UCLA researchers from the Department of Biological Chemistry have developed a chemoproteomic platform for high-coverage, site-specific mapping of small-molecule–protein interactions using a cleavable silyl ether–based photoaffinity probe design.
BACKGROUND: Small molecule drugs rely on specific and selective interactions with protein targets to achieve the desired therapeutic effect. The identification of the binding sites of these interactions relies on affinity labeling followed by proteomic studies. While chemoproteomics coupled with photoaffinity labeling (PAL) is commonly used to map and discover drug-protein interactions across the proteome, existing methods suffer from several limitations. Crosslinked peptides tend to produce convoluted fragmentation patterns during tandem mass spectrometry (MS/MS), resulting in inaccurate site assignment. Other methods rely on bulky and hydrophobic labeling groups, making recovery of modified peptides inefficient. As such, existing methods fail to provide precise binding site resolution and do not enable comparison of structurally distinct drug candidates. There remains an unmet need for a labeling method that allows precise and quantitative mapping of small-molecule binding sites across the proteome.
INNOVATION: UCLA researchers from the Department of Biological Chemistry have developed a novel chemoproteomic platform for the labeling of small molecule-protein interaction sites. This technology, called Silyl Ether Enables Chemoproteomic Interaction and Target Engagement (SEE-CITE), relies on the incorporation of a fully functionalized and chemically cleavable silyl ether linker into the photoaffinity probe structure. The reported SEE-CITE probes covalently label target proteins upon UV activation, and the bulky labeling moiety can be subsequently cleaved under mild acidic conditions. This labels the protein with a minimal barcode that is subsequently used for precise peptide identification by mass spectrometry. The reported approach enables high resolution mapping of drug binding sites across the proteome and direct quantitation of relative binding at individual sites between chemically distinct compounds. SEE-CITE can revolutionize the drug discovery process by providing a powerful tool to delineate novel interaction sites on therapeutically relevant protein targets.
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ADVANTAGES:
DEVELOPMENT-TO-DATE: UCLA researchers have benchmarked SEE-CITE using scout fragments and analogs of FDA-approved kinase inhibitors to identify novel binding sites for multiple high impact targets in vitro.
RELEVANT PUBLICATIONS (from the inventors only): Takechi S, Ngo C, Burton N, Villanueva M, Boatner L, Yu F, et al. Silyl Ether Enables High Coverage Chemoproteomic Interaction Site Mapping. ChemRxiv. 2024; doi:10.26434/chemrxiv-2024-21r7b.
KEYWORDS: chemoproteomics; photoaffinity labeling; silyl ether linker; mass spectrometry; protein interaction mapping; drug discovery; off-target profiling; kinase inhibitors; dasatinib; asciminib; BCR-ABL1; STING; COX5A; FECH; proteome-wide target engagement