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

Alveolar echinococcosis (AE) is caused by infection with the fox tapeworm E. multilocularis. The disease affects humans, dogs, captive monkeys, and other mammals, and it is caused by the metacestode stage of the parasite growing invasively in the liver. The current drug treatment is based on non-parasiticidal benzimidazoles. Thus, they are only limitedly curative and can cause severe side effects. Therefore, novel and improved treatment options for AE are needed. Mefloquine (MEF), an antimalarial agent, was previously shown to be effective against E. multilocularis in vitro and in experimentally infected mice. However, MEF is not parasiticidal and needs improvement for successful treatment of patients, and it can induce strong neuropsychiatric side-effects. In this study, the structure-activity relationship and mode of action of MEF was investigated by comparative analysis of 14 MEF derivatives. None of them showed higher activity against E. multilocularis metacestodes compared to MEF, but four compounds caused limited damage. In order to identify molecular targets of MEF and effective derivatives, differential affinity chromatography combined with mass spectrometry was performed with two effective compounds (MEF, MEF-3) and two ineffective compounds (MEF-13, MEF-22). 1'681 proteins were identified that bound specifically to MEF or derivatives. 216 proteins were identified as binding only to MEF and MEF-3. GO term enrichment analysis of these proteins and functional grouping of the 25 most abundant MEF and MEF-3 specific binding proteins revealed the key processes energy metabolism and cellular transport and structure, as well as stress responses and nucleic acid binding to be involved. The previously described ferritin was confirmed as an exclusively MEF-binding protein that could be relevant for its efficacy against E. multilocularis. The here identified potential targets of MEF will be further investigated in the future for a clear understanding of the pleiotropic effects of MEF, and improved therapeutic options against AE.

Dual inhibition of the Echinococcus multilocularis energy metabolism.

Alveolar echinococcosis is caused by the metacestode stage of the zoonotic parasite Echinococcus multilocularis. Current chemotherapeutic treatment options rely on benzimidazoles, which have limited curative capabilities and can cause severe side effects. Thus, novel treatment options are urgently needed. In search for novel targetable pathways we focused on the mitochondrial energy metabolism of E. multilocularis. The parasite relies hereby on two pathways: The classical oxidative phosphorylation including the electron transfer chain (ETC), and the anaerobic malate dismutation (MD). We screened 13 endochin-like quinolones (ELQs) in vitro for their activities against two isolates of E. multilocularis metacestodes and isolated germinal layer cells by the phosphoglucose isomerase (PGI) assay and the CellTiter Glo assay. For the five most active ELQs (ELQ-121, ELQ-136, ELQ-271, ELQ-400, and ELQ-437), EC50 values against metacestodes were assessed by PGI assay, and IC50 values against mammalian cells were measured by Alamar Blue assay. Further, the gene sequence of the proposed target, the mitochondrial cytochrome b, was analyzed. This allowed for a limited structure activity relationship study of ELQs against E. multilocularis, including analyses of the inhibition of the two functional sites of the cytochrome b. By applying the Seahorse XFp Extracellular Flux Analyzer, oxygen consumption assays showed that ELQ-400 inhibits the E. multilocularis cytochrome bc 1 complex under normoxic conditions. When tested under anaerobic conditions, ELQ-400 was hardly active against E. multilocularis metacestodes. These results were confirmed by transmission electron microscopy. ELQ-400 treatment increased levels of parasite-released succinate, the final electron acceptor of the MD. This suggests that the parasite switched to MD for energy generation. Therefore, MD was inhibited with quinazoline, which did not induce damage to metacestodes under anaerobic conditions. However, it reduced the production of succinate compared to control treated parasites (i.e., inhibited the MD). The combination treatment with quinazoline strongly improved the activity of the bc 1 inhibitor ELQ-400 against E. multilocularis metacestodes under anaerobic conditions. We conclude that simultaneous targeting of the ETC and the MD of E. multilocularis is a possible novel treatment approach for alveolar echinococcosis, and possibly also other foodborne diseases inflicted by platyhelminths, which cause substantial economic losses in livestock industry.

Live monitoring of cellular metabolism and mitochondrial respiration in 3D cell culture system using NMR spectroscopy.

BACKGROUND: Because of the interplay between mitochondrial respiration and cellular metabolism, the simultaneous monitoring of both cellular processes provides important insights for the understanding of biological processes. NMR flow systems provide a unique window into the metabolome of cultured cells. Simplified bioreactor construction based on commercially available flow systems increase the practicability and reproducibility of bioreactor studies using standard NMR spectrometers. We therefore aim at establishing a reproducible NMR bioreactor system for metabolic 1H-NMR investigations of small molecules and concurrent oxygenation determination by 19F-NMR, with in depth description and validation by accompanying measures. METHODS: We demonstrate a detailed and standardized workflow for the preparation and transfer of collagen based 3D cell culture of high cell density for perfused investigation in a 5 mm NMR tube. Self-constructed gas mixing station enables 5% CO2 atmosphere for physiological pH in carbon based medium and is perfused by HPLC pump. RESULTS & DISCUSSION: Implemented perfused bioreactor allows detection of perfusion rate dependent metabolite content. We show interleaved dynamic profiling of 26 metabolites and mitochondrial respiration. During constant perfusion, sequential injection of rotenone/oligomycin and 2-deoxy-glucose indicated immediate activation and deactivation of glycolytic rate and full inhibition of oxygen consumption. We show sensitivity to detect substrate degradation rates of major mitochondrial fuel pathways and were able to simultaneously measure cellular oxygen consumption.