Background: Bumped kinase inhibitors (BKIs) are potential drugs for neosporosis treatment in farm animals. BKI-1294 exposure results in the formation of multinucleated complexes (MNCs), which remain viable in vitro under constant drug pressure. We investigated the formation of BKI-1294 induced MNCs, the re-emergence of viable tachyzoites following drug removal, and the localization of CDPK1, the molecular target of BKIs. Methods: N. caninum tachyzoites and MNCs were studied by TEM and immunofluorescence using antibodies directed against CDPK1, and against NcSAG1 and IMC1 as markers for tachyzoites and newly formed zoites, respectively. Results: After six days of drug exposure, MNCs lacked SAG1 surface expression but remained intracellular, and formed numerous zoites incapable of disjoining from each other. Following drug removal, proliferation continued, and zoites lacking NcSAG1 emerged from the periphery of these complexes, forming infective tachyzoites after 10 days. In intracellular tachyzoites, CDPK1 was evenly distributed but shifted towards the apical part once parasites were extracellular. This shift was not affected by BKI-1294. Conclusions: CDPK1 has a dynamic distribution depending on whether parasites are located within a host cell or outside. During MNC-to-tachyzoite reconversion newly formed tachyzoites are generated directly from MNCs through zoites of unknown surface antigen composition. Further in vivo studies are needed to determine if MNCs could lead to a persistent reservoir of infection after BKI treatment.

The synthesis, characterization, and in vitro antiparasitic and anticancer activity evaluation of new conjugates containing two and three dinuclear trithiolato-bridged ruthenium(II)-arene units are presented. Antiparasitic activity was evaluated using transgenic Toxoplasmagondii tachyzoites constitutively expressing β-galactosidase grown in human foreskin fibroblasts (HFF). The compounds inhibited T.gondii proliferation with IC₅₀ values ranging from 90 to 539 nM, and seven derivatives displayed IC₅₀ values lower than the reference compound pyrimethamine, which is currently used for treatment of toxoplasmosis. Overall, compound flexibility and size impacted on the anti-Toxoplasma activity. The anticancer activity of 14 compounds was assessed against cancer cell lines A2780, A2780cisR (human ovarian cisplatin sensitive and resistant), A24, (D-)A24cisPt8.0 (human lung adenocarcinoma cells wild type and cisPt resistant subline). The compounds displayed IC₅₀ values ranging from 23 to 650 nM. In A2780cisR, A24 and (D-)A24cisPt8.0 cells, all compounds were considerably more cytotoxic than cisplatin, with IC₅₀ values lower by two orders of magnitude. Irrespective of the nature of the connectors (alkyl/aryl) or the numbers of the di-ruthenium units (two/three), ester conjugates 6–10 and 20 exhibited similar antiproliferative profiles, and were more cytotoxic than amide analogues 11–14, 23, and 24. Polynuclear conjugates with multiple trithiolato-bridged di-ruthenium(II)-arene moieties deserve further investigation.

Toxoplasma gondii is an apicomplexan parasite that infects and proliferates within many different types of host cells and infects virtually all warm-blooded animals and humans. Trypanosoma brucei is an extracellular kinetoplastid that causes human African trypanosomiasis and Nagana disease in cattle, primarily in rural sub-Saharan Africa. Current treatments against both parasites have limitations, e.g., suboptimal efficacy and adverse side effects. Here, we investigate the potential cellular and molecular targets of a trithiolato-bridged arene ruthenium complex conjugated to 9-(2-hydroxyethyl)-adenine (1), which inhibits both parasites with IC₅₀s below 10⁻⁷ M. Proteins that bind to 1 were identified using differential affinity chromatography (DAC) followed by shotgun-mass spectrometry. A trithiolato-bridged ruthenium complex decorated with hypoxanthine (2) and 2-hydroxyethyl-adenine (3) were included as controls. Transmission electron microscopy (TEM) revealed distinct ultrastructural modifications in the mitochondrion induced by (1) but not by (2) and (3) in both species. DAC revealed 128 proteins in T. gondii and 46 proteins in T. brucei specifically binding to 1 but not 2 or 3. In T. gondii, the most abundant was a protein with unknown function annotated as YOU2. This protein is a homolog to the human mitochondrial inner membrane translocase subunit Tim10. In T. brucei, the most abundant proteins binding specifically to 1 were mitochondrial ATP-synthase subunits. Exposure of T. brucei bloodstream forms to 1 resulted in rapid breakdown of the ATP-synthase complex. Moreover, both datasets contained proteins involved in key steps of metabolism and nucleic acid binding proteins.

The quinolone decoquinate (DCQ) is widely used in veterinary practice for the treatment of bacterial and parasitic infections, most notably, coccidiosis in poultry and in ruminants. We have investigated the effects of treatment of Toxoplasma gondii in infected human foreskin fibroblasts (HFF) with DCQ. This induced distinct alterations in the parasite mitochondrion within 24 h, which persisted even after long-term (500 nM, 52 days) treatment, although there was no parasiticidal effect. Based on the low half-maximal effective concentration (IC₅₀) of 1.1 nM and the high selectivity index of >5000, the efficacy of oral treatment of pregnant mice experimentally infected with T. gondii oocysts with DCQ at 10 mg/kg/day for 5 days was assessed. However, the treatment had detrimental effects, induced higher neonatal mortality than T. gondii infection alone, and did not prevent vertical transmission. Thus, three quinoline-O-carbamate derivatives of DCQ, anticipated to have better physicochemical properties than DCQ, were assessed in vitro. One such compound, RMB060, displayed an exceedingly low IC₅₀ of 0.07 nM, when applied concomitantly with the infection of host cells and had no impact on HFF viability at 10 µM. As was the case for DCQ, RMB060 treatment resulted in the alteration of the mitochondrial matrix and loss of cristae, but the changes became apparent at just 6 h after the commencement of treatment. After 48 h, RMB060 induced the expression of the bradyzoite antigen BAG1, but TEM did not reveal any other features reminiscent of bradyzoites. The exposure of infected cultures to 300 nM RMB060 for 52 days did not result in the complete killing of all tachyzoites, although mitochondria remained ultrastructurally damaged and there was a slower proliferation rate. The treatment of mice infected with T. gondii oocysts with RMB060 did reduce parasite burden in non-pregnant mice and dams, but vertical transmission to pups could not be prevented.

The apicomplexan parasite Neospora caninum is an important causative agent of congenital neosporosis, resulting in abortion, birth of weak offspring and neuromuscular disorders in cattle, sheep, and many other species. Among several compound classes that are currently being developed, two have been reported to limit the effects of congenital neosporosis: (i) bumped kinase inhibitors (BKIs) target calcium dependent protein kinase 1 (CDPK1), an enzyme that is encoded by an apicoplast-derived gene and found only in apicomplexans and plants. CDPK1 is essential for host cell invasion and egress; (ii) endochin-like quinolones (ELQs) are inhibitors of the cytochrome bc₁ complex of the mitochondrial electron transport chain and thus inhibit oxidative phosphorylation. We here report on the in vitro and in vivo activities of BKI-1748, and of ELQ-316 and its respective prodrugs ELQ-334 and ELQ-422, applied either as single-compounds or ELQ-BKI-combinations. In vitro, BKI-1748 and ELQ-316, as well as BKI-1748 and ELQ-334, acted synergistically, while this was not observed for the BKI-1748/ELQ-422 combination treatment. In a N. caninum-infected pregnant BALB/c mouse model, the synergistic effects observed in vitro were not entirely reproduced, but 100% postnatal survival and 100% inhibition of vertical transmission was noted in the group treated with the BKI-1748/ELQ-334 combination. In addition, the combined drug applications resulted in lower neonatal mortality compared to treatments with single drugs.

Tethering known drugs to a metalorganic moiety is an efficient approach for modulating the anticancer, antibacterial, and antiparasitic activity of organometallic complexes. This study focused on the synthesis and evaluation of new dinuclear ruthenium(II)–arene compounds linked to several antimicrobial compounds such as dapsone, sulfamethoxazole, sulfadiazine, sulfadoxine, triclosan, metronidazole, ciprofloxacin, as well as menadione (a 1,4-naphtoquinone derivative). In a primary screen, 30 compounds (17 hybrid molecules, diruthenium intermediates, and antimicrobials) were assessed for in vitro activity against transgenic T. gondii tachyzoites constitutively expressing β-galactosidase (T. gondii β-gal) at 0.1 and 1 µM. In parallel, the cytotoxicity in noninfected host cells (human foreskin fibroblasts, HFF) was determined by an alamarBlue assay. When assessed at 1 µM, five compounds strongly impaired parasite proliferation by >90%, and HFF viability was retained at 50% or more, and they were further subjected to T. gondii β-gal dose-response studies. Two compounds, notably 11 and 13, amide and ester conjugates with sulfadoxine and metronidazole, exhibited low IC₅₀ (half-maximal inhibitory concentration) values 0.063 and 0.152 µM, and low or intermediate impairment of HFF viability at 2.5 µM (83 and 64%). The nature of the anchored drug as well as that of the linking unit impacted the biological activity.