Extended Data Fig. 4: Functional characterization of covalent ligands targeting an essential cysteine C80 in TOE1.
From: Assigning functionality to cysteines by base editing of cancer dependency genes
a, The fragment electrophile ligandability profile for cysteines in TOE1, showing ligandability of C80. b, Quantification of TOE1_C80 engagement by the indicated compounds as measured by cysteine-directed ABPP performed in Ramos cells (20 µM, 3 h). Data are average values ± SEM normalized to DMSO from n = 2 independent experiments. c, Western blot showing recombinantly expressed FLAG-tagged TOE1 in MCC142 cells. GAPDH was used as a loading control. This was repeated in 3 independent experiments with similar results. d, Schematic summarizing the TOE1 deadenylation assay workflow. e, Alignment of TOE1 orthologous proteins in a panel of selected vertebrate species. Only the aligned sequences near human TOE1_C80 are shown. f, Comparison of deadenylase activity of recombinant FLAG-WT- and C80S-TOE1 proteins immunoprecipitated from MCC142 cells. Data represent two independent experiments. g, Quantification of time-dependent TOE1 deadenylation activity as shown in Fig. 4h. Data represent 2 independent experiments. h, Quantification of TOE1_C80 engagement by the indicated compounds (20 µM, 6 h) as measured by monitoring the C80-containing tryptic peptide in IP–MS experiments from FLAG-WT-TOE1-expressing MCC142 cells. Data are average values ± SEM normalized to DMSO from n = 2 independent experiments. i, Schematic for growth competition assay. Briefly, the TOE1-dependent cell line MCC142 was spin-infected with lentiviral vectors expressing codon-optimized FLAG-WT- or C80S-TOE1. After antibiotic (G418) selection, cells were mixed at a 1:1 ratio and treated with WX-02-33 (10 µM) or DMSO and grown for 8 days, after which genomic DNA was extracted and targeted amplicon sequencing was used to quantify the relative frequency of C80S vs WT. j, Relative allele frequency of MCC142 cells expressing WT- or C80S-TOE1 treated with 10 µM WX-02-33 or DMSO. The p value was calculated using two-sided Student’s t test based on n = 3 independent experiments.
