The Plasmodial Surface Anion Channel And Malaria Parasite Nutrient Acquisition

顶复门分子生理学组继续研究疟原虫表面阴离子通道（PSAC），并作出了两项重大贡献。 首先，我们确定并表征了一种新的抗疟药耐药机制，该机制基于通过PSAC（Antimicrob.探员Chemother 52：2346，2008）。 这一发现需要在体外选择对亮抑酶肽（一种广泛特异性半胱氨酸和丝氨酸蛋白酶抑制剂）具有抗性的疟原虫。 由于亮抑酶肽在受感染的细胞内有多个靶点，研究人员认为耐药性不会轻易获得。 我们发现亮肽素耐药性与半胱氨酸蛋白酶活性的上调、该活性的亮肽素敏感性降低或寄生虫基因组中推定的半胱氨酸或丝氨酸蛋白酶的表达水平变化无关。相反，它与PSAC的显著变化相关，如用膜片钳和其他转运试验所测量的。 发现PSAC拮抗剂拮抗亮抑酶肽对寄生虫生长的抑制，表明亮抑酶肽仅在通过PSAC被动摄取后才能接近其细胞内靶标。 抗性寄生虫的通道介导的显着减少亮抑酶肽摄取，并可以完全解释获得性抗性。 通过选择PSAC中的突变减少大体积亲水性抗疟药的被动摄取代表了一种新的抗疟药耐药机制，药物发现和开发计划应考虑这种机制。 第二，我们对PSAC拮抗剂的小分子文库进行了额外的高通量筛选。 这些筛选已经成功地鉴定出具有独特性质的新型拮抗剂。 这些包括具有最高已知亲和力（低纳摩尔）和PSAC抑制特异性的化合物，增加通过PSAC转运的新型激动剂，改变溶质选择性的化合物，以及提供对PSAC结构和功能的新见解的化合物。

The Apicomplexan Molecular Physiology Section continued its studies of the plasmodial surface anion channel (PSAC) and made two significant contributions. First, we identified and characterized a novel antimalarial drug resistance mechanism based on reduced drug uptake via PSAC (Antimicrob. Agents Chemother. 52:2346, 2008). This discovery entailed in vitro selection of malaria parasites resistant to leupeptin, a broad specificity cysteine and serine protease inhibitor. Because leupeptin has multiple targets within infected cells, workers assumed that resistance would not be acquired easily. We found that leupeptin resistance was not associated with upregulation of cysteine protease activity, reduced leupeptin sensitivity of this activity, or expression level changes for putative cysteine or serine proteases in the parasite genome. Instead, it was associated with marked changes in PSAC, as measured with patch-clamp and other transport assays. PSAC antagonists were found to antagonize parasite growth inhibition by leupeptin, suggesting that leupeptin can access its intracellular targets only after passive uptake via PSAC. The resistant parasite's channel mediated significantly reduced leupeptin uptake and could fully account for the acquired resistance. Reduced passive uptake of bulky hydrophilic antimalarial agents via selection of mutations in PSAC represents a novel antimalarial drug resistance mechanism that should be considered by drug discovery and development programs. Second, we carried out additional high-throughput screens of small molecule libraries for PSAC antagonists. These screens have successfully identified novel antagonists with unique properties. These include compounds that have the highest known affinity (low nanomolar) and specificity for PSAC inhibition, novel agonists that increase transport via PSAC, compounds that alter solute selectivity, and compounds that provide new insights into the structure and function of PSAC.