Rapid Chagas Disease Drug Target Discovery Using Directed Evolution in Drug-Sensitive Yeast

ACS Chem Biol. 2017 Feb 17;12(2):422-434. doi: 10.1021/acschembio.6b01037. Epub 2016 Dec 23.


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.

MeSH terms

  • 14-alpha Demethylase Inhibitors / chemistry
  • 14-alpha Demethylase Inhibitors / pharmacology
  • 14-alpha Demethylase Inhibitors / therapeutic use*
  • Amino Acid Sequence
  • Chagas Disease / drug therapy*
  • Chagas Disease / parasitology
  • Crystallography, X-Ray
  • Directed Molecular Evolution*
  • Dose-Response Relationship, Drug
  • Drug Discovery
  • Gas Chromatography-Mass Spectrometry
  • Molecular Docking Simulation
  • Plasmodium falciparum / drug effects
  • Saccharomyces cerevisiae / drug effects
  • Saccharomyces cerevisiae / genetics
  • Spectrophotometry, Ultraviolet
  • Sterol 14-Demethylase / drug effects
  • Trypanocidal Agents / chemistry
  • Trypanocidal Agents / pharmacology
  • Trypanocidal Agents / therapeutic use*
  • Trypanosoma cruzi / drug effects
  • Trypanosoma cruzi / enzymology


  • 14-alpha Demethylase Inhibitors
  • Trypanocidal Agents
  • Sterol 14-Demethylase