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 ch loroquine and the dihydrofolate reductase (DHFR) inhibitors pyrimetham ine 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 tha n the reductase moiety of the P. falciparum bifunctional DHFR-thymidyl ate 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 cyclo guanil, no decrease was found in the level of susceptibility of transf ormed parasites to proguanil, thus providing evidence of intrinsic act ivity of this parent compound against a target other than DHFR, The tr ansformation system described here has the advantage that P. falciparu m drug-resistant lines are uniformly sensitive to methotrexate and wil l complement transformation with existing pyrimethamine-resistance mar kers in functional studies of P. falciparum genes, This system also pr ovides an approach for screening and identifying novel DHFR inhibitors that will be important in combined chemotherapeutic formulations agai nst malaria.