Evaluation of current and emerging anti-malarial medicines for inhibition of Toxoplasma gondii growth in vitro

Toxoplasma gondii is a common zoonotic infection of humans and estimates indicate that 1-2 billion people are chronically infected. Although largely asymptomatic, chronic infection poses risk of serious disease due to reactivation should immunity decline. Current therapies for toxoplasmosis only control acute infection caused by actively proliferating tachyzoites but do not eradicate the chronic tissue cyst stages. As well, there are considerable adverse side effects of the most commonly used therapy of combined sulfadiazine and pyrimethamine. Targeting the folate pathway is also an effective treatment for malaria, caused by the related parasites Plasmodium spp., suggesting common agents might be used to treat both infections. Here we evaluated currently approved and newly emerging medicines for malaria to determine if such compounds might also prove useful for treating toxoplasmosis. Surprisingly, the majority of anti-malarial compounds being used currently or in development for treatment of malaria were only modestly effective at inhibiting in vitro growth of T. gondii tachyzoites. These findings suggest that many essential processes in P. falciparum that are targeted by anti-malarial compounds are either divergent, or non-essential in T. gondii, thus limiting options for repurposing of current antimalarial medicines for toxoplasmosis.


Introduction
Toxoplasma gondii is a common parasite of animals that causes zoonotic infections in humans. It has diverged from its closest relatives by adopting a broad host range reenforced by flexible modes of transmission (1). Toxoplasma gondii is transmitted by cats, where sexual reproduction in the intestine results in shedding of highly resistant oocysts into the environment (2,3). Ingestion of oocysts by rodents, and many other intermediate hosts, results in acute infection that is characterized by initial expansion of a fast growing tachyzoite form that disseminates widely throughout the body. Following a vigorous immune response the parasite differentiates into a slow growing, semidormant form called the bradyzoite, which inhabits tissue cysts in the muscle and brain (4)(5)(6). Human infections are acquired by ingestion of oocysts that contaminate food or water, or by eating undercooked meat that harbors tissue cysts. Toxoplasma gondii is a significant cause of serious food borne infection in the USA (7), and it has also been associated with waterborne outbreaks in North and South America (8). Global serological studies indicate that ~25% of humans are chronically infected, although prevalence rates vary widely in different locations (7). Most human infections with T.
gondii are relatively benign, although they are persistent, as the chronic stages of infection (i.e. bradyzoites within tissue cysts) are not eliminated by the immune response. Additionally, toxoplasmosis can cause serious disease due to congenital infection (9) and in immuncompromised patients (10,11). Additionally, even healthy adults are at risk due to highly pathogenic strain types that are found some regions such as South America (12,13).
Toxoplasma is a member of the phylum Apicomplexa, a group of more than 10,000 known species, most of which are parasitic (14). Other apicomplexan parasites of medical importance include Plasmodium spp., the causative agent of malaria (15), and Cryptosporidium spp., a frequent cause of severe diarrheal disease in young children in developing countries (16). Apicomplexans are most closely related to ciliates and dinoflagellates, but only distantly related to humans, hence they share many key differences from their hosts (17). Although members of the phylum Apicomplexa span 400 mya of evolution (18), they contain many orthologous genes and much of their biology is conserved (19). Among their similar features, Plasmodium and Toxoplasma contain intact pathways for pyrimidine biosynthesis, while they are purine auxotrophs, and these pathways have been the focus of development of inhibitors to combat both infections (20).
Current therapies for treatment of toxoplasmosis rely primarily on inhibition of the folate pathway in the parasite, although antibiotics developed for treating bacterial infections have also been used with some success (21,22). The most commonly used treatment is a combination of sulfa drugs with pyrimethamine (i.e. sulfadiazine and pyrimethamine or trimethoprim with sulfamethoxazole). This combination is highly synergistic as the sulfa drug inhibits dihydropteroate synthase (DHPS) while pyrimethamine inhibits dihydrofolate reductase (DHFR), together disrupting tetrahydrofolate levels and blocking DNA synthesis. The combination of pyrimethamine and sulfa drugs is highly effective in blocking replication of tachyzoites but has no activity on bradyzoites within tissue cysts and therefore does not eliminate chronic infection (23). As well, there are significant adverse effects of this treatment regime due to intolerance or allergic reaction to the sulfa component and bone marrow suppression that requires co-administration of leucovorin (24,25). Additionally, due to potential for inducing developmental defects, this combination is contraindicated during the first two trimesters of pregnancy, but can be effective in reducing clinical severity when given in the third trimester (26). Although alternative therapies such as clindamycin, azithromycin, and other antibiotics, have also been used to treat acute toxoplasmosis, they also do not clear chronic infection (21).
There have been several efforts to identify new drugs for toxoplasmosis based on FDA-approved drugs. Screening of FDA approved drugs has revealed several inhibitors of tachyzoite growth in vitro, most of which were initially developed to treat inflammation (27). Guanabenz, which targets alpha-2 adrenergic receptors and is used for hypertension, additionally shows efficacy in mouse models of toxoplasmosis (28,29). Finally, treatment of infected cells with tamoxifen, an inhibitor of the estrogen receptor, leads to parasite clearance due to an autophagy-related process (30).
Although such compounds provide promising leads, they do not allow selective inhibition of the parasite since they were originally optimized to modulate host processes. Hence, there is a need for new treatments that are more selective, less toxic, and effective at eliminating chronic infection by T. gondii.
One potential source for new drugs to treat toxoplasmosis would be repurposing of medicines that have been developed for malaria, a concept that is based on their shared ancestry and similar biology. One example is the use of pyrimethamine-sulfa drug combinations to treat toxoplasmosis. Similarly, Fansidar (sulfadoxine and pyrimethamine) was historically effective against P. falciparum. However, due to the global spread of anti-folate (31) and chloroquine resistance (32), the first line of treatment for malaria has shifted to the natural product artemisinin, which is a sequiterpene lactone that contains an endoperoxide bridge that is key to its activity (33).
A number of semi-synthetic variants have been produced including artesunate, artemether, and artemisone, which are more soluble prodrug forms that are rapidly converted to dihydroartemisinin in plasma (31). Artemisinin derivatives also inhibit replication of T. gondii in vitro (34)(35)(36) and are partially effective in murine infection models, although they do not eliminate chronic infection (37,38).
Considerable effort has been expended to develop new generation anti-malarials based on large-scale phenotypic high throughout screens (HTS) for inhibition of asexual blood stage forms of P. falciparum (39). A number of the resulting hits were prioritized by the Medicines for Malaria Venture (MMV) based on chemical properties as well as activity to define a core set of compounds for inclusion in the Malaria Box and Pathogen Box projects (https://www.pathogenbox.org/ ). Combined with genomic analyses of evolved resistant mutants, these screening efforts have led to identification of new leads that target essential steps in the parasite (39). One of the first new active malarial compounds to be identified by a screening/genomics approach was the class of compounds known as spiroindolones (40), including the analog KAE609 that proved effective in curing mice of P. berghei infection with a single oral dose (41). Whole genome sequencing of resistant mutants, and subsequent genetic confirmation, indicated that spiroindolones target the cation transporter PfATP4 (42), thereby disrupting sodium transport in the parasite (43). A similar strategy of whole genome sequencing of resistant mutants has led to the identification of several tRNA synthases as targets of potent antimalarial candidate compounds and mutations in the PfCarl (cyclic amine resistance locus) gene that medicates resistance to potent imidazolopiperizines (39).
The availability of the Malaria and Pathogen Box (https://www.pathogenbox.org/ ) collections has made it possible to expand the analysis of these compounds to other pathogens (44). Analysis of compounds in the Malaria Box for inhibition of in vitro growth of T. gondii identified seven compounds with EC 50 ≤ 5 µM including a piperazine acetamide with an EC 50 < 0.19 uM (45). The hit rate of ~ 2% observed in this study is higher than typically seen in typical HTS; however, it might be considered low based on the premise that T. gondii and P. falciparum are members of the same phylum and share much of their underlying biology. Several of the active compounds contain a quinolone moiety, suggesting they may be active due to their resemblance to endochinlike quinolones (46) and atovaquone (47), which act on the bc1 complex and that are active against T. gondii. A second study that also employed the Malaria Box reported a much higher hit rate, with 49 compounds out of ~400 showing EC 50 values ≤ 1 µM when tested for inhibition of tachyzoite growth in vitro (48); the difference in hit rate being attributed to methodology. Although a number of these compounds were also inhibitors of P. falciparum, the overall correlation in potency between these two parasites was low (48). A similar screen for tachyzoite in vitro growth inhibition by compounds in the Pathogen Box identified four compounds with EC 50 values ≤ 1.0 µM and selectivity indices of > 4 (49). Among the more potent compounds identified was buparvaquone, which is a naphthoquinone that also inhibits mitochondrial electron transport. This study also reported that many compounds active against Plasmodium did not show appreciable inhibition of T. gondii.
To complement previous efforts that have focused on the early preclinical leads found in the Malaria and Pathogen Box collections, we focused here on approved medicines for malaria, new anti-malarial candidates or emerging leads that are in the global malaria portfolio, many of which with Medicines for Malaria Venture (MMV) (39,50). Many of these compounds show excellent potential for treatment of malaria, and have advanced through a number of preclinical safety checks and in some cases clinical studies, and include a number of currently used medicines. The rationale for this project was that if active compounds were found among this set, they might be readily repurposed for treatment of toxoplasmosis or give information on biological pathways to target in T. gondii.

Results and Discussion
We tested 81 compounds including a number of current medicines used for treatment of malaria and candidates that are in late preclinical development or undergoing current clinical trials. Although some individual compounds had been tested on T. gondii previously, many are new compounds and this represents the first time this set of compounds has been compared side by side in the same assay. We tested them in parallel using a multi-well plate assay that monitors in vitro growth of T. gondii tachyzoites based on firefly luciferase expression. Initially, we evaluated each of the compounds at a single concentration (10 µM) in triplicate assays to define those that showed > 50 % growth inhibition. Based on this cutoff, 52 compounds were chosen for further analysis based on duplicate 10-point dilution series that were used to define the EC 50 values for growth inhibition. Results of the screen are summarized in Table 1 and cycloguanil (55,56), consistent with previous reports ( Table 1). As expected, sulfa drugs were also less potent including sulfadiazine, sulfamethoxoazole, and dapsone (Table 1), consistent with previous reports of their in vitro activity (52,57,58). The low activity of these molecules vitro may reflect high levels of p-araminobenzoic acid in culture medium, as this metabolite acts competitively with these inhibitors of DHPS.
Although sulfa drugs are not effective when used alone, as part of the current combination therapy they are highly synergistic with pyrimethamine (52,59). Among the potent compounds that have not been reported previously was methylene blue, a phenothiazin dye that is being evaluated as a transmission blocking compound for malaria (50). Also included among the most actives were several antibiotics that target prokaryotic protein synthesis by disrupting ribosomes including the lincosamides clindamycin and mirincamycin, and the macrolide azithromycin (Table 1). Interesting, although doxycycline was active in inhibiting T. gondii growth, tetracycline showed almost no activity, consistent with a previous report (60). A number of antibiotics have previously been shown to be active on T. gondii (59,(61)(62)(63)(64) and their mechanism of action is likely due to inhibition of protein synthesis in the apicoplast (65). The main limitation to use of broad-spectrum antibiotics for treatment of toxoplasmosis is their potent activity on the endogenous bacterial flora in the microbiome leading to disbiosis and gastrointestinal distress, thus increasing the risk of C. difficile infection (66). The use of such agents that target bacteria also increases the risk of unwanted emergence of resistance among other classes of pathogens.
Other potent inhibitors include the endochin-like quinolone ELQ-300 that targets the Qi site in the cytochrome bc1 complex (67) and atovaquone that targets the Qo site (68) in the cytochrome bc1 complex of the mitochondrial respiratory chain (Table 1).
Previous studies have shown that atovaquone is effective in blocking T. gondii replication in vitro and in reducing cyst numbers in chronically infected mice (52,62), as well as suppressing reactivation of chronic infection in an immunocomprmised mouse model (69). However, prior experience with atovoquone in humans with toxoplasmosis includes several examples of therapeutic failure (47,70), possibly due to resistance arising, although the mechanism was not confirmed at the molecular level. Similar to atovoquone, ELQ-300 is potent in inhibiting parasite growth in vitro and in reducing cyst numbers in the brains of chronically infected mice (46). The main issue with ELQ-300 (46), and related quinolone compounds (71), is their low solubility that reduces oral bioavailability. Consequently, efficacy trials in murine models of toxoplasmosis have relied on parenteral administration of the compounds. This limitation has been partially mitigated by production of esterified pro-drugs that get activated in vitro, allowing for oral treatment that was protective in a mouse model for P. yoelli (72). Given that multiple quinolone containing compounds that affect mitochondrial electron transport are active against T. gondii, including against bradyzoites in tissue cysts, this pathway remains an important target for further investigation.
Drugs that have traditionally been used to treat malaria were much less potent in inhibiting T. gondii including both 4-amino and 8-amino quinolines (Table 1).
Chloroquine and a variety of related 4-aminoquniolines are active against asexual parasite stages of Plasmodium that replicate in red blood cells, where these compounds are thought to inhibit hemozoin formation within the parasite's digestive food vacuole (73). Among this class of compounds, bisquinoline and benzylquine were the most active with EC 50 values of less than 1 µM, while other derivatives were less potent against T. gondii (Table 1). These compounds are thought to target hemozoin formation during hemoglobin degradation in Plasmodium (73), and the lack of an analogous digestive pathway in T. gondii may explain the lack of potency of most members of this class. However, the fact that several 4-amino quinolones and 4-anilino compounds (i.e. pyronaridine, amodiaquine) showed modest potency in inhibiting T.
gondii, suggests that they target another important process (Table 1).
A number of 8-aminoquinolones are also effective for treatment of malaria, although their mechanism of action remains uncertain. Primaquine has traditionally been used to treat the dormant hypnozoite stage of P. vivax and it is also effective against P. falciparum gametocytes. The main deficiency of this compound is its toxicity in patients with G6PD deficiency. A number of 8-aminoquinoline derivatives, such as tafenoquine, lack some of the undesirable effects of primaquine and are also being advanced for preventing relapse of P. vivax (74). Unfortunately, the 8-aminoquinolines as a class were largely inactive against T. gondii tachyzoites (Table 1). However, given their differential activity on semi-dormant stages of Plasmodium development (i.e. gametocytes and hypnozoites) it would be interesting to test these compounds on bradyzoites of T. gondii.
Artemisinin derivatives have become the mainstay of combined therapy against severe and uncomplicated malaria (31). Artemsinin is potent across the stages of intraerythrocytic development and this activity has been attributed to hemoglobin degradation and release of free heme, which is thought to activate the endoperoxide bridge, likely forming adducts with multiple targets (33). More recent efforts have focused on completely synthetic endoperoxides, some of which show greater metabolic stability in vivo, and which could reduce reliance on the natural product produced from Artemesia cultivation (75). Consistent with prior studies (35,37), a number of artemisinin derivatives were modestly active in inhibiting T. gondii growth (Table 1, Figure 2). Artemisone and artemether were among the most active derivatives, while deoxyartemesinin was inactive, indicating that activity is dependent on the endoperoxide moiety. Unfortunately, more stable trioxolane synthetic peroxides such as OZ439 and OZ277 were much less active on T. gondii (Table 1, Figure 2). Even the most potent artemisinin derivatives are several orders of magnitude less effective against T. gondii compared to P. falciparum, thus limiting their potential as therapeutic options for toxoplasmosis.
Several different chemical scaffolds have been shown to inhibit the P-type cation translocating ATPase in P. falciparum known as PfATP4, which resides in the parasite plasma membrane where it modulates cytosolic sodium levels by active extrusion of Na + in exchange for H + (76). Among the most potent PfATP4 inhibitors are the spiroindolones that are currently in clinical trials for treatment of infection by P.
falciparum (50). The spiroindolone KAE609 (also known as NITD609), which is highly active on P. falciparum (41)(42)(43), was among the more active molecules studied here for inhibition of T. gondii growth (EC 50 ~ 0.25 µM) (Table 1, Figure 3). KAE609 was substantially more potent than a second spiroindolone analog KAF246 (EC 50 Figure 3), although these two compounds differ only by substitution of Cl for F on the indole group (Figure 3). KAE609 was previously reported to inhibit T. gondii growth in vitro with a 50% decrease in parasite growth at 1 µM (reported as MIC 50 ) and in a mouse model for acute toxoplasmosis when the compound was administered at 100 mg/kg (given orally on the day of infection and the day after infection) (77).
Compound 21A092, which belongs to a different scaffold known as a pyrazoleamide, also targets PfATP4 and is highly active on P. falciparum (78), but was much less potent on T. gondii (Table 1). Unfortunately, even potent spiroindoles like KAE609 show much greater potency on P. falciparum (EC 50 ~1 nM) (42) than T. gondii, despite the fact that the proposed binding site in PfATP4 is highly conserved, including sites that result in resistance when mutated (77). Interestingly, the relative potency between the ATP4 inhibitors (highest to lowest) KAE609, PA92 and SJ733 on P. falciparum appears to match that in T. gondii. A number of other chemical scaffolds have also been shown to affect PfATP4 in P. falciparum and a previous screen of the Malaria Box identified a number of compounds that likely target PfATP4 (76) as inhibitors of T. gondii (48). As many analogs of the spiroindolones, and other scaffolds that act on PfATP4, are available, it may be worth further investigation of this target to identify more potent inhibitors of T. gondii.

Additionally, potent new antimalarial compounds that have been recently identified
showed only modest activity against T. gondii with EC 50 values that ranged from 1 -5 µM (Table 1). Included in this group was KAF156, an imidazolopiperazine that showed promising results in a recent clinical trial of P. falciparum malaria (79). Additionally, several inhibitors of phosphoinositol 4 kinase (PI4K) showed modest (i.e. UTC944), or no appreciable (i.e. KDU691), activity against T. gondii growth, despite having excellent potency against P. falciparum (80). Similarly, cladosporin, which targets lysyl tRNA synthase in P. falciparum (81,82), showed only modest activity against T. gondii ( Table   1). The reasons for the much lower activities observed on T. gondii is uncertain but it may reflect differences in the molecular targets of these compounds or differences in the extent with which these targets play essential roles in the biology of T. gondii vs. P. falciparum. Alternatively, these differences might arise from differences in the intracellular compartment that affect access of the compounds or from a greater number of efflux mechanisms in T. gondii. Regardless of the precise reasons, these differences in sensitivity provide a rationale to explore a more diverse collection of compounds than studied here, with the potential that other analogs within these chemical scaffolds will be found to be more effective on T. gondii.
Targeting conserved and essential pathways may thus offer greater advantage for finding compounds with a broader spectrum of activity. One potential example is pyrimidine biosynthesis that is conserved in both T. gondii and P. falciparum. In particular, Plasmodium lacks pyrimidine salvage enzymes and thus is reliant on biosynthesis for RNA and DNA synthesis. Targeted screens have advanced new triazolopyrimidine compounds as inhibitors of P. falciparum dihydroorotate dehydrogenase (DHODH), thus blocking pyrimidine biosynthesis (83). One such analog DSM265 is active against both liver and blood stages of P. falciparum, shows excellent pharmacokinetic and safety properties, and has advanced to clinical trials for P. falciparum malaria (84). DHODH is also essential in for pyrimidine biosynthesis in T.
gondii, as well as performing another essential function in mitochondria (85). However, DSM265 was not effective at inhibiting T. gondii growth in vitro (Table 1). This difference may reflect that fact that DSM265 has been carefully selected for potency on P. falciparum hence this may reflect a difference in the molecular target, suggesting that other analogs may be more effective. Alternatively this result that may be due to the capacity of T. gondii to salvage uracil (86). Hence, even apparently conserved pathways present challenges for identification of potent inhibitors both due to potential molecular differences in the target and/or alternative metabolic routes in these two parasites.

Conclusions
We evaluated 80 compounds that are used as current therapy for malaria, or which are in late stage development, for their ability to inhibit the growth of the related apicomplexan parasite T. gondii. The most active compounds identified were previously known agents including lincosamide and macrolide antibiotics that target the apicoplast and quinolones that target the bc1 complex in the mitochondria. Consistent with this pattern, artemisinin and related analogs, were modestly potent, while several new generation trioxanes showed very little activity. Moreover, traditional drugs used against malaria including 4-amino and 8-aminoquinolines showed very little activity against T. gondii. Similarly, a number of newly identified compound classes that target novel pathways in Plasmodium showed limited activity against T. gondii including spiroindolones, which inhibit PfATP4, as well as compounds that target PI4K, lysyl tRNA synthase, and others. These findings may suggest that current malaria drugs target pathways that are not conserved in these two parasites, or alternatively that differences in the molecular target will require different analogs to effectively target each of these parasites. Hence, identifying new treatments for toxoplasmosis will require a concerted effort to identify potent inhibitors of essential targets in this organism.

Cell culture and parasite propagation
Tachyzoites of ME49 strain encoding a transgenic copy of firefly luciferase (type II, ME49-FLuc) (87) were continually passaged in confluent monolayers of human foreskin fibroblasts (HFF) cultured in DMEM supplemented with 10% fetal bovine serum (FBS), glutamine (10 mM) and gentamycin (10 µg/mL). To isolate parasites, heavily infected cultures of late-stage vacuoles containing replicating tachyzoites were scraped, force lysed through a 23g needle and residual host cell material removed using a polycarbonate filter (3 micron pore). The parasites were then counted, diluted in fresh culture medium and added to 96-well plates as described below. All HFF and parasite cultures were grown in a 37°C incubator supplemented with 5% CO 2 and were verified to be mycoplasma free using the e-Myco Plus kit (Intron Biotechnology).

Statistics
All results are presented as the average of two or more biological replicates. Linear regression analysis and dose-response inhibition (Log (inhibitor) vs. normalized response -variable slope) or (Log (inhibitor) vs. normalized response) were performed in Prism 7 (GraphPad Software, Inc.).

Ancillary Information
Supporting Information:  Compounds of interest are highlighted including pyrimethamine (red dot), artemisinin and related compounds (yellow), inhibitors of PfATP4 (orange), and antibiotics (green).
See Table 1 for complete EC 50 values.  Table 1 for complete EC 50 values.