Deubiquitinases (DUBs) revert ubiquitin signals through the removal of ubiquitin and are considered promising therapeutic targets. The inhibition of DUBs offers the opportunity to target yet-to-be-drugged proteins for which no binders are available through retaining their degradative ubiquitin signals.
The DUB USP9X has been reported to have oncogenic effects across various cancers including in triple negative breast cancer (TNBC) progression and maintenance. We have generated a focussed library of ~30 WEHI proprietary molecules with nanomolar activity against recombinant USP9X in established orthogonal USP9X in vitro assays. Here, we identify WEHI-092 as our lead compound that potently inhibited USP9X activity with an IC50 of 285 nM in vitro. Further characterisation of this inhibitor showed strong USP9X specificity when screened against a panel of more than 50 other human DUBs. We have identified a potential novel DUB inhibitor binding site through HDX-MS experiments with WEHI-092.
WEHI-092 inhibited USP9X in cells and led to reduced protein levels of oncogenic USP9X targets in a ubiquitin-proteasome dependent manner. The compound efficiently killed cancer cells and reduced colony formation in cell-based TNBC and other cancer models highlighting its potential application as therapeutic pre-clinical stage small molecule inhibitor.
DUBs are dysregulated in various diseases and disorders, yet few selective DUB inhibitors have been reported highlighting the need for novel compounds. Inhibition of USP9X offers a promising strategy for studying its function, unveiling novel substrates, and potentially identifying therapeutic intervention strategies for the treatment of disease.
Future work aims at revealing the inhibition mechanism of WEHI-092 through co-crystallization experiments and uncovering biological, physiological and disease relevance of USP9X-inhibition.