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Demonstration of in vitro Resurrection of Aged Acetylcholinesterase after Exposure to Organophosphorus Chemical Nerve Agents Qinggeng Zhuang, Andrew J Franjesevic, Thomas S Corrigan, William H Coldren, Rachel Dicken, Sydney Sillart, Ashley DeYong, Nathan Yoshino, Justin Smith, Stephanie Fabry, Keegan Fitzpatrick, Travis G. Blanton, Jojo Joseph, Ryan J Yoder, Craig A McElroy, Ozlem Dogan Ekici, Christopher S Callam, and Christopher M. Hadad J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01620 • Publication Date (Web): 05 Jun 2018 Downloaded from http://pubs.acs.org on June 6, 2018

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Journal of Medicinal Chemistry

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Demonstration of in vitro Resurrection of Aged Acetylcholinesterase after Exposure to Organophosphorus Chemical Nerve Agents Qinggeng Zhuang1, Andrew J. Franjesevic1, Thomas S. Corrigan1, William H. Coldren1, Rachel Dicken1, Sydney Sillart1, Ashley DeYong1, Nathan Yoshino1, Justin Smith1, Stephanie Fabry1, Keegan Fitzpatrick1, Travis G. Blanton2, Jojo Joseph1, Ryan J. Yoder2, Craig A. McElroy3, Özlem Dogan Ekici4, Christopher S. Callam1, Christopher M. Hadad1* 1

Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210.

2

Department of Chemistry and Biochemistry, The Ohio State University–Marion, Marion, Ohio 43302.

3

College of Pharmacy, The Ohio State University, Columbus, Ohio 43210.

4

Department of Chemistry and Biochemistry, The Ohio State University–Newark, Newark, Ohio 43055.

ABSTRACT: After inhibition of acetylcholinesterase (AChE) by organophosphorus (OP) nerve agents, a dealkylation reaction, referred to as aging, of the phosphylated serine can occur. When aged, known reactivators of OP-inhibited AChE are no longer effective. Realkylation of aged AChE may provide a route to reverse aging. We designed and synthesized a library of quinone methide precursors (QMPs) as proposed realkylators of aged AChE. Our lead compound (C8) from an in vitro screening, successfully resurrected 32.7% and 20.4% of the activity of methylphosphonate-aged and isopropyl phosphate-aged electric eel AChE, respectively, after 4 days. C8 displays properties of both resurrection (recovery from the aged to the native state) and reactivation (recovery from the inhibited to the native state). Resurrection of methylphosphonate-aged AChE by C8 was significantly pH-dependent, recovering 21% of activity at 4 mM and pH 9 after only 1 day. C8 is also effective against isopropyl phosphate-aged human AChE.

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Introduction Acetylcholinesterase (AChE) is a serine hydrolase found in brain synapses, neuromuscular junctions and erythrocytes. AChE selectively hydrolyzes the neurotransmitter acetylcholine with its Glu-His-Ser catalytic triad. Organophosphorus (OP) compounds phosphylate the catalytic serine of AChE, and inhibition of AChE results in the accumulation of acetylcholine. OP exposure may lead to death due to seizures or respiratory failure caused by persistent nerve impulses.1-3 Thus, OPs are toxic and have been used as pesticides and chemical warfare agents. Every year OP-based pesticides kill approximately 200,000 people worldwide, especially in rural areas of developing countries.1 OP-based chemical warfare agents have been used in armed conflicts such as the Iran-Iraq war4 and the recent Syrian civil war,5 as well as terrorist attacks such as the Tokyo subway sarin attack.6 OP-inhibited AChE can be reactivated by oximes (more likely, their deprotonated form, the oximates), which nucleophilically substitute the phosphylated serine in the active site.7 Exposure of AChE to OP compounds is complicated by an aging process in which loss of the alkyl side chain of the phosphylated serine produces an oxyanion of OP-poisoned AChE.8,9 Oximes, such as 2-pralidoxime (2-PAM), are ineffective against aged AChE. Some OP compounds, such as soman, with an aging half-time (t1/2) of only several minutes, provide only a minimal chance for medical treatment.10 After decades of research, no clinical treatment has been developed to resurrect aged AChE. To reverse aging, realkylation of the phosphylated oxyanion has been proposed as a strategy against this dealkylation process,11 including unsuccessful efforts from 1970 by Steinberg et al.12 as well as more recent efforts led by Quinn.13 With the negative charge on the phosphylated serine being neutralized by some sort of electrophilic realkylation process, oximes should reactivate AChE again. Several types of electrophilic alkylating agents including sulfonates,11 haloketones,12 sulfoniums,14 and methoxypyridiniums13 were evaluated as potential AChE realkylators. Recently, Khavrutskii and Wallqvist suggested the in silico possibility to resurrect aged AChE by β-aminoalcohols, by a direct process without proceeding through a realkylation event.15 However, prior to this report, no experimental evidence has been reported for the efficacy of any drug to realkylate aged AChE to the best of our knowledge.16 Herein, we report the first family of compounds that demonstrate in vitro efficacy. Quinone methides (QMs, SCHEME 1a) can be regarded as carbocations stabilized by resonance delocalization. Quinone methide precursors (QMPs, SCHEME 1a) are derivatives of QMs with a leaving group attached to the partially positively charged carbon. QMPs can be attacked by nucleophiles either directly via SN2 substitution, or via the corresponding QMs as reactive intermediates (SCHEME 1b). Protein and nucleic acid alkylation by QMPs has been reported for years.17-22

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Phosphodiesters and dibutyl phosphate, which structurally resemble the phosphyl group of aged AChE, have been successfully alkylated by QMs, implying the possibility to realkylate and resurrect aged AChE with QMPs and QMs.22-24 Herein we report a series of QMPs intended to be realkylators of aged AChE. Guided by in silico studies, a library of candidate compounds was synthesized. Their activities were characterized by Ellman’s assay, providing up to 32.7% in vitro resurrection of electric eel AChE (eeAChE) for a methylphosphonate and up to 20.4% for a phosphate after 4 days. Resurrection of AChE was confirmed by bottom-up proteomics.

SCHEME 1 Structures and substitution reaction of QMs and QMPs. (a) Typical structures of QMs (top) and QMPs (bottom). (b) Nucleophiles can substitute the leaving group of QMP by either an SN2 reaction or formation of the corresponding QM.

a

b

Results Computation-guided selection of realkylator candidates We previously conducted molecular docking and molecular dynamics (MD) simulations to evaluate the potential orientation of QMPs at the active site of methylphosphonate-aged human AChE (huAChE).25 In this work a larger library of QMPs was studied through this modeling approach using an in silico model of aged huAChE.



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FIGURE 1. A snapshot obtained from a 1 ns MD simulation, demonstrating a protonated QMP (shown in cyan, later called C8) near the active site of aged AChE (wall-eyed stereo). The electrophilic carbon is ~4.2 Å from the phosphylated oxyanion. Hydrogen bonds with short contact distances are shown (green dashed lines).

SCHEME 2 Structures of realkylator candidates

We determined that pyridyl compounds had a higher propensity to be bound in the active site and close to the phosphyl oxyanion as compared to their phenyl analogues.25 Moreover, 3-hydroxypyridine-derived QMPs with the reactive benzylic carbon attached at the 2-position displayed promising interactions. Of the 72 compounds modeled, six of the top-10 compounds (FIGURE S2) were members of that specific 3-hydroxypyridine framework. The top compound had a pyrrolidine leaving group attached to the reactive benzylic carbon (FIGURE 1). Thirteen 3-hydroxypyridine-derived QMPs (C series, SCHEME 2) were thus synthesized via Mannich reactions, and then evaluated by screening against aged AChE. The electrophilic benzylic methylene, hypothesized to be the site of attack by


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nucleophiles (SCHEME 1b), is attached to the 2-position of the pyridine ring, and the leaving groups were various secondary amines.

Screening of Realkylator Library Two representative OPs (SCHEME 3) were used. PiMP (a pinacolyl methylphosphonate ester), a soman analogue, was synthesized as reported by Amitai et al.26 The resulting methylphosphonate-aged AChE is the aging product of any methylphosphonate nerve agent (e.g. sarin, soman, VX as well as many other V-agents and G-agents except for tabun (GA)). We also used the commercially available pesticide DFP (diisopropyl fluorophosphate) to evaluate a phosphate at the serine residue (SCHEME 3). We chose eeAChE as the target enzyme of these studies, considering its commercial availability and affordable cost. SCHEME 3 Structures of OPs used for aging of AChE and the corresponding aged adducts

The aged enzyme was reacted with various concentrations (0.2 – 4 mM) of each QMP at 37°C, pH 8 for 1 d. Without knowing the re-aging t1/2 of the possible realkylated adducts, NH4F (4 mM) was added as a mild and nonselective reactivator.27-30 We posited that once the aged AChE was realkylated, the newly formed “inhibited” AChE could be reactivated by fluoride to the native AChE. The samples were eventually treated with 4 mM of 2-PAM for 1 h to ensure that any realkylated AChE was completely reactivated. Ellman’s assay,31 with acetylthiocholine as a substrate, was carried out to determine AChE activity. Three controls were prepared and analyzed in parallel: a negative control without realkylator; a 2PAM control with the realkylator replaced by 2-PAM; and a positive control with native AChE, rather than aged, and without realkylator.



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a

b

FIGURE 2. Screening of three concentrations of various C series QMPs against (a) methylphosphonate-aged eeAChE and (b) isopropyl phosphate-aged eeAChE. The horizontal solid and the dashed lines mark the negative controls and 2-PAM controls, respectively. The error bars reflect standard deviations from four replicate efforts.

The screening results are shown in FIGURE 2a. The percentage relative activity is based on the positive control. The negative and 2-PAM controls showed negligible background signals, confirming the completion of aging (solid and dashed horizontal lines in FIGURES 2). For this small library, the only difference was the amino leaving group. Compound C2 with an N-(methyl)ethylamino leaving group showed the highest efficacy among C1 to C7, with each of these compounds having a noncyclic amine. Efficacy was compromised when the N-alkyl groups were shortened (C1 vs C2), lengthened (C3,


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C5 and C7 vs C2), or branched (C4 vs C2, and C6 vs C3). Overall, the pyrrolidinyl compound C8 was the most potent candidate. The other candidates with cyclic leaving groups showed lower or even no activity. The screening against isopropyl phosphate-aged AChE (DFP-treated) showed a similar result (FIGURE 2b), but the trend of varying activity with the Nalkyl groups was less obvious. These comparisons demonstrate that pyrrolidine is the optimum among all tested leaving groups. Hence we compared a variety of realkylator candidates with the pyrrolidinyl leaving group (D series in SCHEME 2). The screenings were carried out following the same procedures as for the 3-hydroxypyridine derivatives (C series). C8 and unsubstituted 3hydroxypyridine were used for comparison. As shown in FIGURE S10, D5 is the only active compound of these candidate compounds. D5 is active only against isopropyl phosphate-aged AChE, and is less active than C8. This observation indicates the superiority of the 3-hydroxypyridine framework over other tested scaffolds. C8 was therefore chosen as a lead compound for subsequent investigations.

Kinetics of Resurrection In order to further confirm the reactivity of C8 and determine the rate of reaction, we monitored the resurrection of aged AChE by this compound for days. Out of curiosity, neither 2-PAM nor fluoride was added to the QMP-treated samples for reactivation of realkylated AChE. As the reaction progressed, linearly increasing activity was seen in the isopropyl phosphate-aged AChE sample treated with 4 mM C8 (FIGURE 3a). The negative control and 2-PAM control remained inactive. After four days, the resurrected relative activity was as high as 20.4%. The apparent reaction rate was 0.20% per hour (r2 = 0.9958). Resurrection kinetics of methylphosphonate-aged AChE was monitored similarly. The C8-treated sample displayed resurrected activity, which reached 32.7% after four days (FIGURE 3b). The apparent reaction rate was 0.32% per hour (r2 = 0.9185) and faster than against isopropyl phosphate-aged AChE. The absence of extra reactivators (NH4F or 2-PAM, etc.) in this reaction suggests the inherent reactivation activity of C8, that is, reactivating the realkylated AChE. This is in agreement with reports of reactivation activity of Mannich phenols by Katz et al.,32 Cadieux et al.,33 and Bierwisch et al.34


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b 120

140

100

120 Relative activity (%)

Relative activity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80 60

Negative Control 2-PAM Control QMP Positive Control

40 20

100 80

Negative Control 2-PAM Control QMP Positive Control

60 40 20

0

0

0

20

40

60

80

100

0

20

40

Time (h)

60

80

100

Time (h)

FIGURE 3. Kinetics of realkylation of aged eeAChE by 4 mM C8 at pH 8 over 4 days for (a) isopropyl phosphate-aged (DFP-treated) eeAChE and (b) methylphosphonate-aged (PiMP-treated) eeAChE. The blue dashed lines illustrate the result of linear regression.

Bottom-up Proteomics Besides determining the resurrected AChE activity by Ellman’s assay, confirmation of the reaction between the QMP and aged AChE can also be revealed by mass spectrometry. Bottom-up proteomics35,36 was used to sequence the peptides and differentiate enzyme species with variable modifications at the catalytic serine. We treated isopropyl phosphate-aged eeAChE with C8 for 11 d, and digested it with trypsin. 2-PAM was not applied to the C8-treated sample. The digest was analyzed with LC-MS/MS. The positive, negative and 2-PAM controls were also prepared in parallel, and percentages of AChE species were obtained from the LC peak areas (TABLE 1). TABLE 1. Percentages of eeAChE species after resurrection by C8, as determined by LC-MS/MS

Percentage (%) Sample

Isopropyl phosphate-aged

Methylphosphonate-aged

Native

Aged

Realkylated

Native

Aged

Realkylated

Positive Control

100

0

0

100

0

0

Negative Control

0

100

0

0

100

0

2-PAM Control

0

100

0

0

100

0

C8-treated

15.4

84.6

0

2.1

97.9

0


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Peptide QVTIFGESAGAASVGMHLLSPDSRPK (residues 195–220), was observed in all samples. The catalytic serine (underscored) in the positive control was completely unmodified because the enzyme was native. By contrast, the modification observed in the negative and 2-PAM controls indicated that they were completely aged. The wild-type serine or otherwise modified serine was not observed at this position. Compared to the unmodified peptide, there was a mass shift of 122.0133 Da (C3H7O3P added), which matches the added isopropyl phosphyl moiety. In the C8-treated sample, the unmodified catalytic serine was observed, indicating resurrection induced by the QMP. Realkylated AChE, however, was not directly observed. This again suggests that our QMPs can resurrect aged AChE independent of extra reactivators such as oximes due to their inherent reactivation activity. Another possibility is that the realkylated phosphyl group was lost under used MS conditions. Similar results were obtained with methylphosphonate-aged AChE (TABLE 1).

Reactivation of Inhibited AChE by Five QMPs a

b

140 120 Relative activity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


100

Positive Control 2-PAM (0.04 mM) 3-Hydroxypyridine (0.4 mM) D5 C8 C2 C1 Negative Control

80 60 40 20 0

0

4

8

12

16

20

24

Time (h)

FIGURE 4. Reactivation activity test of four QMPs (4 mM) against eeAChE inhibited by EMP. (a) Structure of EMP. (b) Relative activity of eeAChE as reactivation proceeded.



Four representative QMPs, namely C1, C2, C8, D5 and unsubstituted 3-hydroxypyridine, were reacted with eeAChE inhibited with a VX analogue (EMP, an ethyl methylphosphonate compound, FIGURE 4a) to determine whether they are reactivators. We chose this OP because the aging by VX is slow.37 After inhibition by EMP, the inhibited AChE was cleaned by Sephadex spin columns, then incubated with the chosen QMPs (4 mM) at pH 8.0 and 37°C. Aliquots were taken and cleaned also with Sephadex spin columns, and monitored by Ellman’s assay (FIGURE 4b). All tested QMPs exhibited obvious and similar reactivation activity, regardless of their different performances in the aforementioned realkylation screening. After 24 h of reaction, they reactivated 64%~80% of inhibited AChE. This may explain why C8 can efficiently resurrect aged AChE in the absence of 2-PAM and fluoride. All tested compounds reacted slower than 3-hydroxypyridine and 2-PAM, which reached their respective plateaus within 1 h of reaction, although they were used at only 0.4 and 0.04 mM concentrations, respectively.

pH Effect and EC50 for C8 Activity We performed the resurrection of methylphosphonate-aged eeAChE by 4 mM C8 at four different pH values (6~9) at 37°C for 1 d, with neither 2-PAM nor fluoride being used. The relative activity of C8-resurrected eeAChE increased dramatically with pH (FIGURE 5a) – over 20% for 1 d at pH 9. Similar effects, but less pronounced, were also observed with isopropyl phosphate-aged eeAChE (FIGURE 5b). a

b 8

25

7

Negative Control QMP


20


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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15

10

Negative Control QMP

6 5 4 3 2

5

1 0

6

7

8

9

0

6

7

8

9

pH

pH

FIGURE 5. Resurrection efficiency of 4 mM C8 against aged eeAChE increased with pH within the range of pH 6~9. (a) Methylphosphonate-aged (PiMP-treated) eeAChE. (b) Isopropyl phosphate-aged (DFP-treated) eeAChE.


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Seven concentrations of C8 (0 – 20 mM), were therefore compared at pH 9 against methylphosphonate-aged and isopropyl phosphate-aged eeAChE, in order to determine the EC50. The results (1.21 and 1.02 mM, respectively, Figure S24) illustrate that further optimization in binding affinity is needed for a more effective therapeutic. There are three heteroatoms in C8, forming multiple possible protonation states. Each of them may interact with the aged AChE active site with different orientations, affinities and rates. 1H NMR spectra of C8 displayed shifting signals as the pH changed (FIGURE 6a). The UV-vis spectra also dramatically changed when pH was increased from 6 to 9 (FIGURE 6b), indicating a pKa between 7 and 8.

a

b

3.0 pH6 pH7 pH8 pH9

2.5

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.0 1.5 1.0 0.5 0.0

240

280 320 Wavelength (nm)

360

FIGURE 6. Influence of C8 protonation states on spectra. (a) 1H NMR spectra of the aromatic protons and (b) UV-vis spectra of C8 as pH is varied from 6~9.

a

b

FIGURE 7. (a) The four most probable protonation states of C8 at pH 8-9. (b) A representative snapshot in the MD simulation of C8c (cyan, wall-eyed stereo). Hydrogen bonds with short contact distances are shown (green dashed lines).



The strong pH dependence motivated us to study which isomers might be the active component(s). We compared the four most probable states at pH 8 and 9, namely the neutral, anionic and two zwitterionic forms (C8n, C8a, C8b and C8c, respectively, FIGURE 7a).Each protonation state was individually docked into a number of geometries of aged AChE and subsequent MD simulations were run on the docked conformations; the location of the exchangeable proton has a dramatic effect on the affinity of the ligand toward different zones in the AChE active site (FIGURE S3-S6). The simulations suggest that both zwitterionic forms (C8b and C8c) have stable interactions and well-defined proximity to the anionic oxygen of the phosphylated serine. The neutral form (C8n) also stably binds to the active site, but the benzylic carbon is not oriented for realkylation. The anionic form (C8a) gradually moves away from the active site during the simulation. Calculations at the B3LYP/6-311+G** (SMD, water) level of theory suggest that C8c (pyrrolidinium) has a free energy that is lower than C8b (pyridinium) by 3.1 kcal/mol, hence is a more probable species. Ser203, Tyr337, Tyr341 residues, along with some hydrogen-bonded water molecules, contribute significantly to the binding of C8c by hydrogen bonding and electrostatic attractions (FIGURE 7b).

Resurrection of Aged Human AChE after exposure to DFP

120 Relative activity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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100 80 60 Negative Control QMP Positive Control

40 20 0

0

1

2

3 4 5 Reaction time (d)

6

7

8

FIGURE 8. Kinetics of resurrection of isopropyl phosphate-aged huAChE by 4 mM C8 (pH 9). The green dashed line illustrates the result of linear regression.

The efficacy of C8 was examined against aged huAChE. The activity of isopropyl phosphate-aged (DFP-treated) recombinant huAChE was monitored in the presence of 4 mM of C8 at pH 9 for a week by Ellman’s assay. After 7 d of reac-


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tion (FIGURE 8), 18% of the aged enzyme was resurrected, and the relative activity of huAChE was still increasing. The apparent rate of resurrection was 2.5% per day, as determined by linear regression. Neither 2-PAM nor fluoride were used in this test, again confirming that C8 alone can resurrect aged AChE to the native form.

Discussion and Conclusions With in silico guidance, we synthesized 20 QMPs with various scaffolds and leaving groups, and then evaluated them for their in vitro resurrection activity against methylphosphonate-aged and isopropyl phosphate-aged eeAChE. Multiple QMPs with a 3-hydroxypyridine scaffold, with the reactive “benzylic” carbon attached at the 2-position, proved efficacious. C8 was found to be the lead compound in the screening. Used at 4 mM without the assistance of 2-PAM, C8 successfully resurrected the relative activity of isopropyl phosphate-aged eeAChE to 20.4% and methylphosphonate-aged eeAChE to 32.7% after 4 d of observation (FIGURE 3). This activity for C8 is also reproduced for aged human AChE (FIGURE 8). The activities of C8 against isopropyl phosphate-aged and methylphosphonate-aged eeAChE were also confirmed with bottom-up proteomics. The peptide containing the catalytic serine was sequenced to reveal the modification. In the samples treated with C8, partial resurrection was observed as indicated by the presence of unmodified catalytic serine from native AChE. Realkylated AChE was not directly observed, though no extra reactivator was added. These observations suggest the activity of C8 against both aged AChE and inhibited/realkylated AChE. This hypothesis was confirmed as C8 and three other QMPs were able to reactivate inhibited AChE (FIGURE 4). At this stage, C8 is not a practical drug for clinical post-aging treatment of OP poisoning or in vivo tests. The rate of reaction is slower than ideal, although the concentration was on the millimolar scale. Whether this concentration will cause toxicity also remains unknown. And, 2-4 d of reaction was needed to resurrect ~20% of activity. However, the resurrection rate is increased dramatically when pH is increased to 9, suggesting that structure-activity relationships and a deeper mechanistic understanding may be effective in affording better activity. Calculations suggest that the zwitterionic form of C8, with the pyrrolidinyl nitrogen being protonated, plays a critical role in activity. Finally, this report provides the first in vitro success to substantially resurrect aged AChE via the use of an appropriate realkylator. After almost 70 years of effort, this goal has been achieved, but further work remains to create a therapeutic drug with all of the desired activity and selectivity.



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Experimental Section Computation-guided selection of realkylator candidates Optimizations, molecular docking, and MD simulations were all performed with the initial library of compounds. The three lowest-energy docking poses of each flexible ligand across 13 rigid aged AChE structures were used as starting points for subsequent 1 ns MD simulations. The QMPs were evaluated based on the time throughout the MD simulations in which the reactive benzylic carbon was within close proximity to the anionic O-(P=O) of the aged serine. More experimental details are described in the Supporting Information.

LC-MS characterization of realkylator candidates Mass spectra were obtained using Thermo LTQ Orbitrap (ESI) mass spectrometer. HPLC was performed with Agilent 1100 HPLC system equipped with Phenomenex Luna 5 µm C18 column (150 × 4.6 mm), eluted with mixtures of water and methanol (5%~95% methanol, containing 0.1% formic acid, 0.6 mL/min), and detected by UV-vis detector at 254 nm as well as ESI-orbitrap. The purity of each realkylator candidate was confirmed to be ≥ 95%.

Preparation of Aged AChE Some OPs (mainly the phosphonates, which are commonly seen in G- and V-type warfare agents) are chiral, and the stereoisomers can inhibit and/or age at different rates.38 Inhibited AChE may remain un-aged even after reacting for longer than the apparent t1/2, if the enzyme is inhibited by the slower aging OP stereoisomer. Reactivation of the residual unaged, but inhibited, AChE can interfere with the observation of the resurrection of aged AChE and may lead to artifacts. Thus, AChE must be thoroughly aged and free of inhibited AChE. To ensure complete aging, the methylphosphonateaged AChE (treated with PiMP) was prepared via two rounds of aging, considering the chirality of PiMP. We treated AChE with 2-PAM after the first round of aging, in order to reactivate any residual inhibited AChE. Then PiMP was added to inhibit and age the enzyme again. The fast-aging isomers thus had a second chance to compete with other isomers and react with the enzyme. The amount of residual inhibited or native AChE in the sample was significantly minimized, often

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