Novel Tetrafunctional Probes Identify Target Receptors and Binding Sites of Small-Molecule Drugs from Living Systems

Significant
advancement of chemoproteomics has contributed to uncovering
the mechanism of action (MoA) of small-molecule drugs by characterizing
drug–protein interactions in living systems. However, cell-membrane
proteins such as G protein-coupled receptors (GPCRs) and ion channels,
due to their low abundance and unique biophysical properties associated
with multiple transmembrane domains, can present challenges for proteome-wide
mapping of drug–receptor interactions. Herein, we describe
the development of novel tetrafunctional probes, consisting of (1)
a ligand of interest, (2) 2-aryl-5-carboxytetrazole (ACT) as a photoreactive
group, (3) a hydrazine-labile cleavable linker, and (4) biotin for
enrichment. In live cell labeling studies, we demonstrated that the
ACT-based probe showed superior reactivity and selectivity for labeling
on-target GPCR by mass spectrometry analysis compared with control
probes including diazirine-based probes. By leveraging ACT-based cleavable
probes, we further identified a set of representative ionotropic receptors,
targeted by CNS drugs, with remarkable selectivity and precise binding
site information from mouse brain slices. We anticipate that the robust
chemoproteomic platform using the ACT-based cleavable probe coupled
with phenotypic screening should promote identification of pharmacologically
relevant target receptors of drug candidates and ultimately development
of first-in-class drugs with novel MoA.