title: Target identification by chromatographic co-elution: monitoring of drug-protein interactions without immobilization or chemical derivatization.
abstract: Bioactive molecules typically mediate their biological effects through direct physical association with one or more cellular proteins. The detection of drug-target interactions is therefore essential for the characterization of compound mechanism of action and off-target effects, but generic label-free approaches for detecting binding events in biological mixtures have remained elusive. Here, we report a method termed target identification by chromatographic co-elution (TICC) for routinely monitoring the interaction of drugs with cellular proteins under nearly physiological conditions in vitro based on simple liquid chromatographic separations of cell-free lysates. Correlative proteomic analysis of drug-bound protein fractions by shotgun sequencing is then performed to identify candidate target(s). The method is highly reproducible, does not require immobilization or derivatization of drug or protein, and is applicable to diverse natural products and synthetic compounds. The capability of TICC to detect known drug-protein target physical interactions (K(d) range: micromolar to nanomolar) is demonstrated both qualitatively and quantitatively. We subsequently used TICC to uncover the sterol biosynthetic enzyme Erg6p as a novel putative anti-fungal target. Furthermore, TICC identified Asc1 and Dak1, a core 40 S ribosomal protein that represses gene expression, and dihydroxyacetone kinase involved in stress adaptation, respectively, as novel yeast targets of a dopamine receptor agonist.
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Janet N Y
authors.initials: JN
authors.lastname: Chan
authors.forename: Dajana
authors.initials: D
authors.lastname: Vuckovic
authors.forename: Lekha
authors.initials: L
authors.lastname: Sleno
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Olsen
authors.forename: Oxana
authors.initials: O
authors.lastname: Pogoutse
authors.forename: Pierre
authors.initials: P
authors.lastname: Havugimana
authors.forename: Johannes A
authors.initials: JA
authors.lastname: Hewel
authors.forename: Navgeet
authors.initials: N
authors.lastname: Bajaj
authors.forename: Yale
authors.initials: Y
authors.lastname: Wang
authors.forename: Marcel F
authors.initials: MF
authors.lastname: Musteata
authors.forename: Corey
authors.initials: C
authors.lastname: Nislow
authors.forename: Andrew
authors.initials: A
authors.lastname: Emili
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis / Q000378:metabolism*; D000935:Antifungal Agents / Q000378:metabolism*; D002474:Cell-Free System; D002853:Chromatography, Liquid; D018491:Dopamine Agonists / Q000378:metabolism; D004926:Escherichia coli; D005656:Fungal Proteins / Q000032:analysis / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006877:Hydroxamic Acids / Q000378:metabolism; D007192:Indenes / Q000378:metabolism; D018942:Macrolides / Q000378:metabolism; D013058:Mass Spectrometry; D008727:Methotrexate / Q000378:metabolism; D008780:Methyltransferases / Q000032:analysis / Q000378:metabolism*; D058990:Molecular Targeted Therapy; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis / Q000378:metabolism*; D011485:Protein Binding; D040901:Proteomics; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000378:metabolism*
chemical_list: C111825:ASC1 protein, S cerevisiae; D048868:Adaptor Proteins, Signal Transducing; D000935:Antifungal Agents; D018491:Dopamine Agonists; D005656:Fungal Proteins; D006877:Hydroxamic Acids; D007192:Indenes; D018942:Macrolides; D029701:Saccharomyces cerevisiae Proteins; C003329:sordarin; C012589:trichostatin A; D008780:Methyltransferases; C022670:delta 24-sterol methyltransferase; D017853:Phosphotransferases (Alcohol Group Acceptor); C023132:glycerone kinase; D019204:GTP-Binding Proteins; C035359:monorden; D008727:Methotrexate