Toxoplasma gondii is an apicomplexan parasite that causes toxoplasmosis in animals and humans, a disease of high medical, veterinary and economic importance worldwide (Daher et al., 2021). T. gondii infections have been reported from most mammalian and several bird species, and approximately 25% of the human population is chronically infected with this parasite. However, only a fraction actually develops acute and thus clinical disease (Dunay et al., 2018).

Throughout its life cycle, T. gondii undergoes three distinct infectious stages: (i) a rapidly dividing tachyzoite stage that undergoes a lytic cycle characterized by invasion, intracellular proliferation within a parasitophorous vacuole, egress and infection of neighboring cells. Tachyzoites cause acute toxoplasmosis; (ii) a slowly dividing bradyzoite stage, which forms intracellular tissue cysts surrounded by a cyst wall that protects these parasites from immunological and physiological reactions on part of the hosts, and represents the chronic stage; (iii) the oocyst stage of high tenacity, which is the end-product of a sexual process taking place in the intestinal tissue of the feline definitive host, and which is released into the environment by fecal shedding and undergoes sporulation to form infective sporozoites (Robert-Gangneux and Dardé, 2012). Bradyzoites and sporozoites are orally infective, and differentiate into tachyzoites which disseminate throughout the host. In immune-competent hosts, these tachyzoites encounter a severe immunological response that initiates differentiation into bradyzoites and cyst formation. Thus, infection normally develops asymptomatically or with only mild and transient symptoms. In immune-compromised patients, however, life-threatening effects can occur, as tachyzoites do not undergo bradyzoite differentiation, and thus cause severe acute toxoplasmosis, either through recently acquired infection, or through reactivation of bradyzoites that re-differentiate into rapidly proliferating tachyzoites when immunity is impaired. An additional route of transmission is congenital infection of the placental tissue and the fetus by tachyzoites, that occurs upon primary infection of the mother during pregnancy, possibly leading to abortion, malformations, or neurological symptoms in the fetus such as hydrocephalus, often with lethal outcome. Other events include ocular toxoplasmosis, or long-term effects affecting brain functions (Attias et al., 2020; Daher et al., 2021). Besides being a significant medical problem, T. gondii causes severe diseases in pets and in small ruminants and many other food animal species, thus inflicting significant losses in livestock farming (Lindsay and Dubey, 2020). Even though the burden of toxoplasmosis is one of the highest on the list of all parasitic diseases, there is currently no efficient vaccine on the market, with the exception of a live-attenuated strain (T.gondii S48) that is marketed for sheep only (Wang et al., 2019). Existing drugs used for treatment typically include antifolates such as a combination of pyrimethamine-sulfadiazine or trimethoprim-sulfamethoxazole, and pyrimethamine combined with clindamycin, azithromycin, or atovaquone. These drugs can cause serious adverse effects and are not always efficacious (Dunay et al., 2018; Djurković-Djaković et al., 2019). In addition, currently marketed treatments are only effective against tachyzoites in the acute phase of infection (Alday and Doggett, 2017).

A promising class of compounds that are being developed for the treatment of toxoplasmosis are bumped kinase inhibitors (BKIs), a class of ATP-competitive kinase inhibitors designed to inhibit the activity of calcium-dependent protein kinase 1 (CDPK1) in a variety of apicomplexan parasite species. CDPK1 is crucially involved in host cell invasion and represents an excellent drug target, since this kinase does not exist in mammals (Billker et al., 2009; Choi et al., 2020). BKIs have been designed to fit into a hydrophobic sub-pocket of the CDPK1 ATP binding pocket, which has glycine as a small gatekeeper residue. In contrast, the bulkier gatekeeper residues in the ATP-binding pocket of mammalian kinases impair binding of BKIs, thus minimizing inhibition of kinases in the mammalian host (Van Voorhis et al., 2017). The in vitro and in vivo action of different BKIs have been investigated and several derivatives demonstrated auspicious efficacy against T. gondii and related apicomplexans (Choi et al., 2020; Van Voorhis et al., 2021), including the 5-aminopyrazole-4-carboxamide BKI-1748 (Imhof et al., 2021). BKI-1748 was shown to be safe and efficacious with IC50-values of 43 nM against T. gondii in vitro, and exhibited favorable pharmacokinetic properties after oral application (Huang et al., 2019; Imhof et al., 2021). In addition, treatment with BKI-1748 led to clear inhibition of transplacental transmission, increased pup survival and decreased cerebral infection in a pregnant toxoplasmosis mouse model based on oocyst infection (Imhof et al., 2021). BKI-treatments of T. gondii, and of the closely related Neospora caninum, demonstrated that these drugs affect host cell invasion and egress but do not act parasiticidal. Instead, they induce the formation of multinucleated complexes (MNCs) (Imhof et al., 2021; Winzer et al., 2015, 2020a, 2020b). These MNCs are schizont-like complexes composed of newly-formed zoites that are blocked in the final stage of cytokinesis. These zoites are surrounded by the inner membrane complex but lack the outer tachyzoite plasma membrane and remain intracellular and viable for extended periods of time (Imhof et al., 2021). It is anticipated that the parasite converts into the MNC-stage to evade the drug pressure and to ensure its survival. In line with this, comparative proteomics of MNCs and tachyzoites of the closely related N. caninum showed that MNCs exhibit several upregulated bradyzoite markers (Winzer et al., 2020b), and that MNC formation was a reversible process, with tachyzoites re-emerging out of the complexes and resuming proliferation after drug-discontinuation (Winzer et al., 2020a).

Artemisinin derived from the plant Artemisia annua and its derivatives (ARTs), originally derived from the plant Artemisia annua, are used as a first-line therapy against malaria, caused by Plasmodium sp. Apart from their antimalarial activity, ARTs have been repurposed as potential drugs against diseases caused by other protozoan parasites including Leishmania spp., Trypanosoma spp., Toxoplasma gondii, Neospora caninum, Eimeria tenella, Acanthamoeba castellanii, Naegleria fowleri, Cryptosporidium parvum, Giardia lamblia, and Babesia spp. (Loo et al., 2017). Two of these derivatives, namely the amino-artemisinins artemisone (GC003) and artemiside (GC008), exhibited promising activity when tested in vitro against T. gondii with IC50 values of 50–70 nM (Müller et al., 2023). Upon oral application of artemiside, this compound is rapidly metabolized to the sulfoxide artemisox and then into artemisone (Gibhard et al., 2021; Watson et al., 2021). Artemisone and artemiside have been previously assessed for efficacy against T. gondii infection, either by subcutaneous application in outbred CD1 mice infected with a sublethal dose of T. gondii tachyzoites, or in inbred C57BL/6 female gamma interferon knock-out (IFN-γ ko) mice orally infected with T. gondii tissue cysts. Results were promising in that these two compounds were able to control the infection, but they did not result in a complete cure (Dunay et al., 2009).

In contrast to BKIs that affect invasion and egress and induce MNC-formation, amino-artemisinins such as artemisone induce distinct alterations in the parasite mitochondrion, including the loss of the matrix and cristae, and swelling of the mitochondrial lumen (Müller et al., 2023). Thus, they obviously exhibit differences in their mechanisms of action. Based on these results, we formulate the hypothesis that a combined treatment with both artemisone and BKI-1748 results in synergistic effects. To test this hypothesis, we quantified the in vitro efficacies of both compounds either individually or as a combined treatment against T. gondii tachyzoites. Moreover, we investigate the impact of individual and combined treatments on the parasite morphology and ultrastructure. To verify whether the observed in vitro effects coud be reproduced in vivo, artemiside, the prodrug of artemisone, BKI-1748, and an artemiside/BKI-1748 combination treatment were assessed in a toxoplasmosis mouse model based on oocyst infection.