Biochemical and genetic tests for inhibitors of Leishmania pteridine pathways.
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.
Hardy
L W
LW
Department of Pharmacology and Molecular Toxicology, University of Massachusetts Medical Center, Worcester 01605, USA.
Matthews
W
W
Nare
B
B
Beverley
S M
SM
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D004351:Drug Resistance; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D006801:Humans; D007891:Leishmania / Q000187:drug effects* / Q000235:genetics; D008727:Methotrexate / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000235:genetics; D011621:Pteridines / Q000737:chemistry / Q000378:metabolism*; D011743:Pyrimidines / Q000737:chemistry; D011799:Quinazolines / Q000737:chemistry; D013045:Species Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000235:genetics / Q000378:metabolism*
D005493:Folic Acid Antagonists; D009097:Multienzyme Complexes; D011621:Pteridines; D011743:Pyrimidines; D011799:Quinazolines; C056930:thymidylate synthase-dihydrofolate reductase; C017205:2,4-diaminopyrimidine; C013474:2,4-diaminoquinazoline; D010088:Oxidoreductases; C085592:pteridine reductase; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D008727:Methotrexate