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 Trypanosoma brucei 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 (EC50 values in the range 1-20 nM) against the important veterinary pathogens T. b. brucei, Trypanosoma evansi, Trypanosoma equiperdum, and Trypanosoma congolense, i.e., >5-fold greater potency than the standard drugs. The methylene-linked 2-methyl-4-hydroxybenzoate 2-pyridinyldiphenylphosphonium derivative (1) was the best inhibitor of recombinant TAO (IC50 = 1.3 nM) via a noncompetitive/allosteric mechanism (Ki = 3.46 nM). Remarkably, X-ray crystallography showed that 1 was bound to a site of TAO ∼25 Å from the catalytic pocket. Although 1 demonstrated good safety toward mammalian cells in vitro (selectivity index >2300), it did not fully clear parasitemia in experimental animals, attributable to a high hepatic clearance.