Comparison of the reactivation rates of acetylcholinesterase modified by structurally different organophosphates using novel pyridinium oximes

Abstract

A novel panel of oximes were synthesized, which have displayed varying degree of reactivation ability towards different organophosphorus (OP) modified cholinesterases following insecticide exposures. In the present article, we report a comparative reactivation profile of a series of quaternary pyridinium-oximes for electric eel acetylcholinesterase (EEAChE) inhibited by the organophosphorus (OP) inhibitors methyl paraoxon (MePOX), ethyl paraoxon (POX; paraoxon) and diisopropyl fluorophosphate (DFP) that are distinguishable as dimethoxyphosphoryl, diethoxyphosphoryl and diisopropoxyphosphoryl AChE-OP-adducts. Most of the 59-oximes tested led to faster and more extensive reactivation of MePOX- and POX-inhibited EEAChE as compared to DFP-modified EEAChE. All were effective reactivators of three OP-modified EEAChE conjugates showing 18–21% reactivation for DFP-inhibited AChE and ≥45% reactivation for MePOX- and POX-inhibited EEAChE. Oximes 7 and 8 showed kr values better than pralidoxime (1) for DFP-inhibited EEAChE. Reactivation rates determined at different inhibition times showed no significant change in kr values during 0–90 min incubation with three OP’s. However, a 34–72% decrease in kr for MePOX and POX and > 95% decrease in kr for DFP-inhibited EEAChE was observed after 24 h of OP-exposure (aging).

Graphical Abstract

1. INTRODUCTION

Organophosphorus (OP) nerve agents are among the most toxic compounds prepared by synthetic means. Their production began in Germany prior to World War II (the organophosphate Tabun, 1936) and since that time many other nerve agents and beneficial organophosphorus derivatives have been prepared for military and agricultural uses (Costanzi et al., 2018). The mode of action of these organophosphorus agents is based on the rapid inhibition of acetylcholinesterase (AChE) in which a serine hydroxyl within the active site attacks the OP phosphoryl to form a phosphoserine ester bond that is stable and resistant to hydrolysis (Figure 1A). The subsequent accumulation of the neurotransmitter acetylcholine and over-stimulation of cholinergic receptors result in a generalized cholinergic crisis including a breakdown of neuromuscular function (Bajgar, 2004; De Bleecker, 2008; King and Aaron, 2015; Marrs, 1993; Tanimoto et al., 2017). For this reason, antidotes aimed at reversing these effects have been extensively developed.

Figure 1.

(A). Pathways of inhibition, reactivation and aging of AChE following reaction with different OPs. (B). Select examples of known AChE reactivators (1–4) and oxime reactivators evaluated in this study (5–8).

The standard treatment of OP nerve agent poisoning includes administration of a muscarinic antagonist, e.g. atropine, along with an oxime reactivator of OP-inhibited AChE (Leadbeater et al., 1985). At present, pralidoxime (2-PAM, 1), trimedoxime (TMB-4, 2), obidoxime (3) and HI-6 (4) are used to reactivate OP-inhibited AChE (Figure 1B) but are considered to be ineffective against certain nerve agents (Worek et al., 2004). The mechanism of reactivation of OP-inhibited AChE occurs by nucleophilic attack on the phosphoryl moiety by the oxime oxyanion to displace it from the active site serine (Figure 1A). The efficacy of oximes depends upon the organophosphorylated structure, reactivator structure (Figure 1B) (Kuca and Cabal, 2002), the source of AChE (Worek et al., 2002) and competing post-inhibitory reactions such as ‘aging’ (Shafferman et al., 1996) and dephosphorylation (spontaneous reactivation) (Musilek et al., 2007b). Due to these factors, OP-modified AChE can be difficult to unblock and frequently the deleterious effects cannot be counteracted (Bajgar, 1996). In order to overcome these limitations numerous new oximes have been synthesized and tested during the past few decades (Acharya et al., 2009; Bhattacharjee et al., 2012; Hosseini et al., 2017; Karade et al., 2016; Kassa et al., 2008; Katalinic et al., 2017; Kliachyna et al., 2014; Lorke et al., 2008a; Lorke et al., 2008b; McHardy et al., 2014; Musilek et al., 2008a; Musilek et al., 2007b; Musilek et al., 2008b; Ochoa et al., 2016; Oh et al., 2008; Petronilho Eda et al., 2016; Renou et al., 2013; Sharma et al., 2016; Wei et al., 2017; Wei et al., 2016; Winter et al., 2016) (reviewed recently (Gorecki et al., 2017; McHardy et al., 2017)) but a single effective oxime that is capable of reversing the effects of the major OP-AChE conjugates is lacking.

Diisopropyl fluorophosphate (DFP) is one of the extremely toxic OP agents (Petroianu and Lorke, 2008), and the reactivation of AChE (O,O-diisopropoxyphosphoryl-AChE) inhibited by DFP is difficult compared to O,O-dimethoxy and O,O-diethoxy-phosphoryl modified AChE’s because the former becomes non-reactivatable with time. Therefore, identification of reactivators of DFP-inhibited AChE is considered to be highly challenging. In the past, few efforts have been made towards the discovery of reactivators of DFP inhibited AChE (Acharya et al., 2009; Bhattacharjee et al., 2015; Lorke et al., 2008a; Oh et al., 2008), however, an ideal strategy would be to identify a single reactivator which is capable of reactivating AChE inhibited by various OPs.

In the present study, three organophosphorus inhibitors (OPs) methyl paraoxon (MePOX), ethyl paraoxon (POX) and diisopropyl fluorophosphate (DFP) were used to generate the putative O,O-dimethoxy, O,O-diethoxy and O,O-diisopropoxy-AChE following inhibition, respectively (Figure 1A). The study was aimed to identify one or more oxime reactivators capable of reactivating AChE inhibited by these three OPs. The comparison of reactivation rates of AChE modified by three structurally different OPs was studied using a library of 59 pyridinium oximes, synthesized previously in our laboratory. Because reactivation and aging rates are monitored over many hours, electric eel acetylcholinesterase (EEAChE) was used as it is stable over this experimental time course. Molecular modelling studies were conducted in order to understand the interaction pattern of reactivators at the active site gorge of OP-inhibited AChE.

2. MATERIALS AND METHODS

2.1. Chemicals and reagents.

Chemicals were purchased from Sigma-Aldrich and used without further purification. Electric eel AChE (EEAChE, Type V-S; EC 3.1.1.7), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), acetylthiocholine iodide (ATChI), diisopropyl fluorophosphate (DFP) and pralidoxime (1) were purchased from Sigma. Ethyl paraoxon and methyl paraoxon were synthesized by reaction of p-nitrophenol with diethyl or dimethyl chlorophosphate (Ghanem et al., 2007), purified to > 99% by chromatography, and characterized by NMR and high resolution mass spectrometry. ¹H NMR spectra were recorded on a Varian 400 MHz NMR spectrometer with TMS as the internal standard. Mass spectra were obtained on a Waters LCT Premier time-of-flight mass spectrometer and IR spectra on Nexus 670 FTIR spectrometer. Melting points are uncorrected.

Organophosphates are toxic reagents and should be handled in a well-ventilated hood. Methyl paraoxon, ethyl paraoxon and diisopropyl fluorophosphate may be destroyed and rendered safe by stirring with 1 N NaOH overnight at rt.

2.2. Synthesis.

All mono- and bisquaternary pyridinium oximes used in the present study were synthesized in our laboratory as reported earlier (Bharate et al., 2009, 2010) and were characterized by ¹H NMR, ¹³C NMR, IR and ESI-MS data.

2.3. Inhibition of EEAChE by MePOX, POX and DFP.

Inhibition of EEAChE by three different OPs was performed using previously published methods (Chao et al., 2016; Kaleem Ahmed et al., 2013) adapted from the original spectrophotometric assay by Ellman (Ellman et al., 1961).

2.4. In vitro reactivation.

An EEAChE stock solution (0.2 mg/mL in PBS pH 7.2) was treated with isopropanol, methanol or ethanol (1% v/v) and 1% v/v of DFP (1 mM in isopropanol), methyl paraoxon (100 µM in methanol) or ethyl paraoxon (100 µM in ethanol) as control and experiment vessels. After 20–50 min, 90% AChE inhibition was achieved and halted with a 32-fold dilution (PBS). A 16 µL aliquot from control and experiment vessels was diluted to 20 µL with PBS and incubated for 30 min as activity control and inhibition control. The initial activity (A₀) or inhibition (A_i) was analyzed by adding DTNB (final concentration of 0.3 mM, 200 µL total volume) and ATChI (final concentration of 1 mM, 200 µL total volume). Oxime reactivator (4 µL, 1 ~ 0.01 mM final concentration) was added to a 16 µL aliquot and after 30 min incubation, the reactivated activity was determined by Ellman assay (Ellman et al., 1961) as A_r. The % reactivation was determined with n ≥ 3:

$$\%\hspace{0.17em}\text{reactivation}=\left[\left({\text{A}}_{\text{r}}-{\text{A}}_{\text{i}}\right)/\left({\text{A}}_{\text{o}}-{\text{A}}_{\text{i}}\right)\right]\times 100.$$

2.5. Reactivation kinetics of selected compounds.

Similar experiments as the in vitro reactivation screening were accomplished using different reactivation time points (0–15 min). By plotting ln(reactivation %-age) vs. reactivation time (t), k_r is presented as the negative slope of the plot. ln(reactivation %-age) = −k_r • t. Each experiment was repeated with n ≥ 3. Plots with R² > 0.9 were chosen for k_r calculations. Reactivation kinetics at different OP exposure times (15 min to 24 h) were also performed. After the addition of OP to EEAChE, aliquots of treated enzyme were removed at different time points for determination of reactivation rate constant of selected oximes.

2.6. Molecular docking.

The crystal structure of DFP inhibited acetylcholinesterase (PDB: 5HCU) (Katz et al., 2015) was used for studying interaction of reactivators at the active site. The 3D structure of AChE was retrieved from the protein data bank (www.rcsb.com) and was prepared by protein preparation wizard in Maestro at pH 6.2. The site of molecular docking was defined by constructing the grid considering OP-linked Serine residue as the centroid of grid box. All docking calculations were done using GLIDE XP docking, and ∆G of inhibitors binding to AChE complex was carried out by Prime using end point MMGB/SA method. The validation of the docking protocol was done by performing docking of the original reactivator ligand and next comparing it to the binding pattern with the co-crystallized structure.

2.7. Statistical analyses.

The data were analyzed using Pearson’s correlation and analysis of variance (ANOVA). The result is considered statistically significant if the p value is less than 0.05.

3. RESULTS AND DISCUSSION

We reported a series of monoquaternary and bis-quaternary oximes (Figure 2) showing good reactivation of POX-inhibited AChE (Bharate et al., 2009, 2010). To test whether or not these compounds were effective as reactivators of cholinesterases modified by structurally-different OPs, the reactivation of AChE inhibited by MePOX and DFP was evaluated and compared these results with those obtained by reactivation of POX-modified AChE from a previous report.

Figure 2.

Structures of mono- and bisquaternary pyridinium compounds.

Monoquaternary pyridinium oximes 5 and 9–19 were designed with N-substituents to explore hydrogen bonding opportunities in the active site, for example, carbamoyl, carboxyl and hydroxyl. In other instances, electron rich heterocycles (oximes 20–37) including thiophene, furan and isoxazole were added to the N-alkyl side chains of 6 and 20–37 to investigate interactions with cation-containing (cation-π bonding) or aromatic residues (π–π bonding) lining the gorge and/or near the AChE active site. The oxime group was positioned at 2, 3 and 4 on the pyridinium ring to determine the best regiochemistry for reactivation of AChE using the N-derivatized groups (Bharate et al., 2009). Bisquaternary pyridinium oximes bearing an ether linkage (3, 38–39), 3–4 carbon chain aliphatic linker (2, 40–44, 54–59) and heterocyclic linkers (7–8, 45–53, 60) were synthesized (Bharate et al., 2010).

All 59 compounds were assayed for their ability to reactivate OP-inhibited EEAChE using a modified Ellman assay (Ellman et al., 1961). Methyl paraoxon (MePOX), ethyl paraoxon (POX), and diisopropyl fluorophosphate (DFP) were used as inhibitors and pralidoxime (1), trimedoxime (2) and obidoxime (3) were chosen as reference reactivators. Most of the oximes reactivated MePOX- and POX-inhibited AChE faster and to a greater extent as compared with DFP-inhibited EEAChE (Table 1 and 2).

Table 1.

Percent reactivation of of EEAChE inhibited by methyl paraoxon (MePOX), ethyl paraoxon (POX) and diisopropyl fluorophosphate (DFP) by monoquaternary pyridinium oximes.

Mono-oxime reactivator	% Reactivation (mean± SEM)*
	MePOX-inhibited EEAChE	POX-inhibited EEAChE	DFP-inhibited EEAChE
1	71 ± 3	67 ± 1	43 ± 1
5	73 ± 2	45 ± 3	18 ± 2
6	21 ± 1	34 ± 2	5.6 ± 0.7
9	3.0 ± 0.3	1.0 ± 0.3	0.5 ± 0.03
10	3.4 ± 0.2	4.5 ± 0.1	1.6 ± 0.4
11	12 ± 1	41 ± 3	0
12	6.7 ± 0.4	9.6 ± 0.7	0.9 ± 0.1
13	13 ± 1	43 ± 2	19 ± 2
14	47 ± 2	50 ± 4	14 ± 1
15	12 ± 0	7.6 ± 0.9	0
16	25 ± 2	70 ± 1	14 ± 1
17	3.6 ± 0.4	9.8 ± 0.5	0
18	5.1 ± 0.4	4.5 ± 0.4	0
19	3.7 ± 0.5	15 ± 1	0.5 ± 0.3
20	0	3.0 ± 0.9	0
21	2.8 ± 0.3	12 ± 0	5.9 ± 0.9
22	1.4 ± 0.2	37 ± 2	7.7 ± 0.9
23	0	7.6 ± 0.7	0
24	1.7 ± 0.5	14 ± 1	3.0 ± 0.2
25	14 ± 1	28 ± 2	3.8 ± 0.6
26	2.0 ± 0.1	8.4 ± 1.4	0.5 ± 0.1
27	0	0.5 ± 0.6	0
28	11 ± 1	28 ± 3	2.0 ± 0.9
29	3.0 ± 0.3	0	0
30	2.5 ± 0.6	0	2.0 ± 0.5
31	5.3 ± 1.0	14 ± 2	0
32	22 ± 1	25 ± 3	1.1 ± 0.5
33	5.0 ± 0.1	0	0
34	18 ± 1	24 ± 2	5.7 ± 0.8
35	43 ± 1	19 ± 2	5.0 ± 0.8
36	16 ± 1	19 ± 1	6.2 ± 1.3
37	8.7 ± 0.3	0.7 ± 1.2	0.6 ± 0.1

^*The highest reactivation that could be achieved by the reactivator at various concentrations.

Table 2.

Percent reactivation of EEAChE inhibited by methyl paraoxon (MePOX), ethyl paraoxon (POX) and diisopropyl fluorophosphate (DFP) by bisquaternary pyridinium oximes.

Bis-oxime reactivator	% Reactivation (mean ± SEM)*
	MePOX-inhibited EEAChE	POX-inhibited EEAChE	DFP-inhibited EEAChE
2	73 ± 1	74 ± 4	28 ± 2
3	86 ± 1	80 ± 4	44 ± 3
7	53 ± 0	74 ± 3	19 ± 1
8	47 ± 0	49 ± 4	21 ± 2
38	9.4 ± 0.6	21 ± 1	2.2 ± 0.3
39	5.9 ± 0.2	49 ± 2	1.7 ± 0.3
40	12 ± 1	7.4 ± 2.1	0.2 ± 0.2
41	20 ± 2	60 ± 3	14 ± 1
42	12 ± 0	50 ± 1	1.1 ± 0.5
43	23 ± 1	22 ± 0	5.8 ± 0.4
44	65 ± 2	68 ± 4	28 ± 0
45	29 ± 3	59 ± 5	11 ± 3
46	12 ± 0	13 ± 2	2.2 ± 0.4
47	44 ± 1	47 ± 3	17 ± 0
48	6.4 ± 0.5	27 ± 2	0.9 ± 0.3
49	12 ± 1	80 ± 3	3.5 ± 1.1
50	12 ± 1	77 ± 4	0.2 ± 0.1
51	26 ± 1	71 ± 1	0.5 ± 0.3
52	8.0 ± 0.3	13 ± 1	0
53	28 ± 2	52 ± 3	7.9 ± 1.0
54	8.8 ± 0.5	28 ± 2	0.6 ± 0.3
55	20 ± 1	58 ± 3	14 ± 2
56	77 ± 2	77 ± 4	34 ± 1
57	42 ± 1	58 ± 4	7.5 ± 0.9
58	18 ± 1	38 ± 2	6.0 ± 0.5
59	77 ± 1	50 ± 1	27 ± 2
60	10 ± 1	60 ± 3	14 ± 1

^*The highest reactivation that could be achieved by the reactivator at various concentrations.

3.1. Reactivation of dimethoxyphosphoryl AChE.

Inhibition of EEAChE by MePOX leads to the formation of a dimethyl phosphoryl AChE conjugate, (MeO)₂P(O)-O-AChE, at the active site serine and several oximes were able to displace this adduct and restore AChE activity. When (MeO)₂P(O)-O-AChE was reacted with the monoquaternary pyridinium oximes, pralidoxime 1, the carboxymethyl analog 5, hydroxyethyl analog 14, and 4-methoxy benzyl analog 35, AChE enzyme was restored to 71, 73, 47 and 43% levels. Compounds 6, 16 and 32 also showed significant (21, 25 and 22%) reactivation of (MeO)₂P(O)-inhibited EEAChE. Several bisquaternary pyridinium oximes reactivated MePOX-inhibited EEAChE, amongst which the heterocycles-linked compounds 7, 8 and 47 restored activity to 54, 47 and 44%, respectively.

3.2. Reactivation of diethoxyphosphoryl AChE.

Similarly, EEAChE was inhibited by POX, where the loss of enzyme activity is associated with diethyl phosphorylation (EtO)₂P(O)-O-AChE of the active site serine. The monoquaternary pyridinium oximes 5, 11, 13, 14 and 16 showed > 40% reactivation of (EtO)₂P(O)-inhibited EEAChE with several monoquaternary oximes 6, 22, 25, 28, 32 and 34 showing more modest reactivation ranging from 24–37%. Bisquaternary oximes 7, 8, 45, 47, 56, 57, and 59 were previously shown to produce ≥ 47% reactivation of POX-inhibited EEAChE (Bharate et al., 2009). In particular, the bis-quaternary oximes 56 and 57 bearing aliphatic three- and four-carbon linkers, respectively, showed the best reactivation (>70%) of POX inhibited AChE.

3.3. Reactivation of diisoproxyphosphoryl AChE.

Inhibition of EEAChE by DFP leads to the formation of a diisopropyl phosphoryl AChE conjugate (iPrO)₂P(O)-O-AChE with the active site serine. All the oximes tested showed diminished reactivation of DFP-inhibited EEAChE((iPrO)₂P(O)-EEAChE) as compared to MePOX- and POX-inhibited AChE. Of all the monoquaternary pyridinium compounds tested, pralidoxime 1 was the most potent reactivator of (iPrO)₂P(O)-EEAChE showing a 42% recovery of activity. Amongst newly synthesized monoquaternary compounds, 5, 13, 14 and 16 showed 18, 17, 14 and 13% reactivation of DFP-inhibited EEAChE. Amongst bisquaternary compounds tested, obidoxime 3 was the most potent reactivator of DFP-inhibited EEAChE leading to 44% reactivation. Trimedoxime 2 and its structural analogs 44, 56 and 59 also showed some ability to reactivate DFP-inhibited EEAChE with 28, 28, 35 and 27% reactivation, respectively. Compounds bearing heterocyclic linkers 7, 8, 45 and 47 showed 19, 21, 11 and 17% reactivation of DFP-inhibited EEAChE.

Literature precedence also indicates that reactivation of (iPrO)₂P(O)-AChE is difficult. Recently Kuca and coworkers (Malinak et al., 2018) have tested a series of bis-quaternary oximes for reactivation of (iPrO)₂P(O)-AChE. The best reactivator from their efforts was trimedoxime which could reactivate (iPrO)₂P(O)-AChE by 10%; however none of their new oxime could reactivate AChE by more than 5%. Another bis-quaternary mono-oxime reported from same group (Musilek et al., 2011) could also reactivate DFP-inhibited AChE maximum by 17%. Hadad’s group (Zhuang et al., 2018) have recently prepared a series of quinone methide precursors as potential reactivators of DFP inhibited aged-AChE. The best compound from their efforts could do 18% reactivation of 7-days aged AChE. Another group (Berberich et al., 2016) identified structurally different bis-quaternary pyridinium mono-oxime via virtual screening, which could reactivate the DFP-inhibited AChE by 58%. This indicates that one should attempt new scaffolds to find out potent reactivators of DFP-inhibited AChE.

Molecular modeling was then performed for mono-quaternary compound 5 and bis-quaternary compound 7 with DFP-inhibited AChE, to understand possible interactions or binding orientations of these reactivators with the OP and/or active site residues. The use of AChE structure without OP attached to SER 203 would not provide an accurate rendition for oxime interactions with the active site gorge. Therefore, the DFP inhibited AChE structure (PDB: 5HCU)(Katz et al., 2015) was selected for molecular modeling, and not the AChE without any OP attached to SER 203 residue. Docking results indicated that the mono-quaternary oxime 5 enters deep into the active site gorge, reaching near the SER 203 residue bearing’-O-P(O)(O)-O-isopropyl’ moiety of DFP. The OH of the oxime group shows H-bonding interaction with the carbonyl oxygen of GLY 120 residue of the catalytic anionic site (CAS). The carboxylic acid group of compound 5 also showed H-bonding with TYR 337 of choline-binding site. The pyridine-ring and quaternary nitrogen of reactivator 5 displayed π-π and cation-π interaction with the TRP 86 residue of the choline binding site. Because of the shorter length of mono-quaternary oxime 5, it does not show any interaction with residues of peripheral anionic site (PAS). This is clearly visible in the surface view of compound 5 interactions with AChE (Figure 3c). In contrary, the bis-quaternary oxime 7 was found to interact with residues of CAS as well as PAS. The oxime OH group of 7 showed H-bonding interaction with TYR 133, GLH 202 and GLY 120 of CAS, whereas oxime OH of another pyridine ring showed H-bonding interaction with ARG 296 residue of PAS. The central furan ring of compound 7 showed a strong π-π interaction with TYR 124 and TYR 341 residues, of PAS (Figure 3a, 3b, 3d). The π-π interaction with TRP 86 was also observed. Overall, compound 7 displayed the most favorable orientation to displace OP and best interactions with the active site of DFP inhibited AChE leading to reactivation.

Figure 3.

Molecular docking of reactivators 5 and 7 with DFP-inhibited AChE (PDB: 5HCU). (a) Surface view of the AChE enzyme showing active site gorge. The residues are colored differently according to their charge, wherein the yellow, blue and red color represents hydrophobic, positively charged and negatively charged residues, respectively. (c and d) The surface view and interaction map of mono-quaternary oxime 5 with DFP inhibited AChE. (b and e) The surface view and interaction map of bis-quaternary oxime 7 with DFP inhibited AChE. Hydrogen bonds and π-π interactions are represented by dotted lines.

3.4. Comparison of the reactivation profile of oxime reactivating agents for three different OP inhibited AChE’s.

A comparison of % reactivation conducted by select mono- and bisquaternary pyridinium oxime reactivating agents on (MeO)₂P(O)-, (EtO)₂P(O)- and (iPrO)₂P(O)-inhibited EEAChE is shown in Figures 4A and 4C. Three reactivation trends were identified: (a) oximes 6, 16, 22, 25, 45 and 57 reactivated EEAChE in the order POX > MePOX > DFP; (b) oximes 1, 2, 3, 7 and 8 reactivated EEAChE in the order POX ≈ MePOX > DFP and oxime 5 reactivated EEAChE in the order MePOX>POX> DFP. Oxime 5 showed 73, 45 and 18% reactivation of MePOX-, POX- and DFP-inhibited EEAChE, respectively with the reactivation trend roughly correlating with the inhibitor size.

Figure 4.

Comparison of % reactivation (A, C) and reactivation rate constant (k_r) (B, D) of select mono- and bis-quaternary pyridinium oximes following inhibition of EEAChE by MePOX, POX, and DFP. Bars are represented as mean ± SEM.

The oxime-mediated reactivation rate constant (k_r) characterizes the dissociation of the enzyme from the phosphorylated-oxime) and was determined for selected mono- (5, 6, 16, 22, 25) and bisquaternary (2, 3, 7, 8, 45, 57) pyridinium oximes. The k_r was calculated using the equation from the linear portion of activity curves (0–15 min) (Thompson et al., 1992) and summarized in the Supplementary Material (Table S1) for the three OP-inhibited EEAChE’s. A majority of the oximes exhibited higher reactivation rates for POX modified AChE than MePOX and DFP treated AChE. Two common trends of k_r were found for the structurally-different OP-inhibited AChE types. Oximes 1, 5, 6, 16, 25, 2, 45 and 57 showed reactivation rate preference in the order POX>MePOX> DFP. Oximes 7 and 8, however, reactivated MePOX- and DFP-inhibited AChE at similar rates but slower than POX-inhibited (POX>MePOX ≈ DFP). Oximes 3 and 22 were exceptions with reactivation rate preferences in the order DFP >POX>MePOX and POX> DFP >MePOX, respectively. These two oximes showed greater reactivation rate for the sterically congested DFP-inhibited AChE than MePOX inhibited AChE. Likewise, obidoxime (3) has a larger k_r for DFP-inhibited AChE than POX treated AChE. The comparison of k_r values of selected oximes for three different OP-modified AChE’s is shown in Figure 4B and 4D.

Newly synthesized bisquaternary compounds 7 and 8 showed reactivation rates (k_r 0.017 and 0.015 min⁻¹) comparable to pralidoxime (1, k_r 0.0139 min⁻¹) for DFP-inhibited EEAChE while the monoquaternary oxime 5 (k_r 0.0101 min⁻¹) was slightly less effective than pralidoxime (1). Oximes 5, 6, 7, 8, 45 and 57 all showed greater k_r values (0.0642–0.0339 min⁻¹) than obidoxime (3, k_r 0.033 min⁻¹) for POX-inhibited EEAChE. The top five reactivators for each OP-inhibited AChE were: 2>1>5>3>8 (MePOX); 1>2>57>7>8 (POX); 3>2>7>8>1 (DFP). Based on these k_r findings, oximes 5, 7 and 8 were the best new reactivators for EEAChE inhibited by the three structurally different OP’s.

Certain OP-modified AChE’s undergo time-dependent, spontaneous dealkylation or ester hydrolysis in a process termed ‘aging’. Aging results in a phosphate monoanion at the serine hydroxyl that is recalcitrant to reactivate, and therefore, oximes capable of restoring activity to OP-aged AChE would be of high therapeutic value. In this study, the reactivation of aged AChE using a control and one new mono- and bisquaternary oxime was studied at different OP exposure times. Monoquaternary oximes 1 and 5 and bisquaternary pyridinium oximes 3 and 7 were examined as possible reactivators using different time points ranging from 0–90 min and a 24 h time point (Figure 5). After 24 h, the % decrease in k_r (from the highest k_r value) was found to be greatest for DFP treated AChE followed by POX- and MePOX-treated AChE. These % decreases in k_r values for all four oximes were found in the range of 34–64% for (MeO)₂P(O)-EEAChE; 48–72% for (EtO)₂P(O)-EEAChE and 95–97% for (iPrO)₂P(O)-EEAChE. The % decrease in k_r value for MePOX and POX treated AChE for obidoxime (3) was less (34 and 49%) compared with other oximes (59–72%). A similar trend of % decrease in k_r value after 24 h was noticed for MePOX and POX treated AChE’s: 1 ≈ 5 > 7 > 3 but for DFP treated AChE, all compounds showed >95% decrease in k_r after 24 h of OP exposure. After 24 h of inhibition, DFP-inhibited AChE was barely reactivatable by any of the oximes with k_r values of 0.00071 (1), 0.0016 (3), 0.00065 (5) and 0.00093 min⁻¹ (7) with less than 4% reactivation. The % reactivation and k_r are OP-, oxime- and exposure time-dependent (p<0.01).

Figure 5.

Comparison of reactivation rate constant (k_r) of compounds 1, 5, 3 and 7 for (MeO)₂P(O)-EEAChE (), (EtO)₂P(O)-EEAChE () and (iPrO)₂P(O)-EEAChE () at different OP exposure times. Bars are represented as mean ± SEM.

To investigate the decrease in reactivation rate for DFP-treated AChE from 0–24 h, the reactivation kinetics at different time points viz. 2, 4, 8, 12, 16, 20 and 24 h was also carried out using oximes 3 and 7. The effect of aging on reactivation rate and % reactivation of DFP-inhibited AChE is shown in Figure 6. The trend of reactivation rate with an increase in DFP exposure time was found to be similar for both oximes. The % decrease in reactivation rate for (iPrO)₂P(O)-EEAChE for oximes 3 and 7 was as follows: 3/7 (time) - 31/29 (2 h); 46/48 (4 h); 75/72 (8 h); 87/83 (12 h); 92/90 (16 h); 95/94 (20 h); 97/96 (24 h). A similar trend was followed by % reactivation at different time intervals. The percent decrease in reactivation for oximes 3 and 7 is as follows: 3/7 (time) - 14/17 (2 h); 37/39 (4 h); 64/65 (8 h); 79/79 (12 h); 86/86 (16 h); 91/92 (20 h); 95/95 (24 h). Both the % reactivation and k_r are oxime compound- and exposure time-dependent (p<0.05).

Figure 6.

The effect of aging on reactivation rate and % reactivation of DIP AChE using two reactivators 3 () and 7 () The data points are represented as mean ± SEM.

Organophosphorus compounds MePOX, POX and DFP differ structurally and vary in the ability to inhibit AChE; POX>MePOX> DFP (Dettbarn et al., 1999; Lorke et al., 2008a; Petroianu and Kalasz, 2007). Inhibition of AChE by these OP’s leads to the formation of dimethyl, diethyl and diisopropyl phosphoryl conjugates at the active site serine. A series of new oxime structures were examined for their ability to reactivate the three different OP-AChE conjugates and certain trends were uncovered worthy of note. Oximes with aliphatic side chains mostly showed concentration-dependent reactivation of EEAChE while compounds with heterocyclic linkers showed better reactivation at lower concentrations (10⁻⁴ M and 10⁻⁵ M) compared with higher concentration (10⁻³ M) (Musilek et al., 2007a; Musilek et al., 2006). Analogs bearing the oxime in the 2- and 4-position were better reactivators than those with oximes in the 3-position. Furthermore, the bis-quaternary oximes were better reactivators than the mono-quaternary. For example, the mono-quaternary oximes bearing thiophen-2-yl linker (6, 20, 21) are weaker reactivators in comparison to bis-quaternary oximes bearing similar linker - thiophen-2,5-yl (45, 46, 47). In case of bis-quaternary oximes, the type of linker connecting both pyridinium rings plays crucial role in its reactivation ability. As shown in Figure 2, the library of bis-quaternary oximes used in the present study comprises variation in the type of linker and the position of oxime on pyridinium rings. On comparing the % reactivation by bis-quaternary 2-oximes varying in the type of linker (compounds 38, 40, 42, 45, 48, and 51), it was observed that pyridinium 2-oximes bearing electron rich heterocycles (45, 48, 51) are superior reactivators than those bearing aliphatic ether or alkyl chain linkers (38, 40, 42). However, in the case of bis-quaternary 4-oximes, the situation was found exactly opposite. The bis-quaternary 4-oximes bearing ether or alkyl chain linkers (2, 3, 44) are superior reactivators in all three OP-inhibited AChEs, compared to those bearing electron rich heterocycles (7, 47, 53). The reason for this opposite observation for 2-versus 4-oximes lies in the length of central linker. In case of bis-quaternary 4-oximes, because of the presence of oxime group at para-position, it adds extra length to the molecule, in comparison to that of bis-quaternary 2-oximes. Therefore, the 3–4-atom linker (e.g. 2, 44) was found to be optimal for bis-quaternary 4-oximes; and further addition of extra length results in decreased reactivation activity; e.g. the compound 47 has 5-atom linker, and it shows 44, 47, and 17% reactivation of MePOX, POX and DFP-inhibited AChE, which is significantly lower than compounds 2 and 44. In contrast, in case of bis-quaternary 2-oximes, the compounds bearing 5-atom linker (e.g. 45, 48, 51) are superior than those bearing 3- or 4-atom linker (e.g. 38, 40, 42). Specifically, in case of DFP-inhibited AChE, the 4-oximes are superior than 2-oximes (2 versus 40; and 44 versus 42).

Practically, the reactivators bearing only one oxime group is enough for reactivation, however numerous bis-quaternary bis-oximes have been reported in literature. Our study has found that the presence of additional oxime group on the second pyridinium ring does not provide any advantage in terms of its reactivation activity. Rather, the bis-quaternary compounds bearing only one oxime group were found to be superior reactivators than those bearing oxime-group on both pyridinium rings (54 versus 40; and 56 versus 2). Although the structure-reactivation relationship indicates the key requirements for ideal reactivator; however it is very difficult to find out the real reason of different efficacy of oximes for AChE’s inhibited by structurally different OPs (Gorecki et al., 2016; Kuca et al., 2006).

Among various parameters which are crucial for dephosphorylation of phosphorylated AChE, the acid dissociation constant (pKa) of the oxime group of reactivator is very important (Valiveti et al., 2015). The oxime group of the reactivator makes a strong nucleophilic attack on the phosphorous atom to break the covalent bond between ‘OP’ and ‘serine OH’ group. For this, the ionization of oxime into oxyanion is an important step in the reactivation of OP inhibited AChE. Therefore, the pKa value of oxime group is considered as a crucial factor in design of reactivators. In order to understand the correlation between pKa of oxime and reactivation efficacy, pKa values were calculated for the oxime reactivators, which are tabulated in the supplementary information (Table S2). It has been observed that the 4-pyridinium oximes have pKa in the range of 6.36–6.99; the 2-pyridinium oximes have pKa in the range 6.99–7.38; whereas the pKa of 3-pyridinium oximes were found in the range of 7.70–8.14. The higher pKa values of the 3-pyridinium oximes resulted in poorer tendency of the oxime dissociation into the oxyanion (correlation coefficient r=−0.31), which could the probable reason that majority of the 3-pyridinium oximes have lower reactivation activity. The most potent reactivators 3 and 7 have pKa values of 6.69 and 6.83.

The best reactivator among the monoquaternary oximes tested was 5 that showed 73, 45 and 18% reactivation of (MeO)₂P(O)-, (EtO)₂P(O)- and (iPrO)₂P(O)-modified EEAChE, respectively. The bisquaternary oximes with aliphatic linkers, 3 and 56 showed better reactivation activity with 86, 80, 44% and 77, 80, 35% reactivation for (MeO)₂P(O)-, (EtO)₂P(O)- and (iPrO)₂P(O)-modified EEAChE, respectively. Amongst bisquaternary compounds bearing heterocyclic linkers, 7 and 8 showed 54, 52, 19% and 47, 52, 21% reactivation for (MeO)₂P(O)-, (EtO)₂P(O)- and (iPrO)₂P(O)-modified EEAChE, respectively. The larger percentage of reactivated (MeO)₂P(O)-and (EtO)₂P(O)-modified AChE by these oximes is likely due to ease of access to the less sterically-congested active site serine and lower extent of aging as compared to (iPrO)₂P(O)-modified AChE.

When phosphorylated enzyme undergoes aging, the protein is considered irreversibly inhibited (Casida and Quistad, 2004; Franjesevic et al., 2018; Quinn et al., 2017) (Figure 1A). As expected, the inhibition, reactivation and non-reactivation rates, and mechanisms are dependent on the structure and reactivity of the phosphorus ester ligands (Fukuto, 1990). OP-AChE conjugates that contain dimethyl or diethyl esters generally reactivate more readily, whereas OP-AChE conjugates that contain larger or branched alkyl groups (e.g. isopropyl) are more prone to undergo aging (Clothier and Johnson, 1979; Jennings et al., 2003; Millard et al., 1999). We studied the effect of certain oximes on aging by observing the change in the k_r value as the OP exposure time was increased. For POX-inhibited AChE, the highest k_r values were achieved within the first 30 min following inhibition while for MePOX-inhibited AChE, the highest k_r was obtained at 1 h. During the initial 90 min period there was only a gradual decrease in reactivation rate for all three OP-treated AChE’s. The increase in k_r values for MePOX-inhibited treated EEAChE with increasing exposure time was likely caused by the contribution of spontaneous reactivation to the k_r since dimethyl phosphorylated enzyme spontaneously reactivates 6 times faster than aging (Jennings et al., 2003).

Our results showed that DFP-modified EEAChE underwent complete aging after 24 h, which could not be reactivated by oximes. A 50% decrease in reactivation rate (as well as % reactivation) was observed in between 4–8 h. This was supported by a reported aging half-life (6.7 h) of (iPrO)₂P(O)-AChE conjugate (Jennings et al., 2003). (EtO)₂P(O)-inhibited AChE (half life_spont.react. = 70 h; half life_aging= 99 h) (Jennings et al., 2003) undergoes aging at a slower rate, which is reactivatable even after 24 h. Similarly, (MeO)₂P(O)-inhibited AChE (half life_spont.react. = 0.7 h; half life_aging= 4.2 h) (Jennings et al., 2003; Worek et al., 1997) undergoes slower aging and was reactivatable even after 24 h. Although the aging half-life for (MeO)₂P(O)-inhibited EEAChE is significantly shorter than for (EtO)₂P(O)-inhibited AChE, the spontaneous reactivation of the (MeO)₂P(O)-modified enzyme proceeds more rapidly than aging, which slows down the overall aging of (MeO)₂P(O)-AChE. In contrast, (iPrO)₂P(O)-AChE conjugates undergo faster aging than spontaneous reactivation. Overall, (iPrO)₂P(O)-inhibited AChE undergoes aging at a faster rate than (MeO)₂P(O)- and (EtO)₂P(O)-AChE conjugates, which becomes non-reactivatable after 24 h of DFP exposure time.

Although the present study was limited to the comparison of reactivation rates of AChE’s inhibited by three structurally different OPs using a library of pyridinium oximes; however, the understanding of differences between AChE’s from different species is also one of the crucial factor in the successful discovery of effective antidotes for human use. The similarity of AChEs from different species has been studied using 3D-structure alignments (Wiesner et al., 2007), and by studying the effect of inhibitors or reactivators (Berry, 1971; Kuca et al., 2005; Worek et al., 2002). Structurally, AChEs from different species are very similar (Wiesner et al., 2007); however, numerous studies have indicated that reactivation potency of oximes to reactivate OP-inhibited AChE varies from species to species (Kuca et al., 2005; Worek et al., 2002). Amongst, various non-human AChE’s, the EEAChE has been routinely used as a model enzyme in the initial phase of inhibitor/reactivator discovery and lead optimization, because of its high structural similarity with HuAChE (Salih et al., 1994) and high stability under longer experimental conditions. Despite of the high sequence identities among AChEs from different species, the reactivation property of oximes varies from species to species; therefore there is a need of careful evaluation of non-human reactivation data before extrapolation to humans.

4. CONCLUSION

In conclusion, all the tested oximes showed better reactivation profile for MePOX and POX modified EEAChE compared with DFP modified EEAChE. The monoquaternary oxime 5 and bisquaternary oximes 7 and 8 were the best reactivators of EEAChE inhibited by all three OP structure types. These compounds showed k_r values greater or comparable to pralidoxime (1) for DFP-inhibited EEAChE.

Highlights.

• A comparative reactivation profile of a series of 59 quaternary pyridinium-oximes

• Three organophosphorus (OP) inhibitors were used for AChE inhibition

• Oximes 7 and 8 showed kr values better than pralidoxime for DFP-inhibited AChE

• Faster reactivation of MePOX- and POX-inhibited AChE than DFP-modified AChE

ABBREVIATIONS

OP

organophosphorus compound

MPO

methyl paraoxon

EPO

ethyl paraoxon

DFP

diisopropyl fluorophosphates

EEAChE

electric eel acetylcholinesterase

PBS

phosphate buffer solution

DMP

dimethyl phosphoryl

DEP

diethyl phosphoryl

DIP

diisopropyl phosphoryl