Methyl Scanning and Revised Binding Mode of 2-Pralidoxime, an Antidote for Nerve Agent Poisoning

Article abstract of DOI:10.1021/acsmedchemlett.9b00586

Organophosphorus nerve agents (OPNAs) inhibit acetylcholinesterase (AChE) and, despite the Chemical Weapons Convention arms control treaty, continue to represent a threat to both military personnel and civilians. 2-Pralidoxime (2-PAM) is currently the only therapeutic countermeasure approved by the United States Food and Drug Administration for treating OPNA poisoning. However, 2-PAM is not centrally active due to its hydrophilicity and resulting poor blood-brain barrier permeability; hence, these deficiencies warrant the development of more hydrophobic analogs. Specifically, gaps exist in previously published structure activity relationship (SAR) studies for 2-PAM, thereby making it difficult to rationally design novel analogs that are concomitantly more permeable and more efficacious. In this study, we methodically performed a methyl scan on the core pyridinium of 2-PAM to identify ring positions that could tolerate both additional steric bulk and hydrophobicity. Subsequently, SAR-guided molecular docking was used to rationalize hydropathically feasible binding modes for 2-PAM and the reported derivatives. Overall, the data presented herein provide new insights that may facilitate the rational design of more efficacious 2-PAM analogs.

Full text of DOI:10.1021/acsmedchemlett.9b00586

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Letter
Methyl Scanning and Revised Binding Mode of 2‑Pralidoxime, an
Antidote for Nerve Agent Poisoning
Adriana Gambino, James C. Burnett, and Kazunori Koide*
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ABSTRACT: Organophosphorus nerve agents (OPNAs) inhibit acetylcholines-
terase (AChE) and, despite the Chemical Weapons Convention arms control
treaty, continue to represent a threat to both military personnel and civilians. 2-
Pralidoxime (2-PAM) is currently the only therapeutic countermeasure approved
by the United States Food and Drug Administration for treating OPNA
poisoning. However, 2-PAM is not centrally active due to its hydrophilicity and
resulting poor blood−brain barrier permeability; hence, these deficiencies warrant
the development of more hydrophobic analogs. Specifically, gaps exist in
previously published structure activity relationship (SAR) studies for 2-PAM,
thereby making it difficult to rationally design novel analogs that are
concomitantly more permeable and more efficacious. In this study, we
methodically performed a methyl scan on the core pyridinium of 2-PAM to
identify ring positions that could tolerate both additional steric bulk and
hydrophobicity. Subsequently, SAR-guided molecular docking was used to rationalize hydropathically feasible binding modes for 2-
PAM and the reported derivatives. Overall, the data presented herein provide new insights that may facilitate the rational design of
more efficacious 2-PAM analogs.
KEYWORDS: Drug design, medicinal chemistry, structure−activity relationships, molecular modeling, antidotes, nerve agents
cetylcholinesterase (AChE) catalyzes the hydrolysis of
acetylcholine, a neurotransmitter responsible for sending
dephosphylate inhibited AChE with a greater efficacy than the
currently used antidotes shown in Figure 1b.
A
activation signals resulting in muscle contraction.1 Structurally,
the enzyme’s active site, a catalytic triad consisting of Ser-203,
Glu-334, and His-447 (human AChE numbering), is buried in
a 20 Å deep binding gorge lined primarily by aromatic
residues.2 The nucleophilic Ser-203 residue, in conjunction
with its Glu and His partners, is responsible for the
homeostatic hydrolysis of acetylcholine.
With respect to 2-pralidoxime (2-PAM) efficacy, the
majority of previously conducted synthetic studies have
focused either on developing bis-pyridinium oximes (via
alteration of the N-alkyl group) or on modifying the
electrostatic character of the core pyridinium.5−7 However,
these modifications appear to have been driven by synthetic
accessibility rather than establishing systematic and rational
structure−activity relationships (SAR)8 and, therefore, fail to
clearly delineate optimal substitution patterns for AChE active
site complementarity. To our knowledge, no studies to date
have systematically explored SAR for alkyl group substitution
on the pyridinium ring of 2-PAM, i.e., with the position of the
alkyl group being the only variable. Therefore, in the current
work, we conducted a methyl scan on the pyridinium ring of 2-
PAM to (1) systematically probe the steric and electrostatic
constraints of the AChE binding site to provide a foundation
for the rational, structure-based design of new analogs and (2)
determine whether the active site can tolerate an additional
Organophosphorus nerve agents (OPNAs) (Figure 1a) are
some of the most toxic of synthetic substances; in small
laboratory animals (e.g., rabbits, guinea pigs, and mice), 24 h
subcutaneous LD50’s range from 10 to 165 μg/kg.3
Mechanistically, these agents phosphylate the catalytic Ser-
203 residue, which consequently inhibits AChE’s function.4,5
Subsequently, toxic levels of neurotransmitter accumulate in
the synaptic cleft and overstimulate cholinergic receptors.
Resulting pathophysiology includes seizures, respiratory arrest,
and often death.4
Current antidotes for OPNA poisoning (Figure 1b) provide
only 24−34% reactivation of AChE in the peripheral nervous
system; none offer global protection against OPNAs.4,5 While
there are treaties and diplomatic efforts attempting to prevent
the use of such agents, OPNAs are still being used during both
acts of terrorism and, more recently, military conflicts.
Consequently, there remains the need for antidotes that will
more effectively pass the blood−brain barrier (BBB) and
Special Issue: Medicinal Chemistry: From Targets to
Therapies
Received: December 9, 2019
Accepted: January 9, 2020
Published: January 9, 2020
https://dx.doi.org/10.1021/acsmedchemlett.9b00586
© XXXX American Chemical Society
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Figure 1. AChE inhibitors and clinically used antidotes: (a) Structures of organophosphorus nerve agents. (b) Structures of clinically used
antidotes. (c) Structure of AChE inhibitor, paraoxon ethyl, used in this study.
alkyl group on the pyridinium ring. The results of this study
would allow for the rational design of a new, more
hydrophobic, and therefore more BBB permeable, therapeutic
candidate than 2-PAM.11−13 We chose 2-PAM because this is
the only antidote approved by the United States Food and
Drug Administration and because the simpler structure
provides a more tractable template than related dimeric
compounds, such as HI-6. For example, if we installed a methyl
group in HI-6, the biological results would be uninterpretable
because the biological results would be a sum of two
modifications. However, the results presented herein will, in
future studies, be extended to further optimize dimeric analogs,
since we will understand the impact of a single methylation
substitution.
Methyl scanning involves synthesizing a series of com-
pounds in which a methyl group is systematically added to
each modifiable position of a parent structure. This is a viable
experimental method to delineate regions of a drug that are
important or unimportant for biological activity, affording
opportunities for structural modifications to improve potency
and/or modify bioavailablilty.9 Specifically, for 2-PAM, we
hypothesized that synthesizing the analogs shown in Figure 2
and subsequently testing their reactivation potencies against
AChE from human (hAChE) and Electrophorus electricus (eel
AChE) pretreated with an organophosphorus-based inhibitor
would provide a better understanding of how such reactivators
interact with surrounding AChE binding site residues, and
hence provide a rational basis for future synthetic efforts.
Although paraoxon ethyl (Figure 1c) may not fully represent
nerve agents,10−12 it has been used in many studies as a
simulant of OPNAs6,13−27 due to stringent government
controls and, therefore, was used in the current study.
Moreover, as shown in Table S2, by simply adding a methyl
group, computational calculations predict slight increases in
both ALogP and BBB permeability, and a slight decrease in
solubility (i.e., an indicator of increased lipophilicity).
The compounds shown in Figure 2 were synthesized via the
route shown in Scheme 1. The first synthetic step involved the
N-oxidation of commercially available dimethylpyridines
following a method reported by Limnios and Kokotos (54−
69% yields).28 Subsequently, a Boekelheide rearrangement29
afforded the corresponding hydroxymethylpyridines in 40−
96% yields. The oxidation of the alcohols with MnO2
generated the corresponding aldehydes in 42−77% yields,
and these aldehydes were then condensed with hydroxylamine
to form the oximes in 52−86% yields. Finally, N-methylation
with MeI provided the final products in 21−77% yields. N-
Methylation of the 3-, 4-, and 5-methyl precursors was facile
and went to completion. The N-methylation of the 6-methyl
precursor required longer reaction time and required
purification due to the formation of a side product.
With these four 2-PAM analogs in hand, Ellman’s assay was
employed to determine their reactivation potencies.30 The
enzyme, either hAChE or eel AChE, was incubated with
paraoxon ethyl for 10 min before being added to a clear 96-
well plate containing the oximes, Ellman’s reagent, and
acetylthiocholine. First, dose-dependent reactivation was
determined by measuring the absorbance at 405 nm with
hAChE (Figure 3a). At 18.8 μM, 3-Me-2-PAM is approximately
13% as efficient as 2-PAM as a reactivator. The relative
reactivation efficiencies for 4-Me-2-PAM, 5-Me-2-PAM, and 6-
Me-2-PAM were 67%, 34%, and 43%, respectively. When we
compared the relative efficiency of reactivation at 75 μM, 3-, 4-,
Figure 2. Structures of target C-methylated 2-PAM analogs for this
study.
B
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a
Scheme 1. Synthesis of C-Methylated 2-PAM Analogs
a
t
Reagents and conditions: (a) 2,2,2-trifluoroacetophenone (10 mol %), MeCN (1.5 equiv), 30% aq H2O2 (5 equiv), BuOH, aq K2CO3, 23 °C,
54−69%; (b) TFAA (3 equiv), DCM, 23 °C, 40−96%; (c) MnO2 (5 equiv), DCE, reflux, 42−77%; (d) K2CO3 (1.2 equiv), NH2OH·HCl (1
t
equiv), MeOH, H2O, reflux, 52−86%; (e) MeI (10 equiv), acetone, 21−55 °C, 21−77%. Bu = tertiary butyl, TFAA = trifluoroacetic anhydride,
Me = methyl, DCM = dichloromethane, DCE = 1,2-dichloroethane.
Figure 3 were caused by the inhibition of AChE by the oxime.
Interestingly, we reproducibly observed the bell-shape
reactivation trends with 4-Me-2-PAM shown in Figure 3c
and questioned if this might stem from compound aggregation.
However, UV−vis spectroscopic analysis of 4-Me-2-PAM
based on Beer’s law indicated that this was not the case
(Supporting Information Table S1, Figure S2, and Figure S3).
Therefore, we are not able to account for the bell-shape trends
at this time at this point. However, with regard to therapeutic
viability, the downward trend may not be relevant, as the
compound concentration in vivo, especially in the central
nervous system, might not exceed 100 μM.
Molecular modeling studies were performed to aid in
rationalizing the AChE reactivation differences observed for
the methyl scan analogs, which could not be explained based
solely on analyses of changes in electrostatic potential
(Supporting Information Figure S4). For AChE coordinates,
PDB entry 5HFA was used.31 This is the human form of the
enzyme inhibited by paraoxon and is cocrystallized with 2-
PAM in an unproductive binding mode. Specifically, in the
Figure 3. Reactivation of AChE by methyl scan analogs. (a) Dose
response curve at 60 min with hAChE. (b) 18.8 μM oxime with
unmodified 5HFA coordinates, the pyridinium nitrogen of 2-
PAM is in the same location as observed for the acetylcholine
quaternary nitrogen in the active site cation box. This allows 2-
PAM to be engaged in both face-to-face π-stacking and a
cation−π interaction with Trp-86. However, while beneficial as
a starting point for modeling studies, the oxime moiety in the
5HFA cocrystal is pointed away from phosphylated Ser-203,
with the antidote’s oxygen atom located 8.42 Å away from the
Ser-203-bound phosphorus atom (Supporting Information
Figure S5a). Such a 2-PAM position neither rationalizes its
reactivation efficacy nor the reactivation efficacies of the
methyl scan analogs, hence the term “unproductive binding”
(Supporting Information Figure S5b).
With respect to the species choice for the modeling studies,
it is important to note that the human 5HFA X-ray structure,
versus an Electrophorus electricus (eel) structure, was employed,
despite using eel AChE as a secondary in vitro experiment to
demonstrate consistency across species. The rationale for this
choice was 2-fold: First, the highest available resolution apo-
state X-ray structures for the two species are virtually identical
(with a root-mean-square deviation (including α-helices,
sheets, and loops) of only 0.642 Å (Supporting Information
Figure S6a)), as are all species from phylum Chordata for
which an AChE X-ray structure has been solved (Supporting
Information Figure S6b). Second, residues surrounding the
binding site are identical in human and eel (Supporting
Information Figure S6c) AChE, as well as in other species
(Supporting Information Figure 6d).
hAChE. (c) Dose response curve at 20 min with eel AChE. (d) 18.8
μM oxime with eel AChE.
5-, and 6-Me-2-PAM were 20%, 85%, 37%, and 62%,
respectively.
The kinetic data with 18.8 μM oximes are shown in Figure
3b. Our linear regression analysis suggests that the relative
rates of 3-, 4-, 5-, and 6-Me-2-PAM (with 2-PAM being 100%)
are 11%, 68%, 32%, and 45%, respectively. This is consistent
with the above analysis with these oximes at 18.8 μM.
The dose response curve of the methyl scan analogs for eel
AChE (Figure 3c) showed that 4-Me-2-PAM and 6-Me-2-
PAM were nearly equivalent to 2-PAM until approximately
150 μM concentrations. At concentrations greater than 150 μM,
the reactivation decreased for both analogs. 5-Me-2-PAM
showed moderate reactivation efficiency, while 3-Me-2-PAM
only slightly reactivated the inhibited enzyme at high
concentrations. At 18.8 μM, both 4-Me-2-PAM and 6-Me-2-
PAM reactivated paraoxon-ethyl-inhibited AChE (Figure 3d)
and were found to be nearly equivalent to 2-PAM. Moreover,
5-Me-2-PAM showed reduced reactivation efficiency, while 3-
Me-2-PAM showed negligible reactivation (Figure 3b). At 18.8
μM, the relative efficiencies of reactivation for 3-, 4-, 5-, and 6-
Me-2-PAM in comparison to 2-PAM were 13%, 90%, 44%, and
87%, respectively.
The four analogs were also incubated with free hAChE and
eel AChE to determine whether any of them might directly
inhibit the enzyme. As Supporting Information (SI) Figure S1
shows, none significantly did so, thereby excluding the
possibility that weaker reactivation potencies observed in
With AChE reactivation in mind, we considered a more
therapeutically relevant, productive binding mode for 2-PAM,
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Figure 4. Binding mode of 2-PAM and its methyl scan derivatives. PDB entry 5HFA31 was used for all modeling and is shown in pale cyan cartoon.
Select residues surrounding the active site are shown with green carbons, the carbons of both paraoxon-bound Ser-203 and paraoxon are shown in
yellow carbons, and the paraoxon phosphorus is colored in orange in panels b−f. Yellow dashes indicate the interaction between the compound’s
oxime oxygen and the paraoxon phosphorus prior to transesterification. (a) Proposed 2-PAM (magenta carbons) binding mode that places the
oxime group in an orientation that is plausible for SN2 attack on the phosphorus atom of paraoxon when it is covalently bound to Ser-203. The
proposed 2-PAM binding mode is superimposed onto that of covalently bound acetylcholine derivative KTA (orange carbons (both ligand and
Trp-86), PDB entry 2HA032) to show similarities in key, atomic-level positioning. (b) The same binding mode of 2-PAM as shown in (a), but
without KTA, and with 2-PAM hydrogens shown. (c) The 3-methyl (cyan carbon) of 3-Me-2-PAM forces the oxime moiety to adopt an out-of-
plane relative to the pyridinium ring. This conformation is predicted to significantly perturb the derivative’s ability to avoid engaging in unfavorable
contacts with surrounding residues to achieve a binding orientation that allows for AChE reactivation. For example, in one potential binding pose,
the 3-methyl would be engaged in a highly unfavorable hydrophobic-polar clash with Tyr 337 (red dashes, distance 2.4 Å). (d) The 4-methyl
substituent (cyan carbon) of 4-Me-2-PAM points out the AChE active site and into the sterically unhindered AChE binding gorge. (e) The 5-
methyl (cyan carbon) of 5-Me-2-PAM is located in a position within 3.4 Å of the side chain phenol oxygen of Tyr-337, resulting in a moderate
hydrophobic-polar clash (pink dashes) that would perturb optimal binding. (f) The 6-methyl (cyan carbon) of 6-Me-2-PAM points into a
hydrophobic location in the AChE binding site and is engaged in a favorable hydrophobic contact with the benzene ring of the Trp 86 side chain.
with the oxime oxygen atom placed within a reasonable
distance from the phosphorus atom of the covalently bound
paraoxon for the transesterification process. To aid in
repositioning the 2-PAM orientation (i.e., versus the
orientation observed in cocrystal 5HFA), a second X-ray
structure, PDB entry code 2HA0, was used as a guide. This
structure contains ketoamyltrimethylammonium (KTA), an
acetylcholine mimetic, which forms a hemiketal bond with Ser-
203.32 Hence, it provided a good approximation and distance
required for an SN2 attack by 2-PAM on the paraoxon
phosphorus. Therefore, the 5HFA and 2HA0 X-ray structures
were superimposed (Supporting Information Figure S7). Next,
we manually repositioned 2-PAM, via translational and
rotational adjustments, over the three-dimensional coordinates
of KTA, taking special care to position the oxime oxygen atom
of 2-PAM directly over the KTA methyl group covalently
bound to the hydroxy group of Ser-203 and the nitrogen atom
of the 2-PAM pyridinium ring over the KTA ammonium
nitrogen (Figure 4a). Following this alignment, the entire
2HA0 X-ray structure (including KTA) was completely
removed, and iterative rounds of energy refinement, atom−
atom contact scoring, and manual adjustments (to remove van
der Waals violations) were performed to optimize the 2-PAM
binding position in the PDB entry 5HFA structure (see the SI
for method specifics). The energy refined binding mode for 2-
PAM is shown in Figure 4b. With respect to the more
applicable, pre-AChE rescue 2-PAM binding mode, three
features are notable: First, the pyridinium nitrogen is engaged
in a cation−π interaction with the Trp-86 side chain indole in a
location that is comparable to that of the ammonium ion of
bound KTA (i.e., the positive charge of 2-PAM is well
positioned in the aromatic box motif of the binding site
(Figures 4a and 4b)). Second, the pyridinium ring is engaged
in favorable edge-to-face stacking with Trp-86. Third, the
oxime oxygen is properly oriented and within a reasonable
distance (2.3 Å) for SN2 attack on the paraoxon phosphorus
that is covalently bound to Ser-203.
Figure 5. Summary of methyl scan study.
Our revised 2-PAM binding mode was iteratively methylated
on the 3-, 4-, 5-, and 6-positions of the drug’s pyridinium. Each
derivative was energetically refined in the binding site (see the
SI for method specifics and SI Table S3 for quantitative, final
HINT scores for active analogs 4-Me-2-PAM, 6-Me-2-PAM,
and 2-PAM as a control for comparison). Subsequently, the
optimized 2-PAM binding mode and those of the methyl scan
analogs were compared to rationalize the reactivation data
shown in Figure 3. Figure 4c shows a proposed binding mode
of the inactive 3-Me-2 PAM derivative. Conformational
analysis indicates that the 3-position methyl forces the oxime
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moiety of this analog out of the pyridinium plane. This twisted
conformation significantly perturbs the derivative’s ability to
achieve an orientation and distance that properly positions the
oxime oxygen near the phosphorus atom of Ser203-bound
paraoxon. When the oxime is oriented as observed for the
optimized 2-PAM model (Figure 4b), the 3-methyl substituent
experiences a highly unfavorable hydrophobic-polar clash with
the hydroxy group of Tyr-124. Subsequently, if the pyridinium
ring of 3-Me-2-PAM is rotated to moderate this highly
unfavorable residue interaction, the 3-methyl is engaged in an
unfavorable hydrophobic-polar and steric clash with the side
chain of Tyr-337. Finally, when attempting to ameliorate both
of these steric/electrostatic incompatibilities, the position of
the 3-methyl-2-PAM oxime moiety cannot orient properly to
perform the SN2 attack required to liberate Ser 203 from
covalently bound paraoxon. Hence, due to its conformation,
the 3-Me-2-PAM derivative is trapped in a negative feedback
cycle that obviates its ability to act as an AChE rescue agent.
In contrast, the binding mode of 4-Me-2-PAM (Figure 4d)
is promising for the incorporation of more structurally diverse
substituents to improve 2-PAM binding with AChE and/or to
increase BBB permeability. Specifically, the model indicates
that the 4-methyl substituent points into sterically unhindered
coordinate space that is mainly surrounded by aromatic
residues of the enzyme’s binding gore. Hence, our computa-
tional analysis shows that this position on the 2-PAM
pyridinium ring is optimal for further derivatizations.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00586.
Synthetic and biological experimental procedures,
computational methods, supplementary figures, and
NMR spectra (PDF)
AUTHOR INFORMATION
■
Corresponding Author
Kazunori Koide − University of Pittsburgh, Pittsburgh,
Pennsylvania; orcid.org/0000-0001-8894-8485;
Email: koide@pitt.edu
Other Authors
Adriana Gambino − University of Pittsburgh, Pittsburgh,
Pennsylvania
James C. Burnett − University of Pittsburgh, Pittsburgh,
Pennsylvania
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.9b00586
Funding
This work was financially supported by the US Defense Threat
Reduction Agency (HDTRA1-16-1-0041).
Notes
The proposed binding mode of less potent 5-Me-2-PAM
indicates that the 5-methyl group would experience a
moderately unfavorable hydrophobic-polar clash with the
side chain of Tyr-337 (Figure 4e). In contrast, the binding
mode of 6-Me-2-PAM (Figure 4f) suggests that the 6-methyl
group points into a desolvated subsite and is engaged in
favorable hydrophobic contacts with the benzene component
of the Trp-86 side chain. However, this subsite is sterically
limited; therefore, when this derivative binds, Trp-86, which is
located in a loop composed of residues Met-85 to Arg-90, is
forced to slightly reorient to accommodate the bulk of the
methyl group. Consequently, due to this structural adjustment,
the binding affinity of 6-Me-2-PAM is less favorable than that
of 2-PAM. Finally, as observed in Figure 4f, the pocket for this
methyl position is in the interior of the acetylcholine binding
site, and substituents larger than a methyl group will result in
unfavorable steric clashes with surrounding residues.
In conclusion, a systematic methyl scanning study on 2-
PAM was conducted for the first time, because although the 4-
position appeared to be the most common position for the
derivatization of 2-PAM, there was no convincing study to
validate such an approach. This study revealed that the 4- and
6-positions can tolerate methylation, while the 3-position
cannot. 5-Me-2-PAM showed reduced reactivation, indicating
that this is not an ideal position for parent compound
derivatization to develop more potent antidotes. The
reactivation efficiency of 5- and 6-Me-2-PAM slightly differed
between eel and hAChE. Varying reactivation profiles have
previously been observed across species.15 A pictorial summary
of the data is shown in Figure 5. Finally, computational
analyses rationalized the assay results and indicated that the 4-
position of 2-PAM provides the most promising location for
future efforts to synthesize new derivatives to improve either
binding affinity, AChE reactivation, and/or BBB permeability.
The authors declare no competing financial interest.
ABBREVIATIONS
■
OPNAs,organophosphorus nerve agents; AChE,acetylcholines-
terase; 2-PAM,2-pralidoxime; SAR,structure activity relation-
ship; BBB,blood−brain barrier; KTA,ketoamyltrimethylammo-
nium; PX,paraoxon
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