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 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.

Keywords: Bumped kinase inhibitors; Calcium dependent protein kinase inhibitor; Neosporosis; Oocysts; Pregnancy; Tachyzoites; Toxoplasmosis; Treatment.

Graphical abstract

Fig. 1

Structure and in vitro activities of BKI-1748 against N. caninum (Nc-β-gal) and T. gondii (Tg-β-gal). (A) molecular structure of BKI-1748, MW = molecular weight; EC₅₀ = concentration of half maximal proliferation inhibition; *toxicity levels against human foreskin fibroblasts (HFF) and zebrafish embryos were determined previously [22]. (B,C) Dose-response curves for Neospora and Toxoplasma tachyzoites grown in HFF.

Fig. 2

Immunofluorescence staining of HFF monolayers infected with N. caninum NcSpain-7 (A) and T. gondii Me49 tachyzoites (B) after treatment with 2.5 μM BKI-1748 for 2, 6 and 9 days. As a control, HFF were infected with the respective parasites but not treated and were fixed and stained after 2 days of incubation at 37 °C/5% CO₂. SAG1 is labelled in green, anti-IMC1 staining is red, and nuclei are stained blue with DAPI. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

SEM micrographs of N. caninum tachyzoites and MNCs after exposure of infected HFF with BKI-1748. (A,B) non-treated tachyzoites, (C–F) BKI-1748 treated MNCs. The framed area in (A) is enlarged in (B), and the framed area in (E) is shown at higher magnification in (F); api = apical complex, small arrows point towards tachyzoites, the horizontal arrows indicate the conoid. Bars in (A) = 2.4 μM; (B) = 0.35 μm; (C) = 0.45 μm; (D) = 0.6 μm; (E) = 0.9 μm; (F) = 0.35 μM.

Fig. 4

Transmission electron microscopy of N. caninum tachyzoites and MNCs. TEM micrographs of tachyzoites within a parasitophorous vacuole (A), and MNCs after 24 h (B,C), 48 h (D) and 72 h of exposure to BKI-1748 (E,F). (F) is a higher magnification view of (E); T indicates individual tachyzoites in BKI-treated cultures; n = nucleus; v = cytoplasmic vacuole; large arrows point towards newly formed apical complexes, dg = dense granules; rop = rhoptries; mito = mitochondrion; mic = micronemes; px = parasitophorous vacuole matrix; apg = amylopectin granules; small arrows indicate separation between nuclear membrane and the cytoplasm. Bars in (A) = 1.2 μm; (B) = 0.8 μm; (C) = 0.6 μm; (D) = 0.25 μm; (E) = 0.5 μm; (F) = 0.3 μm.

Fig. 5

TEM micrographs of N. caninum MNCs after 96 h treatment with BKI-1748. (A) is a low magnification view of an MNC infected human foreskin fibroblast, the framed areas are magnified in (B) and (C). (B) shows a cluster of nuclei, surrounded by secretory organelles and amylopectin granules, and micronemes are also arranged in tightly packed clusters: n = nucleus; v = cytoplasmic vacuole, large arrows point towards apical complexes, dg = dense granules; rop = rhoptries; mito = mitochondrion; mic = micronemes; px = parasitophorous vacuole matrix; apg = amylopectin granules; small arrows indicate separation between nuclear membrane and the cytoplasm. Scale bars in (A) = 2 μm; (B) = 0.5 μm; (C) = 0.75 μm; (D) = 0.6 μm.

Fig. 6

BKI-1748 concentrations measured 1 h after the final treatment at day 5 of 5 mg/kg/day and 20 mg/kg/day. For each dosage, blood of six mice was retrieved through the tail vein and blood plasma was collected to measure the drug concentration by LC-MS/MS.

Fig. 7

BKI-1748 treatment in the pregnant neosporosis mouse model. (A) Survival curve of pups and cerebral parasite burden of non-pregnant mice (B) and dams (C). Survival rates at each time point were plotted in Kaplan-Meier graphs and curves were compared by the Log-rank (Mantel-Cox) test. Differences between BKI-1748 and positive control curves were highly significant (P < 0.0001). BALB/c mice were experimentally infected with 10⁵ NcSpain-7 tachyzoites and treated with BKI-1748 for 5 days at 20 mg/kg/day in corn oil or were treated with corn oil alone (C+). C- was not infected and treated with corn oil only. Brains of all mice were collected directly after euthanasia and parasite burden of non-pregnant mice and dams was quantified by real-time PCR. Values are shown as box plots. No statistically significant differences in the cerebral parasite burden were observed between BKI-1748-treated mice compared to the C+ group (neither in non-pregnant mice nor in dams).

Fig. 8

BKI-1748 treatment in the pregnant toxoplasmosis mouse model. (A) Survival curves of pups and cerebral parasite burden of non-pregnant mice (B) and dams (C). Survival rates at each time point were plotted in Kaplan-Meier graphs and curves were compared by the Log-rank (Mantel-Cox) test. Differences between BKI-1748 and positive control curves were highly significant (P < 0.0001). CD1 mice were infected with 50 oocysts of the TgShSp1 strain and treated with 20 mg/kg BKI-1748, while C+ was infected and treated with corn oil alone. C- was not infected and treated with corn oil only. All mice were euthanized 30 days p.p. After euthanasia, brains were aseptically collected, and cerebral parasite burden of non-pregnant mice and dams was quantified by real-time PCR. Values are depicted as box plots. Parasite burden between groups were compared by the Kruskal-Wallis test, followed by the Mann-Whitney-U test. Differences between BKI-1748-treated and non-treated groups were statistically significant (*P < 0.0127, **P < 0.0095).

Fig. 9

Ocular parasite burden of non-pregnant mice (A) and dams (B) infected with 50 oocysts of the TgShSp1 strain and treated with BKI-1748 at 20 mg/kg/day for 5 days, or with corn oil alone (C+). C- was not infected and treated with corn oil only. Eyes were removed after euthanasia and DNA was extracted and quantified by real-time PCR. Values are shown in box plots. Ocular parasite burden between groups were compared by the Kruskal-Wallis test, followed by the Mann-Whitney-U test. Differences between BKI-1748-treated and non-treated groups were statistically significant in non-pregnant mice (**P < 0.0048) but not in dams.