Viewpoint Cite This: J. Med. Chem. 2018, 61, 7032−7033
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Resurrection Biology: Aged Acetylcholinesterase Brought Back to Life Daniel M. Quinn*
J. Med. Chem. 2018.61:7032-7033. Downloaded from pubs.acs.org by 5.188.216.64 on 08/24/18. For personal use only.
Department of Chemistry, The University of Iowa, Iowa City, Iowa 52242, United States ABSTRACT: Organophosphorus agents such as sarin and soman that phosphylate the active site serine of the enzyme acetylcholinesterase are notorious and pernicious, not only because they have been used by tyrants to effect mass murder of their own populations but also because they are sought by terrorists to inflict mass casualties on civilian populations. These threats underscore the need to develop effective antidotes against such agents. Phosphylation of acetylcholinesterase produces two adducts, an initial neutral adduct that can be reactivated with oxime nucleophiles, and a subsequent monoanionic adduct (called aged acetylcholinesterase) which has proven over two generations to be impervious to reactivation. This Viewpoint discusses a recent article in the journal that describes the first successful efforts to resurrect the activity of aged acetylcholinesterase.
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RESURRECTION OF AGED ACHE: A BRIDGE TOO FAR?1 Organophosphorus (OP) chemical warfare agents rapidly and irreversibly phosphylate the active site serine of the enzyme acetylcholinesterase (AChE), and sufficient doses produce a dire toxidrome that arises from accumulation of the neurotransmitter acetylcholine (ACh) in neuromuscular junctions (NMJs) in the peripheral nervous system and in synapses in the central nervous system (CNS). Inhibition of NMJ AChE by OP agents may lead to death via cardiorespiratory failure, and inhibition of the enzyme in the CNS results in neurotoxicolgical sequelae that in survivors manifest as epileptic seizures and neurobehavioral deficits. Figure 1 outlines the chemical events that ensue when AChE is exposed to the infamous nerve agents sarin and soman. After binding of the OP agent to the active site, nucleophilic attack by the γO of Ser203 displaces the fluoride leaving group and leads to the formation of a neutral phosphyl-AChE adduct. The rates of spontaneous hydrolysis of such adducts are far too slow to be therapeutically useful. However, Wilson and Ginsburg2 reported in 1955 that the oxime 2-pyridinealdoximine methiodide (2-PAM) is a rapid nucleophilic reactivator of OP inhibited AChE, and indeed 2-PAM is the only FDA approved oxime antidote for exposure to neurotoxic OP compounds. Oxime reactivators, however, are not the end of the antidote story. After formation of the initial phosphylAChE adduct, a second reaction occurs in which an alkyl group is hydrolytically cleaved, thereby producing a monoanionic adduct that has come to be known as aged-AChE (see Figure 1). Under physiological conditions, this aging reaction occurs in soman-inhibited AChE in just a few minutes, and therefore oximes are virtually worthless as antidotes to soman poisoning. Despite extensive attempts to reactivate aged-AChE since 1955, all efforts have been notable failures. It is logical to expect that acylation or alkylation of the aged adduct will return the adduct to an uncharged phosphyl-AChE adduct that is susceptible to oxime reactivation. However, aged-AChE has laughed at logic and reason! The literature, both published and © 2018 American Chemical Society
Figure 1. Interaction of AChE with phosphonyl fluoride inhibitors. Amino acid numbering is that of human AChE. The corresponding residues in Torpedo californica AChE are Ser200 and His440. The reactions in this figure are representative of those that occur when AChE is inhibited by a wide range of OP agents, which include commercial pesticides and chemical warfare agents: R = isopropyl (sarin); R = (CH3)3CCHCH3 (soman).
antecdotal (very few unsuccessful efforts are published, an obvious failure of scientific journalism), is a graveyard of efforts to resurrect aged-AChE. Indeed, is resurrection of aged-AChE simply a “bridge too far”?1 Given the considerable scientific and historical challenges posed by the trenchant unreactivity of aged-AChE, the results described in the recent publication from the laboratory of Chris Hadad and colleagues at Ohio State University are Received: July 16, 2018 Published: August 23, 2018 7032
DOI: 10.1021/acs.jmedchem.8b01122 J. Med. Chem. 2018, 61, 7032−7033
Journal of Medicinal Chemistry
Viewpoint
astonishing.3 The Hadad team has used a combination of approaches that is time-honored among medicinal chemists and pharmacologists to tackle this seemingly insurmountable challenge: computational biochemistry, synthetic medicinal chemistry, enzymology, proteomics. They have concentrated their efforts on a class of compounds called “quinone methide precursors”, QMPs. In aqueous media QMPs generate, after expulsion of a leaving group, an avaricious electrophile that quickly traps neighboring nucleophiles. The trick, therefore, is to find QMPs that are selective in their reaction with the AChE active site and that thereby might not show appreciable and undesirable off-target effects. The QMP called C8 (Figure 2) in the article by Hadad et al.3 appears to fit the bill.
close structural analog of the quasi-tetrahedral transition state in the deacylation stage of catalysis. Because enzymes derive their catalytic power from transition state stabilization, it is logical to expect that AChE will similarly stabilize the aged adduct. Figure 3 shows that AChE makes four H-bonds with the apical oxygens of the anionic phosphyl adduct, three in the oxyanion hole, and one with His440. These H-bond interactions are estimated to reduce the pKa of a the conjugate acid of the aged phosphonate monoanion by 4 pK units, to about −2.4 Therefore, in resurrecting aged AChE, Hadad et al.3 have succeeded in alkylating a profounding weak nucleophile in aqueous media, which makes their achievement yet more remarkable. The resurrection of aged AChE by Hadad et al.3 opens the door just a bit on a challenging new venture in biological structure/function/reactivity. Though admirable levels of resurrection have been realized, the reaction is still much too slow to be medicinally useful. However, if the QMP reaction with aged AChE can be rendered 2 orders of magnitude faster, then development of an effective antidote would be within reach. This challenge amounts to reducing the activation free energy of the QMP reaction by around 2.8 kcal/mol. The history of medicinal chemistry and pharmacology is rife with drug developments that have exceeded this mark. Hadad and his team have the tools and the expertise to make it so.
Figure 2. C8, the most reactive of 20 QMPs described by Hadad et al.3
Two aged enzymes were generated by reacting Electrophorus electricus AChE with diethylfluorophosphate and with a relatively safe to handle analog of the nerve agent soman; the respective aged adducts have an isopropylphosphate anion or methyphosphonate anion attached to the active site serine. Both of these adducts showed resurrection of activity on incubation with C8 for 4 days (20.4% and 32.7%, respectively). An added bonus was that C8 (and other QMPs) was itself a reactivator of OP-inhibited AChE. Consequently, QMPs offer the promise of development of a single antidote that not only alkylates aged AChE (the resurrection reaction) but also nucleophilically displaces the phosphyl adduct from the active site (the reactivation reaction, a la oximes).
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected].
REFERENCES
(1) “A bridge too far” is a literary allusion to a valiant though futile campaign by allied forces in 1944, “Operation Market Garden”, to break through Nazi lines at Arnhem, The Netherlands: Ryan, C. A Bridge Too Far; Simon and Schuster: New York, 1974. (2) Wilson, I. B.; Ginsburg, S. A powerful reactivator of alkylphosphate-inhibited acetylcholinesterase. Biochim. Biophys. Acta 1955, 18, 168−170. (3) Zhuang, Q.; Franjesevic, A. J.; Corrigan, T. S.; Coldren, W. H.; Dicken, R.; Sillart, S.; DeYong, A.; Yoshino, N.; Smith, J.; Fabry, S.; Fitzpatrick, K.; Blanton, T. G.; Joseph, J.; Yoder, R. J.; McElroy, C. A.; Ekici, Ö . D.; Callam, C. S.; Hadad, C. M. Demonstration of in vitro resurrection of aged acetylcholinesterase after exposure to organophosphorus chemical nerve agents. J. Med. Chem. 2018, DOI: 10.1021/acs.jmedchem.7b01620 (4) Quinn, D. M.; Topczewski, J.; Yasapala, N.; Lodge, A. Why is aged acetylcholinesterase so difficult to reactivate? Molecules 2017, 22, 1464−1469.
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PARTING SHOTS Perusal of the 3D structure of aged AChE underscores just how monumental the resurrection of aged AChE is (see Figure 3). As the author of this viewpoint has argued,4 the trenchant unreactivity of aged AChE arises because the aged adduct is a
Figure 3. Active site of aged Torpedo californica AChE. Atom colors are carbon (green), nitrogen (blue), oxygen (red), and phosphorus (orange). Dashed lines show H-bond interactions of the phosphonyl monoanion in the oxyanion hole (Gly118, Gly119, Ala201) and with His440. This representation was constructed with Pymol version 0.98rc5 from requisite coordinates in PDB file 1CFJ. 7033
DOI: 10.1021/acs.jmedchem.8b01122 J. Med. Chem. 2018, 61, 7032−7033
