Identification of inhibitors of Plasmodium falciparum phosphoethanolamine methyltransferase using an enzyme-coupled transmethylation assay
Identification of inhibitors of Plasmodium falciparum phosphoethanolamine methyltransferase using an enzyme-coupled transmethylation assay
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DOI：
10.1186/1471-2091-11-4
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发表时间：
2010-01-19
期刊：
影响因子：
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通讯作者：
Ben Mamoun, Choukri
中科院分区：
文献类型：
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作者：
Bobenchik, April M.;Choi, Jae-Yeon;Ben Mamoun, Choukri
背景：恶性疟原虫的磷乙醇胺甲基转移酶(PfPMT)是新近发现的仅存在于一些原生动物、线虫、蛙类和植物中的磷乙醇胺甲基转移酶(PMT)家族的成员，它参与主要膜磷脂磷脂酰胆碱的合成。PMT酶催化磷乙醇胺氮原子的S-腺苷甲硫氨酸依赖的三步甲基化生成磷胆碱。在恶性疟原虫中，这种活性是丝氨酸合成磷脂酰胆碱途径中的限制性步骤，在人红细胞内寄生虫的发育、复制和生存中发挥着重要作用。结果：我们采用了一种酶偶联甲基化试验来筛选PfPMT的潜在抑制剂。除了已知的抑制PfPMT的十六烷基三甲基铵外，两种化合物十二烷基三甲基铵和阿莫地喹在体外也被发现抑制PfPMT的活性。有趣的是，PfPMT活性不被阿莫地喹类似物、氯喹或其他氨基喹啉、氨基醇或组胺甲基转移酶抑制剂抑制。使用酵母作为替代系统，我们发现与野生型细胞不同，依赖PfPMT生存的酵母突变株对阿莫地喹敏感，其磷脂酰胆碱的生物合成被该化合物抑制。此外，核磁共振滴定研究表明，阿莫地喹与PfPMT之间的相互作用具有特异性和浓度依赖性。结论：阿莫地喹在体外和酵母中都是PfPMT的抑制剂，以及该化合物与该酶特异性相互作用的生物物理证据将为该药物的类似物的开发奠定基础，从而特异性地抑制该酶和可能的其他PMTs。
Background: The phosphoethanolamine methyltransferase, PfPMT, of the human malaria parasite Plasmodium falciparum, a member of a newly identified family of phosphoethanolamine methyltransferases (PMT) found solely in some protozoa, nematodes, frogs, and plants, is involved in the synthesis of the major membrane phospholipid, phosphatidylcholine. PMT enzymes catalyze a three-step S-adenosylmethionine-dependent methylation of the nitrogen atom of phosphoethanolamine to form phosphocholine. In P. falciparum, this activity is a limiting step in the pathway of synthesis of phosphatidylcholine from serine and plays an important role in the development, replication and survival of the parasite within human red blood cells.Results: We have employed an enzyme-coupled methylation assay to screen for potential inhibitors of PfPMT. In addition to hexadecyltrimethylammonium, previously known to inhibit PfPMT, two compounds dodecyltrimethylammonium and amodiaquine were also found to inhibit PfPMT activity in vitro. Interestingly, PfPMT activity was not inhibited by the amodiaquine analog, chloroquine, or other aminoquinolines, amino alcohols, or histamine methyltransferase inhibitors. Using yeast as a surrogate system we found that unlike wildtype cells, yeast mutants that rely on PfPMT for survival were sensitive to amodiaquine, and their phosphatidylcholine biosynthesis was inhibited by this compound. Furthermore NMR titration studies to characterize the interaction between amoidaquine and PfPMT demonstrated a specific and concentration dependent binding of the compound to the enzyme.Conclusion: The identification of amodiaquine as an inhibitor of PfPMT in vitro and in yeast, and the biophysical evidence for the specific interaction of the compound with the enzyme will set the stage for the development of analogs of this drug that specifically inhibit this enzyme and possibly other PMTs.