Addressing the Intracellular Vestibule of the Plasmodial Lactate Transporter PfFNT by p-Substituted Inhibitors Amplifies In Vitro Activity

Inhibition of the lactate transporter PfFNT is a valid novel mode of action against malaria parasites. Current pyridine-substituted pentafluoro-3-hydroxy-pent-2-en-1-ones act as substrate analogs with submicromolar EC₅₀ in vitro, and >99.7% activity in mice. The recently solved structure of a PfFNT-inhibitor complex visualized the binding mode. Here, we extended the inhibitor layout by series of amine- and anilide-linked pyridine p-substituents to generate additional interactions in the cytoplasmic vestibule. Virtual docking indicated hydrogen bonding to Tyr31 and Ser102. Fluorescence cross-correlation spectroscopy yielded respectively enhanced target affinity. Strikingly, the in vitro activity increased by 1 order of magnitude to 14.8 nM at negligible cytotoxicity. While <i>p</i>-amine substitutions were rapidly metabolized, the more stable <i>p</i>-acetanilide cleared 99.7% of parasites at 4 × 50 mg kg^-1 in a mouse malaria model. Future stabilization of the p-substitution against metabolism may translate the gain in in vitro potency to the in vivo situation.