Dual-stage inhibition of <i>Plasmodium falciparum</i> by a <i>Skeletocutis</i> derived fungal metabolite targeting Pyruvate Kinase II

Abstract Plasmodium falciparum resistance to current first line treatments is threatening at-risk populations and underscores the urgent need for novel therapeutic targets and drugs. P. falciparum pyruvate kinases I and II are two essential enzymes with distinct roles and subcellular localizations within the parasite. Pf PyrKI is cytosolic, while Pf PyrKII is found in the apicoplast, a specific organelle of Apicomplexa , where it is required for the production of (d)NTPs essential for apicoplast maintenance. We identify skeletocutin E, a Basidiomycete-derived metabolite, as a specific inhibitor of Pf PyrKII. Skeletocutin E inhibits in vitro the activity of Pf PyrKII with an IC 50 of 0.52 ± 0.08 µM through a mixed inhibition mechanism and does not affect the activities of three human pyruvate kinases. Structure–activity relationship analyses using synthetic skeletocutin E analogues allowed us to identify the molecular determinants of this inhibition. Furthermore, determination of the quaternary structure of Pf PyrKII by mass photometry, showed that this enzyme exists as monomers, dimers, and tetramers in equal proportions, revealing its singularity compared to other pyruvate kinases. Interestingly, skeletocutin E does not alter the distribution of the complexes, indicating that it does not interact at the subunit interfaces. Importantly, skeletocutin E inhibits P. falciparum growth in both blood and liver stages, with IC₅₀ values of 3.56 ± 0.50 µM in red blood cells and 3.70 ± 0.74 µM in primary human hepatocytes. Together, these findings establish Pf PyrKII as a druggable antimalarial target and identify skeletocutin E as a promising lead compound for the rational development of dual-stage antimalarial therapies.