Biological Testing of Organophosphorus-Inactivated

Article pubs.acs.org/crt

Biological Testing of Organophosphorus-Inactivated Acetylcholinesterase Oxime Reactivators Identified via Virtual Screening Jason A. Berberich,† Terry R. Stouch,‡ Sankar Manepalli,§,∥ Emilio Xavier Esposito,⊥ and Jeffry D. Madura*,§,∥ †

Department of Chemical, Paper and Biomedical Engineering, College of Engineering and Computing, Miami University, 64 P Engineering Building, 650 East High Street, Oxford, Ohio 45056, United States ‡ Science For Solutions, LLC, 6211 Kaityln Court, Princeton Junction, New Jersey 08550, United States § Center for Computational Sciences, Department of Chemistry & Biochemistry, Duquesne University, 600 Forbes Avenue, 308 Mellon Hall, Pittsburgh, Pennsylvania 15282, United States ∥ Department of Chemistry & Biochemistry, Duquesne University, 600 Forbes Avenue, 308 Mellon Hall, Pittsburgh, Pennsylvania 15282, United States ⊥ exeResearch, LLC, 32 University Drive, East Lansing, Michigan 48823, United States ABSTRACT: There is a pressing need for new therapeutics to reactivate covalently inactivated acetylcholinesterase (AChE) due to exposure to organophosphorus (OP) compounds. Current reactivation therapeutics (RTs) are not broadspectrum and suffer from other liabilities, specifically the inability to cross the blood−brain-barrier. Additionally, the chemical diversity of available therapeutics is small, limiting opportunities for structure−activity relationship (SAR) studies to aid in the design of more effective compounds. In order to find new starting points for the development of oximecontaining therapeutic reactivators and to increase our base of knowledge, we have employed a combination of computational and experimental procedures to identify additional compounds with the real or potential ability to reactivate AChE while augmenting and complementing current knowledge. Computational methods were used to identify previously uninvestigated oxime-containing molecules. Experimentally, six compounds were found with reactivation capabilities comparable to, or exceeding, those of 2-pralidoxime (2-PAM) against a panel of AChE inactivated by paraoxon, diisopropylfluorophosphate (DFP), fenamiphos, and methamidophos. One compound showed enhanced reactivation ability against DFP and fenamiphos, the least tractable of these OPs to be reactivated.

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INTRODUCTION

The catalytic active site of AChE is at the bottom of a long, narrow gorge and contains a catalytic triad1 composed of the catalytic serine, a glutamic acid, and a histidine; S203, E334, and H447, respectively. OPs act through covalent modification of a key catalytic serine residue and results in the inactivation of the enzyme. Therapeutics act by reacting with and subsequently removing this adduct, the OP-AChE conjugate, and consequently reactivating AChE. Each different OP forms a unique OP-AChE conjugate. The nature of the OP-conjugates dictates how and the extent to which the reactivation therapeutics (RTs) are able to reactivate the inactivated AChE.2 The X-ray crystal structures for several OP-AChE conjugates have been solved. The deep and constricted path to the catalytic triad makes it difficult for RTs to access to the OP-conjugate,

Poisoning by organophosphorus (OPs) agents is caused by their reaction with acetylcholinesterase (AChE) resulting in covalent modification and inactivation. The inactivation due to some OPs is reversible, but the modifications due to other OPs are less tractable. Many inactivating OPs are already known, and it is considered inevitable that new, even more potent, and potentially less tractable OPs will arise. Although some therapeutics are available, most fall short of the desired efficacy, and none is able to adequately reactivate a broad spectrum of OPs. Broad-spectrum therapeutics are desired due to the diversity of OP-AChE conjugates and the need for rapid treatment following exposure to OP compounds since there is seldom time to identity the offending substance. Consequently, there is a strong need for new therapeutics; however, the slow progress in the discovery of new therapeutics suggests a need for different approaches for their discovery. © XXXX American Chemical Society

Received: June 6, 2016

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DOI: 10.1021/acs.chemrestox.6b00198 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Chemical Research in Toxicology

compounds with interesting physicochemical and biological properties or even enhanced reactivation ability could be a significant advancement for the field and aid medicinal chemists in the development of the next generation of RTs for organophosphate poisoning. Our approach was straightforward. Molecular similarity and dissimilarity calculations were used to identify within publicly available databases unique commercially available compounds that, although having some similarity to the known 2 and 4pyridyl-aldoxime and ketoxime RTs, would also have chemically interesting variations. Molecular compound/protein “docking,” was further used to appraise their essential ability to fit within the AChE gorge and approach the binding site of inactivated AChE. Those that passed these criteria and were commercially available were assayed for their ability to reactivate human AChE inactivated by four pesticides: fenamiphos, methamidophos, diisopropylfluorophosphate (DFP), and paraoxon (Figure 1). It is well-known that the reactivation of OP-

reactivate the AChE, and allow the new therapeutic-agent conjugate to exit the gorge. From the perspective of rational drug design and structure− activity relationships (commonly referred to as SAR), the currently known potent AChE RTs are limited in structural diversity. These established RTs are positively charged quaternary amines that have difficulty permeating the blood− brain barrier to reactivate inactivated AChE.3−5 A new collection of amidine-oximes6−8 designed, synthesized, and tested by Cashman and co-workers possess the ability to cross the blood−brain barrier due to their zwitterionic nature at physiological pH. Overcoming of the blood−brain barrier impediment is important, but each of the aforementioned problems individually hinders the development and advancement of a therapeutic. When these obstacles are encountered in combination, as is the case for OP-inactivated AChE, prevailing over a single or portion of the noted hurdles is considered a success. The approved and commonly used oxime-containing RTs, such as 2-pralidoxime (2-PAM) and HI-6, tend to be modifications on a theme; the same known moieties are connected in different ways using linkers of varying length and composition. Although a number of other structurally related compounds are known, there is very limited publicly available information on their activity as reactivators. Consequently, for the most effective application of rational drug design and QSAR approaches, there is a need to increase the amount of reactivator-based data and its diversity. Exciting advancements have been made with respect to nonsymmetrical RTs such as those able to interact with the peripheral anionic site (PAS) and are able to reactivate tabuninactivated hAChE more efficiently than obidoxime (2.3×), HI6 (2.8×), and trimedoxime (5×).9 Unfortunately, these pyridinium-4-aldoximes result in reactivation byproducts with high affinities for AChE and thus result in an extended inhibition period.10 The design, synthesis, and in vivo testing of two RTs possessing xylene and naphthalene linkers, while sharing structural features with K203 and trimedoxine, were able to reactivate tabun-inactivated AChE, yet the established RT K203 demonstrated greater reactivation and therapeutic efficacy.11 The work of Gupta et al. strengthens the findings of Kassa et al.11 and demonstrates that oxime RTs with aliphatic links are better than those with xylene linkers for the reactivation of paraoxon inactivated AChE.12 A series of imidazolium aldoximes show promise due to their lower hAChE inhibition tendencies compared to that of obidoxime, HI-6, MMB-4, and TMB-4.13 The in vitro reactivation of sarin, VX, and tabun-inactivated hAChE by 1013 of the imidazolium aldoxime RTs demonstrated that three of the RTs were comparable or better for the reactivation of these OPinactivated hAChE than obidoxime, HI-6, MMB-4, and TMB4. The impressive reactivation ability and lower hAChE inhibition is noteworthy. Here, we report the results of combined computational and experimental approaches to leverage known information about RTs to identify compounds that have not yet been considered as reactivators or at least do not have human AChE reactivation data publicly available. Our goal was to find compounds related to known RTs that would add increased diversity in activity, structure, and properties to the currently available data in order to support the development of QSAR models and other rational drug design efforts that would ultimately aid in the design and development of new RTs. The identification of

Figure 1. Four organophosphate pesticides used in this study: methamidophos, fenamiphos, diisopropylfluorophosphate (DFP), and paraoxon; 2D and 3D depictions are provided.

inactivated AChE is strongly dependent on the structure of the phosphyl moiety;14 thus, these four pesticides were selected due to their varying structures and ease of reactivation. In order to determine the compounds’ own innate inhibitory potential, they were also assayed for inhibition of apo-AChE, the nonphosphorylated AChE. Six compounds with reactivating ability were identified that had not been previously considered as OP-conjugate reactivators at the time of this study and did not have publicly available human AChE reactivation data. These compounds contain structural variations that add to the current molecular and biological information, thus increasing the available structure−activity data to support the design of new RTs.

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METHODS

Molecular Databases. The ZINC15 and PubChem16 databases were searched for 2- and 4-pyridinealdoximes and pyridineketoximes for which the oxime functional group was pendant (i.e., the oxime moiety was not within a cyclic-ring). We retained these features as the core structure for our similarity search as consistent and assumed that they were essential features of this particular class of known reactivators. The chemical representation of the oxime and pyridoxyl moieties within PubChem was not consistent within themselves or with other databases including ZINC. This difference in representation made comparisons difficult, and thus, the rest of our discussions involve compounds extracted from the ZINC database (downloaded July 2009) where the oxime and pyridoxyl moieties were consistently archived and had the additional benefit of being commercially available, a main purpose of ZINC.

B


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Chemical Research in Toxicology Cheminformatics. Calculation of molecular similarity between compounds is commonly used for the search for compounds with properties, including biological activities, similar to those of another molecule. Conversely, calculation of dissimilarity, a related approach, is a means of finding diverse molecules. Molecular similarity and dissimilarity searches of the compounds derived from ZINC in comparison to the known reactivators were performed using Pipeline Pilot, version 8.17 The known RTs, 2-pralidoxime (2-PAM), obidoxime, HI-6, and HLö-7 (Figure 2) were used in the similarity

dissimilarity using the same software. Dissimilarity is implemented in PipeLine Pilot as a cluster analysis followed by selection of cluster centers using a maximum dissimilarity method. The resultant compounds were visually inspected for the most interesting and unique structural features that would contribute structural information to the SAR. For example: ZINC compounds 0117551 and 4695229 were chosen for the presence of 1 or 2 chlorine atoms in the pyridine ring, respectively, which could have effects in terms of bulk, electrostatics, and influence on the reactivity of the reactive oxime moiety. Similarly, compound 2547525 was selected because it contained 4 fluorine atoms. Compound 3895757 had substantial bulk extended beyond the pyridine, whereas compounds 4981419, 5120967, and 5184166 possess bulk positioned as, effectively, substituents of the pyridine ring and direct at the active site. The six ZINC compounds (#1−6) that became the focus of this work provide variation distal to the pyridine ring likely occupying the pocket closer to the exterior (the PAS) and potentially serving as an anchor for the compound as a whole. The few remaining compounds express substantial variation in substitution of the pyridine ring including the positioning of the oxime moiety relative to the pyridine nitrogen and substitution on the pyridine nitrogen itself. Docking Protocol. The X-ray structure of mouse acetylcholinesterase (mAChE) inhibited by methamidophos (PDB ID: 2jge;20,21 reported resolution is 2.6 Å, Rvalue of 0.199, and RFree value of 0.233) was downloaded from the RCSB.20 The methamidophos-inactivated murine protein was used since at the time, no structure of a human protein inactivated by an agent relevant to this study was available. The mouse and human isozymes of AChE share the same fold (α/βhydrolase) and have a sequence identity and similarity of 88% and 97%, respectively, and most importantly, the amino acids lining the binding site and the gorge leading to the binding site are identical. We felt this sequence and structural similarity were sufficient for a rough evaluation of the ability of the compounds to fit within the AChE gorge and interact with phosphorylated S203, since this step was used only as an approach to remove compounds that were too large to fit.

Figure 2. Four oxime-containing therapeutic reactivators used in the similarity analysis. searches. The compounds extracted from the ZINC collection were compared to known oxime reactivators for similarity as calculated using the ECFP6 fingerprints18 and the Tanimoto metric.19 Those compounds with maximum similarity were selected based on a slight breakpoint in the similarity at a value of 0.45 (on the standard similarity score scale of 0, no similarity, to 1.0 identity). In other words, compounds with a Tanimoto similarity score of greater than 0.45 were progressed to further analysis. Since our screening capacity was limited and in order to focus on compounds with the greatest range of structural features, the most diverse representatives of the resultant compounds were then determined by calculation of their

Table 1. 18 Compounds Obtained from the Cheminformatic and Docking Studiesa

a

Compounds examined experimentally are numbered #1−#6. C


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of the OP agent), 20 μL aliquots of the inhibited hAChE were transferred to cuvettes containing phosphate buffer (0.1 M, pH 7.4), DTNB, and an oxime (100 μM; total volume 3.1 mL). After incubating for 60 min (at 37 °C), the hAChE activity was determined by adding ATCI. Data Analysis. The data analysis was performed as in Worek et al.26,27 Enzyme rates were corrected for spontaneous and oximeinduced hydrolysis of acetylthiocholine. All data were compared to controls that were performed in an identical manner without inactivation. The approach of de Jong and Wolring28 was used to calculate percent reactivation:

The MOE 2010.1022 program was used to prepare the protein and subsequent molecular docking. The A chain of the protein structure, associated water molecules, and SGR203 (the PDB nomenclature for the phosphorylated S203, i.e., o-(R)-methyl phosphoroamidate-Lserine) were retained, while a coincidental triethylene glycol and the modified residue SGX203 (the other stereoisomer of the phosphorylated S203, the methamidophos conjugate, o-(S)-methyl phosphoroamidate-L-serine version that was modeled into the crystal structure 2jge) were removed. Hydrogen atoms were added via Protonate3D,23 and atomic partial charges were assigned based on the Amber 99 molecular mechanics force field.24 An energy minimization protocol was used to remove steric and structural strain of the added hydrogens, although the heavy atoms were fixed to the experimentally determined coordinates. The compounds identified by the cheminformatics analysis and visual selection were docked using MOE’s Triangle Matcher placement algorithm and the “London dG” scoring function.22 Our acceptance criteria for a molecule were straightforward. The potential RT needed to fit within the gorge near the catalytic triad and the oxime functional group needed to be within 6 Å of the phosphorus atom of the OP-conjugate; a distance observed in several OP:AChERT crystal structures. Further, docking followed by the distance criteria was employed to ensure a productive interaction could be made between the oxime and the OP-conjugate while providing a tolerance for protein flexibility. The goal of the docking was simply to use it as a coarse screen to remove compounds that could not fit into the gorge. We were not attempting to evaluate any intermolecular interactions between the oxime and AChE. Experimental Bioassay Protocol. Materials and Supplies. Human acetylcholinesterase (hAChE) was purchased from SigmaAldrich. The enzyme is the amphiphilic form that has been extracted from human erythrocytes using Triton X-100 and purified by affinity chromatography. The enzyme comes as a solution in 20 mM HEPES, pH 8.0 with 0.1% Triton X-100. 2-Pralidoxime (2-PAM) was purchased from Sigma-Aldrich (2-pyridinealdoxime methiodide: SA#, P60205; MW, 137.16 g/mol). The pyridoximine compounds identified by our studies were purchased from Asinex (using the naming employed in Table 1): # 1, BAS#, BAS02975882; MW, 356.47 g/mol; # 2, BAS#, BAS02975743; MW, 432.57 g/mol; # 3, BAS#, BAS02975887; MW, 328.42 g/mol; # 4, BAS#, BAS02975491; MW, 322.41 g/mol; # 5, BAS#, BAS02975869; MW, 308.38 g/mol; # 6, BAS#, BAS02975868; MW, 342.44 g/mol. All pesticides were purchased from ChemService, Inc. (www.chemservice.com), and stock solutions were prepared in isopropyl alcohol. Enzyme Activity Determination. Acetylcholinesterase activity was measured spectrophotometrically at 405 nm using the modified Ellman assay25 used by Worek et al.14 The assay was performed using a mixture with a final concentration of 0.45 mM acetylthiocholine (substrate) and 0.3 mM DTNB (chromogen) in 0.1 M phosphate buffer (pH 7.4). hAChE activity was determined by measuring the change in absorbance at 405 nm over 5 min. Inactivation of AChE by Organophosphorus Compounds. Human acetylcholinesterase (in 0.1 M phosphate buffer, pH 7.4) was incubated with pesticide solution for 1 h at 37 °C. The hAChE activity was measured to determine the extent of inhibition. Excess pesticide was removed (extracted) using hexane. Control experiments were performed to verify the removal of inhibitory pesticide by demonstrating the inability to inhibit acetylcholinesterase after incubation for 60 min. Enzyme was also incubated with isopropyl alcohol as a control to account for solvent induced inactivation. Effect of Oximes on AChE Activity. Human acetylcholinesterase was incubated in phosphate buffer (0.1 M, pH 7.4) containing DTNB and an oxime (100 μM). The hAChE activity was determined after incubation for 1 h at 37 °C by addition of acetylthiocholine. The rate of enzyme-catalyzed hydrolysis of acetylthiocholine in the presence of the oxime was compared to the rate of enzyme catalyzed hydrolysis without oxime and was reported as percent relative activity. Reactivation of Acetyl Cholinesterase by Oximes. Acetylcholinesterase reactivation experiments were performed using the method described by Worek et al.26,27 At 0 min (i.e., 30 min after the addition

percent reactivation =

AIR t × A /AR − AI × 100 A − AI

where A = control activity, AR = activity of the control enzyme incubated with the oxime, AI = the activity of the inactivation enzyme (hAChE), and AIRt = activity of the inactivation hAChE after incubation with the oxime for time t.

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RESULTS AND DISCUSSION The ZINC database yielded 1088 pyridineoximes, 633 2pyridyl, and 455 4-pyridyl, a total of 2176 oxime compounds. These compounds contained a range of substitutions on the pyridine nitrogen atom, including the incorporation of pyridine ring into bicyclic systems, and a variety of substitutions on both the carbon and nitrogen atoms of the oxime moieties. The noted substitutions provided variations in the size and shape of RTs while modifying gross physicochemical properties to probe the RTs’ reactivation abilities. Using the criteria described in the Methods sections, a similarity search of the 2176 ZINC compounds against the specified RT structures returned several hundred hits. Many of the resultant molecules had a strong resemblance to the known RTs of Figure 1. To remove duplication and extremely similar compounds from the hit set, a dissimilarity search was used to eliminate the most similar compounds, reducing the hit list to 60 compounds. Visual inspection of these compounds for the most diverse and chemically interesting variations in size, shape, gross electronic properties, and unique chemical features, based on medicinal chemistry experience and the goal to identify compounds that would contribute favorably to further (Q)SAR studies, resulted in a set of 18 compounds (Table 1). Examples of the resulting diversity are (i) three of the molecules contain halogen substitutions on the pyridine ring which influences the electronic nature of the ring and oxime; (ii) two molecules have the pyridine moiety incorporated into a quinoline ring which provides additional bulk near the reactive center; and (iii) two other molecules increase bulk by including relatively large substituents on the pyridinium nitrogen atom. An essential component of any RT is the ability to fit within the gorge and place the reactive oxime moiety within reasonable proximity to the phosphorylated S203 of the inactivated AChE. Consequently, the 18 ZINC molecules identified by the cheminformatics approach were docked in the hAChE as described in the Methods section. All 18 molecules are able to fit comfortably within the gorge and within a reasonable proximity to the OP-conjugate according to our criteria and docking analysis. The ligands did not experience conformational strain or steric clashes with the protein, and the oxime moiety was positioned within 6 Å of the S203’s OPconjugate. Unfortunately, despite the compounds being extracted from the ZINC database, only 6 were conveniently available for purchase (labeled as “oxime #” in Table 1). Somewhat contrary D


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Figure 3. Residual activity of human erythrocyte acetylcholinesterase after incubation with 100 μM oximes at 37 °C for 1 h. Error bars represent the standard error of five experiments.

Table 2. Experimental Results reactivation (%)a relative activity (%) 2PAM oxime #1 oxime #2 oxime #3 oxime #4 oxime #5 oxime #6

75.0 25.3 15.3 53.7 48.4 54.8 45.3

± ± ± ± ± ± ±

7 2 1 8.5 3.25 6 4

a

paraoxon

DFP

fenamiphos

± ± ± ± ± ± ±

9±4 28 ± 7 58 ± 23 22 ± 11 12 ± 2 15 ± 5 17 ± 4

6±0 25 ± 4 41 ± 12 19 ± 6 9 ± 13 15.5 ± 2 17 ± 16

16 46 37 41 23 28 41

10 12 25 NA 6 2 12

methamidophos 35 58 66 49 46 65 67

± ± ± ± ± ± ±

NA NA NA NA NA NA NA

a

Note: The values in the table are percent reactivation (percent of the inhibited enzyme activity that was regained) and percent relative activity (100% indicates no inhibition by the oxime).

Figure 4. Reactivation of organophosphate inhibited human erythrocyte acetylcholinesterase after incubation with 100 μM oximes at 37 °C for 1 h. Error bars represent the standard error of duplicate experiments (except for methamidophos).

indication of human in vitro AChE reactivation ability for the original 18 compounds. It should be noted that compound oxime #6 was found in a patent29 describing in vivo animal models as discussed below. The compounds were assayed to determine the rate of acetylthiocholine hydrolysis in the presence of 100 μM of the oxime (Figure 3 and Table 2). All oximes showed some inhibition of the enzyme (100% relative activity indicates no inhibition) with 2-PAM inhibiting the enzyme the least. Oximes #1 and #2 showed significant inhibition (≥75%) of AChE activity yet are structural homologues of oxime #6 (∼45% relative activity). All six compounds show the ability to

to our search and desire for maximum structural diversity, these compounds can be considered structural homologues. However, even within these six compounds there are interesting structural variations adding unique diversity to the overall collection of known reactivators. These varying features include (i) increased bulk at the pyridinium nitrogen atom; (ii) three different ring systems distal to the pyridinium; (iii) variation with regards to the positioning and substitution of the second, distal, positively charged (in five cases quaternary) nitrogen atom is evidenced; and (iv) all compounds exhibit an aryl amide functionality, a linkage readily amenable to combinatorial chemistry. Extensive search of the literature did not provide any E


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computational methods, we feel the methods stand on their own and that our approach can be further refined and implemented to yield additional and unique compounds. The computational methods can be applied to the evaluation of compounds to indicate their potential reactivating ability against OP-conjugates of interest. The work presented here achieved our main objective of finding reactive compounds that add novel structural and biological information to the known pyridinium-oxime-based class of hAChE reactivators. Further, the reactivation ability of several compounds is promising enough to suggest that they could be further refined to improve OP-inactivated hAChE reactivation and decrease apo AChE inhibition.

reactivate at a comparable or superior level than 2-PAM across the spectrum of OP-inactivated AChEs (Figure 4 and Table 2). The reactivation values trend well with the literature14 with the ease of reactivation rank reported as methamidophos > paraoxon > DFP ∼ fenamiphos. Unfortunately, for any immediate consideration as therapeutics, the inhibition of apo AChE by these potential RTs, oximes #1 and #2, was also determined to be greater than 2-PAM, while the percent relative activity difference between 2-PAM and oximes #3 through #6 is smaller. Perhaps not surprising, all of the compounds were found to have reasonable ability to reactivate the AChE inactivated by methamidophos, the most tractable of the agents examined. However, the substantial potency of oxime #2 against DFP and fenamiphos, the least tractable of the OP-conjugates studied here, is substantial and could provide structural information leading to a better understanding of the molecular features important for reactivation and inhibition of hAChE for this series of oxime containing compounds. Our reported experimental results led us to the following hypotheses. Oximes #1 and #2 bind strongly to apo AChE due to the hydrophobic functional groups attached to the nitrogen of the pyridine ring. The binding is also aided by the interactions between the quaternary nitrogen and the peripheral anionic site near the mouth of AChE’s gorge. Finally, the strong binding of oximes #1 and #2 aids in orienting the oxime functional group for reactivation. Oximes #3 through #6 do not inhibit AChE to the extent of oximes #1 and #2, although these oximes are better at reactivating than 2PAM. This difference is due to the length and composition of the compound distal to the oxime functional group. This results in decreased interactions with the mouth region of the gorge. The experimental evidence also indicates that these compounds might be good reactivators for paraoxon, DFP, fenamiphos, and methamidophos OPs because they are better able to orient the oxime functional group with the OP-conjugate to initiate the reactivation. Adding support for the value of the identified compounds, compound #6 was previously shown29 to have potency in cats poisoned by VX and rats poisoned by sarin, soman, and VX. Although the cat and rat study presented in vivo versus in vitro assay results, did not use the human protein as our study did, and used a different collection of OP agents than us, the overall message is the same; this series of compounds shows promise as potential reactivation therapeutics.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +1-412-396-4129. E-mail: [email protected]. Funding

This work was supported by ICx-Agentase and the U.S. Department of Defense’s Joint Project Manager for Nuclear, Biological, and Chemical Contamination Avoidance. J.D.M. and S.M. were funded by ICx-Agentase, Inc. contract “Modeled CHOlinesterase ReactivatorS (MoCHORS)” CBDIF-07THER-01. T.R.S. was funded by Grant number G0900048 Task order 04 of the G0900048 MoChoRS project from Duquesne University. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS AChE, acetylcholinesterase; OP, organophosphorus containing compounds; RT, reactivating therapeutic; QSAR, quantitative structure−activity relationship

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REFERENCES

(1) Shafferman, A., Kronman, C., Flashner, Y., Leitner, M., Grosfeld, H., Ordentlich, A., Gozes, Y., Cohen, S., Ariel, N., and Barak, D. (1992) Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding. J. Biol. Chem. 267, 17640−17648. (2) Antonijevic, B., and Stojiljkovic, M. P. (2007) Unequal efficacy of pyridinium oximes in acute organophosphate poisoning. Clin. Med. Res. 5, 71−82. (3) Karasova, J. Z., Pohanka, M., Musilek, K., Zemek, F., and Kuca, K. (2010) Passive diffusion of acetylcholinesterase oxime reactivators through the blood-brain barrier: influence of molecular structure. Toxicol. In Vitro 24, 1838−1844. (4) Karasova, J. Z., Stodulka, P., and Kuca, K. (2010) In vitro screening of blood-brain barrier penetration of clinically used acetylcholinesterase reactivators. J. Appl. Biomed. 8, 35−40. (5) Voicu, V. A., Bajgar, J., Medvedovici, A., Radulescu, F. S., and Miron, D. S. (2010) Pharmacokinetics and pharmacodynamics of some oximes and associated therapeutic consequences: a critical review. J. Appl. Toxicol. 30, 719−729. (6) Kalisiak, J., Ralph, E. C., and Cashman, J. R. (2012) Nonquaternary Reactivators for Organophosphate-Inhibited Cholinesterases. J. Med. Chem. 55, 465−474. (7) Kalisiak, J., Ralph, E. C., Zhang, J., and Cashman, J. R. (2011) Amidine-Oximes: Reactivators for Organophosphate Exposure. J. Med. Chem. 54, 3319−3330. (8) Okolotowicz, K. J., Dwyer, M., Smith, E., and Cashman, J. R. (2014) Preclinical Studies of Noncharged Oxime Reactivators for Organophosphate Exposure. J. Biochem. Mol. Toxicol. 28, 23−31. (9) Kliachyna, M., Santoni, G., Nussbaum, V., Renou, J., Sanson, B. T., Colletier, J.-P., Arboléas, M., Loiodice, M., Weik, M., Jean, L.,

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CONCLUSIONS We provide in vitro results for six oxime-containing compounds not previously assayed for the reactivation of human AChE inactivated by four organophosphorus pesticides. The compounds’ inhibitory activity against apo human AChE was also determined. The six compounds showed similar or increased reactivation potential compared to that of 2-PAM for all four of the OP-hAChE conjugates. Compound #2, with a benzyl group attached to the nitrogen of the pyridine ring, showed substantially enhanced reactivation activity against DFP and fenamiphos, two OP agents considered particularly intractable. These compounds were identified using the straightforward application of common computational methods and protocols using prior knowledge of known oxime-based reactivating therapeutics. The results demonstrate the capabilities of these methods to identify previously overlooked compounds. Although the authors have substantial experience with these F


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Biological Testing of Organophosphorus-Inactivated

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