Emerson Lyndal R, Nau Martin E, Martin Rodger K, Kyle Dennis E, Vahey Maryanne, Wirth Dyann F
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Antimicrob Agents Chemother. 2002 Mar;46(3):787-96. doi: 10.1128/AAC.46.3.787-796.2002.
Chloroquine is one of the most effective antimalarials, but resistance to it is becoming widespread. However, we do not fully understand either the drug's mode of action or the mechanism of resistance. In an effort to expand our understanding of the mechanism of action and resistance associated with chloroquine, we used Saccharomyces cerevisiae as a model eukaryotic system. To aid in the discovery of potential drug targets we applied the transcriptional profiling method to identify genes transcriptionally responsive to chloroquine treatment in S. cerevisiae. Among the genes that were differentially expressed with chloroquine treatment were a number of metal transporters involved in iron acquisition (SIT1, ARN2, ARN4, and SMF2). These genes exhibit similar expression patterns, and several are known to be regulated by AFT1, a DNA binding protein, which responds to iron levels in the cell. We investigated the role of chloroquine in iron metabolism by using a variety of approaches, including pharmacological, genetic, and biochemical techniques. For these experiments, we utilized yeast lacking the major iron uptake pathways (FET3 and FET4) and yeast deficient in SIT1, encoding the major up-regulated iron siderophore transporter. Our experiments show that yeast genetically or environmentally limited in iron availability has increased sensitivity to chloroquine in pharmacological assays and that the addition of iron rescues these cells from chloroquine killing. 55FeCl3 accumulation was inhibited in the presence of chloroquine, and kinetic analysis demonstrated that inhibition was competitive. These results are consistent with deprivation of iron as a mechanism of chloroquine killing in yeast.
氯喹是最有效的抗疟药物之一，但对其的耐药性正在广泛传播。然而，我们对该药物的作用模式或耐药机制都尚未完全了解。为了扩展我们对与氯喹相关的作用机制和耐药性的理解，我们使用酿酒酵母作为真核生物模型系统。为了有助于发现潜在的药物靶点，我们应用转录谱分析方法来鉴定酿酒酵母中对氯喹处理有转录反应的基因。在经氯喹处理后差异表达的基因中，有一些参与铁摄取的金属转运蛋白（SIT1、ARN2、ARN4和SMF2）。这些基因表现出相似的表达模式，并且已知其中几个受AFT1调控，AFT1是一种DNA结合蛋白，对细胞内的铁水平有反应。我们通过使用多种方法，包括药理学、遗传学和生物化学技术，研究了氯喹在铁代谢中的作用。对于这些实验，我们使用了缺乏主要铁摄取途径（FET3和FET4）的酵母以及缺乏编码主要上调的铁载体转运蛋白的SIT1的酵母。我们的实验表明，在药理学试验中，铁可用性在遗传上或环境上受到限制的酵母对氯喹的敏感性增加，并且添加铁可使这些细胞免受氯喹杀伤。在氯喹存在的情况下，55FeCl3的积累受到抑制，动力学分析表明这种抑制是竞争性的。这些结果与铁剥夺作为酵母中氯喹杀伤机制是一致的。
