Why Is Aged Acetylcholinesterase So Difficult to Reactivate?

molecules

Review Why is Aged Acetylcholinesterase So Difﬁcult to Reactivate?

Daniel M. Quinn *, Joseph Topczewski, Nilanthi Yasapala and Alexander Lodge

Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA; [email protected] (J.T.); [email protected] (N.Y.); [email protected] (A.L.) * Correspondence: [email protected]; Tel.: +1-319-335-1335

Received: 1 August 2017; Accepted: 29 August 2017; Published: 4 September 2017

Abstract: Organophosphorus agents are potent inhibitors of acetylcholinesterase. Inhibition involves successive chemical events. The ﬁrst is phosphylation of the active site serine to produce a neutral adduct, which is a close structural analog of the acylation transition state. This adduct is unreactive toward spontaneous hydrolysis, but in many cases can be reactivated by nucleophilic medicinal agents, such as oximes. However, the initial phosphylation reaction may be followed by a dealkylation reaction of the incipient adduct. This reaction is called aging and produces an anionic phosphyl adduct with acetylcholinesterase that is refractory to reactivation. This review considers why the anionic aged adduct is unreactive toward nucleophiles. An alternate approach is to realkylate the aged adduct, which would render the adduct reactivatable with oxime nucleophiles. However, this approach confronts a considerable—and perhaps intractable—challenge: the aged adduct is a close analog of the deacylation transition state. Consequently, the evolutionary mechanisms that have led to transition state stabilization in acetylcholinesterase catalysis are discussed herein, as are the challenges that they present to reactivation of aged acetylcholinesterase.

Keywords: acetylcholinesterase; organophosphorus agent; inhibition; reactivation; aging; transition state

1. Introduction Figure1 outlines the various chemical reactions that ensue when acetylcholinesterase (AChE) is exposed to organophosphorus (OP) inhibitors. These reactions are of considerable medicinal and national security interest, since OP agents that inhibit AChE (such as sarin in Figure1) are acute neurotoxins. Events in recent years underscore this concern: Iraq used tabun against Iranian troops in the Iran–Iraq war [1], terrorists have used sarin against civilians [2], tyrants have used sarin to mass murder their own people [3], and a dictator ordered the execution of his own brother with VX [4]. The seminal chemical reaction between sarin and AChE in Figure1 is attack of the active site serine at phosphorus with concomitant displacement of the ﬂuoride leaving group. This initial neutral adduct is an analog of the transition states in the acylation stage of AChE catalysis [5], and its production is accelerated by the catalytic machinery of the active site; i.e., the Ser-His-Glu catalytic triad, the oxyanion hole, and the acyl binding site [6,7]. The initial adduct is trenchantly unreactive toward spontaneous hydrolysis (cf. Figure1, Nu: = H 2O), a reaction that therefore is of no medicinal import. The lack of hydrolytic reactivity of the initial adduct is rationalized herein in terms of the resemblance of the adduct to the transition states of the acylation stage of AChE catalysis. However, judiciously designed oxime nucleophiles can readily dephosphylate the initial adducts, as shown in Figure1 (Nu: = ArCH=NOH). 2-Pyridinealdoxime methiodide (2-PAM) is a component of the FDA approved standard countermeasure that is in use in the United States [8]. Unfortunately, the initial neutral adduct may convert to a monoanionic aged adduct via a dealkylation reaction, as shown in Figure1. This aging reaction is accelerated by cation-π interaction between Trp86 of the active site and the

Molecules 2017, 22, 1464; doi:10.3390/molecules22091464 www.mdpi.com/journal/molecules Molecules 2017, 22, 1464 2 of 6 and the alkyl fragment that departs in the carbocationic transition state [9]. The aged adduct is remarkablyalkyl fragment unreactive. that departs Despite in the considerable carbocationic effort transitions over statethe last [9]. two The generations, aged adduct no is remarkablypracticable meansunreactive. for reactivating Despite considerable the aged adduct efforts have over been the last found, two and generations, therefore nono practicablemedicinal agents means are for availablereactivating for the aged aged AChE. adduct Why have is beenaged found,AChE andso diff thereforeicult to noreactivate? medicinal The agents answers are available to this question for aged AChE.are framed Why in is the aged following AChE so passages difﬁcult in to reactivate?terms of the The structural answers and to thisenergetic question features are framed of the inaged the AChEfollowing adduct, passages and inwill terms hopefully of the structuralinform future and effo energeticrts to featuressolve the of knotty the aged problem AChE that adduct, reactivation and will ofhopefully aged AChE inform poses. future efforts to solve the knotty problem that reactivation of aged AChE poses.

His447 His447 Ser203 Ser203

H+ + F- O- H NNHO O- H NN O Glu334 Glu334 CH3 O O (CH3)2CHO P CH3 (CH ) CHO P F 3 2 O O Neutral Adduct Reactivation

H2O (CH3)2CHOH

His447 H+ + Q His447 Ser203 Ser203

O- H NNH O - Glu334 O H NN+ H O Glu334 O - O O P CH3 - O O Aged Adduct Nu = H2O Q = (CH3)2CHO P CH3 O

O N CHAr

Nu = Oxime Q = (CH3)2CHO P CH3 O

Figure 1. InhibitionInhibition of of AChE AChE by by the orga organophosphorusnophosphorus nerve agent sarin.

2. Discussion Consideration of the selective pressure that guidedguided the evolution of the catalytic power of AChE provides a framework for understanding the hydrolytichydrolytic stability of the initial phosphyl adduct and the remarkable unreactivity of aged AChE. The physiologicalphysiological context in which AChE operates is cholinergic neurotransmission in the centralcentral andand peripheralperipheral nervousnervous systems.systems. Since cholinergic neurotransmission occurs occurs on on a a millis millisecondecond to to second second time time scale, scale, it itis isapparent apparent that that the the evolution evolution of theof the catalytic catalytic power power of AChE of AChE has has been been beset beset with with the the“need “need for forspeed”. speed”. Indeed, Indeed, AChE AChE is among is among the mostthe most potent potent of biocatalysts of biocatalysts that accelerate that accelerate hydrol hydrolysisysis reactions. reactions. The second-order The second-order rate constant rate constant kcat/Km exceeds 109 M−1·s−1 at9 low−1 ionic−1 strength [10], while the turnover number kcat > 104 s−1 [11]. For4 k−cat1 /Km, kcat/Km exceeds 10 M ·s at low ionic strength [10], while the turnover number kcat > 10 s [11]. the rate constant is prominently diffusion controlled; i.e., the enzyme is functioning at the “speed For kcat/Km, the rate constant is prominently diffusion controlled; i.e., the enzyme is functioning at the “speedlimit” of limit” biological of biological catalysis catalysis [12]. [A12 ].yet A more yet more telling telling analysis analysis arises arises from from consideration consideration of of rate constants in the thermodynamic cycle of Figure2 2.. TheThe cyclecycle isis numericallynumerically informedinformed byby thethe catalyticcatalytic constants of AChE catalysis, and by Wolfenden’s determinationdetermination of the rate constant for nonenzymic neutral hydrolysis of acetylcholine, kun = 7.2 × 10−9 −s9−1 [13].−1 These values allow one to calculate the neutral hydrolysis of acetylcholine, kun = 7.2 × 10 s [13]. These values allow one to calculate catalytic acceleration effected by the enzyme when substrate concentration is << Km, a ratio that the catalytic acceleration effected by the enzyme when substrate concentration is << Km, a ratio that Wolfenden callscalls thethe catalyticcatalytic proﬁciencyproficiency ofof thethe enzymeenzyme [[14]:14]: k k catK (1) Catalytic Proficiency = Km =1.4×1017 M−1 Catalytic Proﬁciency = k = 1.4 × 10 M (1) kun The reciprocal of the catalytic proficiency is the thermodynamic dissociation constant of the acylation transition state, KTS, from which one can calculate the free energy of dissociation of the acylation transition state at T = 298 K, as in Equation (2):

Molecules 2017, 22, 1464 3 of 6

The reciprocal of the catalytic proﬁciency is the thermodynamic dissociation constant of the acylation transition state, KTS, from which one can calculate the free energy of dissociation of the acylation transition state at T = 298 K, as in Equation (2):

∆GTS = −RTlnKTS = 98 kJ/mol (23 kcal/mol) (2)

Molecules 2017, 22, 1464 3 of 6 This analysis shows that, for AChE, and indeed for any enzyme catalyzed reaction, catalytic power must derive from transition state stabilization. For AChE, the acylation transition state is 98 kJ/mol ΔG = −RTlnK =98kJ/mol(23kcal/mol) (2) more stable than the transition state of the spontaneous hydrolysis of acetylcholine, an observation This analysis shows that, for AChE, and indeed for any enzyme catalyzed reaction, catalytic that can be interpreted in terms of the elements of molecular recognition that the enzyme brings to power must derive from transition state stabilization. For AChE, the acylation transition state is 98 kJ/mol bear on themore acylation stable than transition the transition state state [6]. of Athe similar spontaneous analysis hydrolysis can beof acetylcholine, considered an when observation AChE operates under conditionsthat can be of interpreted substrate in saturation; terms of the i.e.,elements kcat ofis molecular rate limiting: recognition that the enzyme brings to bear on the acylation transition state [6]. A similar analysis can be considered when AChE operates under conditions of substrate saturation; i.e., kcat is rate klimiting: Catalytic Acceleration = cat = 1.4 × 1012 (3) k = un=1.4×10 (3) Since it is likely that kcat is rate limited by the deacylation stage of catalysis [15], one can calculate Since it is likely that kcat is rate limited by the deacylation stage of catalysis [15], one can calculate 12 the stabilizationthe stabilization of the deacylation of the deacylation transition transition state state as as− −RTln(1.4RTln(1.4 × 10×12)10 = −69) =kJ/mol−69 (− kJ/mol17 kcal/mol). (−17 kcal/mol). E + A EA Km

ku kcat

E + TSu TSE KTS

PE+P Figure 2. Thermodynamic cycle for estimation of transition state stabilization in AChE catalysis. E, Figure 2. Thermodynamic cycle for estimation of transition state stabilization in AChE catalysis. E, EA, EA, A, P, TSu, and TSE are respectively free enzyme, Michaelis complex, substrate, product, transition

A, P, TSu, andstate TSof theE are spontaneous respectively hydrolysis free enzyme,reaction, and Michaelis transition complex,state of the substrate,AChE-catalyzed product, reaction. transition Km state of the spontaneousand kcat are hydrolysisthe respective reaction,Michaelis constant and transition and turnover state number of the of AChE-catalyzedthe AChE-catalyzed reaction.reaction; Km and kcat are theku respective and KTS are Michaelis respectively constant the rate and constant turnover of th numbere spontaneous of the hydrolysis AChE-catalyzed reaction and reaction; the ku and dissociation constant of the enzymic transition state. KTS are respectively the rate constant of the spontaneous hydrolysis reaction and the dissociation constant ofHow the enzymicis the notable transition transition state. state stabilization that AChE effects related to inhibition by OP agents? This is a question that was posed by Ashani and Green in 1982 [16], and that is approached Howherein is the from notable structural transition and energetic state stabilizationperspectives. The that analysis AChE that effects follows relatedis informed to by inhibition the availability by OP agents? This is a questionin the literature thatwas of crystal posed struct byures Ashani of both andaged Greenand neutral in 1982 phosphyl-AChE [16], and adducts that is [17–20]. approached Consider herein from structuralthe and acylation energetic transition perspectives. state for TheAChE analysis catalyze thatd hydrolysis follows of is acetylthiocholine informed by the (ATCh). availability By in the measuring β-deuterium secondary isotope effects on kcat/Km, Quinn and collaborators showed that literature ofthe crystal bond order structures between the of bothγO of agedSer203 andof human neutral AChE phosphyl-AChE and the carbonyl carbonadducts of ATCh [17 –is20 0.8]. ± Consider0.2 the acylation transitionin the acylation state transition for AChE state catalyzed [5]. Moreover, hydrolysis the π-bond of acetylthiocholine of the ATCh carbonyl (ATCh). function By is measuring β-deuteriumextensively secondary broken. isotope These respective effects onbonds kcat have/K mbond, Quinn lengths and of 1.45 collaborators Å and 1.38 Å, as showed was found that in the bond order betweena simple the computationalγO of Ser203 model of ofhuman the acylation AChE transition and state the carbonyl[5]. The corresponding carbon of distances ATCh in is the 0.8 ± 0.2 in neutral phosphyl adduct that results when T. californica AChE is inhibited by VX are 1.57 Å and 1.47 Å. π the acylationThe transitionsimilarity among state angles [5]. Moreover, about the erstwhile the -bond carbonyl of carbon the ATCh of the carbonyltransition state function model isand extensively broken. Thesethe phosphorus respective of the bonds neutral have VX adduct bond are lengths yet more ofremarkable. 1.45 Åand For example, 1.38 Å, in as the was transition found state in a simple computationalmodel, model the bond of angle the acylationfor nucleophile transition oxygen to state carbonyl [5]. carbon The corresponding to carbonyl oxygen distances is 106.6°, while in the neutral phosphylin adduct the VX thatadduct results the corresponding when T. angle californica is 109.5°;AChE in the istransition inhibited state bymodel VX the are acyl 1.57 methyl Å andto 1.47 Å. carbonyl carbon to carbonyl oxygen angle is 113.0°, while in the VX adduct the corresponding angle The similarityis 114.7°. among The notable angles similarity about thebetween erstwhile transition carbonyl state structural carbon features of the and transition those of the state initial model and the phosphorusphosphyl of adduct the neutral suggests VXthat adductthe phosphyl are yetadduct more is trenchantly remarkable. unreactive For toward example, spontaneous in the transition state model, the bond angle for nucleophile oxygen to carbonyl carbon to carbonyl oxygen is 106.6◦, while in the VX adduct the corresponding angle is 109.5◦; in the transition state model the acyl methyl Molecules 2017, 22, 1464 4 of 6 to carbonyl carbon to carbonyl oxygen angle is 113.0◦, while in the VX adduct the corresponding angle is 114.7◦. The notable similarity between transition state structural features and those of the initial phosphyl adduct suggests that the phosphyl adduct is trenchantly unreactive toward spontaneous hydrolysis, because AChE stabilizes the adduct in much the same way that it stabilizes the acylation transitionMolecules state. 2017, 22, 1464 4 of 6 A comparable analysis can be considered for the aged enzyme. Though a detailed structure of the deacylationhydrolysis, transition because stateAChE is stabilizes not available, the adduct Quinn in mu andch the coworkers same way havethat it shownstabilizes by the measurements acylation transition state. of β-deuterium secondary isotope effects on kcat that for the deacylation stage of cholinesterase A comparable analysis can be considered for the aged enzyme. Though a detailed structure of catalysis the Michaelis complex that accumulates on the enzyme in the steady state is a tetrahedral the deacylation transition state is not available, Quinn and coworkers have shown by measurements intermediate [21–23]. In nonenzymic ester hydrolysis the tetrahedral intermediate is at least 11 kcal/mol of β-deuterium secondary isotope effects on kcat that for the deacylation stage of cholinesterase (46 kJ/mol)catalysis less the stableMichaelis than complex the ester that from accumulates which it comeson the enzyme [23]. Therefore, in the steady the tetrahedralstate is a tetrahedral intermediate in theintermediate deacylation [21–23]. stage of In AChE nonenzymic catalysis ester is stabilized hydrolys byis atthe least tetrahedral 46 kJ/mol intermediate (11 kcal/mol). is at Thisleast analysis11 showskcal/mol that the (46 stabilizationkJ/mol) less stable of the than tetrahedral the ester from intermediate which it comes is a [23]. large Therefore, fraction the of tetrahedral the 69 kJ/mol stabilizationintermediate of the in the deacylation deacylationtransition stage of AChE state catalysis that was is stabilized calculated by at from least the46 kJ/mol thermodynamic (11 kcal/mol).cycle of FigureThis 1analysis. Consequently, shows that itthe is stabilization unsurprising of that,the te despitetrahedral two intermediate generations is a large of effort fraction by medicinalof the chemists,69 kJ/mol a nucleophilic stabilization antidoteof the deacylation for aged transition AChE has state yet that to was be found.calculated The from trenchant the thermodynamic unreactivity of agedcycle AChE of arisesFigure because1. Consequently, the aged it adductis unsurprising is a close that, structural despite two analog generations of the tetrahedralof effort by medicinal intermediate chemists, a nucleophilic antidote for aged AChE has yet to be found. The trenchant unreactivity of in the deacylation stage of catalysis, and of the transition states for formation and decomposition of aged AChE arises because the aged adduct is a close structural analog of the tetrahedral intermediate the intermediate. in the deacylation stage of catalysis, and of the transition states for formation and decomposition of Athe seemingly intermediate. obvious approach to reactivate aged AChE is to synthesize and evaluate putative medicinalA agents seemingly that obvious can realkylate approach theto reactivate monoanionic aged AChE aged isadduct, to synthesize which and would evaluate produce putative anew a neutralmedicinal phosphyl agents adduct that can that realkylate can be reactivatedthe monoanionic by nucleophilic aged adduct, medicinal which would reagents, produce such anew as 2-PAM. a Accordingly,Topczewskineutral phosphyl adduct and Quinnthat can [24 be] reportedreactivated that by various nucleophilic substituted medicinal 2-methoxy-1-methylpyridiniums reagents, such as 2-PAM. wereAccordingly, reactive as methyl Topczewski transfer and agents Quinn to the [24] methoxyl reported methyphosphonate that various substituted anion in the2-methoxy-1- reaction shown in Figuremethylpyridiniums3. This reaction were was reactive chosen as asmethyl an analog transfer of agents methyl to transferthe methoxyl to the methyphosphonate aged AChE adduct. The 2-methoxy-1-methylpyridiniumanion in the reaction shown in Figure agents 3. This alkylated reaction the was phosphonate chosen as an anionanalog with of methyl a range transfer of rates to that the aged AChE adduct. The 2-methoxy-1-methylpyridinium agents alkylated the phosphonate anion was described by a multiple linear free energy relationship, with the most reactive agent (Y = 3-F in with a range of rates that was described by a multiple linear free energy relationship, with the most Figurereactive3) effecting agent 40% (Y = methyl 3-F in transferFigure 3) in effecting 10 min. 40 Despite% methyl these transfer very promisingin 10 min. modelDespite reaction these very results, nonepromising of the 2-methoxy-1-methylpyridinium model reaction results, none of the reagents 2-methoxy-1-methylpyridinium gave a 2-PAM reactivatable reagents phosphyl gave a adduct2- with agedPAM reactivatable human AChE. phosphyl adduct with aged human AChE.

Y = H, 4-CN, 5-CN, 6-CN, 3-F, 5-F, 6-F, 5-NO2, 5-CF3, 6-CF3

FigureFigure 3. Substituted 3. Substituted 2-Methoxy-1-methylpyridiniums 2-Methoxy-1-methylpyridiniums as as phosphonate phosphonate anion anion methylating methylating agents. agents. 1 Reactions were run in DMSO-d6 at 25◦ °C and were monitored by H-NMR1 spectroscopy [24]. Reactions were run in DMSO-d6 at 25 C and were monitored by H-NMR spectroscopy [24]. The secret behind the unreactivity of aged AChE toward methylation with 2-methoxy-1- The secret behind the unreactivity of aged AChE toward methylation with 2-methoxy-1- methylpyridinium reagents again surely lies in the fact that the aged adduct is a close structural methylpyridinium reagents again surely lies in the fact that the aged adduct is a close structural analog analog of the deacylation transition state. Consider the following analysis. The pKa of the conjugate of theacid deacylation of methoxyl transition methylphosphonate state. Consider anion, measured the following by 31P-NMR analysis. spectroscopy, The p Kis a1.9of (Topczewski, the conjugate J.J.; acid 31 of methoxylQuinn, D.M., methylphosphonate unpublished observation) anion, measured. As Harel et by al. [6]P-NMR discussed, spectroscopy, the contribution is 1.9 of (theTopczewski, oxyanion J.J.; Quinn,hole D.M., to transition unpublished state observation).stabilization in As AChE Harel catalysis et al. [ 6is] discussed,at least 21 thekJ/mol contribution (5 kcal/mol). of theOne oxyanion can hole toreasonably transition expect state that stabilization this stabilization in AChE will catalysis result in isa atcomparable least 21 kJ/molstabilization (5 kcal/mol). of the anionic One can reasonablyphosphyl expect adduct that of this aged stabilization AChE, which will would result lower in a comparable the conjugate stabilization acid pKa of ofthe the aged anionic adduct phosphyl by 4 pK units. The decreased pKa of the phosphyl adduct should in turn decrease the reactivity of the adduct of aged AChE, which would lower the conjugate acid pKa of the aged adduct by 4 pK units. adduct as a methyl transfer nucleophile to an extent that is accommodated by a Brönsted relationship [25]: The decreased pKa of the phosphyl adduct should in turn decrease the reactivity of the adduct as a methyl transfer nucleophile to an extent that is=10 accommodated by a Brönsted relationship [(4)25 ]:

In this equation, k0 is the rate constant for methyl transfer measured in the model reaction of Figure 3, knuc is the expected rate constant for aged AChE, ΔpKa = −4 as discussed above, and β is a

Molecules 2017, 22, 1464 5 of 6

k nuc = 10βnuc∆pKa (4) k0

In this equation, k0 is the rate constant for methyl transfer measured in the model reaction of Figure3, k nuc is the expected rate constant for aged AChE, ∆pKa = −4 as discussed above, and β is a measure of the degree of methyl transfer that has been achieved in the transition state of the methyl transfer reaction. Since methyl transfer is between oxyanions, it is reasonable to estimate that β = 0.5. Consequently, in aged AChE, methyl transfer will occur at least 100-fold more slowly than in the model reaction. For the most reactive of the 2-methoxy-1-methypyridiniums of Figure3, which had a half-life of 20 min, this analysis predicts that in aged AChE the half-life will be at least 2000 min = 33 h, which is much too slow to be of use in a therapeutic context. It should be noted that the aqueous pKa of methoxyl methanephosphonate has been used in this analysis for reactivity in the model reaction in DMSO. It is well known that conjugate acid pKas of oxyanions are elevated in DMSO versus aqueous solution, and therefore this analysis certainly underestimates the unreactivity of aged AChE. Nonetheless, the general conclusion remains. Because the aged adduct is a close structural mimic of the tetrahedral intermediate in the deacylation stage of catalysis, and of the transition states leading to and from the intermediate, the intrinsic nucleophilic reactivity of the aged enzyme is compromised by the elements of molecular recognition that have evolved as the catalytic power of AChE has evolved.

3. Conclusions The analysis discussed herein suggests that intrinsic unreactivity is a major factor in the inability of medicinal chemists to find an antidote to aged AChE over the last 60 years. The challenge that this problem presents is considerable. As discussed herein, the unreactivity of aged AChE is a result of the evolution of the catalytic power of the enzyme itself. However, there is reason to be optimistic that a solution to this conundrum will be found. An integrated effort that involves medicinal chemistry, biochemistry, and computational chemistry may well reveal additional elements of molecular recognition, reactivator design, and reactivator mechanism that experimenters can harness to produce agents that can covalently modify aged AChE, albeit with sufficient reactivity to be useful in a therapeutic context.

Acknowledgments: The work described in reference 24 was supported by the CounterACT Program, National Institutes of Health Office of the Director, and the National Institute of Neurological Disorders and Stroke, Grant No. 5 R21 NS076430. Conflicts of Interest: The authors declare no conflict of interest.

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