The conjugation of
proteins with
ubiquitin plays numerous regulatory roles through both proteasomal-dependent and nonproteasomal-dependent functions. Alterations in ubiquitylation are observed in a wide range of pathologic conditions, including numerous
malignancies. For this reason, there is great interest in targeting the
ubiquitin-
proteasome system in
cancer. Several classes of
proteasome inhibitors, which block degradation of ubiquitylated
proteins, are widely used in research, and one,
Bortezomib, is now in clinical use. Despite the well-defined and central role of the
ubiquitin-activating enzyme (E1), no cell permeable inhibitors of E1 have been identified. Such inhibitors should, in principle, block all functions of ubiquitylation. We now report 4[4-(5-nitro-
furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-
benzoic acid ethyl
ester (PYR-41) as the first such inhibitor. Unexpectedly, in addition to blocking ubiquitylation,
PYR-41 increased total sumoylation in cells. The molecular basis for this is unknown; however, increased sumoylation was also observed in cells harboring temperature-sensitive E1. Functionally,
PYR-41 attenuates
cytokine-mediated
nuclear factor-kappaB activation. This correlates with inhibition of nonproteasomal (Lys-63) ubiquitylation of
TRAF6, which is essential to
IkappaB kinase activation.
PYR-41 also prevents the downstream ubiquitylation and proteasomal degradation of
IkappaBalpha. Furthermore,
PYR-41 inhibits degradation of p53 and activates the transcriptional activity of this
tumor suppressor. Consistent with this, it differentially kills transformed p53-expressing cells. Thus,
PYR-41 and related pyrazones provide proof of principle for the capacity to differentially kill transformed cells, suggesting the potential for E1 inhibitors as
therapeutics in
cancer. These inhibitors can also be valuable tools for studying ubiquitylation.