Potent and selective activity of a combination of thymidine and 1843U89, a folate-based thymidylate synthase inhibitor, against Plasmodium falciparum

Abstract

Unlike mammalian cells, malarial parasites are completely dependent on the de novo pyrimidine pathway and lack the enzymes to salvage preformed pyrimidines. In the present study, first, it is shown that 1843U89, even without polyglutamylation, is a potent folate-based inhibitor of purified malarial parasite thymidylate synthase. The binding was noncompetitive with respect to methylenetetrahydrofolate, and 1843U89 had a K(i) of 1 nM. The compound also had potent antimalarial activity in vitro. Plasmodium falciparum cells in culture were inhibited by 1843U89, with a 50% inhibitory concentration of about 70 nM. The compound was effective against drug-sensitive as well as drug-resistant clones of P. falciparum. As predicted by the biochemistry of the parasite, the potent inhibition of parasite proliferation by 1843U89 could not be reversed with 10 microM thymidine. In contrast, in the presence of 10 microM thymidine, mammalian cells were unaffected by 1843U89 even at concentrations as high as 0.1 mM, thus offering a selectivity window of more than 1,000-fold. On this basis, folate-based thymidylate synthase inhibitors may represent a powerful additional tool that can be used to combat drug-resistant malaria.

FIG. 1

Comparison of structures of the substrate methylenetetrahydrofolate to the folate-based TS inhibitors D1694 and 1843U89.

FIG. 2

Inhibition of malarial parasite TS by folate-based inhibitors. (A) Comparison of enzyme inhibition by 1843U89 and D1694. The substrates were used at a subsaturating concentration of 2 μM for dUMP and 35 μM for methylenetetrahydrofolate (MTHF). Each assay was performed with 20 U of TS activity. (B) Double-reciprocal plot to assess inhibition of malarial parasite TS by various amounts of 1843U89 (1.25, 2.5, 5, and 10 nM) in the presence of a fixed concentration of 50 μM dUMP and between 25 and 200 μM methylenetetrahydrofolate. (C) Secondary plot to estimate the strength of binding between 1843U89 and malarial parasite TS.

FIG. 3

Inhibition of P. falciparum proliferation and mouse L1210 cell proliferation by 1843U89 in the presence of 10 μM thymidine. (A) Antiproliferative activity of P. falciparum (open squares) and mouse L1210 cells (open circles) in the absence of thymidine. (B) Antiproliferative activity of P. falciparum (closed squares) and mouse L1210 cells in the presence of 10 μM thymidine (closed circles).