Comparative assessment of the effects of bumped kinase inhibitors on early zebrafish embryo development and pregnancy in mice

Abstract

Bumped kinase inhibitors (BKIs) are effective against a variety of apicomplexan parasites. Fifteen BKIs with promising in vitro efficacy against Neospora caninum tachyzoites, low cytotoxicity in mammalian cells, and no toxic effects in non-pregnant BALB/c mice were assessed in pregnant mice. Drugs were emulsified in corn oil and were applied by gavage for 5 days. Five BKIs did not affect pregnancy, five BKIs exhibited ~15-35% neonatal mortality and five compounds caused strong effects (infertility, abortion, stillbirth and pup mortality). Additionally, the impact of these compounds on zebrafish (Danio rerio) embryo development was assessed by exposing freshly fertilised eggs to 0.2-50 μM of BKIs and microscopic monitoring of embryo development in a blinded manner for 4 days. We propose an algorithm that includes quantification of malformations and embryo deaths, and established a scoring system that allows the calculation of an impact score (S_i) indicating at which concentrations BKIs visibly affect zebrafish embryo development. Comparison of the two models showed that for nine compounds no clear correlation between S_i and pregnancy outcome was observed. However, the three BKIs affecting zebrafish embryos only at high concentrations (≥40 μM) did not impair mouse pregnancy at all, and the three compounds that inhibited zebrafish embryo development already at 0.2 μM showed detrimental effects in the pregnancy model. Thus, the zebrafish embryo development test has limited predictive value to foresee pregnancy outcome in BKI-treated mice. We conclude that maternal health-related factors such as cardiovascular, pharmacokinetic and/or bioavailability properties also contribute to BKI-pregnancy effects.

Keywords: Apicomplexan parasite; Bumped kinase inhibitor; CDPK1; Embryo development; Pregnancy; Zebrafish.

Figure 1.. Setup of the zebrafish embryo development assay.

A: Freshly fertilized eggs are transferred into a petri dish containing the test solution, viable eggs are selected microscopically, and 24 eggs containing viable embryos are transferred into a 24 well plates (1 egg per well). 20 wells contain 1 ml of BKI solution at a defined concentration (T), 4 wells serve as internal control (iC); B: 24 well-plate setup. T = BKI test concentration; nC = negative control (E3 medium/osmosis water); iC = internal control (E3 medium/osmosis water); sC = solvent control (DMSO); pC = positive control: dichloroaniline (4 g/L in water) (adapted from [31; 34])

Figure 2.. Normal development of the zebrafish embryo and BKI-induced malformations observed during test period.

A-E: normal zebrafish embryo development at 0.75, 24, 48, 72 and 96h post fertilization. F-N show representative images of embryonal alterations that were observed during BKI-exposure. F: coagulated embryo (observed with BKI-1796, 24hpf/50μM) ;G: absence of eye bud (*) and tail non-detachment(→) (BKI-1796, 24h/50 μM); H: pericardial edema (→) (BKI-1517, 24h/50μM); M and I: body deformation(→)and pericardial edema(*) (BKI-1757, 24h/5 μM); J: tail curvature(→)and half-hatched embryo (*) (BKI 1517, 24h/20μM); K: curved tail (→) and pericardial edema (*) (BKI-1796, 24h/40μM); L: back curvature (scoliosis→) (BKI-1750, 24h/10 μM); M: head deformation(→) (1796, 24h/10μM); N: curved tail(→)(BKI-1796, 24h/10 μM).

Figure 3.. Proposed algorithm for the zebrafish embryo development assay score calculation.

Every compound concentration receives a score based on the negative control and the solvent control. S_neg = negative control score (no compound, no DMSO); S_DMSO = solvent control score (no compound, 0.01% DMSO); S_assay = assay score (score for each compound concentration); S_mean = control mean score; S_i = impact score; N_e = number of embryos introduced per test item (20 per compound and concentration); N_d = number of dead embryos after 96hpf (score = −1 for each dead embryo); N_m: number of embryonal malformations observed after 96hpf (score = − 0.5 for each malformation, including non-hatched embryos at 96hpf).