Potentiating effect of pyrimethamine and sulfadoxine against dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant Plasmodium chabaudi

Abstract

Dihydrofolate reductase was partially purified from a pyrimethamine-sensitive Plasmodium chabaudi clone and a pyrimethamine-resistant clone derived from it and used in a study of the inhibitory effect of pyrimethamine and sulfadoxine, both alone and in combination. Kinetic analysis of the inhibitory effect of sulfadoxine against the enzyme from pyrimethamine-sensitive and -resistant parasites revealed that the drug inhibited the former enzyme competitively, with an inhibition constant (K(is)) of 0.7 ± 0.4 mM, but inhibited the latter enzyme noncompetitively, with K(is) and K(ii) of 8.9 ± 1.2 and 4.1 ± 1.2 mM, respectively. Previous studies also showed competitive inhibition by pyrimethamine on the former enzyme and noncompetitive inhibition on the latter enzyme, with some 200-fold-lower affinity. Sulfadoxine and pyrimethamine exhibited a mutually potentiating effect on the enzyme activity, as revealed by the concave isoboles and the fractional inhibitions of less than unity. A potentiating effect was observed for the enzymes from both sources and was not dependent on the degree of the purification of the enzyme. Our results can be explained by assuming simultaneous binding of two inhibitors on the enzyme.