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.

Spatial visualization of drug uptake and distribution in Fasciola hepatica using high-resolution AP-SMALDI mass spectrometry imaging

“…The tissue distribution and uptake kinetics of TCBZ clearly differ from the kinase inhibitor imatinib that we analyzed in F. hepatica by AP-SMALDI MSI in a previous study (Morawietz et al 2020). Imatinib was already detectable within the fluke section after 20 min of incubation as was TCBZ, yet imatinib was not only present in surface-near tissue, but also in parts of the intestinal region indicating an early oral drug uptake.…”

“…However, its intense and widespread use has led to the spread of resistance in numerous countries (Fairweather et al 2020;Webb und Cabada 2018). Finding alternative treatment options is highly demanded, and several investigators are engaged in developing and testing novel compounds as drug candidates against liver flukes such as Fasciola hepatica (Edwards et al 2015;Machicado et al 2019;Morawietz et al 2020;O'Neill et al 2015;Kirchhofer et al 2012). Understanding how drugs distribute and accumulate in this parasite is important to reveal (i) whether the uptake route is oral or via its surface, (ii) to answer how fast uptake occurs into the parasite, (iii) how this relates to the onset of vitality loss, (iv) to add knowledge on the mode of action and if the drug accumulates in particular parasite organs, and (v) to study the metabolization of a drug within the parasite, e.g., to bioactive or inactive products.…”

“…In addition, the spatial resolution of PET is limited to the low millimeter-range making drug distribution studies on such small samples impossible (Watakabe et al 2020). Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a cuttingedge method in the fields of life sciences and biomedicine that we recently adopted and optimized for the purpose of drug imaging in trematode parasites (Mokosch et al 2021;Morawietz et al 2020). MALDI MSI is a powerful technique that integrates molecular imaging and advanced image analysis.…”

Drug Repurposing and De Novo Drug Discovery of Protein Kinase Inhibitors as New Drugs against Schistosomiasis

“…Imatinib has been described as a potent drug against adult and juvenile S. mansoni, affecting morphology, pairing, and survival [85] (Figure 3). Beyond that, imatinib has also shown activity against S. japonicum [86], Echinococcus multilocularis [87], Fasciola hepatica [88], Brugia malayi [89], Loa loa [90], Leishmania major [91], Giardia lamblia [92], and it prevented erythrocyte egress of Plasmodium falciparum [93]. In a case report, a single 600 mg dose of imatinib was even shown to reduce the number of microfilariae in a patient infected with L. loa [94].…”

In Vitro Evaluation of Thioredoxin Reductase Inhibitor (Auranofin) Activity in Comparison With Triclabendazole on Adult Fasciola Gigantica

“…Seventy-five adult F. gigantica worms of nearly equal size were selected and classified into five groups (fifteen worms each) as follows: G1: Non-drug-exposed worms (Control). G2: Worms were exposed to TCBZ at a concentration of 20μL/mL (TCBZ-exposed) after Nassef et al (2014). G3: Worms were exposed to auranofin at a concentration of 3μg/ ml (Aur 3μg/ml) after Song et al (2012).…”