title: A "clickable" MTX reagent as a practical tool for profiling small-molecule-intracellular target interactions via MASPIT.
abstract: We present a scalable synthesis of a versatile MTX reagent with an azide ligation handle that allows rapid γ-selective conjugation to yield MTX fusion compounds (MFCs) appropriate for MASPIT, a three-hybrid system that enables the identification of mammalian cytosolic proteins that interact with a small molecule of interest. We selected three structurally diverse pharmacologically active compounds (tamoxifen, reversine, and FK506) as model baits. After acetylene functionalization of these baits, MFCs were synthesized via a CuAAC reaction, demonstrating the general applicability of the MTX reagent. In analytical mode, MASPIT was able to give concentration-dependent reporter signals for the established target proteins. Furthermore, we demonstrate that the sensitivity obtained with the new MTX reagent was significantly stronger than that of a previously used non-regiomeric conjugate mixture. Finally, the FK506 MFC was explored in a cellular array screen for targets of FK506. Out of a pilot collection of nearly 2000 full-length human ORF preys, FKBP12, the established target of FK506, emerged as the prey protein that gave the highest increase in luciferase activity. This indicates that our newly developed synthetic strategy for the straightforward generation of MFCs is a promising asset to uncover new intracellular targets using MASPIT cellular array screening.
authors: Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Gent, Belgium.
authors: Martijn D P
authors: MD
authors: Risseeuw
authors: Dries J H
authors: DJ
authors: De Clercq
authors: Sam
authors: S
authors: Lievens
authors: Ulrik
authors: U
authors: Hillaert
authors: Davy
authors: D
authors: Sinnaeve
authors: Freya
authors: F
authors: Van den Broeck
authors: José C
authors: JC
authors: Martins
authors: Jan
authors: J
authors: Tavernier
authors: Serge
authors: S
authors: Van Calenbergh
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000737:chemistry* / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D057930:Click Chemistry; D061565:Cycloaddition Reaction; D057809:HEK293 Cells; D006801:Humans; D008727:Methotrexate / Q000737:chemistry* / Q000633:toxicity; D009025:Morpholines / Q000737:chemistry; D011687:Purines / Q000737:chemistry; D050796:STAT3 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D054852:Small Molecule Libraries / Q000737:chemistry*; D013237:Stereoisomerism; D016559:Tacrolimus / Q000737:chemistry; D013629:Tamoxifen / Q000737:chemistry; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D046888:Tissue Array Analysis
chemical_list: D000964:Antimetabolites, Antineoplastic; D009025:Morpholines; D011687:Purines; D050796:STAT3 Transcription Factor; D054852:Small Molecule Libraries; D013629:Tamoxifen; D013762:Tetrahydrofolate Dehydrogenase; D016559:Tacrolimus; D008727:Methotrexate; C484369:2-(4-morpholinoanilino)-6-cyclohexylaminopurine