Abstract
Target identification involves deconvoluting the protein target of a pharmacologically active, small-molecule ligand, a process that is critical for early drug discovery yet technically challenging. Photoaffinity labelling strategies have become the benchmark for small-molecule target deconvolution, but covalent protein capture requires the use of high-energy ultraviolet light, which can complicate downstream target identification. Thus, there is a strong demand for alternative technologies that allow for controlled activation of chemical probes to covalently label their protein target. Here we introduce an electroaffinity labelling platform that leverages the use of a small, redox-active diazetidinone functional group to enable chemoproteomic-based target identification of pharmacophores within live cell environments. The underlying discovery to enable this platform is that the diazetidinone can be electrochemically oxidized to reveal a reactive intermediate useful for covalent modification of proteins. This work demonstrates the electrochemical platform to be a functional tool for drug-target identification.
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Data availability
Data supporting the main findings of this work are available within the article and Supplementary Information. Crystallographic data for the structures reported in this article have been deposited at the Cambridge Crystallographic Data Centre, under the deposition number CCDC 2093827 (11). Copies of the data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/. Source data are provided with this paper.
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Acknowledgements
The work was supported by the National Science Foundation Center for Synthetic Organic Electrochemistry CHE-2002158 (exploration and development of electrochemically active functional group), National Institutes of Health grant GM-118176 (synthesis of elaborated probes for biological studies) and gifts from Merck & Co., Inc., Kenilworth, NJ, USA (synthesis of elaborated probes for biological studies). G.N.H. was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), grant numbers 419055018/HE 8427/1-1. A.F.S. was supported by the Lundbeck Foundation (grant number R208-2015-3354), E.R.-C. was supported by the Galician Programme for Research, Innovation and Growth for 2018. We thank D.-H. Huang and L. Pasternack (Scripps Research) for assistance with NMR spectroscopy; and J. Chen, B. Sanchez and E. Sturgell (Automated Synthesis Facility, Scripps Research) for purification of compounds and acquisition of HRMS data. We thank T. Wyche (Merck & Co., Inc.) for assistance with HRMS data, S. Ingale (Merck & Co., Inc.) for assistance with peptide synthesis, and J. Oh (Merck & Co., Inc.) for helpful discussions.
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R.C.O., O.O.F. and P.S.B. conceptualized the study. Y.K., G.N.H., A.F.S., J.C.V., E.R.-C. and P.S.B. designed and performed chemical experiments. K.A.R., L.A.A., A.K.O., L.R.R., R.C.O. and O.O.F. designed and performed biological experiments. All the authors contributed to data analysis. Y.K., K.A.R., R.C.O., O.O.F. and P.S.B. wrote the paper.
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K.A.R., L.A.A., A.K.O., L.R.R., R.C.O., and O.O.F. are/were employees of Merck and Co., Inc. during the preparation of this manuscript.
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Unprocessed western blot.
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Peptide mapping spectral count.
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Gene Ontology data.
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Cell assay data.
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Kawamata, Y., Ryu, K.A., Hermann, G.N. et al. An electroaffinity labelling platform for chemoproteomic-based target identification. Nat. Chem. 15, 1267–1275 (2023). https://doi.org/10.1038/s41557-023-01240-y
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DOI: https://doi.org/10.1038/s41557-023-01240-y
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