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 it varied from 0.04 to 0.37 nM, as measured at 72 h of culture (Table 1).
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 and Toxoplasma gondii tachyzoites, respectively [28, 29]. In addition, a previous study demonstrated ELQ-300 IC50 values of 15.4 and 23.1 nM for P. knowlsei and P. falciparum, respectively . 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. In addition, the IC50 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 IC50 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 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 parasite 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. Notably, while the calculated IC100 of T. equi is unexpectedly high (500 times higher than the IC50) (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 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 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 [29-31]. It was recently shown that genetic alterations in the Qi 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 . 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 Qo and Qi 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 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 IC100 doses of ELQ-300 and ELQ-316 in in vitro cultures. For the bovine PBMC experiment, ELQ-300 IC100 of 4.3 nM and ELQ-316 of 3.92 nM were used, respectively, whereas for the equine PBMC experiment, ELQ-300 IC100 of 5.94 nM and ELQ-316 IC100 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, significant 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 IC100, lack significant toxic effect on in vitro cultivated bovine and horse PBMC.