Biochemical and genetic tests for inhibitors of Leishmania pteridine pathways
Biochemical and genetic tests for inhibitors of Leishmania pteridine pathways
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DOI：
10.1006/expr.1997.4207
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发表时间：
1997-11-01
影响因子：
2.1
通讯作者：
Beverley, SM
中科院分区：
文献类型：
--
作者：
Hardy, LW;Matthews, W;Beverley, SM
抗叶酸突变体的原生动物寄生虫利什曼原虫的研究提供了有用的信息，如基因扩增和突变的遗传过程和知识的独特功能的蝶啶代谢途径在这个原始的真核生物。新型双功能二氢叶酸还原酶-胸苷酸合酶（DHFR-TS）是一种重要的酶，但大多数DHFR-TS抑制剂作为潜在药物的前景不大。利什曼原虫具有一种新的替代性蝶啶还原酶（PTR 1），对甲氨蝶呤相对不敏感。我们已经提出PTR 1作为代谢旁路的能力，从而调节药物对DHFR-TS活性的抑制，这可能是许多抗叶酸剂疗效不佳的原因。在这项工作中，我们已经寻找L.主要PTR 1从74个化合物的集合。最有效的抑制剂也进行了测试，对L。鉴定了主要的DHFR-TS和人DHFR，以及对PTR 1单独或对所有三种还原酶显示良好活性的几种化合物。测试这些化合物对野生型前鞭毛体的活性，并且是PTR 1和DHFR-TS两者的有效抑制剂的那些（但不是仅对PTR 1有活性的那些）显示出良好的效力。生长抑制试验。缺乏PTR 1或DHFR-TS（ptr 1（-）和dhfr-TS（-）敲除）或过表达PTR 1的主要突变体被用作“遗传筛选”以评估这两种蝶啶还原酶是否是体内靶。值得注意的是，只有一种化合物显示出甲氨蝶呤样抑制模式。六种化合物显示出良好的抑制利什曼原虫生长，无论PTR 1或DHFR-TS水平。这些发现表明，利什曼原虫细胞含有多种抗叶酸剂的多个靶点，除了DHFR-TS和PTR 1外，还有一个或多个重要的靶点。这强调了结合生化和遗传筛选的必要性，以合理设计利什曼原虫的化疗策略。(C)北京：科学出版社.
The study of antifolate-resistant mutants of the protozoan parasite Leishmania has provided useful information about genetic processes such as gene amplification and mutation and knowledge of the unique features of the pteridine metabolic pathway in this primitive eukaryote. The novel bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is an essential enzyme, yet most DHFR-TS inhibitors show little promise as potential drugs. Leishmania possess a novel alternative pteridine reductase (PTR1) which is relatively insensitive to methotrexate. We have proposed that the ability of PTR1 to serve as a metabolic bypass and thus modulate drug inhibition of DHFR-TS activity may be responsible for the poor efficacy of many antifolates. In this work, we have sought inhibitors of L. major PTR1 from a collection of 74 compounds. The most potent inhibitors were also tested against L. major DHFR-TS and human DHFR and several compounds showing good activity for PTR1 alone, or for all three reductases, were identified. The activity of these compounds was tested against wild-type promastigotes, and those which were potent inhibitors of both PTR1 and DHFR-TS (but not those active against only PTR1) showed good potencies. Growth inhibition tests of L. major mutants, lacking PTR1 or DHFR-TS (ptr1(-) and dhfr-ts(-) knockouts) or overexpressing PTR1, were used as a ''genetic screen'' to assess whether these two pteridine reductases were targets in vivo. Remarkably, only one compound showed a methotrexate-like pattern of inhibition. Six compounds showed good inhibition of Leishmania growth regardless of PTR1 or DHFR-TS levels. These findings suggest that Leishmania cells contain multiple targets for a diverse set of antifolates, with one or more significant targets in addition to DHFR-TS and PTR1. This emphasizes the necessity of combined biochemical and genetic screens in efforts to rationally design chemotherapeutic strategies in Leishmania. (C) 1997 Academic Press.