Activity and efficacy of the bumped kinase inhibitor BKI-1708 in vitro and in non-pregnant and pregnant toxoplasmosis and neosporosis mouse models

Toxoplasma gondii and Neospora caninum are major worldwide morbidity-causing pathogens. Among the compound classes being currently developed against apicomplexan parasites, bumped kinase inhibitors (BKIs) – optimized to target the apicomplexan calcium-dependent protein kinase 1 (CDPK1) – proved to be safe and highly active in vitro and in vivo. The structure of BKI-1708 is based on the same central scaffold as BKI-1748, which showed proven in vitro activity and in vivo efficacy against both N. caninum and T. gondii. When applied in vitro concomitantly to infection, BKI-1708 exhibits IC50 values of 120nM for T. gondii and 480nM for N. caninum and does not affect HFF at concentrations up to 20µM. However, electron microscopy and immunofluorescence established that exposure of tachyzoite-infected cells to 2.5μM BKI-1708 in vitro induces the formation of multinucleated schizont-like complexes (MNC) - characterized by continued nuclear division and enclosing intracellular zoites lacking the outer plasma membrane. These zoites are unable to finalize cytokinesis. Treatments of zebrafish (Danio rerio) embryos during the first 96h following egg hatching showed that BKI-1708 did not affect early embryo development at concentrations up to 2μM. Treatments of mice with BKI-1708 at 20mg/kg/day during five consecutive days resulted in plasma levels ranging from 0.14 to 4.95μM, establishing an average threshold bellow 2μM. In vivo efficacy was evaluated by applying the compound at 20mg/kg/day from day 9-13 of pregnancy in mice experimentally infected with N. caninum (NcSpain-7) tachyzoites or T. gondii (TgShSp1) oocysts. This resulted in significantly decreased cerebral parasite loads and reduced vertical transmission in both models without drug-induced pregnancy interference. Thus, BKI-1708 is highly efficacious and pregnancy-safe in these mouse models, and certainly suitable for further trials.