LASSBio-1586, an N-acylhydrazone derivative, attenuates nociceptive behavior and the inflammatory response in mice

Article abstract of DOI:10.1371/journal.pone.0199009

Pain and inflammation are complex clinical conditions that are present in a wide variety of disorders. Most drugs used to treat pain and inflammation have potential side effects, which makes it necessary to search for new sources of bioactive molecules. In this paper, we describe the ability of LASSBio-1586, an N-acylhydrazone derivative, to attenuate nociceptive behavior and the inflammatory response in mice. Antinociceptive activity was evaluated through acetic acid-induced writhing and formalin-induced nociception tests. In these experimental models, LASSBio-1586 significantly (p<0.05) reduced nociceptive behavior. Several methods of acute and chronic inflammation induced by different chemical (carrageenan, histamine, croton oil, arachidonic acid) and physical (cotton pellet) agents were used to evaluate the anti-inflammatory effect of LASSBio-1586. LASSBio-1586 exhibited potent anti-inflammatory activity in all tests (p<0.05). Study of the mechanism of action demonstrated the possible involvement of the nitrergic, serotonergic and histamine signaling pathways. In addition, a molecular docking study was performed, indicating that LASSBio-1586 is able to block the COX-2 enzyme, reducing arachidonic acid metabolism and consequently decreasing the production of prostaglandins, which are important inflammatory mediators. In summary, LASSBio-1586 exhibited relevant antinociceptive and anti-inflammatory potential and acted on several targets, making it a candidate for a new multi-target oral anti-inflammatory drug.

Full text of DOI:10.1371/journal.pone.0199009

RESEARCH ARTICLE
LASSBio-1586, an N-acylhydrazone derivative,
attenuates nociceptive behavior and the
inflammatory response in mice
1
Juliane Cabral Silva , Raimundo Gonc¸alves de Oliveira Junior , Mariana Gama e Silva ,
1
1
´
Erica Martins de Lavor , Juliana Mikaelly Dias Soares , Sarah Raquel Gomes de Lima-
1
1
´
1
Saraiva , Tamara Coimbra Diniz , Rosemairy Luciane Mendes , Edilson Beserra
1
2
ˆ
de Alencar Filho , Eliezer Jesus de Lacerda Barreiro , Lıdia Moreira Lima , Jackson
2
3
3
´
Roberto Guedes da Silva Almeida1,3
*
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
´
1 Nucleo de Estudos e Pesquisas de Plantas Medicinais (NEPLAME), Universidade Federal do Vale do São
´
Francisco, Petrolina, Brasil, 2 Colegiado de Farmacia, Universidade Federal do Vale do São Francisco,
Petrolina, Brasil, 3 Instituto Nacional de Ciência e Tecnologia de Farmacos e Medicamentos (INCT-
INOFAR), Universidade Federal do Rio de Janeiro, Laboratorio de Avaliac¸ão e Sıntese de Substancias
Bioativas (LASSBio), Rio de Janeiro, Brasil
´
´
´
ˆ
* jackson.guedes@univasf.edu.br
OPEN ACCESS
Abstract
´
Citation: Silva JC, Oliveira Junior RGd, Silva MGe,
Pain and inflammation are complex clinical conditions that are present in a wide variety of
disorders. Most drugs used to treat pain and inflammation have potential side effects, which
makes it necessary to search for new sources of bioactive molecules. In this paper, we
describe the ability of LASSBio-1586, an N-acylhydrazone derivative, to attenuate nocicep-
tive behavior and the inflammatory response in mice. Antinociceptive activity was evaluated
through acetic acid-induced writhing and formalin-induced nociception tests. In these experi-
mental models, LASSBio-1586 significantly (p<0.05) reduced nociceptive behavior. Several
methods of acute and chronic inflammation induced by different chemical (carrageenan, his-
tamine, croton oil, arachidonic acid) and physical (cotton pellet) agents were used to evaluate
the anti-inflammatory effect of LASSBio-1586. LASSBio-1586 exhibited potent anti-inflam-
matory activity in all tests (p<0.05). Study of the mechanism of action demonstrated the pos-
sible involvement of the nitrergic, serotonergic and histamine signaling pathways. In addition,
a molecular docking study was performed, indicating that LASSBio-1586 is able to block the
COX-2 enzyme, reducing arachidonic acid metabolism and consequently decreasing the pro-
duction of prostaglandins, which are important inflammatory mediators. In summary, LASS-
Bio-1586 exhibited relevant antinociceptive and anti-inflammatory potential and acted on
several targets, making it a candidate for a new multi-target oral anti-inflammatory drug.
´
Lavor EMd, Soares JMD, Lima-Saraiva SRGd, et al.
(2018) LASSBio-1586, an N-acylhydrazone
derivative, attenuates nociceptive behavior and the
inflammatory response in mice. PLoS ONE 13(7):
e0199009. https://doi.org/10.1371/journal.
pone.0199009
Editor: Gautam Sethi, National University
Singapore Yong Loo Lin School of Medicine,
SINGAPORE
Received: December 20, 2017
Accepted: May 24, 2018
Published: July 30, 2018
Copyright: © 2018 Silva et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: The authors received specific funding of
´
Conselho Nacional de Desenvolvimento Cientıfico e
Introduction
´
Tecnologico (Process CNPq 465249/2014-0),
Pain is one of the most serious societal problems; it limits productivity and reduces quality of
life as well as has high social and economic impacts. Pain is a complex experience involving
emotional and neurobiological factors that normally indicate the presence of tissue damage to
INCT-INOFAR (Instituto Nacional de Ciência e
´
Tecnologia de Farmacos e Medicamentos) for this
work. The project is coordinated by Prof. Eliezer
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
1 / 19

LASSBio-1586 attenuates nociceptive behavior and inflammatory response in mice
Jesus de Lacerda Barreiro. The funders had no role
in study design, data collection and analysis,
decision to publish, or preparation of the
manuscript.
the organism. The pain response is established by the participation of several neurogenic or
inflammatory mediators. During potential injury, chemical agents (histamine, bradykinin,
prostaglandins, serotonin and nitric oxide) can activate and/or sensitize nociceptive fibers,
contributing to the induction of pain and inflammation. In this context, inflammation can be
understood to be a fundamental defense reaction of the body against the invasion of patho-
gens, injury and other noxious stimuli. Redness, warmth, swelling and pain are the classic clin-
ical features of inflammation [1–3].
Competing interests: The authors have declared
that no competing interests exist.
Currently, the pharmacological treatments used for the management of pain and inflamma-
tion include non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, central analge-
sics (opioids) and adjuvants drugs (anticonvulsants, antidepressants). Despite the great
diversity of available anti-inflammatory and analgesic drugs, their side effects—for instance
gastrointestinal disorders and ineffectiveness for some conditions—require a continuous
search for new bioactive molecules. Therefore, management of pain/inflammation continues
to be a major challenge for modern medicine. Thus, many studies focus on the search for new
compounds as therapeutic alternatives. Neurotransmitters, receptors, and pharmacological
targets have attracted much interest from the scientific community [4–6].
To explore new agents for the treatment of painful and inflammatory conditions, N-acylhy-
drazone (NAH) derivatives were studied in various pain and inflammation models. NAH is
considered to be a privileged structure, as it is present in many compounds with diverse phar-
macological activities. LASSBio-1586 is an example of an NAH derivative that was developed
through molecular modification strategies. In a previous investigation, LASSBio-1586 was
shown to inhibit microtubule polymerization, demonstrating a broad in vitro and in vivo anti-
proliferative profile with satisfactory selectivity for cancer cells [7].
In this sense and following the principle of drug repurposing, we describe in this paper the
antinociceptive and anti-inflammatory potential of LASSBio-1586 in various experimental
models. In addition, a docking study was performed with some of the main targets of the
inflammatory process.
Materials and methods
The N-acylhydrazone derivative LASSBio-1586 was obtained as previously described [7].
Briefly, methyl 3,4,5-trimethoxybenzoate was treated with hydrazine hydrate in ethanol to give
3,4,5-trimethoxybenzohydrazide. This key intermediate was treated with benzaldehyde in eth-
anol (7 ml) containing one drop of 37% hydrochloric acid. The mixture was stirred at room
temperature for 1 hour and was then poured into ice. The precipitate was filtered and dried to
give LASSBio-1586 in good yield. This compound was characterized, and its purity was deter-
mined using the methodologies described by Amaral and coworkers [7].
Animals and ethics statement
All experiments were conducted using 8-week-old male Swiss mice (Mus musculus) (30–35 g).
The total number of animals involved in this study was 300 animals, and each individual was
involved in a single painfull procedure (not reused). The animals were kept in groups of six ani-
mals (n = 6) in polypropylene cages at a temperature of 22 1˚C and relative humidity of 60–
80% with a light/dark cycle of 12:12 h (start 06:00 and end 18:00) and free access to food (Purina
Labina1) and water. This study was performed in accordance with the Conselho Nacional para
o Controle de Experimentac¸ão Animal (CONCEA, Brazil) and complied with the recommenda-
tions of the International Association for the Study of Pain [8]. The protocol was approved by
´
the Comitê de Etica no Uso de Animais of the Universidade Federal do Vale do São Francisco
(CEUA-UNIVASF, Brazil) with authorization number 0013/021014. All efforts were made to
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
2 / 19

LASSBio-1586 attenuates nociceptive behavior and inflammatory response in mice
minimize animal suffering. At the end of the experiments, the animals were anesthetized with
60 mg/kg ketamine plus 7.5 mg/kg xylazine intraperitoneally and euthanized by cervical dislo-
cation. For administration of all substances intraperitoneally syringes of 1 ml with needle of 13
x 0.45 mm were used. For the oral route a gavage needle was used.
Acetic acid-induced writhing test
The writhing test was chosen as a classic model to assess the analgesic or anti-inflammatory
properties of new agents [9]. This test was performed using the method described by Collier
and collaborators [10] with modifications. Mice intraperitoneally (i.p.) received 10 ml/kg of a
0.9% acetic acid solution [11]. The amount of abdominal writhing was recorded for 10 min,
beginning 5 min after administration of the acetic acid solution [12]. Writhing reflexes were
defined as contractions of the abdominal muscles and pelvic rotation, followed by hind limb
extension. Animals were divided into six groups of six animals each (n = 36) and were treated
orally (p.o.) with LASSBio-1586 (10, 20 and 40 mg/kg) and saline (negative control) 1 h before
the nociceptive agent. Indomethacin (20 mg/kg, i.p.) and morphine (10 mg/kg, i.p.) were used
as reference drugs and administered 30 min before the acetic acid solution.
Formalin-induced nociception test
The formalin test was performed as described by Hunskaar and Hole [13]. Animals were
divided into six groups of six animals each (n = 36) and were treated with saline (p.o.), LASS-
Bio-1586 (10, 20 and 40 mg/kg, p.o), indomethacin (20 mg/kg, i.p.) and morphine (10 mg/kg,
i.p.) were given 1 h prior to the formalin injection. A formalin solution (2.5% in 0.9% sterile
saline; 20 μl/animal) was injected into the right hind paw of mice [14]. Immediately after the
formalin injection, animals were placed back in the chambers with a mirror and were observed
for 30 min. The amount of time (in seconds) spent licking and biting the injected paw was
measured as an indicator of pain. The formalin injection produced a biphasic nociceptive
response: (I) an acute phase, 5 min after formalin injection, followed by a quiescent period of
approximately 10 min and (II) a longer lasting tonic phase 15 to 30 min after this period [15].
To verify the possible involvement of the nitrergic and serotonergic systems in the pharma-
cological effect of LASSBio-1586, animals were divided into four groups of six animals each
(n = 24) and were pretreated with the respective antagonists N(G)-Nitro-L-arginine methyl
ester (L-NAME, 10 mg/kg, i.p) and ondansetron (0.5 mg/kg, i.p) 30 min before treatment with
LASSBio-1586 (40 mg/kg, p.o) [16].
Leukocyte migration to the peritoneal cavity induced by carrageenan
Leukocyte migration was induced with 250 μl of 1% carrageenan (i.p.) into the peritoneal cav-
ity of mice 1 h after administration of saline (p.o.) and LASSBio-1586 (10, 20 and 40 mg/kg, p.
o) and 0.5 h after injection of dexamethasone (2 mg/kg, i.p.). Animals (n = 30) were eutha-
nized, as described above, 4 h later, and the peritoneal cavity was washed with 3 ml of a saline
solution containing 1 mM EDTA [17]. The collected fluid was centrifuged (3000 rpm for 6
min) at room temperature. Subsequently, 10 μl of this suspension were dissolved in 200 μl of
Turk solution, and a total cell count was performed using a Neubauer chamber. The results are
expressed as the number of leukocytes/ml [18].
Carrageenan-induced hind paw edema
Mice were divided into six groups of six animals each (n = 36) and were pretreated with LASS-
Bio-1586 (10, 20 and 40 mg/kg, p.o), saline (p.o.) or indomethacin (20 mg/kg, i.p.) 1 h before
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
3 / 19

LASSBio-1586 attenuates nociceptive behavior and inflammatory response in mice
subcutaneous injection of carrageenan (2.0% ƛ-carrageenan) or saline (0.9%) into the right
hind paw of animals at a volume of 20 μl/animal [19,20]. The mice pedal volume up to the
ankle joint was measured using a plethysmometer (PanLab LE 7500, Spain) 0, 1, 2, 3, 4 and 5 h
after administration of carrageenan as described previously [21]. Inhibition of the paw edema
was calculated by edema = (foot volume at measurement time—foot volume at time zero)/foot
volume at time zero.
Histamine-induced hind paw edema
To evaluate the involvement of histaminic receptors, mice were pretreated with LASSBio-1586
(40 mg/kg, p.o) or saline (p.o.) 1 h before the subcutaneous injection of histamine (100 μg/
paw) or saline (0.9%) into their right hind paw at a volume of 20 μl/animal [22]. The volume
was measured 0, 30, 60, 90 and 120 min after injection of histamine or saline [23].
Croton oil-induced ear edema
In this model, inflammation was induced using 20 μl of 5% croton oil (v/v) in acetone on the
inner and outer surfaces of the right ears, while the left ear received 20 μl of acetone. Animals
(n = 30) were euthanized, as described above, six hours later, and a six-mm diameter plug was
removed from both the treated and untreated ears with a punch. The edematous response was
measured as the weight difference between the two plugs. Animals were pretreated with LASS-
Bio-1586 (10, 20 and 40 mg/kg, p.o), saline (p.o.) or indomethacin (20 mg/kg, i.p.) 1 h before
application of the irritant agent [24,25].
Arachidonic acid-induced ear edema
One hour after treatment with LASSBio-1586 (10, 20 and 40 mg/kg, p.o), saline (p.o.) or indo-
methacin (20 mg/kg, i.p.) edema was induced by applying 20 μl of arachidonic acid to the
inner and outer surface of the right ear, while the left ear received 20 μl of acetone and was
used as a control. Thirty minutes after application of arachidonic acid, mice (n = 30) were
euthanized as described above, and a six-mm diameter plug was removed from both the
treated and untreated ears with a punch and weighed [26,27].
Leukocyte migration into subcutaneous air pouches induced by
carrageenan
Mice (n = 30) were pretreated with LASSBio-1586 (10, 20 and 40 mg/kg, p.o), saline (p.o.) or
dexamethasone (2 mg/kg, i.p.) 1 h before subcutaneous injection of carrageenan (1.0% ƛ-carra-
geenan) into the air pouch. The air pouch was formed by injecting 10 ml of sterile air subcuta-
neously into the dorsal thoracic region. On day 6, carrageenan (0.25 ml) was injected into the
pouch [28,29]. Six hours after this injection, the pouch was washed with 3 ml of PBS, and the
collected fluid was centrifuged (3000 rpm for 6 min). Total cells were counted using a Neu-
bauer chamber [30].
Cotton pellet-induced granuloma
A granuloma was induced by sterile cotton pellets (10 0.5 mg) implanted subcutaneously on
the backs of mice 30 min after administration of saline (p.o.), LASSBio-1586 (10, 20 and 40
mg/kg, p.o.) and indomethacin (20 mg/kg, p.o.). Mice were divided into five groups of six ani-
mals each (n = 30). These animals were treated orally for six consecutive days. On day 7, the
animals were anaesthetized and cotton pellets were removed surgically; any extraneous tissues
were removed. The removed pellets were dried overnight at 60˚C and weighed [31–34].
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
4 / 19

LASSBio-1586 attenuates nociceptive behavior and inflammatory response in mice
Physico-chemical properties and ADMET profile
The physico-chemical properties and ADMET profile of LASSBio-1586 was predicted in silico
in comparison with the anti-inflammatory drugs indomethacin and meloxicam, using the
ACD/Percepta Program.
Molecular docking analysis
The X-ray crystallographic structure of murine COX-2 enzyme, complexed with meloxicam
(MXM), was obtained from the RCSB Protein Data Bank (PDB ID: 4M11) [35, 36]. This struc-
ture was chosen because of its structural similarity between LASSBio-1586 and the well-known
NSAID meloxicam, which is most selective for the COX-2 isoform of cyclooxygenase. An anal-
ysis was performed using Autodock tools with the (ADT) v1.5.4 and Autodock v4.2 programs
[37] (Autodock, Autogrid, Autotors, Copyright-1991–2000) produced by the Scripps Research
Institute. The structure of LASSBio-1586 was initially constructed using ACD/ChemSketch
12.01 software [38]. Using the GaussView 6.0 and Gaussian 09 packages [39, 40], the 3D struc-
ture was adjusted similarly to the previously obtained crystal structure [7], followed by geo-
metric optimization using the semi-empirical PM3 method. The structure of MXM was
obtained directly from the original pdb complex. The preparation of docking simulations with
LASSBio-1586 and MXM followed the same procedure. Gasteiger charges and polar hydro-
gens were assigned to protein and ligands. Nonpolar hydrogens were merged. Two water mol-
ecules present in the binding site [35] were kept and edited using the UCSF Chimera package
[41]. The ligands were considered to be flexible upon analysis, and the rotatable bonds were
chosen automatically by the program. To calculate the affinity maps used by Autodock, a grid
point box with dimensions of 40 Å x 40 Å x 40 Å was initially chosen, with spacing of 0.375 Å
between the points and centered on the ligand molecule (native MXM). A conformational
search was performed with the Lamarckian Genetic Algorithm (LGA) [42]. The initial popula-
tion was 150, with a maximum number of generations of 27,000. The maximum number of
energy evaluations was 2,500,000 (long). The mutation and crossover rates were chosen as 0.02
and 0.8, respectively. At the end of the calculations, several conformations were placed into dif-
ferent clusters of similarity considering the binding energy and RMSD (Root Mean Square
Deviation). The lowest-energy conformation of the more populated cluster was considered to
be the most reliable solution. Based on the procedures described above, “redocking” was con-
ducted considering the native MXM. The objective of this step was to determinate the accuracy
of the docking procedure in this system, evaluating the RMSD (Root Mean Square Deviation)
between the native and post-redocking conformation of MXM. The same step was conducted
for LASSBio-1586 to view its possible interaction modes and binding energies.
Statistical analysis
The results are presented as the mean standard error of the mean (SEM), and statistical anal-
ysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s test.
Values of p<0.05 were considered statistically significant. All analyses were performed using
GraphPad Prism1 6.0 (Graph Pad Prism Software, Inc., San Diego, CA, USA).
Results and discussion
Initially, the antinociceptive potential of LASSBio-1586 was evaluated using the acetic acid-
induced nociception test. In this model, LASSBio-1586 reduced nociceptive behavior in a
dose-dependent manner, decreasing the number of writhings by up to 88.97% at the highest
dose tested (40 mg/kg), as shown in Fig 1. This preliminary test indicated that LASSBio-1586
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
5 / 19

LASSBio-1586 attenuates nociceptive behavior and inflammatory response in mice
Fig 1. Effect of LASSBio-1586 (10, 20 and 40 mg/kg, p.o.), morphine (10 mg/kg, i.p.) and indomethacin (20 mg/kg, i.p.)
in the acetic-acid-writhing-induced nociception test in mice (n = 6, per group). Values are expressed as the mean SEM,
where a indicates p<0.05, significantly different from the control group, according to ANOVA, followed by Tukey’s test.
https://doi.org/10.1371/journal.pone.0199009.g001
could be a promising antinociceptive agent. However, the acetic acid-induced writhing test is
quite unspecific. Intraperitoneal administration of the chemical agent induces activation of
nociceptors and stimulates the release of a variety of painful and inflammatory mediators,
including histamine, bradykinin, serotonin, glutamate, noradrenaline, substance P, nitric
oxide and prostaglandins [10]. For this reason, it is not possible to specify the nociceptive path-
ways in which the molecule acts. From this perspective, the formalin-induced nociception test
was performed.
The formalin test is an experimental model that can verify the two distinct phases of a noci-
ceptive episode. The first phase comprises a nociceptive response elicited by central-acting
mediators, including those that activate serotonergic, muscarinic, vanilloid and glutamatergic
receptors. In the second phase, there is a predominance of inflammatory mediators, mainly
histamine, bradykinin and prostaglandins [13]. In this test, LASSBio-1586 reduced nociceptive
behavior in both phases. LASSBio-1586 (10, 20 and 40 mg/kg) showed 49.01%, 49.67% and
67.77% of normal antinociceptive activity, respectively, in the first phase (Fig 2A). Similar to
that observed for indomethacin, LASSBio-1586 promoted a better pharmacological effect in
the second phase of the test, reaching 96.74% of antinociceptive activity at a dose of 20 mg/kg
(Fig 2B).
When animals were pretreated with ondansetron or L-NAME, the pharmacological effect
of LASSBio-1586 (40 mg/kg, p.o.) was completely reversed in the first phase of the test (Fig 3),
suggesting that its central antinociceptive response was involved at least in part in the seroto-
ninergic and nitrergic systems. Serotonin [5-hydroxytryptamine (5-HT)] has long been con-
sidered to have an important role in the control of pain in the central nervous system,
particularly through descending inhibition. Recently, many pharmacological investigations
have focused on the role played by 5-HT in acute or chronic pain conditions, as well as the
identification of the respective 5-HT receptors that are involved. In general, 5-HT1A and
5-HT7 receptor agonists may be promising therapeutic agents for the treatment of pain states
[43–45].
Recent pharmacological reports have shown that nitric oxide (NO) in the nociceptive
response has a dual effect on the regulation of pain mechanisms and can act as a nociceptive or
antinociceptive agent, depending on the cell conditions. Experimental data have demonstrated
that NO inhibits nociception in the peripheral and central nervous systems. Normally, the
analgesic action of NO depends on an intracellular signaling pathway that includes the
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
6 / 19

LASSBio-1586 attenuates nociceptive behavior and inflammatory response in mice
Fig 2. Effect of LASSBio-1586 (10, 20 and 40 mg/kg, p.o.), morphine (10 mg/kg, i.p.) and indomethacin (20 mg/kg, i.p.) in the
first (A) and second (B) phases of the formalin-induced nociception test in mice (n = 6, per group). Values are expressed as the
mean SEM, where a indicates p<0.05, significantly different from the control group, according to ANOVA, followed by Tukey’s
test.
https://doi.org/10.1371/journal.pone.0199009.g002
formation of cyclic GMP, activation of PKG and consequent opening of K+ channels. The
opening of these channels increases the K+ current, leading to hyperpolarization of nociceptive
neurons [46,47]. From this point of view, LASSBio-1586 induces production of NO and conse-
quently inhibits the nociceptive response.
Although LASSBio-1586 reduced nociceptive behavior in both phases of the formalin test,
the pharmacological response was more intense in the second phase, indicating that the anti-
nociceptive effect of our NAH derivative was related to a reduction in inflammation. In this
context, we conducted several tests to explore the anti-inflammatory potential of LASSBio-
1586. At first, the anti-inflammatory effect of LASSBio-1586 was evaluated in models of acute
inflammation, such as the leukocyte migration to the peritoneal cavity induced by carrageenan
test. In this method, LASSBio-1586 reduced leukocyte migration in a dose-dependent manner
(Fig 4). At the highest dose (40 mg/kg, 75.89%), the anti-inflammatory effect was equivalent to
that observed for dexamethasone (2 mg/kg, 76.96%).
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
7 / 19

LASSBio-1586 attenuates nociceptive behavior and inflammatory response in mice
Fig 3. Effect of LASSBio-1586 (40 mg/kg, p.o.), LASSBio-1586 (40 mg/kg, p.o.) + ondansetron (OND 0.5 mg/kg, i.p.) and LASSBio-1586 (40 mg/kg,
p.o.) + L-NAME (10 mg/kg, i.p.) in the first (A) and second (B) phases of the formalin-induced nociception test in mice (n = 6, per group). Values are
expressed as the mean S.E.M., where a indicates p<0.05, significantly different from the control group, according to ANOVA, followed by Tukey’s test.
https://doi.org/10.1371/journal.pone.0199009.g003
In the carrageenan-induced hind paw edema model, LASSBio-1586 significantly decreased
(p<0.05) paw edema at all doses tested, especially at 1, 2 and 3 hours after treatment, suggest-
ing strong anti-inflammatory activity, as shown in Fig 5. In the first few hours of the test,
inflammatory mediators are released in response to carrageenan injection. Histamine is one of
the first mediators produced since it is a fast-acting vasoactive amine that promotes vessel dila-
tion from local circulation, which is essential for exudation and edema formation [19].
In this sense, to assess the role of histaminic receptors in the anti-inflammatory effect of
LASSBio-1586, a similar protocol was performed using histamine as a paw edema inducer. Fig
6 shows that LASSBio-1586 (40 mg/kg, p.o.) significantly reduced (p<0.05) histamine-induced
paw edema at 30, 60 and 90 minutes, suggesting the involvement of histamine receptors in its
anti-inflammatory effect.
The anti-inflammatory effect of LASSBio-1586 was also evaluated against the topical appli-
cation of inflammatory agents. In the croton oil-induced ear edema test, animals treated with
Fig 4. Effect of LASSBio-1586 (10, 20 and 40 mg/kg, p.o.) and dexamethasone (2 mg/kg, i.p.) on leukocyte migration into
the peritoneal cavity induced by carrageenan in mice. Values are expressed as the mean S.E.M. (n = 6, per group), where a
indicates p<0.05, significantly different from the control group, according to ANOVA, followed by Tukey’s test.
https://doi.org/10.1371/journal.pone.0199009.g004
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
8 / 19

LASSBio-1586 attenuates nociceptive behavior and inflammatory response in mice
Fig 5. Effect of LASSBio-1586 (10, 20 and 40 mg/kg, p.o.) and indomethacin (20 mg/kg, i.p.) on paw edema induced by
carrageenan in mice. The sham group was treated only with saline, whereas the control group received saline and carrageenan.
Values are expressed as the mean S.E.M. (n = 6, per group), where a indicates p<0.05, significantly different from the control
group, according to ANOVA, followed by Tukey’s test.
https://doi.org/10.1371/journal.pone.0199009.g005
LASSBio-1586 presented a significant reduction in the weight of edema at all tested doses. A
similar result was found for indomethacin-treated mice, as shown in Fig 7. These findings
indicate the ability of LASSBio-1586 to attenuate the inflammatory process, induced by various
chemical agents and routes of administration, making it a strong candidate for a new anti-
inflammatory drug.
Chemical-induced ear edema is an extremely versatile experimental model, and several
agents can be used to investigate the involvement of various signaling pathways. Given the
importance of prostaglandin production to maintain inflammation and promote the sensitiza-
tion of nociceptive fibers [48,49], we conducted this experiment using arachidonic acid as an
inducer of ear edema. In this test, LASSBio-1586 reduced ear edema at higher doses (20 and 40
Fig 6. Effect of LASSBio-1586 (40 mg/kg, p.o.) on paw edema induced by histamine in mice. The sham group was treated only
with saline, whereas the control group received saline and histamine. Values are expressed as the mean S.E.M. (n = 6, per group),
where a indicates p<0.05, significantly different from the control group, according to ANOVA, followed by Tukey’s test.
https://doi.org/10.1371/journal.pone.0199009.g006
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
9 / 19

LASSBio-1586 attenuates nociceptive behavior and inflammatory response in mice
Fig 7. Effect of LASSBio-1586 (10, 20 and 40 mg/kg, p.o.) and indomethacin (20 mg/kg, i.p.) on croton oil-induced ear
edema test in mice. Values are expressed as the mean S.E.M (n = 6, per group), where a indicates p<0.05, significantly
different from the control group, according to ANOVA, followed by Tukey’s test.
https://doi.org/10.1371/journal.pone.0199009.g007
mg/kg, p.o.), demonstrating approximately 47.93 and 51.53% of anti-inflammatory activity
(Fig 8).
Arachidonic acid is a major product obtained after degradation of membrane phospholip-
ids by the phospholipase A2 enzyme. Once produced, arachidonic acid serves as a substrate for
cyclooxygenase enzymes, producing a diversity of prostaglandins (PGE2, PGI2, PGD2, PGF2)
[49,50]. The significant reduction in ear edema promoted by LASSBio-1586 indicates its
potential for inhibiting the degradation of arachidonic acid into inflammatory mediators, such
as prostaglandins.
To examine the hypothesis involving inhibition of prostaglandins production in greater
detail, we performed a molecular docking study to investigate the interaction of LASSBio-1586
with the COX-2 cyclooxygenase isoform. From a molecular standpoint, it is interesting that
LASSBio-1586 is structurally similar to the well-known nonsteroidal anti-inflammatory drug
(NSAID) meloxicam, a drug that has high selectivity for COX-2. The similarity includes
molecular length, presence of aromatic rings spaced by a chain with an amide-like bond in
similar regions and tendency of the molecules to be planar due to conjugated double extension
(Fig 9).
Fig 8. Effect of LASSBio-1586 (10, 20 and 40 mg/kg, p.o.) and indomethacin (20 mg/kg, i.p.) on the arachidonic acid-
induced ear edema test in mice. Values are expressed as the mean S.E.M (n = 6, per group), where a indicates p<0.05,
significantly different from the control group, according to ANOVA, followed by Tukey’s test.
https://doi.org/10.1371/journal.pone.0199009.g008
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
10 / 19

LASSBio-1586 attenuates nociceptive behavior and inflammatory response in mice
Fig 9. 2D comparative representation of the NSAID meloxicam and LASSBio-1586.
https://doi.org/10.1371/journal.pone.0199009.g009
In recent years, the crystallographic structure of murine COX-2 (Mus musculus) complexed
with meloxicam was deposited in the online PDB database. The enzyme contains four mono-
mers with identical residues that are complexed with four meloxicam molecules. The confor-
mation of the “A” monomer was considered for docking procedures. The redocking of native
MXM presented an RMSD = 0.61 Å, with Binding Energy = −9.82 Kcal/mol, for the best pose.
This result validates the use of AutoDock for this complex, as the maximum value accepted in
the literature to the RMSD is 2.0 Å [51]. Fig 10 shows that the redocking and native conforma-
tions are very similar.
Fig 10. Native (magenta) and post-redocking (cyan) poses of MXM/murine COX-2 complex. RMSD = 0.61 Å. Binding Energy = −9.82 Kcal/mol.
https://doi.org/10.1371/journal.pone.0199009.g010
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
11 / 19

LASSBio-1586 attenuates nociceptive behavior and inflammatory response in mice
The best docking poses presented by LASSBio-1586 provided interesting insight into the
structure and analgesic/anti-inflammatory activity relationships. Fig 10 show a sequence of
images, and the five best poses for LASSBio-1586 are very similar (Fig 11A). The pose with the
lowest binding energy (−7.84 Kcal/mol) is also shown (Fig 11B), as is a comparison performed
by overlapping MXM and LASSBio-1586 after docking (Fig 11C).
Oxicams are a structurally unique class of NSAIDs. Studies of crystal complexes of meloxi-
cam and murine COX-2 [35] showed that this class interacts in a novel mode compared to
other NSAIDs (naproxen, diclofenac, among others), which occupy the entire channel that
interacts with the enzyme, as mediated by two water molecules. In the structure proposed in
Fig 11C, we observe that the hydrazone group of LASSBio-1586 is arranged in the same man-
ner as the meloxicam amide group, establishing the same water-mediated interactions: the
alpha-nitrogen of hydrazone in LASSBio-1586 occupies a similar position as that of the nitro-
gen of meloxicam and is able to interact with Ser-530; the carbonyl oxygen of hydrazone in
LASSBio-1586 occupies a similar position as the amide oxygen in MXM, which binds Arg-120
and Tyr-355 via another ordered water molecule [35], reinforcing the idea that meloxicam and
LASSBio-1586 have a similar pattern of recognition (Fig 11). However, it is also known that
oxicam drugs contain a heteroatom at the 1’ position of the carboxamide five-membered ring
(a nitrogen atom in meloxicam, Fig 9), which establishes water-mediated interactions with
Tyr-385 and Ser-530 [35]. The absence of this interaction in LASSBio-1586 could explain its
less effective binding energy compared to the redocking pose of MXM.
Binding of meloxicam is accompanied by an exclusive interaction between the phenyl ring
and side chain of Leu-531 [35]. Other amino acids complete this hydrophobic pocket as Leu-
534, Ile-345 and Val-344. This molecular volume of occupation is observed in the best poses of
docked LASSBio-1586, considering the trimethoxy-substituted aromatic ring (Fig 11). In the
same way, the original crystallographic article indicated the interaction between Phe-518 and
the methyl group at the 4’ position of the meloxicam thiazole ring as being an important inter-
action with COX-2. Observing the proposed structure in Fig 11, we note that the unsubstituted
phenyl ring of LASSBio-1586 occupies the same region as methyl thiazole, with an additional
possible π-stacking interaction involving the aromatic ring of Phe-518. These crystallographic
and theoretical data at the molecular level corroborate the hypothesis of the analgesic/anti-
inflammatory effect of LASSBio-1586 in mice, at least in part via the COX-2 inhibition
pathway.
Finally, we evaluated the anti-inflammatory potential of LASSBio-1586 in chronic inflam-
mation models. Fig 12 shows the pharmacological response of LASSBio-1586 on leukocyte
migration into subcutaneous air pouches induced by carrageenan. In this test, our NAH deriv-
ative significantly reduced the number of leukocytes at all doses tested (10, 20 and 40 mg/kg, p.
o.), presenting 50.63, 77.39 and 87.03% of the untreated anti-inflammatory effect, respectively.
Similarly, LASSBio-1586 also decreased cotton pellet-induced granuloma, as demonstrated in
Fig 13. These findings demonstrate the ability of LASSBio-1586 to combat the inflammatory
response induced by different agents and at several stages, making it a strong candidate for a
new oral anti-inflammatory drug.
Concerning the toxicity, preliminary studies have shown that LASSBio-1586 was capable of
preventing microtubule polymerization and showed an extensive in vitro antiproliferative pro-
file, as well as a better selectivity index, indicating improved selective cytotoxicity toward can-
cer cells unlike non-tumor cells [7]. This may suggest that the LASSBio-1586 has low toxicity
to non-tumor cells, presenting a good safety profile in vitro. However, we agree that future tox-
icological evaluation is necessary to confirm the potential of LASSBio-1586 as a candidate
drug for the treatment of inflammatory diseases and pain disorders.
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
12 / 19

LASSBio-1586 attenuates nociceptive behavior and inflammatory response in mice
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
13 / 19

LASSBio-1586 attenuates nociceptive behavior and inflammatory response in mice
Fig 11. Results of the docking procedures for LASSBio-1586. (A) Conformations obtained from the most populous and
stable cluster in the binding site of COX-2; (B) most stable docked conformation for LASSBio-1586 (Binding Energy = −7.84
Kcal/mol); (C) native conformation of meloxicam (magenta) and most stable conformation for LASSBio-1586 (cyan).
https://doi.org/10.1371/journal.pone.0199009.g011
Fig 12. Effect of LASSBio-1586 (10, 20 and 40 mg/kg, p.o.) and dexamethasone (2 mg/kg, i.p.) on leukocyte migration into
subcutaneous air pouches induced by carrageenan in mice. Values are expressed as the mean S.E.M (n = 6, per group), where
a indicates p<0.05, significantly different from the control group, according to ANOVA, followed by Tukey’s test.
https://doi.org/10.1371/journal.pone.0199009.g012
Fig 13. Effect of LASSBio-1586 (10, 20 and 40 mg/kg, p.o.) and indomethacin (20 mg/kg, p.o.) on cotton pellet-induced
granuloma. Values are expressed as the mean S.E.M. (n = 6, per group), where a indicates p<0.05, significantly different from the
control group, according to ANOVA, followed by Tukey’s test.
https://doi.org/10.1371/journal.pone.0199009.g013
Regarding to the physico-chemical properties and ADMET profile, as demonstrated in
Table 1, no violation to Lipinski rule of five (Ro5) was found for LASSBio-1586, indicating
that this compound has properties that would make it a likely orally active drug in humans
[52].
However, LASSBio-1586 was predicted to be an insoluble drug once compared to indo-
methacin and meloxicam. Regarding their comparative ADMET profile, they were predicted
to be highly absorbed (HIA = 100%), highly permeable (Pe > 7 ×10−6 cm/s) and extensively
bound to plasma protein (PPB > 90%). All compounds have showed great oral bioavailability
in silico (F = 80–99%). The main differences amongst LASSBio-1586, indomethacin and
meloxicam rely on their metabolic stability in human liver microsomes (HLM) and their
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
14 / 19

LASSBio-1586 attenuates nociceptive behavior and inflammatory response in mice
Table 1. Comparative in silico physico-chemical properties and ADMET profile of LASSBio-1586 and the anti-inflammatory drugs indomethacin and meloxicam.
Predicted PropertiesÃ
Compounds
LASSBio-1586
Indomethacin
Meloxicam
MW (g/mol)
H-Donors
H-Acceptors
Rotatable Bonds
TPSA
314.34
357.79
351.40
1
1
2
6
5
7
6
4
2
69.15
68.53
136.22
LogP
2.44
4.02
2.38
Solubility
Caco-2
0.002 mg/ml
Pe = 231 ×10−6 cm/s
2.5 mg/ml
Pe = 129 ×10−6 cm/s
5.96 mg/ml
Pe = 233 ×10−6 cm/s
HIA
100%
80%
93%
-2.72
0.49
0.52
0.42
100%
99%
99%
-4.32
0.26
0.23
0.27
100%
96%
99%
-5.24
0.32
0.41
0.21
F (oral)
PPB
CNS score
HLM
hERG
AMES
ÃDetermined in silico using the ACD/Percepta Program.
MW = molecular weight; H-Donors = hydrogen bond-donors; H-Acceptors = hydrogen bond-acceptors; TPSA = topological polar surface area; LogP = the logarithm of
the drug partition coefficient between n-octanol and water; Caco-2 = human epithelial cell line Caco-2; HIA = human intestinal absorption; F = Bioavailability;
CNS = central nervous system; HLM = human liver microsomes; hERG = the human Ether-à-go-go-Related Gene; AMES = Ames test = Salmonella typhimurium
reverse mutation assay.
https://doi.org/10.1371/journal.pone.0199009.t001
ability to penetrate in CNS. As depicted in Table 1, indomethacin and meloxicam were pre-
dicted as stable in HLM (scores of 0.26 and 0.32, respectively), while an undefined result was
found for LASSBio-1586 (Score = 0.49). Both anti-inflammatory drugs have been predicted as
non-penetrant to CNS (scores of -4.32 and -5.24, respectively) whereas LASSBio-1586 was
defined as able to penetrate CNS. To have some clues about the toxicity of LASSBio-1586, the
ability of this compound to inhibit hERG (the human Ether-à-go-go-Related Gene), and its
mutagenic profile (i.e. probability of a positive Ames test) were predicted. The results were
converted into classification scores, and an undefined hERG and mutagenic activities (score >
0.33 and ꢀ 0.67) were predicted for LASSBio-1586. Taken together, those in silico results sug-
gest an adequate pharmacokinetic profile for LASSBio-1586, meanwhile the in silico approach
could not predicted its toxicological profile.
Conclusion
Based on the results, LASSBio-1586 showed significant antinociceptive and anti-inflammatory
activities in all of the experimental models. Its mechanism of action appears to involve the
serotonergic, nitrergic and histaminic signaling pathways, in addition to inhibition of COX-2,
as demonstrated by the molecular docking study. In summary, LASSBio-1586 has emerged as
a strong candidate for a multi-target anti-inflammatory drug.
Supporting information
S1 Dataset. Raw data.
(RAR)
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
15 / 19

LASSBio-1586 attenuates nociceptive behavior and inflammatory response in mice
Author Contributions
´
Conceptualization: Juliane Cabral Silva, Raimundo Gonc¸alves de Oliveira Junior, Mariana
´
Gama e Silva, Erica Martins de Lavor, Juliana Mikaelly Dias Soares, Sarah Raquel Gomes de
ˆ
Lima-Saraiva, Tamara Coimbra Diniz, Rosemairy Luciane Mendes, Eliezer Jesus de Lacerda
´
Barreiro, Lıdia Moreira Lima, Jackson Roberto Guedes da Silva Almeida.
´
Funding acquisition: Eliezer Jesus de Lacerda Barreiro, Lıdia Moreira Lima.
´
Investigation: Juliane Cabral Silva, Raimundo Gonc¸alves de Oliveira Junior, Mariana Gama e
´
Silva, Erica Martins de Lavor, Juliana Mikaelly Dias Soares, Sarah Raquel Gomes de Lima-
ˆ
Saraiva, Tamara Coimbra Diniz, Rosemairy Luciane Mendes, Edilson Beserra de Alencar
´
Filho, Eliezer Jesus de Lacerda Barreiro, Lıdia Moreira Lima, Jackson Roberto Guedes da
Silva Almeida.
´
Methodology: Juliane Cabral Silva, Raimundo Gonc¸alves de Oliveira Junior, Mariana Gama e
´
Silva, Erica Martins de Lavor, Juliana Mikaelly Dias Soares, Sarah Raquel Gomes de Lima-
ˆ
Saraiva, Tamara Coimbra Diniz, Rosemairy Luciane Mendes, Edilson Beserra de Alencar
´
Filho, Eliezer Jesus de Lacerda Barreiro, Lıdia Moreira Lima, Jackson Roberto Guedes da
Silva Almeida.
´
Project administration: Eliezer Jesus de Lacerda Barreiro, Lıdia Moreira Lima.
´
Resources: Eliezer Jesus de Lacerda Barreiro, Lıdia Moreira Lima.
´
Writing – original draft: Juliane Cabral Silva, Raimundo Gonc¸alves de Oliveira Junior, Mari-
´
ana Gama e Silva, Erica Martins de Lavor, Juliana Mikaelly Dias Soares, Sarah Raquel
ˆ
Gomes de Lima-Saraiva, Tamara Coimbra Diniz, Rosemairy Luciane Mendes, Edilson
´
Beserra de Alencar Filho, Eliezer Jesus de Lacerda Barreiro, Lıdia Moreira Lima, Jackson
Roberto Guedes da Silva Almeida.
´
Writing – review & editing: Juliane Cabral Silva, Raimundo Gonc¸alves de Oliveira Junior,
´
Mariana Gama e Silva, Erica Martins de Lavor, Juliana Mikaelly Dias Soares, Sarah Raquel
ˆ
Gomes de Lima-Saraiva, Tamara Coimbra Diniz, Rosemairy Luciane Mendes, Edilson
´
Beserra de Alencar Filho, Eliezer Jesus de Lacerda Barreiro, Lıdia Moreira Lima, Jackson
Roberto Guedes da Silva Almeida.
References
1. Pace MC, Mazzariello L, Passavanti MB, Sansone P, Barbarisi M, Aurilio C. Neurobiology of pain. J
Cell Physiol. 2006; 209: 8–12. https://doi.org/10.1002/jcp.20693 PMID: 16741973
2. Brito RG, dos Santos PL, Quintans JS, de Lucca Ju´nior W, Arau´jo AA, Saravanan S, et al. Citronellol, a
natural acyclic monoterpene, attenuates mechanical hyperalgesia response in mice: Evidence of the
spinal cord lamina I inhibition. Chem Biol Interact. 2015; 239: 111–117. https://doi.org/10.1016/j.cbi.
2015.06.039 PMID: 26141506
3. Almeida AAC, Silva RO, Nicolau LAD, Brito TV, Sousa DP, Barbosa ALR, et al. Physio-pharmacological
Investigations about the Anti-inflammatory and Antinociceptive Efficacy of (+)-Limonene Epoxide.
Inflammation. 2017; 40: 511–522. https://doi.org/10.1007/s10753-016-0496-y PMID: 28091830
4. Wang Y, Chen P, Tang C, Wang Y, Li Y, Zhang H. Antinociceptive and anti-inflammatory activities of
extract and two isolated flavonoids of Carthamus tinctorius L. J Ethnopharmacol. 2014; 151: 944–950.
https://doi.org/10.1016/j.jep.2013.12.003 PMID: 24333963
5. Souza GV, Simas AS, Bastos-Pereira AL, Frois GRA, Ribas JLC, Verdan MH, et al. Antinociceptive
Activity of the Ethanolic Extract, Fractions, and Aggregatin D Isolated from Sinningia aggregata Tubers.
PLoS One. 2015; 10(2): e0117501. https://doi.org/10.1371/journal.pone.0117501 PMID: 25719394
6. Xu J, Zhao Q, Wei L, Yang Y, Xu R, Yu N, et al. Phytochemical Composition and Antinociceptive Activity
of Bauhinia glauca subsp. hupehana in Rats. PLoS One. 2015; 10(2): e0117801. https://doi.org/10.
1371/journal.pone.0117801 PMID: 25658740
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
16 / 19

LASSBio-1586 attenuates nociceptive behavior and inflammatory response in mice
7. do Amaral DN, Cavalcanti BC, Bezerra DP, Ferreira PM, Castro RP, Sabino JR, et al. Docking, synthe-
sis and antiproliferative activity of N-acylhydrazone derivatives designed as combretastatin A4 ana-
logues. PLoS One. 2014; 9: e85380. https://doi.org/10.1371/journal.pone.0085380 PMID: 24614859
8. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain.
1983; 16: 109–110. PMID: 6877845
9. Mohamad AS, Akhtar MN, Zakaria ZA, Perimal EK, Khalid S, Mohd PA, et al. Antinociceptive activity of
a synthetic chalcone, flavokawin B on chemical and thermal models of nociception in mice. Eur J Phar-
macol. 2010; 647: 103–109. https://doi.org/10.1016/j.ejphar.2010.08.030 PMID: 20826146
10. Collier HO, Dinneen LC, Johnson CA, Schneider C. The abdominal constriction response and its sup-
pression by analgesic drugs in the mouse. Brit J Pharmacol Chemother. 1968; 32: 295–310.
´
11. Silva JC, Araujo CS, Lima-Saraiva SRG, Oliveira-Junior RG, Diniz TC, Wanderley CW, et al. Antinoci-
ceptive and anti-inflammatory activities of the ethanolic extract of Annona vepretorum Mart. (Annona-
ceae) in rodents. BMC Complement Altern Med. 2015; 15: 197. https://doi.org/10.1186/s12906-015-
0716-2 PMID: 26104689
12. Aoki M, Tsuji M, Takeda H, Harada Y, Nohara J, Matsumiya T, et al. Antidepressants enhance the anti-
nociceptive effects of carbamazepine in the acetic acid-induced writhing test in mice. Eur. J. Pharmacol.
2006; 550: 78–83. https://doi.org/10.1016/j.ejphar.2006.08.049 PMID: 17027750
13. Hunskaar S, Hole K. The formalin test in mice: dissociation between inflammatory and non-inflamma-
tory pain. Pain. 1987; 30: 103–114. PMID: 3614974
14. Santos DA, Fukuib MJ, Nanayakkarac NPD, Khan SI, Sousa JP, Bastos JK, et al. Anti-inflammatory
and antinociceptive effects of Baccharis dracunculifolia DC (Asteraceae) in different experimental mod-
els. J Ethnopharmacol. 2010; 127: 543–550. https://doi.org/10.1016/j.jep.2009.09.061 PMID:
19808087
15. Holanda ADV, Asth L, Santos AR, Guerrini R, Soares-Rachetti VP, Calo’ G, et al. Central adenosine A1
and A2A receptors mediate the antinociceptive effects of neuropeptide S in the mouse formalin test.
Life Sciences. 2015; 120: 8–12. https://doi.org/10.1016/j.lfs.2014.10.021 PMID: 25447449
16. Leite LHI, Leite GO, Coutinho TS, Sousa SDG, Sampaio RS, Costa JGM, et al. Topical Antinociceptive
Effect of Vanillosmopsis arborea Baker on Acute Corneal Pain in Mice. J Evid Based Complementary
Altern Med. 2014; 2014: 1–6.
17. Pereira JG, Mesquita JX, Aragão KS, Franco AX, Souza MHLP, Brito TV, et al. Polysaccharides iso-
lated from Digenea simplex inhibit inflammatory and nociceptive responses. Carbohyd Polym. 2014;
108: 17–25.
18. Melo MS, Guimarães AG, Santana MF, Siqueira RS, Lima ACB, Dias AS, et al. Anti-inflammatory and
redox-protective activities of citronellal. Biol Res. 2011; 44: 363–368. https://doi.org//S0716-
97602011000400008 PMID: 22446600
19. Winter CA, Risley EA, Nuss GW. Carrageenan-induced oedema in hind paw of rat as an assay for anti-
inflammatory drugs. Proc Soc BioI Med. 1962; 111: 544–547.
20. Yamazaki Y, Yasuda K, Matsuyama T, Ishihara T, Higa R, Sawairi T, et al. A Penicillium sp. F33 metab-
olite and its synthetic derivatives inhibit acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltrans-
ferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-induced paw edema in
mice. Biochem Pharmacol. 2013; 86: 632–644. https://doi.org/10.1016/j.bcp.2013.06.021 PMID:
23817078
21. Huang GJ, Pan CH, Liu FC, Wu TS, Wu CH. Anti-inflammatory effects of ethanolic extract of Antrodia
salmonea in the lipopolysaccharide-stimulated RAW246.7 macrophages and the k-carrageenan-
induced paw edema model. Food Chem Toxicol. 2012; 50: 1485–1493. https://doi.org/10.1016/j.fct.
2012.01.041 PMID: 22326970
22. Castardo JC, Prudente AS, Ferreira J, Guimarães CL, Monache FD, Filho VC, et al. Anti-inflammatory
effects of hydroalcoholic extract and two biflavonoids from Garcinia gardneriana leaves in mouse paw
oedema. J Ethnopharmacol. 2008; 118: 405–411. https://doi.org/10.1016/j.jep.2008.05.002 PMID:
18555627
23. Molyva D, Kalokasidis K, Poulios C, Dedi H, Karkavelas G, Mirtsou V, et al. Rupatadine effectively pre-
vents the histamine-induced up regulation of histamine H1R and bradykinin B2R receptor gene expres-
sion in the rat paw. Pharmacol Rep. 2014; 66: 952–955. https://doi.org/10.1016/j.pharep.2014.06.008
PMID: 25443720
24. Barbosa AGR, Oliveira CDM, Lacerda-Neto LJ, Vidal CS, Saraiva RA, da Costa JG, et al. Evaluation of
chemical composition and antiedematogenic activity of the essential oil of Hyptis martiusii Benth. Saudi
J Biol Sci. 2015; 24: 355–361. https://doi.org/10.1016/j.sjbs.2015.10.004 PMID: 28149173
25. Oliveira RB, Chagas-Paula DA, Secatto A, Gasparoto TH, Faccioli LH, Campanelli AP, et al. Topical
anti-inflammatory activity of yacon leaf extracts. Braz. J. Pharmacogn. 2013; 23: 497–505.
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
17 / 19

LASSBio-1586 attenuates nociceptive behavior and inflammatory response in mice
26. Leite GO, Leite LHI, Sampaio RS, Araruna MKA, Rodrigues FFG, de Menezes IRA, et al. Modulation of
topical inflammation and visceral nociception by Vanillosmopsis arborea essential oil in mice. Biomed
Prev Nutr. 2011; 1: 216–222.
27. Ibrahim B, Sowemimo A, Rooyen A, Van de Venter M. Anti-inflammatory, analgesic and antioxidant
activities of Cyathula prostrata (Linn.) Blume (Amaranthaceae). J. Ethnopharmacol. 2012; 141: 282–
289. https://doi.org/10.1016/j.jep.2012.02.032 PMID: 22387161
28. Karram EH, Harper GP. Collagen Degradation Within Subcutaneous Air Pouches In Vivo: The Effects
of Proteinase Inhibitors. J Pharmacol Toxicol Methods. 1995; 34: 97–102. PMID: 8563038
29. Ozdol NC, Melli M. Formation of 8-isoprostaglandin F2α and prostaglandin E2 in carrageenan-induced
air pouch model in rats. Eur J Pharmacol. 2004; 506: 189–197. https://doi.org/10.1016/j.ejphar.2004.
10.050 PMID: 15588740
30. Maleki-Dizaji N, Eteraf-Oskouei T, Fakhrjou A, Maljaie SH, Garjani A. The effects of 5HT3 receptor
antagonist granisetron on inflammatory parameters and angiogenesis in the air-pouch model of inflam-
mation. Int Immunopharmacol. 2010; 10: 1010–1016. https://doi.org/10.1016/j.intimp.2010.05.013
PMID: 20646986
´
´
31. Marroquin-Segura R, Flores-Pimentel M, Carreon-Sanchez R, Garcia-Burciaga MM, Mora-Guevaraa
JLA. The effect of the aqueous extract of Helietta parvifolia A. Gray (Rutaceae) stem bark on carra-
geenan-induced paw oedema and granuloma tissue formation in mice. J Ethnopharmacol. 2009; 124:
639–641. https://doi.org/10.1016/j.jep.2009.06.004 PMID: 19524657
32. Zhang CX, Dai ZR, Cai QX. Anti-inflammatory and antinociceptive activities of Sipunculus nudus L.
extract. J Ethnopharmacol. 2011; 137: 1177–1182. https://doi.org/10.1016/j.jep.2011.07.039 PMID:
21807085
33. Xu Q, Wang Y, Guo S, Shen Z, Wang Y, Yang L. Anti-inflammatory and analgesic activity of aqueous
extract of Flos populi. J Ethnopharmacol. 2014; 152: 540–545. https://doi.org/10.1016/j.jep.2014.01.
037 PMID: 24508857
34. Meshram GG, Kumar A, Rizvi W, Tripathi CD, Khan RA. Evaluation of the anti-inflammatory activity of
the aqueous and ethanolic extracts of the leaves of Albizzia lebbeck in rats. J Tradit Complement Med.
2015; 2015: 1–4.
35. Xu S, Hermanson DJ, Banerjee S, Ghebreselasie K, Clayton GM, Garavito RM, et al. Oxicams Bind in a
Novel Mode to the Cyclooxygenase Active Site via a Two-water-mediated H-bonding Network. J Biol
Chem. 2014; 289: 6799–6808. https://doi.org/10.1074/jbc.M113.517987 PMID: 24425867
36. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, et al. The Protein Data Bank.
Nucleic Acids Res. 2000; 28: 235–242. Available from: http://www.rcsb.org. PMID: 10592235
37. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ. Autodock4 and Auto-
DockTools4: automated docking with selective receptor flexiblity. J Comput Chem. 2009; 16: 2785–
2791.
38. ACD/ChemSketch, version 12.01, Advanced Chemistry Development, Inc., Toronto, ON, Canada,
www.acdlabs.com, 2015. Available from: http://www.acdlabs.com.
39. GaussView, Version 6, Dennington R, Keith TA, Millam JM. Semichem Inc., Shawnee Mission, KS,
2016.
40. Gaussian 09, Revision A.03, Frisch M.J, Trucks GW, Schlegel HB, Scuseria GE, Robb M A, Cheese-
man JR, et al. Gaussian, Inc., Wallingford CT, 2016.
41. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF Chimera—a
visualization system for exploratory research and analysis. J Comput Chem. 2004; 25: 1605–12.
https://doi.org/10.1002/jcc.20084 PMID: 15264254
42. Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, et al. Automated docking using a
Lamarckian genetic algorithm and an empirical binding free energy function. J Comp Chem. 1998; 19:
1639–1662.
43. Bardin L. The complex role of serotonin and 5-HT receptors in chronic pain. Behav Pharmacol. 2011;
22: 390–404. https://doi.org/10.1097/FBP.0b013e328349aae4 PMID: 21808193
44. Abbott FV, Hong Y, Blier P. Activation of 5-HT2A receptors potentiates pain produced by inflammatory
mediators. Neuropharmacology. 1996; 35: 99–110. PMID: 8684602
45. Brenchat A, Romero L, Garcia M, Pujol M, Burgueno J, Torrens A, et al. 5-HT7 receptor activation inhib-
its mechanical hypersensitivity secondary to capsaicin sensitization in mice. Pain; 141: 239–247.
https://doi.org/10.1016/j.pain.2008.11.009 PMID: 19118950
46. Cury Y, Picolo G, Gutierrez VP, Ferreira SH. Pain and analgesia: The dual effect of nitric oxide in the
nociceptive system. Nitric Oxide. 2011; 25: 243–54. https://doi.org/10.1016/j.niox.2011.06.004 PMID:
21723953
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
18 / 19

LASSBio-1586 attenuates nociceptive behavior and inflammatory response in mice
47. Cunha TM, Roman-Campos D, Lotufo CM, Duarte HL, Souza GR, Verri-Junior WA, et al. Morphine
peripheral analgesia depends on activation of the PI3Kgamma/AKT/nNOS/NO/KATP signaling path-
way. Proc Natl Acad Sci USA. 2010; 107: 4442–4447. https://doi.org/10.1073/pnas.0914733107 PMID:
20147620
48. Gold MS, Gebhart GF. Nociceptor sensitization in pain pathogenesis. Nat Med. 2010; 16: 1248–1257.
https://doi.org/10.1038/nm.2235 PMID: 20948530
49. Ricciotti E, FitzGerald GA. Prostaglandins and Inflammation. Arterioscler Thromb Vasc Biol. 2011; 31:
986–1000. https://doi.org/10.1161/ATVBAHA.110.207449 PMID: 21508345
50. Schreiber R, Zechner R. Lipolysis meets inflammation: arachidonic acid mobilization from fat. J Lipid
Res. 2014; 55: 2447–2449. https://doi.org/10.1194/jlr.C055673 PMID: 25332433
51. Trott O, Olson AJ. Software news and update AutoDock Vina: improving the speed and accuracy of
docking with a new scoring function, efficient optimization, and multithreading. J Comp Chem. 2010; 31:
455–461.
52. Lipinski CA. Lead- and drug-like compounds: The rule-of-five revolution. Drug Discov Today Technol
2004; 1: 337–341. https://doi.org/10.1016/j.ddtec.2004.11.007 PMID: 24981612
PLOS ONE | https://doi.org/10.1371/journal.pone.0199009 July 30, 2018
19 / 19