The Plasmodial Surface Anion Channel And Malaria Parasite Nutrient Acquisition

Apicomplexan分子生理学研究组继续对原虫表面阴离子通道(PSAC)进行研究，并做出了两项重大贡献。 首先，我们确定并表征了一种新的基于PSAC(抗微生物药)药物摄取减少的抗疟疾耐药性机制。化学其他探员。52：2346,2008)。这一发现需要在体外筛选出对亮肽素具有抗药性的疟疾寄生虫，亮肽素是一种广泛特异性的半胱氨酸和丝氨酸蛋白酶抑制剂。由于亮肽素在感染细胞内有多个靶点，工作人员认为抗药性不会轻易获得。我们发现，亮氨酸抗性与半胱氨酸蛋白酶活性上调、半胱氨酸蛋白酶敏感性降低或寄生虫基因组中可能的半胱氨酸蛋白酶或丝氨酸蛋白酶的表达水平变化无关。相反，它与PSAC的显著变化有关，正如膜片钳和其他转运分析所测量的那样。PSAC拮抗剂能拮抗亮肽素对寄生虫生长的抑制作用，提示亮肽素只有在通过PSAC被动摄取后才能到达其细胞内靶点。抗药性寄生虫的通道介导显著降低了亮肽素的摄取，并可完全解释获得性抗药性。通过选择PSAC突变减少大量亲水性抗疟疾药物的被动摄取代表了一种新的抗疟疾耐药机制，应该在药物发现和开发计划中加以考虑。 其次，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识别出具有独特性质的新型拮抗剂。这些化合物包括对PSAC具有最高亲和力(低纳摩尔)和特异性的化合物，通过PSAC增加转运的新型激动剂，改变溶质选择性的化合物，以及为PSAC的结构和功能提供新见解的化合物。

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.