Mapping the Protein Interaction Landscape for Fully Functionalized Small-Molecule Probes in Human Cells

Phenotypic screening provides a means
to discover small molecules
that perturb cell biological processes. Discerning the proteins and
biochemical pathways targeted by screening hits, however, remains
technically challenging. We recently described the use of small molecules
bearing photoreactive groups and latent affinity handles as fully
functionalized probes for integrated phenotypic screening and target
identification. The general utility of such probes, or, for that matter,
any small-molecule screening library, depends on the scope of their
protein interactions in cells, a parameter that remains largely unexplored.
Here, we describe the synthesis of an ∼60-member fully functionalized
probe library, prepared from Ugi-azide condensation reactions to impart
structural diversity and introduce diazirine and alkyne functionalities
for target capture and enrichment, respectively. In-depth mass spectrometry-based
analysis revealed a diverse array of probe targets in human cells,
including enzymes, channels, adaptor and scaffolding proteins, and
proteins of uncharacterized function. For many of these proteins,
ligands have not yet been described. Most of the probe–protein
interactions showed well-defined structure–activity relationships
across the probe library and were blocked by small-molecule competitors
in cells. These findings indicate that fully functionalized small
molecules canvas diverse segments of the human proteome and hold promise
as pharmacological probes of cell biology.