The Plasmodial Surface Anion Channel And Malaria Parasite Nutrient Acquisition

2010年，Apicomplexan分子生理科为针对原生生物表面阴离子通道(PSAC)的药物发现做出了重大贡献。该通道的一些抑制剂已经存在多年，但由于这些化合物的PSAC亲和力较低，因此一直没有用于药物开发；它们还抑制人类离子通道或转运体，因此对抗疟疾的开发没有足够的特异性。为了解决这些问题，我们开发了一种PSAC活性的微型检测方法，并进行了高通量的抑制剂筛选。从合成和天然产物库中筛选了大约70,000个化合物，揭示了来自多个结构类别的抑制剂，包括两个新的和有效的杂环支架。单通道膜片钳研究表明，这些化合物直接作用于PSAC，进一步暗示了它们在不同溶质运输中的作用。然后，我们使用一种含有噻吩酮的单一支架的衍生物来检查PSAC活性是否对细胞内寄生虫起重要作用。这类化合物在通道抑制和体外寄生虫杀灭之间显示出统计上的显著相关性，为PSAC作为药物靶点提供了化学验证。这些新的抑制剂应该是重要的研究工具，并可能成为急需的抗疟疾药物的起点。 Apicomplexan分子生理科还通过鉴定赋予寄生虫对水溶性抗疟疾药物耐药性的新的PSAC突变体，为了解抗疟疾机制做出了贡献。此前，我们发现两种这样的毒素，杀菌素S和亮胃素，能够选择PSAC活性改变的突变寄生虫，表明通过减少通道介导的毒素摄取而获得抗性。2010年，我们确定了这些突变通道的性质取决于施加的选择压力：仅用亮肽素或杀菌素S选择产生的Psac突变株不能保护细胞内的寄生虫不被另一种毒素杀死。亮肽素在杀菌素S抗性突变体中的通透性相对保存，与其在体外保留对亮肽素的敏感性一致。随后两种毒素的体外选择产生了一个双突变寄生虫，在PSAC中有额外的变化，为新的抗疟疾耐药机制提供了额外的证据。对这些突变体的鉴定显示，每个突变体上都有一个单一的保守通道，尽管具有不同的门控特性。这些发现与一个共同的通道相一致，该通道调节离子、营养物质和毒素的吸收。该通道的门控和选择性属性可以根据体外选择压力进行修改。我们认为PSAC有一个复杂的选择性过滤器，不同于细菌和高等生物中的模型离子通道。

In 2010, the Apicomplexan Molecular Physiology Section made a significant contribution to drug discovery targeting the plasmodial surface anion channel (PSAC). A number of inhibitors of this channel have been available for many years, but have not been pursued for drug development because those compounds have low PSAC affinity; they also inhibit human ion channels or transporters and therefore have inadequate specificity for antimalarial development. To address these concerns, we developed a miniaturized assay for PSAC activity and carried out a high-throughput inhibitor screen. Approximately 70,000 compounds from synthetic and natural product libraries were screened, revealing inhibitors from multiple structural classes including two novel and potent heterocyclic scaffolds. Single-channel patch-clamp studies indicated that these compounds act directly on PSAC, further implicating a proposed role in transport of diverse solutes. We then used derivatives of a single thiazepinone-containing scaffold to examine whether PSAC activity serves an essential role for the intracellular parasite. This family of compounds exhibited a statistically significant correlation between channel inhibition and in vitro parasite killing, providing chemical validation of PSAC as a drug target. These new inhibitors should be important research tools and may be starting points for much-needed antimalarial drugs. The Apicomplexan Molecular Physiology Section has also contributed to the understanding of antimalarial resistance mechanisms by identifying new PSAC mutants that confer parasite resistance to water-soluble antimalarials. Previously, we found that two such toxins, blasticidin S and leupeptin, are able to select for mutant parasites with altered PSAC activities, suggesting acquired resistance via reduced channel-mediated toxin uptake. In 2010, we determined that the properties of these mutant channels depend on the applied selective pressure: the PSAC mutants generated by selection with either leupeptin or blasticidin S alone do not protect the intracellular parasite from killing by the other toxin. Leupeptin permeability in the blasticidin S-resistant mutant is relatively preserved, consistent with retained in vitro susceptibility to leupeptin. Subsequent in vitro selection with both toxins generated a double mutant parasite having additional changes in PSAC, providing additional evidence for a novel antimalarial resistance mechanism. Characterization of these mutants revealed a single conserved channel on each mutant, albeit with distinct gating properties. These findings are consistent with a shared channel that mediates uptake of ions, nutrients and toxins. This channel's gating and selectivity properties can be modified in response to in vitro selective pressure. We propose that PSAC has a complex selectivity filter different from those of model ion channels in bacteria and higher organisms.