Novel pyridinium oximes: Synthesis, molecular docking and in vitro reactivation studies

Article abstract of DOI:10.1039/c4ra14696d

A computational approach has been attempted for the screening of 4-pyridoxinium (4P) ring based reactivators for paraoxon inhibited AChE. The oxime molecules were designed with the common 4P skeleton and varying the carbon linkers. Initially, the AChE binding capability was assessed by molecular docking with PDB:2WHP and 3ZLV, which showed important interactions with Ser298, Try124 and Trp286. These computational results were validated by an in vitro AChE binding assay, which showed binding affinities in the range of 10-90%. Finally, reactivation potency was calculated as % reactivation on paraoxon inhibited eelAChE in the concentration range of 10^-5 to 10^-7 M. It was observed that introduction of an aliphatic linker attached to 4-pyridoxime has a high binding affinity and hence, may act as a good reactivator as compared to the aromatic pyridoximes.

Full text of DOI:10.1039/c4ra14696d

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PAPER
Novel pyridinium oximes: synthesis, molecular
docking and in vitro reactivation studies†
Cite this: RSC Adv., 2015, 5, 23471
Pooja,ab S. Aggarwal,ab A. K. Tiwari, V. Kumar,a R. Pratap,b G. Singhb
a
*
a
*
and A. K. Mishra
A computational approach has been attempted for the screening of 4-pyridoxinium (4P) ring based
reactivators for paraoxon inhibited AChE. The oxime molecules were designed with the common 4P
skeleton and varying the carbon linkers. Initially, the AChE binding capability was assessed by molecular
docking with PDB:2WHP and 3ZLV, which showed important interactions with Ser298, Try124 and
Trp286. These computational results were validated by an in vitro AChE binding assay, which showed
binding affinities in the range of 10–90%. Finally, reactivation potency was calculated as % reactivation
on paraoxon inhibited eelAChE in the concentration range of 10ꢀ5 to 10ꢀ7 M. It was observed that
introduction of an aliphatic linker attached to 4-pyridoxime has a high binding affinity and hence, may
act as a good reactivator as compared to the aromatic pyridoximes.
Received 17th November 2014
Accepted 24th February 2015
DOI: 10.1039/c4ra14696d
www.rsc.org/advances
compared to 2 and 3 pyridoximes. It was also observed that the
positive charge on the nitrogen of the pyridoxime ring makes it
1. Introduction
more potent for reactivation but it increases the hydrophilicity
of the oxime moiety making it less lipophilic to cross the blood–
brain barrier (BBB), which is the most important lipoidal
membrane. Therefore, various modications have been done
and different non-ionic oxime derivatives were reported in the
literature, but it was found that they have less reactivation
potency, as compared to ionic oximes. This implies that the
positive charge on pyridoxime ring is important. Furthermore,
carbon chain linker was introduced at the –N position of pyr-
idoxime derivatives to give lipophilicity to the molecule for
Acetylcholinesterase (AChE) is a very important enzyme for the
hydrolysis of neurotransmitter acetylcholine at the cholinergic
synaptic site. It has two binding sites, rst is the active site
known as the A-site and second is a peripheral anionic site
known as the PAS or P-site. Hydrolysis of acetylcholine takes
place via hydrolyzing the serine residue by the active site of the
AChE enzyme (Scheme 1).1–6 The major inhibitors of AChE are
nerve gas agents, which include different classes of organo-
phosphorus (OP) compounds which bind with the –OH of the
serine residue and block the action of AChE towards the
hydrolysis of ACh into choline and acetic acid.7–9 This blockage
by these OP agents is responsible for the high level of ACh at the
cholinergic sites leading to failure of the neurotransmission
process and can cause various neurological diseases of the CNS.
The most well known OP agents are sarin, soman, tabun, VX
and paraxon (Fig. 1). To inhibit the action of OP agents and to
reactivate OP-inhibited AChE, various pyridoxime derivatives
(Fig. 2) have been synthesized and reported in the literature e.g.,
2-PAM, HI-6 and obidoxime.10–13 Pyridoxime derivatives have
substitution (hydroxoiminomethyl) at their 2, 3 or 4 positions.
They may interact at the active site and break the covalent bond
formed between the OP agents and –OH of the serine residue of
OP-inhibited AChE. 4-Pyridoxime derivatives were observed to
have high potency to act as reactivators of OP-inhibited AChE as
aDivision of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear
Medicine and Allied Sciences, Brig. S. K. Mazumdar Road, Delhi-110054, India.
E-mail: anjani7797@rediffmail.com; akmishra63@gmail.com
bDepartment of Chemistry, University of Delhi, Delhi-110054, India
† Electronic supplementary information (ESI) available: ESI contains associated
gures and tables. See DOI: 10.1039/c4ra14696d
Scheme 1 Mechanism of AChE inhibition by OP agents.
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Fig. 1 Organophosphorus (OP) as nerve agents.
Fig. 2 Representative pyridoxime derivatives as AChE reactivators.
enhancing its permeability. The studied chain with the 4-pyr- explored species in preliminary studies. Though other studies
idoxime are alkyl, alkoxyl, and aromatic carbon chains.14,15 It have been also performed previously, to show variation in the
was found that as the carbon chain length increases, lip- binding and reactivation potency of oximes with different
ophilicity and permeability to cross BBB increases and % species.17–19
reactivation towards the OP-inhibited AChE also increases. The
4-pyridoxime derivatives have higher lipophilicity when having aliphatic and aromatic carbon chain linker attached at
compared with 2-PAM, e.g., 30% for 4-octyl-PAM.1
the nitrogen (–N) of the pyridoxime moiety. These derivatives
Here, we report the synthesis of six 4-pyridoxime derivatives
These oximes have been studied on the species like were selected on the basis of preliminary computational studies
rodents, which are similar to human.16 We used eelAChE due and comparison with the previously synthesized oxime deriva-
to the easy and readily availability, which is one of the most tives. Finally, in vitro AChE binding and reactivation study was
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performed to see the efficacy of these molecules for further phenyl acetic acid, 2-bromomethylnaphthalene, N-(2-bro-
applications.
moethyl) phthalimide, 2-bromoacetophenone, paraoxon, eel
acetylcholinesterase(eelAChE)enzyme, acetylthiocholineiodide,
(DTNB), purchased from Sigma-Aldrich Co, USA. Thin layer
chromatography on aluminum plates coated with silica gel 1160,
2. Experimental
2.1 Chemicals
F254, CHCl3, acetone, isopropanol, sodium monophosphate, and
4-Pyridine carboxaldehyde, hydroxylamine hydrochloride,
1,3-dibromo propane, 1-bromo butyrl chloride, 4-bromomethyl
sodium diphosphate purchased from Merck, Germany.
Six different oximes were synthesized on the basis of
preliminary computational prediction which are presented in
Fig. 3. The synthetic schemes are shown as Scheme 2 and 3.
2.2 Synthesis of pyridine-4-carbaldehyde oxime (4P)
A solution of hydroxylamine hydrochloride (9.34 mmol, 650 mg)
in water was stirred on a magnetic stirrer. The pH of the reac-
tion mixture was maintained at 6–7 throughout the reaction by
using 1 M NaOH solution.20 Aqueous solution of 4-pyridine
carbaldehyde (4.67 mmol, 0.44 mL) was added dropwise.
ꢁ
Reaction mixture was kept on heating at 50 C for 12 h. Aer
that solvent was evaporated on rota evaporator and nal
product was dried under vacuum.
2.3 Synthesis of 1-(3-bromo-propyl)-4-(hydroxyimino-
methyl)-pyridinium (4PBP)
Pyridoxinium ion was synthesized by using 4-pyridoxime
(2.03 mmol, 250 mg) in dried CHCl3 and 1,3-dibromo propane
(10.15 mmol, 1.18 mL) in CHCl3.21 This reaction mixture was
stirred rst at room temperature for 10 min aer that allowed at
reux condition of temperature 60 ꢁC for 72 h. White precipitate
was appeared in the reaction mixture, which was ltered and
dried under vacuum.
2.4 Synthesis of 1-(4-bromo-butyryl)-4-(hydroxyimino-
methyl)-pyridinium (4PBB)
4PBB was synthesised similar to the procedure for 4PBP by
adding 4-bromo butyryl chloride (2.23 mmol, 0.28 mL) in CHCl3
in place of 1,3-dibromo propane. Reaction mixture was stirred
at room temperature for 1 h aer that white precipitates
appeared in the reaction mixture. It was ltered and solvent was
evaporated under vacuum on rota evaporator.
Fig. 3 Structures of newly developed oxime reactivators. #[a] 4OA,
4ON, 4OPh [b] 4PBP, 4PBB, 4PCB.
2.5 Synthesis of 1-(4-carboxymethyl-benzyl)-4-
(hydroxyimino-methyl)-pyridinium (4PCB)
4PCB was synthesised by the reaction between 4-pyridoxime
(2.03 mmol, 250 mg) in dried acetone and 4-(bromo methyl)
phenyl acetic acid (3.65 mmol, 1.680 g).22 Reaction mixture was
stirred at reuxing for 28 h aer that precipitates appeared in
the reaction mixture. It was ltered and solvent was evaporated
under vacuum on rota evaporator.
2.6 Synthesis of 4-(hydroxyimino-methyl)-1-(2-oxo-2-phenyl-
ethyl)-pyridinium (4OA)
In a two neck round bottom ask, reaction of 4-pyridoxime
(1.00 mmol, 124 mg) with 2-bromoacetophenone (2.50 mmol,
497.6 mg) was added in dry acetone. Reux conditions were
Scheme 2 Synthetic scheme for monoquaternary aliphatic linker
oximes.
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Scheme 3 Synthetic scheme for monoquaternary aromatic linker oximes.
˚
maintained for 15 h. The progress of the reaction was checked crystallographic resolution less than or equal to 2.5 A and
using TLC.
known experimental binding data. Molecular modeling inves-
tigations were carried out using an advanced molecular docking
program GLIDE, 5.8.25 During the docking process, initially
GLIDE performs a complete systematic search of the confor-
mational, orientation and positional space of the docked ligand
eliminating unwanted conformations using scoring followed by
energy optimization. Preparation of the protein for docking
included removal of unnecessary heteroatom and solvent
coupled with addition of hydrogen atoms, bond order for crystal
2.7 Synthesis of 4-(hydroxyimino-methyl)-1-naphthalen-2-
ylmethyl-pyridinium (4ON)
Synthesis of ‘4-(hydroxyimino-methyl)-1-naphthalen-2-ylmethyl-
pyridinium’ (4ON) was carried out by a substitution reaction of
4-pyridoxime with bromo-functionalized naphthalene moiety.
4-Pyridoxime (1.00 mmol, 123.7 mg) was added in a two neck
round bottom ask and dissolved in dry acetone. Then 2-bro-
momethylnaphthalene (2.50 mmol, 552.7 mg) was added to the
reaction mixture and stirring at reux was continued for 15 h.
The TLC was monitored at regular intervals.
˚
ligand and protein were adjusted and minimized up to 0.30 A
RMSD.
2.10 Binding study with AChE
2.8 Synthesis of 1-[2-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-
ethyl]-4-(hydroxyimino-methyl)-pyridinium (4OPh)
Mixture of 750 mL enzyme (eelAChE, 0.1 mg in 20 mL sodium
phosphate buffer of pH 7.4), 750 mL acetylthiocholine iodide as
a substrate (550 mM) and 750 mL oxime solution in the range of
concentration 1 mM to 1 nM was incubated for 12 h at 25 ꢁC. A
parallel experiment was running in which 750 mL buffer was
used in place of test compound in the enzyme solution as a
positive control. In each concentration sample 750 mL DTNB
solution (80 mM, prepared in phosphate buffer of pH 7.4) was
added and absorbance was recorded at 412 nm at every 5 min
for 30 min.26,27
To a solution of 4-pyridoxime (1.00 mmol, 123.7 mg) in dry
acetone, N-(2-bromoethyl) phthalimide (2.50 mmol, 635.2 mg)
was added. The reaction mixture was kept at stirring under
reux conditions for 15 h and the completion of the reaction
was checked by TLC.
2.9 Computational approach for AChE interaction
The X-ray crystal structure of sarin phosphonylated (PDB entry
Enzyme activity was calculated by using the formula:
23
˚
2WHP, having resolution of 2.20 A) and tabun complexed
Ai
acetylcholinesterase (PDB entry 3ZLV, having resolution of
% AChE activity ¼
ꢂ 100
Ao
2.50 A)24 were used for molecular docking studies. The following
˚
criteria were kept in mind before choosing the protein–ligand where, Ao and Ai are the absorbance for the positive control and
complexes: non-covalent binding between protein and ligand, test compound respectively.
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propane as shown in Scheme 2. The product was obtained as
brown precipitate with moderate yield of 60.34% and its struc-
2.11 Reactivation study towards paraoxon inhibited AChE
To study the reactivation on inhibited AChE, the foremost
necessity was to know about the inhibited enzyme activity. So,
30 mL enzyme was incubated with 10 mL paraoxon (10ꢀ2 to 10ꢀ5
M) for 15 min to achieve >95% inhibition. Thereaer, a mixture
of 40 mL inhibited enzyme, 40 mL acetylthiocholine iodide and
40 mL PBS buffer was incubated for 15 min. Then, absorbance
(Ai) was measured at 412 nm by adding 40 mL DTNB at different
time intervals, 0 min, 15 min, 30 min, 45 min and 60 min.
For reactivation study, a mixture of 40 mL inhibited enzyme,
40 mL acetylthiocholine iodide and 40 mL test oxime (10ꢀ3 to
10ꢀ8 M) was incubated for 15 min and absorbance (Ar) was
recorded at 412 nm by adding 40 mL DTNB at different time
interval. A parallel positive control experiment was done by
taking 40 mL enzyme and 40 mL buffer in place of inhibited
enzyme and test oxime respectively.1,15,26
1
ture was conrmed by H NMR, 13C NMR and mass spectros-
copy (shown as S2†).
1H NMR (400 MHz, D2O): d ¼ 8.75 (d, 2H, ArH), 8.66 (s, 1H,
ArH), 8.25 (d, 2H, ArH), 4.65 (s, D2O), 3.43 (t, 2H, –CH2), 2.66
(t, 2H, –CH2), 2.14 (m, 2H, –CH2), 13C NMR (100 MHz, D2O): d ¼
149.19 (–CH]NOH), 146.23 (ArC), 144.62 (ArC), 125.13 (ArC),
57.70, 32.61, 31.58, m/z (ESI-LCMS): calculated mass [M+] ¼
243.01, [M + H+] ¼ 244.9, with M: (M + 2) : 1 : 1 bromo isotopic
pattern.
3.3 Synthesis of 4PBB
N-Alkylation reaction between 4-pyridoxime and 4-bromo
butyryl chloride yields N-(4-bromo-butyryl)-4-pyridoxime with
59.24% as shown in Scheme 2. Its structure was conrmed by
1H NMR, 13C NMR and mass spectroscopy (shown as S3†).
1H NMR (400 MHz, D2O): d ¼ 8.74 (d, 2H, ArH), 8.66 (s, 1H,
ArH), 8.59 (d, 2H, ArH), 4.65 (s, D2O), 3.42 (t, 2H, –CH2), 2.66 (t,
2H, –CH2), 2.41 (m, 2H, –CH2), 13C NMR (100 MHz, D2O): d ¼
183.22 (–C]O), 146.78 (–CH]NOH), 141.49 (ArC), 130.47 (ArC),
124.17 (ArC), 30.33, 27.94, 26.48, m/z (ESI-LCMS): calculated
mass [M+] ¼ 271, [M + H+] ¼ 271.3, with M: (M + 2) : 1 : 1 bromo
isotopic pattern.
% AChE reactivation was calculated by using the formula:
% AChE reactivation ¼ (Ar ꢀ Ai/Ao ꢀ Ai) ꢂ 100
where, Ar, Ai and Ao are the absorbance of test oxime, inhibited
enzyme and positive control found experimentally.
2.12 Fluorometric assay with human serum albumin (HSA)
Binding constant of the oximes with HSA was calculated by the
uorometric experiments. The uorescence intensity was recor-
ded at lem ¼ 350 nm at the xed lex ¼ 280 nm for the different
concentration of oximes from 4 ꢂ 10ꢀ4 to 10ꢀ5 M in PBS of pH ¼
7.4 at 25 ꢁC while the concentration of HSA was xed at 2 ꢂ 10ꢀ5
M. The change in intensity at 350 nm (tryptophan) was used to
calculate the binding constant (K).28,29 The binding constant was
calculated by the Stern–Volmer equation:30
3.4 Synthesis of 4PCB
N-Alkylation between 4-pyridoxime and 4-bromomethyl phenyl
acetic acid gives N-(4-carboxymethyl-benzyl)-4-pyridoxime in the
form of yellow colored compound as shown in Scheme 2.
78.45% yield was obtained and structure conrmation was done
by 1H NMR, 13C NMR and mass spectroscopy (shown as S4†).
1H NMR (400 MHz, D2O): d ¼ 8.70 (d, 2H, ArH), 8.19 (s, 1H,
–CH), 8.01 (d, 2H, ArH), 7.22–7.31 (dd, 4H, ArH), 5.62 (s, 2H,
–CH2), 4.65 (s, D2O peak), 3.59 (s, 2H, –CH2); 13C NMR (100
MHz, D2O): d ¼ 176.63 (–COOH), 148.93 (–CH]NOH), 146.22,
144.48, 136.23, 131.58, 130.65, 129.48, 124.91 (ArC's), 63.86,
40.39, m/z (ESI-LCMS): calculated mass [M+] ¼ 271.11, [M+] ¼
271.3, [M + Na+] ¼ 294.
Fo
¼ 1 þ Ksv½Qꢃ
F
where Fo and F denotes the uorescence intensity in the
absence and presence of quencher pyridoxime, respectively. Ksv
is the Stern–Volmer quenching constant (or binding constant)
and [Q] is the concentration of quencher.
3. Result and discussion
3.1 Synthesis of 4P
3.5 Synthesis of 4OA
4-(Hydroxyimino-methyl)-1-(2-oxo-2-phenyl-ethyl)-pyridinium was
synthesized by the N-alkylation reaction between 4-pyridoxime
and 2-bromoacetophenone as shown in Scheme 3. The product
was obtained by evaporating solvent as a white powder in
moderate yield of 72.01% and its structure has been conrmed
by 1H NMR, 13C NMR and mass spectroscopy (shown as S5†).
1H NMR (CDCl3, 400 MHz): d values (ppm) ¼ 4.19 (s, 2H,
CH2), 7.51–7.69 (m, 3H, ArH), 7.94–7.96 (d, 2H, ArH), 8.04–8.07
(m, 1H, ArH) 8.25 (s, 1H, CH]N), 8.69–8.71 (d, 2H, ArH), 8.88
(s, 1H, ArH). 13C NMR (CDCl3, 100 MHz): d values (ppm) ¼ 60.4
(CH2), 114.6 (ArCH), 128.1 (ArCH), 128.5 (ArCH), 129.5 (ArCH),
132.7 (ArCH), 133.6 (ArCH), 135.8 (ArCH), 143.3 (ArCH), 143.9
4-Pyridoxime was synthesized by the hydrolysis reaction
between 4-pyridine carbaldehyde and hydroxylamine as shown
in Scheme 2. The product was obtained in high yield of 97.17%
1
by evaporating solvent and its structure was conrmed by H
NMR, 13C NMR and mass spectroscopy (shown as S1†).
1H NMR (400 MHz, DMSO): d ¼ 7.5 (d, 2H, ArH), 8.1 (d, 1H,
–CH proton), 8.5 (d, 2H, ArH), 13C NMR (100 MHz, DMSO): d ¼
120.94 (ArC), 141.05 (ArC), 146.73 (ArC), 150.68 (–CH]NOH), m/z
(ESI-LCMS): calculated mass [M+] ¼ 122.05, [M + H+] ¼ 123.4.
3.2 Synthesis of 4PBP
N-(3-Bromo-propyl)-4-pyridoxime was synthesized by the (ArC), 144.6 (ArC), 145.8 (CH), 192.1 (C]O). ESI-MS: m/z ¼
N-alkylation reaction between 4-pyridoxime and 1,3-dibromo 241.2 [M]+.
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breakage was previously formed by OP agents. Oximes showed
binding with Ser298 of AChE by hydroxyimino moiety via
hydrogen bonding interaction, except for aromatic linker
attached oximes (4OA, 4ON and 4OPh). In 4PBP and 4PBB extra
interactions were observed including p–p interaction with the
3.6 Synthesis of 4ON
4-(Hydroxyimino-methyl)-1-naphthalen-2-ylmethyl-pyridinium
was synthesized by the N-alkylation reaction between 4-pyr-
idoxime and 2-bromomethylnaphthalene as shown in Scheme 3.
The product was obtained by evaporating solvent as a pale yellow
powder in moderate yield of 65.12% and its structure has been
conrmed by 1H NMR, 13C NMR and mass spectroscopy
(shown as S6†).
1H NMR (CDCl3, 400 MHz): d values (ppm) ¼ 2.91 (s, 2H,
CH2), 7.27–7.7.81 (m, 7H, ArH), 8.17–8.19 (d, 2H, ArH), 8.45 (s,
1H, CH]N), 8.91–8.93 (d, 2H, ArH). 13C NMR (CDCl3, 100 MHz):
d values (ppm) ¼ 64.3 (CH2), 124.7 (ArCH), 125.5 (ArCH), 127.2
(ArCH), 127.5 (ArCH), 127.7 (ArCH), 128.1 (ArCH), 128.9 (ArCH),
129.5 (ArCH), 130.2 (ArCH), 132.8 (ArC), 133.1 (ArC), 144.3 (ArC),
146.0 (ArC), 148.7 (CH). ESI-MS: m/z ¼ 263.3 [M]+.
3.7 Synthesis of 4OPh
1-[2-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-ethyl]-4-(hydroxyimino-
methyl)-pyridinium (1 g) was synthesized by the N-alkylation
reaction between 4-pyridoxime and N-(2-bromoethyl)phthali-
mide as shown in Scheme 3. The product was obtained by
evaporating solvent as a white powder in moderate yield of
1
61.34% and its structure has been conrmed by H NMR, 13C
NMR and mass spectroscopy (shown as S7†).
1H NMR (CDCl3, 400 MHz): d values (ppm) ¼ 4.18–4.21 (t, 2H,
CH2), 4.76–4.79 (t, 2H, CH2), 7.71–7.75 (m, 4H, ArH), 8.04–8.06 (d,
2H, ArH), 8.24 (s, 1H, CH]N), 8.75–8.77 (d, 2H, ArH). 13C NMR
(CDCl3, 100 MHz): d values (ppm) ¼ 38.3 (CH2), 59.6 (CH2), 123.7
(ArCH), 124.9 (ArCH), 130.7 (ArCH), 135.1 (ArCH), 144.9 (ArC),
146.0 (ArC), 149.4 (CH), 169.7 (C]O). ESI-MS: m/z ¼ 296.1 [M]+.
3.8 Computational approach for AChE interaction
Molecular modeling studies were performed on synthesized
oxime molecules 4PBP, 4PBB, 4PCB, 4OA, 4ON and 4OPh for the
justication of their interaction with OP-inhibited hAChE. It is
important for a reactivator of AChE to bind with the enzyme
which was revealed in the form of Glide Gscore. Binding affinity
of synthesized 4-pyridoximes varies by changing the alkyl chain
substituted to the pyridinium nitrogen of the ring. It was
observed that the Glide Gscore for aliphatic chain alkyl group
attached to pyridoximes are high (ꢀ9.920, ꢀ11.021 for 4PBP and
ꢀ10.793, ꢀ10.902 for 4PBB) as compared to the aromatic
(ꢀ9.951, ꢀ9.760 for 4PCB, ꢀ10.250, ꢀ8.927 for 4OA, ꢀ8.639,
ꢀ8.261 for 4ON and ꢀ9.202, ꢀ8.244 for 4OPh) with both the
PDB's 2WHP and 3ZLV (detail shown in S8†). This variation in
Glide Gscores is due to difference in the type of linkers in each
oxime which affect the binding with various amino acid resi-
dues. These synthesized oximes showed binding with Trp286
and Tyr124 via p–p interaction with the aromatic moiety of
amino acid and pyridinium ring, which is an important inter-
action for oximes to reactivate nerve gas inhibited AChE.26
These amino acid residues are present at peripheral anionic
binding site of enzyme AChE. It is known that attack of OP on
the –OH of serine residue present at the esteric site is important
Fig. 4 2D interaction diagrams for synthesized oximes with 2WHP (a)
for any reactivator to bind with the serine residue and its bond 4PBB (b) 4OA.
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Fig. 5 2D interaction diagrams for synthesized oximes with 3ZLV (a) 4PBP (b) 4PBB.
amino acid residue Tyr72. While, aromatic linker attached
pyridoximes showed less interaction as compared to aliphatic
linker due to rigidity in the structure. The 2-D diagrams of
oximes with PDB 2WHP and 3ZLV were shown in Fig. 4 and 5,
respectively. These results were compared with the well known
AChE reactivator 2-PAM (2D interaction shown in Fig. 6). The
reliability of the docking program Glide used in this study was
veried evaluating its performances in reproducing the geom-
etry of bound inhibitors taken from cocrystallized complexes
(2WHP and 3ZLV). The docking calculation reproduces the
actual orientation of these molecules as shown in S9.† This was
further validated by MD simulation and MMGBSA analysis.
Furthermore, to validate this computational binding there in
vitro binding and reactivation potency were studied towards
OP-inhibited AChE.
MD simulations. MD simulation was performed as per our
previous work on same pdb.31 Model system was placed inside
orthorhombic box containing explicit water molecules modeled
as TIP3P. Three sodium ions (Na+) ions were added to neutralize
the whole system which was relaxed before simulation and
energy of the system is minimized by force eld OPLS2005. This
is followed by submission of a system containing 55 394 atoms,
to MD simulation which was subsequently carried out by using
following procedure: rst, default 1.2 ns run of MD simulation
at constant temperature of 300 K and with all atoms having
positions restrained except for the water molecules inside the
box under the NPT ensemble. This initial MD was necessary in
order to allow the equilibration of the solvent molecules around
the protein. Another production run was carried out which
included: full MD simulations of 5 ns at 300 K with no restric-
tions with 2 fs of integration time. Aer completion, simula-
tions quality and event analysis was done.
MM-GBSA analysis. The MM-GBSA analysis was used to
calculate ligand binding energies based on docking complex, by
¨
using the MM-GBSA technology available in Schrodinger's tool
¨
Prime (Schrodinger, LLC, New York, NY, 2013). This analysis
was done by including poser viewer le of glide docked
complexes and by assuming the default parameters in the
panel. The binding energy is calculated according to the
equation:
DGbind ¼ DEMM + DGsolv + DGSA
where the term DEMM comes from difference between mini-
mized energies of protein–oxime complexes and individual
minimized energies of protein and oxime. DGsolv is a difference
between GBSA energies of complexed protein–oxime and unli-
ganded 2-protein and oxime. DGSA is the difference in surface
area energies for the complex and the sum of the surface area
Fig. 6 2D interaction diagrams for known AChE reactivator 2-PAM
with (a) 2WHP (b) 3ZLV.
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energies for the protein and oxime. The Prime's contribution of
DGSA to the DGbind comes from the non-polar contribution to
the solvation energy due to the surface area.
MMGBSA energy prole with 3ZLV and 2WHP are shown in
S10 and S11† along with 3D interactive poses in S12† and
simulation interactions for the NPT ensemble 4PBB-2WHP
presented in S13† for 5 ns.
3.9 Binding study with AChE
An oxime can only reactivate AChE if it goes to the site of OP-
inhibited AChE and bind to inhibit it. To get more insight of
the reactivation of inhibited AChE, binding affinity of the
synthesized oximes was evaluated with eelAChE by measuring
their inhibitory activity using Ellman method. Binding experi-
ment was performed in which eelAChE was incubated with
different concentrations (10ꢀ3 to 10ꢀ9 M) of oximes for 12 h to
achieve the maximum inhibition. Binding affinity was also
measured for the precursor 4-pyridoxime to compare. The in
vitro data is shown in Fig. 7. It was observed that all the
synthesized oximes showed change in binding affinity by
changing the concentration. Out of six synthesized oximes,
4PBB and 4PCB showed highest binding affinity. At the
concentrations 10ꢀ9 to 10ꢀ7 M, 4PBP have less inhibitory
activity as compared to 4PBB and 4PCB. But at the concentra-
tions 10ꢀ6, 10ꢀ4 and 10ꢀ3 M it showed inhibitory activity of 1%,
1% and 50% for 4PBP, 15%, 80% and 95% for 4PBB, 40%, 92%
and 60% for 4PCB, 36%, 29% and 74% for 4OA, 44%, 45% and
52% for 4ON, 30%, 87% and 89% for 4OPh and 10%, 40% and
60% for precursor 4P respectively. It was also observed that at
the biological concentration of 10ꢀ9, 10ꢀ8 and 10ꢀ7 M these
oximes do not show much efficient inhibitory activity as
compared to the concentration of 10ꢀ6, 10ꢀ4 and 10ꢀ3 M. They
showed optimum inhibitory activity at the concentration range
of 10ꢀ4 M.
Fig. 7 Binding affinity of eelAChE with oximes and 2-PAM at varying
concentration. Binding experiment was performed in eelAChE, which
was incubated with different concentrations (10ꢀ3 to 10ꢀ9 M) of
oximes for 12 h to achieve the maximum inhibition. Binding affinity was
also measured for the precursor 4-pyridoxime for the comparison
purpose and positive control was taken by replacing oxime with buffer.
81.82% for 4PBB, ꢀ32.73%, 32.73%, 21.82% for 4PCB,
ꢀ0.04%, ꢀ0.19%, ꢀ0.03% for 4OA, 3.00%, 5.00%, ꢀ1.00% for
4ON and ꢀ0.04%, 0.02%, ꢀ0.02% for 4OPh at the concen-
tration of 10ꢀ5 M, 10ꢀ6 M and 10ꢀ7 M respectively. These
results were also compared with the precursor 4P which
showed reactivation potency 56.36%, 49.09%, 56.36% for the
same concentration range as for the products. Reactivation
potency was evaluated for the known reactivator 2-PAM which
showed 16.39%, 9.18%, 2.30% reactivation at the concentra-
tion 10ꢀ5 M, 10ꢀ6 M and 10ꢀ7 M respectively. For the aromatic
ring bearing pyridoximes, reactivation was also studied at high
concentration of 10ꢀ3 M and 10ꢀ4 M. It was found that these
oximes able to reactivate paraoxon inhibited AChE at high
3.10 Reactivation study towards paraoxon inhibited AChE
To validate the binding affinity of synthesized pyridoximes with
AChE, in vitro reactivation experiment was demonstrated with
the paraoxon inhibited eel-AChE.10 Enzyme was incubated with
the nerve gas paraoxon about 30 min to achieve maximum
inhibition. Then enzyme activity was determined by performing
well known Ellman assay27 using DTNB as a coloring agent. This
assay was performed on the synthesized pyridoximes (4PBP,
4PBB, 4PCB, 4OA, 4ON and 4OPh) as well as with the synthe-
sized precursor 4P. These reactivation potencies were also
compared with the currently used AChE reactivator 2-PAM. The
results of reactivation experiment were shown in Fig. 8, which
showed reactivation
pyridoximes.
% by varying the concentration of
This experiment showed that the reactivation potency of
the oximes varied by changing the linker at the pyridinium
nitrogen of the ring. It was observed that the reactivation
potency for the synthesized oximes 4PBP and 4PBB were high
as compared to the aromatic ring bearing pyridoximes (4PCB,
4OA, 4ON and 4OPh). Results showed 61.82%, 89.09%,
Fig. 8 Reactivation of paraoxon inhibited eelAChE by oximes and
2-PAM at different concentrations. Reactivation efficacy (%) of the
oximes in comparison to 2-PAM at 60 min against paraoxon inhibited
eelAChE (three concentration of oximes, 0.1 M phosphate buffer, pH
76.36% reactivation potency for 4PBP, 76.36%, 65.45%, 7.4 and temperature 37 ꢁC).
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group was at 2nd position of the pyridinium ring while it was
at 4th position in our synthesized oximes. Synthesized oxime
precursor 4P is also more effective than the 2-PAM at low
concentrations.
Reactivation potency for 2-PAM decreased as the concentra-
tion decreases which showed that they are good reactivators at
the high concentration but not at the low concentration. Both
the aliphatic chain linked with pyridoximes, 4PBP and 4PBB
showed similar reactivation potencies, but if observed at each
concentration then it was found that 4PBB have good reac-
tivation potency (81.82%) at the concentration 10ꢀ7 M whereas
4PBP showed excellent reactivation potency (89.09%) at 10ꢀ6 M.
3.11 HSA binding assay
Interaction of ligand with HSA was analyzed by in vitro uoro-
metric assay. HSA shows emission because of aromatic amino
acids tryptophan, tyrosine and phenylalanine residues present
in it. Quenching of uorescence intensity of tyrosine was
observed if it is present near an amino, a carboxyl group or a
tryptophan, and phenylalanine with very low quantum yield.
Small molecules bound to HSA tend to change in intrinsic
uorescence intensity due to tryptophan residue in the oxime
system. Hence, uorescence quenching was observed by addi-
tion of oxime in the concentration range of 0.4–0.05 mM to the
xed concentration of HSA (0.02 mM) at 350 nm (Fig. 9(a–d)).
This quenching indicates the formation of ligand–HSA complex
which leads to microenvironment change in the protein and
hence showed decrease in uorescence intensity.
The binding constants of the synthesized oximes as well as
precursor 4P were calculated by Stern–Volmer plot and these
data were shown in Fig. 10. Stern–Volmer binding constants
were calculated as 1.17 ꢂ 103 Mꢀ1, 2.29 ꢂ 103 Mꢀ1, 0.90 ꢂ 103
Mꢀ1 and 0.75 ꢂ 103 Mꢀ1 for 4P, 4PBB, 4PBP and 4PCB respec-
tively. These binding constant values suggested that the newly
synthesized oximes have ability to bind with HSA for their
Fig. 9 Emission plot for ligand–HSA fluorescence quenching with
different oximes. # (a) 4P (b) 4PBP (c) 4PBB (d) 4PCB.
concentrations with the % reactivation of, 22.73%, 36.36% for
4PCB at 10ꢀ3 M and 10ꢀ4 M, 20.00%, for 4OA at 10ꢀ3 M,
49.51%, 42.95% for 4ON at 10ꢀ3 M and 10ꢀ4 M and 42.95%,
15.08% for 4OPh at 10ꢀ3 M and 10ꢀ4 M respectively. This data
stated as we move from aliphatic to aromatic linker, reac-
tivation potency decreases and also the synthesized oximes
showed high reactivation potency at low concentration range
between 10ꢀ6 to 10ꢀ7 M. Secondly, these data showed better
reactivation than known reactivator 2-PAM. In 2-PAM, oxime
Fig. 10 Stern–Volmer plot for for determination of the binding constant of the oxime–HSA.
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4. Conclusion
In this study mono-quaternary oximes were synthesized as
reactivator for paraoxon inhibited eelAChE by variation in the
carbon chain attached with the –N position of 4-pyridoxime
moiety. These oximes were studied for their binding affinity
with eelAChE. Further, this binding was validated with the
molecular modeling studies on the basis of Glide Gscore, p–p
interaction and hydrophobic interaction on the active site of
AChE. These synthesized oximes were found to be effective
reactivators towards the paraoxon inhibited eelAChE as
compared to the known reactivator 2-PAM. 4PBB and 4PBP have
high reactivation potency as compared to 4PCB. Furthermore,
they also showed drug likeliness behavior by showing effective
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Acknowledgements
We thank Dr R. P. Tripathi, Director, INMAS, DRDO for
providing the necessary facilities and CSIR for nancial assis-
tance to the rst and second authors during the course of the
research work.
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23480 | RSC Adv., 2015, 5, 23471–23480
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