Preclinical Studies in Anti-Trypanosomatidae Drug Development

Abstract

The trypanosomatid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania are the causative agents of human African trypanosomiasis, Chagas Disease and Leishmaniasis, respectively. These infections primarily affect poor, rural communities in the developing world, and are responsible for trapping sufferers and their families in a disease/poverty cycle. The development of new chemotherapies is a priority given that existing drug treatments are problematic. In our search for novel anti-trypanosomatid agents, we assess the growth-inhibitory properties of >450 compounds from in-house and/or “Pathogen Box” (PBox) libraries against L. infantum, L. amazonensis, L.braziliensis, T. cruzi and T. brucei and evaluate the toxicities of the most promising agents towards murine macrophages. Screens using the in-house series identified 17 structures with activity against and selective toward Leishmania: Compounds displayed 50% inhibitory concentrations between 0.09 and 25 μM and had selectivity index values >10. For the PBox library, ~20% of chemicals exhibited anti-parasitic properties including five structures whose activity against L. infantum had not been reported before. These five compounds displayed no toxicity towards murine macrophages over the range tested with three being active in an in vivo murine model of the cutaneous disease, with 100% survival of infected animals. Additionally, the oral combination of three of them in the in vivo Chagas disease murine model demonstrated full control of the parasitemia. Interestingly, phenotyping revealed that the reference strain responds differently to the five PBox-derived chemicals relative to parasites isolated from a dog. Together, our data identified one drug candidate that displays activity against Leishmania and other Trypanosomatidae in vitro and in vivo, while exhibiting low toxicity to cultured mammalian cells and low in vivo acute toxicity.

Full text

pharmaceuticals
Article
Preclinical Studies in Anti-Trypanosomatidae
Drug Development
Cintya Perdomo 1, Elena Aguilera 2, Ileana Corvo 1, Paula Faral-Tello 3, Elva Serna 4, Carlos Robello 3,5,
Shane R. Wilkinson 6,* , Gloria Yaluff 4and Guzmán Alvarez 1 ,*


Citation: Perdomo, C.; Aguilera, E.;
Corvo, I.; Faral-Tello, P.; Serna, E.;
Robello, C.; Wilkinson, S.R.; Yaluff, G.;
Alvarez, G. Preclinical Studies in
Anti-Trypanosomatidae Drug
Development. Pharmaceuticals 2021,
14, 644. https://doi.org/10.3390/
ph14070644
Academic Editors: Jean Jacques
Vanden Eynde and Annie Mayence
Received: 2 June 2021
Accepted: 30 June 2021
Published: 5 July 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Laboratorio de Moléculas Bioactivas, Departamento de Ciencias Biológicas, CENUR Litoral Norte,
Universidad de la República, Paysandú60000, Uruguay; cuquis266@gmail.com (C.P.);
ilecorvo@gmail.com (I.C.)
2Grupo de Química Orgánica Medicinal, Instituto de Química Biológica, Facultad de Ciencias,
Universidad de la República, Montevideo 11400, Uruguay; elepao168@gmail.com
3Institut Pasteur de Montevideo, Unidad de Biología Molecular, Montevideo11400, Uruguay;
pfaral@pasteur.edu.uy (P.F.-T.); robello@pasteur.edu.uy (C.R.)
4Departamento de Medicina Tropical, Instituto de Investigaciones en Ciencias de la Salud,
Universidad Nacional de Asunción, San Lorenzo 2169, Paraguay; elvsern@hotmail.com (E.S.);
gloriayaluff@yahoo.com (G.Y.)
5Departamento de Bioquímica, Facultad de Medicina, Universidad de la República,
Montevideo11800, Uruguay
6Queen Mary Pre-Clinical Drug Discovery Group, School of Biological and Chemical Sciences,
Queen Mary University of London, London E1 4NS, UK
*Correspondence: s.r.wilkinson@qmul.ac.uk (S.R.W.); guzmanalvarezlqo@gmail.com (G.A.)
Abstract:
The trypanosomatid parasites Trypanosoma brucei,Trypanosoma cruzi and Leishmania are the
causative agents of human African trypanosomiasis, Chagas Disease and Leishmaniasis, respectively.
These infections primarily affect poor, rural communities in the developing world, and are respon-
sible for trapping sufferers and their families in a disease/poverty cycle. The development of new
chemotherapies is a priority given that existing drug treatments are problematic. In our search for
novel anti-trypanosomatid agents, we assess the growth-inhibitory properties of >450 compounds
from in-house and/or “Pathogen Box” (PBox) libraries against L. infantum, L. amazonensis, L. brazilien-
sis, T. cruzi and T. brucei and evaluate the toxicities of the most promising agents towards murine
macrophages. Screens using the in-house series identified 17 structures with activity against and
selective toward Leishmania: Compounds displayed 50% inhibitory concentrations between 0.09 and
25
µ
M and had selectivity index values >10. For the PBox library, ~20% of chemicals exhibited
anti-parasitic properties including five structures whose activity against L. infantum had not been
reported before. These five compounds displayed no toxicity towards murine macrophages over the
range tested with three being active in an
in vivo
murine model of the cutaneous disease, with 100%
survival of infected animals. Additionally, the oral combination of three of them in the
in vivo
Cha-
gas disease murine model demonstrated full control of the parasitemia. Interestingly, phenotyping
revealed that the reference strain responds differently to the five PBox-derived chemicals relative to
parasites isolated from a dog. Together, our data identified one drug candidate that displays activity
against Leishmania and other Trypanosomatidae
in vitro
and
in vivo
, while exhibiting low toxicity to
cultured mammalian cells and low in vivo acute toxicity.
Keywords:
anti-trypanosomatid; arylidene ketones; thiazolidene hydrazines; Pathogen box;
veterinary
isolates
1. Introduction
Neglected tropical diseases (NTD) represent a series of infections that cause illness in
more than 1.7 billion people worldwide [
1
]. They are prevalent in low-income populations
that live in developing areas of Latin America, Africa and Asia, and are responsible for
Pharmaceuticals 2021,14, 644. https://doi.org/10.3390/ph14070644 https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021,14, 644 2 of 19
approximately 150,000 deaths per year [
2
]. Additionally, it can limit the productivity in the
workplace and make it difficult for the infected people to earn a living. It can impair the
physical and cognitive development of children, and can lead to pathologies that result
in social stigma. Together, these all contribute to trapping vulnerable individuals and
their dependents in a disease/poverty cycle. Perversely, and for many NTDs, appropriate
screening programs and cheap, simple to use prophylaxis and/or curative treatments
are available but until recently the widespread implementation of such schemes has not
been forthcoming. Driven by World Health Organization, Drugs for Neglected Diseases
initiative, the Bill and Melinda Gates Foundation and other charity organizations, the true
impact of NTDs on communities least able to deal with these major public health issues was
brought to the attention of governments in the developed world and the pharmaceutical
industry. This has promoted the implementation of prevention, control and treatment
programs with the WHO launching a roadmap that aims to eliminate or eradicate 20 NTDs
by 2030 [1].
Three NTDs, human African trypanosomiasis (HAT), Chagas disease and leishmani-
asis, are caused by protozoa belonging to the family Trypanosomatidae [
1
]. The tsetse fly
transmitted zoonotic infection, HAT is prevalent in 36 countries across sub-Saharan Africa
and caused by subspecies of Trypanosoma brucei. In mammals, this parasite is found at
extracellular sites being restricted in the early stages of infection to the host’s bloodstream
and lymphatic system, where it causes relapsing fever, before eventually crossing the
blood-brain barrier and gaining access to the cerebral spinal fluid, where it elicits diverse
mental, neurological, sensory, and motor manifestations [
3
]. In contrast, Chagas disease is
caused by Trypanosoma cruzi, an intracellular parasite that can invade any nucleated cell in
any mammalian host. This zoonotic disease is transmitted by blood-sucking triatomine
insects and endemic in 21 countries across Latin America. In the initial, acute stage of the
disease, patients are generally asymptomatic although if symptoms are provoked, they
tend to be non-specific, mild and flu-like (fever, fatigue, body aches, and headaches) [
4
].
As such, many Chagas disease cases generally remain undiagnosed [
5
] and in about 70%
of situations, the infection does not progress any further. However, in the remaining in-
stances, the disease can reactivate with the patient entering the chronic stage. Here, cardiac
and gastrointestinal clinical manifestations arise leading to a multitude of heart-related
conditions (arrythmias, conductive defects, cardiomegaly, etc.), megaesophagus and/or
megacolon disorders that are invariably fatal [
4
]. Estimates indicate that up to 8 million
people are currently infected with T. cruzi [
1
] and although incidence at endemic sites has
decreased significantly over the last 20 years, the number of cases in non-endemic regions
(United States, Australia, Europe, and Japan) driven by population migration, modern
medical practices and congenital transfer, have increased: Estimates suggest that there are
about 325,000 and 100,000 cases in the US and Europe, respectively [
6
], indicating that
Chagas disease has truly emerged as a global infection.
The third NTD caused by a member of the Family Trypanosomatidae is leishmaniasis.
This disease represents a group of sandfly transmitted zoonotic diseases caused by different
Leishmania that dependent on the protozoal species, and host immune response affect the
skin, mucosa, and/or viscera. As with T. cruzi,Leishmania are intracellular parasites but
unlike their trypanosome counterpart, infect only macrophages and neutrophils. Infec-
tions are generally chronic, have low morbidity and moderate mortality, and based on
the targeted area, can be disfiguring. Leishmaniasis is prevalent in 98 countries across
5 continents with an estimated 1.3 million new cases occurring each year. About one-third
of new infections correspond to the life-threatening, visceral form of the disease, caused by
Leishmania donovani, a human-to-human transmitted species that predominates in Africa
and the Indian sub-continent, or Leishmania infantum (or its synonym Leishmania chagasi), a
pathogen that can spread from animals (particularly canines) to humans and is found in
the Mediterranean basin and Americas. These parasites promote widespread damage to
the host’s internal organs that weaken the patient’s immune system and make them prone
to secondary infections such as pneumonia, tuberculosis, dysentery or AIDS [7].

Pharmaceuticals 2021,14, 644 3 of 19
With no prospect of prophylaxis, curative chemotherapies currently represent the only
way to treat Trypanosomatidae infections. However, their use can be problematic. For
example, toxic and sometimes fatal side effects are encountered when using the difficult
to dispense, arsenic-containing anti-HAT Melarsoprol, while alternative therapies based
around Eflornithine and Nifurtimox may be expensive, require high dosages, have complex
administration routes, are carcinogenic or have limited efficacy [
8
]). Likewise, drugs
based on phosphocholine (Miltefosine), antimony (Glucantime and Pentostam), cyclic
antibiotics (Amphotericin B), aminoglycosides (Paromomycin), and aromatic diamidines
(Pentamidine) can be used against leishmaniasis but again there are concerns about the
efficacy, cost, and adverse effects of such treatments [
9
]. To complicate this situation, strains
that are non-responsive to treatment have emerged, limiting the usefulness of certain
therapies in some regions [10–12].
Against this backdrop, there is an urgent need for new therapies targeting trypanoso-
mal and leishmanial diseases, not only in humans, but also in animal reservoirs. Here, we
evaluate the growth inhibitory properties of two chemical series, an in-house chemical
collection and/or an open-access Pathogen Box (PBox) library, against Trypanosomati-
dae parasites.
2. Results and Discussion
The in-house chemical collection was tested against L. infantum,L. amazonensis and T.
brucei, and outcomes compared with the previously reported anti-T. cruzi activities [
13
–
16
]:
Data summarizing the
in vitro
and
in vivo
activity against T. cruzi, toxicology profiles, and
potential mechanism of action of the four best hits arising from the previous studies are
shown in Table 1. For the PBox library [
17
], we tested the growth inhibitory properties
of the collection against a reference strain of L. infantum and an isolate obtained from the
latest canine Leishmaniasis outbreak in the World [
18
,
19
]. We also collate information
about commercial availability, literature reference citations, and other associated biological
activity arising from promising hits following extensive literature reviews and database
searches (PubChem, Scifinder
®
, Reaxys
®
, patents, etc). The hits identified here provide
new chemical starting points for novel drugs targeting NTDs caused by Trypanosomatidae
and could help individuals escape from the disease/poverty cycle.
2.1. Chemistry
The compounds used in this study can be made using simple one or two-step, en-
vironmentally friendly synthetic procedures (For thiazolidene hydrazine and arylidene
ketone synthesis see Supplementary Information: Materials and Methods). The methods
employed are readily scalable and facilitate the manufacture of small (grams) to large (kilo-
grams) of each chemical and at low cost. Together, the chemistry involved in synthesizing
chemicals from the in-house and PBox collections met several of the requirements expected
when developing a new drug designed to target NTDs.
2.2. Antiparasitic Activity In Vitro
The growth inhibitory properties of more than 450 molecules derived from two
chemical collections were tested against Leishmania spp or T. brucei. For the PBox library,
approximately 20% of compounds were shown to have activity against L. infantum although
variation in susceptibility phenotypes displayed by the two parasite strains tested were
noted. For example, compounds A10, E2 and G7 displayed significant growth inhibitory
activity (as judged by EC
50
values of <15
µ
M) against the reference strain but were less
effective towards the canine-derived isolate (EC
50
values of >120
µ
M) while the reciprocal
was observed when testing compounds B5, B11, C3 and H2 (Supplementary Figure S1).
This variation in strain susceptibility is consistent with previous observations [
19
]. The
underlying reason as to why the two L. infantum strains display different susceptibilities
towards an array of unrelated chemotypes has yet to be established but given the adaptable
nature of Leishmania we postulate that life cycle selection processes have helped shape the

Pharmaceuticals 2021,14, 644 4 of 19
parasite’s genome leading to the strains with contrasting gene/protein expression profiles
and the observed phenotypic variations [
20
–
22
]. This highlights that when screening for
novel chemotherapeutics targeting a trypanosomatid parasite, multiple strains of the same
species obtained from different sources should be tested.
Table 1.
Anti-T. cruzi activity and general toxicological profile of the four best hits identified from our in-house chemi-
cal collection a.
Compound a
314 1019 796 266
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 3 of 19
humans and is found in the Mediterranean basin and Americas. These parasites promote
widespread damage to the host’s internal organs that weaken the patient’s immune sys-
tem and make them prone to secondary infections such as pneumonia, tuberculosis,
dysentery or AIDS [7].
With no prospect of prophylaxis, curative chemotherapies currently represent the
only way to treat Trypanosomatidae infections. However, their use can be problematic.
For example, toxic and sometimes fatal side effects are encountered when using the dif-
ficult to dispense, arsenic-containing anti-HAT Melarsoprol, while alternative therapies
based around Eflornithine and Nifurtimox may be expensive, require high dosages, have
complex administration routes, are carcinogenic or have limited efficacy [8]). Likewise,
drugs based on phosphocholine (Miltefosine), antimony (Glucantime and Pentostam),
cyclic antibiotics (Amphotericin B), aminoglycosides (Paromomycin), and aromatic di-
amidines (Pentamidine) can be used against leishmaniasis but again there are concerns
about the efficacy, cost, and adverse effects of such treatments [9]. To complicate this
situation, strains that are non-responsive to treatment have emerged, limiting the use-
fulness of certain therapies in some regions [10–12].
Against this backdrop, there is an urgent need for new therapies targeting trypa-
nosomal and leishmanial diseases, not only in humans, but also in animal reservoirs.
Here, we evaluate the growth inhibitory properties of two chemical series, an in-house
chemical collection and/or an open-access Pathogen Box (PBox) library, against Trypa-
nosomatidae parasites.
2. Results and Discussion
The in-house chemical collection was tested against L. infantum, L. amazonensis and
T. brucei, and outcomes compared with the previously reported anti-T. cruzi activities
[13–16]: Data summarizing the in vitro and in vivo activity against T. cruzi, toxicology
profiles, and potential mechanism of action of the four best hits arising from the previous
studies are shown in Table 1. For the PBox library [17], we tested the growth inhibitory
properties of the collection against a reference strain of L. infantum and an isolate ob-
tained from the latest canine Leishmaniasis outbreak in the World [18,19]. We also collate
information about commercial availability, literature reference citations, and other asso-
ciated biological activity arising from promising hits following extensive literature re-
views and database searches (PubChem, Scifinder®, Reaxys®, patents, etc). The hits iden-
tified here provide new chemical starting points for novel drugs targeting NTDs caused
by Trypanosomatidae and could help individuals escape from the disease/poverty cycle.
Table 1. Anti-T. cruzi activity and general toxicological profile of the four best hits identified from our in-house chemical
collection a.
Compound a
314 1019 796 266
Activity in vitro anti-T. cruzi (multiple strains)
bEC50 0.72 μM
Amastigotes EC50 0.60 μM epimastigotes EC50 5.0 μM epimastigotes EC50 > 0.25 μM
amastigotes
Selectivity index > 100 (EC50 mammalian cell/EC50 T. cruzi)
Mechanism of action
Cruzipain
cIC50 4.3 μM
triosephosphate isomerase
IC50 86 nM unknown unknown
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 3 of 19
humans and is found in the Mediterranean basin and Americas. These parasites promote
widespread damage to the host’s internal organs that weaken the patient’s immune sys-
tem and make them prone to secondary infections such as pneumonia, tuberculosis,
dysentery or AIDS [7].
With no prospect of prophylaxis, curative chemotherapies currently represent the
only way to treat Trypanosomatidae infections. However, their use can be problematic.
For example, toxic and sometimes fatal side effects are encountered when using the dif-
ficult to dispense, arsenic-containing anti-HAT Melarsoprol, while alternative therapies
based around Eflornithine and Nifurtimox may be expensive, require high dosages, have
complex administration routes, are carcinogenic or have limited efficacy [8]). Likewise,
drugs based on phosphocholine (Miltefosine), antimony (Glucantime and Pentostam),
cyclic antibiotics (Amphotericin B), aminoglycosides (Paromomycin), and aromatic di-
amidines (Pentamidine) can be used against leishmaniasis but again there are concerns
about the efficacy, cost, and adverse effects of such treatments [9]. To complicate this
situation, strains that are non-responsive to treatment have emerged, limiting the use-
fulness of certain therapies in some regions [10–12].
Against this backdrop, there is an urgent need for new therapies targeting trypa-
nosomal and leishmanial diseases, not only in humans, but also in animal reservoirs.
Here, we evaluate the growth inhibitory properties of two chemical series, an in-house
chemical collection and/or an open-access Pathogen Box (PBox) library, against Trypa-
nosomatidae parasites.
2. Results and Discussion
The in-house chemical collection was tested against L. infantum, L. amazonensis and
T. brucei, and outcomes compared with the previously reported anti-T. cruzi activities
[13–16]: Data summarizing the in vitro and in vivo activity against T. cruzi, toxicology
profiles, and potential mechanism of action of the four best hits arising from the previous
studies are shown in Table 1. For the PBox library [17], we tested the growth inhibitory
properties of the collection against a reference strain of L. infantum and an isolate ob-
tained from the latest canine Leishmaniasis outbreak in the World [18,19]. We also collate
information about commercial availability, literature reference citations, and other asso-
ciated biological activity arising from promising hits following extensive literature re-
views and database searches (PubChem, Scifinder®, Reaxys®, patents, etc). The hits iden-
tified here provide new chemical starting points for novel drugs targeting NTDs caused
by Trypanosomatidae and could help individuals escape from the disease/poverty cycle.
Table 1. Anti-T. cruzi activity and general toxicological profile of the four best hits identified from our in-house chemical
collection a.
Compound a
314 1019 796 266
Activity in vitro anti-T. cruzi (multiple strains)
bEC50 0.72 μM
Amastigotes EC50 0.60 μM epimastigotes EC50 5.0 μM epimastigotes EC50 > 0.25 μM
amastigotes
Selectivity index > 100 (EC50 mammalian cell/EC50 T. cruzi)
Mechanism of action
Cruzipain
cIC50 4.3 μM
triosephosphate isomerase
IC50 86 nM unknown unknown
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 3 of 19
humans and is found in the Mediterranean basin and Americas. These parasites promote
widespread damage to the host’s internal organs that weaken the patient’s immune sys-
tem and make them prone to secondary infections such as pneumonia, tuberculosis,
dysentery or AIDS [7].
With no prospect of prophylaxis, curative chemotherapies currently represent the
only way to treat Trypanosomatidae infections. However, their use can be problematic.
For example, toxic and sometimes fatal side effects are encountered when using the dif-
ficult to dispense, arsenic-containing anti-HAT Melarsoprol, while alternative therapies
based around Eflornithine and Nifurtimox may be expensive, require high dosages, have
complex administration routes, are carcinogenic or have limited efficacy [8]). Likewise,
drugs based on phosphocholine (Miltefosine), antimony (Glucantime and Pentostam),
cyclic antibiotics (Amphotericin B), aminoglycosides (Paromomycin), and aromatic di-
amidines (Pentamidine) can be used against leishmaniasis but again there are concerns
about the efficacy, cost, and adverse effects of such treatments [9]. To complicate this
situation, strains that are non-responsive to treatment have emerged, limiting the use-
fulness of certain therapies in some regions [10–12].
Against this backdrop, there is an urgent need for new therapies targeting trypa-
nosomal and leishmanial diseases, not only in humans, but also in animal reservoirs.
Here, we evaluate the growth inhibitory properties of two chemical series, an in-house
chemical collection and/or an open-access Pathogen Box (PBox) library, against Trypa-
nosomatidae parasites.
2. Results and Discussion
The in-house chemical collection was tested against L. infantum, L. amazonensis and
T. brucei, and outcomes compared with the previously reported anti-T. cruzi activities
[13–16]: Data summarizing the in vitro and in vivo activity against T. cruzi, toxicology
profiles, and potential mechanism of action of the four best hits arising from the previous
studies are shown in Table 1. For the PBox library [17], we tested the growth inhibitory
properties of the collection against a reference strain of L. infantum and an isolate ob-
tained from the latest canine Leishmaniasis outbreak in the World [18,19]. We also collate
information about commercial availability, literature reference citations, and other asso-
ciated biological activity arising from promising hits following extensive literature re-
views and database searches (PubChem, Scifinder®, Reaxys®, patents, etc). The hits iden-
tified here provide new chemical starting points for novel drugs targeting NTDs caused
by Trypanosomatidae and could help individuals escape from the disease/poverty cycle.
Table 1. Anti-T. cruzi activity and general toxicological profile of the four best hits identified from our in-house chemical
collection a.
Compound a
314 1019 796 266
Activity in vitro anti-T. cruzi (multiple strains)
bEC50 0.72 μM
Amastigotes EC50 0.60 μM epimastigotes EC50 5.0 μM epimastigotes EC50 > 0.25 μM
amastigotes
Selectivity index > 100 (EC50 mammalian cell/EC50 T. cruzi)
Mechanism of action
Cruzipain
cIC50 4.3 μM
triosephosphate isomerase
IC50 86 nM unknown unknown
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 3 of 19
humans and is found in the Mediterranean basin and Americas. These parasites promote
widespread damage to the host’s internal organs that weaken the patient’s immune sys-
tem and make them prone to secondary infections such as pneumonia, tuberculosis,
dysentery or AIDS [7].
With no prospect of prophylaxis, curative chemotherapies currently represent the
only way to treat Trypanosomatidae infections. However, their use can be problematic.
For example, toxic and sometimes fatal side effects are encountered when using the dif-
ficult to dispense, arsenic-containing anti-HAT Melarsoprol, while alternative therapies
based around Eflornithine and Nifurtimox may be expensive, require high dosages, have
complex administration routes, are carcinogenic or have limited efficacy [8]). Likewise,
drugs based on phosphocholine (Miltefosine), antimony (Glucantime and Pentostam),
cyclic antibiotics (Amphotericin B), aminoglycosides (Paromomycin), and aromatic di-
amidines (Pentamidine) can be used against leishmaniasis but again there are concerns
about the efficacy, cost, and adverse effects of such treatments [9]. To complicate this
situation, strains that are non-responsive to treatment have emerged, limiting the use-
fulness of certain therapies in some regions [10–12].
Against this backdrop, there is an urgent need for new therapies targeting trypa-
nosomal and leishmanial diseases, not only in humans, but also in animal reservoirs.
Here, we evaluate the growth inhibitory properties of two chemical series, an in-house
chemical collection and/or an open-access Pathogen Box (PBox) library, against Trypa-
nosomatidae parasites.
2. Results and Discussion
The in-house chemical collection was tested against L. infantum, L. amazonensis and
T. brucei, and outcomes compared with the previously reported anti-T. cruzi activities
[13–16]: Data summarizing the in vitro and in vivo activity against T. cruzi, toxicology
profiles, and potential mechanism of action of the four best hits arising from the previous
studies are shown in Table 1. For the PBox library [17], we tested the growth inhibitory
properties of the collection against a reference strain of L. infantum and an isolate ob-
tained from the latest canine Leishmaniasis outbreak in the World [18,19]. We also collate
information about commercial availability, literature reference citations, and other asso-
ciated biological activity arising from promising hits following extensive literature re-
views and database searches (PubChem, Scifinder®, Reaxys®, patents, etc). The hits iden-
tified here provide new chemical starting points for novel drugs targeting NTDs caused
by Trypanosomatidae and could help individuals escape from the disease/poverty cycle.
Table 1. Anti-T. cruzi activity and general toxicological profile of the four best hits identified from our in-house chemical
collection a.
Compound a
314 1019 796 266
Activity in vitro anti-T. cruzi (multiple strains)
bEC50 0.72 μM
Amastigotes EC50 0.60 μM epimastigotes EC50 5.0 μM epimastigotes EC50 > 0.25 μM
amastigotes
Selectivity index > 100 (EC50 mammalian cell/EC50 T. cruzi)
Mechanism of action
Cruzipain
cIC50 4.3 μM
triosephosphate isomerase
IC50 86 nM unknown unknown
Activity in vitro anti-T. cruzi (multiple strains)
bEC50 0.72 µM
Amastigotes EC50 0.60 µM epimastigotes EC50 5.0 µM epimastigotes EC50 > 0.25 µM
amastigotes
Selectivity index > 100 (EC50 mammalian cell/EC50 T. cruzi)
Mechanism of action
Cruzipain
cIC50 4.3 µM
triosephosphate isomerase
IC50 86 nM unknown unknown
Stability in vitro (microsomal, plasma, other solutions)
High low moderate high
Toxicology and Efficacy
Ames Test (mutagenicity)
No no unknown no
Micronucleus test in mice (Genotoxicity)
No unknown unknown no
Acute oral toxicity (up and down test)
dLD50 > 2000 mg/kg
in mice unknown unknown LD50 > 2000 mg/kg
in mice
Full control of the parasitemia in vivo at 50 mg/kg in the murine model of Chagas disease
a
Data were taken from [
13
–
15
].
b
EC
50
refers to the effective concentration of compound that inhibits cell growth by 50%
c
IC
50
the
concentration of compound required to inhibit enzyme activity by 50%,
d
LD
50
the concentration of a compound that kills 50% of the
animals tested in the in vivo acute oral toxicity assay.
Of those PBox library compounds that did display anti-L. infantum properties, 5 com-
pounds (
MMV272144
[1-(3-Methoxyphenyl)-5-(methylsulfonyl)-1H-tetrazole],
MMV688761
[N-(1,3-Benzothiazol-2-yl)-4-methylsulfonyl-3-nitro-N-[[(2R)-oxolan-2-yl]methyl]benzamide],
MMV688768
[2-Methyl-3-[(R)-(4-methylpiperazin-1-yl)-thiophen-2-ylmethyl]-1H-indole],
MMV688763
[4-Chloro-5-{[5-(methylamino)-1,3,4-thiadiazol-2-yl]sulfanyl}-2-phenyl-2,3-
dihydropyridazin-3-one] and
MMV021013
[N-Cyclohexyl-6-cyclopropyl-2-pyridin-2
-ylpyrimidin-4-amine]) represented novel activities whose effect on this parasite had
not been previously described, exhibiting equal potency towards the two strains tested
(Table 2).
Further phenotyping revealed these structures had no growth-inhibitory effect on
murine macrophages over the concentration range tested (up to 50
µ
M) with most having
selectivity index values (calculated as a ratio of the EC
50
against the mammalian line to the
EC50 against the parasite) equivalent or superior to Miltefosine (Table 2).

Pharmaceuticals 2021,14, 644 5 of 19
Table 2. Leishmanicidal and selectivity properties of five novel hits identified from the PBox library.
Compounds
EC50 (µM) Selectivity Index b
L. infantum a
MΦCytotoxicity aMΦ/Reference MΦ/Veterinary
Reference Veterinary
MMV272144 2.4 ±0.3 1.2 ±0.1 >50 >21 >42
MMV688761 4.9 ±0.1 3.9 ±0.3 >50 >10 >13
MMV688768 9.8 ±0.5 6.8 ±0.1 >50 >5 >7
MMV688763 2.1 ±0.2 0.9 ±0.1 >50 >24 >56
MMV021013 0.4 ±0.1 0.3 ±0.1 >50 >125 >167
Miltefosine c5.3 ±0.1 4.1 ±0.1 50 ±7 10 12
a
Compounds were screened against reference (MHOM/BR/2002/LPC-RPV) and veterinary isolate (MCAN_UY_2015_gPL8) strains of L.
infantum while mammalian cytotoxicity assessed using J774.1 murine macrophages (M
Φ
). The Leishmania EC
50
values are means
±
standard
deviation from assays performed in triplicate.
b
The selectivity index corresponds to the fold difference in EC
50
values of J774.1 murine
macrophages versus the reference or veterinary isolate L. infantum strains.
c
For comparative purposes, the L. infantum EC
50
data for
Miltefosine were taken from [19].
As these five compounds represent potential leads to treat visceral leishmaniasis,
informatic searches were performed to explore the synthetic route for each chemical and
evaluate for any synthetic difficulties and accessibility issue that may need to be addressed:
Additional information above these five hits including structure/biological activities/LD50
values, are given in Supplementary Table S1. This indicated that
MMV272144
has a
simple synthetic route but has a low
in vivo
absorption and low metabolic stability while
MMV688763
is reported to have pharmacokinetic problems (data provided by the DNDi
program, non-published). In the case of
MMV021013
, its potential as an anti-leishmanial
agent has previously been reported with it displaying significant activity against the L.
donovani form found in a mammalian host [
23
,
24
]. Together with the observation that it
can be cheaply synthesized and exhibits low cytotoxicity,
MMV021013
was proposed to be
a good candidate for future in vivo studies [23,24].
Extending the screens to an in-house thiazolidene hydrazine series revealed that of the
20 compounds tested, 13 were deemed active (EC
50
values < 20
µ
M) against the L. infantum
reference strain, with
266
,
314
and
1147
also displaying growth inhibitory properties against
the canine-derived isolate (Table 3): These three compounds represent the only structures
to have activity against the veterinary isolate. To gauge how this library affects other
trypanosomatids, their potency toward L. amazonensis and T. brucei was determined or, for
L. braziliensis and T. cruzi, EC
50
values sourced from published data [
13
,
15
,
25
]. This revealed
that
266
and
314
, displayed growth inhibitory effects (EC
50
values < 20
µ
M) against all
the parasite species tested with their pharmacological profiles making them candidates
for the treatment of leishmaniasis, Chagas disease and HAT. For
872
, activities against
T. cruzi,T. brucei and three Leishmania isolates were observed (
872
displayed no growth
inhibitory effect on the L. infantum canine-derived isolate) while
873
,
887
,
911
,
909
,
1115
and
1153
exhibited trypanocidal properties. Other compounds of note include
1102
, which
in terms of its effect on Leishmania, behaved similarly to
872
, and
1147
, which affects both
L. infantum strains and L. braziliensis but not L. amazonensis.

Pharmaceuticals 2021,14, 644 6 of 19
Table 3. Susceptibility of Trypanosomatidae parasites to thiazolidene hydrazines.
Chemical Code EC50 ±SD (µM) a,b
Lin–Ref Lin–Vet L. amaz L. brazils cT. cruzi cT. brucei
Nifurtimox d6.0 ±1.0 10.0 ±2.0 6.0 ±2.0 7.0 ±2.0 1.44.0 e
Glucantime d26.0 ±1.0 18.0 ±2.0 20.0 ±9.0
Miltefosine d5.3 ±0.1 f4.1 ±0.1 f5±2 8 ±2
1385 9.0 ±1.0 19.0 ±3.0
1109
266 2.0 ±0.2 9.0 ±1.0 7.0 ±1.0 20.0 ±2.0 1.6 ±0.5 5.0 ±1.0
872 10.0 ±5.0 10.0 ±1.0 8.0 ±2.0 3.0 ±0.5 6.0 ±1.0
873 0.09 ±0.02 14.0 ±3.0
1153 <3.0 6.0 ±1.0
295 3.5 ±0.2
133
877 15 ±3 10 ±1
1134
314 1.3 ±0.5 2.5 ±1.0 12.0 ±5.0 4.0 ±1.0 3.1 ±0.2 5.0 ±1.0
1112 1.5 ±0.2 1.2 ±0.2 12.0 ±3.0
1115 11.0 ±4.0 7.0 ±2.0
901 1.2 ±0.3 5.0 ±1.0
1119 <6.0 10.0 ±2.0 12.0 ±1.0
1102 5.0 ±2.0 16.0 ±4.0 14.0 ±2.0 1.6 ±0.3 14.0 ±2.0
1140
912 <0.4 18.0 ±5.0
903 3.0 ±1.0 12.0 ±2.0 17.0 ±5.0
263 <0.3 5.0 ±1.0
909 10.0 6.0 ±1.0 19.0 ±1.0
1366 12.0 ±2.0
1367
1369 8.0 ±2.0 15.0 ±5.0
1222
1219
1147 5.0 ±1.0 9.0 ±2.0 <12.0
1097
a
Growth inhibitory effect as judged by mean EC
50 ±
standard deviation of thiazolidene hydrazines (structures in Figure S3) on L. infantum
reference (Linf–ref) and veterinary (Lin–vet) strains, L. amazonensis (L. amaz),L. braziliensis (L. brazils), T. cruzi or T. brucei.
b
Green boxes
represent compounds with anti-parasitic activity (EC
50
values < 20
µ
M) with the EC
50
value indicated. Red boxes signify compounds with
no anti-parasitic activity (EC
50
values > 20
µ
M). Clear boxes represent compounds whose activity was not determined.
d
control drugs
c,e,f
Data from [13,16,19,23,25] respectively.
For the thiazolidene hydrazine series a qualitative analysis of the structure-activity
relationship was performed (Figure 1). We note that the resonance of electrons with
the opening of the furan ring is a determinant of the biological activity in Leishmania
spp., suggesting that this structural motif is part of the pharmacophore of the molecules.
For example, comparing
1109,
which does not have a double bond conjugated to the
furan ring, to
266
, only the latter has biological activity. If we vary the electron density
throughout this conjugation by the incorporation of a methyl group (
873
), we observe
that the activity decreases significantly in Leishmania spp., but not in T. cruzi. Comparing
the furan-containing
266
with
1134,
its thiophene equivalent, a decrease in leishmancidal
activity was observed possibly due to the lower probability of opening of the aromatic ring
in thiophene compare to the furan ring [24]. This suggests a mechanism of action with an
opened furan ring, as a toxic reactive biotransformed molecule.

Pharmaceuticals 2021,14, 644 7 of 19
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 6 of 19
that the activity decreases significantly in Leishmania spp., but not in T. cruzi. Comparing
the furan-containing 266 with 1134, its thiophene equivalent, a decrease in leishmancidal
activity was observed possibly due to the lower probability of opening of the aromatic
ring in thiophene compare to the furan ring [24]. This suggests a mechanism of action
with an opened furan ring, as a toxic reactive biotransformed molecule.
Figure 1. Structure-Activity Relationship a structure striping to decode the possible pharmacophore. This figure showing
the changes in the antiparasitic activity related to little structure modification of the 266 compound. The red arrow minds
a decrement in the antiparasitic activity.
Also, if we compare these molecules with compound 901, the latter has better in
vitro activity against T. cruzi but it has synthetic and biological activity problems because
of tautomerization. The family of selenium compounds arises from the substitution of the
sulfur atom by its bioisosteric selenium in the family of thiazoles described above. Four
compounds were evaluated, of which 1147 was the one with the best biological activity,
being their EC50 in L. infantum (ref and clinical isolate) of 5 and 9 μM, respectively.
However, these compounds have more steps and synthetic difficulties, and lower stabil-
ity than the parental ones.
Table 3. Susceptibility of Trypanosomatidae parasites to thiazolidene hydrazines.
Chemical Code EC50 ± SD (μM) a;b
Lin–Ref Lin–Vet L. amaz L. brazils c T. cruzi c T. brucei
Nifurtimox d 6.0 ± 1.0 10.0 ± 2.0 6.0 ± 2.0 7.0 ± 2.0 1.44.0 e
Glucantime d 26.0 ± 1.0 18.0 ± 2.0 20.0 ± 9.0
Miltefosine d 5.3 ± 0.1 f 4.1 ± 0.1 f 5 ± 2 8 ± 2
1385 9.0 ± 1.0 19.0 ± 3.0
1109
266 2.0 ± 0.2 9.0 ± 1.0 7.0 ± 1.0 20.0 ± 2.0 1.6 ± 0.5 5.0 ± 1.0
872 10.0 ± 5.0 10.0 ± 1.0 8.0 ± 2.0 3.0 ± 0.5 6.0 ± 1.0
873 0.09 ± 0.02 14.0 ± 3.0
1153 <3.0 6.0 ± 1.0
295 3.5 ± 0.2
ONNN
S
Cl
ONNN
S
Cl
NN
S
N
OCl
NNN
SS
Cl
266
873
1109
1134
N
N
H
S
N
O
Cl
901
Figure 1.
Structure-Activity Relationship a structure striping to decode the possible pharmacophore. This figure showing
the changes in the antiparasitic activity related to little structure modification of the
266
compound. The red arrow minds a
decrement in the antiparasitic activity.
Also, if we compare these molecules with compound
901
, the latter has better
in vitro
activity against T. cruzi but it has synthetic and biological activity problems because of
tautomerization. The family of selenium compounds arises from the substitution of the
sulfur atom by its bioisosteric selenium in the family of thiazoles described above. Four
compounds were evaluated, of which
1147
was the one with the best biological activity,
being their EC
50
in L. infantum (ref and clinical isolate)of 5 and 9
µ
M, respectively. However,
these compounds have more steps and synthetic difficulties, and lower stability than the
parental ones.
In the case of 20 curcuminoid-based structures (Table 4),
799
,
795
and
796
were active
against all the trypanosomatids strains tested with three others (
809
,
223
and
1019
) display-
ing anti-parasitic properties towards the L. infantum, L. amazonensis,L. braziliensis and T.
cruzi reference strains: The three latter compounds showed no trypanosomicidal effect on
the canine-derived L. infantum or T. brucei. From a pharmacological perspective, this family
of compounds is often overlooked even though they do exhibit biological activities against
a range of different organisms, including Leishmania, primarily because their classical
structure is metabolically unstable [26].
We postulate that the metabolic stability of two of the best hits,
795
and
796
, may
be increased as they contain furan or thiophene ring structures at the end of the carbon
chain linker, respectively instead of phenolic groupings as found in classical curcuminoids.
Even so, both structures are still considered Pan-Assay Interference Compounds (PAINS)
because they also have groupings that can function as Michael acceptors with these able to
nonspecific toxicity. However, despite extensive efforts, we were unable to identify what
these nonspecific activities are for the compounds used here [14,27–29].

Pharmaceuticals 2021,14, 644 8 of 19
Table 4. Susceptibility of Trypanosomatidae parasites to curcuminoids.
Chemical Code EC50 ±SD (µM) a,b
Lin–Ref Lin–Vet L. amaz L. brazils cT. cruzi cT. brucei
Glucantime d26.0 ±1.0 18.0 ±2.0 20.0 ±9.0
Curcumin 5.0 ±1.0 6.0 ±1.0 6.0 ±1.0 8.0 ±1.0
797 11.0 ±2.0
799 <0.3 12.0 ±3.0 5.0 ±1.0 4.2 ±0.9 5.0 ±1.0 1.7 ±0.5
800 14.0 15.0 ±1.0
795 5.0 ±1.0 10.0 ±2.0 10.0 ±6.0 6.0 ±2.0 24.0 ±2.0 1.8 ±0.5
793 <0.3 5.0 ±0.7 17.0 ±2.0
809 3.0 ±1.0 16.0 ±2.0 6.0 ±1.0 8.0 ±2.0
1223 <0.3 18.0 ±4.0 16.0 ±4.0 5.0 ±2.0
1019 <0.3 7.0 ±1.0 13.0 ±7.0 0.6 ±0.2
1282
796 4.0 ±0.5 10.0 ±2.0 8.0 ±2.0 4.0 ±0.5 5.0 ±0.8 0.6 ±0.1
1387 16.0 ±2.0 0.7 ±0.1
1414 12.0 ±1.0 16.0
798 3.0 ±0.5 19.0 ±5.0 13 ±1
1018 <0.3 11.0 ±3.0 0.04 ±0.01 15.0 ±2.0
1245 <0.3 16.0 ±1.0 0.6 ±0.2 16.0 ±3.0
a
Growth inhibitory effect as judged by average EC
50
values
±
standard deviation of thiazolidene hydrazines (structures in Figure S4) on L.
infantum reference strain (Linf–ref), L. infantum veterinary isolate (Lin–vet), L. amazonensis (L. amaz),L. braziliensis (L. brazils), T. cruzi or T.
brucei.
b
Green boxes represent compounds with anti-parasitic activity (EC
50
values < 20
µ
M) with the determined EC
50
value shown. Red
boxes signify compounds with no anti-parasitic activity (EC
50
values > 20
µ
M). Clear boxes and nd represent compounds whose activity
was not determined. cData from [14]dcontrol drug.
2.3. Toxicology
This section is divided into three parts:
in vitro
toxicity in mammalian cells and
analysis of selectivity; genotoxicity by micronucleus test and acute oral toxicity
in vivo
. In
Table 5the nonspecific cytotoxicity of the compounds with the best antiparasitic activity
was determined. For this, it was taken as a criterion that the compound has activity in
the five species of parasites analyzed (T. cruzi,T. brucei, L braziliensis,L. amazonensis, and
L. infantum) or that they are very active compounds in L. infantum. Compounds with a
selectivity index greater than 10 were considered good and safe. This was performed by
comparing the EC
50
in macrophage cells (relevant cells
in vivo
infection with L. infantum)
and the EC
50 in vitro
cultures of L. infantum, using the reference strain and the circulating
isolate in Uruguay.
Table 5. Cytotoxicity and selectivity index of reference drugs and selected molecules.
Chemical Code EC50 ±SD (µM) aSelectivity Index b
MΦfFibroblasts MΦ/L. amaz MΦ/Lin–Ref MΦ/Lin–Vet
Nifurtimoxc200 ±9 nd 33 33 20
Glucantimec15 ±1 nd 0.83 0.57 Nd
Miltefosinec50 ±7 nd 10 56 10
266 d60 ±6 405 ±10 9 30 7
872 d66 ±7 319 ±16 7 8 2
314 d30 ±5 346 ±9 3 23 12
Curcumin e10 ±2 nd 2 2 Nd
795 e115 ±2 114 ±6 11 23 11
809 e33 ±8 543 ±6 2 6 Nd
796 e38 ±7 158 ±5 5 10 4
799 e115 ±8 nd 23 >63 10
a
Growth inhibitory effect as judged by average EC
50
values
±
standard deviation against murine macrophages (M
Φ
) and fibroblasts
(NCTC929). nd represents not determined.
b
selectivity index represents a ratio of the EC50 value against the mammalian cell line/EC50
against the L. infantum reference strain (Linf–ref), L. infantum veterinary isolate (Lin–vet) or L. amazonensis (L. amaz) (parasite data in
Tables 3and 4)
. nd represents not determined.
c
control drugs.
d,e
thiazolidene hydrazine or curcuminoid-based structures, respectively.
fMΦJ774.1 macrophages or differentiated THP-1 monocytes.

Pharmaceuticals 2021,14, 644 9 of 19
Of the 9 selected compounds with antiparasitic activity, 5 have selectivity indices
greater than 10. However, all of them proved to be more selective than Glucantime,
one of the reference drugs. Within the thiazolidene hydrazine family, of the two active
hits,
314
was less selective than
266
. Furthermore, concerning the circulating strain in
Uruguay,
314
was equally selective as Miltefosine, the drug used in the treatment of
visceral Leishmaniasis [
30
], and was active in all the parasitic species analyzed. Within
the curcuminoids family, we see that curcumin is not selective compared to the active
derivatives
795
,
796,
and
799
. Despite being compounds that are usually considered PAIN,
curcuminoids show that selective molecules can be found. Furthermore, compounds
796
and
799
showed biological activity in all the species of kinetoplastid studied. For all the
compounds analyzed, the cytotoxicity towards fibroblasts was lower than for macrophages,
which is another reason why the selectivity index was estimated using the latter cell type.
Additionally, for compounds
796
and
314,
we analyze the cytotoxicity in human red blood
cells and human macrophages at 50
µ
M and we did not see any inhibition of the cells
grown. These results suggest a selective action against parasites and a large therapeutic
ratio (more than 10 times) for these compounds.
In Table 6, the result of the micronucleus test is presented, used to evaluate geno-
toxicity with a treatment at a fixed dose in mice of 150 mg/kg of weight of compound
796
. A negative control is used to administer only the vehicle and positive control of
intraperitoneal treatment with 40 mg/Kg of Cyclophosphamide, a known genotoxic agent.
This assay is included in those recommended by the FDA to predict DNA toxicity during
the drug development process. Furthermore, a prediction using the Toxicity Estimation
Software Tool (TEST) for mutagenicity of
796
was negative. In previous works by our
group, this test had already been carried out on compounds
266
and
314
, finding that
they are not genotoxic. They also have a negative AMES test that evaluates the mutagenic
capacity of the molecules [13,16].
Table 6. Micronucleus test for compound 796 (150 mg/kg).
Treatment aNumber EPMn bNumber EPC cMedia Mn/Mouse ±SD d
Control 19 5000 4 ±1
796 21 5000 5 ±1
Cyclophosphamide 180 5000 36 ±2
a
Five identical assays are performed at independent times.
b
Total polychromatic micronucleated erythrocytes
(EPMn) found in the 5 assays.
c
Polychromatic erythrocytes (EPC) were observed in total.
d
Percentage of
polychromatic micronucleated erythrocytes (EPMn) ±standard deviation.
Finally, the acute toxicity in mice of compound
796
was evaluated by the up and
down test, resulting in an LD50 > 2000 mg/kg of weight. Hit compounds
266
and
314
were
previously evaluated in our group with this test, obtaining the same result [13,16].
Table 7shown the pharmacokinetic parameters that are commonly evaluated in the
early stages of drug development, for the leading compounds and the reference drugs
for Leishmaniasis and Chagas disease, Miltefosine and Benznidazole, respectively. All
the evaluated compounds are poorly soluble in water (same as Miltefosine) and have
solubility between 10 and 100 times less than Benznidazole. Likewise, Miltefosine and
our compounds have high lipophilicity. On the other hand, the molecules studied show
greater penetrability through the skin, a characteristic that may be advantageous for the
topical administration of drugs for the treatment of cutaneous Leishmaniasis. Finally, it
is interesting to note that the only compound that is predicted to cross the blood–brain
barrier is hit
796
, which suggests that it could be active against T. brucei brain invasion and
used for the treatment of sleeping sickness.

Pharmaceuticals 2021,14, 644 10 of 19
Table 7. Prediction of pharmacokinetic parameters using SwissADME software.
Compound Solubility
(mg/mL)
Gastrointestinal
Absorption
BBB
Permeability
Penetrability
of Skin (cm/s) Bioavailability Lipophilicity
(LogP)
Miltefosine 1.9 ×10−3Low no −4.0 0.55 3.8
Benznidazole 2.3 High no −7.2 0.55 0.5
314 3.5 ×10−3High no −6.3 0.55 4.2
266 2.2 ×10−3High no −5.2 0.55 4.8
796 3.9 ×10−2High yes −5.3 0.55 3.7
Because of the low stability of curcuminoid compounds, we performed a metabolic
stability study with compound
796.
It was detected after 4 h without the emergence of
any other metabolites measured under TLC analyses (see supporting information). The
4 h stability is one of the
in vitro
parameters recommended in the international drug
development guidelines [
31
,
32
]. Compounds
799
and
795
were degraded before 1 h, so
those compounds will not be considered further, due to their lesser metabolic stability.
2.4. In Vivo Proof of Concept
Three compounds were selected to evaluate their
in vivo
efficacy in a murine model of
cutaneous Leishmaniasis (
796, 314, 266
) and the results are presented Figure 2. This model
was used for its simplicity and because it allows a significant reduction in the number of
animals required for the test compared to the visceral mouse model of L. infantum in vivo
infection. The visceral mouse model requires the sacrifice of the animals to follow the
parasites throughout the treatment, getting in a cost of a large number of animals than the
cutaneous model. Furthermore, we observed that in general, the active compounds behave
similarly in the Leishmania species that cause skin and visceral disease studied in this work
(L. amazonensis and L. infantum, respectively). To support this observation we perform a
single dose study at 10
µ
M in the amastigote form of L. infantum for
796, 314, 266
and we
found an inhibition % of 90, 85, and 69, respectively.
For the
in vivo
model of cutaneous disease, 1
×
10
7
amastigote parasites of L. amazonen-
sis PH8 were inoculated in the upper part of the left paw of the mice. As shown in Figure 3,
on day 8 post-infection treatment was started for 14 days with the mentioned compounds
and the reference drug, Glucantime. The mice were sacrificed 30 days post-infection. The
antiparasitic effect was evaluated by two methods: the weekly evolution of the diameter
of the infected paw throughout the experiment (Figure 2C) and the quantification of the
parasite load in the infected area at the end of the experiment (Figure 2A).
Of the three leading compounds,
796
presented more than 50% suppression of para-
sites, behaving similar to the drug Glucantime but administered at a lower dose (1.3 times
less). The % of suppression of the parasites for the other molecules was around 30%, but
those molecules were administered at half of the dose of
796
. Regarding the mechanism of
action, we perform experiments with Triosephosphate isomerase from L. mexicana, because
compound
796
is analog to a potent triosephosphate isomerase inhibitor of T. cruzi [
14
],
but this compound at 50
µ
M was not an inhibitor of this enzyme (see in supporting infor-
mation). On the other hand, in Figure 2C we can see that when treatment with compounds
796
and
266
begins, the diameter of the paw begins to decrease, indicating a decrease
in inflammation that is assumed to be directly proportional to the parasite load or at an
anti-inflammatory effect.

Pharmaceuticals 2021,14, 644 11 of 19
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 11 of 19
Figure 2. In vivo assay of cutaneous leishmaniasis. (A) Infections with L. amazonensis were established for 1 week in the
right hind footpad of BALB/c mice.** and *** are statistically significant difference (0.1 and 0.05 respectively). Treatments
of selected compounds were administered (see (B)) 1 week post-infection and maintained for 2 weeks. Animals were
euthanized two weeks after cessation of treatment and the total parasite loads found in the lesion for the total of the
animals for each treatment. (B) Details relating to the administration of each compound. (C) The right hind footpad of
BALB/c mice was infected with L. amanuensis. Disease progression was monitored by measurement of the lesion diameter
over a period of 28 days. Orally administered treatment of selected compounds (see key) was initiated 1 week
post-infection and maintained for 2 weeks (blue bar). The insert represents an expanded image of lesion development
from day 14 to 28 post-infection. The combination slope corresponds to an orally administered mixture of 266, 314 and
796.
Of the three leading compounds, 796 presented more than 50% suppression of par-
asites, behaving similar to the drug Glucantime but administered at a lower dose (1.3
times less). The % of suppression of the parasites for the other molecules was around
30%, but those molecules were administered at half of the dose of 796. Regarding the
mechanism of action, we perform experiments with Triosephosphate isomerase from L.
mexicana, because compound 796 is analog to a potent triosephosphate isomerase inhib-
itor of T. cruzi [14], but this compound at 50 μM was not an inhibitor of this enzyme (see
in supporting information). On the other hand, in Figure 2C we can see that when
treatment with compounds 796 and 266 begins, the diameter of the paw begins to de-
crease, indicating a decrease in inflammation that is assumed to be directly proportional
to the parasite load or at an anti-inflammatory effect.
We were surprised that low leishmanicidal activity in vivo was observed with the
administration of compound 314. This compound was the one with the best response in
the in vivo model of infection with T. cruzi [16], reducing more than 60% of parasitemia
with 100% survival (with a mean of 60% survival in the untreated group, Figure 3) at the
same dose evaluated in the Leishmaniasis in vivo assay. This could be partly because we
observed great variability in the final appearance of the formulations in the preparation
Figure 2. In vivo
assay of cutaneous leishmaniasis. (
A
) Infections with L. amazonensis were established for 1 week in the
right hind footpad of BALB/c mice.** and *** are statistically significant difference (0.1 and 0.05 respectively). Treatments
of selected compounds were administered (see (
B
)) 1 week post-infection and maintained for 2 weeks. Animals were
euthanized two weeks after cessation of treatment and the total parasite loads found in the lesion for the total of the animals
for each treatment. (
B
) Details relating to the administration of each compound. (
C
) The right hind footpad of BALB/c
mice was infected with L. amanuensis. Disease progression was monitored by measurement of the lesion diameter over
a period of 28 days. Orally administered treatment of selected compounds (see key) was initiated 1 week post-infection
and maintained for 2 weeks (blue bar). The insert represents an expanded image of lesion development from day 14 to
28 post-infection. The combination slope corresponds to an orally administered mixture of 266,314 and 796.
We were surprised that low leishmanicidal activity
in vivo
was observed with the
administration of compound
314
. This compound was the one with the best response in
the
in vivo
model of infection with T. cruzi [
16
], reducing more than 60% of parasitemia
with 100% survival (with a mean of 60% survival in the untreated group, Figure 3) at the
same dose evaluated in the Leishmaniasis
in vivo
assay. This could be partly because we
observed great variability in the final appearance of the formulations in the preparation of
the vehicle with compound
314
, which we could not improve due to solubility problems.
This is decisive in the correct absorption
in vivo
and the concomitant antiparasitic effect.
Therefore, the observed variability between trials possibly reflects pharmacokinetic prob-
lems, since this compound would share the pharmacophore with hit
266
. Additionally, the
dose of compound 314 was half that of 796, and higher doses could be more effective.

Pharmaceuticals 2021,14, 644 12 of 19
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 12 of 19
of the vehicle with compound 314, which we could not improve due to solubility prob-
lems. This is decisive in the correct absorption in vivo and the concomitant antiparasitic
effect. Therefore, the observed variability between trials possibly reflects pharmacoki-
netic problems, since this compound would share the pharmacophore with hit 266. Ad-
ditionally, the dose of compound 314 was half that of 796, and higher doses could be
more effective.
Glucantime behaves similarly to our active compound 796. When the drug admin-
istration is stopped, the diameter of the paw continues to increase, so the treatment time
used was not enough to clear all the parasites. This shows that it is necessary to adjust the
pharmacokinetic parameters and optimize the time and dose of administration of the
compounds to obtain a full elimination of the infection. It should be noted that the an-
ti-leishmanial effect of these compounds was obtained by oral treatment, which repre-
sents the preferred method of administration compared to the more invasive routes that
require the use of injectable. Additionally, this oral administration gives information
about the distribution, because the parasites are in the lesion at the dermic area, then the
distribution seems to be systemic.
There is evidence that the co-administration of thiazolidene hydrazines and curcu-
minoids have synergistic effects on T. cruzi [33], so we decided to evaluate the three
leading compounds at lower doses in the same formulation on the same murine
leshmniasis model and also in the Chagas disease murine model (Figure 3). The dose of
the most active compound 796 was reduced 10 times while the dose of hits 314 and 266
was reduced by half. In this experiment (Figure 2A), we observe higher parasite sup-
pression than the monotherapy (796 at 203 μmol/kg), and because the dose was 10 times
less than monotherapy we conclude that there is a synergic effect between those com-
pounds. We will perform isobolograms in vitro to find which combination and the op-
timal proportion between these compounds are producing the synergic effect. The tryp-
anosomicidal activity in the murine model of Chagas disease was also synergic because
the parasitemia was fully controlled by the reduced doses of this compound compared to
the monotherapy (Figure 3).
Figure 3. In vivo assay in the Chagas disease acute murine model. The oral administration was the same as in the in
vivo experiment on Leishmania. The black bars indicate the 50 of parasitemia reduction with relation to the parasitemia
peak in the vehicle control (group of infected animals treated only with the vehicle).
Figure 3. In vivo assay in the Chagas disease acute murine model.
The oral administration was the same as in the
in vivo
experiment on Leishmania. The black bars indicate the 50 of parasitemia reduction with relation to the parasitemia peak in
the vehicle control (group of infected animals treated only with the vehicle).
Glucantime behaves similarly to our active compound
796
. When the drug admin-
istration is stopped, the diameter of the paw continues to increase, so the treatment time
used was not enough to clear all the parasites. This shows that it is necessary to adjust
the pharmacokinetic parameters and optimize the time and dose of administration of the
compounds to obtain a full elimination of the infection. It should be noted that the anti-
leishmanial effect of these compounds was obtained by oral treatment, which represents
the preferred method of administration compared to the more invasive routes that require
the use of injectable. Additionally, this oral administration gives information about the
distribution, because the parasites are in the lesion at the dermic area, then the distribution
seems to be systemic.
There is evidence that the co-administration of thiazolidene hydrazines and curcumi-
noids have synergistic effects on T. cruzi [
33
], so we decided to evaluate the three leading
compounds at lower doses in the same formulation on the same murine leshmniasis model
and also in the Chagas disease murine model (Figure 3). The dose of the most active
compound
796
was reduced 10 times while the dose of hits
314
and
266
was reduced by
half. In this experiment (Figure 2A), we observe higher parasite suppression than the
monotherapy (
796
at 203
µ
mol/kg), and because the dose was 10 times less than monother-
apy we conclude that there is a synergic effect between those compounds. We will perform
isobolograms
in vitro
to find which combination and the optimal proportion between these
compounds are producing the synergic effect. The trypanosomicidal activity in the murine
model of Chagas disease was also synergic because the parasitemia was fully controlled by
the reduced doses of this compound compared to the monotherapy (Figure 3).
3. Material and Methods
3.1. Cell Culturing
L. amazonensis and L. infantum (MHOM/BR/2002/LPC-RPV) were obtained from
Fiocruz (Collection of Oswaldo Cruz Foundation, Rio de Janeiro, Brazil), while a L. in-
fantum line (MCAN_UY_2015_gPL8) was isolated from a dog suffering from VCL [
19
].
Leishmania promastigotes were cultured at 28
◦
C in RPMI-1640 supplemented with glucose
(0.7% (w/v)), ornithine (0.1% (w/v)), fructose (0.4% (w/v)), malate (0.6% (w/v)), fumarate
(0.05% (w/v)), succinate (0.06% (w/v)), heat-inactivated fetal bovine serum (HI-FBS; 20%
(v/v)), vitamins, and amino acids solution (Gibco, NY, USA). Once established, parasites

Pharmaceuticals 2021,14, 644 13 of 19
in the exponential phase were transferred and cultured at 28
◦
C in an axenic medium
consisting of BHI-Tryptose supplemented with FBS (10% (v/v)), hemin (2
×
10
−5
mg/mL),
glucose (0.03% (w/v)), streptomycin (2.0
×
10
−4
g/mL), ampicillin (1.3
×
10
−4
g/mL).
Leishmania metacyclic parasites were harvested from promastigote cultures as described
previously [
18
]. These were used to infect differentiated human acute monocytic leukemia
(THP-1) cells at a ratio of 20 parasites per mammalian cell. The infected monolayers were
incubated overnight at 37
◦
C under a 5% (v/v) CO
2
atmosphere in a mammalian growth
medium and then washed with RPMI-1640 to remove residual parasites. Leishmania
amastigote parasites were maintained in differentiated THP-1 cells at 37
◦
C under a 5%
(v/v) CO2atmosphere in RPMI-1640 medium.
T. brucei brucei bloodstream form trypomastigotes (MITat 427 strain; clone 221a) were
cultured at 37
◦
C under a 5% (v/v) CO
2
atmosphere in modified Iscove’s medium supple-
mented with 10% (v/v) HI-FBS as described previously [34].
The J774.1 (ATCC
®
TIB-67
™
) murine macrophage line was grown at 37
◦
C under a
5% (v/v) CO2atmosphere in DMEM medium containing L-glutamine (4 mM) and HI-FBS
(10% (v/v)) [16].
The THP-1 (ATCC
®
TIB-202) human acute monocytic leukemia line was grown at 37
◦
C
under a 5% (v/v) CO
2
atmosphere in RPMI-1640 medium containing 2-mercaptoethanol
(50
µ
M) and HI-FBS (10% (v/v)). Differentiation of THP-1 to produce macrophage-like
cells was carried out using phorbol 12-myristate-13-acetate (Sigma-Aldrich, Deisenhofen,
Germany) [18].
3.2. In Vitro Antiparasitic Activity
All growth inhibition assays were performed in a 96-well plate format. Leishmania
promastigotes (2
×
10
4
cells) or T. b. brucei BSF trypomastigotes (2
×
10
3
cells) were
seeded in 200
µ
L growth medium containing different concentrations of the compound.
The parasites were used at the exponential estate at the growing curve. Compounds
were dissolved in dimethylsulfoxide (DMSO). After culturing the parasites at 28
◦
C for
2 days (Leishmania) or 37
◦
C under a 5% (v/v) CO
2
atmosphere for 2 days (T. b. brucei),
resazurin (9
µ
g for Leishmania or 2.5
µ
g for T. b. brucei) (Sigma Aldrich, Deisenhofen,
Germany) was added to each culture and the plates incubated for a further 6–8 h. Cell
densities were determined by monitoring the fluorescence of each culture using a Gemini
Fluorescent Plate Reader (Molecular Devices, CA, USA) at an excitation wavelength of
530 nm, an emission wavelength of 585 nm and a filter cut off at 550 nm. The change in
fluorescence resulting from the reduction of resazurin is proportional to the number of live
cells. The effective concentration of a compound that inhibits cell growth by 50% (EC
50
)
was established using OriginLab8.5
®
sigmoidal. All assays were performed in triplicate on
two experimental repeats.
Differentiated THP-1 monocytes (30,000 cells on an 18-mm round glass coverslip)
were infected with Leishmania metacyclic parasites (30,000 parasites). Following incubation
overnight at 37
◦
Cina5%(v/v) CO
2
atmosphere, the cultures were washed twice in a
growth medium to remove noninternalized parasites and the supernatant was replaced
with a fresh growth medium containing the compound under investigation. Compound-
treated infections were incubated for a further 2 days at 37
◦
C under a 5% (v/v) CO
2
atmosphere. Phosphate buffered saline-washed cells were fixed in 95% (v/v) ethanol,
stained with 10% (v/v) Giemsa, visualized with Leica DMRA2 light microscope (Wetzlar,
Germany) using a X100 oil immersion objective and images captured using a Retiga EXi Fast
1394 digital camera (Teledyne Imaging, AZ, USA). Infectivity was assessed by determining
the number of infected cells and the number of parasites per infected cell. Infectivity
index calculation = (% of infected cells
×
(amastigotes per cell))/number of total counted
cells [18].

Pharmaceuticals 2021,14, 644 14 of 19
3.3. Nonspecific In Vitro Cytotoxicity in Mammalian Cells
Adhered J774.1 macrophages or differentiated THP-1 monocytes (1
×
10
4
cells) were
cultured in the compound-containing medium for 48 h to the compounds. Cell viability
was then assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) colorimetric assay [
1
]. In this analysis, MTT was added to a final concentration of
0.1 mg/mL to each well, and the culture was incubated at 37
◦
C for 3 h. The medium was
removed, any formazan crystals dissolved in solubilization buffer (glycine (10 mM); NaCl
(10 mM); EDTA pH10.5 (0.05 mM) in DMSO) and the absorbance at 560 nm determined. The
effective compound concentration that inhibits cell growth by 50% (EC
50
) was established
using OriginLab8.5®sigmoidal. All assays were performed in triplicate [35].
To assess a compound’s hemolytic activity the Red Blood Cell Lysis Assay (national
blood bank) was performed. A compound-containing erythrocyte (2% (w/v)) suspension
prepared in phosphate-buffered saline pH 7.4 was incubated for 24 h at 37
◦
C and the
amount of hemoglobin released into the supernatant determined spectrophotometrically
using a Varioskan TM Flash Multimode Reader (Thermo ScientificTM, MA, USA) set to
a wavelength of 405 nm. The % hemolysis was determined using the follows equation:
% released hemoglobin = [(A
1−
A
0
)/(A
1water
)]
×
100, where A
1
and A
0
represent the
absorbance at 405 nm of the test sample at 0 and 24 h, and A
1water
the absorbance at 405 nm
of water at 24 h. The experiments were performed by quintuplicate. Amphotericin B (final
concentration of 1.5 µM) was used as a positive control [13].
3.4. Vehicles/Formulation Preparation
The compounds (at the doses described in the appropriate section) used for the
in vivo
assay were vehiculated in a mixture composed of a surfactant (10%) containing Eumulgin
HRE 40 (polyoxyl-hydrogenated castor oil), sodium oleate, and soya phosphatidylcholine
(8:6:3), and an oil phase (10%) containing cholesterol and PBS (80%). For formulation,
cholesterol, Eumulgin HRE 40, and phosphatidylcholine previously pulverized in mortar
were dissolved in chloroform and the solvent was evaporated under vacuum to dryness.
In parallel, sodium oleate was dissolved in phosphate buffer and left in an orbital shaker
for 12 h at room temperature. The latter was then added to the evaporated residue, and
the mixture was homogenized and placed in an ultrasonic bath at full power for 30 min
and kept at room temperature until use [
13
]. The volume used as a single dose on 0.2 mL,
and then 30 mL of the compound at the respective doses were prepared. The doses were
prepared according to the mouse weight at the time of the experiment.
3.5. In Vivo Micronucleus Test
For the
in vivo
micronucleus test, approximately three-month-old CD-1 male mice
were housed in polycarbonate cages at room temperature (25
◦
C) and a photoperiod of
12 h throughout the study. The selected compound/vehicle was orally administered twice,
at days one and two, to groups of five mice at a dose of 150 mg/kg of body weight.
Mice were sacrificed 24 h after the last administration, and the bone marrow prepared
for evaluation as described with slight modification [
16
]. At least two slides of the cell
suspension per animal were made. The air-dried slides were stained with Giemsa stain
(5% in phosphate buffer, pH 7.4) and examined at 1000
×
magnification. Small round
or oval bodies, the size of which ranged from about 1/5 to 1/20 of the diameter of a
polychromatic erythrocyte (PCE), were counted as micronuclei. A total of 1000 PCEs were
scored per animal by the same observer for determining the frequencies of micronucleated
polychromatic erythrocytes (MNPCEs). Cyclophosphamide (50 mg/kg) administered
intraperitoneally (i.p.) 24 h before mouse sacrifice, was used as a positive control. For
statistical analysis, the homogeneity of variances of data was tested by the analysis of
variance (ANOVA) test (p< 0.05) using the EpiInfo (3.5.1) software.

Pharmaceuticals 2021,14, 644 15 of 19
3.6. In Vivo Acute Oral Toxicity in Mice
The
in vivo
50% lethal dose (LD
50
) of selected compounds using healthy young adult
male B6D2F1 mice (30 days old, 25 to 30 g) was determined according to the guidelines
of the Organization for Economic Cooperation and Development (OECD). Initially, the
compound was dissolved in the vehicle described above (see Section 2.4) and was ad-
ministered at 2000 mg/kg, by orogastric cannula, to one animal. The animal was fasted,
maintained, and observed for 48 h. If the mouse survived, another animal received the
same dose, and 48 h later, a third animal. If there were no signs of toxicity, the experiment
was halted 14 days after administration, with the euthanasia of the animals according to
the OECD guidelines. Observations of the general status of the organs were performed
after sacrifice. The PROTOX software was used to predict the LD
50
of the compounds
(http://tox.charite.de/protox_II/, accessed on 5 October 2018) [
16
]. The mutagenicity
prediction was using the Toxicity Estimation Software Tool (TEST) [36].
3.7. In Vivo Anti-Leishmania Studies in Cutaneous Mice Model
Golden hamsters (Mesocritus auratus) were used to maintain L. amazonensis infections
with the parasites passage every 6 to 8 weeks. BALB/c mice (female and male) supplied by
the IFFA-CREDO, Lyon, France, and bred at the Instituto de Investigaciones en Ciencias
de la Salud, Asuncion, Paraguay, were inoculated in the right hind footpad with
2×106
L. amazonensis amastigotes in 100
µ
L PBS obtained from donor hamsters. Disease pro-
gression was monitored by the measurement of lesion diameters for 7 to 12 weeks. In all
experiments, treatment was initiated 1 or 2 weeks after inoculation, when infection had
become established and lesions obvious. Two days before the administration of the drug,
the mice were randomly divided into groups of eight. N-Methylglucamine antimonate
was administered subcutaneously to the BALB/c mice (100 mg/kg) for 20 days while
the selected compound was administered orally at 50 mg/kg body weight. The animals
were euthanized two weeks after cessation of treatments to assess parasitological loads
in the infected footpad. Briefly, the mice were sacrificed, and the lesions of the infected
footpad excised, weighed, and homogenized in a tissue glass grinder and the homogenate
suspended in 1 mL RPMI-1640 (Gibco, NY, USA) supplemented with 10% (v/v) FBS, glu-
tamine (29.4
µ
g/mL), penicillin (100 U/mL), and streptomycin (100
µ
g/mL). Following
incubation at 27
◦
C for seven days, cultures were examined with an inverted microscope
(Olympus, Tokyo, Japan) at a magnification of
×
400. The number of parasites per gram in
the lesion was calculated by the following equation: Parasite burden = geometric mean of
the number of parasites in each duplicate/(number of microscope field counted
×
weight
of lesion
×
(25,000) hemocytometer correction factor) [
37
]. The mean and standard de-
viations were calculated by using OriginPro9 and GraphPad Prism5. Comparisons of
parasite suppression in the infected footpads of the untreated and drug-treated groups
were performed by Student’s t-test. One-way ANOVA (as a non-parametric statistical test)
was also used on ranks.
3.8. In Vivo Studies in the Acute Model of Chagas Disease in Mice
Three-month-old Balb/c mice (day 0) were infected with infected blood from mice at
the beginning of the peak of parasitemia (parasitemia greater than 1.0
×
10
6
p/mL), with the
CL Brener clone of T. cruzi (10,000 parasites per mouse), was inoculated intraperitoneally.
Parasitemia was followed from the fourth day post-infection and until all the mice in the
group were positive. Parasitemia measurements were made by the Hemoconcentration
and counting micro method. Once all the mice were positive, the treatment was started.
The treatment lasts 15 consecutive days, the compounds were administered orally by
intragastric cannula once a day. Parasitemia was monitored weekly, at 30 and 60 days
200
µ
L of blood was extracted from the mouse tail for serological tests (ELISA test to detect
antigens of T. cruzi). Day 60 was the end of the experiment and the animals were sacrificed.
Hemoconcentration and counting micro method; the mice were bled by pricking the tail
and taking the blood with capillaries. One capillary per mouse draws 8–18 mm in height

Pharmaceuticals 2021,14, 644 16 of 19
of blood [
37
]. The millimeters were converted to
µ
L by a table previously described and
calibrated in the laboratory. The capillaries were centrifuged at 3000
×
gfor 40 s. The
parasitic load in the capillary was observed by optical microscopy (OM) in the capillary
the Red Blood Cells (GR)) were distributed at one end and the supernatant serum, at the
interface are the trypomastigotes. Then the capillary was cut a few mm on the interface
towards the GR part were spread on a slide, covered with a coverslip and the parasites
were counted in 50 fields (OM at 40
×
magnification), the total number of microscopic
fields was calculated and the factor corresponding to 1/50 of this total number of fields.
This factor, multiplied by the number of trypomastigotes counted in each slide, gives
us the number of trypanosomes per 5 mm
3
and is finally compared with the figures of
the control animals. For serology, they were taken in the same way 4 capillaries filled
with blood were cut and stored in tubes to perform the ELISA. The mean and standard
deviation were calculated by using OriginPro9 and GraphPadprisma5. Comparisons of
parasite suppression were performed by the analysis of variance (two-way ANOVA as a
non-parametric statistical test).
3.9. Liver Fraction Stability Studies and Calculation of Pharmacokinetic Parameters
For the determination of the stability in the different fractions, (cytosolic and microso-
mal of rat hepatocytes) the different proteins present in them were used. The fractions were
prepared according to the previously reported protocol [38]. The protein concentration in
the different fractions was determined by the Sigma bicinchoninic acid (BCA) assay, as sug-
gested in the manual. The final concentration of the molecules in the aqueous medium was
400
µ
M and prepared from a stock in DMSO of 40 mM. The solutions were homogenized
and incubated at 37
◦
C at 10 min, 30 min, 1 h, 2 h, 3 h and 4 h. (by TLC). For this, it was in-
cubated at 37
◦
C in a volume of 1 mL: 2.5
µ
L of 30 mM Magnesium Chloride (MgCl); 2.5
µ
L
of 40 mM Nicotinamide Adenosine Dinucleotide Phosphate (NADP+); 5
µ
L of 350 mM
Glucose 6- Phosphate (Glu6P); 5
µ
L of Glucose 6- phosphate dehydrogenase (Glu6PD)
50 U/mL, 5
µ
L of the stock of 40 mM compounds and the volume of the Phosphate Buffer
(pH = 7) must be such that cytosolic (CF) and microsomal (MF) fraction FC and FM present
a final protein concentration of 0.1 mg/mL. After that, the stability of compounds
796
and
1019
in the cytosolic (FCF) and microsomal (FMF) fraction of rat hepatocytes was
evaluated at different times (1–4 h). Thin-layer chromatography of ethyl acetate extracts
was conducted to evaluate the presence of decomposition products. The mobile phase
used for these TLCs was n-hexane: ethyl acetate (7: 3).
Predictions were made using the open-access SwissADME software (http://www.
swissadme.ch accessed on 5 of October 2018), a tool that allows the prediction of different
pharmacokinetic parameters such as water solubility, gastrointestinal absorption, skin
penetrability, lipophilicity, bioavailability, etc. [
39
] (in supporting information). The Swis-
sADME software input uses the SMILES codes of the molecules, which were generated
with the ChemBioOffice 2010 program.
4. Conclusions
We identified five drug candidates for
in vivo
studies in the visceral Leishmaniasis
model from PBox. These candidates can be used as inspiration for new and potent re-
designed molecules. We suggested a new function or tool for the drug collection such as
PBox in the molecular characterization of a new cell strain. Additionally, we showed a lack
of representation of reference strains of L. infantum in the drug discovery process.
Of the 50 compounds evaluated from our chemical collection, we conclude that three
of the leading compounds (
796
,
266
,
314
), were found to have good antiparasitic activity in
different species of trypanosomatids. Furthermore, these molecules have low nonspecific
cytotoxic effects and did not show genotoxic or mutagenic effects. In addition to these
encouraging results, they are safe, showing 100% survival in the acute toxicity model
in vivo
. None of the 400 compounds from the PBox have multi antiparasitic activity against
T. cruzi,T. brucei,L. amazonensis,L. braziliensis, and L. infantum as our chemical collection has.

Pharmaceuticals 2021,14, 644 17 of 19
Regarding the mechanism of action of compound
796
, it should be noted that triosephos-
phate isomerase (TIM) does not appear to be the molecular target of the compound, re-
garding the low inhibition effect showed in LmTIM and TbTIM at 42.5% and 61% after
treatment with 50
µ
M (in supporting information). Therefore this matter remains elusive
and more studies are needed to assess this. Concerning the pharmacokinetic parameters of
the compounds that were tested in the
in vivo
model, it is observed that the predictions
were similar to the parameters shown by Miltefosine. Some are even estimated to have
higher levels of gastrointestinal absorption and skin absorption, desirable qualities for a
drug to be used in the treatment of Leishmaniasis.
It is important to mention that the oral administration of the compounds used in the
in vivo
model has many advantages in this type of disease, where the cost of the drug must
be low and its route of administration simple. Additionally, the treatment has to be suitable
for use in dogs.
Compound
796
was the most promising compound with effective control of the Leish-
mania parasite infection
in vivo
. Additionally, the synergic effect was observed between
the two chemical groups (curcuminoids and thiazolidene hydrazines). Taken together, the
results obtained encourage us to continue with the clinical phase of experimentation in the
canine Leishmaniasis model of our leading compounds.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10
.3390/ph14070644/s1, Figure S1. Susceptibility of L. infantum towards compounds in the PBox library,
Table S1. General activity profiles previously reported of the five hits identified in the phenotypic
screening, Table S2. Compounds structures and compound preparation and characterization, Table
S3. Activity of compound 796 in Triosephosphate isomerase from L. mexicana (LmTIM) and T. brucei
(TbTIM).
Author Contributions:
The manuscript was written through the contributions of all authors. Con-
ceptualization, I.C. and C.R.; methodology, C.P. and E.A.; software, G.A.; validation, P.F.-T., G.Y.
and E.S.; formal analysis, G.A.; investigation, C.P., E.A. and P.F.-T.; resources, C.R. and G.Y.; data
curation, S.R.W.; writing—original draft preparation, G.A. and S.R.W.; writing—review and editing,
S.R.W.; visualization, G.A.; supervision, G.A., C.R. and G.Y.; project administration, G.A.; funding
acquisition, G.A. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by CSIC (Comisión Sectorial de Investigación Científica) I+D
2016 program number ID435 and ID35 (grant).
Institutional Review Board Statement:
The study was conducted according to the guidelines of the
Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of
Universidad Nacional de Asuncion (Leishmania
in vivo
model, that include the use of hamsters
and mouse, it is the same protocol: GY_P46/2018/PY and for the Chagas
in vivo
model was this:
GY_P38/2016/PY).
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Acknowledgments: Pathogen Box was kindly provided by Medicines for Malaria Ventures.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the result.
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