Direct amidinium-phosphate interactions guide AT-selective DNA binding by a potent antileishmanial drug.

Neglected tropical diseases caused by kinetoplastid parasites remain a major global health challenge, with current chemotherapies offering limited efficacy and safety. JNII40 is a promising bis(pyridine-2-carboxamidine) ligand with potent antiparasitic activity and selectivity in vivo against Leishmania parasites. The compound specifically targets AT-rich regions of DNA, predominant in the mitochondrion of these parasites. We used NMR spectroscopy to determine the high-resolution structure of a DNA duplex containing four consecutive AT repeats in complex with JNII40. The structure reveals that JNII40 binds deeply into the DNA minor groove through a network of direct and water-mediated hydrogen bonds, including direct interactions between amidine protons and DNA phosphate oxygens. These contacts, supported by extensive intermolecular NOEs, explain the high affinity and sequence selectivity toward AT-rich regions observed by calorimetry and NMR. Restrained molecular dynamics simulations highlight the critical role of two ordered water molecules that bridge the ligand and DNA, preserving the geometry of the complex. Ligand insertion induces a noticeable narrowing of the minor groove. Altogether, these findings provide a structural framework for the rational drug design of selective DNA-targeting molecules against kinetoplastid parasites such as Leishmania.