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

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. 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. 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. 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 those of genera 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]. As a general rule, Babesia and Theileria are not eliminated in surviving animals and therefore can 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]. The threat of EP imposes severe and costly restrictions in the transportation of high-performance horses between endemic and non-endemic areas for participation 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].
Babesia bovis and B. bigemina are two main causative agents of bovine babesiosis (BB), an acute and persistent economically important disease of cattle that typically causes 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 the cytoadhesion of parasite-infected RBC in the brain capillaries, similar to that seen in 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 animals aged < 1 year 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 losses due to babesiosis in adult vaccine-susceptible animals that need to be transported from non-endemic Babesia areas to endemic ones. 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 first-choice methods to manage animals with acute BB and EP [10,11]. 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 in attempts to achieve clearance of the parasites, which is a usual occurrence for valuable horses affected by EP [12]. In addition to toxic side effects, and although specific resistance to imidocarb by Babesia and Theileria parasites has not yet been 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) compounds 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 been shown to be highly effective against different species and multiple stages of Plasmodium [18,19]. Importantly, the ELQ-300 and ELQ-316 compounds have been selected as preclinical anti-malaria candidates based on their reasonable oral bioavailability at efficacious doses, long half-life and metabolic stability [18,19]. A recent study also demonstrated the efficacy of ELQ prodrugs combined with atovaquone to treat experimental babesiosis caused by Babesia microti in the immunodeficient mouse model [20]. Data from the study reported here showed that the combined therapy of ELQ and atovaquone resulted in complete clearance of the parasite, with no disease recrudescence even > 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.

Synthesis of ELQ-300 and ELQ-316
The ELQ-300 and ELQ-316 compounds were synthesized as previously described [19,21] (Additional file 1: Figure 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 to be > 99% by proton-nuclear magnetic resonance spectroscopy 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 room temperature until use. Working solutions were freshly prepared in parasite culture medium before being added 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 in culture media containing different concentrations of ELQ-300 or ELQ-316 (range 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 flow cytometric analysis. Fresh culture medium (150 µl/well) containing the 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 flow cytometry, as previously described [22,23]. The half maximal (50%) inhibitory concentration (IC 50 ) values were calculated for ELQ-300 and ELQ-316 at 24, 48, and 72 h of incubation by extrapolation, with a 50% reduction of the PPE in the wells containing the ELQs compared with the positive control wells using nonlinear regression (GraphPad Prism ver. 8.0.2 for Windows; Graphpad Software Inc., San Diego, CA, USA). Similarly, maximal (100%) inhibitory concentration (IC 100 ) values were also calculated at 72 h.

Flow cytometric analysis for detection of parasite growth in cultures
The PPE of parasite cultures was determined by flow cytometry, as previously described [26,27]. Briefly, 5 µl of the cultures was collected from the bottom of the wells and centrifuged at 450 g for 1 min at 4 ℃. The supernatant was discarded and the cell pelle washed twice with 150 μl of phosphate buffer saline (PBS) pH 7.2, following which the cell pellet was suspended in 200 μl of 25 μg/ μl hydroethidine (HE) (Invitrogen, Carlsbad, CA, USA), incubated in 5% CO 2 in an incubator at 37 ℃ for 20 min in the dark and ten washed twice with 200 μl of PBS to remove the excess HE. The supernatant was then discarded, and the cell pellet was suspended in 200 μl of fresh PBS. The suspended cells were analyzed by flow cytometry using a Guava ® easyCyte flow cytometer (Luminex Corp., Austin, TX, USA) at a ratio of 800-1000 cells/µl with 20,000 events collected. The results were analyzed by FCS Express v6 (De Novo Software, Glendale, CA, USA). 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 100 on parasite growth
In vitro growth inhibition assays were performed over a period of 8 days using a starting PPE of 0.2 and 2%. Parasites were grown in the presence of calculated ELQ-300 or ELQ-316 IC 100 values. Cultures growing in medium in the absence of the ELQs and non-infected RBC maintained in medium only were used as positive and negative controls, respectively. Culture medium containing the respective compound concentrations was replaced daily, 150 µl medium per well, for a period of 72 h, following which the parasites were cultivated in media only and split every 48 h for a period of 8 days. The PPE was evaluated at 24, 48, and 72 h, and 8 days of culture by flow cytometry.

Cytotoxicity assay
Cytotoxicity of ELQ-300 and ELQ-316 in ex vivo peripheral blood mononuclear cells (PBMC) of bovine and equine were examined by exposing cells to the compounds at their calculated IC 50 and IC 100 . For the bovine PBMC experiment, ELQ-300 IC 50 and IC 100 were 0.56 nM and 4.3 nM, respectively. And for the ELQ-316 IC 50 and IC 100 were 0.07 nM and 3.92 nM, respectively. For the equine PBMC experiment, ELQ-300 IC 50 and IC 100 were 0.23 and 5.94 nM, respectively, and ELQ-316 IC 50 and IC 100 were 0.11 and 6.18 nM, respectively. Viability of bovine and horse PBMC was evaluated by monitoring cell metabolic activity using a colorimetric assay. Briefly, peripheral blood was collected from healthy cattle and horses via jugular venipuncture into Vacutainer ® tubes containing ACD (acid citrate dextrose) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and PBMC were isolated using Histopaque ® (Sigma-Aldrich) per standard protocol. Cells were then plated at 2 × 10 4 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 Applied Science, Penzberg, Germany) was added to the cell cultures following the manufacturer's protocol at 24, 48, and 72 h after exposure to the ELQ compounds. Absorbance at 440 nm was measured using an enzyme-linked immunosorbent assay (ELISA) plate reader at 4 h after adding the WST-1 reasgent 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-Aldrich) and Draxxin ® [22] were used as a positive control.

Statistical analysis
Growth of parasites in culture was analyzed using oneway analysis of variance (GraphPad Prism version 8.0.2 for Windows; Graphpad Software Inc.). Values of P < 0.05 were considered to be statistically significant in terms of the effect of the ELQs on the parasite growth. Significant differences in PBMC viability were measured by Student's t-test, and P values < 0.05 were considered to be significant.

Ethical Statement
The in vitro cultures of B. caballi, T. equi, B. bovis and B. bigemina require erythrocyte and serum equine and cattle donors. The protocols used for the bleeding of the horse and cattle donors for were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Idaho (Project title: Bovine and Equine Bleeding, UI IACUC # 2020-42).

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 (Figs. 1a-d, 2a-d). In addition, the inhibitory effect of ELQ-300 and ELQ-316 was found to be dose-dependent for all four parasites tested. The calculated IC 50 and IC 100 values of ELQ-300 and ELQ-316 for each parasite are shown in Table 1. Overall, comparisons of the IC 50 values among all parasites tested indicate increased susceptibility to ELQ-316 compared to ELQ-300. The ELQ-316 IC 50 varied from 0.002 to 0.1 nM, while the ELQ-300 IC 50 varied from 0.04 to 0.37 nM, as measured at 72 h of culture (Table 1).
Interestingly, our calculated values of IC 50 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 IC 50 values of 7.97, 0.66 and 0.35 nM were established for Besnoitia besnoiti and Toxoplasma gondii tachyzoites, respectively [28,29]. In addition, a previous study demonstrated ELQ-300 IC 50 values of 15.4 and 23.1 nM for Plasmodium knowlsei and P. falciparum, respectively [15]. In addition to 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. The IC 50 values obtained for ELQ-300 and ELQ-316 are also lower than those shown with anti-babesial drugs in recently published studies, but in the same IC 50 range of imidocarb dipropionate for B. bovis and B. bigemina (Additional file 2: Table S1).
ELQ-300 and ELQ-316 consistently 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 of the four parasites tested and thus 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, we conclude that 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. Notably, while the calculated IC 100 of T. equi is unexpectedly high (500-fold higher than the IC 50 ) ( Table 1), we cannot rule out 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 efficiency 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 ELQ-300 IC 100 , regardless of their initial PPE (P < 0.05) (Fig. 3a, c, d). Nonetheless, the addition of ELQ-300 to B. bigemina cultures at 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 in which the parasites were grown in culture in the presence of ELQ-300 for 3 days, following which the cultures were split 1:10 and maintained in media free of the drug for an additional 5 days of culture. 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,  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 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, with the exception of 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 to the ELQ drugs. However, it may be speculated that such susceptibility  bovis, b B. bigemina c B. caballi, d Theilera equi. "0" represents parasites in the absence of ELQ-300. Assays were carried out in triplicate, and the error bars indicate standard error deviation for each ELQ-300 concentration tested. Asterisk (*) represents statistically significant difference between cultures grown without and with ELQ-300 at P < 0.05, using Student's t-test. nRBC Non-infected erythrocytes, PPE percentage of parasitized erythrocytes can be due to variations/mutations in the cytochrome bc 1 target sequence that affect ELQ binding, differential uptake or elimination of the drugs or a combination of these factors (Additional file 3: Figure S2) [29][30][31]. It was recently shown that genetic alterations in the Q i binding site of the cytochrome bc 1 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) (Additional file 2: Table S2). Overall, the results presented here set the rationale for further studies to alter and/or knockdown 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  B. bovis, b B. bigemina, c B. caballi, d T. equi. "0" represents parasites in the absence of ELQ-316. Assays were carried out in triplicate, and the error bars indicate standard error deviation for each ELQ-316 concentration tested. Asterisk (*) represents statistically significant difference between cultures grown without and with ELQ-316 at P < 0.05, using Student's t-test  (Fig. 5a, b). In addition, a significant increase (P <0.05) in cell proliferation was observed in bovine and horse PBMC exposed to ConA for 24 and 48 h, respectively (Fig. 5a, b), indicating adequate sensitivity for the WST-1 proliferation assay used in this study. Taken together, results of the cell viability study revealed that ELQ-300 and ELQ-316, at their respective IC 100 , lack a significant toxic effect on bovine and horse PBMC cultured in vitro. 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 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 this aspect needs further examination.

Conclusions
Overall, the results presented here demonstrate that both of the 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, the IC 100 doses of the ELQ drugs did not significantly affect the viability of in vitro-cultured cattle and horse PBMC. Collectively, the 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. However, it will be important to confirm their mechanisms of action, as well as the drugs's potential to select for resistant strains.
Further studies in vivo in horses and bovines are needed to evaluate the efficacy of ELQ-300 and ELQ-316 against acute and chronic BB and EP. Bovine and horse PBMC assays were carried out in triplicate and the error bars indicate standard error deviation. Asterisk (*) represents statistically significant differences compared to PBMC cultivated in medium only at P < 0.05, using Student's t-test