Common Molecular Targets of a Quinolone Based Bumped Kinase Inhibitor in Neospora caninum and Danio rerio

Abstract

Neospora caninum is an apicomplexan parasite closely related to Toxoplasma gondii, and causes abortions, stillbirths and/or fetal malformations in livestock. Target-based drug development has led to the synthesis of calcium-dependent protein kinase 1 inhibitors, collectively named bumped kinase inhibitors (BKIs). Previous studies have shown that several BKIs have excellent efficacy against neosporosis in vitro and in vivo. However, several members of this class of compounds impair fertility in pregnant mouse models and cause embryonic malformation in a zebrafish (Danio rerio) model. Similar to the first-generation antiprotozoal drug quinine, some BKIs have a quinoline core structure. To identify common targets in both organisms, we performed differential affinity chromatography with cell-free extracts from N. caninum tachyzoites and D. rerio embryos using the 5-aminopyrazole-4-carboxamide (AC) compound BKI-1748 and quinine columns coupled to epoxy-activated sepharose followed by mass spectrometry. BKI-binding proteins of interest were identified in eluates from columns coupled to BKI-1748, or in eluates from BKI-1748 as well as quinine columns. In N. caninum, 12 proteins were bound specifically to BKI-1748 alone, and 105 proteins, including NcCDPK1, were bound to both BKI-1748 and quinine. For D. rerio, the corresponding numbers were 13 and 98 binding proteins, respectively. In both organisms, a majority of BKI-1748 binding proteins was involved in RNA binding and modification, in particular, splicing. Moreover, both datasets contained proteins involved in DNA binding or modification and key steps of intermediate metabolism. These results suggest that BKI-1748 interacts with not only specific targets in apicomplexans, such as CDPK1, but also with targets in other eukaryotes, which are involved in common, essential pathways.

Differential Affinity Chromatography Coupled to Mass Spectrometry: A Suitable Tool to Identify Common Binding Proteins of a Broad-Range Antimicrobial Peptide Derived from Leucinostatin

“…Then, the columns were separated, and the binding proteins were eluted with 50 mM acetic acid (5 mL per column). The eluates were lyophilized and stored at −80 °C [ 14 , 15 ].…”

“…In earlier studies, we have successfully identified common targets of thiazolides, a class of broad anti-infective and anti-cancer drugs, in protozoan parasites [ 10 , 11 ], as well as host cells [ 12 , 13 ], by affinity chromatography, followed by gel electrophoresis and the identification of major bands by mass spectrometry (MS). In recent studies [ 14 , 15 ], we have alleviated the limitations of this method by subjecting cell-free extracts from parasites and suitable host cells to differential affinity chromatography (DAC) coupled to MS. This approach allows the identification of entire proteomes binding to compounds of interest but not to (closely related) ineffective compounds and not to single proteins anymore [ 14 ].…”

“…This approach allows the identification of entire proteomes binding to compounds of interest but not to (closely related) ineffective compounds and not to single proteins anymore [ 14 ]. Thereby, common pathways in parasites, as well as in mammalian hosts, which are touched by a given (class of) compounds, can be identified [ 15 ]. In an ideal situation, compounds interfering with essential pathways such as protein biosynthesis will be discarded or improved before subjecting them to in vivo trials.…”

Investigation of the mechanism of action of mefloquine and derivatives against the parasite Echinococcus multilocularis

“…(c) Differential affinity chromatography (DAC) Differential affinity chromatography was performed similarly as described previously (Anghel et al, 2021;Müller et al, 2022aMüller et al, , 2022b. Parasite extracts were passed first through a mock column (Küster et al, 2015) followed by a column containing effective or ineffective drugs and at a flow rate of 0.25 ml/min.…”

“…Differential affinity chromatography (DAC) is based on pull-downs of cell free extracts on columns coated with effective compounds or ineffective derivatives followed by nano-liquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS) of the eluates. In recent studies, DAC has been used to identify molecular targets of ruthenium complexes in Toxoplasma gondii and Trypanosoma brucei ( Anghel et al, 2021 ), and to identify molecular targets of a bumped kinase inhibitor in Neospora caninum and Danio rerio ( Müller et al, 2022a ) and proteins binding to Leucinostatin-like peptides ( Müller et al, 2022b ).…”