Evaluation of the activities of pyrimethamine analogs against Plasmodium vivax and Plasmodium falciparum dihydrofolate reductase-thymidylate synthase using in vitro enzyme inhibition and bacterial complementation assays

Abstract

Pyrimethamine analogs were examined as potential agents against vivax malaria using a bacterial surrogate system carrying Plasmodium vivax dihydrofolate reductase-thymidylate synthase (PvDHFR-TS), in which the PvDHFR complemented chemically knocked out host dihydrofolate reductase. The system was initially tested with P. falciparum dihydrofolate reductase-thymidylate synthase and was found to have good correlation with the parasite-based system. The 50% inhibitory concentrations derived from PvDHFR-TS-dependent bacteria were correlated with their corresponding inhibition constants (Ki) from an enzyme inhibition assay, pointing to the likelihood that the potent enzyme inhibitors will also have potent antimalarial activities. Active compounds against both wild-type and S58R S117N (SP21) double-mutant P. vivax include analogs with structures which can avert a steric clash with the asparagine (S117N) side chain of the mutant, similar to those found for homologous Plasmodium falciparum mutants, raising the possibility that the same compounds can be developed against both types of antifolate-resistant malaria. This rapid and convenient drug screening system should be useful for development of new antifolates against P. vivax, for which a continuous culture system is not yet available.

FIG. 1.

BL21(DE3)pLysS complemented with pETpvDHFR-TS and pETpvSP21 (A and B) selected in MM containing 100 μg ml⁻¹ ampicillin and 34 μg ml⁻¹ chloramphenicol and BL21(DE3) complemented with pETpfTM4 and pETpfK1 (C and D) grown in MM containing 100 μg ml⁻¹ ampicillin. Cultures were grown in the absence (A and C) or presence (B and D) of 4 μM Tmp. pET17b indicates cell containing vector without the Plasmodium gene.

FIG. 2.

Graphics show inhibition of Pyr analogs. (A) Correlation of IC₅₀s derived from bacterial complementation assay and in vitro culture of P. falciparum assay. (B) Relationship between IC₅₀s obtained from cell-based assay systems (either parasite [square] or bacteria [triangle]) and the K_i values from the in vitro enzyme inhibition assay. Closed and open symbols are data points derived from the wild type and the mutant, respectively (data with K_i values and IC₅₀s greater than 60 nM and 50 μM were excluded from the plot). Each data point is the average from at least three independent experiments (each performed in triplicate).

FIG. 3.

Inhibition profile of Pyr library. Panels A and B show relationships of IC₅₀s derived from the bacterial assay system and K_i values from the in vitro enzyme assay of WT PvDHFR-TS and the SP21 mutant, respectively. Inset (B) shows the data used for calculating r² (data with K_i and IC₅₀ greater than 7 nM and 50 μM were excluded from calculation). (C) Relationship of IC₅₀s between bacteria expressing DHFR-TS of P. falciparum or P. vivax. (D) Relationship of K_i values tested against PvDHFR-TS and PfDHFR enzymes. For C and D, closed and open symbols are data points derived from wild types and mutants (SP21 for P. vivax and C59R S108N for P. falciparum), respectively. Each data point is the average from at least three independent experiments (each performed in triplicate). Pf, P. falciparum; Pv, P. vivax.