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PLoS Pathog. 2015 Jul 17;11(7):e1005058. doi: 10.1371/journal.ppat.1005058. eCollection 2015 Jul.

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

Author information

1
Department of Genetics and Neglected Diseases, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
2
Department of Medicinal Chemistry, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
3
Novartis Institutes for BioMedical Research, Novartis Campus, Basel, Switzerland.
4
Small Molecule Discovery Center and Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, United States of America.
5
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California, United States of America.

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.

PMID:
26186534
PMCID:
PMC4506092
DOI:
10.1371/journal.ppat.1005058
[Indexed for MEDLINE]
Free PMC Article

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