Improved Deconvolution of Protein Targets for Bioactive Compounds Using a Palladium Cleavable Chloroalkane Capture Tag

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.