Cited by 102Advanced Proteomics Techniques and ApplicationsProtein Interaction Studies and Fluorescence AnalysisMass Spectrometry Techniques and Applications

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

Gao, Jinxu, Mfuh, Adelphe, Amako, Yuka, Woo, Christina M.·2018·Journal of the American Chemical Society

Abstract

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.

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Crossref · 10.1021/jacs.7b11639

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  • Equipping noncovalent fragments with a photoreactive diazirine moiety enables their proteome-wide target identification without prerequisite of a particular amino acid in or nearby a ligandable site.

    “Equipping noncovalent fragments with a photoreactive diazirine moiety similarly enables their proteome-wide target identification without the prerequisite of a particular amino acid in or nearby a ligandable site.98−100”

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Authors
Gao, J., Mfuh, A., Amako, Y., & Woo, C. M.
Affiliations
Harvard University (US)
Corresponding author
Woo, Christina M.
Venue
Journal of the American Chemical Society
Published
2018-03-15
Volume
140
Issue
12
Pages
4259-4268
ISSN
  • 0002-7863 (ISSN-L)
  • 1520-5126
OpenAlex
W2794389997
Cited by
102
References
49
Citations by year
2026: 6 · 2025: 12 · 2024: 17 · 2023: 8 · 2022: 12 · 2021: 19 · 2020: 11 · 2019: 12 · 2018: 4 · 2017: 1
FWCI
4.97
Type
journal article
Language
en
Open access
closed
Locations
  • Journal of the American Chemical Society (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • Harvard University
  • Burroughs Wellcome Fund
  • Uehara Memorial Foundation
  • Murata Science Foundation
Topics
  • Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Interaction Studies and Fluorescence Analysis
  • Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
Keywords
nonsteroidal anti-inflammatory drugs, photoaffinity labeling, protein binding sites, polypharmacology, histone H2A, histone H2B, NF-κB, AP-2, proteasome, Jurkat cells, K562 cells, protein-protein interaction
MeSH terms
Anti-Inflammatory Agents, Non-Steroidal, Binding Sites, Humans, Peptides, Proteins, Molecular Structure, Jurkat Cells, Photoaffinity Labels, K562 Cells, Small Molecule Libraries

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