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

Abstract

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 EC 50 s 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.

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 treatment with 10 mg/kg ELQ-334 and 10 mg/kg ELQ-422 resulted in only little neonatal mortality (two out of 11 and one out of 17 pups, respectively). As previously reported, BKI-1748 applied at 20 mg/kg/day did not impair fertility rates or pup survival ( Imhof et al, 2021 ). However, the combination treatments appeared to induce neonatal pup mortality to a higher degree (mortality of five out of 11 and three out of 19 pups for the combined ELQ-334 + BKI-1748 and ELQ-422 + BKI-1748 treatments, respectively), which raised some safety concerns.…”

“…Three hours after infection, coverslips were treated either with 1 μM ELQ-422, 2.5 μM BKI-1748, or 1 μM ELQ-422 + 2.5 μM BKI-1748, and the cultures were further maintained in the presence of the drugs. Immunofluorescence staining was carried out as described ( Imhof et al, 2021 ). Briefly, at different time points, the coverslips were washed once in PBS, fixed in 3% paraformaldehyde in PBS (pH 7.2) for 10 min, and permeabilized in a 1:1 solution of pre-cooled methanol/acetone at −20 °C ( Alaeddine et al, 2013 ; Guionaud et al, 2010 ; Winzer et al, 2015 ).…”

“…The placebo group received 100 μl corn oil for five consecutive days. One hour after the last administration of compound, blood was collected from the tail vein, and transferred to heparin-coated tubes, which were inverted carefully followed by centrifugation (10 min at 1000× g ) and plasma was collected and stored at −20 °C for drug level analysis ( Imhof et al, 2021 ). During the experimental period, mice were examined daily and weighed several times during pregnancy to determine the number of pregnant animals and to detect possible abortions (which would result in rapid weight loss).…”

“…BKI-1748 and ELQ-316 concentrations in mouse plasma samples were determined as previously described ( Hulverson et al, 2019 ; Imhof et al, 2021 ). Briefly, BKI-1748 and ELQ-316 were extracted from plasma using acetonitrile/0.1% formic acid with an internal standard and a standard curve was prepared for quantification.…”

“…After mating, (day 0) female mice were randomly assigned to seven groups: ELQ-334 (n = 14), ELQ-422 (n = 14), BKI-1748 (n = 14), ELQ-334 + BKI-1748 (n = 14), ELQ-422 + BKI-1748 (n = 14), positive control (corn oil treatment and infection, n = 14), and negative control (corn oil treatment without infection, n = 6). NcSp-7 tachyzoites were transferred from HFF cells to BALB/c dermal fibroblasts and maintained at 37 °C and 5% CO 2 three days prior to infection ( Imhof et al, 2021 ). On day 7, tachyzoites were harvested and prepared for infection as previously described ( Arranz-Solís et al, 2015 ).…”