Activity in vivo of anti-Trypanosoma cruzi compounds selected from a high throughput screening.

Abstract

Novel technologies that include recombinant pathogens and rapid detection methods are contributing to the development of drugs for neglected diseases. Recently, the results from the first high throughput screening (HTS) to test compounds for activity against Trypanosoma cruzi trypomastigote infection of host cells were reported. We have selected 23 compounds from the hits of this HTS, which were reported to have high anti-trypanosomal activity and low toxicity to host cells. These compounds were highly purified and their structures confirmed by HPLC/mass spectrometry. The compounds were tested in vitro, where about half of them confirmed the anti-T. cruzi activity reported in the HTS, with IC50 values lower than 5 µM. We have also adapted a rapid assay to test anti-T. cruzi compounds in vivo using mice infected with transgenic T. cruzi expressing luciferase as a model for acute infection. The compounds that were active in vitro were also tested in vivo using this assay, where we found two related compounds with a similar structure and low in vitro IC50 values (0.11 and 0.07 µM) that reduce T. cruzi infection in the mouse model more than 90% after five days of treatment. Our findings evidence the benefits of novel technologies, such as HTS, for the drug discovery pathway of neglected diseases, but also caution about the need to confirm the results in vitro. We also show how rapid methods of in vivo screening based in luciferase-expressing parasites can be very useful to prioritize compounds early in the chain of development.

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Activity In Vivo of Anti-Trypanosoma cruzi Compounds
Selected from a High Throughput Screening
Grasiella Andriani1, Anne-Danielle C. Chessler2, Gilles Courtemanche3, Barbara A. Burleigh2, Ana
Rodriguez1*
1Division of Medical Parasitology, Department of Microbiology, New York University School of Medicine, New York, New York, United States of America, 2Immunology
and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America, 3Infectious Diseases Unit, Sanofi-Aventis, Toulouse, France
Abstract
Novel technologies that include recombinant pathogens and rapid detection methods are contributing to the development
of drugs for neglected diseases. Recently, the results from the first high throughput screening (HTS) to test compounds for
activity against Trypanosoma cruzi trypomastigote infection of host cells were reported. We have selected 23 compounds
from the hits of this HTS, which were reported to have high anti-trypanosomal activity and low toxicity to host cells. These
compounds were highly purified and their structures confirmed by HPLC/mass spectrometry. The compounds were tested
in vitro, where about half of them confirmed the anti-T. cruzi activity reported in the HTS, with IC50 values lower than 5 mM.
We have also adapted a rapid assay to test anti-T. cruzi compounds in vivo using mice infected with transgenic T. cruzi
expressing luciferase as a model for acute infection. The compounds that were active in vitro were also tested in vivo using
this assay, where we found two related compounds with a similar structure and low in vitro IC50 values (0.11 and 0.07 mM)
that reduce T. cruzi infection in the mouse model more than 90% after five days of treatment. Our findings evidence the
benefits of novel technologies, such as HTS, for the drug discovery pathway of neglected diseases, but also caution about
the need to confirm the results in vitro. We also show how rapid methods of in vivo screening based in luciferase-expressing
parasites can be very useful to prioritize compounds early in the chain of development.
Citation: Andriani G, Chessler A-DC, Courtemanche G, Burleigh BA, Rodriguez A (2011) Activity In Vivo of Anti-Trypanosoma cruzi Compounds Selected from a
High Throughput Screening. PLoS Negl Trop Dis 5(8): e1298. doi:10.1371/journal.pntd.0001298
Editor: Timothy G. Geary, McGill University, Canada
Received June 10, 2011; Accepted July 18, 2011; Published August 30, 2011
Copyright: ? 2011 Andriani et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was partially supported by NIH grant 1R03 MH085673-01. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Ana.Rodriguez@nyumc.org
Introduction
It is estimated that around 100 million people live with the risk
of infection with T. cruzi in endemic areas in Latin America, with
approximately 8 million already infected. The considerable influx
of immigrants from Latin American countries to USA, Canada
and Europe has also made Chagas disease an important health
issue in these countries [1].
Although Chagas disease was discovered more than one
hundred of years ago, the medicines available for treatment have
serious drawbacks. The two drugs current in use, Benznidazole
and Nifurtimox that were released in the 70’s, present toxic side
effects and low efficacy in some strains [2]. It was believed that
both of them were only efficient for the treatment of the acute
phase but recent studies suggest that chagasic patients in the
chronic phase of the disease treated with Benznidazole show
reduced disease progression and increased negative seroconversion
than the untreated patients [3].
In an advanced position in the pipeline for future anti-T. cruzi
treatments there is only Posaconazole, an oral antifungal that is
currentlyinthemarketandhasbeentestedsuccessfullyinmice[4]and
humans [5] infected with T. cruzi. Given the limitations of the current
available treatments and the low number of candidates undergoing
clinical tests, the development of new anti-T. cruzi compounds
combining broad and high efficacy with low toxicity is an urgent need.
About ten years ago, the advent of high-throughput screening
(HTS) technology revolutionized the process of early drug
development, enabling researchers to rapidly collect enormous
amounts of data and explore compound libraries with unprece-
dented thoroughness. Even if this technology has not yielded the
expected increase in the number of licencesed medicines in the
market, it still is considered a fundamental tool in early drug
development in the pharmaceutical industry [6].
Additional developments in the field of drug discovery include
luminescent reporter gene assays, which appear as the most
prominent type of reporter gene assay used in biomolecular and
pharmaceutical development laboratories. The success of these
techniques is due to the high signal associated with luciferases,
which makes them ideal for high throughput screening (HTS) in
vitro applications, but also for the possibility of adapting these
assays for in vivo screening [7].
Major changes are being introduced in the field of Chagas
disease drug discovery since the development of recombinant T.
cruzi parasites to be used as tools for drug screening. The first
example is a transgenic T. cruzi strain expressing the reporter
enzyme b-galactosidase [8] that has allowed performing a HTS for
compounds active against T. cruzi infection of host cells (Pubchem
AID:1885). Screening of drugs in T. cruzi mouse models has
also been made much more rapid and simple with the use of
fluorescent [9] or luminescent [10] recombinant parasites.
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Recombinant parasites expressing luciferase are already available
for several species and have been used effectively for drug
discovery in Leishmania [11,12].
In this work we describe the continuation of a chemical HTS
against T. cruzi trypomastigote infection of host cells. Re-testing of
some of the HTS hits for in vitro anti-T. cruzi activity revealed that
approximately half of them did not confirm the activity. Screening
of the active compounds in a mouse model of acute Chagas
resulted in the finding of one molecular structure with high anti-
trypanosomal activity in mice.
Materials and Methods
Ethical statement
Animal studies were approved by the Institutional Animal Care
and Use Committee of New York University School of Medicine
(protocol #81213). This protocol adheres to the guidelines of the
Association For Assessment and Accreditation Of Laboratory
Animal Care International (AAALAC).
Compound selection identification and purification
The compounds were selected from a HTS campaign per-
formed by the Broad Institute, as part of the MLPCN (Molecular
Libraries Probe Centers Network) T. cruzi inhibition project. The
results from a HTS for T.cruzi trypomastigote infection of host cells
were made available at Pubchem (AID: 1885). This HTS was
performed by screening of 303,286 molecules (the NIH collection)
form where 4,065 hits were selected by their activity against T.
cruzi trypomastigote infection. These compounds were further
assayed to determine their IC50 (Pubchem AID: 2044) and their
toxicity to host NIH-3T3 cells (Pubchem AID: 2010).
Compounds were selected from the hits of this HTS among the
ones with reported IC50,1.2 mM and at least 100 fold activity
versus toxicity All the compounds selected for this analysis had
toxicity activity .60 mM. Chromatographic analyses were per-
formed to determine the degree of purification (all compounds
were .90% pure except for CID-563075 and CID-2234099 that
were 87 and 88% pure, respectively). Electrospray ionization mass
spectrometry was performed to confirm compound identification.
Finally, compounds were dissolved in DMSO at 10 mM
concentration.
T. cruzi and mammalian cells cultures
LLC-MK2 and NIH/3T3 cells were cultivated in DMEM
supplemented with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml
streptomycin, and 0.292 mg/ml glutamine (Pen-Strep-Glut) at
37uC and 5% CO2atmosphere.
T. cruzi parasites from the Tulahuen strain stably expressing the
b-gal gene (clone C4) [8] and T. cruzi Y strain expressing the firefly
Luciferase gene were kept in culture by infection of LLC-MK2
every 5 or 6 days in DMEM with 2% FBS and 1% Pen-Strep-Glut
at 37uC and 5% CO2atmosphere.
Trypomastigotes forms were released in the supernatant of
infected LLC-Mk2 and harvested between days 5 and 7. The
harvested medium was centrifuged for 7 min at 1,237 g and, in
order to eliminate the amastigotes, the trypomastigotes forms were
allowed to swim out of the pellet for at least 3 h. The parasites
were counted in a Neubauer Chamber and 10 million trypomas-
tigotes were used to infect 1 million LLC-MK2 cells plated in a
75 cm2culture flask.
T. cruzi in vitro inhibition assay
Between 5 to 7 days after the infection, NIH/3T3 cells and T.
cruzi Tulahuen expressing b-galactosidase [8] were harvested,
centrifuged and washed with DMEM without phenol red
supplemented with 2% FBS and Pen-Strep-Glut. The phenol
red needed to be eliminated in order to avoid interference with the
assay absorbance readings at 590 nM. NIH/3T3 cells (50,000 per
well) were seeded in 96-well plates 2 h before addition of purified
T.cruzi trypomastigotes (50,000 per well) and the compounds for
testing at the maximum concentration of 50 mM, therefore the
DMSO percentage was never higher than 0.5%. This concentra-
tion of DMSO was tested repeatedly and it does not affect the
viability of the parasites. Each determination was performed in
duplicate. Amphotericin B (Sigma-Aldrich) was used as positive
control at a final concentration of 4 mM. Negative and positive
controls were carried in every plate. After 4 days, 50 ml of PBS
containing 0.5% of the detergent NP40 and 100 mM Chlorophe-
nol Red-b-D-galactoside (CPRG) (Sigma) were added per well.
Plates were incubated at 37uC for 4 h and absorbance was read at
590 nm using a Tecan Spectra Mini plate reader.
The absorbance obtained was proportional to the viability of the
parasite. The value of IC50 was determined using Graph Prism
Software.
Generation and characterization of luciferase-expressing
T. cruzi trypomastigotes
The firefly luciferase gene (luc) was used to replace the GFP in
the T. cruzi episomal expression vector, pTREX-GFP [13], a
modified version of pRIBOTEX-GFP [14]. T. cruzi (Y strain)
epimastigotes maintained at 28uC in LIT medium were transfect-
ed with 10 mg of pTREX-luc using a nucleofector transfection
system (T-cell protocol; AMAXA) and selected with 200 mg/ml
G418 for 4 weeks. Parallel transfections with pTREX-GFP
demonstrated that under similar selection conditions .95% of
parasites are strongly positive for GFP after 4 weeks (not shown).
Mammalian-infective metacyclic trypomastigotes were harvested
from stationary phase epimastigote cultures and enriched
following passage over DEAE-cellulose/PBS pH.8.0 as routinely
performed [15]. Tissue culture trypomastigotes were harvested
from monkey kidney epithelial cells, LLcMK2, monolayers
infected with Y-luc metacyclic trypomastigotes. Relative luciferase
activity in Y strain epimastigotes grown in the presence of 200 mg/
ml G418 and in mammalian infective trypomastigotes harvested
from infected monolayers after the second passage through
Author Summary
Chagas is a devastating disease affecting about 100 million
people in Latin America. The drugs available for treatment
against the causative agent, the parasite Trypanosoma
cruzi, have associated toxicity and are not completely
effective against the chronic form of the disease, which is
the most common presentation in the clinic. There is a
great need for new drugs against this disease. Novel
technologies in drug development are now being applied
for the search of new compounds against Chagas. Taking
advantage of a high throughput screening performed
recently to identify compounds active against T. cruzi
replication in host cells in vitro, we have selected 23
compounds, which have been re-tested to selected active
ones. We have also adapted a transgenic T. cruzi
expressing luciferase, which allows for direct visualization
when mice are infected. These parasites have been used to
establish a model for acute Chagas disease useful for drug
testing in mice. Using this method, we have tested the
activity of the selected compounds and found two
compounds with strong anti-T. cruzi activity in mice.
Activity In Vivo of Anti-T. cruzi Compounds
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mammalian cells (ie. 2 weeks in the absence of drug selection) was
similar ,2.56106RLU/106parasites.
T. cruzi in vivo inhibition assay
Trypomastigotes forms from transgenic T. cruzi Y strain
expressing firefly Luciferase were purified, diluted in PBS and
injected i.p. in Balb/c mice (105trypomastigotes per mouse).
Three days after infection the mice were anesthesized by either i.p.
injection of 300 mg/kg of Xylazine and 3500 mg/kg of Ketamine
or by inhalation of isofluorane (controlled flow of 1.5% isofluorane
in air was administered through a nose cone via a gas anesthesia
system). Mice were injected with 150 mg/kg of D-Luciferin
Potassium-salt (Goldbio) dissolved in PBS. Mice were imaged 5 to
10 min after injection of luciferin with an IVIS 100 (Xenogen,
Alameda, CA) and the data acquisition and analysis were
performed with the software LivingImage (Xenogen). One day
later (4 days after infection) treatment with compounds at 5 mg/
kg/day or vehicle control (DMSO in PBS) was started by i.p.
injection in groups of 5 mice and continued daily for the indicated
number of days. On the days indicated, mice were imaged again
after anesthesia and injection of luciferin as described above.
Parasite index is calculated as the ratio of parasite levels in treated
mice compared to the control group and is multiplied by 100. The
ratio of parasite levels is calculated for each animal dividing the
luciferase signal one day after the end of the 5 day treatment (day 9
of infection) by the luciferase signal one day before the beginning
of treatment (day 3 of infection).
Immunofluorescence assay
The compound CID-12402750 was selected for this assay due
to its activity against T. cruzi in vivo. NIH-3T3 cells plated on
coverslips were infected with T. cruzi Tulahuen expressing b-
galactosidase and incubated with or without drug at 5, 10, 50 or
100 times the value of the IC50 obtained in the in vitro assay
(IC50=0.11 mM). After 3 days, they were fixed with 4% of
paraformaldehyde, rinsed with PBS, permeabilized for 15 min in
PBS with 0.1% Triton X-100 (Sigma-Aldrich) and blocked for
20 min in PBS with 10% goat serum, 1% bovine serum albumin,
Figure 1. Inhibition of T.cruzi growth by 23 compounds selected from the HTS. The IC50 reported from the HTS available at Pubchem and
the IC50 determined in our laboratory are shown.
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100 mM glycine and 0.05% sodium azide. The cells were
incubated for 1 h at room temperature with a polyclonal rabbit
anti-T. cruzi at 1:2,000 dilution. After rinsing, they were incubated
for 1 h at a 1:800 dilution with an Alexa FluorH 488 goat anti-
rabbit IgG secondary antibody (Molecular Probes, Invitrogen).
DAPI was used to stain the DNA and the coverslips were mounted
on Mowiol. Cells were analyzed using an inverted Olympus IX70
microscope with a 606oil objective.
Statistical analysis
Data were analyzed using Prism (v. 4.0c, GraphPad). t-test was
performed. Statistics were considered significant if P,0.05 or
P,0.01, as indicated.
Results
The results of the first high throughput screening (HTS) to
identify molecules effective against T.cruzi trypomastigote infection
of host cells were used to select 23 compounds with reported
IC50,1.2 mM and at least 100 fold activity versus toxicity for
further analysis. The quality control of these compounds was made
by HPLC/MASS.
We first tested the activity of the 23 selected compounds against
T. cruzi trypomastigote infection of host cells using a similar assay
and the same parasite and host cells that were used in the HTS
(see methods). Our tests showed a higher value for the IC50 for the
majority of the compounds when compared to the ones reported
in the HTS (Pubchem, AID: 2044), with 11 compounds showing
no detectable activity against T. cruzi (Fig. 1).
We then selected all compounds that showed activity in our in
vitro assay for testing of anti-T. cruzi activity in mice (except for
CID 1473168, which was not available). For this purpose, we
adapted a rapid method for drug testing in mouse models using
recombinant T. cruzi expressing the firefly luciferase gene in an
episomal expression vector.
We generated a recombinant T.cruzi expressing luciferase (Y-
luc),which presented good infectivity and stability. We find that
peak parasitemia was comparable for both WT and Y-luc parasites
(data not shown) and that Y-luc trypomastigotes harvested from
blood on day 7 of infection exhibited comparable levels of
luciferase expression as epimastigotes maintained on drug selection
or trypomastigotes that were used to inoculate the mice (Fig. 2).
Given that T. cruzi amastigotes divide every 12 h and the
intracellular infection cycle is 4–5 days, we estimate that these in
vivo passaged Y-luc parasites were free from drug selection for at
least 30 generations (when including time to generate trypomas-
tigotes in culture) [11]. Stable expression of luciferase from
pTREX-luc for a minimum of 7 days in vivo gives us an adequate
window of time to assess the effects of small molecule inhibitors on
acute T. cruzi infection in vivo. Parasite loads were measured at
different days after infection by injecting luciferin, the substrate of
luciferase, followed by imaging and quantification of the
luminescence signal with an IVIS Lumina imager. Focusing on
the area of highest intensity signal (red and turquoise), it is clear
that luminescent T. cruzi are concentrated in the intraperitoneal
cavity (site of injection) (Fig. 3A). Following the signal for 10 days
post-inoculation demonstrates that there is a clear indication of
parasite migration from the injection site (peritoneal cavity) to
distal sites, perhaps spleen and liver (Fig. 3A).
Using this recombinant parasite, we infected two groups of five
Balb/c mice and followed the course of infection over 13 days. To
determine whether this method would be useful for testing of
drugs, one of the groups was treated with benznidazole, while
control group was injected with vehicle control. A reduced signal
was obtained in the group treated with benznidazole (Fig. 3A,B).
Even if variation between individual animals is high, as expected
in this type of in vivo experiments, values between groups are
significantly different after only two days of treatment and
maintain different levels of infection for the 6 days of treatment.
We then used a modification of this protocol with quantification
of the parasite loads only at days 3 and 9 after infection, which
corresponds to five days of treatment (Fig. 4A) to test the activity of
the eleven compounds selected from the in vitro assay (Fig. 1). We
found that treatment with some of the compounds had no activity
on parasite levels (index close to 100) and others even resulted in
increased parasite loads (index higher than 100), possibly because
they interfere with the immune response of the mice. However,
two of the compounds tested, CID-24892493 and CID-12402750,
resulted in severe decreases in the levels of T. cruzi in mice that
were significantly different from their control group (Fig. 4B,C).
No toxic effects were apparent on the mice on visual observation.
These two compounds are closely related, they belong to the 1-(4-
Halogeno-benzyl)-2,4,6-triphenyl-pyridinium series and are differ-
ing only in the nature of halogen on the para position of the benzyl
(Fluorine for CID-24892493 and Chlorine for CID-12402750).
To get a better understanding of the anti-T. cruzi effect
observed, wenextdetermined
12402750 could inhibit T. cruzi replication within mammalian
host cells. We infected cells for 2 h, rinsed away the remaining free
trypomastigotes and, after adding the compound at concentrations
between the IC5 and the IC100, we incubated cells for 3 days to
allow for amastigote proliferation. In control cells, amastigotes
homogenous in size were distributed throughout the cytoplasm of
the host cells (Fig. 5A). Treatment with CID-12402750 resulted in
infected cells containing only a few amastigotes of average size
(Fig. 5B,C), suggesting that this compound interferes with
proliferation of amastigotes.
whether compoundCID-
Discussion
The use of novel pharmaceutical technologies for neglected
diseases is opening new possibilities for drug development in this
Figure 2. Stable expression of luciferase after in vitro or in vivo
passaging of T. cruzi trypomastigotes. Luciferase activity in 16105
epimastigotes (Epi) under continued drug selection (G418 200 mg/ml)
(Epi); 16105trypomastigotes (Tryp); 16105trypomastigotes 2 weeks
after differentiation into metacyclics, removal of drug selection and in
vitro passage through LLCMK2 cells and subsequent in vivo passage in
mice where luciferase activity was measured in 105trypomastigotes
acquired from the blood of an infected 6 week-old Balb/c mouse 1
week post infection (in vivo trypo).
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area. As an example of this, the first HTS performed for Chagas
disease (Pubchem AID: 1885) represents a major advance in this
field. However, our results illustrate the need for confirmation of
the HTS results, since as much as 11 hits from the HTS out of 23
selected did not show activity against T. cruzi in our hands. Since
the same parasite and host cells and were used for the HTS and for
testing in our laboratory, and the screening assay is also very
similar (see methods), it is likely that the reason for the
discrepancies resides in the chemical compounds used for analysis.
The quality control of the compounds used in our laboratory was
checked by HPLC followed by mass spectrometry and therefore
we can be confident that the chemical identity and the purity of
the compounds was optimal.
Another recent advance in the drug development field is the
development of new methods for screening of compounds in
animal models. Testing of compounds for activity in mice was
always considered a labor intensive and expensive step in the chain
of pre-clinical drug development and therefore was left at the end
of the time line. The availability of sensitive imaging techniques
and transgenic parasites, either fluorescent or luminescent [9,10]
and the Y-luc parasite described here, allow for rapid testing of
relatively high number of compounds. Compared to traditional
methods that require bleeding of infected mice and counting
parasites in a haemocytometer, intraperitoneal injection of the
luciferase substrate and imaging requires considerably less time,
with the additional advantage that there is no manipulation of
infected blood. Detection of parasites expressing luciferase is also
more sensitive than conventional counting of parasites in blood
samples. In our model of infection, Balb/c mice infected with T.
cruzi Y strain, we are not able to detect parasitemia by manual
counting in peripheral blood at any time after infection with 105
parasites, but inoculation of the same amount of Y-luc parasites
allows to follow the course of infection (Fig. 4). The stability of the
Y-luc parasite and the sensitivity of detection allows for screening
Figure 3. Method for testing anti-T.cruzi compounds in mice.
Groups of five mice were infected with T. cruzi trypomastigotes
expressing luciferase and imaged on the indicated days after infection.
Treatment with benznidazole (5 mg/kg/day, i.p.) started on day 4. (A)
One representative mouse of each group is shown. (B) Quantification of
luminescence signal from infected control or benznidazole treated
mice. Results are expressed as average 6 standard deviation (*, P,0.05;
**, P,0.01).
doi:10.1371/journal.pntd.0001298.g003
Figure 4. Test for activity in vivo of compounds active in vitro.
Groups of five mice were infected with T. cruzi and treated with
different compounds following the protocol shown in (A). (B)
Quantification of parasite infection levels in the groups of mice treated
with the different compounds is expressed as T. cruzi index.
Compounds are identified by their CID. Results are expressed as
average 6 standard deviation (*, P,0.05). (C) One representative mouse
of each group treated with compounds CID-12402750 and CID-
24892493.
doi:10.1371/journal.pntd.0001298.g004
Figure 5. Compound CID-12402750 shows trypanostatic activ-
ity in vitro. NIH-3T3 fibroblasts were incubated with T. cruzi
trypomastigotes for 2 h before washing of extracellular T. cruzi and
addition of drugs. Cells were incubated for 3 days, stained with an anti-
T. cruzi antibody and DAPI to visualize DNA. Control infection (A) or
infection in the presence of compound CID-12402750 at IC5 (B) and
IC100 (C) concentration. These are representative images from a total of
50 fields observed in each condition. Scale bar: 10 mm.
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