Atovaquone, chloroquine, primaquine, quinine and tetracycline: antiproliferative effects of relevant antimalarials on Neospora caninum

Abstract

Neospora caninum is an apicomplexan parasite that causes abortion in cattle, resulting in significant economic losses. There is no commercial treatment for neosporosis, and drug repositioning is a fast strategy to test possible candidates against N. caninum. In this article, we describe the effects of atovaquone, chloroquine, quinine, primaquine and tetracycline on N. caninum proliferation. The IC50 concentrations in N. caninum were compared to the current information based on previous studies for Plasmodium and Toxoplasma gondii, correlating to the described mechanisms of action of each tested drug. The inhibitory patterns indicate similarities and differences among N. caninum, Plasmodium and T. gondii. For example, atovaquone demonstrates high antiparasitic activity in all the analyzed models, while chloroquine does not inhibit N. caninum. On the other hand, tetracycline is effective against Plasmodium and N. caninum, despite its low activity in T. gondii models. The repurposing of antimalarial drugs in N. caninum is a fast and inexpensive way to develop novel formulations using well-established compounds.

Construction of luciferase-expressing Neospora caninum and drug screening

“…Consistent with previously published studies [22][23], atovaquone had an IC 50 value of 0.004 ± 0.0007 µM and pyrimidine had an IC 50 value of 0.314 ± 0.1040 µM. To assess the cytotoxicity of these drugs on HFF cells, dilutions of the drugs were sequentially added to the HFF cells and cell viability was determined using CCK-8 reagent.…”

“…The results show that atovaquone and pyrimethamine have dose-dependent inhibitory effects on Nc1-Luc, which can be re ected by a reduced level of luminescent signaling. Pereira et al [22][23] demonstrated that atovaquone and pyrimethamine inhibited the growth of N. caninum with IC 50 values of 0.008 ± 0.002 µM and 0.312 ± 0.017µM, respectively. This is consistent with our ndings, in which atovaquone and pyrimethamine inhibited Nc1-Luc strains with IC 50 values of 0.004 ± 0.0007 µM and 0.314 ± 0.1040 µM, respectively, demonstrating the high susceptibility of Nc1-Luc strains to drug evaluation.…”

“…This is consistent with our ndings, in which atovaquone and pyrimethamine inhibited Nc1-Luc strains with IC 50 values of 0.004 ± 0.0007 µM and 0.314 ± 0.1040 µM, respectively, demonstrating the high susceptibility of Nc1-Luc strains to drug evaluation. Moreover, Pereira et al [22][23] used the N. caninum expressing β-galactosidase (Nc1-LacZ), which required incubation of the lysates with the substrate for 4 h, whereas our Nc1-Luc strain could be detected immediately after the lysates were mixed with the substrate. Therefore, this method using luciferase reduces the time required to screen compounds against N. caninum in vitro.…”

“…Therefore, we next focused on testing the Nc1-Luc strain as a tool to evaluate the e cacy of anti-N. caninum therapeutics. Since atovaquone and pyrimethamine have been shown to effectively inhibit the growth of N. caninum [22,23], penicillin is ineffective against protozoa [24]. We selected atovaquone and pyrimethamine as positive drugs and penicillin G potassium as a negative drug to test their inhibitory effects on N. caninum using the Nc1-Luc strain.…”