Anti-trypanosomal 8-hydroxyl naphthyridines are chelators of divalent transition metals

Lack of information regarding the mechanisms of action (MoA) or specific molecular targets of phenotypically active compounds can prove a barrier to their development as chemotherapeutic agents. Here, we report the results of our orthogonal genetic, molecular and biochemical studies to determine the MoA of a novel 7-substituted 8-hydroxy-1,6-naphthyridine (8-HNT) series that display promising activity against Trypanosoma brucei and Leishmania donovani. High-throughput loss-of-function genetic screens in T. brucei highlighted two probable zinc transporters associated with resistance to these compounds. These transporters localized to the parasite Golgi. Directed by these findings, the role of zinc and other divalent cations in the MoA of these compounds was investigated. 8-HNT compounds were found to directly deplete intracellular levels of Zn2+ while the addition of exogenous Zn2+ and Fe2+ reduced the potency of compounds from this series. Detailed biochemical analyses confirmed that 8-HNT compounds bind directly to a number of divalent cations, predominantly Zn2+, Fe2+ and Cu2+, forming 2:1 complexes with one of these cations. Collectively, our studies demonstrate transition metal depletion, due to chelation, as the MoA of the 8-HNT series of compounds. Strategies to improve the selectivity of 8-HNT compounds are discussed.