Hardy LW, Matthews W, Nare B, Beverley SM
Biotech 2, University of Massachusetts Medical Center, Worcester, Massachusetts, 01605, U.S.A.
Exp Parasitol. 1997 Nov;87(3):158-70.
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. Copyright 1997 Academic Press. Copyright 1997 Academic Press
对原生动物寄生虫利什曼原虫抗叶酸突变体的研究，为基因扩增和突变等遗传过程提供了有用信息，也让人们了解了这种原始真核生物中蝶啶代谢途径的独特特征。新型双功能二氢叶酸还原酶-胸苷酸合成酶（DHFR-TS）是一种必需酶，但大多数DHFR-TS抑制剂作为潜在药物的前景不佳。利什曼原虫拥有一种新型的替代蝶啶还原酶（PTR1），它对甲氨蝶呤相对不敏感。我们提出，PTR1作为代谢旁路并调节药物对DHFR-TS活性的抑制作用的能力，可能是许多抗叶酸药物疗效不佳的原因。在这项工作中，我们从74种化合物中寻找大利什曼原虫PTR1的抑制剂。对最有效的抑制剂也进行了针对大利什曼原虫DHFR-TS和人DHFR的测试，并鉴定出了几种仅对PTR1或对所有三种还原酶都有良好活性的化合物。测试了这些化合物对野生型前鞭毛体的活性，那些对PTR1和DHFR-TS都有强效抑制作用的化合物（但不包括仅对PTR1有活性的化合物）显示出良好的效力。利用缺乏PTR1或DHFR-TS（ptr1(-)和dhfr-ts基因敲除）或过表达PTR1的大利什曼原虫突变体的生长抑制试验作为“遗传筛选”，来评估这两种蝶啶还原酶在体内是否为靶点。值得注意的是，只有一种化合物表现出类似甲氨蝶呤的抑制模式。六种化合物无论PTR1或DHFR-TS水平如何，都对利什曼原虫生长有良好抑制作用。这些发现表明，利什曼原虫细胞含有多种抗叶酸药物的靶点，除了DHFR-TS和PTR1外还有一个或多个重要靶点。这强调了在合理设计利什曼原虫化疗策略的努力中，联合生化和遗传筛选的必要性。版权所有1997年学术出版社。版权所有1997年学术出版社
