Antileishmanial activity of 4-phenyl-1-[2-(phthalimido-2-yl)ethyl]-1H-1,2,3-triazole (PT4) derivative on Leishmania amazonensis and Leishmania braziliensis: In silico ADMET, in vitro activity, docking and molecular dynamic simulations

Article abstract of DOI:10.1016/j.bioorg.2020.104437

Organic compounds obtained by click chemistry reactions have demonstrated a broad spectrum of biological activities being widely applied for the development of molecules against pathogens of medical and veterinary importance. Cutaneous leishmaniasis (CL),

Full text of DOI:10.1016/j.bioorg.2020.104437

Journal Pre-proofs
Antileishmanial effects of 4-phenyl-1-[2-(phthalimido-2-yl)ethyl]-1H-1,2,3-
triazole (PT4) derivative on Leishmania amazonensis and Leishmania brazil-
iensis: In silico ADMET, in vitro activity, docking and molecular dynamic
simulations
Vanderlan Nogueira Holanda, Welson Vicente da Silva, Pedro Henrique do
Nascimento, Sérgio Ruschi Bergamachi Silva, Paulo Euzébio Cabral Filho,
Shalom Porto de Oliveira Assis, César Augusto da Silva, Ronaldo
Nascimento de Oliveira, Regina Celia Bressan Queiroz de Figueiredo, Vera
Lucia de Menezes Lima
PII:
S0045-2068(20)31735-1
DOI:
https://doi.org/10.1016/j.bioorg.2020.104437
YBIOO 104437
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
22 July 2020
30 September 2020
26 October 2020
Please cite this article as: V. Nogueira Holanda, W. Vicente da Silva, P. Henrique do Nascimento, S. Ruschi
Bergamachi Silva, P. Euzébio Cabral Filho, S. Porto de Oliveira Assis, C. Augusto da Silva, R. Nascimento de
Oliveira, R. Celia Bressan Queiroz de Figueiredo, V. Lucia de Menezes Lima, Antileishmanial effects of 4-
phenyl-1-[2-(phthalimido-2-yl)ethyl]-1H-1,2,3-triazole (PT4) derivative on Leishmania amazonensis and
Leishmania braziliensis: In silico ADMET, in vitro activity, docking and molecular dynamic simulations,
Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104437
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Antileishmanial effects of 4-phenyl-1-[2-(phthalimido-2-yl)ethyl]-1H-1,2,3-triazole
PT4) derivative on Leishmania amazonensis and Leishmania braziliensis: In silico
ADMET, in vitro activity, docking and molecular dynamic simulations
(
a,b
b
Vanderlan Nogueira Holanda , Welson Vicente da Silva , Pedro Henrique do
b
c
d
Nascimento , Sérgio Ruschi Bergamachi Silva , Paulo Euzébio Cabral Filho , Shalom
e
f
g
Porto de Oliveira Assis , César Augusto da Silva ; Ronaldo Nascimento de Oliveira ;
b,1
a,*,1
Regina Celia Bressan Queiroz de Figueiredo , Vera Lucia de Menezes Lima
aLaboratório de Lipídios e Aplicação de Biomoléculas em Doenças Prevalentes e
Negligenciadas. Departamento de Bioquímica, Centro de Biociências, Universidade
Federal de Pernambuco, Avenida Professor Moraes Rego, 1235, 50670-901 Recife, PE,
Brazil;
bLaboratório de Biologia Celular de Patógenos, Instituto Aggeu Magalhães,
Departamento de Microbiologia, Avenida Professor Moraes Rego, 1235, 50670-901
Recife, PE, Brazil;
cInstituto do Cérebro, Universidade Federal do Rio Grande do Norte, Av. Nascimento
de Castro, 2155 - Morro Branco, 59056-450 Natal, RN, Brazil;
dDepartamento de Biofísica e Radiobiologia, Universidade Federal de Pernambuco,
Avenida Professor Moraes Rego, 1235, 50670-901 Recife, PE, Brazil;

eNúcleo de Pesquisas em Ciências Ambientais e Biotecnologia, Universidade Católica
de Pernambuco, Rua do Príncipe, 526, 50050-900 Recife, PE, Brazil;
fColegiado de Medicina, Universidade Federal do Vale do São Francisco, Avenida José
de Sá Maniçoba, s/n - Campus Universitário, 56304-205, Petrolina, PE;
gLaboratório de Síntese de Compostos Bioativos, Departamento de Química,
Universidade Federal Rural de Pernambuco, Rua Dom Manuel de Medeiros, s/n - Dois
Irmãos, 52171-900, Recife, PE, Brazil
1Both authors contributed equally to this work
*
Corresponding author: Dr. Vera Lucia de Menezes Lima. Laboratório de Lipídios e Aplicação
de Biomoléculas em Doenças Prevalentes e Negligenciadas. Departamento de Bioquímica,
Centro de Biociências, Universidade Federal de Pernambuco, Avenida Professor Moraes Rego,
1
235, 50670-901 Recife, PE, Brazil. E-mail addres: lima.vera.ufpe@gmail.com (V. L. M. Lima)

Abstract
Organic compounds obtained by the click chemistry reactions have demonstrated a
broad spectrum of biological activities being widely applied for the development of
molecules against pathogens of medical and veterinary importance. Cutaneous
leishmaniasis (CL), caused by intracellular protozoa parasite of genus Leishmania,
comprises a complex of clinical manifestations that affect the skin and mucous
membranes. The available drugs for the treatment are toxic and costly, with long
periods of treatment, and the emergence of resistant strains has been reported. In this
study we investigated the in vitro effect of a phthalimide-1,2,3-triazole derivative, the 4-
Phenyl-1- [2-(phthalimido-2-yl)ethyl]-1H-1,2,3-triazole (PT4) obtained by click
chemistry, on mammalian cells and L. amazonensis and L. braziliensis, the causative
agent of CL in Brazil. In silico ADMET evaluation of PT4 showed that this molecule
has good pharmacokinetic properties with no violation of Lipinski’s rules. The in vitro
assay showed that PT4 was selective for both Leishmania species than to mammalian
cells. This compound also has a low cytotoxicity to mammalian cells with CC > 500
5
0
μM. Treatment of promastigote forms with different concentrations of PT4 resulted in
ultrastructural alterations, such as plasma membrane wrinkling, shortening of cell body,
increased cell volume and cell rupture. The molecular dynamic simulation results
showed that PT4 interacts with Lanosterol 14 α-demethylase from Leishmania, an
essential enzyme of lipid synthesis pathway in this parasite. Our results demonstrated
PT4 was effective against both species of Leishmania. PT4 caused a decrease of
mitochondrial membrane potential and increased production of reactive oxygen species,
which may lead to parasite death. Taken together, our results show pointed PT4 as
promissory therapeutic agent against CL.

Keywords: Leishmaniasis, Chemotherapy, Phthalimide, 1,2,3-triazole, click chemistry.
Introduction
1
Leishmaniasis is a complex of prevalent and neglected diseases caused by
protozoa of the genus Leishmania. The clinical manifestations of leishmaniasis range
from potential fatal visceral leishmaniasis (Le Rutte et al., 2019) to the most common
cutaneous leishmaniasis (CL), which is characterized by the appearance of skin and
mucous membrane lesions (Blum et al., 2013). CL occurs in several countries of Latin
America, in the Mediterranean, Middle East, and Central Asia. This disease is
characterized by lesions that can lead to anatomical disfigurement of the nasal septum
and ears, depending on the immune status of patient and on the Leishmania species
(Couto et al., 2014; Berbert et al., 2018). Furthermore, an intense inflammatory
response, characteristic of CL, may lead to tissue damage, ulcer formation, scarring,
which ultimately result in social stigmatization (Maspi et al., 2016).
Regardless of the clinical manifestations of Leishmaniasis, pentavalent
antimonials such as sodium stibogluconate and meglumine antimoniate have been used
since the early twentieth century as the first choice for the treatment of this illness
(Sundar and Chakravarty, 2015). Despite of their efficacy, these drugs require long-term
parenteral administration, are expensive and have adverse reactions, such as
cardiotoxicity and acute renal failure (Shanehsaz and Ishkhanian, 2016). In addition, the
appearance of parasite resistance has also been reported (Gervazoni et al., 2018). Due to
the limitations related to their pharmacokinetics and bioavailability, these drugs are not
used in topical formulations. Pentamidine and amphotericin B are introduced as second-
choice drugs against leishmaniasis therapy for the treatment of refractory cases.
However, these drugs also have high cost and toxicity, imposing important health
concerns (Oliveira et al., 2011). In this regard, the search for new safe and economically
viable therapeutic agents against leishmaniasis is still necessary.

Triazoles and their derivatives have attracted the attention of the scientific
community due to their interesting properties as: anti-inflammatory (Tariq et al., 2018),
antimicrobial (Zhang, 2019), antifungal (Xie et al., 2017), anticancer (Gholampour et
al., 2019) and antimalarial (Chu et al., 2019). The heterocyclic rings of triazoles bind
with high affinity to a variety of enzymes and receptors via non-covalent interactions
(Shao et al., 2011). These properties make 1,2,3-triazoles compounds very attractive,
not only as bioactive compounds, but also as molecular blocks for organic synthesis and
as functionalizes in the materials science (Dheer et al., 2017; Alves et al., 2018). In the
last decades, with the establishment of click chemistry reactions, the synthesis of 1,2,3-
triazoles derivatives and hybrid molecules was substantially improved. The click
chemistry refers to a group of reactions that are low cost, fast, simple to perform,
versatile, regiospecific, giving high product yield (Hein et al., 2008). Furthermore, these
reactions use solvents that are inoffensive and can be easily removed (Kolb et al.,
2
001).
As the triazoles derivatives, heterocyclic molecules belonging to the phthalimide
class have been also attracted attention due to their relevant biological activities such as:
anticonvulsive (Kamiński et al., 2011), antioxidant (Karthik et al., 2015), analgesic
(
Alanazi et al., 2015), antimicrobial (Pan et al., 2016), and anti-inflammatory
Banarouei et al., 2019; Batista et al., 2019). Among the interesting chemical properties
(
of phthalimides and their N-substituted derivatives, the presence of both hydrophobic
aryl ring, (-CONH), which acts as a hydrogen donor, and an electron donor system (C =
N) contributes for the biological activities of these molecules (Kushwaha and Kaushik,
2
016).
Because the infection with Leishmania spp. lead to an exacerbate inflammatory
response (Rodrigues et al., 2015), the anti-inflammatory propriety of phthalimide and

their derivatives, allied to its putative leishmanicidal potential, could be a useful
strategy to combat leishmaniasis infection.
The combination, by click chemistry reactions, of chemical groups having anti-
inflammatory and antiparasitic action is promising for the prospection of molecules
candidates for CL therapy. In this regard, previous study carried out by our group has
identified a hybrid molecule phthalimide-1,2,3-triazole, 4-Phenyl-1-[2- (phthalimido-2-
yl) ethyl]-1H-1,2,3-triazole (PT4) as having a potent in vivo anti-inflammatory activity
(Assis et al., 2019). Thus, in this work we selected this molecule to evaluate its
leishmanicidal potential against the Leishmania amazonensis and Leishmania
braziliensis, the main etiologic agents of CL in Brazil.
2
Materials and Methods
2
.1 Synthesis and obtaining of 2-[2-(4-phenyl-1H-1,2,3-triazol-1-yl)ethyl]-1H-
isoindole-1,3(2H)-dione (PT4). The strategy for the synthesis of 2-[2-(4-phenyl-1H-
,2,3-triazol-1-yl)ethyl]-1H-isoindole-1,3(2H)-dione (PT4) derivative was presented in
1
Scheme 1. The compound was obtained as previously described (Sirion et al., 2010).
The infrared spectra were recorded with an IFS66 Bruker spectrophotometer using KBr
1
13
discs. H and C NMR were obtained on Varian Unity Plus 400 MHz spectrometer
using DMSO-d as the solvent. The compounds were purified using column
6
chromatography on Merck silica gel 60 (70-230 mesh), with a system hexane:EtOAc
(1:1) ratio. The purity of fractions was monitored in TLC analysis on GF254 plate. The
azide and 1,2,3-triazoles compounds were prepared according to the procedure
described by Silva et al. (2012).

Scheme 1. Click reaction for PT4 synthesis
2
.2 In Silico ADMET assay. Prediction of absorption, distribution, metabolism,
excretion and toxicity (ADMET) parameters of PT4 and the reference drugs for
leishmaniasis were performed using the online SwissADME
http://www.swissadme.ch/) platform of the Swiss Institute of Bioinformatics,
(
PROTOX-II Server (http://tox.charite.de/protox_II / index.php? site=home) and pkCSM
server (http://biosig.unimelb.edu.au/pkcsm/). The physical-chemical parameters,
potential for oral bioavailability, similarities to other compounds already described were
also predicted using OSIRIS Data Explorer and DataWarrior version 4.7 software
(http://www.openmolecules.org/datawarrior/) and compared with the descriptors
obtained for reference drugs.
2
.3 Cytotoxicity Assay in Mammalian Cells. The cytotoxic potential of PT4 on
macrophages (obtained from Balb/c mice peritoneal exudate (mPEC) and from J774A.1
®
®
ATCC TIB-67 ™ linage) and fibroblast (ATCC PCS-201-018) were assayed by MTT
methodology, as established by Mosmann (1983) with slight modifications. J774
5
macrophages, mPEC and fibroblasts (5 × 10 cells / well) were plated in 96-well plates
containing 100 μL of the RPMI (for macrophage) or DMEM (for fibroblast) medium
supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C for 3 h in a

5
% atmosphere of CO . After this, the adhered cells were incubated in the absence or
2
presence of PT4 (78.5-1,256.5 µM) in culture medium for 48h. After incubation time,
the cells were washed with HBSS buffer and incubated in RPMI containing 5 mg/mL 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St.
Louis, MO, USA) for 3h at 37 °C in the absence of light. The medium was then
discarded and 100 μL of an acidified isopropanol solution (HCl 0.04 N + absolute
isopropanol) was added to each well to solubilize the formazan crystals. The
absorbance was read spectrophotometrically in a microplate spectrophotometer (Bio-
®
Rad , California, USA) at 540 nm. Each assay was performed in triplicate in three
independent experiments. The cytotoxic concentration for 50% of cells (CC ) was
5
0
calculated by linear regression using the IBM SPSS Statistics 25.
2
.4 Parasites. The promastigote forms of Leishmania amazonensis (LTB0016) and
Leishmania braziliensis (LTB2903) were maintained in Schneider's medium (Sigma-
Aldrich, USA) supplemented with 10% of bovine fetal serum and 100 IU penicillin and
1
00 IU / mL streptomycin, at 26 °C. Exponential-phase promastigotes were used in all
experiments. Amastigote forms were obtained from Balb/c peritoneal macrophages
infected with the stationary growth promastigote forms of the parasites.
2
.5 In vitro effects of PT4 on promastigotes and amastigote of L. amazonensis and L.
6
braziliensis. L. amazonensis and L. braziliensis promastigotes (10 cells / mL) were
incubated in Schneider's medium in the presence or the absence of PT4 (314.1- 19.6
μM) for 48h. The culture density was determined by direct counting in a Neubauer
chamber. The concentration of PT4 compound capable of inhibiting the growth of the
culture by 50% (IC ) was determined by linear regression analysis using the IBM SPSS
5
0

Statistics 25, after 48 hours of drug treatment. To investigate the effect of PT4 on
6
intracellular amastigote forms, mPEC (10 ꢀcells/mL) were plated in 24-well plates and
maintained at 37 ° C for 3h in a 5% CO atmosphere. Subsequently, the adhered cells
2
were infected with promastigotes of L. amazonensis and L. braziliensis, at 15: 1
parasite/cell ratio, at 37 °C for 14h. The non-internalized parasites were removed by
washing and then the infected cells were incubated for 24 h with PT4 at concentrations
corresponding to ½ IC , IC or 2x IC values, determined for promastigote forms of
5
0
50
50
each species. Infected-cells cultured in drug-free medium were used as a negative
control. Cells infected and treated with pentamidine (30 μM) were used as a positive
control. After the incubation time, treated and untreated cells were washed with PBS,
fixed with methanol and stained with Giemsa. The percentage of infected cells was
determined by counting 150 randomly chosen macrophages in duplicate. The survival
index was determined by multiplying the number of infected cells by the mean number
of amastigotes per cell. The concentration that inhibited amastigote (IC /amastigote)
5
0
growth within the macrophages was determined by regression analysis as described
above. These assays were performed in duplicate in three independent experiments.
2
.6. Scanning Electron Microscopy. For identify putative morphological changes
induced by PT4 treatment on the parasites, scanning electron microscopy analysis were
performed. For this, promastigote forms of L. amazonensis and L. braziliensis treated or
not with IC or 2x IC of PT4, for 48 h were harvest by centrifugation, washed with
5
0
50
0
.1 M phosphate buffer, pH 7.2, and then fixed for 2 h with 2.5% glutaraldehyde/4%
paraformaldehyde solution, in 0.1 M phosphate buffer at pH 7.2. After the fixation, cells
were allowed to adhere to coverslips previously coated with poly-L-lysine. The samples
were then post-fixed with a 1% osmium tetroxide/0.8% potassium ferricyanide/5 mM

CaCl in 0.1 M cacodylate buffer solution, at pH 7.2, for 1 hour in dark condition. After
2
post-fixation, the samples were washed in the same buffer, dehydrated in graded ethanol
series, critical-point-dried with CO , coated with a 20 nm-thick gold layer and observed
2
with a JEOL T-200 scanning electron microscope.
2
.7 Docking and Molecular Dynamic (MD) Simulations. The structure of PT4 was
built with Avogadro software (Hanwell et al., 2012) and optimized using the PM3 semi-
empirical method (Stewart, 1989) implemented in Orca 4.2 (Neese, 2012). Molecular
docking was performed based on the confrontation between optimized PT4 structure
and the sterol 14-α-demethylase enzyme (CYP51) from Leishmania infantum. The
docking analysis was carried out using Autodock Vina software (Trott and Olson,
2
010). The crystal structure of sterol 14-α-demethylase was obtained from Protein Data
Bank (PDB) under the code 3L4D (Hargrove et al., 2011). Water molecules were
removed, and polar hydrogens were added. The best binding poses were assessed in a
3
0 x 30 x 30 Å docking cube centered inside of the substrate binding cavity. The two
most energetically favorable structures obtained from docking were selected as initial
conformations for MD simulations. These calculations and analyses were performed for
two binding poses, name PT4 and PT4 , using the GROMACS 2018.4 (Abraham et
A
B
al., 2015; Pronk et al., 2013) software package with CHARMM36 force field (Huang et
al., 2017). The force field topology and parameters for PT4 were obtained from
CHARMM-GUI module (Kim et al., 2017). The systems were solvated by 17879 SPC
+
-
water molecules and counter ions Na and Cl were added to neutralize the charge and
maintain the system at a concentration of 150 mM. The protonation states of amino acid
sidechains were verified by PROPKA 3.0 (Olsson et al., 2011) software assuming
physiological pH. Each system was submitted to the energy minimization step using

steepest descent algorithms and the equilibration period (EP) of 125 ps was made with
NPT ensemble. The production runs were carried out in the NVT ensemble with a total
time simulation of 100 ns for each system. The V-rescale thermostat (Bussi, Donadio
and Parrinello, 2007) was used to maintain the system temperature at 310K. Other MD
parameters and covalent bonds involving hydrogen atoms in the protein and water
molecules were restrained by LINCS and SETTLE algorithms, respectively (Miyamoto
and Kollman, 1992; Hess et al., 1997). The Leap-Frog algorithm (Van Gunsteren and
Berendsen, 1988) was applied to integrate the motion equation with a time step of 2.0
fs. The long-range interactions were treat using Particle-Mesh Ewald sum (PME)
(Darden, York and Pedersen, 1993) with a 1.2 nm cutoff distance. The Interaction
Potential Energy (IPE) and hydrogen bond evaluations were performed as we
previously described (Silva et al., 2017). The two systems, PT4 and PT4 , present the
A
B
same number of particles and the only difference is the PT4 disposition into the binding
cavity.
2
.8 Analysis of PT4 on mitochondrial membrane potential and reactive oxygen
species production. To determine the effect of PT4 on mitochondrial membrane
potential (ΔΨm), L. amazonensis and L. braziliensis promastigotes were treated with 1x
or 2x IC of PT4 for 48h. After this, treated and control parasites were incubated with
50
1
0 μg/mL of rhodamine 123 (Sigma Aldrich, St. Louis, USA) for 15 min. Changes in
the mitochondrial membrane polarization were measured using the Variation Index (VI)
obtained by the equation (MT − MC) / MC where MC is the median fluorescence
intensity of the control and MT the median fluorescence intensity of the treated cells.
The mitochondrial reactive oxygen species production (ROS) production was assayed
using the sensitive-hydroethidine-analogue mitochondrial targeted probe, MitoSOX 3,8-

phenanthridinediamine,
5-(6′-triphenylphosphoniumhexyl)-5,6
dihydro-6-phenyl
(Invitrogen - Molecular Probes, USA). The cells were loaded with 2 mL of 5 μM
MitoSOX reagent and incubated for 10 min at 26 °C, protected from light. After
incubation, the parasites were washed three times with HBSS buffer. H O (10 μM) was
2
2
used as a positive control. For both analyses, the data collection was performed in a BD
Accuri™ C6 Plus flow cytometer (BD Biosciences, USA), in a corresponding channel
detection for each fluorescent probe. Twenty thousand events per sample were
analyzed.
2
.9 Ethical Considerations. The present research was carried out in accordance with the
ethical principles adopted by Brazilian legislation 11,794 / 2008 and approved by the
Instituto Aggeu Magalhães / Fundação Oswaldo Cruz Ethics Committee on Animal
Research (No. 146/2019).
3
Results and Discussion
The organic synthesis of new chemotherapeutic compounds against neglected
diseases, including leishmaniasis, may be hindered when the methods used are
complexes, time-consuming, expensive and have low possibility of large-scale
production. In a previous work we used click chemistry methodology to efficiently
synthetize alkyl-substituted phthalimide 1H-1,2,3-triazole derivatives. Among them,
the compound 2-[2-(4-phenyl-1H-1,2,3-triazol-1-yl)ethyl]-1H-isoindole-1,3(2H)-dione
(PT4) derivative presenting an important anti-inflammatory activity (Assis et al., 2019)
being selected for the analysis of its leishmanicidal potential on the etiological agents of
CL in Brazil. This hybrid phtalimide-1,2,3 triazole compound was obtained with 93%

o
yield and presented the following characteristics colorless solid; mp 187-189 C (Lit.
o
1
1
57-158 C); Rf 0.4 (EtOAc-Hexane, 1:1); IR (KBr)  3126, 2952, 1774, 1716, 1464,
max
-1
1
13
432, 1396, 1231, 1078, 765, 720 cm . The H and C NMR are in accordance with
previously reported data (Sirion et al., 2010).
In the search of new compounds against leishmaniasis the drug-likeness and
absorption, distribution, metabolism and excretion (ADMET) proprieties of drug
candidates should be considered (Lasing et al., 2020). In silico ADMET is useful and
low-cost method for accurate prediction of pharmacokinetic parameters and their
possible interactions with cells, enzymes and biological membranes (Vijayakumar et al.,
2
019). In this regard, in silico ADMET platforms were used to characterize the
pharmacokinetics profile of PT4 comparing with the reference drugs glucantime (GLU),
pentamidine (PEN), miltefosine (MIL) e amphotericin B (AMB) (Table 1).
Table 1. Analysis of pharmacokinetic and toxicological parameters (ADMET) of PT4
and the reference drugs used in the treatment of CL.
Parameter
PT4
GLU
PEN
MIL
AMB
Lipinski Rules Violation
No
Yes
No
No
Yes
Physicochemical
properties
HBA (≤10)
HBD (≤5)
LogP (≤5)
4
0
9
7
4
4
4
0
18
12
2.23
-2.90
2.72
3.83
-0.39

MW (≤500) g/mol
318.33
365.98
340.42
407.57
924.08
n-ROTB (≤10)
TPSA (A²)
4
6
10
20
3
68.09
167.55
118.20
68.40
319,61
Absorption
BBB
Yes
High
No
No
Low
No
No
High
No
No
Low
Yes
No
Low
Yes
GIA
P-GP substrate
Skin permeability (cm/s)
Metabolism
-6.85
-11.34
-6.56
-3.97
-11.94
CYP450 2C9 inhibitor
CYP450 2D6 inhibitor
CYP450 2C19 inhibitor
CYP450 3A4 inhibitor
CYP450 1A2 inhibitor
Excretion
Yes
No
No
No
No
No
No
Yes
Yes
No
No
No
No
No
No
No
No
No
No
No
Yes
No
No
Yes
No
Total Clearance (log
ml/min/kg)
0
.117
No
0.895
No
0.814
Yes
1.112
No
-1.495
No
Renal OCT2 substrate
Toxicity
DL (mg/Kg)
4700
3500
500
246
100
5
0
Subtitle: PEN - Pentamidine; GLU - glucantime; MIL - miltefosine; AMB - amphotericin B.
Physicochemical properties: HBA - Number of hydrogen force acceptors; HBD - Number of

hydrogen donors; LogP - Octanol-water partition coefficient (lipophilicity); MW - Molecular
Weight; n-ROTB - Number of rotary connections; TPSA- polar surface area. Absorption: BBB -
blood-brain barrier; GIA - gastrointestinal absorption; P-gp - permeability glycoprotein; Log Kp
-
Skin permeation. Metabolism: CYP450 - Cytochrome P450 Enzyme; Toxicity: MUT -
mutagenic; TUM - tumorigenic; IRR - annoying; LD - lethal dose up to 50%.
5
0
The Lipinski´s rule-of-five is an empirical rule of thumb which describe the
physicochemical parameters to determine drug-likeness and fitter hit molecules
(Lipinski et al., 1997). These rules state that an orally active drug does not has more
than two violation of the following parameters: 1) No more than 5 hydrogen bond
donors; 2) No more than 10 hydrogen bond acceptors; 3) a molecular mass less than 500
Daltons.; 4) an octano-water partition coefficient LogP not greater than 5. According to
our predictive analysis the reference drug Glucantime presented 1 violation (NH or
OH>5) and Amphotericin B (AmB) had three violations (MW>500, N or O>10 and NH
or OH>5). On the other hand, PT4 fulfil all the requirements of Lipinsk´s rule of five as
a candidate for development of oral formulation against cutaneous leishmaniasis.
Ocatanol-water partition coefficient (LogP) is a measure of hydrophobicity of a given
compound. The value of LogP for PT4 was 2.23 whereas the reference drug Miltefosine
was the most lipophilic molecule with LogP value of 3.83. It well known that the
hydrophobicity is pivotal for drug absorption, bioavailability, hydrophobic drug
receptor interactions, metabolism and cytotoxicity (Ali Khan et al., 2013). PT4
presented a topological polar surface (TPSA) of 68.09 Å, which favor its permeability
and oral bioavailability. It has been assumed by various predictive studies that the
bioavailability and oral permeability properties decrease as the value of TPSA increases
(Lagorce et al., 2017). For polarity, the optimal values of bioavailability should range
between 20 and 130Å. (Daina, Michielin and Zoete, 2017). Both reference drugs

Glucantime and Amphotericin B presented higher TPSA, which impaired their passive
diffusion through the BBB. The combination of the number of rotatable bound count
with the TPSA reflects the flexibility of PT4. It has been demonstrated that higher
TPSA/numbers of rotatable bounds can decrease the bioavailability of drugs on rat
model (Lagorce et al., 2017). Although PT4, Glucantime and AmB have low number of
rotatable bounds, the higher values of TPSA found for the reference drugs, probably
contribute for their low bioavailability profile. Additionally, in the case of CNS
permeation by passive diffusion, the TPSA must be below 80 Å (Table 1).
Cytochrome P450 (CYP450) enzymes are essential for detoxification of
foreign chemicals and drug metabolism in mammalian cells (Manikandan and Nagini,
2
018). Our results showed PT4 as potential inhibitors of three CYP450 enzymes
(CYP450 2C9, CYP450 C19, and CYP450 1A2) whereas Pentamidine was found as a
potential inhibitor of CYP450 2C9 and CYP450 2D6. None of CYP450 enzymes were
predicted to be inhibited by Glucantime, Miltefosine and Amphotericin B. Drugs
interact with the CYP450 system in several ways. The metabolic interaction between
CYP450 and drugs or xenobiotic are referred to as either inhibitors or inducers (Lynch
and Price, 2007). It well documented that commercially available drugs containing
triazole groups, such as Itraconazole and Posaconazole antifungals, are substrates and
inhibitors CYPs (Amsden and Gubbins, 2017). To varying extents, two enzymes in the
CYP3A family catalyze the metabolism of all triazole-containing drugs: CYP3A4 and
CYP3A5. Interestingly, PT4 was not predicted to inhibit CYP450 3A4, suggesting that
this compound, as other triazoles derivatives can be efficiently metabolized by this
enzyme. Although PT4 has been predicted as inhibitors of some CYP450 enzymes, the
extent to which PT4 affects these enzymes may be influenced by factors as its ability to
bind the enzyme (reversibly or irreversibly), the dose and CYP450 enzyme

polymorphism. Therapeutically, further studies are needed to better understand the
nature of PT4 interactions with human CYPs enzymes and other drugs.
When compared with the reference drugs for leishmaniasis, PT4 have a
reduced risk of toxicity. The predicted lethal dose for murine model (LD ) was 4,700
5
0
mg/kg, which were at least 1.34 and 470 higher than those found for Glucantime (3500
mg/Kg) and Amphotericin B (100 mg/Kg), the less and most toxic among the reference
drugs tested. Overall, our in silico ADMET prediction showed that PT4 has a desirable
drug-like properties and favorable safety profile.
Our in silico ADMET results prompted us to further investigate the in vitro
effects of this molecule on Leishmania amazonensis and Leishmania braziliensis, as
well as its cytotoxic potential on mammalian cells. For analysis of cytotoxicity of PT4
on mammalian cells, two cell types were chosen: Macrophages (peritoneal and J774)
and fibroblast cells. Leishmania spp. is mandatory intracellular pathogens, which
require macrophages for its survival and multiplication (Filardy et al., 2014). Like
macrophages, fibroblasts show a high degree of injury during the development of
cutaneous and/or mucosal lesions in cutaneous leishmaniasis (Bogdan et al., 2000). Our
results showed that PT4 has an inhibitory effect on the viability of all mammalian cells
tested. However, only at the higher concentration, PT4 was able to significantly
decrease the percentage of viable peritoneal macrophages and fibroblast. None of PT4
concentrations tested was able to inhibit the viability of treated cells by 100% (Figure
1
). The peritoneal macrophages were the most resistant to PT4 treatment, whereas J774
was the most susceptible. The CC /48h was 981.37, 521.47 and 895.17 µM for
5
0
peritoneal macrophages, J774A.1 cells and fibroblasts, respectively.

Figure 1. Effect of PT4 on mammalian cells: (a) mPEC, (b) J774A macrophages and
c) fibroblasts. Values represent the mean ± standard deviation of three independent
(
experiments in triplicate. * significant differences at p < 0.05. compared to untreated
control.
The PT4 significantly inhibited the growth of promastigote forms of both
Leishmania species tested, in dose-dependent manner (Figure 2) for both species. L.
amazonensis was more susceptible to PT4 treatment than L. braziliensis with an
estimated IC50 of 70.46 and 181.73 μM, respectively. The biochemical/molecular
differences in the susceptibility to drug between Leishmania species are well
documented in the literature (Croft et al., 2002) and can be partially explained by their
distinct division rate, differential permeability, difference in plasma membrane
composition and intracellular targets (Coelho et al., 2016).
Figure 2. Effect of PT4 on (a) promastigote forms of L. amazonensis and (b)
Leishmania braziliensis. Values represent the mean ± standard deviation of three
independent experiments in triplicate. *significant differences at p < 0.05 compared to
untreated control.

The PT4 also significantly inhibited the amastigote survival inside
macrophages at all concentrations tested (Figure 3), with IC values of 125.18 and
5
0
2
33.18 μM for L. amazonensis and L. braziliensis, respectively. As observed for
promastigote, the amastigote of L. amazonensis was more susceptible to the PT4
treatment. In both species, however, the amastigote was more resistant to this compound
than promastigotes Although PT4 significantly inhibited the survival of amastigote
inside the macrophages, only at the higher concentration (314.14 μM) this molecule was
able exhibit an inhibitory activity similar to the reference drug pentamidine (PMD) at 30
μM (Figure 3). However, it is important to emphasize that pentamidine has serious
collateral effects and decreasing efficacy in prolonged treatment (Sundar and
Chakravarty, 2015).

Figure. 3. Effect of PT4 on intracellular amastigote forms of (a) L. amazonensis and (b)
L. braziliensis. (c) Representative image of Giemsa staining showing the presence of
intracellular amastigotes (black arrow) in macrophages treated or not with PT4. Values
represent the mean ± standard deviation of three independent experiments in duplicate
compared to untreated control, *p < 0.05.
The main drawback of the chemotherapy leishmaniasis is the lack of specificity
of currently available drugs used for the treatment, which often results in high toxicity
to mammalian cells (Nagle et al., 2014). Thus, the selectivity index (SI), i.e. the ratio of

CC , host cell cytotoxicity and IC against the parasite, is an important parameter to be
5
0
50
taken in account in the search of new leishmanicidal agents (Papadopoulou et al., 2014).
Our results showed that PT4 was selective against both evolutive forms of parasite. The
SI values were 13.94 and 5.40 (for promastigotes) and 7.84 and 4.21(for amastigotes) of
L. amazonensis and L. braziliensis, respectively (Table 2).
Table 2. Evaluation of the leishmanicidal effect and cytotoxicity of PT4
CC50
IC50 (pro)
µM
IC50 (ama)
µM
Cell Type
Selective Index
µM
Pro
L.b L.a L.b
13.9 5.4 7.8 4.2
7.4 2.8 4.1 2.2
12.7 4.9 7.1 3.8
Ama
L.a
mPEC
J774A.1
Fib
982.43±5.6
-
-
521.47±1.2
-
-
895.17±1.2
-
-
La
-
-
70.46±3.5
181.72±5.5
125.05±3.2
233.18±3.7
Lb
-
-
Subtitle: mPEC - Peritoneal macrophages; CC - cytotoxic concentration in 50% of cells; IC -
5
0
50
5
0% inhibitory concentration of parasites; Pro - promastigotes; Ama - amastigotes, L.a –
Leishmania amazonensis and L.b – Leishmania braziliensis.
To investigate the effects of PT4 on the ultrastructure of L. amazonensis and L.
braziliensis promastigote forms, scanning electron microscopy (SEM) was performed
(Figure 4). Control cells had a smooth plasma membrane, spindle-shaped body and
elongated flagellum (Figures 4A-B). Drastic morphological changes could be observed

in PT4-treated cells as compared to control cells. The level of cell injuries was strongly
dependent on the concentration of PT4 tested, in both Leishmania amazonensis (Figures
4
B, 4D and 5F) and Leishmania braziliensis (Figures 4A, 4C and 4E). The most
prominent changes observed were the increase in the cell volume (Figure 5F) with
shortening and rounding of the cell body (Figure 4C). An interesting finding was the
increased presence of extracellular vesicles (EVs) of different sizes budding from the
cell body and flagellum in treated cells (Figures 4C and 4E). EVs are a population of
lipid-enclosed spherical vesicles containing bioactive components such as soluble
proteins, carbohydrates, lipids and genetic material from parental cell (Mardahl et al.,
2
019). Commonly, EVs are classified based on their biogenesis and size into exosomes
(
30-150 nm in diameter), microvesicles (100-1000 nm in diameter) and apoptotic bodies
100-5000 nm in diameter). Microvesicles and exosomes have been shown to be
(
constitutively release by several protozoan parasites, including Leishmania species and
T. cruzi as a mechanism of both parasite-host and parasite-parasite communication
(Mardahl et al., 2019).
The increase in the EVs resembling microvesicles and exosomes in cell treated
with PT4 suggests that besides their role in parasite communication EVs may also
participate in the response of parasite to drugs. The large EVs, also observed budding
from PT4-treated parasites, could correspond to apoptotic bodies induced by PT4
treatment (Khajah and Luqmani, 2016). PT4-treated parasites also presented incomplete
division, with abnormal septation and short flagellum. These results suggest that PT4
may also have a cytostatic effect on both species of Leishmania.

Figure 4. Ultrastructural changes induced by PT4 on L. braziliensis (A, C and E) and L.
amazonensis (B, D and F) promastigotes. Control cells (A-B), cells treated with 1x the
value of IC (C-D) and 2x the value of IC (E-F).
5
0
50
Ultrastructural changes similar to those found in our study were also observed in
Leishmania spp. treated with triazole inhibitors of ergosterol biosynthesis (De Macedo-

Silva et al., 2015). Trypanosomatids and fungi are strongly dependent on ergosterol and
other 24-alkyl sterols that were not found in mammalian cells (Mccall et al., 2015,
Emami et al., 2017). The Cytochrome P450 (CYP) is the superfamily of protoheme
containing monooxygenases, found in a large number of organisms. Among P450
enzymes, sterol 14α-demethylase (CYP51), an essential enzyme in sterol biosynthesis,
has been pointed as an important drug target in trypanosomatids (Lapesheva et al.,
2
011). It has been demonstrated that commercially available anti-fungal azoles, as
posaconazole and fluconazole, that inhibit the ergosterol biosynthesis in fungi are also
effective against T. cruzi and Leishmania parasites (Chen et al., 2010). To investigate
whether PT4 could also be an inhibitor of Leishmania CYP51 in silico docking of PT4
against this enzyme was performed.
Because the crystal structure of CYP51 of L. amazonensis and L. braziliensis
was not available on the protein data bank (PDB) we used the CYP51 of L. infantum
(CYP51Li, Protein data bank entry: 3L4D) as a template for molecular docking assays
and MD simulations. This protein presented 97% and 95%, of amino acid sequence
similarity with two hypothetical orthologue proteins of L. amazonensis and L.
braziliensis, respectively. The sequence similarity reaches 100% if we compare only the
residues from the active site (Hargrove et al., 2011). Previous study revealed that
CYP51 is highly conserved between kinetoplastids. Chen et al., (2010) reported that a
CYP51Tc from T. cruzi and CYP51Tb from T. brucei share 83% sequence identity.
Furthermore, Leishmania CYP51 are 72–78% identical to that of T. cruzi and T. brucei.
The high level of conservation of CYP51 in these parasites makes possible to
extrapolate structural features that can be modeled to facilitate drug discovery and
development (Chen et al., 2010).

By using CYP51L.i as receptor and the models generated from docking
analysis, the MD simulations were carried out for identifying the PT4 intermolecular
interaction profile. The interaction potential energy (IPE) calculations were performed
to evaluate the contribution of each residue of the binding cavity and channel entrance
of CYP51 receptor with the ligand. Based on non-hydrogen atoms of PT4 as reference,
the RMSD analysis showed (Figure 5a) a structural stabilization around 70 ns, hence,
the IPE and hydrogen bond analysis were made in the last 30 ns for both systems. The
highest variation on RMSD values evidenced the motions of inhibitor inside the binding
cavity and consequent settle down. The independent MD simulations taken in account
two possible entering modes of PT4 molecule to an internal region of the cavity. These
modes were obtained from docking analysis and indicated the first approaching of
1
,2,3-triazole or phthalimide moiety to the channel of the cavity, named respectively
PT4 and PT4 systems, as shown in figure 5b.
A
B
An 8 Å sphere centered on PT4 molecule was used for IPE calculation over
time. This volume encompasses 62 neighboring (average) residues during the simulated
time for PT4 and PT4 , although most of them interact with energy very close to zero.
A
B
Applying an arbitrary cutoff of IPE lower than -0.5 kcal/mol, the number of residues
interacting with PT4 is 17 and 16 for PT4 and PT4 , respectively. This total includes
A
B
the heme group. All these most relevant residues identified by IPE calculation were
presented in Table 3. They constitute the substrate channel entrance and the binding
cavity described previously for the sterol 14α-demethylase receptor of L. infantum
(Hargrove et al., 2011).
Table 3. Average (standard deviation) of Interaction Potential Energy (IPE) for each
residue of CYP51L.i interacting with PT4 and PT4 systems over time.
A
B

IPE (kcal/mol)
Residue
PT4A
PT4B
Tyr102
Phe104
Met105
Phe109
Tyr115
Leu126
Val212
Ala286
Phe289
Ala290
Leu355
Met357
Met359
Met459
Val460
Heme
-2.84 (1.26)
-1.48 (0.59)
-1.59 (0.55)
-0.61 (0.35)
-4.16 (1.25)
-0.53 (0.37)
-0.79 (0.64)
-0.66 (0.46)
-1.35 (0.60)
-2.81 (0.82)
-1.82 (0.61)
-2.41 (0.96)
-1.11 (0.53)
-2.91 (1.58)
-1.45 (0.40)
-8.59 (2.13)
-5.45 (0.96)
-2.66 (0.61)
-1.97 (0.86)
-0.96 (0.56)
-3.54 (2.14)
-0.54 (0.41)
-1.19 (0.46)
-0.88 (0.54)
-0.99 (0.61)
-0.89 (0.54)
-2.42 (0.98)
-3.39 (2.06)
-1.10 (0.76)
-1.88 (0.79)
-0.86 (0.42)
-14.71 (3.87)
The analysis of IPE reveals that PT4 interacts with CYP51 residues in a similar
way to other antifungal azoles (Urbina et al., 2000). As PT4, these nitrogenated
heterocyclic compounds, contain a basic atom, which easily coordinates with heme iron
(Figure 5c). The calculated IPE were -8.59 ± 2.13 kcal/mol and -14.71 ± 3.87 kcal/mol
for PT4 and PT4 . For PT4 orientation, the interaction occurs through triazole
A
B
A
nitrogen with an average distance of 4.60 ± 0.39 Å and for PT4 , the nitrogen of
B
phthalimide moiety interacts with an average distance of 4.52 ± 0.38 Å. This strong
interaction with the heme group plays a key role in replacing the steroid substrate
becoming unfeasible for the iron reduction and consequent oxidative removal of the
1
4α-methyl group from the sterol core.

Among other more interacting residues presented in Table 1, Tyr102, Phe109,
Tyr115 (helix B',B'/C loop), Leu126 (helix C), Ala286, Phe289, Ala290 (helix I),
Leu355, Met357, Met359 (β4 strand 1-4) are constituents of substrate binding cavity
(SBC) while Val212 (helix F''), Met459, Val460 (β4 hairpin) belongs to substrate
channel entrance (SCE), as shown in figure 5b. Taken together, the PT4 and PT4
A
B
orientations showed similar interactions with SBC and SCE residues. Besides heme
interaction, the most energetically favorable interactions take place between PT4 and a
tyrosine pair belonging to the binding cavity, with -2.84 ± 1.26 kcal/mol and -4.16 ±
1
.26 kcal/mol for PT4 -Tyr102 and PT4 -Tyr115 respectively, and -5.45 ± 0.96
A A
kcal/mol and -3.54 ± 2.14 kcal/mol for PT4 -Tyr102 and PT4 -Tyr115. Together with
B
B
Arg360, His420 and Arg123, both Tyr102 and Tyr115 are conserved heme-contacting
residues that formed hydrogen bonds with heme propionates through their hydroxyl side
chains at least 99.4% over simulated time. The conserved position of these tyrosine
residues near to heme group and consequent proximity with heme-inhibitors is
important for the stabilization of cavity binding via side-chain interactions through
benzene ring or hydroxyl moiety. The IPE values with the other SBC residues showed
their extended wrap regarding PT4 presence. In addition to helix B',B'/C loop contacts,
β4 strand 1-4 and helix I structures form noncovalent interactions with benzene-like
moieties of PT4 whatever entering orientation. It is noteworthy the interactions with
Ala286, Phe289, Ala290 (helix I) because the high proximity with this region could be
related to the better activity of heme-coordinated inhibitors. This helix acts as an
anchoring point for ligand avoiding their replacement in the active site by the substrate
(Lepesheva and Waterman, 2011).
Concerning the PT4 orientation inside the binding cavity, no significant
differences were found on the spatial features and energetic values (Figure 5c). The total

IPE between PT4 and all surrounding residues showed similar values, -43.9 ± 3.99
kcal/mol and -49.3 ± 5.06 kcal/mol for PT4 and PT4 , respectively. Due to method
A
B
limitations, a bond breaking/forming cannot be assessed, hence, the FeHeme-NPT4 bond
formation has not been described. This information could be properly evaluated by
using a quantum method on further investigations. It has been already demonstrated that
inactivation of the CYP51 in Leishmania parasites can interfere with membrane
stability, increasing its fluidity, causing collapse in the membrane lipid rafts and altered
morphology (Xu et al., 2014).
Figure 5. MD simulation analysis and graphical representations. A) Root Mean Square
Deviations (RMSD) for PT4A (green) and PT4B (orange) and one-nanosecond running
averages of the deviations versus time. B) Graphical representation of the simulated
system with PT4A and PT4B orientation (tubes), substrate channel entrance (red
surface), substrate binding cavity (yellow surface), PT4 model inside the cavity (green
tubes), heme group (blue surface) and chain A tertiary structure of sterol 14α-
demethylase (CYP51) (gray cartoon). C) Superimposed view of PT4-Heme group
interactions. PT4A and PT4B were differentiated through carbon coloring green and
orange tubes respectively. Iron, oxygen, nitrogen and hydrogen atoms were represented
by black sphere, red, blue and white tubes.

Previous work has shown that inhibition of sterol biosynthesis by triazole
derivatives, alone or in combination, affects the mitochondrial function of L.
amazonensis promastigotes by inducing a dissipation of the of mitochondrial membrane
potential and increased ROS production (Macedo-Silva et al., 2015). To investigate
whether PT4 was able to interfere with the physiology of L. braziliensis and L.
amazonensis single mitochondrion, promastigote forms were treated or not with IC50
and 2x IC of PT4 and submitted to staining with the fluorescent probes, Rhodamine
5
0
1
23 (for evaluation of mitochondrial membrane potential) and MitoSOX (for evaluation
of mitochondrial ROS production). The treatment of parasites with PT4 caused a
decrease in the fluorescence intensity for Rho 123 in both species and concentration
tested (Table 4). Consistently, the treatment of parasites with PT4 also leading a