μMap Photoproximity Labeling Enables Small Molecule Binding Site Mapping

The characterization of ligand binding modes is a crucial
step
in the drug discovery process and is especially important in campaigns
arising from phenotypic screening, where the protein target and binding
mode are unknown at the outset. Elucidation of target binding regions
is typically achieved by X-ray crystallography or photoaffinity labeling
(PAL) approaches; yet, these methods present significant challenges.
X-ray crystallography is a mainstay technique that has revolutionized
drug discovery, but in many cases structural characterization is challenging
or impossible. PAL has also enabled binding site mapping with peptide-
and amino-acid-level resolution; however, the stoichiometric activation
mode can lead to poor signal and coverage of the resident binding
pocket. Additionally, each PAL probe can have its own fragmentation
pattern, complicating the analysis by mass spectrometry. Here, we
establish a robust and general photocatalytic approach toward the
mapping of protein binding sites, which we define as identification
of residues proximal to the ligand binding pocket. By utilizing a
catalytic mode of activation, we obtain sets of labeled amino acids
in the proximity of the target protein binding site. We use this methodology
to map, in vitro, the binding sites of six protein targets, including
several kinases and molecular glue targets, and furthermore to investigate
the binding site of the STAT3 inhibitor MM-206, a ligand with no known
crystal structure. Finally, we demonstrate the successful mapping
of drug binding sites in live cells. These results establish μMap
as a powerful method for the generation of amino-acid- and peptide-level
target engagement data.