The Impact of BKI-1294 Therapy in Mice Infected With the Apicomplexan Parasite Neospora caninum and Re-infected During Pregnancy.

Exposure of Neospora caninum tachyzoites to BKI-1294 in vitro results in the formation of long-lived multinucleated complexes (MNCs). However, in vivo treatment of BALB/c mice with BKI-1294 shortly after N. caninum infection during pregnancy was safe and profoundly reduced pup mortality and vertical transmission. We hypothesized that the formation of MNCs could trigger immune responses that contribute to BKI efficacy in vivo. In this study, mice were first vaccinated with a sublethal dose of N. caninum tachyzoites and were treated with BKI-1294. We then investigated the effects of these treatments after mating and re-infection during pregnancy. Effects on fertility, pup survival, vertical transmission, and parasite load in dams were evaluated. Cytokines in sera or splenocyte culture supernatants were assessed by either ELISA or the Luminex™ 200 system, and humoral immune responses against tachyzoite and MNC antigens were compared by ELISA, Western blotting and immunoproteomics. Our results showed that BKI-1294 treatment of live-vaccinated mice reduced the cerebral parasite load in the dams, but resulted in higher neonatal pup mortality and vertical transmission. In live-vaccinated mice, cytokine levels, most notably IFN-y, IL-10, and IL-12, were consistently lower in BKI-1294 treated animals compared to non-treated mice. In addition, comparative Western blotting identified two protein bands in MNC extracts that were only recognized by sera of live-vaccinated mice treated with BKI-1294, and were not found in tachyzoite extracts. We conclude that treatment of live-vaccinated mice with BKI-1294 influenced the cellular and humoral immune responses against infection, affected the safety of the live-vaccine, and decreased protection against re-infection and vertical transmission during pregnancy

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

Photonic and Optomechanical Thermometry

Temperature is one of the most relevant physical quantities that affects almost all processes in nature. However, the realization of accurate temperature standards using current temperature references, like the triple point of water, is difficult due to the requirements on material purity and stability of the environment. In addition, in harsh environments, current temperature sensors with electrical readout, like platinum resistors, are difficult to implement, urging the development of optical temperature sensors. In 2018, the European consortium Photoquant, consisting of metrological institutes and academic partners, started investigating new temperature standards for self-calibrated, embedded optomechanical sensor applications, as well as optimised high resolution and high re- liability photonic sensors, to measure temperature at the nano and meso-scales and as a possible replacement for the standard platinum resistant thermometers. This article presents an overview of the results obtained with sensor prototypes that exploit photonic and optomechanical techniques for sensing temperatures over a large temperature range (5 K to 300 K). Different concepts are demon- strated, including ring resonators, ladder-like resonators and suspended membrane optomechanical thermometers, highlighting initial performance and challenges, like self-heating that need to be overcome to realize photonic and optomechanical thermometry applications.This work was carried out under the 17FUN05 PhotOQuanT project, which has received funding from the EMPIR program, co-financed by the Participating States and the European Union’s Horizon 2020 research and innovation progra

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

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 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

Neospora caninum calcium-dependent protein kinase 1 is an effective drug target for neosporosis therapy.

Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis

In Vitro Activities of MMV Malaria Box Compounds against the Apicomplexan Parasite Neospora caninum, the Causative Agent of Neosporosis in Animals.

(1) Background: Neospora caninum is a major cause of abortion in cattle and represents a veterinary health problem of great economic significance. In order to identify novel chemotherapeutic agents for the treatment of neosporosis, the Medicines for Malaria Venture (MMV) Malaria Box, a unique collection of anti-malarial compounds, were screened against N. caninum tachyzoites, and the most efficient compounds were characterized in more detail. (2) Methods: A N. caninum beta-galactosidase reporter strain grown in human foreskin fibroblasts was treated with 390 compounds from the MMV Malaria Box. The IC50s of nine compounds were determined, all of which had been previously been shown to be active against another apicomplexan parasite, Theileria annulata. The effects of three of these compounds on the ultrastructure of N. caninum tachyzoites were further investigated by transmission electron microscopy at different timepoints after initiation of drug treatment. (3) Results: Five MMV Malaria Box compounds exhibited promising IC50s below 0.2 µM. The compound with the lowest IC50, namely 25 nM, was MMV665941. This compound and two others, MMV665807 and MMV009085, specifically induced distinct alterations in the tachyzoites. More specifically, aberrant structural changes were first observed in the parasite mitochondrion, and subsequently progressed to other cytoplasmic compartments of the tachyzoites. The pharmacokinetic (PK) data obtained in mice suggest that treatment with MMV665941 could be potentially useful for further in vivo studies. (4) Conclusions: We have identified five novel compounds with promising activities against N. caninum, the effects of three of these compounds were studies by transmission electron microscopy (TEM). Their modes of action are unknown and require further investigation

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

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