Endochin-like quinolone-300 and ELQ-316 inhibit Babesia bovis, B. bigemina, B. caballi and Theileria equi

doi:10.21203/rs.3.rs-58018/v1

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Endochin-like quinolone-300 and ELQ-316 inhibit Babesia bovis, B. bigemina, B. caballi and Theileria equi

https://doi.org/10.21203/rs.3.rs-58018/v1

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published 03 Dec, 2020

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Background: The most common apicomplexan parasites causing bovine babesiosis are Babesia bovis and B. bigemina, while B. caballi and Theileria equi are responsible for equine piroplasmosis. Treatment and control of these diseases are usually achieved using potentially toxic chemotherapeutics, such as imidocarb diproprionate, but drug-resistant parasites are emerging, and alternative effective and safer drugs are needed. Endochin-like quinolones (ELQ)-300 and ELQ-316 proved safe and efficacious against related apicomplexans, such as Plasmodium spp., and ELQ-316 was also effective against B. microti, without showing toxicity in mammals.

Methods: Inhibitory effects of ELQ-300 and ELQ-316 were assessed on the growth of cultured B. bovis, B. bigemina, B. caballi and T. equi. Percentage of parasitized erythrocytes was measured by flow cytometry. Effect of the ELQ drugs on the viability of actively replicating horse and bovine peripheral blood mononuclear cells (PBMC) was assessed by ELISA.

Results: We calculated IC₅₀ ranging from 0.04 to 0.37 nM for ELQ-300, and from 0.002 to 0.1 nM for ELQ-316 at 72 hr among all cultured parasites tested. None of the parasites tested were able to replicate in cultures in the presence of the ELQs-300 and ELQ-316 at IC₁₀₀, which range from 1.3 to 5.7 nM for ELQ-300 and from 1.0 to 6.0 nM for ELQ-316 at 72 hours. Neither ELQ-300 nor ELQ-316 altered the viability of equine and bovine PBMC at their IC₁₀₀ in in vitro testing.

Conclusions: ELQ-300 and ELQ-316 have a significant inhibitory activity on the main parasites responsible for bovine babesiosis and equine piroplasmosis at doses that are tolerable to host cells. These ELQ drugs may be viable candidates for developing alternative protocols for the treatment of bovine babesiosis and equine piroplasmosis.

Parasitology

Bovine babesiosis

equine piroplamsosis

Babesia bovis

Babesia bigemina

Babesia caballi

Theileria equi

endochin-like quinolones

ELQ-300

ELQ-316

Tick-borne diseases caused by apicomplexan hemoparasites, such as Babesia and Theileria, impose serious economic impact on the cattle and horse industries worldwide [1, 2]. Babesiosis and theileriosis share similar acute disease signs, including loss of weight, anorexia and fever. Usually, Babesia and Theileria are not eliminated in surviving animals and cause lifelong persistent infections. Important shared features among Babesia and Theileria species include a sexual reproductive cycle in their Ixodes arthropod hosts and asexual reproduction in the red blood cells (RBC) of their vertebrate hosts, a process that results in severe, potentially fatal hemolytic anemia.

Theileria equi and Babesia caballi are the etiological agents of equine piroplasmosis (EP), a disease that affects horses, mules, donkeys, and zebras worldwide [3]. EP imposes severe and costly restrictions in transportation of high-performance horses between endemic and non-endemic areas to participate in equestrian sporting events. No vaccines are currently available against T. equi and B. caballi, and considerable resources have been spent to develop drugs to treat animals against the harmful effects of acute EP and to prevent the loss of performance in chronically infected, high-value horses. Despite these efforts, horses that survive acute infection, especially when caused by T. equi, become persistently infected, asymptomatic carriers, a condition that can be associated with the resurgence of outbreaks of EP worldwide [4].

B. bovis and B. bigemina are two main causative agents of bovine babesiosis (BB), an acute and persistent economically important disease of cattle, that typically cause high mortality [1]. While B. bigemina is usually associated with relatively milder acute hemolytic disease, B. bovis is implicated in a more severe presentation of the acute phase of the disease, characterized by cytoadhesion of parasite-infected RBC in the brain capillaries, which resembles cerebral malaria and often leads to death [1].

Prevention and control of EP and BB have been typically achieved by controlling tick vector populations, the use of live attenuated vaccines in the case of BB, and chemotherapy. The live attenuated vaccines available to prevent acute BB are only recommended for less than 1-year old animals and present several additional constraints, including the risk of reversion to virulence. Furthermore, cattle vaccinated with live attenuated vaccines may also become persistently infected with the parasites, and can serve as a reservoir for tick acquisition and transmission [5]. In addition, live vaccines can cause severe disease to immunocompromised and older cattle, which may be more susceptible to the attenuated vaccine strains [5]. Given these scenarios, some animals vaccinated with live Babesia vaccines also need to be treated with anti-babesial drugs to prevent the development of acute disease caused by virulent escapes within the population of parasites in the attenuated vaccine strains. Currently, babesicidal drugs are the only option available for preventing loses due to babesiosis in adult vaccine-susceptible animals that need to be transported from non-endemic to Babesia endemic areas. Altogether, these aspects highlight the importance of having reliable babesicidal and theilericidal drugs to control the spread of outbreaks and prevent development of acute disease in herds vaccinated with live attenuated Babesia vaccines.

Chemotherapy treatments based on diminazene aceturate and imidocarb dipropionate are the most effective and first-choice methods to manage animals with acute BB and EP [6, 7]. However, the efficacy of these drugs is highly variable and treated animals need to be monitored closely for adverse effects, especially when high doses are used for attempting clearance of the parasites, which is a usual occurrence for valuable horses affected by EP [8]. In addition to toxic side effects, development of drug-resistance by Babesia parasites has been previously demonstrated [9]. Consequently, there is the need to search for new effective and less toxic alternative chemotherapeutics against BB and EP.

Endochin-like quinolone (ELQ) are potent selective inhibitors of the mitochondrial cytochrome bc₁ complex, as demonstrated in Plasmodium falciparum, causative agent of the most severe form of human malaria [10–13]. ELQ compounds have shown to be highly effective against different species and multiple stages of Plasmodium [14, 15]. Importantly, ELQ-300 and ELQ-316 have been selected as pre-clinical anti-malaria candidates, considering their reasonable oral bioavailability at efficacious doses, long half-life, and metabolic stability [14, 15]. A recent study also demonstrated the efficacy of ELQ prodrugs combined with atovaquone to treat experimental babesiosis caused by B. microti in the immunodeficient mouse model [16]. Data from this study showed that the combined therapy of ELQ and atovaquone resulted in complete clearance of the parasite with no disease recrudescence even more than 100 days after discontinuation of the treatment [16]. Based on these conclusive parasite inhibitory results, we evaluated the effect of ELQ-300 and ELQ-316 on the in vitro growth of B. bovis, B. bigemina, B. caballi, and T. equi. Strong inhibition of the development of all these parasites, coupled with the lack of toxic effects on host cells, suggests that these two compounds are promising candidates for future development of novel alternative therapies to control BB and EP.

Synthesis of ELQ-300 and ELQ-316

ELQ-300 and ELQ-316 were synthesized as previously described [15, 17] (Fig. S1). Both compounds were kindly provided by the Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR, USA. Purity of both ELQ derivatives was assessed at > 99% by proton NMR and gas chromatography – mass spectrometry. ELQ-300 and ELQ-316 were diluted in 100% dimethyl sulfoxide (DMSO) to prepare stock solutions. Stock solutions were kept at RT until use. Working solutions were freshly prepared in parasite culture medium before adding to the parasite cultures.

Cultures of B. bovis, B. bigemina, B. caballi and T. equi

B. caballi Puerto Rico strain [18], B. bovis Texas strain [19], B. bigemina Puerto Rico strain [20] and Theileria equi Florida strain [21] were grown in long-term microaerophilous stationary-phase cultures and incubated at 37 °C in an atmosphere of 5% CO₂, 5% O₂ and 90% N₂, as previously described [18–21]. B. bovis and B. bigemina were grown in 96-well plate using 180 µl per well of complete HL-1 culture media (pH 7.2; 2.38 g/L HEPES, 5 mL/L L-glutamine, 60 U/ml of penicillin G, 60 µg/ml of streptomycin, and 0.15 µg/ml of amphotericin B (Sigma)) supplemented with 40% bovine serum. Cultures contained a suspension of 10% and 5% packed cell volume of bovine erythrocytes for B. bovis and B. bigemina, respectively. B. caballi and T. equi were cultured under similar conditions, but culture media were supplemented with 10% or 20% horse serum, respectively. In addition, B. caballi and T. equi cultures contained a suspension of 10% packed cell volume equine erythrocytes.

Parasite Growth Inhibition Assay

Growth inhibition assays using ELQ-300 or ELQ-316 were performed on cultured B. bovis, B. bigemina, B. caballi and T. equi. Parasites were grown as described above, using culture media containing different concentrations of ELQ-300 or ELQ-316 from 0.05 to 50 nM diluted in DMSO at higher volume (0.5%). Parasite cultures in the absence of the ELQ compounds were used as a positive control for parasite growth. Extra wells containing uninfected bovine or equine RBC were prepared and used as negative controls for the flow cytometric analysis. Fresh culture medium (150 µl/well) containing respective ELQs concentrations or DMSO only was added daily to parasite cultures. These experiments were carried out in triplicate for each tested concentration and controls, over a period of 72 hr. Percentage of parasitized erythrocytes (PPE) was monitored daily by flow cytometry, as previously described [22, 23]. Fifty percent inhibitory concentration (IC₅₀) values were calculated for ELQ-300 and ELQ-316 at 24, 48, and 72 hr by extrapolation in which there is a 50% reduction of the PPE in the wells containing the ELQs compared with the positive control well using nonlinear regression (GraphPad Prism 8 software). Similarly, 100% inhibitory concentration (IC₁₀₀) values were also calculated at 72 hr.

Flow cytometric analysis for detection of parasite growth in cultures

PPE of parasite cultures was determined by flow cytometry, as previously described [22, 23]. Briefly, 5 µl of cultures were collected from the bottom of the wells and centrifuged at 450 xg for 1 min at 4 °C. Supernatant was discarded, and cell pellet was washed twice with 150 µl of phosphate buffer saline (PBS) pH 7.2. Then, cell pellet was suspended in 200 µl of 25 µg/µl hydroethidine (HE) (Invitrogen), incubated in 5% CO₂ incubator at 37 °C for 20 min in the dark, and washed twice with 200 µl of PBS to remove the excess of HE. After that, supernatant was discarded, and the cell pellet was suspended in 200 µl of fresh PBS. Then, suspended cells were analysis by flow cytometry using a Guava® easyCyte flow cytometer (Luminex) at a ratio of 800-1,000 cells/µl with 20,000 events collected. Results were analyzed by FCS Express v6 (De Novo Software). Normal, uninfected horse and cattle RBC were used as a negative control for the flow cytometric analysis.

Effect Of Elq-300 And Elq-316 Ic On Parasite Growth

In vitro growth inhibition assays were performed over a period of 8 days using PPE starting at 0.2% and 2%. Parasites were grown in the presence of calculated ELQ-300 or ELQ-316 IC₁₀₀ values. Cultures growing in medium in the absence of ELQs and non-infected RBC maintained in medium only were used as positive and negative controls, respectively. Culture medium containing respective compound concentrations was replaced daily, 150 µl medium per well, for a period of 72 hr. Then, parasites were cultivated in media only and split every 48 hr for a period of 8 days. PPE was evaluated at 24, 48, and 72 hr, and 8 days of culture by flow cytometry.

Cytotoxicity Assay

Cytotoxicity of ELQ-300 and ELQ-316 on ex vivo peripheral blood mononuclear cells (PBMC) of cattle and horses was examined by exposing cells to the compounds at their calculated IC₅₀ and IC₁₀₀. Metabolic activity of PBMC was monitored using a colorimetric assay. Briefly, peripheral blood was collected from healthy cattle and horses via jugular venipuncture into Vacutainer® tubes containing ACD (Becton Dickinson) and PBMC were isolated using Histopaque® (Sigma) per standard protocol. Cells were then plated at 2 × 10⁴ cells/well in 96-well plates in complete Dulbecco’s modified essential medium cDMEM (10% fetal bovine serum, 24 mM of HEPES, 2 mM of L-glutamine, 100 IU/ml penicillin, and 100 ug/ml streptomycin) and incubated with the ELQ compounds. The Cell Proliferation WST-1 reagent (Roche) was added to the cell cultures following the manufacture’s protocol at 24, 48, and 72 hr after exposure to the ELQ compounds. Absorbance at 440 nm was measured using an ELISA plate reader at 4 hr after adding WST-1 to the cells. Cells in cDMEM in the absence of the ELQ compounds and cells exposed to DMSO only (1/400 dilution, which corresponds to the highest volume used on the diluted ELQs) were used as negative controls. PBMC exposed to concanavalin (Con) A diluted in cDMEM (5 µg/ml) (Sigma) and Draxxin® [22] were used as a positive control.

Statistical analysis

Growth of parasites in culture was analyzed using one-way ANOVA (GraphPad Prism 8 software). Values of P < 0.05 were considered statistically significant concerning the effect of the ELQs on the parasite growth. Significant differences in PBMC metabolic activity were measured by Student’s t test, and P values < 0.05 were considered significant.

ELQ-300 and ELQ-316 inhibit the growth of B. bovis, B. bigemina, B. caballi, and T. equi

The effect of ELQ-300 and ELQ-316 on parasite growth, with a starting PPE of 0.2%, was evaluated using seven different concentrations of each compound, ranging from 0.05 to 50 nM. Both tested drugs significantly inhibited (P < 0.05) the growth of B. bovis, B. bigemina, B. caballi, and T. equi (Fig. 1A-D and Fig. 2A-D). In addition, the inhibitory effect of ELQ-300 and ELQ-316 was found to be dose-dependent for all four parasites tested. Calculated IC₅₀ and IC₁₀₀ values of ELQ-300 and ELQ-316 for each parasite are shown in Table 1. Overall, comparisons of the IC₅₀ values among all parasites tested indicate increased susceptibility to ELQ-316 than to ELQ-300. The ELQ-316 IC₅₀ varied from 0.002 to 0.1 nM, while in the ELQ-300 compound varied from 0.04 to 0.37 nM, as measured at 72 hr of culture (Table 1).

Table 1

IC₅₀ and IC₁₀₀ of ELQs 300 and ELQ-316 calculated for *B. bovis*, *B. bigemina*, *B. caballi* and *T. equi* at 72 hr of culture.
	ELQ 300		ELQ 316
Species	IC₅₀ (nM)	IC₁₀₀ (nM)	IC₅₀ (nM)	IC₁₀₀ (nM)
B. bovis	0.09 ± 0.002	4.2 ± 0.10	0.07	3.8 ± 0.12
B. bigemina	0.37 ± 0.19	1.3 ± 0.05	0.05	3.0 ± 0.15
B. caballi	0.19 ± 0.04	5.7 ± 0.24	0.1 ± 0.006	6.0 ± 0.18
T. equi	0.04 ± 0.003	3.36 ± 0.83	0.002	1.0 ± 1.55

Interestingly, our calculated values of IC₅₀ for ELQ-300 and ELQ-316 are in the same range or lower than values estimated for other related apicomplexans in previous studies. ELQ-316 IC₅₀ values of 7.97, 0.66 and 0.35 nM were established for Besnoitia besnoiti, Neospora caninum and Toxoplasma gondii tachyzoites, respectively [24]. In addition, a previous study demonstrated ELQ-300 IC₅₀ values of 15.4 and 23.1 nM for P. knowlsei and P. falciparum, respectively [11]. Besides the acceptable IC₅₀ inhibitory values found for ELQ-300, our study showed even lower ELQ-316 IC₅₀ values for B. bovis, B. bigemina, B. caballi, and T. equi, suggesting that these parasites are also highly susceptible to these two drugs.

Consistently, ELQ-300 and ELQ-316 completely abrogated the growth of all four parasites when tested at their respective IC₁₀₀. The calculated IC₁₀₀ values ranged from 1.3 to 5.7 nM for ELQ-300, and from 1.0 to 6.0 nM for ELQ-316 (Table 1). Overall, B. bigemina, displayed the lowest IC₁₀₀ value out of the four parasites tested, and appears to be the most susceptible to ELQ-300. On the other hand, and based on the IC₁₀₀ values (Table 1), T. equi appears to be more susceptible to ELQ-316 than the other four parasites tested in this study. Taking the IC₅₀ and IC₁₀₀ data together, ELQ-300 and ELQ-316 are able to efficiently inhibit the in vitro growth of B. bovis, B. bigemina, B. caballi, and T. equi blood stages.

Growth inhibitory effect of ELQ-300 and ELQ-316 is independent of initial parasitemia

We then tested whether the efficiency of the compounds is dependent on the parasite initial parasitemia, by comparing the effects of ELQ-300 and ELQ-316, at their respective IC₁₀₀, on the four parasites growing in in vitro cultures with starting PPEs of 0.2% and 2%. Neither B. bovis, B. caballi nor T. equi were able to grow in in vitro cultures in the presence of the IC₁₀₀ ELQ-300, regardless of their initial PPE (P < 0.05) (Fig. 3A, C, and D). Nonetheless, the addition of ELQ-300 to B. bigemina cultures with an initial PPE of 2% did not result in a rapid decrease of parasitemia (Fig. 3B), in contrast to what was found when the initial PPE was 0.2% (Fig. 3B).

Based on these results, a parasite rescue experiment was performed where the parasites were grown in culture in the presence of ELQ-300 for 3 days, then cultures were split 1:10, and maintained in media free of the drug for 5 additional days. Parasite growth was not detected (P < 0.05), by the end of this period of time for B. bovis and T. equi, but that was not the case for B. bigemina and B. caballi (Fig. 3B and C). These results suggest the absence of pre-existing ELQ-300-resistant parasite subpopulations in the B. bovis and T. equi strains with the ability to survive the initial drug-inhibitory treatment among the parasite strains tested. Collectively, these results are consistent with the relatively increased tolerance of B. bigemina and B. caballi to ELQ-300, compared to the other two parasites tested, as shown in Fig. 1B and C.

Interestingly, none of the four species of parasites tested in this study was able to grow in the presence of the ELQ-316 IC₁₀₀ concentration regardless of their initial PPE (P < 0.05) at 72 hr (Fig. 4A-D). The same lack of parasite growth was observed after 8 days in the parasite rescue experiment, except for B. caballi (Fig. 4A-D), independent of the starting PPE. A possible interpretation of these results is that the B. caballi strain used in this study may contain a mix of subpopulations of parasites, each one with distinct degrees of tolerance for ELQ-316. In contrast, the B. bovis, B. bigemina, and T. equi strains used in these experiments appear to be composed of subpopulations that are highly susceptible to ELQ-316. It was beyond the scope of this study to investigate the mechanism involved in the susceptibility for the ELQ drugs. However, one may speculate that such susceptibility can be due to variations/mutations in the cytochrome bc1 target sequence that affect the ELQ binding, differential uptake or elimination of the drugs, or a combination of these factors [25–27]. It was recently showed that genetic alterations in the Q_i binding site of cytochrome bc1 complex (Cytb) of B. microti is associated with resistance to ELQ-316, which suggests that this cytochrome gene is as a potential target for the ELQ drugs [16]. Based on these observations, we performed alignment analysis of the Cytb genes of B. bovis, B. bigemina, B. caballi, and T. equi together with the B. microti Cytb. Our results indicated full conservation of the two canonical Q_o and Q_i binding sites of Cytb in all sequences analyzed and a high level of amino acid identity, which ranged from 47.2 to 49.6% in comparison to B. microti (Fig. 2) (Table S1). It was beyond the scope of this study to determine the mechanism of action of the ELQ drugs in B. bovis, B. bigemina, B. caballi, and T. equi. However, the results presented here set the rationale for further studies to alter and/or knock down the Cytb gene in these parasites and evaluate its potential effect on the susceptibility or resistance to the ELQ drugs.

ELQ-300 and ELQ-316 do not affect viability of equine and bovine PBMC

Cytotoxic assays were performed to assess whether ELQ-300 and ELQ-316 affect the viability of equine and bovine PBMC, which we used as surrogates of nucleated vertebrate host cells. The cytotoxic assays were performed using IC₅₀ and IC₁₀₀ doses of ELQ-300 and ELQ-316 in in vitro cultures. Viability of PBMC was similar regardless of the presence or absence of parasite lethal doses of ELQ-300 or ELQ-316, strongly suggesting that cell viability was not compromised by any of these two drugs under the experimental conditions used in the assays (Fig. 5A and B). In addition, significant increase (P < 0.05) in cell proliferation was observed in bovine and horse PBMC exposed to ConA for 24 hr and 48 hr, respectively (Fig. 5A and B), indicating adequate sensitivity for the WST-1 viability/proliferation assay used in this study. Taken together, results of cell viability revealed that ELQ-300 and ELQ-316, at their respective IC₁₀₀, lack significant toxic effect on in vitro cultivated bovine and horse PBMC.

Overall, results presented here demonstrate that both drugs tested in this study, ELQ-300 and ELQ-316, are efficient in inhibiting the growth of in vitro cultured B. bovis, B. bigemina, B. caballi and T. equi. Importantly, IC₁₀₀ doses of the ELQ drugs did not significantly affect viability of in vitro cultured cattle and horse PBMC. Collectively, findings of this study strongly suggest that ELQ-300 and ELQ-316 can be potentially effective and safe candidates for the development of novel therapies to control BB and EP. Further studies in vivo using horses and bovines are needed to evaluate the efficacy of ELQ-300 and ELQ-316 against acute and chronic BB and EP.

Con: concanavalin

DMSO: dimethyl sulfoxide

ELQ: endochin-like quinolones

HE: Hydroethidine

IC: inhibitory concentration

nRBC: non-infected erythrocytes

PBMC: peripheral blood mononuclear cells

PBS: phosphate buffer saline

PPE: percentage of parasitized erythrocytes

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was funded by the United States Department of Agriculture-Agriculture Research Service Current Research Information System Project No. 2090-32000-039-00D, and the International Development Research Centre (IDRC) (Canada) project 108525-001. Dr. Riscoe’s laboratory receives financial support from the United States Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development Program Award number i01 BX003312. MKR. is a recipient of a VA Research Career Scientist Award (14S-RCS001). Research reported in this publication was also supported by the US National Institutes of Health under award number AI100569 (MKR) and by the U.S. Department of Defense Peer Reviewed Medical Research Program (Log # PR181134 (MKR), and the National Institute of Allergy and Infectious Diseases under award number R01AI141412. This work was also funded by Career Development Award BX002440 and VA Merit Review Award BX004522 to JSD from U.S. Department of Veterans Affairs Biomedical Laboratory Research and Development.

Author’s contribution

MGS, RGB and CES conceived and designed the study. MGS and RGB performed the inhibitory assays and microscopy experiments, analyzed the data and drafted the manuscript. MGS and RGB performed the flow cytometry assay and cytotoxic assay. JSD, MKR, SP, RW, and AN manufactured and provided endochin-like quinolones, and gave technical guidance regarding their use. MGS, RGB, CES, JSD, and MKR wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors would like to acknowledge Paul Lacy for his help with the in vitro B. bovis, B. bigemina and B. caballi cultures and cultures supplies, Johanatan Hyunki and Ji Heon Lee for checking PPE on smears, and Jacob Laughery for providing uninfected bovine erythrocytes and serum, Lowell Kappemeyer for providing the initial in vitro B. caballi and T. equi cultures and the uninfected equine erythrocytes, and Megan Blauert, for animal handling and blood draw.

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Table S1 Amino acid percent identity of cytochrome bc1 complex (Cytb) of Babesia bovis, B. bigemina, B. caballi, and Theileria equi in comparison to B. microti.

	B. bovis	B. bigemina	B. caballi	T. equi
B. microti	49.6%	48.5%	49.6%	47.2%

SilvaandBastosetalFigS1.pdf
Fig. S1 Chemical structures of (A) ELQ-300 and (B) ELQ-316.
SilvaandBastosetalFigS2.pdf
Fig. S2 Alignment of amino acid sequences of the cytochrome bc1 complex (Cytb) of B. bovis (GenBak accession YP_001504108), B. bigemina (GenBak accession BAI66164.1), B. caballi (GenBak accession BAI66167.1), T. equi (GenBak accession XP_025033545.1), and B. microti (GenBak accession MT114078)

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Endochin-like quinolone-300 and ELQ-316 inhibit Babesia bovis, B. bigemina, B. caballi and Theileria equi

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