Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil.
Increasing resistance of Plasmodium falciparum malaria parasites to chloroquine and the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and cycloguanil have sparked renewed interest in the antimalarial drugs WR99210 and proguanil, the cycloguanil precursor. To investigate suggestions that WR99210 and proguanil act against a target other than the reductase moiety of the P. falciparum bifunctional DHFR-thymidylate synthase enzyme, we have transformed P. falciparum with a variant form of human DHFR selectable by methotrexate. Human DHFR was found to fully negate the antiparasitic effect of WR99210, thus demonstrating that the only significant action of WR99210 is against parasite DHFR. Although the human enzyme also resulted in greater resistance to cycloguanil, no decrease was found in the level of susceptibility of transformed parasites to proguanil, thus providing evidence of intrinsic activity of this parent compound against a target other than DHFR. The transformation system described here has the advantage that P. falciparum drug-resistant lines are uniformly sensitive to methotrexate and will complement transformation with existing pyrimethamine-resistance markers in functional studies of P. falciparum genes. This system also provides an approach for screening and identifying novel DHFR inhibitors that will be important in combined chemotherapeutic formulations against malaria.
Fidock
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Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0425, USA.
Wellems
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D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D004261:DNA Replication; D018076:DNA, Complementary; D004351:Drug Resistance / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics; D002727:Proguanil / Q000494:pharmacology*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D014227:Triazines / Q000494:pharmacology*
D000962:Antimalarials; D018076:DNA, Complementary; D014227:Triazines; C006201:BRL 6231; D013762:Tetrahydrofolate Dehydrogenase; D002727:Proguanil; D008727:Methotrexate