Utilization of exogenous folate in the human malaria parasite Plasmodium falciparum and its critical role in antifolate drug synergy

The antifolate combination pyrimethamine/sulphadoxine (PYR/SDX; Fansidar) i s frequently used to combat chloroquine-resistant malaria. Its success depe nds upon pronounced synergy between the two components, which target dihydr ofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) in the folat e pathway, This synergy permits clearance of parasites resistant to either drug alone, but its molecular basis is still unexplained, Plasmodium falcip arum can use exogenous folate, which is normally present in vivo, bypassing SDX inhibition of DHPS and, apparently, precluding synergy under these con ditions, However, we have measured parasite inhibition by SDX/PYR combinati ons in assays in which folate levels are strictly controlled, In parasites that use exogenous folate efficiently, SDX inhibition can be restored by le vels of PYR significantly lower than those required to inhibit DHFR. Isobol ograms show that the degree of synergy between PYR and SDX is highly depend ent upon prevailing folate concentrations and are indicative of PYR acting to block folate uptake and/or utilization. No significant synergy was obser ved at physiological drug levels when PYR/SDX acted on purified DHFR, wheth er wild type or mutant. We conclude that the primary basis for antifolate s ynergy in these organisms arises from PYR targeting a site (or sites) in ad dition to DHFR, which restores DHPS as a relevant target for SDX.