Research School of Biology, Australian National University, Canberra, Australian Capital Territory 2600, Australia.
ACS Infect Dis. 2024 Apr 12;10(4):1185-1200. doi: 10.1021/acsinfecdis.3c00588. Epub 2024 Mar 18.
New drugs with novel modes of action are needed to safeguard malaria treatment. In recent years, millions of compounds have been tested for their ability to inhibit the growth of asexual blood-stage parasites, resulting in the identification of thousands of compounds with antiplasmodial activity. Determining the mechanisms of action of antiplasmodial compounds informs their further development, but remains challenging. A relatively high proportion of compounds identified as killing asexual blood-stage parasites show evidence of targeting the parasite's plasma membrane Na-extruding, H-importing pump, PfATP4. Inhibitors of PfATP4 give rise to characteristic changes in the parasite's internal [Na] and pH. Here, we designed a "pH fingerprint" assay that robustly identifies PfATP4 inhibitors while simultaneously allowing the detection of (and discrimination between) inhibitors of the lactate:H transporter PfFNT, which is a validated antimalarial drug target, and the V-type H ATPase, which was suggested as a possible target of the clinical candidate ZY19489. In our pH fingerprint assays and subsequent secondary assays, ZY19489 did not show evidence for the inhibition of pH regulation by the V-type H ATPase, suggesting that it has a different mode of action in the parasite. The pH fingerprint assay also has the potential to identify protonophores, inhibitors of the acid-loading Cl transporter(s) (for which the molecular identity(ies) remain elusive), and compounds that act through inhibition of either the glucose transporter PfHT or glycolysis. The pH fingerprint assay therefore provides an efficient starting point to match a proportion of antiplasmodial compounds with their mechanisms of action.
需要具有新型作用机制的新药来保护疟疾治疗。近年来，已经测试了数百万种化合物以评估其抑制无性血期寄生虫生长的能力，从而鉴定出了数千种具有抗疟活性的化合物。确定抗疟化合物的作用机制可以为它们的进一步开发提供信息，但仍然具有挑战性。相对较高比例的被鉴定为杀死无性血期寄生虫的化合物表明它们靶向寄生虫的质膜 Na 外排、H 输入泵 PfATP4。PfATP4 的抑制剂会导致寄生虫内部 [Na] 和 pH 的特征变化。在这里，我们设计了一种“pH 指纹”测定法，该测定法可稳健地鉴定 PfATP4 抑制剂，同时还可以检测乳酸盐：H 转运蛋白 PfFNT 的抑制剂（这是一种经过验证的抗疟药物靶标）和 V 型 H ATPase 的抑制剂（该酶被认为是临床候选药物 ZY19489 的可能靶标）。在我们的 pH 指纹测定法和随后的二级测定法中，ZY19489 没有显示出抑制 V 型 H ATPase 调节 pH 的证据，这表明它在寄生虫中具有不同的作用机制。pH 指纹测定法还有可能鉴定质子载体、酸化 Cl 转运蛋白（其分子身份仍未确定）的抑制剂以及通过抑制葡萄糖转运蛋白 PfHT 或糖酵解起作用的化合物。因此，pH 指纹测定法为将一部分抗疟化合物与其作用机制相匹配提供了一个有效的起点。
