Fig. 5: N-phosphono-(S)−3-aminopiperidine-2-ones bind covalently to DPP9.

a X-ray structure of 16 in complex with DPP9 refined to a final resolution of 2.49 Å, shown from two different angles. The ligand is covalently bound to the catalytically active serine S730 residue and is contoured at 1 σ with the refined 2F_o-F_c electron density map. Hydrogen bond interactions are depicted as dashed lines. b ABPP without (FC plotted on x-axis) or after pre-treatment with 50 µM 8, 11 or 16 (competition experiment, FC plotted on y-axis) with 10 µM 9 after click chemistry (n = 4 biologically independent samples). Dashed lines indicate the FC ≥ 2 (9/DMSO) or FC ≤ -2 (Competitor/9) threshold. Black symbols indicate identified protein groups. To identify statistically significant hits from the analysis (marked as a triangle or square), p ≤ 0.01 (two-sided Student’s t-test, permutation-based FDR with 250 randomizations and FDR = 0.05) was applied. c Addition of N-phosphono-(S)−3-aminopiperidine-2-ones block the interaction between endogenous DPP9 and BRCA2. Quantification of PLAs showing MMC-induced DPP9-BRCA2 PLA events in HeLa wild-type cells, in the presence of 10 µM of the respective inhibitory compounds. Cells were treated with 300 nM MMC for 24 h and 10 µM of the indicated inhibitors for 1 h prior to fixation. Each dot represents the number of PLA events in a single cell. Data were analyzed by unpaired two-sided t-test comparisons (****p < 0.0001). To visualize the three biological replicates, each biological replicate is labeled differently: circle, triangle or square. n values indicate total number of cells in each analysis group from 3 independent biological replicates in total: n = 240 for BRCA2 Ab-Ctrl., n = 237 for DPP9 Ab-Ctrl., n = 222 for +Ctrl., n = 202 for 1G244, n = 231 for 8, n = 205 for 16. d Inhibition of DPP8/9 increases cellular sensitivity to genotoxic stress. HeLa wild-type (WT) cells were treated with 1 µM MMC and 10 µM of the respective inhibitors. Cell viability was measured after 72 h, and normalized to WT mock treated cells. DPP9^KO cells and WT cells treated with 1G244 (10 µM) were analyzed as positive controls, whereas WT cells treated with Sitagliptin were used as a negative control. Dashed lines indicate the viability of the WT + MMC cells. The graph shows the mean and error bars indicate the SEM of all individual measurements (n = 18 (HeLa wild-type controls), 17 (HeLa DPP9^KO control) or 9 (inhibitor-treated samples) independent biological replicates). Data were analyzed by a Tukey two-way ANOVA, using a mixed effect analysis (n.s. = not significant, *p = 0.0157, ****p < 0.0001). Source data are provided as a Source Data file.