Assessment of the Activity of Decoquinate and Its Quinoline-O-Carbamate Derivatives against Toxoplasma gondii In Vitro and in Pregnant Mice Infected with T. gondii Oocysts.

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.

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 activity, safety and in vivo efficacy of the novel bumped kinase inhibitor BKI-1748 in non-pregnant and pregnant mice experimentally infected with Neospora caninum tachyzoites and Toxoplasma gondii oocysts.

Bumped kinase inhibitors (BKIs) target the apicomplexan calcium-dependent protein kinase 1 (CDPK1). BKI-1748, a 5-aminopyrazole-4-carboxamide compound when added to fibroblast cells concomitantly to the time of infection, inhibited proliferation of apicomplexan parasites at EC50s of 165 nM (Neospora caninum) and 43 nM (Toxoplasma gondii). Immunofluorescence and electron microscopy showed that addition of 2.5 µM BKI-1748 to infected HFF monolayers transformed parasites into multinucleated schizont-like complexes (MNCs) containing newly formed zoites, which were unable to separate and form infective tachyzoites or undergo egress. In zebrafish (Danio rerio) embryo development assays, no embryonic impairment was detected within 96 h at BKI-1748 concentrations up to 10 µM. In pregnant mice, BKI-1748 applied at days 9-13 of pregnancy at a dose of 20 mg/kg/day was safe and no pregnancy interference was observed. The efficacy of BKI-1748 was assessed in standardized pregnant mouse models infected with N. caninum (NcSpain-7) tachyzoites or T. gondii (TgShSp1) oocysts. In both models, treatments resulted in increased pup survival and profound inhibition of vertical transmission. However, in dams and non-pregnant mice, BKI-1748 treatments resulted in significantly decreased cerebral parasite loads only in T. gondii infected mice. In the T. gondii-model, ocular infection was detected in 10 out of 12 adult mice of the control group, but only in 3 out of 12 mice in the BKI-1748-treated group. Thus, TgShSp1 oocyst infection is a suitable model to study both cerebral and ocular infection by T. gondii. BKI-1748 represents an interesting candidate for follow-up studies on neosporosis and toxoplasmosis in larger animal models.

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.

Activities of Endochin-Like Quinolones Against in vitro Cultured Besnoitia besnoiti Tachyzoites.

Endochin-like quinolones (ELQs) potently inhibit the proliferation of Plasmodium, Toxoplasma, Neospora, and Babesia by targeting the cytochrome b Qo and Qi sites and interfering with oxidative phosphorylation and pyrimidine biosynthesis. The activities of 14 different ELQs were assessed against B. besnoiti tachyzoites grown in human foreskin fibroblasts (HFF) by quantitative real time PCR. The values for 50% proliferation inhibition (IC50) of five ELQs were determined in a 3-days growth assay after an initial screen of 12 ELQs at 0.01, 0.1, and 1 µM. The IC50s of ELQ-121, -136, and -316 were 0.49, 2.36, and 7.97 nM, respectively. The IC50s of ELQs tested against B. besnoiti were higher than IC50s previously observed for P. falciparum and T. gondii. However, the B. besnoiti cytochrome b sequence and the predicted Qo and Qi ELQ binding sites in the Toxoplasma, Neospora, and Besnoitia cytochrome b are virtually identical, suggesting that the differences in ELQ susceptibility are not due to variations in the substrate binding sites. TEM of ELQ-treated parasites primarily demonstrated alterations within the parasite mitochondrion, profound thickening of the nuclear membrane, as well as increased vacuolization within the tachyzoite cytoplasm. Long-term treatment assays of intracellular B. besnoiti with ELQs for up to 20 days followed by the release of drug pressure caused a substantial delay in parasite growth and proliferation while ELQs were present, but parasite proliferation resumed days after ELQs were removed. Interestingly, structural alterations persisted after ELQ removal and parasite proliferation was slowed. These findings provide a basis for further in vivo studies of ELQs as therapeutic options against B. besnoiti infection.

Anti-parasitic dinuclear thiolato-bridged arene ruthenium complexes alter the mitochondrial ultrastructure and membrane potential in Trypanosoma brucei bloodstream forms.

Trypanosoma brucei causes human African trypanosomiasis and Nagana disease in cattle, imposing substantial medical and economic burden in sub-Saharan Africa. The current treatments have limitations, including the requirement for elaborated protocols, development of drug resistance, and they are prone to adverse side effects. In vitro screening of a library of 14 dinuclear-thiolato bridged arene ruthenium complexes, originally developed for treatment of cancer cells, resulted in the identification of 7 compounds with IC50 values ranging from 3 to 26 nM. Complex [(η6-p-MeC6H4Pri)2Ru2(µ2-SC6H4-o-Pri)3]Cl (2) (IC50 = 4 nM) and complex [(η6-p-MeC6H4Pri)2Ru2(µ2-SCH2C6H4-p-But)2(µ2-SC6H4-p-OH)]BF4(9) (IC50 = 26 nM) were chosen for further assessments. Application of complex 2 and 9 at 20 nM and 200 nM, respectively, for 4.5 h induced alterations in the trypanosome mitochondrion as evidenced by immunofluorescence employing an antibody against mitochondrial Hsp70 and Mitotracker labeling. Transmission electron microscopy of parasites taken at 2 and 4h of treatment demonstrated massive alterations in the mitochondrial ultrastructure, while other organelles and structural elements of the parasites remained unaffected. Complex 2 treated trypanosomes exhibited a distorted mitochondrial membrane, and the mitochondrial matrix was transformed into an amorphous mass with different degrees of electron densities. Complex 9 did not notably impair the integrity of the membrane, but the interior of the mitochondrion appeared either completely translucent, or was filled with filamentous structures of unknown nature. Dose- and time-dependent effects of these two compounds on the mitochondrial membrane potential were detected by tetramethylrhodamine ethyl ester assay. Thus, the mitochondrion and associated metabolic processes are an important target of dinuclear thiolato-bridged arene ruthenium complexes in T. brucei.

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 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 ß-galactosidase (Nc-ßgal). 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.

Repurposing of commercially available anti-coccidials identifies diclazuril and decoquinate as potential therapeutic candidates against Besnoitia besnoiti infection.

Repurposing of currently marketed compounds with proven efficacy against apicomplexan parasites was used as an approach to define novel candidate therapeutics for bovine besnoitiosis. Besnoitia besnoiti tachyzoites grown in MARC-145 cells were exposed to different concentrations of toltrazuril, diclazuril, imidocarb, decoquinate, sulfadiazine and trimethoprim alone or in combination with sulfadiazine. Drugs were added either just prior to infection of MARC-145 cells (0 h post infection, hpi) or at 6 hpi. A primary evaluation of drug effects was done by direct immunofluorescence staining and counting. Potential effects on the host cells were assessed using a XTT kit for cell proliferation. Compounds displaying promising efficacy were selected for IC50 and IC99 determination by qPCR. In addition, the impact of drugs on the tachyzoite ultrastructure was assessed by TEM and long-term treatment assays were performed. Cytotoxicity assays confirmed that none of the compounds affected the host cells. Decoquinate and diclazuril displayed invasion inhibition rates of 90 and 83% at 0 h pi and 73 and 72% at 6 h pi, respectively. The remaining drugs showed lower efficacy and were not further studied. Decoquinate and diclazuril exhibited IC99 values of 100 nM and 29.9 µM, respectively. TEM showed that decoquinate primarily affected the parasite mitochondrium, whilst diclazuril interfered in cytokinesis of daughter zoites. The present study demonstrates the efficacy of diclazuril and decoquinate against B. besnoiti in vitro and further assessments of safety and efficacy of both drugs should be performed in the target species.

In vitro efficacy of bumped kinase inhibitors against Besnoitia besnoiti tachyzoites.

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a chronic and debilitating disease that causes systemic and skin manifestations and sterility in bulls. Neither treatments nor vaccines are currently available. In the search for therapeutic candidates, calcium-dependent protein kinases have arisen as promising drug targets in other apicomplexans (e.g. Neospora caninum, Toxoplasma gondii, Plasmodium spp. and Eimeria spp.) and are effectively targeted by bumped kinase inhibitors. In this study, we identified and cloned the gene coding for BbCDPK1. The impact of a library of nine bumped kinase inhibitor analogues on the activity of recombinant BbCDPK1 was assessed by luciferase assay. Afterwards, those were further screened for efficacy against Besnoitiabesnoiti tachyzoites grown in Marc-145 cells. Primary tests at 5µM revealed that eight compounds exhibited more than 90% inhibition of invasion and proliferation. The compounds BKI 1294, 1517, 1553 and 1571 were further characterised, and EC99 (1294: 2.38µM; 1517: 2.20µM; 1553: 3.34µM; 1571: 2.78µM) were determined by quantitative real-time polymerase chain reaction in 3-day proliferation assays. Exposure of infected cultures with EC99 concentrations of these drugs for up to 48h was not parasiticidal. The lack of parasiticidal action was confirmed by transmission electron microscopy, which showed that bumped kinase inhibitor treatment interfered with cell cycle regulation and non-disjunction of tachyzoites, resulting in the formation of large multi-nucleated complexes which co-existed with viable parasites within the parasitophorous vacuole. However, it is possible that, in the face of an active immune response, parasite clearance may occur. In summary, bumped kinase inhibitors may be effective drug candidates to control Besnoitiabesnoiti infection. Further in vivo experiments should be planned, as attainment and maintenance of therapeutic blood plasma levels in calves, without toxicity, has been demonstrated for BKIs 1294, 1517 and 1553.

The single cyclic nucleotide-specific phosphodiesterase of the intestinal parasite Giardia lamblia represents a potential drug target.

BACKGROUND: Giardiasis is an intestinal infection correlated with poverty and poor drinking water quality, and treatment options are limited. According to the Center for Disease Control and Prevention, Giardia infections afflict nearly 33% of people in developing countries, and 2% of the adult population in the developed world. This study describes the single cyclic nucleotide-specific phosphodiesterase (PDE) of G. lamblia and assesses PDE inhibitors as a new generation of anti-giardial drugs. METHODS: An extensive search of the Giardia genome database identified a single gene coding for a class I PDE, GlPDE. The predicted protein sequence was analyzed in-silico to characterize its domain structure and catalytic domain. Enzymatic activity of GlPDE was established by complementation of a PDE-deficient Saccharomyces cerevisiae strain, and enzyme kinetics were characterized in soluble yeast lysates. The potency of known PDE inhibitors was tested against the activity of recombinant GlPDE expressed in yeast and against proliferating Giardia trophozoites. Finally, the localization of epitope-tagged and ectopically expressed GlPDE in Giardia cells was investigated. RESULTS: Giardia encodes a class I PDE. Catalytically important residues are fully conserved between GlPDE and human PDEs, but sequence differences between their catalytic domains suggest that designing Giardia-specific inhibitors is feasible. Recombinant GlPDE hydrolyzes cAMP with a Km of 408 µM, and cGMP is not accepted as a substrate. A number of drugs exhibit a high degree of correlation between their potency against the recombinant enzyme and their inhibition of trophozoite proliferation in culture. Epitope-tagged GlPDE localizes as dots in a pattern reminiscent of mitosomes and to the perinuclear region in Giardia. CONCLUSIONS: Our data strongly suggest that inhibition of G. lamblia PDE activity leads to a profound inhibition of parasite proliferation and that GlPDE is a promising target for developing novel anti-giardial drugs.

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 IC50 values between 4 and 43nM. Three compounds with IC50 values 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.

Characterization of the Activities of Dinuclear Thiolato-Bridged Arene Ruthenium Complexes against Toxoplasma gondii.

The in vitro effects of 18 dinuclear thiolato-bridged arene ruthenium complexes (1 monohiolato compound, 4 dithiolato compounds, and 13 trithiolato compounds), originally designed as anticancer agents, on the apicomplexan parasite Toxoplasma gondii grown in human foreskin fibroblast (HFF) host cells were studied. Some trithiolato compounds exhibited antiparasitic efficacy at concentrations of 250 nM and below. Among those, complex 1 and complex 2 inhibited T. gondii proliferation with 50% inhibitory concentrations (IC50s) of 34 and 62 nM, respectively, and they did not affect HFFs at dosages of 200 µM or above, resulting in selectivity indices of >23,000. The IC50s of complex 9 were 1.2 nM for T. gondii and above 5 µM for HFFs. Transmission electron microscopy detected ultrastructural alterations in the matrix of the parasite mitochondria at the early stages of treatment, followed by a more pronounced destruction of tachyzoites. However, none of the three compounds applied at 250 nM for 15 days was parasiticidal. By affinity chromatography using complex 9 coupled to epoxy-activated Sepharose followed by mass spectrometry, T. gondii translation elongation factor 1α and two ribosomal proteins, RPS18 and RPL27, were identified to be potential binding proteins. In conclusion, organometallic ruthenium complexes exhibit promising activities against Toxoplasma, and the potential mechanisms of action of these compounds as well as their prospective applications for the treatment of toxoplasmosis are discussed.

Two Novel Calcium-Dependent Protein Kinase 1 Inhibitors Interfere with Vertical Transmission in Mice Infected with Neospora caninum Tachyzoites.

We present the effects of two novel bumped kinase inhibitors, BKI-1517 and BKI-1553, against Neospora caninum tachyzoites in vitro and in experimentally infected pregnant mice. These compounds inhibited tachyzoite proliferation of a transgenic beta-galactosidase reporter strain cultured in human foreskin fibroblasts with 50% inhibitory concentrations (IC50s) of 0.05 ± 0.03 and 0.18 ± 0.03 µM, respectively. As assessed by an alamarBlue assay, fibroblast IC50s were above 20 µM; however, morphological changes occurred in cultures treated with >5 µM BKI-1517 after prolonged exposure (>6 days). Treatment of intracellular tachyzoites with 5 µM BKI-1553 for 6 days inhibited endodyogeny by interfering with the separation of newly formed zoites from a larger multinucleated parasite mass. In contrast, parasites treated with 5 µM BKI-1517 did not form large complexes and showed much more evidence of cell death. However, after a treatment duration of 10 days in vitro, both compounds failed to completely prevent the regrowth of parasites from culture. BALB/c mice experimentally infected with N. caninum Spain7 (Nc-Spain7) and then treated during 6 days with BKI-1517 or BKI-1553 at different dosages showed a significant reduction of the cerebral parasite load. However, fertility was impaired by BKI-1517 when applied at 50 mg/kg of body weight/day. At 20 mg/kg/day, BKI-1517 significantly inhibited the vertical transmission of N. caninum to pups and increased the rate of survival of offspring. BKI-1553 was less detrimental to fertility and also provided significant but clearly less pronounced protection of dams and offspring. These results demonstrate that, when judiciously applied, this compound class protects offspring from vertical transmission and disease.

N-terminal fusion of a toll-like receptor 2-ligand to a Neospora caninum chimeric antigen efficiently modifies the properties of the specific immune response.

Immunoprophylactic products against neosporosis during pregnancy should induce an appropriately balanced immune response. In this respect, OprI, a bacterial lipoprotein targeting toll like receptor (TLR)2, provides promising adjuvant properties. We report on the manipulation of the innate and the T-cell immune response through the fusion of OprI with the Neospora caninum chimeric protein Mic3-1-R. In contrast to Mic3-1-R, OprI-MIC3-1-R significantly activated bone-marrow dendritic cells from naïve mice. Mice immunized with OprI-Mic3-1-R induced an immune response with mixed T helper (Th)1 and Th2 properties (high levels of both immunoglobulin (Ig)G1 and IgG2a and of interleukin (IL)-10, IL-12(p70) and interferon-γ responses) whereas Mic3-1-R+saponin induced a clear Th2-biased response (low IgG2a and high IL-4 and IL-10). After mating and challenge with N. caninum, increased expression of interferon-γ was only found in placentas from OprI-Mic3-1-R immunized dams. However, no protection against vertical transmission and neonatal mortality was observed in either of the two groups. These results indicated that more exhaustive studies must be done to elucidate the immune mechanisms associated with transplacental transmission. Antigen linkage to TLR2-ligands, such as OprI, is a useful tool to investigate this enigma by reorienting the innate and adaptive immune responses against other candidate antigens in future studies.

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.

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.

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.

Catechol pyrazolinones as trypanocidals: fragment-based design, synthesis, and pharmacological evaluation of nanomolar inhibitors of trypanosomal phosphodiesterase B1.

Trypanosomal phosphodiesterases B1 and B2 (TbrPDEB1 and TbrPDEB2) play an important role in the life cycle of Trypanosoma brucei, the causative parasite of human African trypanosomiasis (HAT), also known as African sleeping sickness. We used homology modeling and docking studies to guide fragment growing into the parasite-specific P-pocket in the enzyme binding site. The resulting catechol pyrazolinones act as potent TbrPDEB1 inhibitors with IC50 values down to 49 nM. The compounds also block parasite proliferation (e.g., VUF13525 (20b): T. brucei rhodesiense IC50 = 60 nM, T. brucei brucei IC50 = 520 nM, T. cruzi = 7.6 µM), inducing a typical multiple nuclei and kinetoplast phenotype without being generally cytotoxic. The mode of action of 20b was investigated with recombinantly engineered trypanosomes expressing a cAMP-sensitive FRET sensor, confirming a dose-response related increase of intracellular cAMP levels in trypanosomes. Our findings further validate the TbrPDEB family as antitrypanosomal target.

Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro.

Synthetic agonists of TLR9 containing novel DNA structures and R'pG (wherein R=1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T(H)-1-type-immune responses and potently reverse allergen-induced T(H)-2 responses to T(H)-1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5(+) and CD21(+)) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-gamma, IL-10, TGF-beta, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5(+) T cells nor CD21(+) B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-gamma on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-beta and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T(H)-1 immune response including T- and B-cell proliferation.