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

ayatsuda@fcfrp.usp.br 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 IC 50 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


Introduction
Neospora caninum is an obligate intracellular protozoan and a member of the phylum Apicomplexa. Canids are the definitive host of N. caninum, while ruminants are infected by the non-sexual forms of the parasite (Dubey & Schares, 2011;Marugan-Hernandez, 2017). In intermediate hosts, neosporosis causes abortion and impairs fertility, thus strongly affecting livestock productivity (Reichel et al., 2013).
There is no commercial strategy to control neosporosis, despite the recent advances (Anghel et al., 2018;Harmse et al., 2017;Pereira et al., 2020;Sánchez-Sánchez et al., 2018a, b) and the development of management control measures designed to reduce parasite transmission (Reichel et al., 2014). Likewise, there are few options for the treatment of human toxoplasmosis, which is usually treated with compounds (antifolates, clindamycin, and atovaquone) that are toxic, especially to pregnant women (Neville et al., 2015). On the other hand, there is an arsenal of drugs against malaria, targeted to various stages of the parasite, which were developed in response to the side effects (i.e., blue urine and sclera in methylene blue treated patients) or cases of resistance (artemisinin, pyrimethamine, chloroquine) (Bosson-Vanga et al., 2018;Luzzi & Peto, 1993;Takala-Harrison & Laufer, 2015;Wadi et al., 2018). Thus, the application of anti-malarial drugs indicates an interesting source for drug repurposing against N. caninum. For example, methylene blue and analogues, pyrimethamine and artemisinin formulations have been successfully tested on in vitro (Kim et al., 2002;Lindsay & Dubey, 1989;Pereira et al., 2017Pereira et al., , 2018 and in vivo (Pereira et al., 2020) models of N. caninum infection. Likewise, several novel candidates with anti-N. caninum activity were identified from the Malaria Venture (MMV) Pathogen Box, with promising results (Müller et al., 2017(Müller et al., , 2020. Moreover, antimalarial drugs also demonstrate activity against T. gondii (Holfels et al., 1994;Kadri et al., 2014;Kim et al., 2002;Lindsay et al., 1994;McFadden et al., 1997;Secrieru et al., 2020). As members of the same phylum (Apicomplexa), there are several similarities among N. caninum, Plasmodium and T. gondii (Morrissette & Sibley, 2002;Reid et al., 2012), which was also observed when drugs were evaluated.
In this study, the widely used antimalarials quinine, chloroquine, primaquine and atovaquone were tested against N. caninum using LacZ-tagged tachyzoites and were compared with the current information (based on previous studies) about Plasmodium and T. gondii, reinforcing the similarities and differences among them. This will underpin the development of common or exclusive therapeutic strategies based on drug repurposing.

Cytotoxicity
The cytotoxicity of the antimalarial drugs on Vero cells was evaluated by MTT assay (Mosmann, 1983). The drugs were incubated in 96-well plates for 72 h, 37 °C, 5% CO 2 on confluent monolayers of Vero cells in phenol red-free RPMI. After incubation, the supernatant was carefully discarded and the wells were incubated with 100 μL of MTT solution (500 μg/mL) for 4 h, 37 °C, followed by the dilution of formazan crystals with DMSO (Sigma). The plates were read in a spectrophotometer at 570 nm and the percentage of cytotoxicity was calculated (Pereira et al., 2017). The drugs were initially diluted at 20 μM (atovaquone), 1 mM (chloroquine), 2 mM (quinine), 1 mM (primaquine) and 1 mM (tetracycline). Three independent assays were performed for each drug.

Statistical analysis
The percentage of proliferation inhibition and toxicity were calculated using the formula ((ABS control -ABS sample / ABS control )*100), where ABS control and ABS sample represent the mean absorbance of the drug-free control and the absorbance from each drug treatment, respectively. The IC 50 (parasite inhibition) and CC 50 (Vero cell toxicity) were calculated from the proliferation/toxicity percentages using CompuSyn software (CompuSyn, 2017;Chou, 2010). The selective index (CC 50 /IC 50 ) was also calculated from the IC 50 and CC 50 values. The values are presented as the mean of three independent tests ± SD, calculated using the Graphpad Prism 5 software.

Results and Discussion
Among the tested antimalarials, atovaquone showed the lowest IC 50 for N. caninum (0.008 μM, Table 1 and Figure 1A). This concentration was similar to that reported for Plasmodium (0.0007-0.0018 μM) (Basco et al., 1995) and T. gondii (0.007-0.021 μM) (McFadden et al., 1997). Atovaquone combined with proguanil (registered as Malarone®) has been used for the treatment of uncomplicated malaria in non-endemic countries and as a preventive strategy for travelers (Thybo et al., 2004). Atovaquone has also exhibited promising results against retinochoroiditis caused by T. gondii (Harrell & Carvounis, 2014;Pearson et al., 1999). In the Plasmodium model, the molecule causes the mitochondria to collapse, inhibiting electron transport through the cytochrome bc 1 complex (Mather et al., 2005). Indeed, the mutation in the cytochrome bc1 complex is causally associated with atovaquone resistance in malaria patients (Staines et al., 2018). Despite the low CC 50 of atovaquone in Vero cells (3.3 μM), the drug is usually welltolerated (Baggish & Hill, 2002). For example, the CC 50 for HEK293T (human embryonic kidney) is 43 μM (Schuck et al., 2013), indicating a higher susceptibility of Vero cells to the drug, fact also observed in several cell lines of breast cancer (CC 50 : 11-18 μM) (Gupta & Srivastava, 2019). Indeed, there is a report of atovaquone related nephrotoxicity in allogeneic transplanted patients (Mendorf et al., 2015), indicating the susceptibility of some cells of renal origin. As observed in toxoplasmosis and malaria, atovaquone has an interesting potential against neosporosis. Further studies should elucidate the mechanisms of action.
Quinine, chloroquine and primaquine are members of the quinolone family, which have traditionally been used to treat malaria. Quinine was originally extracted from the bark of the Cinchona (quina-quina) tree, used by native inhabitants of South America for the alleviation of malaria symptoms (Achan et al., 2011). The compound Table 1. Parasite inhibitory (IC 50 ) and cytotoxic (CC 50 ) doses of antimalarials in N. caninum. Purified N. caninum (lacZ) tachyzoites were distributed in Vero cell monolayers and incubated with serial dilutions of atovaquone, chloroquine, primaquine, quinine and tetracycline. Proliferation was evaluated by CPRG. In parallel, the cytotoxicity on Vero cells was determined by MTT under the same conditions. The IC 50 and CC 50 concentrations were calculated from dose response-curves, using CompuSyn software. SI: Selectivity index. Quinine 56.6 (± 11.7) 508.5 (± 155.4) 8.9
The manipulation of the methylene blue structure generated pamaquine and quinaquine, the basic compounds for the synthesis of primaquine and chloroquine, respectively (Al-Bari, 2015). Eventually, methylene blue was replaced with chloroquine, mainly due to the absence of visible side effects (green urine and sclera) of the phenothiazinium dyes (Ginimuge & Jyothi, 2010). Moreover, no activity of methylene blue on hepatic stages of Plasmodium sp. was observed, including hypnozoites (Bosson-Vanga et al., 2018), in contrast to its effective in vitro activity against N. caninum (Pereira et al., 2017). Currently, the use of chloroquine is restricted to non-complicated malaria in regions with no prevalence of drug resistance (Mwanza et al., 2016). Primaquine is used to prevent the relapse of Plasmodium vivax and has the unique ability to eliminate the gametocyte form of Plasmodium falciparum (Ashley et al., 2014). Chloroquine susceptible strains of Plasmodium are usually inhibited in doses below 0.1 μM Chehuan et al., 2013;Fall et al., 2015). In Brazil, the recommended treatment for Plasmodium vivax (83.6% of the reported cases) is based on primaquine and chloroquine combinations to control the hypnozoite and trophozoite forms, respectively (Brasil, 2010;Negreiros et al., 2016). Chloroquine is also active against T. gondii, with an IC 50 of 2.25 μM (Kadri et al., 2014). However, chloroquine showed a robust inhibitory effect against N. caninum only at concentrations above 100 μM (Table 1 and Figure 1B). Similarly to quinine, primaquine inhibited N. caninum at 44.4 μM (Table 1 and Figure 1C), whereas no effect has been reported against T. gondii (Holfels et al., 1994). On the other hand, Plasmodium falciparum exhibits higher susceptibility to primaquine (IC 50 range; 0.46-18.9 μM) than N. caninum (Basco et al., 1999;Cabrera & Cui, 2015). Although formulations containing quinine, chloroquine or primaquine have been applied in malaria therapy for more than 50 years, the mechanisms of action are not completely established. The suggested mechanism for quinine and chloroquine is based on the prevention of heme polymerization, converting the toxic molecule to hemozoin (Sullivan et al., 1996). The mechanism of primaquine is not fully understood, but the drug probably interferes with the cellular respiration in Plasmodium, which generates oxygen free radicals and deregulates the electron transport (Fernando et al., 2011). The quinolones showed different effects on N. caninum, Plasmodium and T. gondii, indicating species-specific targets. Firstly, the erythrocyte cycle is absent in N. caninum and T. gondii, requiring future assays to elucidate the mechanism of quinine and chloroquine in coccidian members. The higher activity of quinine and primaquine in N. caninum compared to T. gondii indicates an interesting group of drugs for the control of neosporosis, with independent strategies compared to toxoplasmosis models. Moreover, there is a wide range of available quinolone derivatives (Chu et al., 2019;Gao et al., 2019;Hu et al., 2017;Wang et al., 2019), amplifying the candidate list for testing against N. caninum.
Tetracycline was active against N. caninum (IC 50 19.6 μM, Table 1 and Figure 1E and ineffective against T. gondii at concentrations above 40 μg/mL (83.1 μM) (Chang et al., 1990). Moreover, tetracycline analogues (doxycycline and minocycline) have demonstrated high activity against N. caninum, blocking 100% of the parasite proliferation at doses > 2 μM (Lindsay et al., 1994). For Plasmodium, tetracycline inhibits 50% of in vitro proliferation at concentrations below 9.8 μM (Ye & Van Dyke, 1994). The drug and derivatives (i.e., doxycycline) are applied as slow-acting blood schizonticidal agents in formulations for the treatment of uncomplicated malaria, usually in combination with quinine (Dahl et al., 2006;Gaillard et al., 2015). Tetracycline has an antagonist affinity at the 30S ribosomal subunit of prokaryotes, preventing the attachment of aminoacyl tRNA to the acceptor (A) site of the organelle (Chopra & Roberts, 2001). In Plasmodium, the inhibitory effect of tetracycline is also observed on ribosomes of the parasite apicoplast, inducing the delayed death mechanism (Fichera & Roos, 1997;Ramya et al., 2007;Uddin et al., 2017), and occurs mainly after a cycle of egress and invasion (Botté et al., 2012;Uddin et al., 2017). Apicoplast is an exclusive organelle of the phylum Apicomplexa with a prokaryotic origin and houses singular and essential metabolic pathways to the parasite's survival and virulence, representing a promising target for the control of apicomplexan diseases (Biddau & Sheiner, 2019;Dahl et al., 2006). The differential susceptibility to tetracycline suggests divergences among the apicoplast metabolism of N. caninum, Plasmodium and T. gondii (Haussig et al., 2011). Although the tetracycline delayed death process is well documented in Plasmodium, further experiments are needed to evaluate this phenomenon in N. caninum and T. gondii.
Excepting atovaquone, all antimalarials tested demonstrated low toxicity in Vero cells (CC 50 ≥ 483.5 μM) (Table 1). Chloroquine, primaquine, quinine and tetracycline usually lead to toxicity in non-tumoral lineages at concentrations above 51 μM, 395 μM, 200 μM and 225 μM, respectively (Lelièvre et al., 2012;Davanço et al., 2014;Sanders et al., 2014;Ou at al., 2019). However, we must consider that the CC 50 concentrations vary depending on the cell lineage employed (Florento et al., 2012). Therefore, novel assays focusing on the cytotoxicity on different cells lineages, especially from bovine and canine models, are mandatory to elucidate the safety of antimalarials for the control of neosporosis.
The tested antimalarials (Figure 2) exhibited several similarities and differences against N. caninum, Plasmodium and T. gondii, contributing to a specific comprehension of the metabolic mechanisms of each parasite. Our results indicate the potential of atovaquone for use in in vivo assays, as well as the demand for investigating effective analogues of chloroquine, primaquine, quinine and tetracycline. The repurposing of antimalarials against T. gondii and N. caninum is an interesting way to obtain fast and low-cost candidates, since there is a vast body of information about their efficacy and toxicity in Plasmodium models (Müller et al., 2017(Müller et al., , 2020. The comparison between these related parasites is a valid methodology, which will guide common or exclusive treatment regimens against neosporosis, malaria and toxoplasmosis. Figure 2. Schematic illustration of the antimalarial inhibition pattern on N. caninum, T. gondii and Plasmodium. Antimalarial drugs tested on N. caninum in this study (atovaquone, primaquine, tetracycline, chloroquine and quinine) were grouped according to their inhibitory pattern. Compounds with an IC 50 > 50 μm were separated from those with higher inhibitory activity (IC 50 < 50 μm). This classification was also applied to the same compounds with reported assays against T. gondii and Plasmodium. 1 McFadden et al.