IJP: Drugs and Drug Resistance 10 (2019) 84–90
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IJP: Drugs and Drug Resistance
journal homepage: www.elsevier.com/locate/ijpddr
Screening the Medicines for Malaria Venture Pathogen Box against
piroplasm parasites
T
Arifin Budiman Nugrahaa,b, Bumduuren Tuvshintulgaa, Azirwan Guswantoc,
Dickson Stuart Tayebwaf, Mohamed Abdo Rizka,d, Sambuu Gantuyaa, Gaber El-Saber Batihae,
Amany Magdy Beshbishya, Thillaiampalam Sivakumara, Naoaki Yokoyamaa, Ikuo Igarashia,∗
a
National Research Center for Protozoan Diseases, Obihiro University of Agriculture Veterinary Medicine, Nishi 2 Sen-13, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan
Department of Animal Infectious Diseases and Veterinary Public Health, Faculty of Veterinary Medicine, IPB University, Jl. Agatis, Kampus IPB Dramaga, Bogor, Jawa
Barat, 16680, Indonesia
c
Balai Veteriner Subang (DIC Subang), Jl. Terusan Garuda 33/11 Blok Werasari Dangdeur, Subang, Jawa Barat, 41212, Indonesia
d
Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Mansoura University, Mansoura, 35516, Egypt
e
Department of Pharmacology and Therapeutics, Faculty of Veterinary Medicine, Damanhour University, Al-Beheira, 22511, Egypt
f
Research Center for Tropical Diseases and Vector Control, College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, 7062, Kampala,
Uganda
b
A R TICL E INFO
A BSTR A CT
Keywords:
Pathogen Box
Drug screening
Antipiroplasm agent
Diminazene aceturate (DA) and imidocarb dipropionate are commonly used in livestock as antipiroplasm agents.
However, toxic side effects are common in animals treated with these two drugs. Therefore, evaluations of novel
therapeutic agents with high efficacy against piroplasm parasites and low toxicity to host animals are of paramount importance. In this study, the 400 compounds in the Pathogen Box provided by the Medicines for Malaria
Venture foundation were screened against Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi. A
fluorescence-based method using SYBR Green 1 stain was used for initial in vitro screening and determination of
the half maximal inhibitory concentration (IC50). The initial in vitro screening performed using a 1 μM concentration as baseline revealed nine effective compounds against four tested parasites. Two “hit” compounds,
namely MMV021057 and MMV675968, that showed IC50 < 0.3 μM and a selectivity index (SI) > 100 were
selected. The IC50s of MMV021057 and MMV675968 against B. bovis, B. bigemina, T. equi and B. caballi were
23, 39, 229, and 146 nM, and 2.9, 3, 25.7, and 2.9 nM, respectively. In addition, a combination of MMV021057
and DA showed additive or synergistic effects against four tested parasites, while combinations of MMV021057
with MMV675968 and of MMV675968 with DA showed antagonistic effects. In mice, treated with 50 mg/kg
MMV021057 and 25 mg/kg MMV675968 inhibited the growth of Babesia microti by 54 and 64%, respectively,
as compared to the untreated group on day 8. Interestingly, a combination treatment with 6.25 mg/kg DA and
25 mg/kg MMV021057 inhibited B. microti by 91.6%, which was a stronger inhibition than that by single
treatments with 50 mg/kg MMV021057 and 25 mg/kg DA, which showed 54 and 83% inhibition, respectively.
Our findings indicated that MMV021057, MMV675968, and the combination treatment with MMV021057 and
DA are prospects for further development of antipiroplasm drugs.
1. Introduction
Significant economic impacts of bovine babesiosis and equine piroplasmosis on the cattle and horse industries have been reported,
especially in piroplasmosis-endemic countries. Bovine babesiosis
caused by Babesia bovis and Babesia bigemina decreases meat and milk
production and leads to the death of infected cattle (Yusuf, 2017).
Equine piroplasmosis caused by Babesia caballi and Theileria equi is associated with detrimental effects in horses. Once the horse is infected by
Piroplasmosis is a tick-transmitted disease caused by Babesia and
Theileria parasites. Piroplasmosis affects humans, livestock, and wild
animals worldwide. Generally, piroplasm infection is characterized by
fever, icterus, hemolysis, hemoglobinuria, and death if treatment fails
or is not attempted (Schnittger et al., 2012; Wise et al., 2013).
∗
Corresponding author. National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-13Inada-cho,
Obihiro, 080-8555, Japan.
E-mail address: igarcpmi@obihiro.ac.jp (I. Igarashi).
https://doi.org/10.1016/j.ijpddr.2019.06.004
Received 18 May 2019; Received in revised form 13 June 2019; Accepted 17 June 2019
Available online 19 June 2019
2211-3207/ © 2019 Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
IJP: Drugs and Drug Resistance 10 (2019) 84–90
A.B. Nugraha, et al.
bovis (Texas strain), B. bigemina (Argentine strain), and a USDA strain of
B. caballi and T. equi. The Munich strain of B. microti was used for in vivo
studies.
either or both Babesia caballi and Theileria equi, that animal could remain a carrier for its entire life. Consequently, such horses are restricted
with regard to international movement because they can transmit disease that affects trade and equestrian sport (Knowles, 1996).
There are three widely used strategies for piroplasmosis control:
vaccination, the use of antipiroplasm drugs, and vector control measures. In the past several decades, antipiroplasm drugs, including diminazene aceturate (DA) and imidocarb dipropionate (ID), have played
an important role in the prevention and control of piroplasmosis
(Mosqueda et al., 2012). However, recent studies on Babesia have reported the development of resistance to DA and documented toxic side
effects in ID-treated equines (Hwang et al., 2010; Tuntasuvan et al.,
2003). Furthermore, high levels of ID and DA drug residue in edible
tissue have been reported in treated animals (Belloli et al., 2007;
Mdachi et al., 1995). Therefore, continuous efforts to discover and
develop novel antipiroplasm drugs are urgently needed.
One alternative approach to fast-track the development of novel
antiparasitic agents is large-scale screening of compounds from existing
databases, such as the Medicines for Malaria Venture (MMV) Pathogen
Box. The MMV foundation offers free access to compounds in the MMV
Pathogen Box to researchers all over the world. The activity of compounds in the MMV Pathogen Box has been confirmed against several
diseases, including tuberculosis, malaria, leishmaniasis, trypanosomiasis, helminthiasis, toxoplasmosis, and dengue. Additionally, the
MMV Pathogen Box contains 26 reference compounds. Recently, several researchers have discovered effective compounds in the MMV
Pathogen Box and repurposed them for treatment against other parasitic protozoa, including Toxoplasma gondii, Cryptosporidium parvum,
Giardia lamblia, Neospora caninum, Plasmodium falciparum, and
Trypanosoma spp. (Duffy et al., 2017; Hennessey et al., 2018; Müller
et al., 2017; Spalenka et al., 2018). Moreover, all of the compounds
have been tested for their cytotoxicity on mammalian cells with greater
than fivefold selectivity indexes (http://www.pathogenbox.org/aboutpathogen-box/supporting-information). The present study aimed to
discover potent inhibitors against B. bovis, B. bigemina, B. caballi, and T.
equi by screening 400 compounds from the MMV Pathogen Box.
2.4. In vitro cultures
Babesia bovis and B. bigemina were cultured in purified bovine red
blood cells (RBCs) using Medium 199 (M199) supplemented with 40%
bovine serum. Babesia caballi and T. equi were cultured in purified horse
RBCs. The medium for B. caballi was GIT supplemented with 40% horse
serum, while M199 supplemented with 40% horse serum and hypoxanthine (MP Biomedicals, USA) at a final concentration of 13.6 μg/ml
was used for T. equi cultivation. An antibiotic–antimycotic solution
containing 60 U/ml penicillin G, 60 μg/ml streptomycin, and 0.15 μg/
ml amphotericin B (Sigma-Aldrich, Tokyo, Japan) was added to all of
the media.
2.5. In vitro inhibition assay
Initially, all 400 compounds were tested against B. bovis, B. bigemina, B. caballi, and T. equi at a single concentration of 1 μM. Each
compound was tested in duplicate and repeated in three separate experiments. Compounds that showed over 60% inhibition against all of
the parasites tested were selected for further analysis to determine their
half maximal inhibitory concentration (IC50).
To determine the IC50, each compound was diluted by a twofold
serial dilution ranging from 0.001 to 1 μM. DA was also included for
each experiment as a comparator drug. The medium containing 0.1%
DMSO with infected RBCs (iRBCs) and uninfected RBCs were used as
positive and negative controls, respectively. This experiment was repeated three times in separate experiments.
In vitro inhibition assays were carried out as previously described
(Guswanto et al., 2014; Rizk et al., 2015). Briefly, the parasites were
harvested after 4 days of the initial culture, when the parasitemia
reached more than 3%. The iRBCs were harvested and diluted with
fresh RBCs to make a 1% parasitemia. The experiment was conducted
using 2.5 μl (for B. bovis and B. bigemina) or 5 μl (for B. caballi and T.
equi) of iRBCs. The iRBC was added to each well in triplicate. The volume of culture medium containing the drug was added, up to a total
reaction volume of 100 μl. The plates were incubated at 37 °C with 5%
CO2, 5% O2, and 90% N2. After 96 h, 100 μl of lysis buffer containing
SYBR Green 1 was added to each well and mixed gently by pipetting.
The plates were wrapped in aluminum foil to protect them from light
and kept at room temperature for 6 h. The relative fluorescence values
were measured using a fluorescence spectrophotometer (Fluoroskan
Ascent, Thermo Fisher Scientific, USA) excitation and emission wavelength of 485 nm and 518 nm, respectively. The relative fluorescence
value was set to percentages after subtracting the mean values of the
negative control.
2. Materials and methods
2.1. Pathogen Box compounds
The Pathogen Box consists of 400 compounds provided by the MMV
foundation following a request from our laboratory. The compounds
were delivered in five plates, each containing 80 compounds. Each
compound had a 10 μl volume diluted in 100% dimethyl sulfoxide
(DMSO) to a concentration of 10 mM. In order to prepare a 1 mM stock
solution, 90 μl of DMSO was added to each well and divided into two
identical plates in accordance with MMV instructions. Additionally, DA
(Sigma-Aldrich, Tokyo, Japan) was diluted in Milli-Q water (MQW) to
make a 10 mM stock solution. All compounds were stored at −30 °C
until needed for the experiments. For in vivo studies, MMV021057 was
purchased from Sigma-Aldrich, while MMV675968 was supplied from
MMV in powder form. All compounds were diluted in normal saline
with 4% DMSO and 8% Tween 80 for in vivo studies.
2.6. Combination treatment with MMV675968, MMV021057, and DA
2.2. Reagents
A drug combination assay was performed using a 96-well culture
plate with a single drug assay at the constant ratio in accordance with
Chou
(2006).
The
two-drug
combination
therapy
(MMV675968 + MMV021057, MMV675968 + DA, and
MMV021057 + DA) was performed at concentrations of 0.25 x IC50,
0.5 x IC50, IC50, 2 x IC50, and 4 x IC50. The degree of synergism was
determined as the weighted average of CI values using the formula ((1 x
IC50) + (2 x IC75) + (3 x IC90) + (4 x IC95))/10). The values with <
0.9, 0.90 to 1.10, and > 1.10 indicated synergistic, additive, and antagonistic effects, respectively. Each experiment was repeated in three
separate trials.
A lysis buffer containing Tris (130 mM at pH 7.5), EDTA (10 mM),
saponin (0.016%; w/v), and Triton-X 100 (1.6%; v/v) was prepared and
stored at 4 °C. The 10,000 x SYBR Green 1 nucleic acid stain (Lonza
Rockland Inc., Rockland, USA) was stored at −30 °C. All reagents were
purchased from Sigma-Aldrich (Tokyo, Japan).
2.3. Parasites
Four strains of parasites were used for in vitro studies, including B.
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A.B. Nugraha, et al.
2.7. In vivo inhibition assay
(SI) > 100. The IC50s of MMV021057 on B. bovis, B. bigemina, T. equi,
and B. caballi were 23 ± 8, 39 ± 11, 229 ± 62, and 146 ± 36 nM,
respectively. The IC50s of MMV675968 on B. bovis, B. bigemina, T. equi,
and B. caballi were 2.9 ± 0.3, 3 ± 0.8, 25.7 ± 6.4, and
2.9 ± 0.1 nM, respectively.
The growth-inhibitory effect of the hit compounds was determined
using a mouse model infected with B. microti, as previously described
(Tuvshintulga et al., 2016). Thirty-five BALB/c mice (CLEA Japan Inc.,
Tokyo, Japan) were divided into seven groups, each consisting of five
mice. Groups 1 and 2 were kept as a negative control (uninfected and
untreated) and positive control (infected and untreated), respectively.
Groups 3 and 4 were treated by intraperitoneal (i.p.) injection of
25 mg/kg body weight (BW) MMV675968 and subcutaneous (sc) injection of 50 mg/kg BW MMV021057, respectively. Groups 5 and 6
were treated by i.p. injection of 6.25 mg/kg and 25 mg/kg BW DA,
respectively. Group 7 was injected with a combination of 25 mg/kg BW
MMV021057 + 6.25 mg/kg BW DA by sc and i.p. injection, respectively.
Prior to the start of the in vivo experiments, a frozen stock of B.
microti was recovered from −80 °C and injected intraperitoneally into a
mouse. Subsequently, the parasitemia was monitored every 2 days in a
Giemsa-stained blood smear. When 30% parasitemia was observed in
mice, the mouse was anesthetized, and blood was collected through
cardiac puncture. The blood was then diluted by 1 × PBS to acquire
2 × 107/ml B. microti iRBCs. Subsequently, all mice in the six groups
except the negative control group were injected intraperitoneally with
0.5 ml of inoculum to achieve 1 x 107/ml iRBCs.
Parasitemia was monitored by counting iRBCs every 2 days among
10,000 RBCs in Giemsa-stained blood smears. When approximately 1%
parasitemia was achieved on day 4 post-infection (p.i.), the treatment
was initiated and continued for 5 consecutive days (day 4 to day 8). To
analyze the effects of the treatment, 10 μl of blood from the tail was
collected every 4 days and used to determine the hematological profiles
using an automatic hemocytometer (Celltac α MEK-6450, Nihon
Kohden, Tokyo, Japan). All parameters were monitored until day 30,
and the experiment was repeated twice. The animal experiment was
conducted in accordance with the Regulations for Animal Experiments
of Obihiro University of Agriculture and Veterinary Medicine, Japan
(accession number of Animal experiment: 18-120; Pathogen experiment: 201826).
3.2. Combination treatment
The combination of two drugs including MMV021057,
MMV675968, and DA was performed as described section in section
2.6. A combination of MMV021057 and DA showed synergistic effects
against B. caballi and B. bovis, while an additive effect was observed
against B. bigemina and T. equi. On the other hand, drug combinations of
MMV021057 and MMV675968 and of MMV675968 and DA showed
antagonistic effects in all tested parasites (Table 3 and Supplementary
Fig. S12).
3.3. In vivo inhibition assay
In vivo experiments were conducted to evaluate the chemotherapeutic efficacy of single treatments with 50 mg/kg BW MMV021057,
25 mg/kg BW MMV675968, 25 mg/kg BW DA, and 6.25 mg/kg BW DA
and combination treatments with MMV021057 + DA (25 + 6.25 mg/
kg BW) against B. microti. The groups treated with 50 mg/kg BW
MMV021057 and 25 mg/kg BW MMV675968 showed a higher inhibition level and more rapid reduction of parasitemia than the untreated
group (Fig. 2A). The peak parasitemia in MMV021057- and
MMV675968-treated mice was reached on day 10 p.i., while that in the
untreated and DA-treated group was reached on day 8 p.i. The percentage of growth inhibition of 50 mg/kg BW MMV021057, 25 mg/kg
BW MMV675968 and 25 mg/kg BW DA, compared with that of the
untreated group, was 54, 64, and 83%, respectively. Furthermore, the
inhibitory effect was improved to 91.6% when a combination of 25 mg/
kg BW MMV021057 and 6.25 mg/kg BW DA was used (Fig. 2B). The
hematocrit indexes were stable in the treated groups as compared to the
positive control group (infected and untreated) (Fig. 3).
4. Discussion
2.8. Statistical analysis
Although diminazene aceturate and imidocarb dipropionate are still
readily available for use against piroplasmosis, it has been reported that
they have toxic side effects and leave residue in animal products and
that parasites are developing resistance (Belloli et al., 2007; Tuntasuvan
et al., 2003). Thus, there is a need to find new drugs with potent antipiroplasm activity and low toxic side effects to the host.
In the present study, 400 compounds from the MMV Pathogen Box
were screened against Babesia and Theileria parasites. Nine hit compounds were identified as effective against Babesia and Theileria parasites with IC50s of less than 1 μM. The most interesting hit compounds
were MMV021057 and MMV675968, with IC50s of less than 0.3 μM and
an SI of more than 100. The two hit compounds identified from the
Pathogen Box were fewer than the four hits identified from the Malaria
Box in a previous study under the same conditions (Van Voorhis et al.,
2016). Interestingly, one of the reference compounds, known as buparvaquone, showed a lower IC50 on T. equi than on Babesia parasites.
This finding was comparable to that of a previous report showing that
buparvaquone could be useful for the treatment of equine theileriosis in
the field. However, this drug was unable to clear carrier infections
alone (Zaugg and Lane, 1992).
MMV675968 was the most effective antipiroplasm compound, with
IC50s of less than 10 nM in B. bovis, B. bigemina, and B. caballi, but not in
T. equi. This result revealed that MMV675968 is more effective against
Babesia than Theileria. Moreover, the IC50 of this compound was 5.6
times lower than that of DA. MMV675968 is known as an inhibitor of
the dihydrofolate reductase (DHFR) enzyme in Cryptosporidium. The
mechanism of action (MOA) of MMV675968 has been reported the
The percentage of inhibition on the in vitro studies was calculated in
Microsoft Excel. Data were calculated for the IC50 using a nonlinear
regression sigmoidal dose-response curve fit, available in GraphPad
Prism (GraphPad Software Inc., USA). For combination therapy, the
combination indexes (CIs) were calculated using CompuSyn software.
Statistical analysis of the parasitemia and hematological profiles was
done using Student's t-test. The difference in parasitemia between untreated and drug-treated groups was considered statistically significant
if P < 0.05.
3. Results
3.1. In vitro inhibition assay
The initial screening was performed to identify compounds showing
at least 60% growth inhibition at the initial concentration of 1 μM
against the four parasites tested, namely, B. bovis, B. bigemina, B. caballi,
and T. equi. Out of the 400 compounds, six were active against B. bovis
and B. bigemina (Supplementary Fig. S1). Four compounds were active
against B. caballi and T. equi. Nine compounds were active against all
tested parasites (Table 1). The compound structures of active compound against four tested parasites shown in Fig. 1.
The IC50s of all nine compounds were determined as shown in
Table 2 and Supplementary Figs. S2–S11. Two compounds,
MMV021057 and MMV675968, were selected as hit compounds because they showed an IC50 < 0.3 μM and a selectivity index
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Table 1
The nine lead compounds in all tested parasites after primary screening at 1 μM.
Compound ID
Disease set within the Pathogen Box
Compound class
Mechanism of action in other organisms
MMV021057
MMV689480
MMV676602
MMV688547
MMV688703
MMV010576
MMV688362
MMV688407
MMV675968
Malaria
Reference compound (buparvaquone)
Kinetoplastids
Kinetoplastids
Toxoplasmosis
Malaria
Kinetoplastids
Kinetoplastids
Cryptosporidiosis
β-Methoxyacrylate analogue (azoxystrobin)
Hydroxynaphthoquinone
Milciclib
Bisarylamidine
Trisubstituted pyrrole
2-Amino-3,5-diaryl pyridine
Bisarylamidine
Triazole
Quinazoline-2,4-diamine
Mitochondrial cytochrome bc1 complex
Qo quinone-binding site of mitochondrial cytochrome b
Cyclin-dependent kinase 2 inhibitor
DNA minor groove binding at AT-rich DNA sequences
cGMP-dependent protein kinase
Kinase
DNA minor groove binding at AT-rich DNA sequences
Not available
Dihydrofolate reductase
Fig. 1. Structures of the nine hit compounds identified by the initial in vitro screening using 1 μM as the highest concentration. The most effective compounds are
indicated by underlined letters. These compound structures were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov).
Table 2
Half maximum inhibition concentrations (IC50s).
Compound ID
h
MMV021057
MMV689480
MMV676602
MMV688547
MMV688703
MMV010576
MMV688362
MMV688407
MMV675968h
DAg
IC50 (nM)
B. bovis
B. bigemina
T. equi
B. caballi
23 ± 8
135 ± 41
243 ± 45
143 ± 16
796 ± 57
65 ± 10
74 ± 62
92 ± 33
2.9 ± 0.3
215 ± 3.6
39 ± 11
488 ± 30
327 ± 31
694 ± 173
804 ± 27
30 ± 8
566 ± 118
188 ± 20
3.0 ± 0.8
1050 ± 61
229 ± 62
96 ± 19
468 ± 122
219 ± 97
751 ± 70
480 ± 140
338 ± 99
263 ± 106
25.7 ± 6.4
140 ± 30
146 ± 36
237 ± 93
510 ± 145
562 ± 33
583 ± 199
610 ± 173
262 ± 96
204 ± 113
2.9 ± 0.1
23 ± 6
CC50 (μM)
SIf
> 28a
8.66d
1.1b
> 32b
> 50c
> 10a
> 32b
> 32b
5.5d
> 40e
> 1217; > 717; > 122; > 192
64; 18; 90; 37
5; 3; 2; 2
> 224; > 46; > 146; > 57
> 63; > 62; > 67; > 86
> 153; > 333; > 21; > 16
> 432; > 57; > 95; > 122
> 348; > 170; > 122; > 157
1896; 1833; 214; 1896
> 186; > 38; > 286; > 1739
The cytotoxicity values on HepG2, MRC5, and HL60 were obtained from (http://www.pathogenbox.org/about-pathogen-box/supporting-information).
a
The CC50 values on HepG2 cells.
b
The CC50 values on MRC5 cells.
c
The CC50 values on HL60 cells.
d
The CC50 values on Vero cells (Spalenka et al., 2018).
e
The CC50 values on MDBK cells (Guswanto et al., 2018).
f
Selectivity index (SI) = CC50/IC50 on B. bovis, B. bigemina, B. caballi and T. equi ratio.
g
Diminazene aceturate (DA) as a control drug.
h
Two selected compounds which showed IC50 < 0.3 μM and SI > 100 in four tested parasites.
substituted two-atom linker interacts with Cys 113 of C. hominis-DHFR
enzyme and extends the phenyl group into two different hydrophobic
pockets, therefore, inhibited an enzyme activity (Popov et al., 2006).
DHFR is an essential enzyme in nucleic acid and amino acid and also is
highly conserved in all protozoan parasites, including Babesia and
Theileria (Anderson, 2005; Begley et al., 2011). MMV675968 was also
identified as a potent inhibitor against Plasmodium falciparum and
Toxoplasma gondii, with IC50s of 0.03 μM and 0.02 μM, respectively
(Duffy et al., 2017; Spalenka et al., 2018). In concordance with previous
studies, we assumed that the findings in the current study indicate that
MMV675968 also inhibited the DHFR enzyme in Babesia and Theileria
parasites.
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Table 3
Combination indexes among MMV675968, MMV021057, and DA.
Parasites
Drug combinations
CI value at
IC50
IC75
IC90
IC95
WIa
Degree of synergismb
B. bovis
MMV675968 + MMV021057
MMV675968 + DA
MMV021057 + DA
12.872
5.795
0.691
7.214
3.255
0.646
4.293
2.009
0.615
3.135
1.568
0.602
5.272
2.460
0.624
Antagonistic
Antagonistic
Synergistic
B. bigemina
MMV675968 + MMV021057
MMV675968 + DA
MMV021057 + DA
20.143
15.61
2.05
7.681
6.727
1.303
3.102
2.908
0.872
1.756
1.648
0.68
5.184
4.438
0.999
Antagonistic
Antagonistic
Additive
B. caballi
MMV675968 + MMV021057
MMV675968 + DA
MMV021057 + DA
1.66
0.493
1.162
1.652
1.109
0.989
1.674
2.649
0.842
1.697
4.88
0.755
1.677
3.018
0.869
Antagonistic
Antagonistic
Synergistic
T. equi
MMV675968 + MMV021057
MMV675968 + DA
MMV021057 + DA
27.663
6.877
0.913
21.66
9.54
0.953
16.996
13.357
0.994
14.433
16.878
1.025
17.970
13.354
0.990
Antagonistic
Antagonistic
Additive
a
b
Weight average (WI) of combination index (CI) values.
WI values: synergistic (< 0.90), additive (0.90–1.10), antagonistic (> 1.10).
Fig. 2. The growth-inhibitory effects of
B. microti in mice treated with the test
compounds MMV675968, MMV021057,
and DA and monitored for 30 days. (A)
The parasitemia in mice administered
with single treatments of MMV675968,
MMV021057, and diminazene aceturate
(DA) at doses of 25, 50, and 25 mg/kg
BW, respectively. (B) The parasitemia in
mice administered with combination
treatments of 25 mg/kg BW MMV021057
and 6.25 mg/kg BW DA via subcutaneous
and intraperitoneal injections, respectively. The arrow indicates the 5 consecutive days of treatment beginning on
day 4 and continuing through day 8 p.i.
Each value represents the mean and
standard deviation (S.D.) from two separate experiments of five mice per experimental group. The significant differences
(P < 0.05) between untreated and
MMV021057 or combination-treated
mice are indicated with asterisks.
Fig. 3. The hematocrit levels in uninfected and untreated mice and in B. microti-infected and treated
mice administered with 6.25 mg/kg BW DA, 25 mg/
kg BW DA, 50 mg/kg BW MMV021057, and the
combination of DA and MMV021057 (6.25 + 25 mg/
kg BW). Drug treatment was performed on days 4–8
p.i. (arrow). The hematocrit levels were the mean and
S.D. from two separate experiments of five mice in
each experimental group. Asterisks indicate statistically significant differences (P < 0.05) between infected and treated mice and uninfected and untreated
mice.
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The compound MMV021057, also known as azoxystrobin, was the
second best after MMV675968. MMV021057, also known as a broadspectrum fungicide, has been reported to function as an inhibitor of
the mitochondrial respiration process by binding at the Qo site of
cytochrome b. Eventually, there is blockage of the electron transfer
between cytochrome b and c, which blocks the production of ATP
(Bartlett et al., 2004). In addition, the MOA of MMV021057 (azoxystrobin) has been reported binding at the Qo site in the haem b lproximal region or in the distal site of cytochrome b in Plasmodium
falciparum (Vallieres et al., 2013). Furthermore, MMV021057 that
targeted bc 1 complex was a potent inhibitor of P. falciparum, with
ranged IC50 15–30 nM (Duffy et al., 2017; Witschel et al., 2012). Although further studies are needed to elucidate the exact mode of action, it is possible that MMV021057 exerted this effect against the
growth of Babesia and Theileria.
The present study investigated the growth-inhibitory effects of
MMV675968 and MMV021057 against B. microti in mice. The findings
showed that MMV675968 and MMV021057 inhibited the growth of B.
microti by 64% and 54%, respectively, compared to the untreated
group. Furthermore, the mice treated with MMV675968 and
MMV021057 showed peak parasitemia on day 10, whereas the untreated mice and DA-treated group had peak parasitemia on day 8. Even
though the MMV675968 and MMV021057 single treatments were less
effective than DA against B. microti in mice, they delayed the peak of
parasitemia by 2 days (Fig. 2A). This implied that MMV675968 and
MMV021057 slowed the growth of B. microti and could be potentiated
to increase their efficacy.
To test the effect of potentiation with an effective drug (DA) and
the possibility of reducing the therapeutic dose of DA in the field, we
conducted a combination treatment of 25 mg/kg BW MMV021057
with a lowered dose of DA 6.25 mg/kg BW. Importantly, the combination treatment showed a stronger inhibitory effect than single
treatments with 50 mg/kg BW MMV021057 and 25 mg/kg BW DA.
Comparatively, the MMV021057 and DA combination was more effective than combination treatments of DA with clofazimine, 17DMAG, allicin, and thymoquinone (El-Sayed et al., 2019; Guswanto
et al., 2018; Salama et al., 2014; Tuvshintulga et al., 2017). This
emphasized that MMV021057 in combination with DA could reduce
the toxic effect of DA, since only 6.25 mg/kg BW was used in the
combination therapy. The synergistic and additive effect of other
known cytochrome b inhibitor (atovaquone) also observed when
combined with DA against piroplasm parasites (Guswanto et al.,
2018). This finding strongly suggest that drug targeted to cyctochrome
b could be develop for combination treatment with DA as approved
drug against piroplasm parasites. Moreover, MMV021057 was reported to be nongenotoxic, nonteratogenic, and nonneurotoxic by the
Codex Committee on Pesticide Residues (CCPR). In addition,
MMV021057 is metabolized quickly and excreted in both feces and
urine (FAO, 2005).
The current study identified two hit compounds from the MMV
Pathogen Box that possess potent growth-inhibitory effects against
Babesia and Theileria parasites in vitro. Further analysis in vivo confirmed the effectiveness observed in vitro. Furthermore, one of the
compounds, MMV021057, produced outstanding growth-inhibitory effects when combined with DA, which was superior to the previously
reported combination of DA and other test compounds, namely, clofazimine, 17-DMAG, allicin, and thymoquinone. Taken together, this
study has identified compounds that could be repurposed for treatment
against piroplasmosis. Most importantly, the combination of
MMV021057 and DA could be an alternative treatment with higher
chemotherapeutic efficacy and could reduce the side effects of single
treatment with DA. Future studies are needed to evaluate the mode of
action of MMV675968 and MMV021057 and to evaluate the efficacy of
derivatives related to these compounds against Babesia and Theileria
parasites.
Acknowledgments
We would like to thank the Medicines for Malaria Venture (MMV)
foundation for providing the Pathogen Box compounds. This study was
supported by the Japan Society for the Promotion of Science KAKENHI
Grant Number JP18H02337.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ijpddr.2019.06.004.
Conflicts of interest
The authors declare that they have no competing interests.
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