Ramesh SaiShyam, Cihalova Daniela, Rajendran Esther, Van Dooren Giel G, Maier Alexander G
Research School of Biology, The Australian National University, Canberra, Australia.
Bio Protoc. 2023 Nov 5;13(21):e4863. doi: 10.21769/BioProtoc.4863.
The mitochondrial electron transport chain (ETC) is a multi-component pathway that mediates the transfer of electrons from metabolic reactions that occur in the mitochondrion to molecular oxygen (O). The ETC contributes to numerous cellular processes, including the generation of cellular ATP through oxidative phosphorylation, serving as an electron sink for metabolic pathways such as de novo pyrimidine biosynthesis and for maintaining mitochondrial membrane potential. Proper functioning of the mitochondrial ETC is necessary for the growth and survival of apicomplexan parasites including , a causative agent of malaria. The mitochondrial ETC of is an attractive target for antimalarial drugs, due to its essentiality and its differences from the mammalian ETC. To identify novel ETC inhibitors, we have established a real-time assay to assess ETC function, which we describe here. This approach measures the O consumption rate (OCR) of permeabilized parasites using a Seahorse XFe96 flux analyzer and can be used to screen compound libraries for the identification of ETC inhibitors and, in part, to determine the targets of those inhibitors. Key features • With this protocol, the effects of candidate inhibitors on mitochondrial O consumption in permeabilized asexual parasites can be tested in real time. • Through the sequential injection of inhibitors and substrates into the assay, the molecular targets of candidate inhibitors in the ETC can, in part, be determined. • The assay is applicable for both drug discovery approaches and enquiries into a fundamental aspect of parasite mitochondrial biology.
线粒体电子传递链（ETC）是一条多组分途径，介导线粒体中发生的代谢反应产生的电子向分子氧（O）的转移。ETC参与众多细胞过程，包括通过氧化磷酸化生成细胞ATP，作为从头嘧啶生物合成等代谢途径的电子汇以及维持线粒体膜电位。线粒体ETC的正常功能对于包括疟原虫（疟疾的病原体）在内的顶复门寄生虫的生长和存活至关重要。疟原虫的线粒体ETC因其必要性以及与哺乳动物ETC的差异，成为抗疟药物的一个有吸引力的靶点。为了鉴定新型疟原虫ETC抑制剂，我们建立了一种实时测定法来评估ETC功能，在此进行描述。这种方法使用海马XFe96通量分析仪测量透化疟原虫的氧消耗率（OCR），可用于筛选化合物库以鉴定ETC抑制剂，并部分确定这些抑制剂的靶点。关键特性 • 使用此方案，可实时测试候选抑制剂对透化无性疟原虫线粒体氧消耗的影响。 • 通过在测定中依次注入抑制剂和底物，可部分确定候选抑制剂在ETC中的分子靶点。 • 该测定法适用于药物发现方法以及对寄生虫线粒体生物学基本方面的研究。
