In silico, in vitro and in vivo studies indicate resveratrol analogue as a potential alternative for neuroinflammatory disorders

Article abstract of DOI:10.1016/j.lfs.2020.117538

Inflammaging is known as an imbalance between pro-inflammatory and anti-inflammatory immune mechanisms, being related to the onset of neurological disorders, such as major depression and Alzheimer's disease. Considering the known disadvantages regarding the FDA approved drug to manage such illnesses, resveratrol emerges as a natural drug candidate, despite its low bioavailability. In this study, resveratrol analogues were evaluated for their capacity of inhibiting acetylcholinesterase in silico, in vitro, and in vivo. Molecular docking simulations pointed out RSVA1 and RSVA6 as potent inhibitors, even more than resveratrol. Ellman's assay demonstrated RSVA6 as capable of inhibiting 92.4% of the enzyme activity. Further, male Swiss mice were pretreated with RSVA6 (100 mg kg^?1) 60 min before receiving scopolamine (1 mg kg^?1). The Novel Recognition Object (NOR), Object Location (OLT), and Buried Pellet tests (BPL) demonstrated an RSVA6 neuroprotective effect. In the second round of tests, mice received a single intraperitoneal injection of lipopolysaccharide (0.5 mg kg^?1) 24 h before treatment with RSVA6 (1, 10, and 100 mg kg^?1). The Open Field (OFT), Tail Suspension (TST), and Splash tests (ST) were evaluated. LPS had no significant effect on the crossing and rearing number, indicating an association between the immobility time and anhedonia observed in the TST and ST, respectively, with depressive-like behavior. RSVA6 significantly reduced the depressive-like behavior triggered by LPS in the TST and ST. Altogether, our data suggest RSVA6 as a potential drug candidate for the treatment of neuroinflammatory conditions.

Full text of DOI:10.1016/j.lfs.2020.117538

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In silico, in vitro and in vivo studies indicate resveratrol analogue
as a potential alternative for neuroinflammatory disorders
Pollyana Mendonça de Assis, Amanda Fávero, Jaíne Ferrareis
Menegasso, Raissa Soares Meinel, Gabriel Macedo Marion,
Vinicius Schmitz Pereira Nunes, Priscila Vanessa Zabala Capriles
Goliatt, Adilson David da Silva, Rafael Cypriano Dutra, Nádia
Rezende Barbosa Raposo
PII:
S0024-3205(20)30286-1
https://doi.org/10.1016/j.lfs.2020.117538
LFS 117538
DOI:
Reference:
To appear in:
Life Sciences
Received date:
Revised date:
Accepted date:
16 November 2019
3 March 2020
8 March 2020
Please cite this article as: P.M. de Assis, A. Fávero, J.F. Menegasso, et al., In silico,
in vitro and in vivo studies indicate resveratrol analogue as a potential alternative for
neuroinflammatory
j.lfs.2020.117538
disorders,
Life
Sciences(2020),
https://doi.org/10.1016/
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In silico, in vitro and in vivo studies indicate resveratrol analogue as a potential alternative for
neuroinflammatory disorders
Pollyana Mendonça de Assisa, Amanda Fáveroa, Jaíne Ferrareis Menegassob, Raissa Soares Meinelc, Gabriel
Macedo Mariond, Vinicius Schmitz Pereira Nunesd, Priscila Vanessa Zabala Capriles Goliattd, Adilson David da
Silvac, Rafael Cypriano Dutrab, Nádia Rezende Barbosa Raposoa
a NUPICS, Faculty of Pharmacy, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil
b Laboratory of Autoimmunity and Immunopharmacology (LAIF), Federal University of Santa Catarina, 88906-072,
Araranguá, Santa Catarina, Brazil.
c Chemistry Department, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil
d Graduate Program in Computational Modeling, Department of Computer Science, Federal University of Juiz de
Fora, Juiz de Fora, MG, Brazil
*Corresponding author at:
Rua José Lourenço Kelmer, s/n, Campus Universitário, 36036-900, Juiz de Fora, MG, Brazil. Tel/Fax: +55 32
21023809.
E-mail address: nadiacritt@gmail.com

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Abstract
Inflammaging is known as an imbalance between pro-inflammatory and anti-inflammatory immune
mechanisms, being related to the onset of neurological disorders, such as major depression and Alzheimer's disease.
Considering the known disadvantages regarding the FDA approved drug to manage such illnesses, resveratrol
emerges as a natural drug candidate, despite its low bioavailability. In this study, resveratrol analogues were
evaluated for their capacity of inhibiting acetylcholinesterase in silico, in vitro, and in vivo. Molecular docking
simulations pointed out RSVA1 and RSVA6 as potent inhibitors, even more than resveratrol. Ellman´s assay
demonstrated RSVA6 as capable of inhibiting 92.4% of the enzyme activity. Further, male Swiss mice were
pretreated with RSVA6 (100 mg kg-1) 60 min before receiving scopolamine (1 mg kg-1). The Novel Recognition
Object (NOR), Object Location (OLT), and Buried Pellet tests (BPL) demonstrated an RSVA6 neuroprotective
effect. In the second round of tests, mice received a single intraperitoneal injection of lipopolysaccharide (0.5 mg kg-
1) 24 h before treatment with RSVA6 (1, 10, and 100 mg kg-1). The Open Field (OFT), Tail Suspension (TST), and
Splash tests (ST) were evaluated. LPS had no significant effect on the crossing and rearing number, indicating an
association between the immobility time and anhedonia observed in the TST and ST, respectively, with depressive-
like behavior. RSVA6 significantly reduced the depressive-like behavior triggered by LPS in the TST and ST.
Altogether, our data suggest RSVA6 as a potential drug candidate for the treatment of neuroinflammatory
conditions.
Keywords: resveratrol, acetylcholinesterase, scopolamine, lipopolysaccharide, homology modeling, protein-ligand
docking.

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1. INTRODUCTION
Inflammaging is known as a multifactorial and age-related imbalance between pro-inflammatory and anti-
inflammatory immune mechanisms resulted from lifelong processes of stressor exposures that promote a progressive
increase of the inflammatory status [1-3]. In the brain, aging may be responsible for morphological and functional
changes in neurons and glial cells. Since microglia are the major immune mediators of the central nervous system
(CNS) secreting inflammatory cytokines and reactive oxygen species (ROS), any alteration in activation and
regulation of these cells can lead to a brain chronic inflammatory state [4]. When in this state, the activated
microglia over-produce different pro-inflammatory cytokines (e.g., TNF, IL-1β, and IL-6) [5] consecutively
exposing neurons to various inflammatory mediators that can stimulate neuronal cell injury or death [6]. Recently,
neuron loss has been associated with the release of high mobility group box-1 (HMGB1) protein in the extracellular
milieu. This protein acts as a damage-associated molecular pattern (DAMP) capable of activating inflammatory
processes through receptor for advanced glycation end products (RAGE) and toll-like receptor 4 (TLR4) linkage,
playing an important role in inflammation [7]. Altogether, immunescence associated with inflammaging plus up-
regulation of systemic inflammation may remodulate the neural-immune cells and lead to a chronic low-grade
inflammation in the brain, also known as neuroinflammation [8].
Some evidence has been indicating a relationship between the acceleration of the inflammaging process
and the onset and development of age-related chronic neuroinflammatory and neurodegenerative disorders [9,10],
including Parkinson disease (PD), amyotrophic lateral sclerosis, Alzheimer disease (AD) [11], and depression [5].
According to the “neuroinflammatory hypothesis” of AD, Aβ plaques and intracellular tangles can act as pro-
inflammatory agents, stimulating the proliferation and activation of astrocytes and microglia. Once activated, those
cells produce cytokines responsible for triggering signaling cascades that lead to brain dysfunction and cognitive
deterioration, with further neuronal death [12]. The cholinergic dysfunction, one of the consequences of the loss of
cholinergic innervation, has been attributed as the main cause of early AD symptoms [13]. Not by chance, targeting
the cholinergic system is still an essential approach for developing new strategies for AD patients [14]. Major
depressive disorder (MDD) may be a comorbid condition found in AD patients, and during the past years,
inflammation has been considered a common link between both of those illnesses [15].
However, the current FDA approved drugs for managing neurological and neuropsychiatric disorders have
considerable disadvantages. For AD, for example, at least half of the patients are unresponsive to the
acetylcholinesterase inhibitors (iAChE) (donepezil, rivastigmine, galantamine) [16]. Likewise, the available
antidepressants (i.e., selective serotonin reuptake inhibitors, selective noradrenaline reuptake inhibitors, monoamine
oxidase inhibitors, and tricycles) are associated with many side effects and a variety of clinical responses [17,18].
According to Freires and colleagues [19], the use of animal models had been supported scientific
experimentation by predicting efficacy, mechanistic, and toxicological insights in drug discovery. Scopolamine, a
non-selective muscarinic antagonist, is employed to trigger cognitive impairments related to learning and memory
behaviors in animal models [20]. The similarity between the alteration caused by the drug in the CNS and the early
symptoms observed in AD made the scopolamine a widely accepted pattern to assess new drugs with potential

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cognitive effects [21]. Intraperitoneal injection of lipopolysaccharide (LPS - outer-membrane endotoxin of Gram-
negative bacteria) is known to prompt neuroinflammation [22]. The LPS-induced microglia activation through
TLR4 triggers an innate immune response leading to the sickness behavior, characterized by apathy, relative inertia,
and curling up the body that changes to a depressive-like behavior for up to 24 h later the LPS administration [5,23].
For this reason, LPS-induced systemic inflammation in animal models is commonly used to investigate neurological
disorders [24].
Resveratrol (RSV; 3,5,4’-trihydroxystilbene) is a polyphenol commonly found in the barks of purple grapes
and red wine. In addition to its widely known and studied antioxidant action, RSV still exhibits anti-inflammatory
[25], anticancer [26], cardioprotective [27], and antiprotozoal activities [28,29]. Furthermore, this stilbene has also
been reported as improving cognitive and non-cognitive impairments, such as learning, memory, anxiety, and
depression [30], as well as demonstrated positive results in AD [31].
Despite the well-described biological activities, RSV low bioavailability is still a limiting factor for its use
in pharmacological purposes [28,32,33]. In this sense, the development of RSV analogues may surpass this problem.
For this study, a combination of in silico, in vitro, and in vivo studies was applied in a series of RSV analogues
showing their potential as iAChE, neuroprotective and antidepressant-like, indicating these compounds as possible
candidates for developing new drugs for the treatment of neuroinflammatory disorders.
2. EXPERIMENTAL SECTION
2.1. Materials
All chemicals, organic solvents (ethanol), reagents (aniline and aromatic aldehydes), acetylcholine iodide
(AChI), 5,5´-dithiobis-(2-nitrobenzoic acid) (DTNB), physostigmine (Eserin), dimethyl sulfoxide (DMSO),
scopolamine and lipopolysaccharides (LPS) were purchased from Sigma-Aldrich Chemical Co., St. Louis, USA.
2.2. Chemistry
A series of resveratrol analogues (RSVA1-8) were synthesized utilizing a classical method of imine
formation that consists of the condensation of aniline with several different aromatic aldehydes, using ethanol as
1
solvent at room temperature (Scheme 1). The structure of these compounds was characterized by H and 13C RMN
and melting point (Table 1), being in agreement with the literature.
2.3. In silico experiments
2.3.1. Molecular modeling
Considering the in vitro assays were performed using the commercial AChET obtained from Electrophorus
electricus, and in vivo assays were performed on Mus musculus, we modeled the tetrameric conformation of

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acetylcholinesterase (AChET) of E. electricus (EeAChET), Mus musculus (MmAChET) and Homo sapiens
(HsAChET) to validate the computational studies. The models were then used in molecular docking simulations.
The amino acid sequences of the enzymes were obtained from the UniProt database (UniProt Consortium, 2017):
O42275 (EeAChET), P21836 (MmAChET) and P22303 (HsAChE). Each sequence was submitted to the SignalP
server for signal peptide identification. Once identified, the sequence was removed from the template and, therefore,
not modeled.
Crystallographic structures were searched in the PDB (Protein Data Bank) to find suitable templates for the
construction of the EeAChET, MmAChET, and HsAChET models. Several AChE structures (EC 3.1.1.7) fitted the
search criteria (high identity and similarity with human AChE), but only two presented tetrameric conformation:
1C2O and 1MAA from M. musculus. The 1C2O structure has a 4.5 Å of resolution while 1MAA has 2.9 Å, and both
are classified as a non-planar and antiparallel tetramer [34,35]. An important difference between these structures is
that chains A and D of 1C2O partially blocking the active site of chains B and D, whereas, in structure 1MAA, such
obstruction is not observed showing that the non-planar conformation of the tetramer exhibits significant
conformational flexibility [34]. 1MMA structure presents both better resolution and active site accessibility of the
four chains than 1C2O. Thus, 1MMA was selected as a template to modeling the EeAChET, MmAChET, and
HsAChET tetramers.
In the attempt to understand the active site affinity to acetylcholine (Ach) and other known inhibitors and to
complete the three-dimensional (3D) model of the tetrameric structures, other crystallographic structures were
selected as template: 1MAA, 4M0E (chain B), 4M0F (chain B), 2HA4 (chain b), and 1VZJ. The values of sequence
identity and similarity between HsAChET and templates are 91% / 93% (1MAA), 97% / 97% (4M0E), 97% / 97%
(4M0F), 86% / 91 % (2HA4), and 100% / 100% (1VZJ). For MmAChET, the respective values are 97% / 97%
(1MAA), 84% / 89% (4M0E), 84% / 89% (4M0F), 95% / 95% (2HA4) and 100% / 100% (1VZJ). And for
EeAChET, the respective values are 58% / 70% (1MAA), 57% / 70% (4M0E), 57% / 70% (4M0F), 58% / 70%
(2HA4) and 80% / 98% (1VZJ).
The 4M0E and 4M0F structures of H. sapiens were selected as templates due to the presence of AChE
inhibitors, territrem and dihydrotanshinone. The use of 2HA4 of M. musculus as a template was justified by the
presence of the substrate Ach. The ligands present in the crystallographic structures were kept during the modeling
process of EeAChET and HsAChET. The H. sapiens crystallographic structure 1VZJ is the only one in the PDB
databank that corresponds to a more elongated alpha-helix in the C-terminus region of the AChE. This region is the
result of alternative splicing that maintains the exon 6 in the primary structure of the AChE, forming the AChET
isoform [36]. Also, part of the 1VZJ structure is a stretch of residues that correspond to the PRAD (Proline-Rich
Attachment Domain) sequence, which anchors the tetrameric form of the AChET in the plasma membrane of
neurons in the synapse [36].
All models were built using the Modeller v9.20 [37]. Then, the quality of the models was analyzed by the
Ramachandran plot generated by PROCHECK [38] and Molprobity [39] programs and the ERRAT web server [40].

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2.3.2. Electrostatic profile
An important analysis in the process of structure-based drug design is the electrostatic profile. This analysis
allows verifying the distribution of the electric charges in the protein molecular surface, being able to identify
regions with a higher concentration of positive charges, as well as those with a higher level of negative charges. In
this study, we used PDB2PQR v2.1 and APBS v1.5 softwares to calculate the charge distributions and generate the
electrostatic profile.
2.3.3. ADMET analysis and target prediction
In silico analyzes on absorption, distribution, metabolism, excretion (ADME), and toxicity (T) has become
an essential tool in rational drug design. Such analyzes have received considerable attention from pharmaceutical
scientists, and various predictive models related to ADMET have been reported [41]. The high-throughput and low-
cost nature of these models allow a more streamlined drug development process in which its structural optimization
can be guided based on a parallel investigation of bioavailability and safety along with the activity. For the ADMET
analysis, we used in this work the SwissADME [42].
Molecular target prediction was also made for each ligand to confirm AChE as a possible target. We used the
SwissTargetPrediction [43] and Molinspiration servers for this analysis.
2.3.4. Molecular docking simulations
All ligands and control molecules were drawn in the ChemSketch software and their structures optimized in
the Avogadro software v1.1, using the MMFF94 force field and the steepest descended algorithm. For docking
simulations, the models were initially prepared by the addition of polar hydrogens and generation of pdbqt files in
AutoDock Tools (ADT) v1.5.6. In the second step, the ligands (Table 2) and control molecules (physostigmine,
resveratrol, and acetylcholine) were prepared in ADT, where the partial Gasteiger charges were added, rotatable
bonds were set, and pdbqt files were generated.
The third step consisted in set as flexible a set of residues from the active site during the docking
simulations, considering different classes of ligands found in PDB structures: (i) catalytic triad (HsAChET: E199,
S200, H444; MmAChET: E202, S203, H447; EeAChET: E201, S202, H471); (ii) peripheric anionic site, also
known as PAS (EeAChET: Y71, D73, Y123, W281, Y336; MmAChET: Y72, D74, Y124, W286, Y34; HsAChET,
Y69, D71, Y121, W283, Y338); (iii) oxyanion hole (EeAChET: G120, G121, A203; MmAChET: G121, G122,
A206; HsAChET: G118, G119, A201); and (iv) acyl pocket (EeAChET: F290, F292; F333; MmAChET, F295,
F297; F338; HsAChET: F292, F294, F335).
For all the residues listed above, all bonds were allowed to be rotatable. The exceptions were residues Y71,
D73, Y123, F292 for EeAChET; Y72, D74, Y124, W283, F294 for MmAChET; and Y69, D71, Y121, W283, F294
for HsAChET, where rotation between the alpha and beta carbons were disabled to maintain the maximum number
of rotations allowed by AutoDock Vina.

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The last step consists of setting in chain A, of both models, the center and size of the grid box. The center
coordinates for EeAChET were X=-11, Y=-7, Z=56, with box sizes X=27Å, Y=28Å, and Z=24Å. The center
coordinates for MmAChET were X=-14, Y=-10, Z=58, and box sizes X=20Å, Y=30Å, Z=22Å. For HsAChET, the
center coordinates were X=-22, Y=-11, and Z=57, with box size X=18Å, Y=32Å, and Z=24Å.
Molecular docking was performed with AutoDock Vina [44]. As the AChE catalytic site presents five
structural water molecules, an adaptation was necessary for the program AutoDock Vina. This adaptation consisted
of including the parameters of the water molecule, similar to presented in Autodock Hydrated, in the AutoDock
Vina parameter file.
2.4. In vitro AChE inhibitory assay
The RSV analogues were tested for AChE inhibiting activity using a modified Ellman’s colorimetric
method [45]. A 50 mM Tris-HCl pH 8.0 buffer was prepared for the assay. In 96-well plates, were added 25 μL of
15 mM AChI in water, 125 μL of 3 mM DTNB, 50 μL of the buffer, and 25 μL of the tested molecules dissolved in
MeOH at concentrations ranging from 62.5 to 1000 μg mL-1. Then, 25 μL of AChE solution (0.22 U mL-1) was
added, and the absorbance measured in a spectrophotometer (SpectraMax 190, Molecular Devices, Sunnyvale, CA,
USA) at a wavelength of 415 nm every 15 seconds for 41 cycles at 37 °C. To avoid false-positive results, a parallel
test omitting the DTNB was done. Physostigmine was used as a positive control. All assays were performed in
triplicate. The percentage of inhibition was calculated using the following equation:
% Inhibition = [(E − S)/S] × 100
where E is the enzyme activity without the test compound, and S is the enzyme activity with the tested compound.
IC50 for all analogues was determined using nonlinear regression in Graph Pad Prism version 6.0 for Windows,
Graph Pad Software (San Diego, CA, USA), and it represents in which concentration the hydrolysis of acetylcholine
is inhibited in 50%.
2.5. In vivo studies
2.5.1. Animals
This study was approved by the Ethics Committee on Animal Use (CEUA) of the Federal University of
Santa Catarina (Florianópolis, Brazil) under the number 3914220319 for the LPS-induced depressive and
scopolamine-induced amnesia models. The breeding unit of the same university provided the male Swiss mice (40-
55 g, 8-week old). The animals were maintained under controlled light (07:00 a.m. to 07:00 p.m.), and temperature
(21 ± 1 °C) conditions, and they were allowed to access standard food and water freely. All animal manipulations
have occurred between 9:00 a.m. and 4:00 p.m. For this study, all procedures were performed according to the
National Institute of Health Guidelines for Laboratory Care and Use. All efforts were made to minimize animal
suffering and reduce the number of animals used in the experiment.
2.5.2. Experimental design

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Scopolamine-induced amnesia model: for the scopolamine-induced amnesia model, male Swiss mice were
randomly divided into four experimental groups: i) naive; ii) scopolamine (1 mg kg-1), iii) RSVA6 (100 mg kg-1) and
iv) rivastigmine (1 mg kg-1). RSVA6 (100 mg kg-1, p.o.) and rivastigmine (1 mg kg-1, i.p.) group animals were
treated one hour before scopolamine administration (1 mg kg-1, i.p.). Thirty minutes later, the mice were
behaviorally analyzed by object recognition, object relocation, locomotor activity, and buried pellet tests [46,47].
LPS-induced depressive model: for the LPS-induced depressive model, the animals were randomly
assigned to five experimental groups (n = 5 animals/group) to evaluate the RES analogue RSVA6 antidepressant-
like behavior. LPS (0.5 mg kg-1) was administered intraperitoneally, and 24 h after the injection, mice were treated
with RSVA6 (1, 10, and 100 mg kg-1, p.o.) or imipramine (20 mg kg-1, i.p.). One hour later, the animals were
behaviorally analyzed via the open field, tail suspension, and splash tests.
2.6. Memory and behavioral tests
2.6.1. Open Field Test (OFT)
The OFT was performed before the TST and ST trials, to evaluate the effect on locomotor and exploratory
activities. For the test, a wooden box (40 cm x 60 cm x 50 cm) was used. Its floor was divided into 12 squares, and
temperature, light, and noise were controlled. Mice were placed in the left corner of the field, with permission to
freely explore the floor. The number of crossings (squares crossed by the animal with its four legs) and the number
of rearing (animal standing upright on its riding paws) were registered throughout 6 minutes. At the end of each
session, the floor was cleaned with a 10% ethanol solution [48].
2.6.2. Novel object recognition (NOR)
In the experimental protocol, the animals' memory of recognition is evaluated. Long habituation consists of
three previous presentations on the apparatus. In this protocol, the animals were individually habituated in the open
field for 5 minutes for three days. After 24 hours, they were again exposed to the arena with the presence of two
equal objects placed on the floor, positioned in the same line, and with a distance of 7 cm on both sides of the box
(training session). At this moment, we recorded the investigation time (in seconds) of each of the objects placed in
the apparatus. The animals were removed from the open field, and after 24 hours, they were again exposed to the
arena but replacing one object. Again, we record the exploration time (in seconds) of each of the objects placed in
the box. The following are considered exploration of the object: smelling, touching, or observing the object less than
2 cm away [49,50]. For each animal, recognition memory was assessed via the discrimination index (DI; %),
calculated as follows: DI = TN/(TN + TF) x 100 [TN = time spent exploring the novel object; TF = time spent
exploring the familiar object].
2.6.3. Object location test (OLT)
The object location memory test is used to test local memory in mice [51]. The animals underwent one day
of habituation in the open field apparatus for 5 minutes. On day 2 of the protocol, the rodents were trained for 5

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minutes in the arena with the presence of two equal objects, positioned 12 cm from each other and 11 cm from the
wall. On day 3 (test day), one of the objects was moved from its original position to a new location, and the animals
allowed to explore for 5 minutes. Moreover, we place visual clues on the walls for the animals. All objects were
sanitized at each test to minimize olfactory cues between tests. They are considered as an exploration of the object:
smelling, touching, or observing the object less than 2 cm away. As in the NOR, recognition memory was evaluated
via DI (%). The locomotor activity was analyzed together with the object relocation test since the same apparatus
was used for both tests.
2.6.4. Buried Pellet test (BPT)
In the experimental protocol, we evaluated the memory and olfactory capacity of the animals, 48 hours
before the test each animal had its weight recorded and then underwent a dietary restriction at 90% of body weight.
Before the test and during food restriction, each animal was offered two cereal pallets to be used in the test. The
animals were fasted for 18 hours before training, followed by 1 hour of habituation in the room where the test took
place. The test cage was prepared with 3 cm of fresh litter, with a cereal buried 0.5 cm below in one corner of the
cage. Individually the animals were placed in the cage, and latency time is counted until the animal finds the
sediment. If it does not find in the predetermined period of 300 seconds, the experiment ends, and a latency of 5
minutes will be recorded. The following day (24 hours after), the same training protocol was performed [52-54].
2.6.5. Tail Suspension Test (TST)
The TST is based on suspending the animals 50 cm above the ground by adhesive tape placed 1 cm from
the tip of their tail, in an acoustically and visually isolated place. The immobility time was recorded throughout 6
minutes. The depressive-like behavior status is attributed to those mice that develop an apathetic and immobile
posture when subjected to the short-term stress of being suspended by their tail [17].
2.6.6. Splash Test (ST)
The ST consists of spraying a 10% sucrose solution on the dorsal coat of the mice, and then they are put in
a glass chamber. The sucrose solution dirties the mice's fur, and it leads the animals to initiate grooming behavior.
The latency to begin the grooming was recorded for 5 minutes and used as a measure of self-care and motivational
behavior. A grooming time decrease is associated with anhedonia and depressive-like behavior. After each session,
the chamber was cleaned with a 10% ethanol solution [55].
2.6.7. Statistical analysis
All in vivo experimental results are expressed as the mean ± standard error of the mean (SEM). The one-
way and two-way analysis of variance (ANOVA) was performed to analyze the differences among experimental
groups, and P < 0.05 was defined as statistically significant. The statistical analysis was achieved through Graph Pad
Prism software version 6.0 for Windows, Graph Pad Software (San Diego, CA, USA).

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3. RESULTS
3.1. Chemistry
The synthesis of the RSVA analogues has been carried out as previously described by Paula et al. [28]
through condensation of aniline (1 mM) with several different aromatic aldehydes (1.1 mM), using ethanol as
solvent. The reaction was allowed to stand at room temperature and constant stirring. After 0.5 to 24 hours,
depending on the aldehyde, the compounds were obtained in solid form after the precipitate formed was filtered,
washed in ethanol and dried in an oven.
3.2. In silico studies
3.2.1. AChET models
The models of MmAChET, HsAChET, and EeAChET are homotetramers formed by two antiparallel
homodimers (AB and CD), and the monomers of each dimer are non-planar. The C-terminal region of each
monomer in all models is complete and forms a helix around the PRAD sequence (Fig. 1).
The amino acid sequence of HsAChET shows a high identity and similarity with MmAChET (89% and 93%,
respectively) and EsAChET (60% and 74%, respectively). Similarly, the amino acid sequence alignment between
MmAChET and EeAChET also presents a high identity (60%) and similarity (71%). Alignments can be visualized
in Supplementary Fig. 1.
These values of identity and similarity point to high structural conservation between the three enzymes,
mainly between MmAChET and HsAChET. Such structural conservation is reflected in the similarity between the
profiles of the electrostatic potentials on the outer surface of the three enzymes (Supplementary Fig. 2). The
predominance of electronegative areas has already been highlighted by other works [56,57] as an important
characteristic of AChE.
Structural conservation can also be observed among the residues that form the active site and gorge site of
the studied enzymes. The list of residues, according to Lushington et al. [58] and Cheng et al. [59], is presented
below following the order of EeAChET/MmAChET/HsAChET: (i) active site: Y71/ Y72/ Y69, D73/ D74/ D71,
G120/ G121/ G118, G121/ G122/ G119, Y132/ Y133/ Y130, A203/ A204/ A201, F290/ F295/ F292, E329/ E334/
E331, Y332/ Y337/ Y334; (ii) gorge site: W85/ W86/ W83, Y123/ Y124/ Y121, W281/ W286/ W283, F292/ F297/
F294, F333/ F338/ F335, Y336/ Y341/ Y338; and catalytic triad: E201/ E202/ E199, S202/ S203/ S200, H471/
H447/ H444. This full conservation has an important consequence concerning the electrostatic profile of the active
site that has a predominance of electronegative potential (Fig. 2).
Throughout the 3D modeling of AChET structures, six water molecules were maintained in the active site.
These water molecules were selected based on the structural alignment of the templates and based on the literature
[60].
3.2.2. Molecular docking simulations

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The docking results presented in Table 2 include the binding energy of the compounds, the theoretical
concentration of each compound required to produce half-maximum inhibition of the enzyme (cKi, based on the
binding energy), and the description of the interactions between compounds and residues of AChE from E.
electricus, M. musculus, and H. sapiens. Figure 3 shows all ligands best pose in the catalytic site of EeAChET (Fig.
3a), MmAChET (Fig. 3b), and HsAChET (Fig. 3c).
The RSV analogues interact with residues of histidine and serine from the catalytic triad, tyrosine, and
tryptophan of PAS, and tryptophan of choline-binding site (Table 2 and Fig. 3). It is important to note that even
considering the flexibility of some residues, all docking results of physostigmine in EeAChET presented
conformations in the external binding site (Fig. 3a).
The consensus result of three organisms suggesting that the modifications made in the molecules resulted in
ligands more potent than resveratrol, and point out RSVA1 and RSVA6 as potent inhibitors. A single docking image
was prepared for these two compounds in the three organisms to compare the results (Fig. 4).
3.2.3. ADME predictions
SwissADME and Molinspiration were able to provide a significant amount of data on each compound
analyzed. However, the most critical data are the parameters contained in Lipinski's rule of five (RO5) and the total
polar surface area (TPSA). The results presented in Table 3 show that all compounds presented in this work follow
the RO5, which indicates as minimal the probability of having dissolution and permeation problems. Comparing
RSV and its analogues, the proposed compounds presented similar or even better values than the polyphenol.
3.3. The inhibitory effect of RSV analogues on AChE activity
All RSV derivatives were screened for AChE inhibitory capacity through Ellman’s method with minor
modifications [45] and classified according to their percentage of AChE inhibition [61] as potent (>50% inhibition),
moderate (30-50% inhibition) or inactive/low activity (<30% inhibition). In sequence, the molecules were further
investigated to determine their IC50 values (Table 4).
Considering the analogue RSVA6 presented the best result in docking tests with M. musculus, and in vitro
results pointed to this compound as the most potent AChET inhibitor of E. electricus, we elected RSVA6 to follow
in the in vivo experiments.
3.4. Effects of acute treatment with RSVA6 on memory and behavioral assessments
3.4.1. RSVA6 has a neuroprotective effect on memory in the NOR, OLT, and BPT and does not affect locomotor
activity
The NOR and OLT were conducted to assess the impact of RSVA6 on short-term (Fig. 5a, 5b), spatial
memory (Fig. 5c, 5d) in scopolamine-induced amnesia. In the training phase, there were no significant differences

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(P > 0.05) in the discrimination index (DI) among groups in both NOR (Fig. 5a) and OLT (Fig. 5c). In the NOR test
phase (Fig. 5b), the DI of the scopolamine groups was significantly reduced than that of the naïve group (P < 0.01).
Contrarily, previous treatment with RSVA6 (100 mg kg-1; p.o.) significantly elevated the DI (P < 0.001) compared
to the scopolamine group, result as similar as that one observed to the rivastigmine group. The results from the OLT
test phase (Fig. 5d) show a lower DI for the scopolamine group compared to the naïve group (P < 0.05). However,
RSVA6 was significantly able (P < 0.05) to prevent memory impairment.
The BPT (Fig. 5e) evaluates the olfactory memory of mice. There were no significant differences among
groups during the training phase (P > 0.05). In contrast, animals from the scopolamine group spent more time (P <
0.01) to find the buried pellet than those animals from the naïve group. However, RSVA6 lowered significantly (P <
0.01) the latency in comparison to the scopolamine group, indicating the neuroprotective effect of the RSV
derivative. The OFT was applied to the animals from the scopolamine-induced amnesia model to exclude any
suspect of locomotor impairment due to RSVA6 administration. There were no significant differences among groups
(Fig. 5f).
3.4.2. LPS injection has no effect on the locomotor and exploratory activities, and RSVA6 has antidepressant-like
behavior in the TST and ST
The OFT analyzed the number of crossing and rearing to discard a possible association between immobility
in the behavioral tests and changes in motor activities. The one-way ANOVA revealed no significant alterations in
the crossing number (P > 0.05) and the rearing number (P > 0.05; Fig. 6a and 6b, respectively), indicating that LPS
has not affected either the locomotor and exploratory activities among groups. Consequently, the immobility time
and anhedonia observed respectively in the TST and ST can be associated with depressive-like behavior.
The TST, one of the most commonly used tests for assessing antidepressant-like behavior in mice, was
performed to evaluate the influence of RSVA6 (1, 10, and 100 mg kg-1, p.o.) on the depressive-like behavior
triggered by i.p. LPS (Fig. 6c and 6d). It was observed an increase in the immobility time in those animals exposed
to LPS compared to the naïve group (Fig. 6c). The oral administration of RSVA6 at the highest dose (100 mg kg-1,
p.o.) has significantly reversed the LPS effect (P < 0.01). The same significant effect was observed in those animals
treated with the standard drug (imipramine, mg kg-1, i.p.). From the ST (Fig. 6e and 6f), LPS injection decreased the
grooming time significantly when compared to the control group (Fig. 6e). The treatment with RSVA6 at the highest
dose (100 mg kg-1, p.o.) reversed this effect significantly (P < 0.05), as well as imipramine did. The results
demonstrate an antidepressant-like action of the RES analogue RSVA6.
4. DISCUSSION
Herein, in silico studies carried out for RSV analogues have demonstrated the modifications made in the
molecules resulted in structures more potent than RSV itself in the in vitro assay, which was able to point out
RSVA1, RSVA3, and RSVA6 as potent inhibitors. The AChE active site is composed of three subsites: an anionic, a
stearic, and a peripheral anionic. In the first is located the catalytic site (serine-protease triad), the second is the site

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of choline interaction, and the last one is the site of interaction of quaternary ligands [62]. The AChE mechanism of
action consists of the following steps (Supplementary Fig. 3): (1) ACh approaches the site and a nucleophilic attack
by serine hydroxyl group occurs; (2) an adjustment of electron configuration happens after the attack; (3) water is
produced and leaves the Ser-ACh complex; (4) a nucleophilic attack by the water present in the site onto the acyl-
enzyme, which will cause the deacylation of ACh; (5) and (6) shows the electron configuration where acetic acid is
produced as the final product, and finally, residues return to the apo state [58]. RSV derivatives used in this study
have a similar structure to acetylcholine, such as length, charged atoms, or electronegative atoms/groups at the
second aromatic ring. They make the central region of the ring more positive, improving the chances of interactions
with critical residues where "N+” from ACh interacts. Furthermore, the hydrogens at the first aromatic ring allow
hydrogen bonds with the hydroxyl group from the catalytic serine. Comparing all the results, herein, the RSV
interacts with essential residues for the mechanism of action of AChET, such as S200, from the catalytic triad and
Y121, W283, and Y338, from PAS site (numbering according to H. sapiens). The strong interaction of these
compounds, represented by very low binding energies, shows that these ligands probably compete with
acetylcholine and prevent it from binding to the active site of AChET.
The ADME findings have demonstrated our RSV analogues as having a low probability of dissolution and
permeation problems. According to RO5, a molecule has a higher chance of becoming a drug if there are no more
than 5 hydrogens (H-) bond donors (sum of oxygen-hydrogen and oxygen-nitrogen bonds); no more than 10 H-bond
acceptors (all nitrogen and oxygen); the molecular weight (MW) is less than 500 daltons, and LogP (octanol-water
partition coefficient) is less than 5 [63]. The TPSA is used to evaluate drug transport properties. It is the sum of all
surface areas of polar atoms (oxygen, nitrogen, and attached hydrogen), and it has shown a significant correlation
with passive molecular transport through membranes and, therefore, it allows prediction of transport properties of
drugs in the intestines and blood-brain barrier [64]. The TSPA value is inversely proportional to absorbance. Thus,
values below 140 Ų favor intestine absorbance as long as values below 60 Ų favor blood-brain barrier absorbance
[65]. Considering the brain as AChE local site, these results support our RSV analogues that presented values below
60 Ų. It is important to note that RSV showed a value close to 60.7 Ų and that all the analogues tested had values
below 60 Ų. Respecting all the cited parameters, the chances of poor solubility and permeation and transportation
impairments are less likely.
Dementia and depression are co-morbidities frequently observed in aged people, although the clinical
relationship between both of them is poorly understood yet [66]. In AD, the most prevalent form of dementia in the
elderly, downregulation of acetylcholine levels is one of the disease hallmarks [67] and correlated with illness
severity [68]. Currently, searching for iAChEs is still a strategy to find an efficient treatment for AD since the few
available drugs only alleviate the symptoms, in addition to presenting many side effects [69]. Following the in silico
predictions, we have found analogues that showed higher AChE inhibitory capacity than RSV. RSVA6 had the best
performance and made it to 92.4%, suggesting this compound as a good candidate for in vivo assessments.
In this present study, RSVA6 demonstrated a neuroprotective effect on scopolamine-induced memory
impairments in mice. The olfactory memory was investigated via de BPT, and, as expected, the scopolamine

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injection (1 mg kg-1; i.p.) caused a sensory deficit in the animals even after a period of food deprivation. Both
cholinergic receptor blockade and lesions in cholinergic projection neurons are capable of interfering with the
acquisition of the olfactory associative memory [70]. Interestingly, olfactory dysfunction is commonly observed in
the early stages of AD [71], and one of the causes may be the cholinergic deficit [72]. Although the efficacy (or not)
of iAChE on olfactory dysfunction has not been elucidated yet, Velayudhan and Lovestone [73] demonstrated in an
open study that donepezil therapy (5 - 10 mg/day; p.o.) was able to improve odor identification in AD patients,
suggesting a higher ACh availability as playing a role on that enhancement. Thus, the results showed that
pretreatment with RSVA6 (100 mg kg-1; p.o.) exerted a neuroprotective effect on mice cognition, probably by
inhibiting AChE and, consequently, increasing the ACh concentration at the brain regions involved in the olfactory
processes.
The NOR and OLT tasks were used to assess the spatial and short-term recognition memory of the animals,
taking into account their natural tendency to explore any novelty for longer than everyday items [74]. Animals from
the ESC group (1 mg kg-1; i.p.) were unable to distinguish both the object replaced in OLT and the object relocated
in NOR from the familiar object. A significant reduction in DI compared to the control group was found, in
agreement with previous studies [75-77]. Otherwise, the comparison between the RSVA6 and ESC groups showed a
significant DI increase, suggesting a neuroprotective effect of the RSV analogue on the animals induced to cognitive
impairment. The OFT was conducted to eliminate any interference on locomotor activities, which could be triggered
by RSVA6 administration. No behavioral alterations were observed at all. Taken together, the in silico, in vitro, and
in vivo data set indicates the RSVA6 neuroprotective action through blocking the enzyme AChE and, consequently,
elevating the ACh concentration at the synaptic cleft during neuropathological processes. However, studies
concerning the ACh and AChE levels after RSVA6 administration, as well as the molecule pharmacokinetics, are
encouraged at this point.
Depressive signs or MDD occur in over 20% of the AD patients [78]. Previous observations have suggested
an involvement of the cholinergic system in the depressive process, even though the mechanisms around this
possible relationship are mostly unknown [79]. In a case-control study, Kamath and colleagues [80] found AChE
levels significantly increased in patients who have been diagnosed with depression and had not received any
treatment before the examination. The current drugs taken in the AD treatment are known to play a mild effect on
depression, regardless of the observation of a connection between cognitive worsening and that psychiatric disorder
[81]. From that perspective, RSVA6 significantly diminished the depressive signs developed on mice, and this result
may have some relation to the molecule's ability to inhibit the AChE, even though the animal model here discussed
is the LPS-induced instead of the scopolamine-induced. More tests are encouraged to investigate the mechanisms
behind the results.
Another hypothesis suggested explaining the results found in vivo may be associated with the RSVA6
effect on the immune system. Inflammatory dysfunctions in both peripheral and central nervous systems have been
correlated with psychiatric illness and neurodegeneration disorders like depression and AD [82-84]. A single LPS
systemic challenge is well established as triggering acute systemic inflammation leading to behavioral changes in

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animal models, being considered a model of intensification of MDD [85]. Endotoxin from gram-negative bacteria
was previously identified as toll like receptor 4 ligand (TLR4) [86], and the signaling pathway activated via this
receptor may generate neuroinflammation. TLR4 is a transmembrane protein [87] expressed at microglial cell
surface [88]. The LPS-TLR4 biding mediates the activation of transcription factors, such as factor nuclear kappa B
(NF-κB), and consequently induces production and release of pro-inflammatory cytokines, like as the tumor necrosis
factor (TNF) and interleukin (IL)-1β [89]. Moreover, a previous report demonstrated that acute systemic LPS
injection, besides elevating cytokines levels, increased microglial cells in different brain areas, including frontal
cortex and hippocampus [90]. It means an exacerbation of the immune response triggered by the LPS model.
From the OFT, locomotor and exploratory activities among groups were not affected after LPS injection.
Once it was established, the immobility time and anhedonia detected in the respective TST and ST were associated
with depressive-like behavior. Previously, RSV has demonstrated to be able to reduce the release of pro-
inflammatory factors, as well as the production of prostaglandins and the NF-κB pathway [91-93], as illustrated in
Figure 7. Recently, Finnell et al. [94] demonstrated in vivo a reduction of splenic cells expressing TNF and IL-1β
after a 12-day regimen with RSV (10 or 30 mg/kg/day i.p.). Furthermore, RSV decreased IL-1β and TNF levels in
the locus coeruleus of rats. This anti-inflammatory effect of RSV was previously attributed to its ability to
downregulate TLR4 and NF-κB p65 subunit expression with consequent reduction of cytokines levels [95-97]. In
this present study, RSVA6 was effective in reversing the inflammatory response triggered by LPS. The observation
that small bioactive molecules sharing structural similarities are more likely to share similar targets [43] points
RSVA6 as a potential candidate for more studies exploring its effectiveness on neuroinflammatory pathways.
In summary, scientific evidence point to a relationship between neuroinflammation and a series of
modifications in the brain that may be responsible for the onset of neurological and neuropsychiatric disorders.
Moreover, the cholinergic system seems to be involved in several of those processes, even though little is known
about the mechanisms behind the outbreak of diseases such as AD and MDD. Current pharmacological treatments
are limited, which have either significant adverse effects or low efficacy. Therefore, natural compounds well-known
as anti-inflammatory and immunomodulatory agents may prove useful as a new therapy or adjuvant to the strategies
already taken to handle depression and AD, for example. Many in vitro and in vivo studies have demonstrated RSV
as containing biological properties that could advantageous to treat psychiatric and neurological patients. However,
the clinical effects of RSV are impaired by its low bioavailability in humans [98]. Our set of RSV analogues have
been shown through computational modeling as having similar or even better performance than RSV on dissolution
and permeation. Most importantly, RSVA6 ability to inhibit AChE in silico and in vitro as well as the
neuroprotective and anti-depressant effects observed in vivo made this RSV analogue a potent candidate to elucidate
its molecular mechanisms of action more thoroughly.
Conflict of interest
The authors declare that there is no conflict of interest regarding the publication of this article.

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Acknowledgments
The authors would like to thank the Brazilian agencies Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de
Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), and Universidade Federal de Juiz de Fora (UFJF).
R.C.D. is a recipient of a research productivity fellowship from the CNPq.

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Scheme 1. Synthetic pathway of RSV analogues.
Figure 1: 3D model of tetrameric structure of MmAChET, HsAChET, and EeAChET. (a) Front view of structural
alignment of models in cartoon representation: EeAChE-T (red), MmAChE-T (green), and HsAChE-T (blue). (b) Top view
of chains.

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Figure 2: Electrostatic potential profile of the active site of acethylcholinesterase models: (a) EeAChE-T, (b) MmAChE-T
and (c) HsAChE-T. The electrostatic potential profile of AChET vary from -5 (red) to 5 (blue) kT/e. The image was
generated using PyMOL version 1.8.2, Schrödinger – LLC.
Figure 3: Binding mode for all ligands (best conformations): (a) EeAChET (red), (b) MmAChET (green), and (c)
HsAChET (blue) in cartoon representation. Score values of each docking are presented in Table 2. Ligands are in stick
representation: acetylcholine (red), physostigmine (green), resveratrol (blue), and resveratrol analogues (cyan). The main
active site residues are highlighted by numbered spheres. The images were generated using PyMOL version 1.8.2,
Schrödinger – LLC.

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Figure 4: Best docking results of RSV analogues against acethylcholinesterase. Score values of each docking are presented
in Table 2. The compounds RSVA1 (a, c, e) and RSVA6 (b, d, f) are in sticks representation and (a-b) EeAChET (red), (c-

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d) MmAChET (green), and (e-f) HsAChET (blue) are in cartoon representation. Pharmacophoric characteristics are
represented by spheres: aromatic ring and lipophilic region (gold), hydrogen bond donor (light blue), hydrogen bond
acceptor (purple), hydrogen bond donor and acceptor (blue). The images were generated using PyMOL version 1.8.2,
Schrödinger – LLC.
Figure 5: Effect of RSVA6 treatment using a scopolamine-induced amnesia model in mice. RSVA6 (100 mg kg-1, p.o.) and
rivastigmine (1 mg kg-1, i.p.) were administered 60 minutes prior to scopolamine (1 mg kg-1, i.p.) before the studies. Novel
object recognition (NOR), object relocation (OLT), buried pellet test (BPT) and open field test (OFT) were completed. (a)
discrimination index in object recognition test training, (b) discrimination index in object recognition test, (c) discrimination
index in object location test training, (d) discrimination index in object location test, (e) latency in seconds to find pellet in
the buried pellet test, (f) number of quadrants traveled in open field test. Data are expressed as mean ± standard error of the
mean. Statistical analysis was performed using two-way ANOVA by Bonferroni's post-hoc test. *P < 0.05, **P < 0.01,
***P < 0.001.

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Figure
6:
Influence of RSVA6 treatment on LPS-induced depressive-like behavior. (a) Crossing number and (b) rearing number in
the OFT. (c) Immobility time and (d) number of immobility in the TST. (e) Grooming time and (f) number of grooming in
the ST. Data are shown as Mean±SEM, n=5. #P < 0.05, ##P < 0.01 vs control; *P < 0.05, **P < 0.01 vs LPS.

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Figure 7: Schematic representation of resveratrol anti-inflammatory action on LPS-TLR4 signaling pathway.

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Table 1: Spectral data of RSV analogues.
Compounds
δ CH=N
δ C=N
Melting Point (oC)
Yield (%)
8.72
159.8
220.4-221.6
61.4-62.1
96.8-97.3
89.6-90.7
89.2-90.7
50.7-51.4
53.1-54.2
133.8-134.4
67.0
RSVA1
8.51
8.39
8.80
8.44
8.96
8.43
7.58
159.8
159.9
158.8
160.0
163.5
160.2
159.9
65.0
72.0
75.0
74.0
63.0
63.0
87.0
RSVA2
RSVA3
RSVA4
RSVA5
RSVA6
RSVA7
RSVA8