International Journal of

Molecular Sciences

Article
Virtual Screening of FDA-Approved Drugs against Triose
Phosphate Isomerase from Entamoeba histolytica and Giardia
lamblia Identifies Inhibitors of Their Trophozoite Growth Phase
Alfredo Juárez-Saldivar 1 , Elizabeth Barbosa-Cabrera 2 , Edgar E. Lara-Ramírez 3 , Alma D. Paz-González 1 ,
Ana V. Martínez-Vázquez 4 , Virgilio Bocanegra-García 4 , Isidro Palos 5 , Nuria E. Campillo 6 and
Gildardo Rivera 1, *

1 Laboratorio de Biotecnología Farmacéutica, Centro de Biotecnología Genómica,

Instituto Politécnico Nacional, Reynosa 88710, Mexico; [email protected] (A.J.-S.);
[email protected] (A.D.P.-G.)
2 Sección de Estudios de Posgrado e Investigación, Escuela Superior de Medicina,
Instituto Politécnico Nacional, Ciudad de México 11340, Mexico; [email protected]
3 Unidad de Investigación Biomédica de Zacatecas, Instituto Mexicano Del Seguro Social,
Zacatecas 98617, Mexico; [email protected]



4 Centro de Biotecnología Genómica, Instituto Politécnico Nacional, Reynosa 88710, Mexico;


[email protected] (A.V.M.-V.); [email protected] (V.B.-G.)
5 Unidad Académica Multidisciplinaria Reynosa-Rodhe, Universidad Autónoma Tamaulipas,
Citation: Juárez-Saldivar, A.;
Barbosa-Cabrera, E.; Lara-Ramírez, Carr. Reynosa-San Fernando, Reynosa 88779, Mexico; [email protected]
6 Centro de Investigaciones Biológicas Margarita Salas (CIB-CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain;
E.E.; Paz-González, A.D.;
Martínez-Vázquez, A.V.;
[email protected]
* Correspondence: [email protected]; Tel.: +52-1-8991-601-356
Bocanegra-García, V.; Palos, I.;
Campillo, N.E.; Rivera, G. Virtual
Screening of FDA-Approved Drugs Abstract: Infectious diseases caused by intestinal protozoan, such as Entamoeba histolytica (E. histolytica)
against Triose Phosphate Isomerase and Giardia lamblia (G. lamblia) are a worldwide public health issue. They affect more than 70 million
from Entamoeba histolytica and Giardia people every year. They colonize intestines causing primarily diarrhea; nevertheless, these infections
lamblia Identifies Inhibitors of Their can lead to more serious complications. The treatment of choice, metronidazole, is in doubt due
Trophozoite Growth Phase. Int. J. Mol. to adverse effects and resistance. Therefore, there is a need for new compounds against these
Sci. 2021, 22, 5943. https://doi.org/ parasites. In this work, a structure-based virtual screening of FDA-approved drugs was performed
10.3390/ijms22115943 to identify compounds with antiprotozoal activity. The glycolytic enzyme triosephosphate isomerase,
present in both E. histolytica and G. lamblia, was used as the drug target. The compounds with
Academic Editor: Edmond Dik
the best average docking score on both structures were selected for the in vitro evaluation. Three
Lung Ma
compounds, chlorhexidine, tolcapone, and imatinib, were capable of inhibit growth on G. lamblia
trophozoites (0.05–4.935 µg/mL), while folic acid showed activity against E. histolytica (0.186 µg/mL)
Received: 25 April 2021
Accepted: 28 May 2021
and G. lamblia (5.342 µg/mL).
Published: 31 May 2021
Keywords: protozoa; FDA; virtual screening; drug repositioning; molecular docking
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. 1. Introduction
Intestinal protozoa are eukaryotic unicellular organisms that cause several diseases
to humans and animals. Mainly, they affect developing regions; nevertheless, due to
globalization and the increase of human migration, some of these diseases are becoming a
Copyright: © 2021 by the authors. health threat all over the world [1]. The parasites, Entamoeba histolytica (E. histolytica) and
Licensee MDPI, Basel, Switzerland. Giardia lamblia (G. lamblia), are the major agents causing parasitosis affecting more than
This article is an open access article 70 million people every year [2,3]. E. histolytica causes amoebiasis, characterized by pyrexia,
distributed under the terms and abdominal cramping and dysentery symptoms. This parasite can migrate to other organs,
conditions of the Creative Commons like liver, causing amebic liver abscess [4,5]. G. lamblia colonizes small intestine, causing
Attribution (CC BY) license (https:// giardiasis disease, one of the most common causes of diarrhea in children and adults [6,7].
creativecommons.org/licenses/by/ Other symptoms caused by G. lamblia infection are greasy stool, flatulence and bloating [8].
4.0/).

Int. J. Mol. Sci. 2021, 22, 5943. https://doi.org/10.3390/ijms22115943 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 8

Int. J. Mol. Sci. 2021, 22, 5943 2 of 8

Other symptoms caused by G. lamblia infection are greasy stool, flatulence and bloating
[8]. Some reports indicate up to 10% of coinfection with both parasites in migrant popu-
lations and returning travelers [9,10].
SomeThe mainindicate
reports line of treatment
up to 10%against these parasites
of coinfection with bothisparasites
metronidazole. It has
in migrant been used
populations
for over 60 years, but the
and returning travelers [9,10]. increase of drug resistance leads to higher dose use in treatment,
and therefore
The mainmore line ofsevere side effects
treatment against[8]. Consequently,
these there is a need for
parasites is metronidazole. new
It has andused
been safe
alternatives for amoebiasis
for over 60 years, and giardiasis
but the increase treatment.leads
of drug resistance Onetoofhigher
the maindosestrategies used in
use in treatment,
the
andfinding
therefore ofmore
activesevere
compounds is drug
side effects [8]. repositioning,
Consequently, which
there isreduces
a need forcostnewandand
time in
safe
alternatives
drug [11,12].for
The amoebiasis
incrementand giardiasis
of free availabletreatment.
biologicalOne of the
data andmain strategies
advances used in
in computa-
the finding
tional of active
techniques have compounds
led to several is drug
new repositioning,
ways of virtualwhich reduces
screening, that,cost and time
compared to in
drug [11,12]. The increment of free available biological data and advances
vitro evaluation, have become a cheaper and faster alternative to screen drug libraries. in computational
techniques
Thus, have ledand
ligand-based to several new ways
structure-based of virtual
virtual drugscreening,
repositioningthat,are
compared
widely used to intoday
vitro
evaluation, have become a cheaper and faster alternative to screen drug libraries. Thus,
[13–17].
ligand-based and structure-based
Structure-based methods involve virtualstructural
drug repositioning
information are widely used today
of enzymes and/or [13–17].
other
typesStructure-based
of proteins used methods
as druginvolve
targets.structural
In the lastinformation
decade, new of drug
enzymes and/or
targets have other
been
types of proteins used as drug targets. In the last decade, new drug
studied in the search of compounds against intestinal protozoa [18–23]. Among these drug targets have been
studied in
targets, the search of compounds
triosephosphate isomerase against
(TIM), intestinal protozoa
a glycolytic enzyme, [18–23]. Among
has been these drug
as widely ex-
targets, triosephosphate isomerase (TIM), a glycolytic enzyme, has
plored on many species; nevertheless, studies on E. histolytica and G. lamblia lack been as widely explored
depth
on manyRecently,
[24–26]. species; nevertheless,
new series ofstudies
compoundson E. histolytica
have beenand G. lamblia
reported lack depth
to inhibit [24–26].
TIM from E.
Recently, new
histolytica seriesand
(TIMEh) of compounds
TIM from G. have been (TIMGl)
lamblia reported [27,28].
to inhibit TIM frominE.this
Therefore, histolytica
work,
(TIMEh)
known inhibitor from G. lamblia
and TIMcompounds were (TIMGl)
used to[27,28]. Therefore,
identify structuralincharacteristics
this work, known and inhibitor
main in-
compounds were used to identify structural characteristics and
teractions involved in the binding to TIMEh and TIMGl. Later, a virtual screen of main interactions involved
library
in the
of 1466binding to TIMEh drugs
FDA-approved and TIMGl. Later, a virtual
was performed screen
on this drugoftarget
librarytoofidentify
1466 FDA-approved
new antipro-
drugs was
tozoal drugsperformed on this
with possible drug target to identify
broad-spectrum activity.new antiprotozoal drugs with possible
broad-spectrum activity.
2. Results and discussion
2. Results and Discussion
2.1.
2.1. Docking-Based
Docking-Based Virtual
Virtual Screening on TIMEh
Screening on TIMEh and
and TIMGl
TIMGl
Before
Before starting
starting the
the screening virtual (SV)
screening virtual (SV) of
of the
the FDA
FDA library
library against
against TIMEh,
TIMEh, the
the dock-
dock-
ing
ing of
of aa known
known inhibitor,
inhibitor, compound
compound D4, D4, denominates
denominates 5,5′-[(4-nitrophenyl)methylene]bis-
0
5,5 -[(4-nitrophenyl)methylene]bis-
6-hydroxy-2-mercapto-3-methyl-4(3H)-pyrimidinone)[27],
6-hydroxy-2-mercapto-3-methyl-4(3H)-pyrimidinone)[27], was was used
used both to validate
both to validate the
the
docking protocol and to use this compound as a control compound. The
docking protocol and to use this compound as a control compound. The docking score docking score
obtained
obtained forfor D4
D4 was
was -5.4
-5.4 kcal/mol.
kcal/mol. The
The non-covalent
non-covalent interactions
interactions between
between TIMEh and D4
TIMEh and D4
were calculated using PLIP. As Figure 1 shows, the main interactions were
were calculated using PLIP. As Figure 1 shows, the main interactions were hydrophobic hydrophobic
residues Trp75(B),
with residues Trp75(B),Tyr81(B),
Tyr81(B),Ile108(A)
Ile108(A)and
andGlu111(B).
Glu111(B).TheTheresidue
residueTrp75(B)
Trp75(B)also
also had
had a
aπ-stacking
π-stackinginteraction.
interaction.This
Thiscompound
compoundalso alsoformed
formedtwotwo hydrogen
hydrogen bonds
bonds with Lys77(A)
and Gln115(B).

inhibitor, and
Figure 1. 2D interaction diagram between compound D4, an inhibitor, and TIMEh
TIMEh on
on the
the interface.
interface.

theD4
Once the D4inhibitor
inhibitorwas
wasstudied,
studied, 1466
1466 FDA
FDA drugs
drugs were
were docked
docked against
against TIMEh.
TIMEh. The
The compounds
compounds werewere ranked
ranked based
based on their
on their docking
docking score,
score, andand
thethe
toptop
tenten
areare described
described in
in Table S1. These ten compounds contain several aromatic rings (like D4); therefore,
hydrophobics and π-stacking interactions were expected (Table S2). Risperidone and
iloperidone are antipsychotic drugs with a similar structure composed of a benzoxazole
attached to a fluorine atom and a piperidine ring. On the other hand, folic acid, rilpivirine
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 8

Table S1. These ten compounds contain several aromatic rings (like D4); therefore, hydro-
Int. J. Mol. Sci. 2021, 22, 5943 phobics and π-stacking interactions were expected (Table S2). Risperidone and iloperi- 3 of 8
done are antipsychotic drugs with a similar structure composed of a benzoxazole attached
to a fluorine atom and a piperidine ring. On the other hand, folic acid, rilpivirine and
pralatrexate, used as chemotherapy drugs, share a benzaldehyde and a glutamic acid in
and pralatrexate, used as chemotherapy drugs, share a benzaldehyde and a glutamic
their structures. Interesting, bisacodyl is a tricyclic compound like D4, which also contains
acid in their structures. Interesting, bisacodyl is a tricyclic compound like D4, which also
two methyl acetate that could form a hydrogen bond. According to the interaction profiles
contains two methyl acetate that could form a hydrogen bond. According to the interaction
calculated with PLIP
profiles calculated (Table
with PLIPS2), the most
(Table common
S2), the interactions
most common between
interactions TIMEhTIMEh
between and these
and
structures were π-stacking Trp75, a hydrogen bond with Gln115 and
these structures were π-stacking Trp75, a hydrogen bond with Gln115 and hydrophobichydrophobic inter-
action with Ile108.
interaction with Ile108.
InInthe
thecase
caseofofTIMGl,
TIMGl,thethe
omeprazole
omeprazole drug hashas
drug been previously
been previouslyreported as an
reported aseffec-
an ef-
tive inhibitor [28]. Hence, omeprazole was docked at the interface to identify
fective inhibitor [28]. Hence, omeprazole was docked at the interface to identify the the possible
interactions involved in
possible interactions its binding
involved in itstobinding
TIMGl.to The docking
TIMGl. Thescore for omeprazol
docking was -7.7
score for omeprazol
kcal/mol and its interactions are shown in Figure 2. In this diagram, several
was −7.7 kcal/mol and its interactions are shown in Figure 2. In this diagram, several hy- hydrogen
bonds
drogenwith Arg99(A),
bonds Arg99(B),
with Arg99(A), Met103(B),
Arg99(B), Gln109(B)
Met103(B), and Lys113(A)
Gln109(B) are indicated,
and Lys113(A) as
are indicated,
well as some
as well hydrophobic
as some hydrophobic interactions
interactions with Tyr68(A),
with Tyr68(A), Leu69(A)
Leu69(A)and andGln109(B).
Gln109(B).AAsalt
salt
bridge could be formed with Glu78 from each monomer. Also, Tyr68(B)
bridge could be formed with Glu78 from each monomer. Also, Tyr68(B) interacts through interacts through
π-stacking
π-stackingwithwiththe
thebenzimidazole
benzimidazolemoiety.
moiety.

2Dinteraction
Figure2.2.2D
Figure interactiondiagram
diagrambetween
betweenomeprazole
omeprazoledrug
drugand
andTIMGl
TIMGlon
onthe
theinterface.
interface.

For the 1466 FDA-approved drugs virtual screening on TIMGl, the top ten are outlined
For the 1466 FDA-approved drugs virtual screening on TIMGl, the top ten are out-
in Table S3. These compounds are structurally diverse with the exception of sulfasalazine
lined in Table S3. These compounds are structurally diverse with the exception of sulfasal-
and eltrombopag that share a benzoic acid and an aniline ring. Nevertheless, their interac-
azine and eltrombopag that share a benzoic acid and an aniline ring. Nevertheless, their
tion profile showed some similar interactions (Table S4). The most common interactions
interaction profile showed some similar interactions (Table S4). The most common inter-
are the π-stacking interaction with Tyr68, and hydrogen bonding with Arg99, Gln109
actions are the π-stacking interaction with Tyr68, and hydrogen bonding with Arg99,
andLys113, which are shared with the known inhibitor.
Gln109 InandLys113, which energies
general, binding are shared onwith
TIMGlthewere
known inhibitor.
better than for TIMEh and there were
In general, binding energies on TIMGl were
no compounds ranked within the top ten of both species. better than for TIMEh and
Therefore, there scores
docking were no of
compounds ranked within the top ten of both species. Therefore,
each compound were merged and obtain the average docking score on both TIMEh docking scores of each
and
compound
TIMGl andwerethusmerged
identifyand obtain thewith
compounds average docking
potential scoreeffect.
broad on both TIMEh andwere
Compounds TIMGl re-
and thusbased
ranked identify compounds
on this with potential
average score broadineffect.
and presented Table Compounds
S5. There arewere
somere-ranked
structural
based on this average
characteristics to pointscore
out.and presentedreported
Previously in TableTIM
S5. There are some
inhibitors structural
bound charac-
to the interface
teristics to point out. Previously reported TIM inhibitors bound to the interface
by aromatic interactions [29]. Interestingly, re-ranked compounds have several aromatic by aro-
matic interactions [29]. Interestingly, re-ranked compounds have several
rings in their structure which could bind through π-stacking or π-cation interactions. aromatic rings
inAmong
their structure which could
these compounds, bind through
chlorhexidine π-stacking
is the only one or π-cation
which interactions.
structure contains Among
halogen
these compounds,
atoms, and also has chlorhexidine is the donors,
several H-bond only onewhich
whicharestructure
one of contains
the mainhalogen atoms,
characteristics
and also has several
in omeprazole H-bond
binding donors, which
to TIMGl. are oneand
Pemetrexed of the main
folic acidcharacteristics
have a similar in structure,
omepra-
zole
theybinding
containtoaTIMGl.
glutamic Pemetrexed
acid group and folic acid
attached tohave a similar
a benzoic structure,
acid. they contain
The difference a
is that
pemetrexed has pyrrole[2,3-d]pyrimidine, folic acid has pteridine ring; nevertheless, both
functional groups are suitable for aromatic interaction and hydrogen bonding. Pyrimidine
structure is also present in imatinib structure, along with a benzoic acid. On the other hand,
dolasetron have an indol group, similar to the pyrrolepyrimidine in folic acid. Tolcapone
and arbutamine have another interesting group, which is benzenediol. These structures
Int. J. Mol. Sci. 2021, 22, 5943 4 of 8

showed the common functional groups that could be explore in the early drug design steps
process to develop new TIM inhibitors.

2.2. In Vitro Activity

Due to the fact that TIM is an essential protein for parasite survival, inhibition of TIM
would inhibit cellular growth. On this basis, we considered that compounds selected from
the virtual screening would have an inhibitory effect on both E. histolytica and G. lamblia.
In order to explore a variety of chemical structures against intestinal protozoa, based on
the in silico results, chlorhexidine, tolcapone, imatinib and folic acid were selected for
their evaluation in vitro. Table 1 shows that these compounds were capable of inhibiting
G. lamblia growth and only folic acid has inhibitory activity against both parasites. Only
two of these compounds, chlorhexidine and imatinib, have previous report of activity
against G. lamblia [30]. Selected compounds have a better in vitro activity on G. lamblia;
only folic acid showed inhibitory activity on E. histolytica.

Table 1. Biological activity of four FDA drugs against trophozoites from E. histolytica and G. lamblia.

Compound. IC50 E. histolytica (µg/mL) IC50 G. lamblia (µg/mL)

Metronidazole 0.205 7.8
D4 8.306 ± 1.616 1 -
Omeprazol - 0.025 2
Chlorhexidine > 100 4.93 ± 0.005
Tolcapone > 100 0.05 ± 0.002
Imatinib > 100 3.46 ± 0.005
Folic acid 0.186 ± 0.003 5.34 ± 0.007
1 [26], 2 [28], Half-maximal inhibitory concentration (IC50 ).

Chlorhexidine is an antiseptic compound used to treat bacterial and fungal infection.

Its structure is composed of two chlorophenyl guanide groups linked by a hexamethylene
bridge. As Figure 3 shows, this structure has a higher number of interactions in TIMGl than
TIMEh, which explains the differences on docking score. The high number of hydrogen
bonds represent an interesting feature that is directly related to binding stability along with
the π-stacking interactions with Tyr68B (TIMGl).
Tolcapone is a benzophenone used in the treatment of Parkinson disease. This struc-
ture has a few interactions with TIMEh but several hydrogen bonds with TIMGl. These
interactions could explain why tolcapone was only active against G. lamblia.
In the case of imatinib, an anticancer drug, interactions are mainly by hydrogen bond-
ing and hydrophobic interactions despite the four rings in its structure which commonly
form π-stacking interactions with aromatic residues.
Interestingly, folic acid, a supplement, showed a high number and diverse types of
interactions in both TIMEh and TIMGl. This was the only evaluated compound that showed
inhibitory activity against E. histolytica and G. lamblia, there are some reports considering
the impact of folic acid in parasitic infections, but no conclusion is given about its use
as an antiprotozoal compound [31,32]. Its interaction diagram summarized its potential
binding mechanism in both protozoal TIM. It mainly interacts by hydrogen bonding. Also,
it forms a π-stacking interaction with Trp75B in TIMEh and by π-cation with Arg99 on
TIMGl. Nevertheless, specific inhibition studies on TIMEh and TIMGl along with more
robust computational analysis are needed to validate these findings.
 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 8

Int. J. Mol. Sci. 2021, 22, 5943 5 of 8

Arg99 on TIMGl. Nevertheless, specific inhibition studies on TIMEh and TIMGl along with
more robust computational analysis are needed to validate these findings.

(a) (b)

(c) (d)

(e) (f)

(g) (h)
Figure
Figure 3. 3. Interaction
Interaction diagram
diagram ofof selected
selected compound
compound inin complex:
complex: (a)(a) Chlorhexidine
Chlorhexidine onon TIMEh;
TIMEh; (b)(b) Chlorhexidine
Chlorhexidine onon TIMGl;
TIMGl;
(c) Tolcapone on TIMEh; (d) Tolcapone on TIMGl; (e) Imatinib on TIMEh; (f) Imatinib on TIMGl; (g) Folic acid
(c) Tolcapone on TIMEh; (d) Tolcapone on TIMGl; (e) Imatinib on TIMEh; (f) Imatinib on TIMGl; (g) Folic acid on TIMEh; on TIMEh;
(h) Folic acid on TIMGl. Residues in bold text represent interactions shared with the known inhibitor.
(h) Folic acid on TIMGl. Residues in bold text represent interactions shared with the known inhibitor.

3. Materials and Methods

3. Materials and Methods
3.1.Docking-Based
3.1. Docking-BasedVirtual
Virtual Screening
Screening
Crystalstructures
Crystal structuresofofTIM
TIMproteins
proteinsofofE.E.histolytica
histolyticaand
andG.G.lamblia
lamblia(PDB
(PDBIDs,
IDs,TIMEh:
TIMEh:
1M6J and TIMGl: 4BI7) were retrieved from Protein Data Bank (PDB,
1M6J and TIMGl: 4BI7) were retrieved from Protein Data Bank (PDB, RCSB PDB: Home- RCSB PDB: Homep-
age accessed
page accessed on on88October
October2020).
2020).EachEachstructure
structurewas wasprepared
preparedwithwiththe
thedock
dockprep
prep tool
tool
from
from USFC
USFC Chimera
Chimera software
software (University
(University ofof California,
California, San
San Francisco,
Francisco, CA,CA, USA)
USA) [33].
[33]. InIn
this step, all ions, water molecules and co-crystalized ligands were removed,
this step, all ions, water molecules and co-crystalized ligands were removed, and missing and missing
side
side chains
chains were
were added.
added. TIM
TIM is is
a adimeric
dimericstructure
structure composed
composed ofof a TIM-barrel
a TIM-barrel structure
structure inin
each monomer with the catalytic site being inside this barrel. Due to the high similarityof
each monomer with the catalytic site being inside this barrel. Due to the high similarity
of the catalytic site among all TIM structures, species-specific inactivation is focused on
the dimeric interface. Therefore, conformational search space for docking was defined
Int. J. Mol. Sci. 2021, 22, 5943 6 of 8

as a 20 × 20 × 20 Å box centered in PDB 1M6J at x = 14.620, y = 27.866 and z = 14.108,

and in PDB 4BI7 at x = 5.710, y = −0.023 and z = −28.240. Later, a total of 2454 SDF
structures corresponding to FDA-approved drugs were retrieved from DrugBank (Drug-
Bank|Pharmaceutical Knowledge Base|API Integrations accessed on 8 October 2020) [34].
These structures, along with D4 and omeprazole (inhibitors used as control for TIMEh
and TIMGl, respectively), were split, minimized and converted to Mol2 format using open
babel [35]. Only those with a molecular weight between 100 and 900 Da were used in the
subsequent steps. A total of 1466 was successfully prepared for docking. The rest were
discarded due to its molecular weight, minimizing errors and non-supported atom types.
Additionally, AutoDockTools (ADT/AutoDockTools—AutoDock(scripps.edu) accessed
on 8 October 2020) [36] was used to specify Gasteiger partial charges and AutoDock atom
types to ligands and receptors. Then, docking was performed by Autodock vina (vina) [37]
and in-house python scripts to automate calculations (Figure S1). Finally, the non-covalent
interactions of the docked complexes were calculated with PLIP (protein–ligand interaction
profiler) [38].

3.2. In Vitro Activity

E. histolytica strain HM1-IMSS was grown in TYI-S-33 medium supplemented with
10% heat-inactivated bovine serum (Sigma Adrich, Toluca, Mexico). G. lamblia strain
IMSS:8909:1 trophozoites used in all experiments were cultivated axenically at 37 ◦ C in
TYI-S-33 modified medium supplemented with 10% calf serum and bovine bile. In vitro
susceptibility tests were performed using E. histolytica (6 × 103 ) or G. lamblia (5 × 104 )
trophozoites were incubated for 48 h at 37 ◦ C in the presence of different concentrations
(2.5–200 µg/mL) of pure compounds in DMSO at 2%. After incubation, the trophozoites
were detached by chilling and 50 µL samples of each tube were subcultured in fresh
medium for another 24 h. The final number of parasites was determined in Neubauer.
Then, data were analyzed using probit analysis. The percentage of trophozoites surviving
was calculated by comparison with the growth in the control group. The plot of probit
against log concentration was made, the best straight line was determined by regression
analysis, and the IC50 values were calculated. The regression coefficient, its level of
significance (p < 0.05 indicates significant difference between groups), and correlation
coefficient were calculated and 95% confidence interval (CI) values determined. ADMET
characteristics of selected compounds are shown in Table S3 [34].

4. Conclusions
In this work, virtual screening based on molecular docking was used to identify
potential antiprotozoal compound among FDA-approved drugs that binds to the TIM from
E. histolytica and G. lamblia. Those with a lowest average docking score were selected for
in vitro evaluation. Four compounds, chlorhexidine, tolcapone, imatinib and folic acid,
were capable of inhibiting growth of trophozoite of G. lamblia with an IC50 below standard
treatment. Folic acid also showed activity against E. histolytica. Although these results
are promising, more studies are needed to understand the mechanism underlying the
inhibitory activity of these compounds.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/

10.3390/ijms22115943/s1.
Author Contributions: Conceptualization, A.J.-S. and G.R.; methodology, A.J.-S. and E.B.-C.; re-
sources, N.E.C.; visualization, A.V.M.-V. and I.P.; writing—original draft preparation, A.J.-S.; writing—
review and editing, E.E.L.-R., V.B.-G. and G.R.; project administration, A.D.P.-G. and G.R.; funding
acquisition, G.R. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Secretaria de Investigación y Posgrado del Instituto
Politécnico Nacional, grant number 20210050.
Conflicts of Interest: The authors declare no conflict of interest.
Int. J. Mol. Sci. 2021, 22, 5943 7 of 8

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