title: Rapid Chagas Disease Drug Target Discovery Using Directed Evolution in Drug-Sensitive Yeast.
abstract: Recent advances in cell-based, high-throughput phenotypic screening have identified new chemical compounds that are active against eukaryotic pathogens. A challenge to their future development lies in identifying these compounds' molecular targets and binding modes. In particular, subsequent structure-based chemical optimization and target-based screening require a detailed understanding of the binding event. Here, we use directed evolution and whole-genome sequencing of a drug-sensitive S. cerevisiae strain to identify the yeast ortholog of TcCyp51, lanosterol-14-alpha-demethylase (TcCyp51), as the target of MMV001239, a benzamide compound with activity against Trypanosoma cruzi, the etiological agent of Chagas disease. We show that parasites treated with MMV0001239 phenocopy parasites treated with another TcCyp51 inhibitor, posaconazole, accumulating both lanosterol and eburicol. Direct drug-protein binding of MMV0001239 was confirmed through spectrophotometric binding assays and X-ray crystallography, revealing a binding site shared with other antitrypanosomal compounds that target Cyp51. These studies provide a new probe chemotype for TcCyp51 inhibition.
authors.affiliation: Department of Pediatrics, University of California, San Diego, School of Medicine , La Jolla, California 92093, United States.
authors.forename: Sabine
authors.initials: S
authors.lastname: Ottilie
authors.affiliation: Department of Pediatrics, University of California, San Diego, School of Medicine , La Jolla, California 92093, United States.|Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute , La Jolla, California 92037, United States.
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Goldgof
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego , La Jolla, California 92093, United States.|Cellular Ultrastructure Laboratory, IOC, FIOCRUZ , Rio de Janeiro, Rio de Janeiro, Brazil 21045-360.
authors.forename: Claudia Magalhaes
authors.initials: CM
authors.lastname: Calvet
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego , La Jolla, California 92093, United States.
authors.forename: Gareth K
authors.initials: GK
authors.lastname: Jennings
authors.affiliation: Department of Pediatrics, University of California, San Diego, School of Medicine , La Jolla, California 92093, United States.
authors.forename: Greg
authors.initials: G
authors.lastname: LaMonte
authors.affiliation: Department of Pediatrics, University of California, San Diego, School of Medicine , La Jolla, California 92093, United States.
authors.forename: Jake
authors.initials: J
authors.lastname: Schenken
authors.affiliation: Department of Pediatrics, University of California, San Diego, School of Medicine , La Jolla, California 92093, United States.
authors.forename: Edgar
authors.initials: E
authors.lastname: Vigil
authors.affiliation: Department of Pediatrics, University of California, San Diego, School of Medicine , La Jolla, California 92093, United States.
authors.forename: Prianka
authors.initials: P
authors.lastname: Kumar
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego , La Jolla, California 92093, United States.
authors.forename: Laura-Isobel
authors.initials: LI
authors.lastname: McCall
authors.affiliation: Department of Pediatrics, University of California, San Diego, School of Medicine , La Jolla, California 92093, United States.|Department of Pharmacy, Federal University of Paraná , Curitiba, Paraná, Brazil 80210-170.
authors.forename: Eduardo Soares Constantino
authors.initials: ES
authors.lastname: Lopes
authors.affiliation: Department of Pediatrics, University of California, San Diego, School of Medicine , La Jolla, California 92093, United States.
authors.forename: Felicia
authors.initials: F
authors.lastname: Gunawan
authors.affiliation: Department of Pediatrics, University of California, San Diego, School of Medicine , La Jolla, California 92093, United States.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Yang
authors.affiliation: Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute , La Jolla, California 92037, United States.
authors.forename: Yo
authors.initials: Y
authors.lastname: Suzuki
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego , La Jolla, California 92093, United States.
authors.forename: Jair L
authors.initials: JL
authors.lastname: Siqueira-Neto
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego , La Jolla, California 92093, United States.
authors.forename: James H
authors.initials: JH
authors.lastname: McKerrow
authors.affiliation: Department of Chemistry & Biochemistry, University of California, San Diego , La Jolla, California 92093-0340, United States.
authors.forename: Rommie E
authors.initials: RE
authors.lastname: Amaro
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego , La Jolla, California 92093, United States.
authors.forename: Larissa M
authors.initials: LM
authors.lastname: Podust
authors.affiliation: Department of Biological Sciences, University of Pittsburgh , Pittsburgh, Pennsylvania 15260, United States.
authors.forename: Jacob D
authors.initials: JD
authors.lastname: Durrant
authors.affiliation: Department of Pediatrics, University of California, San Diego, School of Medicine , La Jolla, California 92093, United States.
authors.forename: Elizabeth A
authors.identifier: 0000-0002-4049-2113
authors.initials: EA
authors.lastname: Winzeler
mesh_terms: D058888:14-alpha Demethylase Inhibitors / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use*; D000595:Amino Acid Sequence; D014355:Chagas Disease / Q000188:drug therapy* / Q000469:parasitology; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery; D008401:Gas Chromatography-Mass Spectrometry; D062105:Molecular Docking Simulation; D010963:Plasmodium falciparum / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D058886:Sterol 14-Demethylase / Q000187:drug effects; D014344:Trypanocidal Agents / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use*; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000201:enzymology
chemical_list: D058888:14-alpha Demethylase Inhibitors; D014344:Trypanocidal Agents; D058886:Sterol 14-Demethylase