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

Abstract

eWe 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 (IC 50 s) 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 NcLiv, 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. N eospora caninum is a cyst-forming apicomplexan parasite that is closely related to Toxoplasma gondii but exhibits distinct differences in transmission patterns, virulence, host specificity, immunogenetic aspects, and the pathology it induces. T. gondii causes toxoplasmosis in humans and many domestic and wildlife animals, with great economic impact especially in sheep but also in many other animal species (1). Human toxoplasmosis causes serious pathology in immune-suppressed individuals. In addition, if a seronegative mother acquires primary infection during pregnancy, human toxoplasmosis can lead to abortion, microcephalus and hydrocephalus, and other fetal abnormalities causing intellectual disability (2). N. caninum is a veterinary health problem and represents one of the most important infectious causes of bovine abortion, stillbirth, and the birth of weak calves, with an economic impact of over $1.3 billion (3-5). In addition, N. caninum causes neuromuscular disease in dogs, and neosporosis has also been detected in a wide range of other species of livestock and wild animals worldwide.Despite their differences, an important common feature of these parasites is their ability to invade and replicate within a wide range of cell types and tissues, where they reside in an intracellular parasitophorous vacuole, surrounded by a parasitophorous vacuole me...

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

“…The inhibitory effects of BKI-1517 and BKI-1553 in comparison to BKI-1294 (16,17) against N. caninum tachyzoites were assessed by adding the drugs to the cultures at the time point of infection and then measuring parasite proliferation after 3 days (Fig. 2).…”

“…Based on previously reported PK data (19), we applied BKI-1517 at 50 mg/kg of body weight during 6 days, thus in a scheme similar to the one established previously for BKI-1294 (17). The corresponding placebo group received corn oil.…”

“…One compound of this class, BKI-1294, is effective against cryptosporidiosis in immunosuppressed mice (14) and against toxoplasmosis in vitro and in vivo (15). BKI-1294 is also effective against N. caninum in vitro and in vivo (16) and strongly interferes with the vertical transmission of neosporosis to newborn pups in a pregnant mouse model of neosporosis (17).…”

In Vitro Screening of the Open-Source Medicines for Malaria Venture Malaria Box Reveals Novel Compounds with Profound Activities against Theileria annulata Schizonts

“…Apicomplexans share a number of conserved pathways representing potential drug targets (24)(25)(26). For instance, the cross-reactivity of inhibitors of calcium-dependent protein kinases (CDPKs) against Plasmodium (27), Toxoplasma (28), and Neospora (29,30) and the demonstration of profound in vitro and in vivo activity of BPQ against Neospora caninum infection (31) have underscored a potential role for exploiting the possibility of repurposing drugs among apicomplexans.…”

Vaccines and drugs against Neospora caninum, an important apicomplexan causing abortion in cattle and other farm animals

“…Some BKIs, especially BKI-1294, exhibited excellent effectiveness against N. caninum in vitro and in vivo (Table 3). They are, however, not directly parasitocidal: only after long-term in vitro treatment of infected human 44 A similar induction of bradyzoite antigen expression was observed when treating N. caninum-infected fibroblast monolayers with artemisone and respective derivatives. 45 …”