Small Molecule Interactome Mapping by Photoaffinity Labeling Reveals Binding Site Hotspots for the NSAIDs
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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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”
From Advancing Targeted Protein Degradation via Multiomics Profiling and Artificial Intelligence (2023) · cites this paper as [99]
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Advancing Targeted Protein Degradation via Multiomics Profiling and Artificial Intelligence
Duran-Frigola, M., Cigler, M., & Winter, G. E. (2023) · Journal of the American Chemical Society · doi:10.1021/jacs.2c11098 · Cited by 50 · see citing works
- 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
- Publisher
- American Chemical Society (ACS)
- 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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