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.

Chemogenetic Characterization of Inositol Phosphate Metabolic Pathway Reveals Druggable Enzymes for Targeting Kinetoplastid Parasites

“…In addition, IPMK inhibition was also lethal for intracellular T. cruzi amastigotes, demonstrating the potential value of this drug target in T. cruzi. This extends the short list of validated targets for drug development in T. cruzi , which includes cytochrome b, cruzain, sirtuins and sterol 14α-demethylase (Doyle, et al, 2010; Khare, et al, 2015; McGrath, et al, 1995; Moretti, et al, 2015). Some IPMK inhibitors are also effective against P. falciparum , suggesting that these inhibitors may be broadly used to develop new antiparasitic drugs.…”

Effects of a novel β–lapachone derivative on Trypanosoma cruzi : Parasite death involving apoptosis, autophagy and necrosis

“…Nevertheless it may furnish significant insights for the infection chemotherapy, as β-lapachone was reported to produce similar alterations in T. cruzi epimastigotes, amastigotes and trypomastigotes (Docampo et al., 1978), the developmental forms that multiply within mammalian host cells and spread via blood, respectively. In addition, different antiparasitic compounds may display similar effects upon epimastigotes and trypomastigotes and/or amastigotes, the developmental forms (Urbina et al., 1988, Urbina et al., 1993; Moreira et al., 2013a; Costa et al., 2011, Azeredo et al., 2014, Díaz et al., 2014; Jimenez et al., 2014; Veiga-Santos et al., 2014, Britta et al., 2015, Meira et al., 2015, Volpato et al., 2015, Beer et al., 2016) and the epimastigotes may therefore comprise and/or take part in experimental models (Kessler et al., 2013, Benítez et al., 2014, Sangenito et al., 2014, Wong-Baeza et al., 2015, Khare et al., 2015, Pessoa et al., 2016, Valera Vera et al., 2016). Thus, numerous studies perform screening experiments with epimastigotes and/or trypomastigotes further approach the selected active compounds in intracellular amastigotes (e.g.…”

Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness

“…Next, treated macrophages were washed with the phosphate-buffered saline buffer (PBS; Sigma-Aldrich) supplemented with 0.5 mM magnesium chloride (Sigma-Aldrich) and 0.5 mM calcium chloride (Sigma-Aldrich), fixed with 0.4% paraformaldehyde (Sigma-Aldrich) in PBS, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS, and stained with SYBR Green I nucleic acid stain(Invitrogen, 1:100,000 dilution in PBS) overnight at 4 °C. Image collection and enumeration of macrophage cells and intracellular L. donovani amastigotes was performed using the OPERA QEHS automated confocal microscope system equipped with 20x water immersion objective (Evotec Technologies) and the OPERA Acapella software (Evotec Technologies) as described previously 32 .…”

“…The number of macrophage cells in wells was determined by high content microscopy as described previously 32 .…”

“…T. cruzi epimastigotes cultures resistant to GNF3943 and GNF8000 were generated using a methodology described previously 32 . Briefly, epimastigotes were initially cultured in the presence of compound concentration equivalent to its EC 20 value (GNF3943 EC 20 = 1.5 μM and GNF8000 EC 20 = 0.2 μM in 0.2% DMSO) or 0.2% DMSO (control).…”