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

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. The endochin-like quinolones (ELQ)-300 and ELQ-316 have been proven to be safe and efficacious against related apicomplexans, such as Plasmodium spp., with ELQ-316 also being effective against Babesia microti, without showing toxicity in mammals. Methods The inhibitory effects of ELQ-300 and ELQ-316 were assessed on the growth of cultured B. bovis, B. bigemina, B. caballi and T. equi. The percentage of parasitized erythrocytes was measured by flow cytometry, and the effect of the ELQ compounds on the viability of horse and bovine peripheral blood mononuclear cells (PBMC) was assessed by monitoring cell metabolic activity using a colorimetric assay. Results We calculated the half maximal inhibitory concentration (IC50) at 72 h, which ranged from 0.04 to 0.37 nM for ELQ-300, and from 0.002 to 0.1 nM for ELQ-316 among all cultured parasites tested at 72 h. None of the parasites tested were able to replicate in cultures in the presence of ELQ-300 and ELQ-316 at the maximal inhibitory concentration (IC100), which ranged from 1.3 to 5.7 nM for ELQ-300 and from 1.0 to 6.0 nM for ELQ-316 at 72 h. Neither ELQ-300 nor ELQ-316 altered the viability of equine and bovine PBMC at their IC100 in in vitro testing. Conclusions The compounds ELQ-300 and ELQ-316 showed 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.


Background
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 anemia, loss of weight, anorexia and fever [3]. 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 [4][5][6].
Theileria equi and Babesia caballi are the etiological agents of equine piroplasmosis (EP), a disease that affects horses, mules, donkeys, and zebras worldwide [7]. EP imposes severe and costly restrictions in transportation of high-performance horses between endemic and non-endemic areas to participate in equestrian sporting events [3,6]. 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 [8].
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 parasiteinfected 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, which are in use only in a limited number of countries, 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 [9]. In addition, live vaccines can cause severe disease to immunocompromised and older cattle, which may be more susceptible to the attenuated vaccine strains [9]. 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 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 rst-choice methods to manage animals with acute BB and EP [10,11]. However, the e cacy 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 [12]. In addition to toxic side effects, and although speci c resistance to imidocarb by Babesia and Theileria parasites was not yet documented, the potential for the development of drug-resistance by Babesia parasites to other drugs such as amicarbalide isethionate has been previously recorded [13]. 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 bc 1 complex, as demonstrated in Plasmodium falciparum, the causative agent of the most severe form of human malaria [14][15][16][17]. ELQ compounds have shown to be highly effective against different species and multiple stages of Plasmodium [18,19]. Importantly, ELQ-300 and ELQ-316 have been selected as preclinical anti-malaria candidates, considering their reasonable oral bioavailability at e cacious doses, long half-life, and metabolic stability [18,19]. A recent study also demonstrated the e cacy of ELQ prodrugs combined with atovaquone to treat experimental babesiosis caused by B. microti in the immunode cient mouse model [20]. 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 [20]. 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.

Methods
Synthesis of ELQ-300 and ELQ-316 ELQ-300 and ELQ-316 were synthesized as previously described [19,21] (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.

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 with a starting percentage of parasitized erythrocytes (PPE) of 0.2. 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. Parasite cultures in the presence of DMSO (0.5 μl) and 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 ow cytometric analysis. Fresh culture medium (150 µl/well) containing respective drug concentration was replaced daily to parasite cultures. These experiments were carried out in triplicate for each tested concentration and controls, over a period of 72 h. PPE was monitored daily by ow cytometry, as previously described [22,23]. Fifty percent inhibitory concentration (IC 50 ) values were calculated for ELQ-300 and ELQ-316 at 24, 48, and 72 h by extrapolation in which there is a 50% reduction of the PPE in the wells containing the ELQs compared with the positive control wells using nonlinear regression (GraphPad Prism version 8.0.2 for Windows, San Diego, California, USA). Similarly, 100% inhibitory concentration (IC 100 ) values were also calculated at 72 h.
Flow cytometric analysis for detection of parasite growth in cultures PPE of parasite cultures was determined by ow cytometry, as previously described [26,27]. Brie y, 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% CO 2 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, the supernatant was discarded, and the cell pellet was suspended in 200 μl of fresh PBS. Then, suspended cells were analyzed by ow cytometry using a Guava® easyCyte ow 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 ow cytometric analysis. nM and 6.18 nM, respectively. Viability of bovine and horse PBMC was evaluated by monitoring cell metabolic activity using a colorimetric assay. Brie y, 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 2x10 4 cells/well in 96-well plates in complete Dulbecco's modi ed 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 h after exposure to the ELQ compounds. Absorbance at 440 nm was measured using an ELISA plate reader at 4 h 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.  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 IC 50 and IC 100 values of ELQ-300 and ELQ-316 for each parasite are shown in Table 1 [28,29]. In addition, a previous study demonstrated ELQ-300 IC 50 values of 15.4 and 23.1 nM for P. knowlsei and P. falciparum, respectively [15]. Besides the acceptable IC 50 inhibitory values found for ELQ-300, our study showed even lower ELQ-316 IC 50 values for B. bovis, B. bigemina, B. caballi, and T. equi, suggesting that these parasites are also highly susceptible to these two drugs. In addition, the IC 50 values obtained for ELQ-300 and ELQ-316 are lower than the values shown with anti-babesial drugs in recently published studies, but in the same IC 50 range of imidocarb dipropionate for the B. bovis and B. bigemina (Table S1).
Consistently, ELQ-300 and ELQ-316 completely abrogated the growth of all four parasites when tested at their respective IC 100 . The calculated IC 100 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 IC 100 value out of the four parasites tested, and appears to be the most susceptible parasite to ELQ-300. On the other hand and based on the IC 100 values (Table 1), T. equi appears to be more susceptible to ELQ-316 than the other four parasites tested in this study. Taking the IC 50 and IC 100 data together, ELQ-300 and ELQ-316 are able to e ciently inhibit the in vitro growth of B. bovis, B. bigemina, B. caballi, and T. equi blood stages. Notably, while the calculated IC 100 of T. equi is unexpectedly high (500 times higher than the IC 50 ) ( Table 1), we cannot rule out, however, the possibility that the actual concentration of the drug in the culture well was affected by poor solubility in the culture media.

Growth inhibitory effect of ELQ-300 and ELQ-316 is independent of initial parasitemia
We then tested whether the e ciency of the compounds is dependent on the parasite initial parasitemia, by comparing the effects of ELQ-300 and ELQ-316, at their respective IC 100 , 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 IC 100 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 IC 100 concentration regardless of their initial PPE at 72 h (P <0.05) (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 [29][30][31]. It was recently shown 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].  (Table S2). Overall, 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 the IC 100 doses of ELQ-300 and ELQ-316 in in vitro cultures. For the bovine PBMC experiment, ELQ-300 IC 100 of 4.3 nM and ELQ-316 of 3.92 nM were used, respectively, whereas for the equine PBMC experiment, ELQ-300 IC 100 of 5.94 nM and ELQ-316 IC 100 of 6.18 nM were used, respectively. 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, signi cant increase (P <0.05) in cell proliferation was observed in bovine and horse PBMC exposed to ConA for 24 h and 48 h, respectively ( Fig. 5A and B), indicating adequate sensitivity for the WST-1 proliferation assay used in this study. Taken together, results of cell viability revealed that ELQ-300 and ELQ-316, at their respective IC 100 , lack signi cant toxic effect on in vitro cultivated bovine and horse PBMC. Although the data presented here strongly suggest that these two drugs are appropriate candidates for the treatment of BB and EP, it needs to be pointed out that we did not assess their effect of in vivo, and that our evidence on the effectivity and safety of ELQ-300 and ELQ-316 was obtained from testing the effect of the drugs on parasites growing in culture. In addition, investigation of the mechanism of action of ELQ-300 and ELQ-316 in the parasites studied herein was also beyond the scope of this study, and it needs further examination.

Conclusions
Overall, results presented here demonstrate that both drugs tested in this study, ELQ-300 and ELQ-316, are e cient in inhibiting the growth of in vitro cultured B. bovis, B. bigemina, B. caballi and T. equi. Importantly, IC 100 doses of the ELQ drugs did not signi cantly affect the viability of in vitro cultured cattle and horse PBMC. Collectively, ndings 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.
However, it will be important to con rm their mechanisms of action, and the drugs's potential to select for resistant strains. Further studies in vivo using horses and bovines are needed to evaluate the e cacy of ELQ-300 and ELQ-316 against acute and chronic BB and EP.

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Availability of data and material
The datasets supporting the conclusions of this article are included within the article and its additional les.

Competing interests
The authors declare that they have no competing interests.