Analogs of the Natural Product Chamuvarinin Target the Trypanosomatid FoF1-ATP Synthase Mitochondrial Complex V

Current treatments for trypanosomatid diseases are outdated, increasingly ineffective and associated with severe adverse effects. We recently reported trypanocidal activity of a series of novel synthetic bistetrahydropyran 1,4-triazole (B-THP-T) analogs based on the framework of the natural product chamuvarinin, an acetogenin first synthesized by
our groups. Acetogenins are potent inhibitors of the human mitochondrial Complex I, however our compounds show potent inhibitory activity against bloodstream form Trypanosoma brucei, in which Complex I is absent, thus chamuvarinin must target another protein in the trypanosomatids. The mode of action of our B-THP-T compounds is unknown so this work aimed to identify their target in Leishmania major and T. cruzi. We synthesized a series of B-THP-T analogs for use in photo-affinity labeling (PAL), to covalently tag our target for protein target identification in vivo. We subsequently identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target of our compounds using pull-down experiments. Next we undertook a series of biological analyses to validate this pulldown. By labeling B-THP-T-tagged proteins with the Cy5.5 fluorophore we confirmed that the target is mitochondrial by fluorescence co-localization. Using a luciferase-based ATP quantitation assay we show that B-THP-T compounds inhibit cellular ATP production and that they ablate oxidative phosphorylation. Taken together, these data indicate that our B-THP-T compounds are trypanocidal through their mitochondrial targeting of the FoF1-ATP synthase. We are using genetic manipulation of T. cruzi and L. major FoF1 ATP synthase subunits to validate these findings. With the
target of our bis-tetrahydropyran 1,4-triazoles identified as mitochondrial complex V a structure-based approach can be used to optimize inhibitor potency and specificity.