In vitro susceptibilities of Plasmodium falciparum to compounds which inhibit nucleotide metabolism

Abstract

A unique metabolic feature of malaria parasites is their restricted ability to synthesize nucleotides. These parasites are unable to synthesize the purine ring and must therefore obtain preformed purine bases and nucleosides from the host cell, the erythrocyte. On the other hand, pyrimidines must be synthesized de novo because of the inability of the parasites to salvage preformed pyrimidines. Thus, one would anticipate that the blockage of purine salvage or pyrimidine de novo synthesis should adversely affect parasite growth. This premise was tested in vitro with a total of 64 compounds, mostly purine and pyrimidine analogs, known to inhibit one or more steps of nucleotide synthesis. Of the 64 compounds, 22 produced a 50% inhibition of the growth of the human malaria parasite Plasmodium falkiparum at a concentration of 50 ,uM or less. Inhibition of the growth of chloroquine-resistant clones ofP. fakciparum did not differ significantly from that of the growth of chloroquine-susceptible clones. Two of the compounds which effectively inhibited parasite growth, 6-mercaptopurine and 6-thioguanine, were found to be potent competitive inhibitors of a key purine-salvaging enzyme (hypoxanthine-guanine-xanthine phosphoribosyltransferase) of the parasite.The worldwide resurgence of human malaria during the past decade (60) has been due in part to the rapid spread of Plasmodium falciparum strains resistant to chloroquine and other commonly used antimalarial drugs (21, 41). In view of this resurgence, there is currently an urgent need to develop and identify additional safe and effective drugs (47).A rational approach to drug development is to identify drugs which can be directed at unique metabolic targets in the parasite. One such target is the synthesis of purine and pyrimidine nucleotides. Unlike humans, malaria parasites cannot make purines de novo and must therefore obtain purine bases and nucleosides from the erythrocyte host cell and then metabolize these to nucleotides by salvage pathways (54). Another metabolic distinction between these parasites and humans is that P. falciparum relies exclusively on pyrimidine synthesis de novo because of its inability to salvage preformed pyrimidines (54). Thus, drugs capable of blocking either purine salvage or pyrimidine de novo synthesis would be expected to show strong antimalarial activity.In the present study, we measured the in vitro antimalarial activity of 64 compounds known to inhibit one or more aspects of nucleotide or nucleic acid metabolism. A number of these compounds were also tested for their ability to inhibit a key purine-salvaging enzyme (43)

Comparative complement selection in bacteria enables screening for lead compounds targeted to a purine salvage enzyme of parasites

“…This observation is consistent with the conclusion that the 6 position (19). Previous kinetic analyses of these compounds as inhibitors showed that the K i s (at 0.42 M for thioguanine and 0.55 M for 6-mercaptopurine) were about 1 order of magnitude lower than the reported K i s (at 2.4 M for thioguanine and 3.9 M for 6-mercaptopurine) of the corresponding enzyme from human erythrocytes (14,19). The previous studies of these two compounds permit them to be used as standards for this recombinant screening method.…”

“…Of seven additional compounds that we would predict to have at least some antimalarial activity (compounds 40, 44, 48, and 51 to 54) on the basis of their effects on the growth of bacteria expressing the recombinant P. falciparum HPRT, four (compounds 40, 48, 53, and 54) were included in the antimalarial screens (16) and were found to inhibit the growth of P. falciparum (in the range of 25 to 36%). Of the remaining three compounds, 6-mercaptopurine (compound 51) and 6-thioguanine (compound 52) were previously tested for their antimalarial activities in a separate study (19) and were reported to have 50% inhibitory doses for P. falciparum of approximately 6.2 and 18 M, respectively. With the exception of untested compound 44, all of the compounds identified by the recombinant screens as inhibiting the malarial HPRT have been shown to inhibit the growth of P. falciparum …”

“…Limited progress has been made toward the identification of lead compounds selectively targeted to the purine salvage enzymes of parasites. Exceptions include a report that mercaptopurine and thioguanine selectively inhibit a purine salvage enzyme of the parasite responsible for human malaria (19).…”

Modulatory Effects of 6‐Carboxymethylthiopurine on Activated Murine Macrophages

“…Unlike humans, leishmania parasites cannot make purines de novo and must therefore obtain purine bases and nucleotides from the host cell and then metabolize these to nucleotides by salvage pathway (16). A similar mechanism occurs in malaria parasites, which have also demonstrated significant sensitivity to 6-MP, 6-TG and other compounds (17).…”

Molecular targets of 5-fluoroorotate in the human malaria parasite, Plasmodium falciparum

“…Orotic acid is the only preformed pyrimidine that is utilized by malarial parasites (19). This, in turn, has inspired evaluation of orotic acid analogs as potential antimalarial agents (18,27,(35)(36)(37)(38). 5-Fluoroorotate inhibited the in vitro proliferation of chloroquine-susceptible as well as chloroquine-resistant P. falciparum with a 50% inhibitory concentration of 6 nM (38).…”