RESUMO
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.
RESUMO
The lethal disease alveolar echinococcosis (AE) is caused by the metacestode stage of the fox tapeworm Echinococcus multilocularis. Current chemotherapeutical treatment of AE relies on albendazole and mebendazole, with the caveat that these compounds are not parasiticidal. Drugs have to be taken for a prolonged period of time, often life-long, which can cause adverse effects and reduces the patients' quality of life. In some individuals, benzimidazoles are inactive or cause toxicity, leading to treatment discontinuation. Alternatives to benzimidazoles are urgently needed. Over the recent years, in vivo and in vitro models for low-to-medium throughput drug discovery against AE have been set in place. In vitro drug tests include the phosphoglucose-isomerase (PGI) assay to measure physical damage induced to metacestodes, and viability assays to assess parasiticidal activity against metacestodes and stem cells. In vitro models are also employed for studies on mechanisms of action. In vivo models are thus far based on rodents, mainly mice, and benefits could be gained in future by comparative approaches in naturally infected dogs or captive monkeys. For the identification of novel drugs against AE, a rare disease with a low expected market return, drug-repurposing is the most promising strategy. A variety of chemically synthesized compounds as well as natural products have been analyzed with respect to in vitro and/or in vivo activities against AE. We here review and discuss the most active of these compounds including anti-infective compounds (benzimidazoles, nitazoxanide, amphotericin B, itraconazole, clarithromycin, DB1127, and buparvaquone), the anti-infective anti-malarials (artemisinin, ozonids, mefloquine, and MMV665807) and anti-cancer drugs (isoflavones, 2-methoxyestradiol, methotrexate, navelbine, vincristine, kinase inhibitors, metallo-organic ruthenium complexes, bortezomib, and taxanes). Taking into account the efficacy as well as the potential availability for patients, the most promising candidates are new formulations of benzimidazoles and mefloquine. Future drug-repurposing approaches should also target the energy metabolism of E. multilocularis, in particular the understudied malate dismutation pathway, as this offers an essential target in the parasite, which is not present in mammals.
