PYR-41 is the first cell-permeable inhibitor of ubiquitin-activating enzyme E1, with no activity at E2.

E1:< 10 μM

PYR-41 (50 μM) inhibits activity of ubiquitin-activating enzyme E1 by over 90%. PYR-41 could be a target for nucleophilic attack and potentially reacts with the active site cysteine of E1. PYR-41 efficiently blocks cyclin E degradation. PYR-41 decreases the level of E1fUb thioesters in cells with a IC50 of between 10 and 25 μM, and prevents proteasome inhibitor–induced accumulation of ubiquitylated proteins. PYR-41 increases total sumoylation in cells and in cell harboring temperature-sensitive E1. PYR-41 is able to inhibit both proteasome-dependent and proteasome-independent activities of ubiquitylation. PYR-41 (50 μM) attenuates 1 ng/mL IL-1α-mediated nuclear factor-κB activation by >60% through preventing the downstream ubiquitylation and proteasomal degradation of IκBα. PYR-41 inhibits degradation of p53 and activates the transcriptional activity of p53, which enable its differentially killing transformed p53-expressing cells. [1] PYR-41 blocks ubiquitination reactions but paradoxically leads to the accumulation of high MW ubiquitinated proteins. PYR-41 also has equal or greater inhibitory activity against several deubiquitinases (DUBs) in intact cells and purified USP5 in vitro. PYR-41 also mediates cross-linking of specific protein kinases (Bcr-Abl, Jak2) to inhibit their signaling activity. [1]

Rabbit or mouse E1 (apper 250 ng) is incubated with 32P-ubiquitin in 1× reaction buffer [50 mM Tris (pH 7.4), 0.2 mM ATP, 0.5 mM MgCl2] at room temperature for 15 min. In some experiments, the His-tagged mouse E1 is bound to TALON cobalt affinity resin before carrying out incubations and reactions. Mouse E1 and 32P-ubiquitin are added to the beads in 1× reaction buffer and incubated as for E1 reactions. Samples are heated in nonreducing SDS-PAGE sample buffer and resolved by SDS-PAGE. Thioesters with ubiquitin are visualized by Storm PhosphoImager.