Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease

Abstract

Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.

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.

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 Q_P and Q_N 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.

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.