Fig. 3: GK13S potently inhibits recombinant and cellular UCHL1.
From: Structural basis for specific inhibition of the deubiquitinase UCHL1
a Intact protein mass spectrometry revealed covalent binding of GK13S and GK13R to recombinant UCHL1. UCHL1 (0.8 µM) was treated with compound (1 µM) or DMSO for 2 h. b Inhibitory potencies of indicated compounds, preincubated with UCHL1 for 1 h, determined from a Ubiquitin rhodamine cleavage assay. Data points are shown as mean ± standard deviation (N = 2) from independent experiments. IC50 values were determined from 5 (Cpd158), 3 (GK13S), or 2 (GK13R) independent experiments. Source data are provided as a Source Data file. c kobs/[I] kinetic assay of GK13S binding to UCHL1 at indicated concentrations. Data points are shown as means calculated from N = 3 wells (N = 6 wells for DMSO and blank (i.e., Ubiquitin rhodamine without enzyme)). Rate constants kobs were determined from the plot on the left, and then plotted against inhibitor concentrations as shown on the right for a representative experiment. The kobs/[I] value was determined as the slope and is given as means ± standard deviation calculated from five independent experiments. Source data are provided as a Source Data file. d Thermal shift assay showing the melting temperature (Tm) of UCHL1 (1 µM) pretreated for 1 h with compounds at indicated concentrations. Source data are provided as a Source Data file. e Inhibition of cellular UCHL1. Western blot analysis of endogenous UCHL1 labeled with HA-Ub-VS after treatment of HEK293 cells with either the indicated compounds or DMSO for 24 h. Uncropped versions of gels and blots are shown in the supplementary information.
