title: 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.
authors.affiliation: Department of Genetics and Neglected Diseases, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
authors.forename: Shilpi
authors.initials: S
authors.lastname: Khare
authors.affiliation: Department of Medicinal Chemistry, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
authors.forename: Steven L
authors.initials: SL
authors.lastname: Roach
authors.affiliation: Department of Genetics and Neglected Diseases, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
authors.forename: S Whitney
authors.initials: SW
authors.lastname: Barnes
authors.affiliation: Novartis Institutes for BioMedical Research, Novartis Campus, Basel, Switzerland.
authors.forename: Dominic
authors.initials: D
authors.lastname: Hoepfner
authors.affiliation: Department of Genetics and Neglected Diseases, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
authors.forename: John R
authors.initials: JR
authors.lastname: Walker
authors.affiliation: Department of Medicinal Chemistry, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
authors.forename: Arnab K
authors.initials: AK
authors.lastname: Chatterjee
authors.affiliation: Small Molecule Discovery Center and Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, United States of America.
authors.forename: R Jeffrey
authors.initials: RJ
authors.lastname: Neitz
authors.affiliation: Small Molecule Discovery Center and Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, United States of America.
authors.forename: Michelle R
authors.initials: MR
authors.lastname: Arkin
authors.affiliation: Department of Genetics and Neglected Diseases, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
authors.forename: Case W
authors.initials: CW
authors.lastname: McNamara
authors.affiliation: Department of Genetics and Neglected Diseases, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
authors.forename: Jaime
authors.initials: J
authors.lastname: Ballard
authors.affiliation: Department of Genetics and Neglected Diseases, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
authors.forename: Yin
authors.initials: Y
authors.lastname: Lai
authors.affiliation: Department of Genetics and Neglected Diseases, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
authors.forename: Yue
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Department of Medicinal Chemistry, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
authors.forename: Valentina
authors.initials: V
authors.lastname: Molteni
authors.affiliation: Department of Medicinal Chemistry, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
authors.forename: Vince
authors.initials: V
authors.lastname: Yeh
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California, United States of America.
authors.forename: James H
authors.initials: JH
authors.lastname: McKerrow
authors.affiliation: Department of Genetics and Neglected Diseases, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Glynne
authors.affiliation: Department of Genetics and Neglected Diseases, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
authors.forename: Frantisek
authors.initials: F
authors.lastname: Supek
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D000968:Antimycin A / Q000378:metabolism; D014355:Chagas Disease / Q000188:drug therapy* / Q000235:genetics / Q000382:microbiology*; D045303:Cytochromes b / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport / Q000187:drug effects / Q000276:immunology; D023281:Genomics; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009154:Mutation; D010101:Oxygen Consumption / Q000187:drug effects; D014349:Trypanosoma cruzi / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D000968:Antimycin A; D045303:Cytochromes b