Transgenic pyrimethamine resistantPlasmodium bergheias a model forin vivoanti-DHFR drug testing

Abstract

Inhibitors for Plasmodium falciparum dihydrofolate reductase (DHFR) form an important class of antimalarial drugs widely used for malaria treatment, but have been compromised by development of resistance to the drugs. Mutations in DHFR are the main contributing factors to the resistance. Although new, rationally designed antifolates active against resistant P. falciparum, such as P218, have been developed, the activity against the quadruple mutant P. falciparum (V1/S) has only been demonstrated in vitro, and in vivo activity has only been shown in SCID mice. A convenient in vivo model for antifolate testing is desirable. In this study, the endogenous P. berghei dihydrofolate reductase-thymidylate synthase (Pbdhfr-ts) gene was successfully replaced by quadruple dhfr-ts mutant gene from P. falciparum (N51I+C59R+S108N+I164L). The transgenic parasite gained resistance to pyrimethamine but not to other class of antimalarial drugs. While 30 mg/kg of pyrimethamine could not inhibit the transgenic parasite, P218 could inhibit the transgenic parasite with the ED50 of 0.11±0.02 mg/kg, a level similar to the P. falciparum in SCID mice model. These results demonstrated the validity of our model and showed that P218 was very potent against quadruple Pfdhfr-ts mutant parasite, in vivo.

New insights into antimalarial chemopreventive activity of antifolates

“…This study sought to characterize antifolates as chemopreventive antimalarial drugs, especially against the quadruple mutant Pfdhfr parasite. Using the transgenic P. berghei harboring quadruple mutant Pfdhfr in place of the wild-type Pbdhfr as an in vivo antifolate resistant model, we demonstrated that the quadruple mutation on Pfdhfr does not only leads to PYR treatment failure (Koonyosying et al, 2020) but also confers complete resistance to PYR chemoprevention. Antifolates of the previous generation are then no longer effective for both applications in most malaria endemic areas, thus prompting the development of alternative treatments.…”

“…The benefit of having a well-known target allows characterization of resistance mechanism and rational design of new classes of antifolates that can overcome the resistance (Yuthavong et al, 2012). Although the new class of antifolates such as P218 can inhibit asexual stage and male gametocyte exflagellation of quadruple mutant parasites (Koonyosying et al, 2020;Posayapisit et al, 2021;Yuthavong et al, 2012), the information on how potent these new compounds are for the chemoprevention activity against quadruple mutant parasite has never been experimentally demonstrated due to the lack of reliable liver stage model of the resistant parasite.…”

“…The wild-type Pbdhfr P. berghei ANKA strain 676m1cl1 (MRA-868) was obtained through BEI Resources, NIAID, NIH, contributed by Chris J. Janse and Andrew P. Waters. P. berghei with quadruple dhfr mutant from P. falciparum replacing the wild-type dhfr was generated in previous study (Koonyosying et al, 2020).…”

“…We utilized a transgenic P. berghei harboring P. falciparum dhfr-4M developed by our group (Koonyosying et al, 2020) to determine chemoprevention activity of PYR against quadruple mutant parasite. We have confirmed that the transgenic Pb::Pfdhfr-4M parasite still maintains an ability to develop into mosquito and liver stages (Figure 1).…”

“…Here we used the previously developed transgenic P. berghei with the wild-type Pbdhfr replaced by quadruple mutant Pfdhfr (Koonyosying et al, 2020) to investigate chemoprevention activity of the previous generation as well as the new generation of antifolate compounds, represented by PYR and P218 respectively, against quadruple mutant Pfdhfr parasite. This study also takes an advantage of a highly potent, short half-life properties of P218 to explore stage-specific activity of antifolate compounds against hepatic parasites.…”