Plasmodium vivax (P. vivax, Pv) infection still remains problematic despite intensive efforts on malaria elimination. The persistence of P. vivax is due to low parasiteamia without symptom, relapse and emergence of chloroquine resistant parasites. The fact that pyrimethamine, an antifolate targeting dihydrofolate reductase (PvDHFR) has proven to be effective against wildtype P.vivax (WT) but becomes ineffective by subsequent emergence of pyrimethamine-resistant P. vivax with point mutations in PvDHFR, has made the target attractive for development of new, effective antifolates against P. vivax. However, drug discovery for P.vivax treatment has been hampered by lack of simple continuous P. vivax culture. To overcome such limitation, we have successfully developed a bacterial surrogate model for antifolate screening against PvDHFR. The surrogate model is E. coli BL21(DE3) namely PEcDFTtolC-PvDHFR-TS, in which folA (dhfr), thyA (thymidylate synthase, ts), and tolC genes were knocked out, and its folA and thyA are functionally replaced by Pvdhfr-ts gene. Three types of Pvdhfr-ts (wildtype (WT) and 2 antifolate resistant mutants from clinical isolates with double mutation (DM, S58R+S117T) and quadruple mutation (QM, F57I+S58R+T61M+S117T)) were cloned into PEcDFTtolC. We found that WT is sensitive to pyrimethamine and cycloguanil but resistant to trimethoprim while the mutants are moderately-highly resistant to the drugs. Series of rigid and flexible antifolates developed for P. falciparum have been screened against the cells. The results showed that WT is sensitive to most antifolates while the mutants are resistant to rigid antifolates but sensitive to the flexible ones such as WR99210 and P218. The results are in line with enzyme inhibition assay. Furthermore, ex-vivo P. vivax testing demonstrated that both mutants are sensitive to P218. These results are encouraging for further development of antifolates against P. vivax.
This work is supported by BIOTEC (P1450525 & P1551096) and NSTDA (P1450883 & Core-Researcher Award: P1850116).