Targeting Kinases in Fasciola hepatica: Anthelminthic Effects and Tissue Distribution of Selected Kinase Inhibitors

Abstract

Protein kinases have been discussed as promising druggable targets in various parasitic helminths. New drugs are also needed for control of fascioliasis, a food-borne trematode infection and worldwide spread zoonosis, caused by the liver fluke Fasciola hepatica and related species. In this study, we intended to move protein kinases more into the spotlight of Fasciola drug research and characterized the fasciolicidal activity of two small-molecule inhibitors from human cancer research: the Abelson tyrosine kinase (ABL-TK) inhibitor imatinib and the polo-like 1 (PLK1) inhibitor BI2536. BI2536 reduced viability of 4-week-old immature flukes in vitro, while adult worms showed a blockade of egg production. Together with a significantly higher transcriptional expression of PLK1 in adult compared to immature worms, this argues for a role of PLK1 in fluke reproduction. Both fluke stages expressed ABL1-TK transcripts at similar high levels and were affected by imatinib. To study the uptake kinetic and tissue distribution of imatinib in F. hepatica, we applied matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) for the first time in this parasite. Drug imaging revealed the accumulation of imatinib in different fluke tissues from 20 min to 12 h of exposure. Furthermore, we show that imatinib is metabolized to N-desmethyl imatinib by F. hepatica, a bioactive metabolite also found in humans. Besides the vitellarium, gastrodermal tissue showed strong signal intensities. In situ hybridization demonstrated the gastrodermal presence of abl1 transcripts. Finally, we assessed transcriptional changes of physiologically important genes in imatinib-treated flukes. Moderately increased transcript levels of a gene encoding a multidrug resistance protein were detected, which may reflect an attempt to defend against imatinib. Increased expression levels of the cell cycle dependently expressed histone h2b and of two genes encoding superoxide dismutases (SODs) were also observed. In summary, our pilot study demonstrated cross-stage activity of imatinib but not BI2536 against immature and adult F. hepatica in vitro; a fast incorporation of imatinib within minutes, probably via the oral route; and imatinib-induced expression changes of physiologically relevant genes. We conclude that kinases are worth analyzing in more detail to evaluate the potential as therapeutic targets in F. hepatica.

Keywords: BI 2536; Fasciola hepatica; MALDI mass spectrometry imaging; drug target; imatinib; inhibitors; kinases; superoxide dismutase.

Figure 1

Transcriptional expression of selected kinases in different developmental stages of Fasciola hepatica. Relative expression in 4-week-old immature and 12-week-old adult worms was determined by qRT-PCR and normalization against two reference genes, glutamyl-prolyl-tRNA synthetase (Fheprs), and tubulin-specific chaperone D (Fhtbcd). Data represent the mean ± SEM of three to four biological replicates. Relative expression values were calculated by the formula 2^−ΔCt × f, with f = 1,000 as an arbitrary factor. Significant differences are indicated with *p < 0.05 (Wilcoxon rank sum test). abl1, tyrosine-protein kinase ABL1; plk1, polo-like kinase 1. Data from (18) (modified) under the Creative Commons license (http://creativecommons.org/licenses/by/4.0/).

Figure 2

Treatment with kinase inhibitors affects motility of Fasciola hepatica immature flukes in vitro. Four-week old flukes were treated for 72 h with different concentrations (20–150 μM) of the PLK1 inhibitor BI2536 or the ABL-TK inhibitor imatinib. Motility was assessed every 24 h. Control worms were treated with the inhibitor solvent, DMSO. (A) Representative images taken after 72 h treatment. (B) Motility scores for all timepoints and concentrations (score 3 = normal, 2 = reduced, 1 = severely reduced, 0 = no motility). Data represent the mean ± SEM of two independent experiments with two to four flukes per experiment. Significant differences are indicated with *p < 0.05 (Wilcoxon rank sum test). Triangle indicates a score of zero. Scale bars correspond to 1 mm.

Figure 3

Motility of adult Fasciola hepatica after treatment with kinase inhibitors in vitro. Twelve-week-old flukes were treated for 72 h with different concentrations (20–150 μM) of the PLK1 inhibitor BI2536 or the ABL-TK inhibitor imatinib. Motility was assessed every 24 h. Control worms were treated with the inhibitor solvent, DMSO. (A) Representative images taken after 72 h treatment. (B) Motility scores for all timepoints and concentrations (score 3 = normal, 2 = reduced, 1 = severely reduced, 0 = no motility). Data represent the mean ± SEM of two independent experiments with two to four flukes per group and experiment. Significant differences are indicated with **p < 0.01 (Wilcoxon rank sum test). Scale bars correspond to 5 mm.

Figure 4

Treatment with the PLK1 inhibitor BI2536 interrupts egg production by adult Fasciola hepatica in vitro. Twelve-week-old flukes were treated for 72 h with different concentrations of BI2536 (20–150 μM). Control worms were treated with the inhibitor solvent, DMSO. Eggs produced in the last 24 h of the treatment period were counted. (A) Representative images of eggs produced by one control fluke compared to a fluke treated with 50 μM of BI2536. (B) Relative egg numbers compared to control flukes. Data represent the mean ± SEM of 2 independent experiments with two to four flukes per experiment. Significant differences are indicated with *p < 0.05, **p < 0.01 (Wilcoxon rank sum test). Triangle indicates a score of zero. Scale bars correspond to 5 mm.

Figure 5

Detection of imatinib and its main metabolite in tissues of adult Fasciola hepatica by MALDI mass spectrometry imaging (MSI). Twelve-week-old flukes were treated for 20 min, 4 h, or 12 h with 100 μM imatinib. Compounds and endogenous lipids were detected in transversal sections using AP-SMALDI MSI, and sections were stained with H&E afterwards. (A) Example of an H&E-stained section showing the typical riddled pattern with numerous white spots from which tissue was laser-ablated during MALDI. Different tissues are indicated: gut/gastrodermis (g), tegument (t), uterus (u), and vitellarium (v). (B) For each timepoint of imatinib treatment, two flukes were analyzed, of which one representative is depicted. (i) H&E-stained sections to visualize the fluke‘s tissues after AP-SMALDI MSI measurements. (ii) RGB images from AP-SMALDI MSI showing transversal sections with the following signals: m/z 494.266464 (green, protonated imatinib), m/z 812.618589 [red, protonated PE(41:3), m/z 810.598235; blue, PC(36:1) sodium adduct]. Lipid assignment is based on LIPIDMAPS (36) results. (iii, iv) AP-SMALDI MSI single channel images showing sections depicting the imatinib signal at m/z 494.266464 corresponding to [M+H]⁺ (iii) or the metabolite N-desmethyl imatinib at m/z 480.251261 corresponding to [M+H]⁺ (iv). Arrows indicate compound localized within the intestine or gastrodermis, arrowheads within the vitellarium. Scale bars correspond to 500 μm.

Figure 6

In situ hybridization localizes transcripts of Fhabl1 kinase in gastrodermal tissue of adult Fasciola hepatica. ISH was performed on transverse sections of 12-week-old F. hepatica using digoxygenin-labeled sense probes for Fhabl1 as negative control (A), or anti-sense probes to detect Fhabl1 transcripts (B). Positive, red staining localized to the gastrodermis (arrow). Scale bars correspond to 250 μm (whole sections) or 100 μm (inserts).

Figure 7

Gene expression changes in adult Fasciola hepatica after imatinib treatment. Twelve-week-old flukes were treated for 72 h with 100 or 150 μM imatinib in vitro, and the fold change of transcripts of selected genes relative to DMSO-treated worms was quantified by qRT-PCR. Normalization was done against two reference genes, glutamyl-prolyl-tRNA synthetase (Fheprs) and tubulin-specific chaperone D (Fhtbcd). Of interest were (A) extracellular superoxide dismutase (sodex) and (B) superoxide dismutase (sod) as genes involved in the defense toward oxidative stress, (C) the histone h2b important for DNA duplication during the cell cycle, and (D) a putative multidrug resistance gene (mdr). Data represent the mean ± SEM of two independent experiments with three to five flukes per group. Significant differences vs. control are indicated with *p < 0.05 (Wilcoxon rank sum test).