ANTIFOLATE-RESISTANT MUTANTS OF PLASMODIUM-FALCIPARUM DIHYDROFOLATE-REDUCTASE

Single and multiple mutations at residues 16, 51, 59, 108, and 164 of Plasmodium falciparum dihydrofolate reductase (pfDHFR) have been linke d to antifolate resistance in malaria. We prepared and characterized a ll seven of the pfDHFR mutants found in nature, as well as six mutants not observed in nature, Mutations involving residues 51, 59, 108, or 164 conferred cross resistance to both the antifolates pyrimethamine a nd cycloguanil, whereas mutation of residue 16 specifically conferred resistance to cycloguanil. The antifolate resistance of enzyme mutants found in nature correlated with in vivo antifolate resistance; howeve r, mutants not found in nature were either poorly resistant or had ins ufficient catalytic activity to support DNA synthesis, Thus, specific combinations of multiple mutations at target residues were selected in nature to optimize resistance, Further, the resistance of multiple mu tants was more than the sum of the component single mutations, indicat ing that residues were selected for their synergistic as well as intri nsic effects on resistance, Pathways inferred for the evolution of pyr imethamine-resistant mutants suggested that all multiple mutants emerg ed from stepwise selection of the single mutant, S108N, Thus, we propo se that drugs targeted to both the wild-type pfDHFR and S108N mutant w ould have a low propensity for developing resistance, and hence could provide effective antimalarial agents.