Substrate and inhibitor specificity of Plasmodium nucleoside transporters ENT1 orthologs

Malaria caused by Plasmodium infection poses a serious hazard to human health. Plasmodium falciparum equilibrative nucleoside transporter 1 (PfENT1), which mediates nucleoside uptake, is essential for the growth and proliferation of Plasmodium parasites, suggesting that PfENT1 is a potential antimalarial target. The promising compound GSK4 effectively inhibits the transport activity of PfENT1, thereby restraining the growth of Plasmodium parasites. However, it still needs to be clarified whether Plasmodium ENT1 orthologs have different selectivities for nucleosides and inhibitors. Here, we systematically compared the nucleoside selectivity of Plasmodium ENT1 orthologs from P. falciparum (PfENT1), Plasmodium berghei (PbENT1), and Plasmodium vivax (PvENT1), revealing that Plasmodium ENT1 orthologs present a distinct nucleoside recognition pattern. In addition, GSK4 robustly binds to PfENT1 and PvENT1 from two human-hosted Plasmodium parasites but has a weakened binding affinity for PbENT1 from mouse-hosted Plasmodium parasites. We further structurally optimized the inhibitor and generated three GSK4 analogs. One of the GSK4 analogs presented a slightly increased binding affinity for PfENT1. This optimization represents a promising advancement for antimalarial drug development, providing a novel foundation for future endeavors in antimalarial drug design.