Chaudhry Sheena, Zurbriggen Raphael, Preza Matías, Kämpfer Tobias, Kaethner Marc, Memedovski Roman, Scorrano Nathalie, Hemphill Andrew, Doggett Joseph Stone, Lundström-Stadelmann Britta
Institute of Parasitology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.
Front Vet Sci. 2022 Aug 5;9:981664. doi: 10.3389/fvets.2022.981664. eCollection 2022.
Alveolar echinococcosis is caused by the metacestode stage of the zoonotic parasite . 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 . 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) for their activities against two isolates of 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), EC values against metacestodes were assessed by PGI assay, and IC values against mammalian cells were measured by Alamar Blue assay. Further, the gene sequence of the proposed target, the mitochondrial cytochrome , was analyzed. This allowed for a limited structure activity relationship study of ELQs against , including analyses of the inhibition of the two functional sites of the cytochrome . By applying the Seahorse XFp Extracellular Flux Analyzer, oxygen consumption assays showed that ELQ-400 inhibits the cytochrome complex under normoxic conditions. When tested under anaerobic conditions, ELQ-400 was hardly active against 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 inhibitor ELQ-400 against metacestodes under anaerobic conditions. We conclude that simultaneous targeting of the ETC and the MD of 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.
泡型包虫病由一种人畜共患寄生虫的中绦期引起。目前的化疗选择依赖于苯并咪唑类药物，但其治疗能力有限且会引起严重副作用。因此，迫切需要新的治疗方法。为寻找新的可靶向途径，我们聚焦于该寄生虫的线粒体能量代谢。该寄生虫在此依赖两条途径：包括电子传递链（ETC）的经典氧化磷酸化途径，以及无氧苹果酸歧化（MD）途径。我们通过磷酸葡萄糖异构酶（PGI）测定法和CellTiter Glo测定法，筛选了13种类内chin喹诺酮（ELQs）对两种中绦期分离物和分离的生发层细胞的活性。对于活性最高的5种ELQs（ELQ - 121、ELQ - 136、ELQ - 271、ELQ - 400和ELQ - 437），通过PGI测定法评估其对中绦期的EC值，通过阿拉玛蓝测定法测量其对哺乳动物细胞的IC值。此外，对所提出的靶标线粒体细胞色素的基因序列进行了分析。这使得能够对ELQs针对该寄生虫进行有限的构效关系研究，包括对细胞色素两个功能位点抑制作用的分析。通过应用海马XFp细胞外通量分析仪，耗氧测定表明ELQ - 400在常氧条件下抑制该寄生虫的细胞色素复合物。在无氧条件下进行测试时，ELQ - 400对中绦期几乎没有活性。这些结果通过透射电子显微镜得到证实。ELQ - 400处理增加了寄生虫释放的琥珀酸水平，琥珀酸是MD的最终电子受体。这表明该寄生虫转向MD进行能量生成。因此，用喹唑啉抑制MD，喹唑啉在无氧条件下不会对中绦期造成损伤。然而，与对照处理的寄生虫相比，它降低了琥珀酸的产生（即抑制了MD）。喹唑啉联合处理在无氧条件下显著提高了该寄生虫抑制剂ELQ - 400对中绦期的活性。我们得出结论，同时靶向该寄生虫的ETC和MD可能是泡型包虫病以及可能由扁形虫引起的其他食源性疾病的一种新的治疗方法，这些疾病给畜牧业造成了巨大经济损失。
