Molecular Pathways Affected by Drugs that Disrupt Na+ Homeostasis in Malaria Parasites

每年都有数以亿计的疟疾患者接受抗疟疾药物的治疗，而每一个 单个患者携带数千亿疟疾寄生虫，需要继续喂食 抗疟疾新药的流水线，以对抗可能出现的耐药性。近几年来有几个 新的抗疟疾化合物被发现能够破坏疟疾中的Na+动态平衡 寄生虫。其中四种化合物(螺吲哚、吡唑酰胺、二氢异喹诺酮和噻三唑) 被指定为临床候选药物。值得注意的是，这些药物属于非常不同的化学类别 具有不同的药效团和对疟疾寄生虫生命周期不同阶段的活性。重要的是，所有 这些药物在体内对寄生虫的清除速度很快。对其中几种化合物产生抗药性的寄生虫 显示了P型ATPase PfATP4中的一系列突变，现在被认为是Na+泵。因此， 通过抑制PfATP4的Na+内流被认为是所有这些疾病的共同作用机制 化合物。我们在过去几年的工作表明，虽然PfATP4的突变对于 对于所有这些化合物的抗药性，它们并不总是足以产生全部水平的抗药性。我们 发现抗吡唑胺的寄生虫携带额外的突变，这些突变需要传递完整的 PfATP4突变与抗性有关，提示PfATP4的上位调控成分 活动。我们对Na+流入寄生虫的生理后果的研究揭示了一些 戏剧性的变化表明一条迄今未知的调控途径受到不适当的干扰 寄生虫的胞浆Na+水平。因此，彻底调查受影响的分子通路 破坏疟疾寄生虫的Na+稳态既是必要的，也可能提供进一步的见解 指导未来的药物发现和开发。基因编辑和条件性技术的最新进展 在恶性疟原虫中的基因表达使现在有可能以前所未有的方式解开这些途径 细节。通过应用这些方法，我们将研究PfATP4在维持Na+和 恶性疟原虫的胆固醇稳态。我们将评估与耐药性相关的表型后果 PfATP4的突变，并研究PfATP4以外的基因突变在 结合PfATP4突变。我们将研究一种可能的质膜胆固醇转运蛋白 这是受这些新抗疟疾药物的影响。这些研究将促进我们对新分子的理解 我们已经确认为有效抗疟疾药物开发中的靶点的途径。

With hundreds of millions of malaria cases being treated with antimalarial drugs each year and with each individual patient bearing hundreds of billions of malaria parasites, it is necessary to continue to feed the antimalarial pipeline with new drugs to counter the likely emergence of resistance. In recent years several novel antimalarial compounds have been discovered with the ability to disrupt Na+ homeostasis in malaria parasites. Four of these (a spiroindolone, a pyrazoleamide, a dihydroisoquinolone, and a thiotriazole) have been designated clinical drug candidates. Remarkably, these drugs belong to very different chemical classes with distinct pharmacophores and activity against different stages of malaria parasite life cycle. Importantly, all these drugs show fast clearance of parasites in vivo. Parasites resistant to several of these compounds have shown a range of mutations within a P-type ATPase, PfATP4, that is now believed to be a Na+ pump. Thus, influx of Na+ through inhibition of PfATP4 is considered to be the common mechanism of action for all these compounds. Our work over the last few years has revealed that, while mutations in PfATP4 are necessary for resistance to all of these compounds, they are not always sufficient to generate the full level of resistance. We have found that pyrazoleamide-resistant parasites bear additional mutations, which are required to impart full resistance in conjunction with PfATP4 mutations, suggesting epistatic regulatory components to PfATP4 activity. Our investigations of physiological consequences of Na+ influx into the parasite have revealed some dramatic changes suggesting a hitherto unknown regulatory pathway that is perturbed by inappropriate cytosolic Na+ levels in the parasite. Therefore, a thorough investigation of molecular pathways affected by disruption of Na+ homeostasis in malaria parasites is both necessary and likely to provide further insights to guide future drug discovery and development. Recent advances in technology for gene editing and conditional gene expression in Plasmodium falciparum make it now possible to unravel these pathways in unprecedented details. By applying these approaches, we will investigate the role of PfATP4 in maintenance of Na+ and cholesterol homeostasis in P. falciparum. We will assess phenotypic consequences of resistance-associated mutations in PfATP4, and study the role of mutations in genes other than PfATP4 that affect drug resistance in combination with PfATP4 mutations. We will investigate a putative plasma membrane cholesterol transporter that is affected by these new antimalarials. These studies will advance our understanding of novel molecular pathways that we have validated as targets for potent antimalarial drugs in development.