From the Research School of Biology, Australian National University, Canberra, Australian Capital Territory 2601, Australia.
From the Research School of Biology, Australian National University, Canberra, Australian Capital Territory 2601, Australia
J Biol Chem. 2018 Aug 24;293(34):13327-13337. doi: 10.1074/jbc.RA118.003640. Epub 2018 Jul 9.
The antimalarial activity of chemically diverse compounds, including the clinical candidate cipargamin, has been linked to the ATPase PfATP4 in the malaria-causing parasite The characterization of PfATP4 has been hampered by the inability thus far to achieve its functional expression in a heterologous system. Here, we optimized a membrane ATPase assay to probe the function of PfATP4 and its chemical sensitivity. We found that cipargamin inhibited the Na-dependent ATPase activity present in membranes from WT parasites and that its potency was reduced in cipargamin-resistant PfATP4-mutant parasites. The cipargamin-sensitive fraction of membrane ATPase activity was inhibited by all 28 of the compounds in the "Malaria Box" shown previously to disrupt ion regulation in in a cipargamin-like manner. This is consistent with PfATP4 being the direct target of these compounds. Characterization of the cipargamin-sensitive ATPase activity yielded data consistent with PfATP4 being a Na transporter that is sensitive to physiologically relevant perturbations of pH, but not of [K] or [Ca]. With an apparent for ATP of 0.2 mm and an apparent for Na of 16-17 mm, the protein is predicted to operate at below its half-maximal rate under normal physiological conditions, allowing the rate of Na efflux to increase in response to an increase in cytosolic [Na]. In membranes from a cipargamin-resistant PfATP4-mutant line, the apparent for Na is slightly elevated. Our study provides new insights into the biochemical properties and chemical sensitivity of an important new antimalarial drug target.
化学结构多样的化合物具有抗疟活性，包括临床候选药物西普加敏，其与疟原虫中的 ATP 酶 PfATP4 有关。由于迄今为止未能在异源系统中实现 PfATP4 的功能表达，因此对 PfATP4 的特性进行了描述。在这里，我们优化了膜 ATP 酶测定法来研究 PfATP4 的功能及其化学敏感性。我们发现西普加敏抑制了来自 WT 寄生虫的膜中的 Na 依赖性 ATP 酶活性，并且在西普加敏抗性 PfATP4 突变寄生虫中其效力降低。膜 ATP 酶活性的西普加敏敏感部分被以前显示以类似于西普加敏的方式破坏 中离子调节的“疟疾框”中的 28 种化合物中的所有化合物抑制。这与 PfATP4 是这些化合物的直接靶标一致。西普加敏敏感 ATP 酶活性的特征与 PfATP4 是一种 Na 转运蛋白的特征一致，该蛋白对 pH 的生理相关扰动敏感，但对 [K] 或 [Ca] 不敏感。对于 ATP 的表观 Km 为 0.2 mM，对于 Na 的表观 Km 为 16-17 mM，在正常生理条件下，该蛋白预计以低于其半最大速率运行，从而允许 Na 外流量增加以响应细胞溶质 [Na] 的增加。在来自西普加敏抗性 PfATP4 突变株的膜中，Na 的表观 Km 略有升高。我们的研究为重要的新抗疟药物靶标提供了对其生化特性和化学敏感性的新见解。
