title: Improved Deconvolution of Protein Targets for Bioactive Compounds Using a Palladium Cleavable Chloroalkane Capture Tag.
abstract: The benefits provided by phenotypic screening of compound libraries are often countered by difficulties in identifying the underlying cellular targets. We recently described a new approach utilizing a chloroalkane capture tag, which can be chemically attached to bioactive compounds to facilitate the isolation of their respective targets for subsequent identification by mass spectrometry. The tag minimally affects compound potency and membrane permeability, enabling target engagement inside cells. Effective enrichment of these targets is achieved through selectivity in both their rapid capture onto immobilized HaloTag and their subsequent release by competitive elution. Here, we describe a significant improvement to this method where selective elution was achieved through palladium-catalyzed cleavage of an allyl-carbamate linkage incorporated into the chloroalkane capture tag. Selective tag cleavage provided robust release of captured targets exhibiting different modes of binding to the bioactive compound, including prolonged residence time and covalent interactions. Using the kinase inhibitors ibrutinib and BIRB796 as model compounds, we demonstrated the capability of this new method to identify both expected targets and "off-targets" exhibiting a range of binding affinities, cellular abundances, and binding characteristics.
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Rachel
authors.initials: R
authors.lastname: Friedman Ohana
authors.affiliation: Promega Biosciences LLC , San Luis Obispo, California 93401, United States.
authors.forename: Sergiy
authors.initials: S
authors.lastname: Levin
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Monika G
authors.initials: MG
authors.lastname: Wood
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Kris
authors.initials: K
authors.lastname: Zimmerman
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Melanie L
authors.initials: ML
authors.lastname: Dart
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Marie K
authors.initials: MK
authors.lastname: Schwinn
authors.affiliation: Promega Biosciences LLC , San Luis Obispo, California 93401, United States.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kirkland
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Robin
authors.initials: R
authors.lastname: Hurst
authors.affiliation: Promega Biosciences LLC , San Luis Obispo, California 93401, United States.
authors.forename: H Tetsuo
authors.initials: HT
authors.lastname: Uyeda
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Lance P
authors.initials: LP
authors.lastname: Encell
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Keith V
authors.initials: KV
authors.lastname: Wood
mesh_terms: D000473:Alkanes / Q000737:chemistry*; D002384:Catalysis; D010165:Palladium / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*
chemical_list: D000473:Alkanes; D011506:Proteins; D010165:Palladium