Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, 333 Cedar St., New Haven 06052, USA.
BMC Biochem. 2010 Jan 19;11:4. doi: 10.1186/1471-2091-11-4.
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.
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 wild-type 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.
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.
恶性疟原虫 PfPMT（磷酸乙醇胺甲基转移酶）属于磷酸乙醇胺甲基转移酶（PMT）家族的一个新成员，该家族仅存在于一些原生动物、线虫、青蛙和植物中，其参与主要膜磷脂——磷脂酰胆碱的合成。PMT 酶催化 S-腺苷甲硫氨酸依赖性的三步磷酸乙醇胺氮原子甲基化，形成磷酸胆碱。在恶性疟原虫中，该活性是丝氨酸合成磷脂酰胆碱途径中的限速步骤，在寄生虫在人类红细胞内的发育、复制和存活中发挥重要作用。
我们采用酶偶联甲基化测定法筛选 PfPMT 的潜在抑制剂。除了先前已知抑制 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氨苯砜，也被发现可抑制 PfPMT 的体外活性。有趣的是，PfPMT 活性不受氨苯砜类似物——氯喹或其他氨基喹啉、氨基醇或组氨酸甲基转移酶抑制剂的抑制。我们利用酵母作为替代系统发现，与野生型细胞不同，依赖 PfPMT 存活的酵母突变体对氨苯砜敏感，且该化合物可抑制其磷脂酰胆碱的生物合成。此外，用于表征氨苯砜与 PfPMT 之间相互作用的 NMR 滴定研究表明，该化合物与酶的特异性和浓度依赖性结合。
在体外和酵母中鉴定出氨苯砜是 PfPMT 的抑制剂，以及该化合物与酶的特异性相互作用的生物物理证据，将为开发专门抑制该酶和可能其他 PMT 的此类药物的类似物奠定基础。
Zotero 插件
