Cellular and Molecular Physiology of Bloodstream Malaria Parasites

2022年，抗疟原虫表面阴离子通道(PSAC)是感染疟疾寄生虫的红细胞膜上的一种基本营养和离子通道，Apicomexan分子生理科推进了针对原虫表面阴离子通道的药物开发。PSAC活性和与该通道相连的三元RHopH蛋白复合体在所有被研究的疟原虫物种中都是保守的。由于这个通道在其他属中是不存在的，它似乎是治疗发展的一个重要靶点。在一项研究中，我们使用恶性疟原虫和MBX-2366的衍生物从高通量筛选中产生了一种有效和特定的药物铅，MBX-2366是一种纳米分子亲和型哒嗪酮抑制剂。由于这种筛选缺乏体内疗效所需的生物利用度和稳定性，我们合成了315个衍生物来优化类药物特性，建立靶标特异性，并保持有效的抗寄生虫诱导的通透性。使用一个健壮的迭代流水线，我们产生了MBX-4055，一种对不同的人类寄生虫株有活性的衍生物。MBX-4055改善了口服吸收，具有可接受的体内耐受性和药代动力学。它对由35个人类通道和受体组成的电池也没有活性，并且在扩展的体外选择中对获得性耐药性是难耐的。单分子和单细胞膜片钳显示对PSAC的直接作用。这些研究确定哒嗪酮是一种新的、易处理的抗疟疾支架，具有明确的作用机制。摩尔。《药物学》，出版中(2022)。PMID：35798366 在另一项研究中，我们开发并使用了一种新的插入和表面暴露报告(RISE)来跟踪疟疾寄生虫蛋白向宿主红细胞膜的传递。Rise技术可以通过DNA转染将NanoLuc的11个氨基酸的HiBit小片段插入到目标蛋白中，从而连续无损地跟踪感染细胞上的抗原暴露；通过与LgBit的高亲和力互补来检测细胞表面的HiBit表位标记暴露，以产生发光。我们跟踪了CLAG3的输出和表面暴露，CLAG3是一种与PSAC活性和营养吸收有关的寄生虫蛋白，在人类红细胞中的恶性疟原虫周期中。我们的方法揭示了人口贩运和表面暴露的关键决定因素。C-末端跨膜结构域的移除中止了出口。出乎意料的是，暴露的报告分子大小的某些增加改善了发光信号，但其他变化消除了表面信号，揭示了细胞外表位的大小和电荷都影响膜插入。带有包含多个HiBit表位的较大插入的显著细胞间差异表明，CLAG3在宿主膜上的插入调控复杂。因此，对CLAG3表面暴露的定量、连续跟踪揭示了决定该蛋白在宿主红细胞膜上的运输和插入的多种因素。RISE分析不仅能够从不同的细胞内病原体中确定表面暴露的抗原，而且可以提供膜蛋白递送和表面暴露的拓扑和动力学信息。MBio 13：e0040422(2022)。PMID：35420481

In 2022, the Apicomplexan Molecular Physiology Section Malaria advanced drug development against the plasmodial surface anion channel (PSAC), an essential nutrient and ion channel at the host membrane of erythrocytes infected with malaria parasites. Both PSAC activity and the ternary RhopH protein complex linked to this channel are conserved in all examined Plasmodium species. Because this channel is absent from other genera, it appears to be an important target for therapy development. In one study, we generated a potent and specific drug lead using Plasmodium falciparum, a virulent human pathogen, and derivatives of MBX-2366, a nanomolar affinity pyridazinone inhibitor from a high-throughput screen. As this screening hit lacks the bioavailability and stability needed for in vivo efficacy, we synthesized 315 derivatives to optimize drug-like properties, establish target specificity, and retain potent activity against the parasite-induced permeability. Using a robust, iterative pipeline, we generated MBX-4055, a derivative active against divergent human parasite strains. MBX-4055 has improved oral absorption with acceptable in vivo tolerability and pharmacokinetics. It also has no activity against a battery of 35 human channels and receptors and was refractory to acquired resistance during extended in vitro selection. Single-molecule and single-cell patch-clamp indicate direct action on PSAC. These studies identify pyridazinone as a novel and tractable antimalarial scaffold with a defined mechanism of action. Mol. Pharmacol., in press (2022). PMID: 35798366 In another study, we developed and used a novel Reporter of Insertion and Surface Exposure (RISE) to track delivery of malaria parasite proteins to the host erythrocyte membrane. The RISE technology permits continuous nondestructive tracking of antigen exposure on infected cells though DNA transfection to insert a small 11-amino acid HiBit fragment of NanoLuc into a target protein; HiBit epitope tag exposure at the cell surface is detected through high-affinity complementation with LgBit to produce luminescence. We tracked the export and surface exposure of CLAG3, a parasite protein linked to PSAC activity and nutrient uptake, throughout the Plasmodium falciparum cycle in human erythrocytes. Our approach revealed key determinants of trafficking and surface exposure. Removal of a C-terminal transmembrane domain aborted export. Unexpectedly, certain increases in the exposed reporter size improved the luminescence signal, but other changes abolished the surface signal, revealing that both size and charge of the extracellular epitope influence membrane insertion. Marked cell-to-cell variation with larger inserts containing multiple HiBit epitopes suggests complex regulation of CLAG3 insertion at the host membrane. Quantitative, continuous tracking of CLAG3 surface exposure thus reveals multiple factors that determine this protein's trafficking and insertion at the host erythrocyte membrane. The RISE assay will not only enable confident identification of surface-exposed antigens from divergent intracellular pathogens, but can inform topology and kinetics of membrane protein delivery and surface exposure. mBio 13:e0040422 (2022). PMID: 35420481