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Updated: Jul 17, 2025
Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations
Published on: November 7, 2019
Cellular Validation of a Chemically Improved Inhibitor Identifies Monoubiquitination on OTUB2
Jin Gan1, Jelle de Vries1, Jimmy J L L Akkermans2
1Department of Cell and Chemical Biology, Division of Chemical Biology and Drug Discovery, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands.
Abstract:
Ubiquitin thioesterase OTUB2, a cysteine protease from the ovarian tumor (OTU) deubiquitinase superfamily, is often overexpressed during tumor progression and metastasis. Development of OTUB2 inhibitors is therefore believed to be therapeutically important, yet potent and selective small-molecule inhibitors targeting OTUB2 are scarce. Here, we describe the development of an improved OTUB2 inhibitor, LN5P45, comprising a chloroacethydrazide moiety that covalently reacts to the active-site cysteine residue. LN5P45 shows outstanding target engagement and proteome-wide selectivity in living cells. Importantly, LN5P45 as well as other OTUB2 inhibitors strongly induce monoubiquitination of OTUB2 on lysine 31. We present a route to future OTUB2-related therapeutics and have shown that the OTUB2 inhibitor developed in this study can help to uncover new aspects of the related biology and open new questions regarding the understanding of OTUB2 regulation at the post-translational modification level.
Insights
Researchers developed LN5P45, a novel small-molecule inhibitor targeting ovarian tumor (OTU) deubiquitinase 2 (OTUB2). This inhibitor shows high selectivity and induces OTUB2 monoubiquitination, paving the way for new cancer therapeutics.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Ovarian tumor (OTU) deubiquitinase 2 (OTUB2) is a cysteine protease frequently overexpressed in tumors, driving progression and metastasis.
- Targeting OTUB2 with small-molecule inhibitors is therapeutically significant, but potent and selective options are limited.
Purpose of the Study:
- To develop an improved, potent, and selective small-molecule inhibitor for OTUB2.
- To investigate the mechanism of action and cellular effects of the novel OTUB2 inhibitor.
Main Methods:
- Design and synthesis of a novel OTUB2 inhibitor, LN5P45, featuring a chloroacethydrazide moiety for covalent active-site targeting.
- Assessment of target engagement and proteome-wide selectivity using cellular assays.
- Analysis of OTUB2 post-translational modifications, specifically monoubiquitination at lysine 31, upon inhibitor treatment.
Main Results:
- LN5P45 demonstrated excellent target engagement and high proteome-wide selectivity in living cells.
- LN5P45 and other OTUB2 inhibitors effectively induced monoubiquitination of OTUB2 on lysine 31.
- The study identified a potential therapeutic strategy and highlighted LN5P45's utility in exploring OTUB2 biology.
Conclusions:
- LN5P45 represents a promising lead compound for developing novel OTUB2-targeted cancer therapeutics.
- OTUB2 inhibition influences its post-translational modification status, specifically inducing monoubiquitination at K31.
- This work provides a foundation for further research into OTUB2 regulation and therapeutic targeting in cancer.
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