Cellular and Molecular Physiology of Bloodstream Malaria Parasites

2022年，Apicomplexan分子生理学部门疟疾推进了针对疟原虫表面阴离子通道（PSAC）的药物开发，PSAC是感染疟疾寄生虫的红细胞宿主膜上的必需营养素和离子通道。 这两个PSAC活性和三元RhopH蛋白复合物连接到这个通道是保守的，在所有研究的疟原虫物种。 由于该通道在其他属中不存在，因此它似乎是治疗开发的重要靶点。 在一项研究中，我们使用恶性疟原虫（一种致命的人类病原体）和MBX-2366（一种来自高通量筛选的纳摩尔亲和力哒嗪酮抑制剂）的衍生物产生了一种有效且特异的药物先导。由于这种筛选缺乏体内功效所需的生物利用度和稳定性，我们合成了315种衍生物，以优化药物样性质，建立靶标特异性，并保留对寄生虫诱导的渗透性的有效活性。使用强大的迭代管道，我们生成了MBX-4055，这是一种对不同人类寄生虫菌株具有活性的衍生物。MBX-4055具有改善的口服吸收，具有可接受的体内耐受性和药代动力学。它也对35种人类通道和受体的电池没有活性，并且在延长的体外选择过程中不易获得抗性。单分子和单细胞膜片钳显示对PSAC的直接作用。这些研究确定了哒嗪酮作为一种新的和易处理的抗疟支架，具有明确的作用机制。 摩尔药理学，出版中（2022年）。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）来跟踪疟原虫蛋白质向宿主红细胞膜的传递。 RISE技术通过DNA转染将NanoLuc的小的11个氨基酸HiBit片段插入靶蛋白，允许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表位标签暴露，以产生发光。我们跟踪了CLAG 3的出口和表面暴露，CLAG 3是一种与PSAC活性和营养摄取相关的寄生虫蛋白，在人红细胞中的恶性疟原虫周期中。我们的方法揭示了贩运和表面暴露的关键决定因素。去除C-末端跨膜结构域中止输出。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。含有多个HiBit表位的较大插入物的显著细胞间变异表明CLAG 3插入宿主膜的复杂调节。因此，CLAG 3表面暴露的定量、连续跟踪揭示了决定这种蛋白质在宿主红细胞膜上的运输和插入的多种因素。RISE检测不仅能够可靠地鉴定来自不同细胞内病原体的表面暴露抗原，而且可以告知膜蛋白递送和表面暴露的拓扑结构和动力学。 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