Buparvaquone is active against Neospora caninum in vitro and in experimentally infected mice.

The naphthoquinone buparvaquone is currently the only drug used against theileriosis. Here, the effects of buparvaquone were investigated in vitro and in an experimental mouse model for Neospora caninum infection. In 4-day proliferation assays, buparvaquone efficiently inhibited N. caninum tachyzoite replication (IC50 = 4.9 nM; IC100 = 100 nM). However, in the long term tachyzoites adapted and resumed proliferation in the presence of 100 nM buparvaquone after 20 days of cultivation. Parasiticidal activity was noted after 9 days of culture in 0.5 µM or 6 days in 1 µM buparvaquone. TEM of N. caninum infected fibroblasts treated with 1 µM buparvaquone showed that the drug acted rather slowly, and ultrastructural changes were evident only after 3-5 days of treatment, including severe alterations in the parasite cytoplasm, changes in the composition of the parasitophorous vacuole matrix and a diminished integrity of the vacuole membrane. Treatment of N. caninum infected mice with buparvaquone (100 mg/kg) either by intraperitoneal injection or gavage prevented neosporosis symptoms in 4 out of 6 mice in the intraperitoneally treated group, and in 6 out of 7 mice in the group receiving oral treatment. In the corresponding controls, all 6 mice injected intraperitoneally with corn oil alone died of acute neosporosis, and 4 out of 6 mice died in the orally treated control group. Assessment of infection intensities in the treatment groups showed that, compared to the drug treated groups, the controls showed a significantly higher parasite load in the lungs while cerebral parasite load was higher in the buparvaquone-treated groups. Thus, although buparvaquone did not eliminate the parasites infecting the CNS, the drug represents an interesting lead with the potential to eliminate, or at least diminish, fetal infection during pregnancy

Endochin-Like Quinolones Exhibit Promising Efficacy Against Neospora Caninum in vitro and in Experimentally Infected Pregnant Mice

We report on the efficacy of selected endochin-like quinolones (ELQs) against N. caninum tachyzoites grown in human foreskin fibroblasts (HFF), and in a pregnant BALB/c mouse model. Fourteen ELQs were screened against transgenic N. caninum tachyzoites expressing β-galactosidase (Nc-βgal). Drugs were added concomitantly to infection and the values for 50% proliferation inhibition (IC50) were determined after 3 days. Three compounds exhibited IC50 values below 0.1 nM, 3 ELQs had IC50s between 0.1 and 1 nM, for 7 compounds values between 1 and 10 nM were noted, and one compound had an IC50 of 22.4 nM. Two compounds, namely ELQ-316 and its prodrug ELQ-334 with IC50s of 0.66 and 3.33 nM, respectively, were previously shown to display promising activities against experimental toxoplasmosis and babesiosis caused by Babesia microti in mice, and were thus further studied. They were assessed in long-term treatment assays by exposure of infected HFF to ELQs at 0.5 μM concentration, starting 3 h after infection and lasting for up to 17 days followed by release of drug pressure. Results showed that the compounds substantially delayed parasite proliferation, but did not exert parasiticidal activities. TEM of drug treated parasites detected distinct alterations within the parasite mitochondria, but not in other parasite organelles. Assessment of safety of ELQ-334 in the pregnant mouse model showed that the compound did not interfere in fertility or pregnancy outcome. In N. caninum infected pregnant mice treated with ELQ-334 at 10 mg/kg/day for 5 days, neonatal mortality (within 2 days post partum) was found in 7 of 44 pups (15.9%), but no postnatal mortality was noted, and vertical transmission was reduced by 49% compared to the placebo group, which exhibited 100% vertical transmission, neonatal mortality in 15 of 34 pups (44%), and postnatal mortality for 18 of the residual 19 pups during the 4 weeks follow-up. These findings encourage more research on the use of ELQs for therapeutic options against N. caninum infection

Targeting of the mitochondrion by dinuclear thiolato-bridged arene ruthenium complexes in cancer cells and in the apicomplexan parasite Neospora caninum

A library of 18 dinuclear-thiolato bridged arene ruthenium complexes, some of which with demonstrated activity against cancer cells, was screened for activity against a transgenic Neospora caninum strain that constitutively expresses beta-galactosidase. Initial assessments were done at concentrations of 2500, 250, 25 and 2.5 nM, and 5 compounds were further evaluated with regard to their half maximal proliferation-inhibiting concentration (IC50). Among those, [(η6-p-MeC6H4Pri)2Ru2(μ2-SC6H4-p-CH3)3]Cl (1), [(η6-p-MeC6H4Pri)2Ru2(μ2-SC6H4-p-But)3]Cl (2) and [(η6-p-MeC6H4Pri)2Ru2(μ2-SCH2C6H4-p-But)2(μ2-SC6H4-p-OH)]BF4 (9) inhibited N. caninum proliferation with low C50 values of 15, 5 and 1 nM, respectively, while [(η6-p-MeC6H4Pri)2Ru2(μ2-SC6H4-p-OH)3]Cl (3) and [(η6-p-MeC6H4Pri)2Ru2(μ2-SC6H4-p-mco)3]Cl (5, mco = 4-methylcoumarinyl) were less active (IC50 = 280 and 108 nM, respectively). These compounds did not affect human foreskin fibroblast (HFF) host cells at dosages of 5 μM and above, but impaired proliferation of the human ovarian carcinoma cell line A2780 (IC50 values of 130 nM (1), 30 nM (2), 530 nM (3), 7730 nM (5), 130 nM (9)). A2780 cancer cells were treated with complexes 1, 2, and 5, and biodistribution analysis using inductively coupled plasma mass spectrometry (ICP-MS) showed that most of the drugs accumulated in the mitochondrial fractions. Transmission electron microscopy showed that the parasite mitochondrion is the primary target also in N. caninum tachyzoites, but these compounds, when applied at 200 nM for 15 days in vitro, did not act parasiticidal. Complexes 1, 2 and 9 applied orally at 2 and 10 mg kg−1 day−1 during 5 days in a neosporosis mouse model did not reduce parasite load and did not limit parasite dissemination to the central nervous system. In accordance with these results, ICP-MS carried out on different organs of mice orally administrated with complexes 1 and 9, demonstrated that the drugs were readily absorbed, and after 3 and 48 h, were mainly detected in liver and kidney, but were largely absent from the brain. Thus, dinuclear thiolato-bridged arene ruthenium complexes exhibit interesting activities against N. caninum in vitro, but further modifications of these promising molecules are required to improve their bioavailability and pharmacokinetic properties in order to exert a pronounced and selective effect against N. caninum in vivo

Endochin-like quinolones (ELQs) and bumped kinase inhibitors (BKIs): Synergistic and additive effects of combined treatments against Neospora caninum infection in vitro and in vivo.

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

In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum.

We report on the in vitro effects of the bumped kinase inhibitor 1294 (BKI-1294) in cultures of virulent Neospora caninum isolates Nc-Liverpool (Nc-Liv) and Nc-Spain7 and in two strains of Toxoplasma gondii (RH and ME49), all grown in human foreskin fibroblasts. In these parasites, BKI-1294 acted with 50% inhibitory concentrations (IC50s) ranging from 20 nM (T. gondii RH) to 360 nM (N. caninum Nc-Liv), and exposure of intracellular stages to 1294 led to the nondisjunction of newly formed tachyzoites, resulting in the formation of multinucleated complexes similar to complexes previously observed in BKI-1294-treated N. caninum beta-galactosidase-expressing parasites. However, such complexes were not seen in a transgenic T. gondii strain that expressed CDPK1 harboring a mutation (G to M) in the gatekeeper residue. In T. gondii ME49 and N. caninum Nc-Liv, exposure of cultures to BKI-1294 resulted in the elevated expression of mRNA coding for the bradyzoite marker BAG1. Unlike in bradyzoites, SAG1 expression was not repressed. Immunofluorescence also showed that these multinucleated complexes expressed SAG1 and BAG1 and the monoclonal antibody CC2, which binds to a yet unidentified bradyzoite antigen, also exhibited increased labeling. In a pregnant mouse model, BKI-1294 efficiently inhibited vertical transmission in BALB/c mice experimentally infected with one of the two virulent isolates Nc-Liv or Nc-Spain7, demonstrating proof of concept that this compound protected offspring from vertical transmission and disease. The observed deregulated antigen expression effect may enhance the immune response during BKI-1294 therapy and will be the subject of future studies

Giardia lamblia: missing evidence for a canonical thioredoxin system

Abstract The microaerophilic protozoan parasite Giardia lamblia occurs globally and causes dysentery in humans and animals. Since it is very sensitive to oxygen and reactive oxygen species, G. lamblia disposes over several enzymatic pathways to counter oxidative stress. One of the enzymes involved is thioredoxin reductase (TrxR), a central redox regulator that indirectly reduces peroxiredoxins via thioredoxin, an electron shuttle protein. Interestingly, the components of the TrxR-mediated redox system, including functional thioredoxins, have so far not been described despite their surmised importance for parasite survival. We aimed at filling this gap and attempted to identify functional thioredoxins and other interaction partners of TrxR in G. lamblia. To this end, we conducted database searches and expressed three recombinant candidate thioredoxins in Escherichia coli for ensuing enzyme assays. Further, co-immunoprecipitation experiments were conducted in order to identify further components of the thioredoxin redox network. Finally, the cellular localization of TrxR and peroxiredoxin 1 was determined by immunofluorescence microscopy. Surprisingly, our endeavours did not result in the identification of a functional thioredoxin or other credible interaction partners of TrxR. We, therefore, conclude that there is currently no evidence for a canonical thioredoxin redox network in G. lamblia.</jats:p

In vitro screening of the open source Pathogen Box identifies novel compounds with profound activities against Neospora caninum.

Neospora caninum is a major cause of abortion in cattle and represents an important veterinary health problem of great economic significance. The Medicines for Malaria Venture (MMV) Pathogen Box, an open-source collection of 400 compounds with proven anti-infective properties against a wide range of pathogens, was screened against a N. caninum beta-galactosidase reporter strain grown in human foreskin fibroblasts. A primary screening carried out at 1µM yielded 40 compounds that were effective against N. caninum tachyzoites. However, 30 of these compounds also affected the viability of the host cells. The 10 remaining compounds exhibited ICvalues between 4 and 43nM. Three compounds with ICvalues below 10nM, namely MMV676602, MMV688762 and MMV671636, were further characterized in vitro in more detail with respect to inhibition of invasion versus intracellular proliferation, and only MMV671636 had an impact on intracellular proliferation of tachyzoites. This was confirmed by transmission electron microscopy, showing that the primary target of MMV671636 was the mitochondrion. MMV671636 treatment of experimentally infected mice significantly reduced the number of animals with lung and brain infection, and these mice also exhibited a significantly reduced titer of antibodies directed against N. caninum antigens. Thus, MMV671636 is a promising starting point for the development of a future neosporosis therapy

In vitro effects of new artemisinin derivatives in Neospora caninum-infected human fibroblasts.

From a panel of 34 artemisinin derivatives tested in vitro, artemisone, GC007 and GC012 were most efficacious at inhibiting Neospora caninum replication (IC50 values of 3-54nM), did not notably impair the invasiveness of tachyzoites and were non-toxic for human foreskin fibroblasts (HFFs). Transmission electron microscopy of drug-treated N. caninum-infected HFFs demonstrated severe alterations in the parasite cytoplasm, changes in the composition of the matrix of the parasitophorous vacuole (PV) and diminished integrity of the PV membrane. To exert parasiticidal activity, parasites had to be cultured continuously in the presence of 5μM artemisone or GC007 for 3 weeks. N. caninum tachyzoites readily adapted to a stepwise increase in concentrations (0.5-10μM) of GC012, but not to artemisone or GC007. Drugs induced the expression of elevated levels of NcBAG1 and NcSAG4 mRNA, but only NcBAG1 could be detected by immunofluorescence. Thus, artemisinin derivatives represent interesting leads that should be investigated further

Neospora caninum: Structure and Fate of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294.

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