine as a fairly weak reactivator of acetylcholinesterase inhibited by tetraethyl phosphate, Wilson and Ginsburg sought to design a drug capable of dephosphorylating acetylcholinesterase inhibited by organophosphorus compounds to restore enzyme activity.57,58 As part of these efforts, pyridine-2-aldoxime methiodide, also known as pralidoxime or 2-PAM (Figure 10, 2-PAM), was synthesized by reaction of pyridine-2-aldehyde with hydroxylamine followed by reaction with methyl iodide. 57 The same compound was also independently synthesized in the same manner and recognized as an acetylcholinesterase reactivator by British chemist Albert Green at the Chemical Defence Experimental Establishment in Porton in 1954.57 As structural evidence indicates, 2-PAM, like all other oxime antidotes, reactivates the enzyme through a nucleophilic attack performed by the oximate anion of the antidote to the phosphorus atom of the covalently bound inhibitor, yielding the cleavage of the resulting inhibitor− antidote complex (Figure 11).50

8. CURRENT RESEARCH DIRECTIONS FOR THE DEVELOPMENT OF IMPROVED TREATMENT AND PRETREATMENT OPTIONS As mentioned, the treatment of choice for nerve agent poisoning is a combination of atropine, to antagonize the muscarinic receptors, the antidote 2-PAM, to reactivate the irreversibly inhibited acetylcholinesterase, and diazepam, to counteract convulsions.4 As we also mentioned, military personnel at risk of exposure to nerve agents can be pretreated with the reversible acetylcholinesterase inhibitor pyridostigmine to limit the enzyme pool accessible to nerve agents.62 However, such treatments suffer from several limitations. To name a few: none of the currently known antidotes are capable of reactivating the inhibited acetylcholinesterase once it ages; there is no current broad-spectrum antidote;4 the effects of nerve agents on the nicotinic transmission are not adequately addressed because atropine effectively antagonizes muscarinic but not nicotinic receptors;64 the protective effect of the pretreatment of military personnel with pyridostigmine is limited to outside the CNS because pyridostigmine is unable to cross the blood−brain barrier.4,62 To address these problems, significant research efforts are currently ongoing. 8.1. Improved Antidotes. As we mentioned, the currently available oxime antidotes are not equally effective against all nerve agents. Moreover, they are incapable of regenerating the enzyme after the adduct has aged. To better understand the reasons of these shortcomings, it is worth analyzing the reactivation process in greater detail. Acetylcholinesterase reactivation is highly dependent on the antidote’s ability to properly position itself inside the covalently inhibited catalytic site of the enzyme. This is well illustrated by panel C of Figure 7, which shows the nonaged acetylcholinesterase−sarin adduct with the hydroxyl group of the HI-6 antidote positioned in a way that allows the desired reaction with the bound inhibitor. Moreover, the oxime group of HI-6 forms a hydrogen-bonded tetrad involving Glu334, His447, and a water molecule that makes it a strong enough nucleophile to displace the covalently bound organophosphorus compound.52 Crystallographic evidence also suggests that when the enzyme is in its nonaged form, several πinteractions with aromatic amino acids at the enzyme’s active site gorge play a critical role in the proper orientation of antidotes. For instance, as shown in panel C of Figure 7, the Nalkylpyridinium ring of HI-6 proximal to the oxime forms interactions with Tyr124, Tyr337, and Phe338 in the gorge of the enzyme.52 Given the variety of nerve agents (Figures 2 and 3), it is reasonable to expect that the catalytic site of nonaged acetylcholinesterase will adopt an array of different conformations depending on the nature of the bound agent. Consequently, the effectiveness of a given antidote strongly depends on the agent with which the enzyme has been inhibited.65 As we mentioned, 2-PAM is effective against sarin and VX but offers limited protection against cyclosarin and soman.59 Research is ongoing to address this problem, and antidotes endowed with different selectivity profiles have been identified. For instance, IL6 efficiently reactivates acetylcholinesterase inhibited by soman, cyclosarin, and Russian VX but is not effective against tabun.66 However, broad-spectrum antidotes have not been identified. This is clearly problematic, as it may prove impractical to

Figure 11. Mechanism of action for a general antidote. The nucleophilic attack of the oxime results in phosphonylation of the antidote and reprotonation of Ser203.

Despite its great therapeutic utility, 2-PAM suffers some limitations. Above all, as is the case for all other known antidotes, 2-PAM is only capable of reactivating nerve agent-inhibited acetylcholinesterase in its nonaged form.52 Moreover, as an additional limitation that is also common to all known antidotes, 2-PAM is not equally effective against all nerve agents: while it readily reactivates acetylcholinesterase inhibited by VX and sarin, 2-PAM is considerably less effective against tabun, soman, and cyclosarin.59 Finally, 2-PAM cleaves nonaged nerve agent from acetylcholinesterase in the peripheral nervous system but does not efficiently cross the blood−brain barrier.60 Atropine and 2-PAM are two of the three components of the ATNAA, a kit for the treatment nerve agent poisoning designed by the United States Department of Defense for the U.S. military. The third compound of the kit is the benzodiazepine compound diazepam (Figure 10, diazepam). In particular, diazepam, which is a positive allosteric modulator of the GABAA receptors, is included in the ATNAA to counteract convulsive seizures due to the stimulation of nicotinic receptors in the amygdala.4,61 Beyond the ATNAA, the United States Food and Drug Administration (FDA) approved the reversible acetylcholinesterase inhibitor pyridostigmine for the pretreatment of military personnel at risk of nerve agent exposure (Figure 10, pyridostigmine).62,63 Although the use of an acetylcholinesterase inhibitor to prevent poisoning from nerve agents may seem counterintuitive, pretreatment with pyridostigmine makes the enzyme’s catalytic site less available to nerve agents, thus protecting from their toxicity. However, pyridostigmine too is 880


Review


mechanisms, including competitive antagonism, noncompetitive antagonism, or desensitization of the receptors due to agonist stimulation. Several problems are associated with competitive antagonists, chiefly due to difficulties in administering a dose sufficient to counter the overstimulation of the receptors but not high enough to induce paralysis. Conversely, noncompetitive antagonists appear more promising and have been shown to enhance protection against the nerve agents tabun, sarin, and soman when administered as part of a combination therapy with muscarinic antagonists in guinea pigs.64,71,72 One can speculate that effective antagonists of the nicotinic receptors, by countering the overstimulation of the receptors, might also be able to prevent the consequent desensitization and could therefore counteract the symptoms due to both nicotinic stimulation and blockage. 8.3. Small-Molecule Pretreatment Options. To overcome the shortcomings of pyridostigmine bromide, several pretreatment compounds capable of acting on the CNS are being explored. Research shows that galantamine, a drug approved by the FDA for the treatment of Alzheimer’s disease, is also very effective in preventing the lethal consequences of sarin and soman poisoning in guinea pigs when administered before or soon after exposure to nerve agents, especially when associated with atropine treatment.62,73−76 Galantamine is a reversible inhibitor of acetylcholinesterase that, compared to pyridostigmine, has the advantage of being permeable to the blood−brain barrier. Perhaps more importantly, galantamine also acts as a potentiating allosteric ligand that enhances the response of nicotinic receptors to acetylcholine.77 In this capacity, it is likely that galantamine counteracts the loss of nicotinic signaling due to receptor desensitization caused by their overstimulation. Huperzine A is also a reversible acetylcholinesterase effective in both the central and peripheral nervous systems.78 Although it is not an FDA approved drug, the compound is believed to improve symptoms of Alzheimer’s disease and dementia and has displayed antiseizure effects in animal models.78,79 Although huperzine A does not act as a positive allosteric modulator of nicotinic signaling, a recent study using cynomolgus macaques indicates the compound is approximately 88 times more potent than galantamine and prevents soman toxicity equally well when administered as a pretreatment.78 Furthermore, huperzine A provides greater behavioral safety than galantamine.78 8.4. Protein-Based Pretreatment Options. Bioscavengers. Another line of research, which began in the late 1980s with seminal work conducted at Walter Reed by Doctor and coworkers,80 concerns the development of macromolecular bioscavengers of organophosphorus compounds, which could be used prophylactically to avoid nerve agent poisoning or limit its consequences. Examples of bioscavengers include enzymes, reactive proteins, and antibodies that neutralize circulating free nerve agent before it can bind to and inhibit acetylcholinesterase. Specifically, bioscavengers can be divided into two different classes: stoichiometric bioscavengers, which bind and sequester nerve agents in a fixed ratio, and catalytic bioscavengers, which bind and enzymatically degrade nerve agents. Unlike stoichiometric bioscavengers, which get inactivated upon binding nerve agents, catalytic bioscavengers regenerate after detoxifying a unit of nerve agent to continue scavenging.81 A major challenge in bioscavenger-based approaches to pretreatment is maintaining high enough circulating levels of the high molecular weight substances to afford protection.81 In this regard, catalytic bioscavengers are preferable because they can detoxify more molar equivalents of nerve agent per unit than

establish the nature of the nerve agent before the administration of an antidote. Concerning the development of antidotes that can reactivate the aged form of irreversibly inhibited acetylcholinesterase, research efforts are currently ongoing to understand the structural and physicochemical reasons behind the fact that the currently available antidotes are unable to perform such a task and figure out how such problems could be overcome. Crystallographic studies show how the aging of the enzyme causes a structural rearrangement of the catalytic site that does not allow the currently known antidotes to place themselves in a suitable position to react with the enzyme-bound inhibitor and cleave it from the enzyme (panel D of Figure 7).52 When comparing panels C and D of Figure 7, it appears evident that the HI-6 antidote shows two different binding modes before and after the aging of the enzyme. Beyond the proper positioning of the antidotes, a further question concerns the electronic properties of the enzyme−inhibitor bond, which may change in a way that renders the cleavage of the bond more difficult after the aging of the adduct. Encouragingly, it has been recently shown that a series of N-methyl-2-methoxypyridinium compounds can indeed react with a weakly basic phosphonate anion that can be considered as a model for an aged acetylcholinesterase−nerve agent system.67 While the ability of such compounds to cleave an aged nerve agent from acetylcholinesterase has not yet been reported, the fact that the reaction can occur suggests that N-methyl-2-methoxypyridinium compounds that fit the binding site of the enzyme and position themselves properly might be able perform the sought-after task. To explore this possibility, a recent computational chemistry study compared the estimated free energy barriers for the reaction in water as well as in the context of the aged enzyme, concluding that the barrier is much higher in the enzyme than in water.68 The available crystallographic information, possibly coupled with computational experiments, could be leveraged to rationally identify antidotes that can effectively reactivate the aged form of the irreversibly inhibited enzyme. A final problem of conventional antidotes is given by the fact that they afford minimal reactivation of acetylcholinesterase in the CNS. To bypass this problem, Pro-2-PAM is being explored as an antidote capable of crossing the blood−brain barrier to reactivate central nervous system acetylcholinesterase.60 The pyridyl ring of 2-PAM was replaced with a dihydropyridyl moiety to create a lipid soluble pro-drug that is oxidized to 2-PAM upon entering the central nervous system.60 Early studies indicate that, on average, injection of pro-2-PAM with atropine sulfate protects twice as much as injection of 2-PAM and atropine sulfate against percutaneously administered soman in animals pretreated with pyridostigmine bromide.60 In further efforts to attain centrally active reactivators, several series of oxime compounds capable of crossing the blood−brain barrier have been synthesized. Among them, the zwitterion RS194B (Figure 10) showed good in vitro potency as a reactivator of sarin-inhibited acetylcholinesterase, although lower than the one displayed by 2-PAM. Notably, RS194B displayed the ability to rapidly revert the central effects of sarin poisoning in vivo in a nonhuman primate model. Moreover, it displayed highly favorable pharmacokinetics and low toxicity. Hence, RS194B promises to be an excellent candidate for the development of a new centrally active antidote.69,70 8.2. Antagonists of the Nicotinic Receptors. Several progresses have been made concerning the blockage of nicotinic signaling, which can be achieved through a variety of 881


Review

ACS Chemical Neuroscience stoichiometric bioscavengers.81 An ideal catalytic bioscavenger with a high turnover rate could be administered in a much lower dose at a lower cost than stoichiometric bioscavengers and afford equal or better protection.82 Bioscavengers also pose challenges from an immunological perspective: in this regards, the best candidates are human proteins, which are least likely to precipitate an immune response.82 Fetal bovine serum acetylcholinesterase, or FBS-AChE, was first investigated as a stoichiometric nerve agent bioscavenger in 1987.83 Experiments demonstrated that pretreatment with FBSAChE protects against up to five times the LD50 of soman in rhesus monkeys. The current leading stoichiometric bioscavenger is human butyrylcholinesterase (hBChE). A 200 mg dose of the enzyme is predicted to protect against twice the LD50 of soman in humans; however, large scale production of the bioscavenger as a pretreatment is not possible.82 To overcome this obstacle, recombinant forms of hBChE in several expression systems are being explored, the most promising of which is rhBChE expressed in the milk of transgenic goats.82 Because early goat milk derived rhBChE had a less favorable pharmacokinetic profile than hBChE, researchers developed a PEGylated version.84 This PEGylated version of rhBChE has a longer mean residence time and a more favorable pharmacokinetic profile.81 Mixtures of stoichiometric bioscavengers and oxime reactivators of the selected bioscavenger, referred to as pseudocatalytic bioscavengers, are being explored to reduce the amount of the stoichiometric bioscavenger required to afford protection.82 Several catalytic bioscavengers of potential clinical relevance are under investigation. Early efforts to design catalytic bioscavengers focused on mutagenesis of human cholinesterases to yield enzymes with improved spontaneous reactivation rates through introduction of additional catalytic centers.81 Unfortunately, none of the mutants developed to date are of practical interest as catalytic bioscavengers, as none exhibit sufficient catalytic activity nor offer adequate protection against nerve agent exposure.85 Conversely, very successful results have been obtained with mutants of phosphotriesterases (PTEs), which have shown excellent catalytic properties and the ability to prevent systemic VX toxicity even when administered postexposure.86,87 Among them, an engineered Brevundimonas diminuta PTE mutant dubbed C23 has been shown to efficiently hydrolyze V-series agents in vitro, with a Kcat/Km of 5 × 106 M−1 min−1. Moreover, treatment with C23 following exposure to twice the LD50 of VX was shown to prevent systemic toxicity and mortality in guinea pigs.86 Beyond PTEs, paroxonases are another class of enzymes under study for their ability to hydrolyze phosphotriester compounds. Among them, human paraoxonase 1 (hPON-1) has been explored extensively as a catalytic bioscavenger. This calcium dependent enzyme acts as a lactonase, arylesterase, and phosphotriesterase and is known to readily hydrolyze many nerve agents.82 Animal studies show that intravenously administered hPON-1 offers some protection against tabun, sarin, and soman poisoning.85,88 Research efforts have produced recombinant PON1 (rePON1) variants expressed in Escherichia coli with Kcat/Km values for soman and cyclosarin of 3−5 × 107 M−1 min−1. Despite this progress, work is still needed to improve catalytic efficiency against other nerve agents to make PON1 a clinically viable broad-spectrum pretreatment.88 Another challenge in catalytic bioscavenger development is that existing candidate enzymes with the most desirable catalytic properties are expected to be immunogenic in

humans. As such, gene delivery of evolved PON1 variants is under investigation as a promising pretreatment strategy.82,85

9. SUMMARY AND CONCLUSIONS Although the development of the first nerve agents dates back to the 1930s, these organophosphorus chemical warfare agents are still employed in warfare, terrorism, and assassinations. As we have seen, the high toxicity of the compounds stems from the fact that they interfere with the metabolism of acetylcholine, a key neurotransmitter involved in a plethora of functions in the sympathetic and parasympathetic branches of the nervous system, the motor neurons of the somatic nervous system, and the central nervous system. In particular, they do so by covalently inhibiting acetylcholinesterase, the enzyme deputed to the degradation of acetylcholine after its release by the presynaptic termini of cholinergic neurons. The accumulation of the neurotransmitter results in an initial overexcitation of the above-mentioned systems, followed by a consequent shutdown of the same due to desensitization of acetylcholine muscarinic and nicotinic receptors. Death typically occurs from respiratory failure or seizures. Through this article, after reviewing the physiological aspects of cholinergic transmission as well as the biochemistry of acetylcholinesterase and its inhibition by nerve agents, we surveyed the currently available treatment options and the current research directions for their improvement. As we have seen, the ATNAA, which is the kit used by the U.S. military to treat nerve agent intoxication, contains three components: atropine, which is an antagonist of the muscarinic receptors, 2PAM, which is an antidote that functions by cleaving the bound nerve agent from the catalytic site of the enzyme, and diazepam, a benzodiazepine that acts as a positive allosteric modulator of the GABAA receptors and counteracts convulsive seizures. Moreover, the reversible acetylcholinesterase inhibitor pyridostigmine is available as a pretreatment drug for military personnel at risk of nerve agent exposure. Despite their therapeutic usefulness, these drugs are affected by significant problems. For instance: the known antidotes are incapable of reactivating the inhibited acetylcholinesterase once the adduct ages, which is problematic in general and particularly so for those nerve agents whose adducts tend to age very quickly, such as soman; there is no current broad-spectrum antidote, which is problematic because it is impractical to determine the nature of the nerve agent before administering a suitable drug; the action of the antidote 2-PAM is mostly confined to outside the CNS; the activation of nicotinic receptors by the accumulated acetylcholine is not contrasted by atropine, which is an antagonist only for the muscarinic receptors; the protection offered by the pretreatment drug pyridostigmine is limited to outside the CNS because pyridostigmine is unable to cross the blood−brain barrier. As we discussed, significant research efforts are being made to attempt to address these problems. In particular, studies are being conducted to unravel the reasons behind the inability of the antidotes to reactivate the poisoned enzyme after aging and to explain their lack of universality. Presently, it is not yet clear if obtaining of antidotes devoid of these problems is a feasible goal; given the great benefit that such drugs would provide, further investigation is indeed warranted. Conversely, good progress has been made toward the development of antidotes active in the CNS, for instance with the prodrug Pro-2-PAM. Progress has been made with respect to addressing the nicotinic effects of acetylcholine with the exploration of noncompetitive antagonists of the nicotinic receptors. Finally, significant progress has been 882


Review


(15) Ministry for Europe and Foreign Affairs. (2017) National evaluation: Chemical attack of 4 April 2017 (Khan Sheikhoun) Clandestine Syrian chemical weapons programme, Annex. https:// www.diplomatie.gouv.fr/en/country-files/syria/events/article/ chemical-attack-in-syria-national-evaluation-presented-by-jean-marcayrault (accessed April 19, 2018). (16) OPCW. (2018) OPCW Fact-Finding Mission Continues Deployment to Syria. https://www.opcw.org/news/article/opcw-factfinding-mission-continues-deployment-to-syria/ (accessed April 19, 2018). (17) Olson, K. B. (1999) Aum Shinrikyo: once and future threat? Emerging Infect. Dis. 5, 413−416. (18) Ng, E. (2017) Post-mortem: VX poison killed brother of North Korean leader, Associated Press, October 3, 2017. https://apnews.com/ 90e425dbaf1e44d1ba77e2eea890fc67. (19) Cheng, M. (2018) UK says ex-spy poisoned with Sovietdeveloped nerve agent, Associated Press, March 13, 2018. https:// apnews.com/1a4c95b0e6af4d70b054c8030e177b47. (20) Ashani, Y., Gupta, R., Goldsmith, M., Silman, I., Sussman, J., Tawfik, D., and Leader, H. (2010) Stereo-specific synthesis of analogues of nerve agents and their utilization for selection and characterization of paraoxonase (PON1) catalytic scavengers. Chem.-Biol. Interact. 187, 362−369. (21) Benschop, H., and De Jong, L. (1988) Nerve agent stereoisomers: analysis, isolation and toxicology. Acc. Chem. Res. 21, 368−374. (22) Jang, Y. J., Kim, K., Tsay, O. G., Atwood, D. A., and Churchill, D. G. (2015) Update 1 of: Destruction and Detection of Chemical Warfare Agents. Chem. Rev. 115, PR1−PR76. (23) Kasten, S. A., Zulli, S., Jones, J. L., Dephillipo, T., and Cerasoli, D. M. (2014) Chiral Separation of G-type Chemical Warfare Nerve Agents via Analytical Supercritical Fluid Chromatography. Chirality 26, 817− 824. (24) Kim, K., Tsay, O. G., Atwood, D. A., and Churchill, D. G. (2011) Destruction and detection of chemical warfare agents. Chem. Rev. 111, 5345−5403. (25) Ordentlich, A., Barak, D., Kronman, C., Benschop, H. P., De Jong, L. P., Ariel, N., Barak, R., Segall, Y., Velan, B., and Shafferman, A. (1999) Exploring the active center of human acetylcholinesterase with stereomers of an organophosphorus inhibitor with two chiral centers. Biochemistry 38, 3055−3066. (26) Altmann, H. J., Oelze, S., and Niemeyer, B. (2013) Chemical Agents−Small Molecules with Deadly Properties. CBRN Protection: Managing the Threat of Chemical, Biological, Radioactive and Nuclear Weapons, 67−101. (27) OPCW. (2017) Nerve Agents. https://www.opcw.org/aboutchemical-weapons/types-of-chemical-agent/nerve-agents/ (accessed April 19, 2018). (28) Brown, D. A. (2006) Acetylcholine. British journal of pharmacology 147, S120−S126. (29) Kupchik, Y. M., Barchad-Avitzur, O., Wess, J., Ben-Chaim, Y., Parnas, I., and Parnas, H. (2011) A novel fast mechanism for GPCRmediated signal transduction–control of neurotransmitter release. J. Cell Biol. 192, 137−151. (30) Vernino, S., Hopkins, S., and Wang, Z. (2009) Autonomic ganglia, acetylcholine receptor antibodies, and autoimmune ganglionopathy. Auton. Neurosci. 146, 3−7. (31) McCorry, L. K. (2007) Physiology of the autonomic nervous system. Am. J. Pharm. Educ. 71, 78. (32) Huh, K.-H., and Fuhrer, C. (2002) Clustering of nicotinic acetylcholine receptors: from the neuromuscular junction to interneuronal synapses. Mol. Neurobiol. 25, 79−112. (33) Slutsky, I., Silman, I., Parnas, I., and Parnas, H. (2001) Presynaptic M(2) muscarinic receptors are involved in controlling the kinetics of ACh release at the frog neuromuscular junction. J. Physiol. 536, 717− 725. (34) Picciotto, M. R., Higley, M. J., and Mineur, Y. S. (2012) Acetylcholine as a neuromodulator: cholinergic signaling shapes nervous system function and behavior. Neuron 76, 116−129.

made on the front of the development of pretreatment options: above all, galantamine has been identified as a very promising small-molecule pretreatment option, and a variety of bioscavengers are under development as macromolecular pretreatment options. In conclusion, the currently ongoing research efforts are making good progress in many different directions. Hopefully, these advancements will soon lead to a lower target susceptibility, thus reducing the appeal of nerve agents as chemical weapons.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Stefano Costanzi: 0000-0003-3183-7332 Author Contributions

All authors contributed to the research, the writing of the manuscript, and the preparation of the figures. Funding

This research was supported by funding from American University and The George Washington University. Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) King, A. M., and Aaron, C. K. (2015) Organophosphate and carbamate poisoning. Emergency Medicine Clinics 33, 133−151. (2) Tucker, J. B. (2007) War of nerves: chemical warfare from World War I to Al-Qaeda, pp 24−63, Anchor, New York. (3) Salem, H., Ternay, A. L., and Smart, J. K. (2010) Brief history and use of chemical warfare agents in warfare and terrorism. In Chemical warfare agents: chemistry, pharmacology, toxicology, and therapeutics (Lukey, B. J., Romano, J. A., and Salem, H., Eds.), pp 1−17, Informa Healthcare, London. (4) Moshiri, M., Darchini-Maragheh, E., and Balali-Mood, M. (2012) Advances in toxicology and medical treatment of chemical warfare nerve agents. Daru, J. Pharm. Sci. 20, 81. (5) Rembovskiy, V., Radilov, A., Savelieva, E., Kuznetsov, S., Mindukshev, I., Prokofieva, D., Krivorotova, N., Khlebnikova, N., Jenkins, R., and Goncharov, N. (2015) Russian VX. In Handbook of toxicology of chemical warfare agents (Gupta, R. C., Ed.), 2nd ed., pp 111− 128, Academic Press, Cambridge. (6) Hoenig, S. L. (2007) Compendium of chemical warfare agents, p 100, Springer, New York. (7) Pitschmann, V. (2014) Overall view of chemical and biochemical weapons. Toxins 6, 1761−1784. (8) Vásárhelyi, G., and Földi, L. (2007) History of Russia’s chemical weapons. Academic and Applied Research in Military Science 6, 135−146. (9) Halámek, E., and Kobliha, Z. (2011) Potenciálni ́ bojové chemické látky. Chem. Listy 105, 323−333. (10) Hosseini, S. E., Saeidian, H., Amozadeh, A., Naseri, M. T., and Babri, M. (2016) Fragmentation pathways and structural characterization of organophosphorus compounds related to the Chemical Weapons Convention by electron ionization and electrospray ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom. 30, 2585− 2593. (11) Ellison, D. H. (2007) Handbook of chemical and biological warfare agents, pp 37−42, CRC Press, Boca Raton. (12) Hoenig, S. L. (2007) Compendium of chemical warfare agents, pp 79−80, Springer, New York. (13) Pita, R., and Domingo, J. (2014) The use of chemical weapons in the Syrian conflict. Toxics 2, 391−402. (14) Balali-Mood, M., and Balali-Mood, K. (2008) Neurotoxic disorders of organophosphorus compounds and their managements. Archives of Iranian Medicine 11, 65−89. 883


Review

ACS Chemical Neuroscience (35) Aroniadou-Anderjaska, V., Figueiredo, T. H., Apland, J. P., Qashu, F., and Braga, M. F. (2009) Primary brain targets of nerve agents: the role of the amygdala in comparison to the hippocampus. NeuroToxicology 30, 772−776. (36) Brown, R. E., Basheer, R., McKenna, J. T., Strecker, R. E., and McCarley, R. W. (2012) Control of sleep and wakefulness. Physiol. Rev. 92, 1087−1187. (37) Hasselmo, M. E. (2006) The role of acetylcholine in learning and memory. Curr. Opin. Neurobiol. 16, 710−715. (38) Ordentlich, A., Barak, D., Kronman, C., Flashner, Y., Leitner, M., Segall, Y., Ariel, N., Cohen, S., Velan, B., and Shafferman, A. (1993) Dissection of the human acetylcholinesterase active center determinants of substrate specificity. Identification of residues constituting the anionic site, the hydrophobic site, and the acyl pocket. J. Biol. Chem. 268, 17083−17095. (39) Zhou, Y., Wang, S., and Zhang, Y. (2010) Catalytic reaction mechanism of acetylcholinesterase determined by Born− Oppenheimer ab initio QM/MM molecular dynamics simulations. J. Phys. Chem. B 114, 8817−8825. (40) Colovic, M. B., Krstic, D. Z., Lazarevic-Pasti, T. D., Bondzic, A. M., and Vasic, V. M. (2013) Acetylcholinesterase inhibitors: pharmacology and toxicology. Current neuropharmacology 11, 315−335. (41) Bourne, Y., Radić, Z., Sulzenbacher, G., Kim, E., Taylor, P., and Marchot, P. (2006) Substrate and product trafficking through the active center gorge of acetylcholinesterase analyzed by crystallography and equilibrium binding. J. Biol. Chem. 281, 29256−29267. (42) Sussman, J. L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L., and Silman, I. (1991) Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein. Science 253, 872−879. (43) Silman, I., and Sussman, J. L. (2008) Acetylcholinesterase: how is structure related to function? Chem.-Biol. Interact. 175, 3−10. (44) Ripoll, D. R., Faerman, C. H., Axelsen, P. H., Silman, I., and Sussman, J. L. (1993) An electrostatic mechanism for substrate guidance down the aromatic gorge of acetylcholinesterase. Proc. Natl. Acad. Sci. U. S. A. 90, 5128−5132. (45) Xu, Y. C., Colletier, J. P., Weik, M., Qin, G. R., Jiang, H. L., Silman, I., and Sussman, J. L. (2010) Long Route or Shortcut? A Molecular Dynamics Study of Traffic of Thiocholine within the Active-Site Gorge of Acetylcholinesterase. Biophys. J. 99, 4003−4011. (46) Sanson, B., Colletier, J. P., Xu, Y. C., Lang, P. T., Jiang, H. L., Silman, I., Sussman, J. L., and Weik, M. (2011) Backdoor opening mechanism in acetylcholinesterase based on X-ray crystallography and molecular dynamics simulations. Protein Sci. 20, 1114−1118. (47) Massiah, M. A., Viragh, C., Reddy, P. M., Kovach, I. M., Johnson, J., Rosenberry, T. L., and Mildvan, A. S. (2001) Short, strong hydrogen bonds at the active site of human acetylcholinesterase: proton NMR studies. Biochemistry 40, 5682−5690. (48) Rossodivita, A., Guidotti, M., Ranghieri, M. C., Stikova, E. J., and Cummings, C. E. (2007) Chemical warfare agents − Medical aspects and principles of treatment, Part I − Nerve agents. In Strengthening national public health preparedness and response to chemical, biological and radiological agent threats (Cummings, C. E., and Stikova, E., Eds.), pp 103−111, IOS Press, Amsterdam, Netherlands. (49) Department of the Army. (1974) Edgewood Arsenal Special Report: Chemical Agent Data Sheets, Aberdeen Proving Ground, MD. http://www.dtic.mil/dtic/tr/fulltext/u2/b028222.pdf (accessed April 19, 2018). (50) Sanson, B. t., Nachon, F., Colletier, J.-P., Froment, M.-T., Toker, L., Greenblatt, H. M., Sussman, J. L., Ashani, Y., Masson, P., and Silman, I. (2009) Crystallographic snapshots of nonaged and aged conjugates of soman with acetylcholinesterase, and of a ternary complex of the aged conjugate with pralidoxime. J. Med. Chem. 52, 7593−7603. (51) Ekström, F., Akfur, C., Tunemalm, A.-K., and Lundberg, S. (2006) Structural changes of phenylalanine 338 and histidine 447 revealed by the crystal structures of tabun-inhibited murine acetylcholinesterase. Biochemistry 45, 74−81. (52) Ekström, F., Hörnberg, A., Artursson, E., Hammarström, L.-G., Schneider, G., and Pang, Y.-P. (2009) Structure of HI-6• sarin-

acetylcholinesterase determined by X-ray crystallography and molecular dynamics simulation: reactivator mechanism and design. PLoS One 4, e5957. (53) Hörnberg, A., Tunemalm, A.-K., and Ekström, F. (2007) Crystal structures of acetylcholinesterase in complex with organophosphorus compounds suggest that the acyl pocket modulates the aging reaction by precluding the formation of the trigonal bipyramidal transition state. Biochemistry 46, 4815−4825. (54) Akfur, C., Artursson, E., and Ekström, F. (2011) Nonaged form of mouse acetylcholinesterase inhibited by sarin - update, The Protein Data Bank. https://www.rcsb.org/structure/2Y2V (accessed April 19, 2018). (55) Allgardsson, A., Berg, L., Akfur, C., Hörnberg, A., Worek, F., Linusson, A., and Ekström, F. J. (2016) Structure of a prereaction complex between the nerve agent sarin, its biological target acetylcholinesterase, and the antidote HI-6. Proc. Natl. Acad. Sci. U. S. A. 113, 5514−5519. (56) Worek, F., Thiermann, H., Szinicz, L., and Eyer, P. (2004) Kinetic analysis of interactions between human acetylcholinesterase, structurally different organophosphorus compounds and oximes. Biochem. Pharmacol. 68, 2237−2248. (57) Petroianu, G. (2013) The history of pyridinium oximes as nerve gas antidotes: The British contribution. Die Pharmazie-An International Journal of Pharmaceutical Sciences 68, 916−918. (58) Wilson, I. B. (1958) Designing of a new drug with antidotal properties against the nerve gas sarin. Biochim. Biophys. Acta 27, 196− 199. (59) McDonough, J. H., and Romano, J. A. (2010) Health effects of low-level exposure to nerve agents. In Chemical warfare agents: chemistry, pharmacology, toxicology, and therapeutics (Lukey, B. J., Romano, J. A., and Salem, H., Eds.), pp 71−96, Informa Healthcare, London. (60) DeMar, J. C., Clarkson, E. D., Ratcliffe, R. H., Campbell, A. J., Thangavelu, S. G., Herdman, C. A., Leader, H., Schulz, S. M., Marek, E., and Medynets, M. A. (2010) Pro-2-PAM therapy for central and peripheral cholinesterases. Chem.-Biol. Interact. 187, 191−198. (61) Iha, H. A., Kunisawa, N., Shimizu, S., Tokudome, K., Mukai, T., Kinboshi, M., Ikeda, A., Ito, H., Serikawa, T., and Ohno, Y. (2017) Nicotine Elicits Convulsive Seizures by Activating Amygdalar Neurons. Front. Pharmacol. 8, 57. (62) Alexandrova, E., Alkondon, M., Aracava, Y., Pereira, E., and Albuquerque, E. (2014) Galantamine prevents long-lasting suppression of excitatory synaptic transmission in CA1 pyramidal neurons of somanchallenged guinea pigs. NeuroToxicology 44, 270−278. (63) Haigh, J. R., Adler, M., Apland, J. P., Deshpande, S. S., Barham, C. B., Desmond, P., Koplovitz, I., Lenz, D. E., and Gordon, R. K. (2010) Protection by pyridostigmine bromide of marmoset hemi-diaphragm acetylcholinesterase activity after soman exposure. Chem.-Biol. Interact. 187, 416−420. (64) Turner, S., Chad, J., Price, M., Timperley, C., Bird, M., Green, A., and Tattersall, J. (2011) Protection against nerve agent poisoning by a noncompetitive nicotinic antagonist. Toxicol. Lett. 206, 105−111. (65) Mercey, G., Verdelet, T., Renou, J., Kliachyna, M., Baati, R., Nachon, F., Jean, L., and Renard, P.-Y. (2012) Reactivators of acetylcholinesterase inhibited by organophosphorus nerve agents. Acc. Chem. Res. 45, 756−766. (66) Saxena, A., Luo, C., Chilukuri, N., Maxwell, D. M., and Doctor, B. P. (2010) Novel approaches to medical protection against chemical warfare agents. In Chemical warfare agents: chemistry, pharmacology, toxicology, and therapeutics (Lukey, B. J., Romano, J. A., and Salem, H., Eds.), pp 145−167, Informa Healthcare, London. (67) Topczewski, J. J., and Quinn, D. M. (2013) Kinetic assessment of N-methyl-2-methoxypyridinium species as phosphonate anion methylating agents. Org. Lett. 15, 1084−1087. (68) An, Y., Zhu, Y., Yao, Y., and Liu, J. (2016) Is it possible to reverse aged acetylcholinesterase inhibited by organophosphorus compounds? Insight from the theoretical study. Phys. Chem. Chem. Phys. 18, 9838− 9846. (69) Radic, Z., Sit, R. K., Kovarik, Z., Berend, S., Garcia, E., Zhang, L. M., Amitai, G., Green, C., Radic, B., Fokin, V. V., Sharpless, K. B., and Taylor, P. (2012) Refinement of structural leads for centrally acting 884


Review

ACS Chemical Neuroscience oxime reactivators of phosphylated cholinesterases (vol 287, pg 11789, 2012). J. Biol. Chem. 287, 19337−19337. (70) Rosenberg, Y. J., Mao, L. J., Jiang, X. M., Lees, J., Zhang, L. M., Radic, Z., and Taylor, P. (2017) Post-exposure treatment with the oxime RS194B rapidly reverses early and advanced symptoms in macaques exposed to sarin vapor. Chem.-Biol. Interact. 274, 50−57. (71) Ring, A., Strom, B. O., Turner, S. R., Timperley, C. M., Bird, M., Green, A. C., Chad, J. E., Worek, F., and Tattersall, J. E. (2015) Bispyridinium compounds inhibit both muscle and neuronal nicotinic acetylcholine receptors in human cell lines. PLoS One 10, e0135811. (72) Sheridan, R. D., Smith, A. P., Turner, S. R., and Tattersall, J. E. (2005) Nicotinic antagonists in the treatment of nerve agent intoxication. J. R. Soc. Med. 98, 114−115. (73) Albuquerque, E. X., Pereira, E. F., Aracava, Y., Fawcett, W. P., Oliveira, M., Randall, W. R., Hamilton, T. A., Kan, R. K., Romano, J. A., and Adler, M. (2006) Effective countermeasure against poisoning by organophosphorus insecticides and nerve agents. Proc. Natl. Acad. Sci. U. S. A. 103, 13220−13225. (74) Alexandrova, E. A., Aracava, Y., Pereira, E. F., and Albuquerque, E. X. (2010) Pretreatment of Guinea pigs with galantamine prevents immediate and delayed effects of soman on inhibitory synaptic transmission in the hippocampus. J. Pharmacol. Exp. Ther. 334, 1051− 1058. (75) Gullapalli, R. P., Aracava, Y., Zhuo, J., Neto, E. H., Wang, J., Makris, G., Merchenthaler, I., Pereira, E. F., and Albuquerque, E. X. (2010) Magnetic resonance imaging reveals that galantamine prevents structural brain damage induced by an acute exposure of guinea pigs to soman. NeuroToxicology 31, 67−76. (76) Pereira, E. F., Aracava, Y., Alkondon, M., Akkerman, M., Merchenthaler, I., and Albuquerque, E. X. (2010) Molecular and cellular actions of galantamine: clinical implications for treatment of organophosphorus poisoning. J. Mol. Neurosci. 40, 196−203. (77) Samochocki, M., Höffle, A., Fehrenbacher, A., Jostock, R., Ludwig, J., Christner, C., Radina, M., Zerlin, M., Ullmer, C., and Pereira, E. F. (2003) Galantamine is an allosterically potentiating ligand of neuronal nicotinic but not of muscarinic acetylcholine receptors. J. Pharmacol. Exp. Ther. 305, 1024−1036. (78) Hamilton, L. R., Schachter, S. C., and Myers, T. M. (2017) Time Course, Behavioral Safety, and Protective Efficacy of Centrally Active Reversible Acetylcholinesterase Inhibitors in Cynomolgus Macaques. Neurochem. Res. 42, 1962−1971. (79) Gersner, R., Ekstein, D., Dhamne, S., Schachter, S., and Rotenberg, A. (2015) Huperzine A prophylaxis against pentylenetetrazole-induced seizures in rats is associated with increased cortical inhibition. Epilepsy Res. 117, 97−103. (80) Raveh, L., Ashani, Y., Levy, D., De La Hoz, D., Wolfe, A. D., and Doctor, B. P. (1989) Acetylcholinesterase prophylaxis against organophosphate poisoning. Quantitative correlation between protection and blood-enzyme level in mice. Biochem. Pharmacol. 38, 529−534. (81) Lenz, D. E., Broomfield, C. A., Yeung, D. T., Masson, P., Maxwell, D. M., and Cerasoli, D. M. (2010) Nerve agent bioscavengers: progress in development of a new mode of protection against organophosphorus exposure. In Chemical warfare agents: chemistry, pharmacology, toxicology, and therapeutics (Lukey, B. J., Romano, J. A., and Salem, H., Eds.), pp 175−193, Informa Healthcare, London. (82) Nachon, F., Brazzolotto, X., Trovaslet, M., and Masson, P. (2013) Progress in the development of enzyme-based nerve agent bioscavengers. Chem.-Biol. Interact. 206, 536−544. (83) Wolfe, A. D., Rush, R. S., Doctor, B. P., Koplovitz, I., and Jones, D. (1987) Acetylcholinesterase prophylaxis against organophosphate toxicity. Toxicol. Sci. 9, 266−270. (84) Huang, Y.-J., Huang, Y., Baldassarre, H., Wang, B., Lazaris, A., Leduc, M., Bilodeau, A. S., Bellemare, A., Côté, M., and Herskovits, P. (2007) Recombinant human butyrylcholinesterase from milk of transgenic animals to protect against organophosphate poisoning. Proc. Natl. Acad. Sci. U. S. A. 104, 13603−13608. (85) Masson, P., and Lushchekina, S. V. (2016) Emergence of catalytic bioscavengers against organophosphorus agents. Chem.-Biol. Interact. 259, 319−326.

(86) Bigley, A. N., Mabanglo, M. F., Harvey, S. P., and Raushel, F. M. (2015) Variants of Phosphotriesterase for the Enhanced Detoxification of the Chemical Warfare Agent VR. Biochemistry 54, 5502−5512. (87) Worek, F., Seeger, T., Reiter, G., Goldsmith, M., Ashani, Y., Leader, H., Sussman, J. L., Aggarwal, N., Thiermann, H., and Tawfik, D. S. (2014) Post-exposure treatment of VX poisoned guinea pigs with the engineered phosphotriesterase mutant C23: a proof-of-concept study. Toxicol. Lett. 231, 45−54. (88) Goldsmith, M., Ashani, Y., Simo, Y., Ben-David, M., Leader, H., Silman, I., Sussman, J. L., and Tawfik, D. S. (2012) Evolved stereoselective hydrolases for broad-spectrum G-type nerve agent detoxification. Chem. Biol. 19, 456−466. (89) Singh, B., Prasad, G. K., Pandey, K., Danikhel, R., and Vijayaraghavan, R. (2010) Decontamination of chemical warfare agents. Def. Sci. J. 60, 428. (90) Lillie, S., Hanlon, Jr., E., Kelly, J., and Rayburn, B. (2005) Potential military chemical/biological agents and compounds, Army, Marine Corps, Navy, Air Force, FM 3−11, Vol 9, https://fas.org/irp/doddir/ army/fm3-11-9.pdf.

885


Nerve agents: what they are, how they work, how to counter them

Recommend Documents