Fig. 5: Ubiquitination of BI8626 derivatives occurs in cells.
From: Selective ubiquitination of drug-like small molecules by the ubiquitin ligase HUWE1
a Representative targeted MS² spectrum of a BI8626-modified C-terminal Ub peptide (precursor m/z 278.1506, z = 2), generated by tryptic digestion of cell lysates, following Ub-IP. Annotated fragment ions at m/z 249.6405 (z = 2) and 498.2745 (z = 1) correspond to a glycine–BI8626 conjugate. Additional fragment ions derived from BI8626 itself are observed at m/z 91.0545 (z = 1), 212.6164 (z = 2), 221.1296 (z = 2), 334.1778 (z = 1), 424.2258 (z = 1), and 441.2523 (z = 1), corresponding to characteristic cleavages of the compound. Structural representations of key fragmentation events are indicated, with boxed parts corresponding to the respective fragment ions. b Cartoon of our click chemistry-based strategy for detecting inhibitor ubiquitination in cell lysates. Elements of this figure were created in BioRender. Pohl, P. (2025) https://BioRender.com/sygo5n6. c Proof-of-principle experiment for the click chemistry-based detection of cell-based derivative 6 ubiquitination according to (b). Additionally, the total protein is shown by Coomassie staining (bottom). Compound 7 is clickable, but devoid of a primary amino group, providing a negative control. d Click-chemistry-based detection of cellular derivative 8 ubiquitination in the absence and presence of the UBA1 inhibitor TAK243, analogous to (c). Note that compound 8 is used here instead of 6, for it provided enhanced signal (see Methods). The two sections are part of the same gel, shown with different exposures. e Click-chemistry-based detection of cellular derivative 8 ubiquitination in HEK293T WT and HUWE1 KO cells, analogous to (c). Additionally, the HUWE1 level and an actin-loading control are shown based on IB. f Quantification of the ubiquitinated compound (across all species, including 8-Ub, 8-Ub2 and 8-Ubn) from (e), normalized to WT cells. Data are represented as mean ± SD (n = 4 biological replicates). Statistical analysis with an unpaired, two-sided t-test **p = 0.0036. g Click-chemistry-based detection of cellular derivative 8 ubiquitination in HEK293T HUWE1 KO cells, analogous to (c), upon overexpression of the indicated HUWE1 variants. C4341S catalytically inactive. The HUWE1 level and an actin-loading control are shown based on IB. Source data are provided as a Source Data file.
