Cellular and Molecular Physiology of Bloodstream Malaria Parasites

2022年，apiccomplex an Molecular Physiology Section Malaria推进了针对疟原虫感染红细胞宿主膜上必需营养物质和离子通道plasmodial surface阴离子通道（PSAC）的药物开发。PSAC活性和与该通道相连的三元RhopH蛋白复合物在所有被检测的疟原虫物种中都是保守的。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，我们利用恶性疟原虫（一种致命的人类病原体）和MBX-2366（一种高通量筛选的纳米摩尔亲和力吡嗪酮抑制剂）的衍生物产生了一种强效和特异性的药物先导物。由于这种筛选结果缺乏体内有效性所需的生物利用度和稳定性，我们合成了315个衍生物来优化药物样特性，建立靶点特异性，并保持对寄生虫诱导的渗透性的有效活性。使用稳健的迭代管道，我们生成了MBX-4055，一种对不同的人类寄生虫菌株具有活性的衍生物。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。它也对35种人类通道和受体没有活性，并且在延长的体外选择中难以获得性耐药。单分子和单细胞膜片钳表明直接作用于PSAC。这些研究确定吡嗪酮是一种新的、易于处理的抗疟疾支架，具有明确的作用机制。摩尔。杂志。，出版（2022）。PMID: 35798366

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