The ubiquitin-proteasome system (UPS) is critical for the regulation of cellular homeostasis. Conjugation of the 76-amino acid protein ubiquitin to substrate proteins serves as a post-translational signal modulating a multitude of cellular processes and protein fates, most importantly marking substrates for degradation by the proteasome. Deubiquitinases (DUBs) revert ubiquitin signals by removing ubiquitin from substrate proteins, leading to protein stabilisation, and contributing to maintaining a pool of free ubiquitin in the cell.
Targeted protein degradation (TPD) harnessing the UPS has been gaining growing interest in recent years as it offers strategies to target yet-to-be-drugged proteins in disease. DUBs are of interest to the TPD field and are emerging as drug targets through the development of specific DUB inhibitors. Blockade of DUB activity can preserve substrate ubiquitination leading to the degradation of the substrate protein by the proteasome. Targeted manipulation of substrate protein levels requires the identification of substrate-specific DUBs.
We have established a CRISPR/Cas9-based arrayed screening platform to identify DUBs that regulate the oncogenic transcription factor ERG implicated in prostate cancer and leukemia. Using a K-562 tool cell line expressing luciferase-tagged ERG, we performed high-throughput screening with a direct readout of protein levels.
Evidence from others suggests an involvement of the DUB USP9X in the regulation of ERG. In an orthogonal approach, we have developed a novel small molecule inhibitor that potently inhibits purified USP9X activity in vitro. Our compound WEHI-092 is highly potent with an IC50 of <1 µM in vitro against USP9X in a fluorescence-based DUB activity assay. Further characterisation of this inhibitor showed strong USP9X specificity when screened against a panel of other mammalian and viral DUBs.
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.