Small Molecule Interactome Mapping by Photoaffinity Labeling Reveals Binding Site Hotspots for the NSAIDs

Many
therapeutics elicit cell-type specific polypharmacology that
is executed by a network of molecular recognition events between a
small molecule and the whole proteome. However, measurement of the
structures that underpin the molecular associations between the proteome
and even common therapeutics, such as the nonsteroidal anti-inflammatory
drugs (NSAIDs), is limited by the inability to map the small molecule
interactome. To address this gap, we developed a platform termed small
molecule interactome mapping by photoaffinity labeling (SIM-PAL) and
applied it to the in cellulo direct characterization of specific NSAID
binding sites. SIM-PAL uses (1) photochemical conjugation of NSAID
derivatives in the whole proteome and (2) enrichment and isotope-recoding
of the conjugated peptides for (3) targeted mass spectrometry-based
assignment. Using SIM-PAL, we identified the NSAID interactome consisting
of over 1000 significantly enriched proteins and directly characterized
nearly 200 conjugated peptides representing direct binding sites of
the photo-NSAIDs with proteins from Jurkat and K562 cells. The enriched
proteins were often identified as parts of complexes, including known
targets of NSAID activity (e.g., NF-κB) and novel interactions
(e.g., AP-2, proteasome). The conjugated peptides revealed direct
NSAID binding sites from the cell surface to the nucleus and a specific
binding site hotspot for the three photo-NSAIDs on histones H2A and
H2B. NSAID binding stabilized COX-2 and histone H2A by cellular thermal
shift assay. Since small molecule stabilization of protein complexes
is a gain of function regulatory mechanism, it is conceivable that
NSAIDs affect biological processes through these broader proteomic
interactions. SIM-PAL enabled characterization of NSAID binding site
hotspots and is amenable to map global binding sites for virtually
any molecule of interest.