Uncovering the Unusual Inhibition Mechanism of a Trypanosome Alternative Oxidase Inhibitor Displaying Broad-Spectrum Activity against African Animal Trypanosomes.

The glucose-dependent respiration of bloodstream forms of the parasite depends on an unusual and essential mitochondrial electron-transport system, consisting of glycerol-3-phosphate dehydrogenase and the trypanosome alternative oxidase (TAO). We report here the discovery of an allosteric inhibitor of TAO that displays highly potent activity (EC values in the range 1-20 nM) against the important veterinary pathogens , , , and , i.e., >5-fold greater potency than the standard drugs. The methylene-linked 2-methyl-4-hydroxybenzoate 2-pyridinyldiphenylphosphonium derivative () was the best inhibitor of recombinant TAO (IC = 1.3 nM) via a noncompetitive/allosteric mechanism ( = 3.46 nM). Remarkably, X-ray crystallography showed that was bound to a site of TAO ∼25 Å from the catalytic pocket. Although demonstrated good safety toward mammalian cells (selectivity index >2300), it did not fully clear parasitemia in experimental animals, attributable to a high hepatic clearance.