Figure 1: VLX1570 inhibits and binds to proteasome DUBs in vitro.
(a) Structure of VLX1570 and b-AP15. The α,β-unsaturated carbonyls required for activity are marked with red filled circles and the Michael acceptor at the acrylamide is denoted with a green circle. (b) Inhibition of active-site-directed labeling of proteasomal deubiquitinases. Purified 19S proteasomes (5 nM) were pre-treated with DMSO or the indicated concentrations of VLX1570 (in DMSO) for 10 min at room temperature, followed by labeling with HA-Ub-VS and immunoblotting. (c) VLX1570 binding to USP14. Left: readings show binding signal in RU plotted against time in seconds. Higher concentrations clearly give rise to a higher binding signal. Right: points taken 10 seconds from the stop of injection in the reading to the left, plotted against the concentration on a linear scale. VLX1570 saturates the binding site in USP14 approaching a max signal and the fit of the data approaches the Rmax. The dissociation constant KD is read from the fit where the 1:1 binding isotherm has the value of 50% of the Rmax.
