Synergistic activity of RSL3 and Pyrimethamine to inhibit the proliferation of <i>Plasmodium falciparum</i>

Malaria tropica, caused by <i>Plasmodium falciparum</i> (<i>P. falciparum</i>), remains a global health challenge with limited therapeutic options. In mammalian cells, the small-molecule compound RAS-selective lethal 3 (RSL3) induces ferroptosis via lipid peroxidation. In this study, we demonstrate that RSL3 synergizes with Pyrimethamine, an inhibitor of <i>P. falciparum</i> dihydrofolate reductase (DHFR), to suppress parasite proliferation in red blood cells (RBCs). A similar synergistic effect was observed with Cycloguanil, a structural analog of Pyrimethamine, but not with other DHFR inhibitors or alternative agents that induce ferroptosis in nucleated mammalian cells. Notably, Ferrostatin-1, an antagonist of lipid peroxidation, largely failed to rescue parasite growth in the presence of RSL3, possibly suggesting a mechanism distinct from canonical ferroptosis. These findings suggest that the synergy may involve unidentified targets of RSL3 and Pyrimethamine in <i>P. falciparum</i>, divergent from those described in mammalian systems. Moreover, RSL3 and related compounds could serve as promising adjuvants to enhance the antimalarial efficacy of Pyrimethamine and potentially overcome drug resistance.