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. 2023 Feb 8;14:686. doi: 10.1038/s41467-023-36246-0

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Hit molecule WH-9943-103C inhibited deubiquitination activity of VCPIP1 in Ub-Rho cleavage assay after 6 h incubation (n = 1). b WH-9943-103C inhibited VCPIP1 (highlighted in green) selectively out of a panel of 41 purified recombinant DUBs after 15 min incubation. c WH-9943-103C displayed in-cell target engagement as determined for DUB labeling by ABP then visualized on a Western blot. d WH-9943-103C labeled recombinant VCPIP1 with 1:1 stoichiometry as read out by intact protein mass spectrometry. Labeled ion envelope is shown in blue, unlabeled ion envelope from DMSO control is shown in red. e CE-MS/MS identified the catalytic cysteine of VCPIP1 to be covalently modified by WH-9943-103C, red and blue glyphs adjacent to the peptide sequence indicate y- and b-type fragment ions, respectively, detected in the MS/MS spectrum. The y-ion series confirms catalytic cysteine modification (C*). f A proteome-wide competitive binding survey of WH-9943-103C activity (50µM) indicated off-target modification of 39 (red dots, FDR <1%, >3-fold competition relative to DMSO) out of 24,579 unique cysteines detected (p-values for all detected ratios derived from maximum likelihood estimation and corrected by the Benjamini-Hochberg method). The blue box indicates >4.5-fold competition relative to DMSO, the observed fold competition for VCPIP1. This includes 4 cysteines that were previously characterized as ‘hyper-reactive’ (red stars). Source data are provided as a source data file.