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

Dennis Imhof,Nicoleta Anghel,Pablo Winzer,Vreni Balmer,Jessica Ramseier,Kai Hänggeli,Ryan Choi,Matthew A Hulverson,Grant R Whitman,Samuel L.M Arnold,Kayode K Ojo,Wesley C Van Voorhis,J Stone Doggett,Luis M Ortega-Mora,Andrew Hemphill

doi:10.1016/j.ijpddr.2021.05.001

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.

Highlights

The apicomplexan parasites Neospora caninum and Toxoplasma gondii cause important diseases in farm animals and have an enormous global economic impact
Immunofluorescence staining of N. caninum (Fig. 2A) or T. gondii infected Human foreskin fibroblasts (HFF) (Fig. 2B), demonstrated that Bumped kinase inhibitors (BKIs)-1748 treatment did not just inhibit proliferation of tachyzoites but induced the formation of intracellular multinucleated complexes (MNCs), similar to what has been observed earlier with BKI-1294 (Winzer et al, 2020b)
These multinucleated schizont-like complexes (MNCs) were enclosed by the major surface antigen SAG1 and contained zoites that underwent active proliferation, as evidenced by labeling with an antibody directed against the inner membrane complex 1 (IMC1)

Summary

Introduction

The apicomplexan parasites Neospora caninum and Toxoplasma gondii cause important diseases in farm animals and have an enormous global economic impact. Cycles comprised of three distinct stages: (i) sporozoites, encapsulated in oocysts that are formed within their respective definitive hosts (feline for T. gondii, canine for N. caninum) and which are shed with the feces; (ii) rapidly proliferating tachyzoites causing acute disease that can be vertically transmitted; and (iii) slowly proliferating bradyzoites, which form tissue cysts, and persist for years without clinical signs (Dubey et al, 2017). Conversion to bradyzoites occurs, and cysts are formed mostly within the brain and muscle tissue (Webster and Dubey, 2010; Dubey et al, 2017). At this stage, the parasites do not cause disease. No cases of human N. caninum infection have been reported to date, but neosporosis-induced abortion and disease in cattle and other ruminants is a major economic burden (Aguado-Martínez et al, 2017)

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Conclusion