title: Identification of potent inhibitors of the Trypanosoma brucei methionyl-tRNA synthetase via high-throughput orthogonal screening.
abstract: Improved therapies for the treatment of Trypanosoma brucei, the etiological agent of the neglected tropical disease human African trypanosomiasis, are urgently needed. We targeted T. brucei methionyl-tRNA synthetase (MetRS), an aminoacyl-tRNA synthase (aaRS), which is considered an important drug target due to its role in protein synthesis, cell survival, and its significant differences in structure from its mammalian ortholog. Previous work using RNA interference of MetRS demonstrated growth inhibition of T. brucei, further validating it as an attractive target. We report the development and implementation of two orthogonal high-throughput screening assays to identify inhibitors of T. brucei MetRS. First, a chemiluminescence assay was implemented in a 1536-well plate format and used to monitor adenosine triphosphate depletion during the aminoacylation reaction. Hit confirmation then used a counterscreen in which adenosine monophosphate production was assessed using fluorescence polarization technology. In addition, a miniaturized cell viability assay was used to triage cytotoxic compounds. Finally, lower throughput assays involving whole parasite growth inhibition of both human and parasite MetRS were used to analyze compound selectivity and efficacy. The outcome of this high-throughput screening campaign has led to the discovery of 19 potent and selective T. brucei MetRS inhibitors.
authors.affiliation: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Pedró-Rosa
authors.affiliation: Department of Medicine, University of Washington, Seattle, WA, USA.
authors.forename: Frederick S
authors.initials: FS
authors.lastname: Buckner
authors.affiliation: Department of Medicine, University of Washington, Seattle, WA, USA.
authors.forename: Ranae M
authors.initials: RM
authors.lastname: Ranade
authors.affiliation: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Eberhart
authors.affiliation: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors.forename: Franck
authors.initials: F
authors.lastname: Madoux
authors.affiliation: Department of Medicine, University of Washington, Seattle, WA, USA.
authors.forename: J Robert
authors.initials: JR
authors.lastname: Gillespie
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA, USA.
authors.forename: Cho Yeow
authors.initials: CY
authors.lastname: Koh
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: Steven
authors.initials: S
authors.lastname: Brown
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Lohse
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA, USA.
authors.forename: Christophe L M
authors.initials: CL
authors.lastname: Verlinde
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA, USA.
authors.forename: Erkang
authors.initials: E
authors.lastname: Fan
authors.affiliation: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bannister
authors.affiliation: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA.
authors.forename: Louis
authors.initials: L
authors.lastname: Scampavia
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA, USA.
authors.forename: Wim G J
authors.initials: WG
authors.lastname: Hol
authors.affiliation: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA spicert@scripps.edu.
authors.forename: Timothy
authors.initials: T
authors.lastname: Spicer
authors.affiliation: The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA Amgen, Inc., Thousand Oaks, CA.
authors.forename: Peter
authors.initials: P
authors.lastname: Hodder
mesh_terms: D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods* / Q000592:standards; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057166:High-Throughput Screening Assays / Q000379:methods* / Q000592:standards; D006801:Humans; D020128:Inhibitory Concentration 50; D008718:Methionine-tRNA Ligase / Q000037:antagonists & inhibitors*; D058069:Neglected Diseases / Q000188:drug therapy; D054852:Small Molecule Libraries; D014346:Trypanosoma brucei brucei / Q000187:drug effects* / Q000201:enzymology*; D014353:Trypanosomiasis, African / Q000188:drug therapy
chemical_list: D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries; D008718:Methionine-tRNA Ligase
keywords: Trypanosoma brucei; aminoacyl-tRNA synthetases; high-throughput screening; human African trypanosomiasis; orthogonal screening