The acetogenic-based bis-tetrahydropyran 1,4-triazole, JM425 potentially targets the trypanosomatid mitochondrial complex V

The acetogenin family of polyketides produced by annonaceacous plants has broad anti-microbial activity through its potent inhibition of mitochondrial complex I. We recently reported [Florence, Chem. Med. Chem. 9;2548 (2014)] the trypanocidal activity of a series of novel synthetic bis-tetrahydropyran 1,4-triazole (B-THP-T) analogues based on the framework of the acetogenin chamuvarinin. These compounds are active against both bloodstream and procyclic (insect) forms of Trypanosoma brucei, the parasite responsible for African sleeping sickness, also known as Human African Trypanosomiasis (HAT). T. brucei lacks functional complex I, so this study aimed to identify the target of our B-THP-T compounds in this important parasite. We performed pull-down experiments with a cross-linkable B-THP-T compound (ALF072) and identified the alpha- and beta- subunits of the FoF1 ATP synthase (mitochondrial complex V) as potential targets in the procyclic form. Next we undertook a series of biological analyses to validate this pull-down. Using a luciferase-based ATP quantitation assay we show that our lead B-THP-T compound (JM425) inhibits cellular ATP production from proline metabolism in the procyclic form, and specifically ablates oxidative phosphorylation in isolated procyclic form mitochondria, but has little effect on substrate-level ATP production. Imaging with MitoTracker dye revealed that JM425 elevates the procyclic mitochondrial membrane potential, consistent with inhibition of mitochondrial complex V, and support our pull-down of the regulatory (alpha) and catalytic (beta) subunits of the FoF1 ATP synthase. Complex V is essential for both bloodstream and procyclic T. brucei and is an attractive drug target. 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.