Fig. 2.

Celastrol was identified as a covalent inhibitor against the peroxidase activity of PRDX1. a Peroxidase activity inhibition of PRDX1 with Celastrol incubation. Celastrol was incubated with recombinant PRDX1 for 1.5 h at different concentrations. The inhibition rate was calculated from initial reaction slope of each assay well as shown in Supplementary Fig. 2a. The IC₅₀ value of Celastrol against PRDX1 is 0.29 ± 0.009 µM. Peroxidase activity inhibition of PRDX2 (b), PRDX3 (c), PRDX4 (d), PRDX5 (e), and PRDX6 (f) with Celastrol incubation. Celastrol was incubated with recombinant PRDX2-6 as in (a). The inhibition rates were calculated from initial reaction slope in Supplementary Fig. 2b–f for PRDX2~PRDX6. The IC₅₀ values of Celastrol are 3.79 ± 0.26 µM for PRDX2, 6.67 ± 0.52 µM for PRDX3, above 100 µM for PRDX4, 2.30 ± 0.13 µM for PRDX5, and 27.26 ± 0.39 µM for PRDX6, respectively. The inhibitions of peroxidase activity by Celastrol were calculated from triplicate experiments. All data is shown in mean ± SEM. g The binding affinity of Celastrol with recombinant PRDX1 determined by the SPR assay. h Click chemistry labeling of recombinant PRDX1 through alkynylated Celastrol. Covalently bounded PRDX1 was labeled with click chemistry reaction through alkynyl group. “Input” represents PRDX1 without any incubation. “DMSO” represents PRDX1 incubated with DMSO. “Input” and “DMSO” were set as negative control. Click chemistry labeling was blotted with streptavidin HRP, and the amount of PRDX1 in each sample was blotted with anti-PRDX1 primary antibody. i Mass spectra analysis of recombinant PRDX1 before and after Celastrol binding. The upper spectra was the sample of PRDX1 incubated with DMSO, while the lower spectra was the sample of PRDX1 incubated with Celastrol for 1.5 h. Main mass peak of ligand-free PRDX1 was shown in red, while peak of PRDX1-Celastrol complex was shown in cyan. The exact mass of Celastrol is 450.277. j Click chemistry labeling of recombinant wild type (WT) PRDX1 and four mutants. Alkynylated Celastrol (10 µM) was incubated with WT PRDX1 and four mutants for 1 h and blotted. WT PRDX1 incubated with alkynyl warhead was set as negative control