Fig 1.
GNF7686 clears T. cruzi amastigotes from infected 3T3 cells.
A) Structures of GNF7686, benznidazole and prototypic cytochrome b inhibitors used in this study. B) Growth curves of wild-type and GNF7686-resistant T. cruzi epimastigotes. C) Microscopy images of NIH/3T3 cells infected with T. cruzi after 48 hour treatment with 0.2% DMSO, 5 μM benznidazole or 1 μM GNF7686.
Table 1.
Potency of GNF7686 and prototypic cytochrome b Inhibitors on wild-type (WT) and GNF7686-resistant (DR) T. cruzi morphological forms.
Fig 2.
Chemogenomic profiling in S. cerevisiae suggests cytochrome b as the target of GNF7686 in yeast.
A) HIP profile of GNF7686. B-D) Alignment of the HIP profile of GNF7686 with profile of cytochrome b inhibitor strobilurin (B), F-type ATPase inhibitor venturicidin (C), and microtubule-binding fungicide benomyl (D).
Fig 3.
GNF7686 resistance-conferring mutation in T. cruzi cytochrome b structure.
A) Secondary structure of yeast cytochrome b (adapted from Ding et al. 2006 and Fisher et al. 2008, [50, 57]) with amino acid sequence stretches that form QP and QN ubiquinol-binding sites highlighted (green and violet, respectively). The amino acid indicated by a green arrow corresponds to the L197F mutation (equivalent to L198F in S. cerevisiae) present in the GNF7686-resistant T. cruzi strain. B) Alignment of S cerevisiae and T. cruzi cytochrome b amino acid sequence around L197F mutation. Identical amino acids are highlighted in black and conserved substitutions are highlighted in grey.
Table 2.
Potency of GNF7686 and prototypic cytochrome b inhibitors in L. donovani, T. brucei, P. falciparum and S. cerevisiae proliferation assays.
Fig 4.
GNF7686 inhibits cellular respiration and cytochrome b function in T. cruzi.
A) T. cruzi epimastigote respiration was monitored using the MitoXpress-Xtra HS phosphorescent probe (see Methods) in the presence of 0.2% DMSO (control) and various concentrations of GNF7686. Oxygen consumption rates per 106 T. cruzi epimastigotes cells are shown. Plotted values were derived from three biological repeats (n = 3) with duplicate technical repeats in either wild-type or evolved GNF7686-resistant (DR) parasites. B) Oxygen consumption IC50 values for prototypic cytochrome b inhibitors in T. cruzi epimastigote respiration assay on wild-type and GNF7686-resistant (DR) parasites. Shown GNF7686 IC50 values were derived from the experiment shown in the (A) panel. C) Mitochondrial complex III activity was monitored in digitonin-solubilized T. cruzi epimastigotes (both wild-type and evolved GNF7686-resistant (DR) parasite strains) utilizing a coupled decylubiquinol oxidation / cytochrome c reduction reaction in the presence of a compound (GNF7686 or antimycin A). IC50 values were determined based on three biological repeats (n = 3) with triplicate technical repeats in either wild-type or evolved drug-resistant (DR) parasites relative to 0.2% DMSO conditions. D) High selectivity of GNF7686 for T. cruzi cytochrome b is reflected in the absence of inhibition of rat mitochondrial respiration by this compound up to 25 μM concentration. For comparison, antimycin A potently inhibits mammalian mitochondrial respiration. Oxygen consumption rates per 1 mg of total mitochondrial protein are shown.