Action of pyrimethamine and related drugs againstPlasmodium knowlesi in vitro

Abstract

A dihydrofolate reductase has been isolated from free-parasite preparations of the primate malarial parasite,Plasmodium knowlesi. Its properties are similar to those reported for the enzyme from the rodent malarial parasite,P. berghei,even though the base compositions of the DNA of these two species are quite different. TheKmsfor substrate and cofactor are 3 × 10−6Mand 1 × 10−6Mrespectively and the molecular weight is ˜ 200000. Concentrations of pyrimethamine and trimethoprim as low as 1 × 10−9Mand 3 × 10−8Mrespectively are sufficient to cause 50% inhibition of enzyme activity. The sensitivity to inhibition by pyrimethamine and trimethoprim of growth of cultures ofP. knowlesihas also been investigated. Preliminary experiments showed that it was only the schizont stage that was susceptible to the action of the drugs and that in their presence, normal nuclear divisions and segmentation did not occur and subsequently, no reinvasion of fresh red cells took place. The minimum concentrations of drug required to produce these effects were 10−9Mfor pyrimethamine and 10−7Mfor trimethoprim. Thus, there is a close correlation between the concentrations of pyrimethamine and trimethoprim required to inhibit the dihydrofolate reductase in a cell-free system and the growth of the parasitein vitro. Pyrimethamine (10−9M) did not, however, affect the incorporation of radioactive precursors into DNA, RNA or protein of schizont stage parasites until after morphological damage could be seen and reinvasion was complete in control cultures. The time courses of incorporation of [14C]algal protein hydrolysate into protein in the presence (10−9M) or absence of pyrimethamine are the same as those described recently with immune serum. The possibility is thus raised as to whether pyrimethamine and immune serum act in the same way.One of us (P.I.T.) received financial assistance from the World Health Organization. We thank Dr F. Hawking for many helpful discussions, Miss Jane Dunnett and Mr T. Scott-Finnigan for technical assistance and Dr O. D. Standen for samples of pyrimethamine and trimethoprim.