2795

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Current Topics in Medicinal Chemistry, 2019, 19, 2795-2804

RESEARCH ARTICLE
ISSN: 1568-0266
eISSN: 1873-4294

Search for Potential Inducible Nitric Oxide Synthase Inhibitors with

Favorable ADMET Profiles for the Therapy of Helicobacter pylori Impact
Factor:
3.442

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Ricardo Pereira Rodrigues1,*, Juliana Santa Ardisson1, Rita de Cássia Ribeiro Gonçalves1,2,
Tiago Branquinho Oliveira3, Vinicius Barreto da Silva4, Daniel Fábio Kawano5 and
Rodrigo Rezende Kitagawa1,2

1
Graduate Program in Pharmaceutical Sciences, Health Sciences Center - CCS, Federal University of Espirito Santo -
UFES, Marechal Campos Av., 1468, Vitoria 29043-900, ES, Brazil; 2Department of Pharmaceutical Sciences, Federal
University of Espirito Santo-UFES, Marechal Campos Av., 1468, Vitoria 29043-900, ES, Brazil; 3Department of Phar-
macy, Federal University of Sergipe (UFS-SE), Av. Marechal Rondon s/n, Jd. Rosa Elze, Sao Cristovao 49100-000, SE,
Brazil; 4Department of Biomedicine and Pharmacy, Pontifical Catholic University of Goiás, 74605-140 Goiania-GO,
Current Topics in Medicinal Chemistry

Brazil; 5Faculty of Pharmaceutical Sciences, University of Campinas, Rua Cândido Portinari 200, 13083-871 Campi-
nas-SP, Brazil

Abstract: Background: Helicobacter pylori is a gram-negative bacterium related to chronic gastritis, peptic
ulcer and gastric carcinoma. During its infection process, promotes excessive inflammatory response, increas-
ing the release of reactive species and inducing the production of pro-inflammatory mediators. Inducible Nitric
Oxide Synthase (iNOS) plays a crucial role in the gastric carcinogenesis process and a key mediator of in-
flammation and host defense systems, which is expressed in macrophages induced by inflammatory stimuli. In
chronic diseases such as Helicobacter pylori infections, the overproduction of NO due to the prolonged induc-
tion of iNOS is of major concern.
Objectives: In this sense, the search for potential iNOS inhibitors is a valuable strategy in the overall process
ARTICLE HISTORY
of Helicobacter pylori pathogeny.

Received: July 18, 2019
Revised: September 04, 2019
Accepted: October 04, 2019
DOI:
10.2174/1568026619666191112105650
Methods: In silico techniques were applied in the search of interesting compounds against Inducible Nitric
Oxide Synthase enzyme in a chemical space of natural products and derivatives from the Analyticon Discov-
ery databases.
Results: The five compounds with the best iNOS inhibition profile were selected for activity and toxicity pre-
dictions. Compound 9 (CAS 88198-99-6) displayed significant potential for iNOS inhibition, forming hydro-
gen bonds with residues from the active site and an ionic interaction with heme. This compound also displayed
good bioavailability and absence of toxicity/or from its probable metabolites.
Conclusion: The top-ranked compounds from the virtual screening workflow show promising results regard-
ing the iNOS inhibition profile. The results evidenced the importance of the ionic bonding during docking se-
lection, playing a crucial role in binding and positioning during ligand-target selection for iNOS.

Keywords: Molecular docking, Virtual screening, Helicobacter pylori, Inducible nitric oxide synthase, Natural product re-
search, Gastric carcinogenesis.

1. INTRODUCTION ized by the production of reactive oxygen (ROS) and reac-

Helicobacter pylori is one of the main causes of gastric
cancer, being categorized as a group I carcinogen and repre-
senting the third cause of deaths related to cancers in the
world [1, 2]. The development of gastric cancer is promoted
by lesions in the mucosa and in the cellular DNA associated
with the inflammatory response. This condition is character
*Address correspondence to this author at the Department of Pharmaceuti-
cal Sciences, Health Sciences Center - CCS, Federal University of Espírito
Santo - UFES, Av. Marechal Campos 1468, 29047-105 Vitoria-ES, Brazil;
Tel: 55-27-3335-7307; E-mail: ricardo.p.rodrigues@ufes.br
tive nitrogen (nitric oxide - NO) species resulting from the
infiltration of defense cells and the increased expression of
proinflammatory cytokines such as IL-6, IL-8 and tumor
necrosis factor (TNF-α) [3 ,4].
Nitric oxide (NO) is a small, diffusible and transient
molecule produced from the amino acid L-arginine by three
nitric oxide synthase (NOS) enzymes. Endothelial (eNOS)
and neuronal (nNOS) isoenzymes are constitutively ex-
pressed and regulated by Ca+2 ions, providing that NO sig-
naling is associated with vasodilation, thermoregulation,
neuroprotection and endocrine function.

1873-4294/19 $58.00+.00 © 2019 Bentham Science Publishers

2796 Current Topics in Medicinal Chemistry, 2019, Vol. 19, No. 30
The inducible, non-sensitive NOS isoenzyme (iNOS) is
expressed in response to cytokines or pathogens and pro-
duces NO at a high rate to kill bacteria, viruses and tumor
cells. Excess of NO from iNOS has been implicated in in-
flammation, rheumatoid arthritis, inflammatory bowel dis-
ease, immune-type diabetes, stroke, cancer, thrombosis and
infection. Overproduction of NO by iNOS (and nNOS) has
also been associated with neurodegenerative disorders, in-
cluding Parkinson's and Alzheimer's disease, as well as mul-
tiple sclerosis [5, 6].
The iNOS enzyme produces NO when it is induced by
cytokines and lipopolysaccharides through the substrates L-
arginine and oxygen, having as an electron donor NADPH
(nicotinamide dinucleotide phosphate and adenine), while
the electrons are donated to the iron heme at the active site of
iNOS [7]. The iNOS isoform can produce NO at toxic levels
to the individual's cell and tissues, which is related to the
development of several pathologies, including cancer. There-
fore, experimental substances capable of inhibiting this en-
zyme are of increasing clinical importance [4, 6].
Several substances with the ability of inhibiting the pro-
duction of this enzyme have already been reported: methyl
(3S)-3-{2-[(1,3-benzodioxol-5-ylmethyl)amino]-2-oxoeth-
yl}-4-[2 -(1H-imidazol-1-yl)pyrimidin-4-yl]piperazine-1-
carboxylate (1), N-(3-aminomethyl) benzyl)acetamidine in-
hibitor, 1400W (2), Nω-Nitro-L-arginine methyl ester, L-
NAME (3) and the natural substrate, L-arginine (4) (Fig. 1)
[8-10].
O
N 2.2. 3-D Shape and Electrostatic Similarity Search
O
N
H N
N The bioactive conformation of 1 at the iNOS binding site
N
N was used for the development of a 3-D shape-based model
O with ROCS software [16] and also used as a query for the
N
O NH2 HN
O the MDL MOL file as the 3-D input. The top-ranked solu-
1 2
H2N NH H2N
HN HN
O HN NO2 O NH
OCH3 OH
3 4
Fig. (1). Chemical structures of the iNOS inhibitors methyl (3S)-3-
{2-[(1,3-benzodioxol-5-ylmethyl)amino]-2-oxoethyl}-4-[2-(1H-
imidazol-1-yl) pyrimidin-4-yl]piperazine-1-carboxylate (1), N-(3-
aminomethyl)benzyl) acetamidine (2), Nω-Nitro-L-arginine methyl
ester (3) and the natural substrate, L-arginine (4).
The L-NAME is a non-selective inhibitor of the NOS iso-
forms and acts inhibiting the enzyme through competition
with L-arginine [10], while 1 and 2 selectively inhibit the
iNOS enzyme [8, 9]. The substrate L-arginine 4, fits into the
binding cavity in a position that its guanidino group is copla-
nar to the heme, also allowing the establishment of two hy-
drogen bonds with the amino acids glutamate and trypto-
phan, being these residues critical points of interaction for
the substrate at the iNOS binding site. The iron at the heme
group is coordinated with the amino acid cysteine at the top
of the binding site and this heme is also stabilized by van der
Walls interactions with the side chains of the amino acids
tryptophan and phenylalanine [7].
Rodrigues et al.
Concerning the search and development of novel iNOS
inhibitors, natural products are a valuable source of biologi-
cally relevant molecular entities. Natural products-inspired
screening databases can help to transfer biologically relevant
information (e.g., pharmacophores) to synthetically more
accessible molecules, improving the biological activity of
these synthetic small molecules [11].
The drug discovery optimization provided by in silico
ADMET methods aiming the development of active sub-
stances provides the reduction of research costs, such as or-
ganic solvents, drugs and reagents, which are used for the
isolation, purification of substances and the execution of
bioassays. It is, therefore, the integration between in vivo, in
vitro and in silico methods within the scope of drug design,
providing a more extensive search in its spectrum of activity.
2. MATERIALS AND METHODS
2.1. Data Preparation
The chemical structures were drawn with Marvin Sketch
software [12] and saved in MOL and SDF file formats for
post-processing analysis [13]. The 3-D chemical structures
were minimized with MMFF94 force field of Avogadro
software [14]. Conformer generation was performed with
OMEGA software [15]. The protein binding site analysis
was over the co-crystallographic murine iNOS protein-ligand
complex 1DD7. The database used for virtual screening was
Analyticon Discovery from ZINC (http://zinc.docking.org).
NH electrostatic similarity search with EON software [17], using
tions from ROCS and EON were filtered with PASS [18]
and DEREK [19] software for ADMET predictions.
NH2
2.3. Molecular Docking
Docking calculations were conducted with GOLD 5
software [20] analysing the pose-binding over the crystallo-
graphic protein-ligand complex of iNOS protein, (PDB ID
1DD7), obtained from the Protein Data Bank server [21],
using the CHEMSCORE score function [22], centered in a
10 Å radius sphere over the coordinates of the central ligand.
Docking runs were set to 10 for the average virtual screening
and the highest score solutions were saved for visual inspec-
tion at protein binding site and used for pose-binding selec-
tion of the top-ranked compounds.
2.4. ADMET Predictions
The Prediction of Activity spectra of substances (PASS)
was used to select the most promising iNOS inhibitors and
the Meteor software to predict the metabolic pathways for
the top-ranked compounds. Simulations included phase I/II
biotransformations processed until the 3rd generation of
probable metabolites (minimum likelihood), where they are
conjugated or have low log P values, being more likely to be
excreted [19].
Search for Potential Inducible Nitric Oxide Synthase Inhibitors
The analysis of toxicophoric groups regarding the parent
structure and their proposed metabolites was carried out us-
ing the software Derek in order to model the toxicity end-
points, including carcinogenicity, mutagenicity, genotoxic-
ity, skin sensibilization, teratogenicity, airways hyperreactiv-
ity, hepatotoxicity, neurotoxicity, among others. Meteor and
Derek identify metabolic reactions and toxicophoric groups
(structural moieties that can exert toxic effects in a specific
compound), respectively, through a knowledge-based system
wherein structure-property correlations are searched from a
database built from relevant literature concerning metabolic
and toxicological data [19].
Molecular Discovery VolSurf Plus was then used to es-
timate the main pharmacokinetic parameters of these com-
pounds: the distribution coefficient, hydrosolubility, plasma-
protein binding, CYP450 3A4 metabolic stability, Caco-2
permeability and blood-brain barrier permeability. The soft-
ware calculates molecular descriptors based on the three-
dimensional GRID molecular interaction fields and has been
successfully used to predict the ADMETox properties for a
number of drugs [23].
The hydrosolubility was expressed as - Log[Soly], where
Soly is the solubility in water (mol/L) at 25ºC. Compounds
are predicted to have a poor solubility when - Log[Soly] <
−4, a medium solubility when − 4 ≤ - Log[Soly] < −1 and a
high solubility when - Log[Soly] ≥ −1. The cytochrome
P450 3A4 isoform (CYP3A4) metabolic stability estimates
the final concentration of a compound incubated for 60 min-
utes with a fixed concentration of the enzyme at 37°C, where
compounds with a final concentration greater than or equal
to 50% are considered as stables [23].
Caco-2 cell permeabilities were expressed as transformed
values where a positive index expresses that the compound is
Fig. (2). Shape-based model developed by ROCS software evidencing the ring system (green), the acceptor (red) and the donor (blue)
spheres. Hydrophobic motifs were indicated by light orange spheres.3.2. ROCS validation.
Current Topics in Medicinal Chemistry, 2019, Vol. 19, No. 30 2797
well-absorbed and a negative value indicates that the com-
pound is poorly absorbed. Compounds are assumed as brain-
penetrating for LogBB > 0.5, with moderate permeation for
0.5 ≤ LogBB < 0, with reduced ability to cross the BBB for 0
≤ LogBB < −0.3 or very little brain-penetrating when LogBB
< −0.3 [23].
3. RESULTS AND DISCUSSION
3.1. Virtual Screening Based on Three-dimensional
Shape and Electrostatic Similarity
The shape-based model was developed based on the bio-
active conformation of the crystallographic inhibitor 1,
which occupies part of the L-arginine binding site and due to
its bulky shape also blocks the dimerization process of iNOS
by accessing part of an allosteric site ROCS identifies simi-
larities among the chemical structures from the database and
the query molecule based on their three-dimensional shapes.
Its algorithm is based on a series of implementations of mo-
lecular shape comparison, in order to find and identify the
maximum overlap of volumes between two chemical struc-
tures [15, 24, 25]. Subsequently, EON was employed to rank
the chemical structures by its Electrostatic Tanimoto Combo
Score (ET combo) that is the sum of the measures of the
Poisson-Boltzmann Electrostatic Tanimoto Coefficient (ET
pb) and the Shape Tanimoto Escore (ST), comparing the
electrostatic potential of input molecules and its overlapped
volume against a query [16, 22].
The model was optimized and weighted by a dataset of
30 highly active compounds and 998 inactive compounds
(Fig. 2; Fig. S1, from supplemental material).
The Receiver Operating Characteristic (ROC) curve
method was used for the quality control plotting actives
2798 Current Topics in Medicinal Chemistry, 2019, Vol. 19, No. 30
against inactives (decoys). The calculated area under the
curve (AUC) of 0.86 represents greater selectivity in favor of
the actives, giving an indication of how the actives and inac-
tives are ranked during a ROCS run. It describes the prob-
ability that a randomly chosen active has a higher score than
a randomly chosen inactive and thus, high values are desir-
able in order to obtain a selective model. In the present sce-
nario, the actives were retrieved first since they score most
highly (Fig. 3).
1.00
0.75
Fraction decoys found
0.50
0.25
0.00
0.00 0.25 0.50
Fraction actives found
Fig. (3). ROC curve for the theoretical validation of the 3D shape-
based model.
3.2. Docking Validation and Analyses of Interactions
The reference complex (PDB ID: 1DD7) was chosen for
docking since its crystallographic ligand (1) has the ability to
bind to the natural substrate interaction region of L-arginine
and is a bulky binder, that promotes a conformational change
in the iNOS structure that prevents the protein from adopting
its active conformation. The main binding site interactions
observed for the crystallographic inhibitor comprises an
ionic bond with the heme iron complex, at 2.2 Å, a hydrogen
bond with amino acid Tyr485 and hydrophobic contributions
stabilizing the main chain with the amino acids Gln257,
Val346, Phe363, Asn364, Gly365, Trp366 and Tyr367 (Fig. 4).
Fig. (4). Interactions of 1 at iNOS binding site: cysteine residue and
the nitrogen of imidazolic ring of 1 coordinates the iron of heme
group. Some amino acids were hidden for better visualization of the
image.
Rodrigues et al.
The pose prediction accuracy of the docking software
was evaluated by redocking analysis, extracting the co-
crystal ligand from this file, leaving only the protein that was
prepared for the docking calculations. Once the simulation
was performed, the docking poses were compared to the pro-
tein-ligand complex by measuring the Root Mean Square
Deviation (RMSD) obtaining a value below 2 Å (RMSD =
1.503 Å) (Fig. 5).
0.75 1.00
Fig. (5). Redocking of 1 at iNOS binding site: RMSD = 1.503 Å;
PDB ID: 1DD7. Docking pose is colored in white, whereas the
crystallographic inhibitor’s carbon atoms colored in gray.
The shape-based model was searched against a collection
of Natural Products from AnalytiCon Discovery, which
comprises almost 30,000 structures, being more than 5,000
of natural products (http://zinc.docking.org/catalogs/
acdiscnp) and the remaining (nearly 25,000) of natural de-
rivatives (http://zinc.docking.org/catalogs/acdiscnd).
To study the binding modes of these compounds, analy-
ses of their intermolecular interactions with the iNOS were
performed, using the results from the crystallographic ligand
1 as the reference for the top 100 solutions retrieved from the
shape-based virtual screening. By visual inspection, 20 com-
pounds with the best binding profiles were selected and their
most probable biological activities were ascribed using the
PASS software, in which the biological spectrum is pre-
dicted based on the probability of a structure being active
(Pa) or inactive (Pi) based on the similarity with a database
of active compounds. We selected the top-5 iNOS inhibitors
both based on the predictions of iNOS inhibition per se but
also considering some related pathophysiological scenarios
such as inflammation or cancer (Fig. 6, Table 1).
In this context, biological activities related to direct iNOS
inhibition were ascribed to all the reference compounds, ex-
cept for the crystallographic ligand, which was categorized
only as an antineoplastic compound (Table 1). For the
screened compounds, only compound 9 was suggested as a
direct iNOS inhibitor and all the remaining compounds were
categorized only with actions possibly related to iNOS inhi-
bition.
Search for Potential Inducible Nitric Oxide Synthase Inhibitors Current Topics in Medicinal Chemistry, 2019, Vol. 19, No. 30 2799

 

 



Fig. (6). Docking poses of the top compounds. A) ZINC000035464678_41 (5) Docking score = 44.2, ΔG (kJ/mol) = -53.88; B)
ZINC000013378197_42 (6) Docking score = 39.77, ΔG (kJ/mol) = -45.49; C) ZINC000031156495_28 (7) Docking score = 35.90, ΔG
(kJ/mol) = -38.39; D) ZINC000104648178_49 (8) Docking score = 35.58, ΔG (kJ/mol) = -37.03; E) ZINC000014760235_44 (9) Docking
score = 34.77, ΔG (kJ/mol) = -39.94.
2800 Current Topics in Medicinal Chemistry, 2019, Vol. 19, No. 30 Rodrigues et al.

Table 1. Predicted biological activities for the reference ligands (1 - 4) and the top-5 putative iNOS inhibitors. Predictions were
performed in the PASS software and reflect the probability of a structure being active (Pa) or inactive (Pi).

Compound CAS Number PASS Prediction (Pa/Pi)

O
N
O
N
H N
N
N
N
Antineoplastic: non-Hodgkin's lymphoma (0.476/0.047)
O 934535-20-3
N Antineoplastic: solid tumors (0.315/0.078)
O
O
(1)

iNOS inhibitor (0.699/0.002)

NH2 HN NH 180001-34-7 NOS inhibitor (0.69/0.002)
Anti-inflammatory, intestinal (0.273/0.095)
(2)
H2 N NH
HN Inducible nitric-oxide synthase inhibitor (0.091/0.018)
O HN NO2 1040747-63-4 Nitric-oxide synthase inhibitor (0.22/0.005)
OCH3
Arginine 2-monooxygenase inhibitor (0.918/0.003)
(3)
H2 N NH2
HN Inducible nitric-oxide synthase inhibitor (0.281/0.004)
O NH 74-79-3 Nitric-oxide synthase inhibitor (0.671/0.002)
OH
Arginine 2-monooxygenase inhibitor (0.986)
(4)

O
O Superoxide dismutase inhibitor (0.258/0.157)
O Arginine 2-monooxygenase inhibitor (0.268/0.157)
OH 1212365-37-1
Anti-inflammatory, intestinal (0.386/0.027)
Antineoplastic (0.672/0.031)
(5)
O OH
Arginine 2-monooxygenase inhibitor (0.452/0.06)
257301-36-3 Anti-inflammatory (0.552/0.043)
HO OH
Anti-inflammatory, intestinal (0.461/0.011)
(6)

O
O OH
O Antineoplastic alkaloid (0.341/0.025)
156002-98-1 Antineoplastic (bone cancer) (0.266/0.018)
HO O
Antineoplastic (multiple myeloma) (0.263/0.128)
OH
O
(7)
HO
OH OH
O Antineoplastic alkaloid (0.338/0.026)
OH 1212337-84-2 Antineoplastic (bone cancer) (0.252/0.03)
O Antineoplastic (gastric cancer) (0.136/0.075)

(8)
O
O
O Inducible nitric-oxide synthase inhibitor (0.062/0.049)
O 88198-99-6 Non-steroidal anti-inflammatory agent (0.208/0.094)
O O Antineoplastic (0.582/0.049)
(9)
Search for Potential Inducible Nitric Oxide Synthase Inhibitors Current Topics in Medicinal Chemistry, 2019, Vol. 19, No. 30 2801

The binding modes of the selected screened compounds
presented a binding pattern coordinated by the ionic interac-
tions with the two nickel ions of the binding site. Compound
9 presented a binding mode with 2 hydrogen bonding to the
residues Tyr485 (2.6 Å) and Met368 (2.7 Å) and an ionic inter-
action with iron from heme similar to the reference inhibitor,
1 (Fig. 4). Therefore, both the analyses of the predicted bio-
logical spectra and of toxicity endpoints suggest a safety and
efficacy profile for compound 9 that is comparable to the
reference iNOS inhibitors 1-4.
3.3. Prediction of Pharmacokinetics and Toxicity Profiles
To assess the pharmacokinetic profiles of the potential
iNOS inhibitors and the reference ligands 1-4, we performed
in silico predictions based on the 3-D structures of the com-
pounds (Table 2). The distribution coefficients for 2-4 and 7
indicate relative hydrophilic behaviors, with good water
solubility (- log [Soly] ≥ −1) predicted only for 2 and 4,
while compound 5 displayed an opposite profile. Com-
pounds 1, 6, 8 and 9 fitted in the ideal range predicted for
drugs with good bioavailability (1 < LogD7.5 < 3), with a
moderate water solubility (−4 ≤ - log [Soly] < −1) predicted
for 8. Compounds 1, 2, 5, 6, 8 and 9 are expected to have
good permeability (as reflected by the positive scores) in
human colon adenocarcinoma cells (Caco-2), which repre-
sent an in vitro model for the estimation of in vivo absorption
from the intestinal epithelial cells [23, 26].
Because of the relative low lipophilicity (except for 5),
most of the compounds displayed low to moderate affinities
for plasma proteins and all displayed low volumes of distri-
bution (VD < 3L). Most of the compounds are also expected
to undergo low first-pass metabolism catalyzed by the
CYP450 system, 3A4 isoform, except for 1, 5 and 9, which,
hypothetically, could yield toxic metabolites [23].
Considering this, we also predicted the main toxicity
endpoints for the nine compounds (Table 3), which included
carcinogenicity, mutagenicity, genotoxicity, teratogenicity,
hepatotoxicity and skin sensibilization). The DEREK soft-
ware identifies toxicophoric groups using a knowledge-based
system, wherein the structure-property correlations are
searched from a database built from relevant literature con-
cerning the toxicological data [19].
Analyses of the toxicological profiles for the parent drugs
suggest the reference iNOS inhibitors as safe substances,
except for 3, where the nitro group was suggested as a trig-
ger for carcinogenicity, mutagenicity and chromosome dam-
age (Table 3). However, 1, 2 and 4 were subsequently evalu-
ated for the generation of probable toxic metabolites and 1 is
expected yield a compound with a risk for the development
of hepatoxicity and skin rashes after the oral administration
[27-29].
Concerning the screened inhibitors, compound 9 was the
only substance free from toxicity endpoints for the parent
structure and the probable metabolites, while compound 7,
conversely, was prompted with seven different potential tox-
icities. This particular substance has an epoxide as a sub-
structure, which tend to be irritating to the skin and to the
eyes [28]. Epoxides are also strong alkylating agents be-
cause, after ring-opening, they form a reactive ion that alky-
lates the DNA. Consequently, exposure to such chemicals is
strongly contraindicated during pregnancy [29].
All the remaining triggers for toxicities for compounds 5-
8 are associated with a phenol or some structurally-related
group (e.g. catechol or p-alkylphenol). For compounds with
these substructures, skin sensitization results from the gen-
eration of phenolic radicals that subsequently reacts with
skin proteins, primarily at the ortho-positions of the phenol
group [30] and the same can be inferred for mitochondrial
dysfunction both for phenols and alkylphenols [31].
However, the most problematic triggers were obtained
for compounds containing the p-alkylphenol substructure
mainly because of the risk of hepatotoxicity, since alkylphe-
nols are known to significantly affect the liver function and
enzyme levels, causing centrilobular necrosis and cholestasis

Table 2. Main predicted pharmacokinetic parameters for the reference ligands (1 - 4) and the top-5 putative iNOS inhibitors.

Partition Coefficient Hydrosolubility Volume of Albumin CYP3A4 Metabolic Caco−2

Compound
(LogD7.5) - log [Soly] Distribution (L) Binding (%) Stability (%) Permeability

1 1.13 -4.63 -0.25 65 40 0.11

2 -4.29 -0.59 0.24 45 100 0.18

3 -1.72 -1.07 0.25 32 100 -0.26

4 -5.88 0.78 0.11 11 100 -0.90

5 4.16 -4.85 0.22 85 31 1.20

6 2.67 -4.44 -0.02 70 55 0.33

7 0.71 -4.33 -0.36 69 68 -0.32

8 2.27 -3.90 0.15 59 70 0.10

9 2.25 -4.07 -0.15 78 37 0.98

2802 Current Topics in Medicinal Chemistry, 2019, Vol. 19, No. 30 Rodrigues et al.

Table 3. Main toxicity endpoints calculated for the reference ligands (1 - 4), the top-5 putative iNOS inhibitors and some of their
main probable metabolites.

Toxicophore at the Expected Toxicities for the Main Toxic Metabolite Expected Toxicities for the
Compound
Parent Structure Parent Structure Metabolite
O OH

O HN O

O HN Hepatotoxicity (2-
N
NH N
Aminopyrimidine) and skin
1 Not observed Not observed
sensitisation (formaldehyde
O N
donor)
N
O
O

2 Not observed Not observed Not observed Not observed

3 Nitro group
4 Not observed
5 Substituted phenol
6 p-Alkylphenol
Epoxide, phenol and p-
7 -
alkylphenol
Catechol and p-
8 sensitisation (catechol), hepato-
alkylphenol
9 Not observed
Carcinogenicity, mutagenicity
- -
and chromosome damage
Not observed Not observed Not observed
Skin sensitisation - -
Hepatotoxicity, chromosome
- -
damage and skin sensitisation
Developmental toxicity, skin
sensitisation, ocular irritation
(epoxide), mitochondrial dysfunc-
-
tion (phenol), chromosome dam-
age, hepatotoxicity, (p-
alkylphenol)
Chromosome damage and skin
- -
toxicity (p-alkylphenol)
Not observed Not observed Not observed

in experimental animals with depleted glutathione levels CONSENT FOR PUBLICATION

[32].
Not applicable.

CONCLUSION AVAILABILITY OF DATA AND MATERIALS

iNOS enzyme is related to a number of pathologies such The data supporting the findings of the article is available
as inflammatory diseases, autoimmune, septic shock, hemor- at the corresponding authors's research group at
rhagic shock, rheumatoid arthritis, osteoarthritis, multiple https://drive.google.com/open?id=1znBuzOXrZYkTYLCFsF
sclerosis. The search for new potentially active substances rUCHd8uBFdGpt0.
for this target is of major importance. The use of natural
products-inspired databases can help to obtain potentially FUNDING
relevant chemical structures with biologically interesting This study was financed in part by the “Coordenação de
chemical features. Based on the activity predictions and Aperfeiçoamento de Pessoal de Nível Superior - Brasil
docking calculations, compound 9 was the most promising (CAPES)” - Finance Code 001.
one with a binding mode similar to the reference compound
2 without relevant toxicity endpoints or toxic metabolites CONFLICT OF INTEREST
and can be used as starting point to the drug development
process in the search for new highly active derivatives. The authors declare no conflict of interest, financial or
otherwise.
ETHICS APPROVAL AND CONSENT TO PARTICI-
PATE ACKNOWLEDGEMENTS

Not applicable. The authors thanks Coordenação de Aperfeiçoamento de

Pessoal de Nível Superior (CAPES); National Postdoctoral
HUMAN AND ANIMAL RIGHTS program (PNPD/CAPES); Fundação de Amparo à Pesquisa
No animals/humans were used for studies that are the ba- do Estado do Espírito Santo (FAPES); Conselho Nacional de
sis of this research. Desenvolvimento Científico e Tecnológico (CNPq), Funda-
ção de Amparo à Pesquisa do Estado de Goiás (FAPEG )
Search for Potential Inducible Nitric Oxide Synthase Inhibitors
and Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP).
SUPPLEMENTARY MATERIAL
Supplementary material is available on the publisher’s
web site along with the published article.
REFERENCES
[1] Sugano, K.; Tack, J.; Kuipers, E.J.; Graham, D.Y.; El-Omar, E.M.;
Miura, S.; Haruma, K.; Asaka, M.; Uemura, N.; Malfertheiner, P.
Kyoto global consensus report on Helicobacter pylori gastritis. Gut,
2015, 64(9), 1353-1367.
[http://dx.doi.org/10.1136/gutjnl-2015-309252] [PMID: 26187502]
[2] Ferlay, J.; Soerjomataram, I.; Dikshit, R.; Eser, S.; Mathers, C.;
Rebelo, M.; Parkin, D.M.; Forman, D.; Bray, F. Cancer incidence
and mortality worldwide: sources, methods and major patterns in
GLOBOCAN 2012. Int. J. Cancer, 2015, 136(5), E359-E386.
[http://dx.doi.org/10.1002/ijc.29210] [PMID: 25220842]
[3] Wroblewski, L.E.; Peek, R.M., Jr; Wilson, K.T. Helicobacter pylori
and gastric cancer: factors that modulate disease risk. Clin. Micro-
biol. Rev., 2010, 23(4), 713-739.
[http://dx.doi.org/10.1128/CMR.00011-10] [PMID: 20930071]
[4] Chen, A.L. Role of the Helicobacter pylori-induced inflammatory
response in the development of gastric cancer. J. Cell. Biochem.,
2014, 114(3), 491-497.
[DOI: 10.1002/jcb.24389.]
[5] Huang, Z.; Fu, J.; Zhang, Y. Nitric oxide donor-based cancer ther-
apy: advances and prospects. J. Med. Chem., 2017, 60(18), 7617-
7635.
[http://dx.doi.org/10.1021/acs.jmedchem.6b01672] [PMID:
28505442]
[6] de Oliveira, G.A.; Cheng, R.Y.S.; Ridnour, L.A.; Basudhar, D.;
Somasundaram, V.; McVicar, D.W.; Monteiro, H.P.; Wink, D.A.
Inducible nitric oxide synthase in the carcinogenesis of gastrointes-
tinal cancers. Antioxid. Redox Signal., 2017, 26(18), 1059-1077.
[http://dx.doi.org/10.1089/ars.2016.6850] [PMID: 27494631]
[7] Alderton, W.K.; Cooper, C.E.; Knowles, R.G. Nitric oxide syn-
thases: structure, function and inhibition. Biochem. J., 2001, 357(Pt
3), 593-615.
[http://dx.doi.org/10.1042/bj3570593] [PMID: 11463332]
[8] McMillan, K.; Adler, M.; Auld, D.S.; Baldwin, J.J.; Blasko, E.;
Browne, L.J.; Chelsky, D.; Davey, D.; Dolle, R.E.; Eagen, K.A.;
Erickson, S.; Feldman, R.I.; Glaser, C.B.; Mallari, C.; Morrissey,
M.M.; Ohlmeyer, M.H.J.; Pan, G.; Parkinson, J.F.; Phillips, G.B.;
Polokoff, M.A.; Sigal, N.H.; Vergona, R.; Whitlow, M.; Young,
T.A.; Devlin, J.J. Allosteric inhibitors of inducible nitric oxide syn-
thase dimerization discovered via combinatorial chemistry. Proc.
Natl. Acad. Sci. USA, 2000, 97(4), 1506-1511.
[http://dx.doi.org/10.1073/pnas.97.4.1506] [PMID: 10677491]
[9] Fedorov, R.; Hartmann, E.; Ghosh, D.K.; Schlichting, I. Structural
basis for the specificity of the nitric-oxide synthase inhibitors
W1400 and Nomega-propyl-L-Arg for the inducible and neuronal
isoforms. J. Biol. Chem., 2003, 278(46), 45818-45825.
[http://dx.doi.org/10.1074/jbc.M306030200] [PMID: 12954642]
[10] Peterson, D.A.; Peterson, D.C.; Archer, S.; Weir, E.K. The non
specificity of specific nitric oxide synthase inhibitors. Biochem.
Biophys. Res. Commun., 1992, 187(2), 797-801.
[http://dx.doi.org/10.1016/0006-291X(92)91266-S] [PMID:
1382421]
[11] Rodrigues, T.; Reker, D.; Schneider, P.; Schneider, G. Counting on
natural products for drug design. Nat. Chem., 2016, 8(6), 531-541.
[http://dx.doi.org/10.1038/nchem.2479] [PMID: 27219696]
[12] MarvinSketch (Version 16), Drawing Module. Developed by Che-
mAxon, 2016.
[13] Dalby, A.; Nourse, J.G.; Hounshell, W.D.; Gushurst, A.K.I.; Grier,
D.L.; Leland, B.A.; Laufer, J. Description of several chemical
Current Topics in Medicinal Chemistry, 2019, Vol. 19, No. 30 2803
structure file formats used by computer programs developed at mo-
lecular design limited. J. Chem. Inf. Model., 1992, 32, 244-255.
[http://dx.doi.org/10.1021/ci00007a012]
[14] Hanwell, M.D.; Curtis, D.E.; Lonie, D.C.; Vandermeersch, T.;
Zurek, E.; Hutchison, G.R. Avogadro: an advanced semantic
chemical editor, visualization, and analysis platform. J. Chemin-
form., 2012, 4(1), 17.
[http://dx.doi.org/10.1186/1758-2946-4-17] [PMID: 22889332]
[15] Hawkins, P.C.; Skillman, A.G.; Warren, G.L.; Ellingson, B.A.;
Stahl, M.T. Conformer generation with OMEGA: algorithm and
validation using high quality structures from the Protein Databank
and Cambridge Structural Database. J. Chem. Inf. Model., 2010,
50(4), 572-584.
[http://dx.doi.org/10.1021/ci100031x] [PMID: 20235588]
[16] Hawkins, P.C.D.; Skillman, A.G.; Warren, G.L.; Ellingson, B.A.;
Stahl, M.T. ROCS 3.2.1.4: OpenEye Scientific Software, Santa Fe,
NM. 2014.
[17] EON 2.2.0.5: OpenEye Scientific Software, Santa Fe, NM.
[18] Filimonov, D.A.; Druzhilovskiy, D.S.; Lagunin, A.A.; Gloriozova,
T.A.; Rudik, A.V.; Dmitriev, A.V.; Pogodin, P.V.; Poroikov, V.V.
Computer-aided prediction of biological activity spectra for chemi-
cal compounds: opportunities and limitations. Biomed. Chem. Res.
Methods, 2018, 1.
[DOI: 10.18097/BMCRM00004]
[19] Greene, N.; Judson, P.N.; Langowski, J.J.; Marchant, C.A. Knowl-
edge-based expert systems for toxicity and metabolism prediction:
DEREK, StAR and METEOR. SAR QSAR Environ. Res., 1999,
10(2-3), 299-314.
[http://dx.doi.org/10.1080/10629369908039182] [PMID:
10491855]
[20] Verdonk, M.L.; Cole, J.C.; Hartshorn, M.J.; Murray, C.W.; Taylor,
R.D. Improved protein-ligand docking using GOLD. Proteins,
2003, 52(4), 609-623.
[http://dx.doi.org/10.1002/prot.10465] [PMID: 12910460]
[21] Kirchmair, J.; Markt, P.; Distinto, S.; Schuster, D.; Spitzer, G.M.;
Liedl, K.R.; Langer, T.; Wolber, G. The Protein Data Bank (PDB),
its related services and software tools as key components for in
silico guided drug discovery. J. Med. Chem., 2008, 51(22), 7021-
7040.
[http://dx.doi.org/10.1021/jm8005977] [PMID: 18975926]
[22] Jones, G.; Willett, P.; Glen, R.C.; Leach, A.R.; Taylor, R. Devel-
opment and validation of a genetic algorithm for flexible. J. Mol.
Biol., 1997, 267(3), 727-48.
[DOI: 10.1006/jmbi.1996.0897]
[23] Bergström, C.A.S.; Haeberlein, M.; Norinder, U. Computational
Absorption Prediction; Drug Bioavailability., 2009.
[24] Markt, P.; Petersen, R.K.; Flindt, E.N.; Kristiansen, K.; Kirchmair,
J.; Spitzer, G.; Distinto, S.; Schuster, D.; Wolber, G.; Laggner, C.;
Langer, T. Discovery of novel PPAR ligands by a virtual screening
approach based on pharmacophore modeling, 3D shape, and elec-
trostatic similarity screening. J. Med. Chem., 2008, 51(20), 6303-
6317.
[http://dx.doi.org/10.1021/jm800128k] [PMID: 18821746]
[25] Kearnes, S.; Pande, V. ROCS-derived features for virtual screen-
ing. J. Comput. Aided Mol. Des., 2016, 30(8), 609-617.
[http://dx.doi.org/10.1007/s10822-016-9959-3] [PMID: 27624668]
[26] Kerns, E.H.; Di, L. Drug-like properties: concepts, structure, de-
sign, and methods. from ADME to toxicity optimization, 1st ed;
Academic Press: San Diego, 2008.
[27] Martinat, C.; Amar, C.; Dansette, P.M.; Leclaire, J.; Lopez-Garcia,
P.; Cao, T.D.; N’Guyen, H.N.; Mansuy, D. In vitro metabolism of
isaxonine phosphate: formation of two metabolites, 5-
hydroxyisaxonine and 2-aminopyrimidine, and covalent binding to
microsomal proteins. Eur. J. Pharmacol., 1992, 228(1), 63-71.
[PMID: 1397069]
[28] Kanerva, L.; Elsner, P.; Wahlberg, J.E.; Maibach, H. Handbook of
occupational dermatology; Springer: Berlin, 2000.
[http://dx.doi.org/10.1007/978-3-662-07677-4]
2804 Current Topics in Medicinal Chemistry, 2019, Vol. 19, No. 30
[29] US Environmental Protection Agency. Guideline for human expo-
sure assessment. Risk Assess. Forum, 2016, p. 1-199.
[30] Barratt, M.D.; Basketter, D.A. Possible origin of the skin sensitiza-
tion potential of isoeugenol and related compounds. (I). Prelimi-
nary studies of potential reaction mechanisms. Contact Dermat.,
1992, 27(2), 98-104.
[http://dx.doi.org/10.1111/j.1600-0536.1992.tb05217.x] [PMID:
1395636]
Rodrigues et al.
[31] Mehta, R.; Chan, K.; Lee, O.; Tafazoli, S.; O’Brien, P.J. Drug-
Associated Mitochondrial Toxicity., 2008.
[http://dx.doi.org/10.1002/9780470372531.ch3]
[32] Powell, C.J.; Connolly, A.K. The site specificity and sensitivity of
the rat liver to butylated hydroxytoluene-induced damage. Toxicol.
Appl. Pharmacol., 1991, 108(1), 67-77.
[http://dx.doi.org/10.1016/0041-008X(91)90269-K] [PMID:
2006506]