Harnessing Ubiquitylation for Drug Discovery

Deubiquitylating enzymes (DUBs) detach small ubiquitin modifications from protein substrates in eukaryotic cells. DUBs regulate many aspects of cell biology such as cell cycle progression, cell division, and differentiation.¹ The role of DUBs in pathophysiology has evolved remarkably over the past several years. Defects in ubiquitylation processes have been linked to sporadic and genetic syndromes, as well as cancers and neurological disorders.^2,3

There is considerable interest in exploiting components of the ubiquitylation machinery and in developing small-molecule DUB inhibitors as therapeutic agents despite the challenges that this represents. This article highlights some of these challenges and recent technological innovations that are helping scientists overcome them.

DUB challenges

Although DUBs have distinct catalytic pockets that may serve as potential binding sites for novel compounds, researchers face obstacles in developing small molecules that inhibit the activity of DUBs. A key challenge has been the identification of compounds that can selectively distinguish between related DUB subtypes and simultaneously, have chemical properties that allow their development for clinical use.

Moreover, the catalytic activity of most DUBs requires a reactive thiol group. So, the value of most standard assays to identify inhibitors is reduced due to the ability of nonselective redox or alkylating compounds to generate false positives in these assays.⁴ Other complexities include substrate-mediated catalysis and allosteric modulation of DUB activity—where a compound binds at a regulatory site other than the active site of the enzyme. Many DUBs alternate between different catalytically competent and incompetent conformations.^5,6

Finally, DUBs often exhibit specificity for ubiquitin chains and their protein substrates. Hence, to improve the likelihood of identifying genuine inhibitors, researchers need to design primary and orthogonal assays, using endogenous substrates to address ubiquitin-linkage specificity for each DUB. These pose challenges for the design of predictive biochemical assays and the development of selective DUB inhibitors as drugs.

Emerging technologies

Despite such challenges, emerging companies have developed screening methods to identify and characterize small-molecule inhibitors for DUBs over the past few years. Many of these compounds are now moving toward or are in clinical evaluation.¹

For example, DUBprofiler, a proprietary assay developed by Ubiquigent, enables the discovery and development of DUB modulators through rapid determination of the selectivity and potency of DUB inhibitors.⁷ The platform has supported the identification of potent inhibitors of USP7 (ubiquitin-specific protease 7) that induce the tumor suppressor p53 in cancer cell lines.⁸ More recently, KSQ Therapeutics has initiated clinical trials for a USP1 DUB inhibitor for cancer patients. The compound was screened on DUBprofiler.⁹

“More than a decade ago, scientists at Ubiquigent recognized the emerging need for a robust platform that could support the development of DUB-targeting compounds. DUBprofiler remains the gold standard as one of the most highly cited and comprehensive DUB profiling assays available. Comprising an enzyme panel representative of the entire human DUB family, it is the most efficient way to identify novel DUB inhibitors and establish their selectivity and potency,” said Sheelagh Frame, PhD, CSO at Ubiquigent.

Frame added, “DUBprofiler is complemented by our REDOXprofiler assay. REDOXprofiler efficiently counter-screens and eliminates compounds that have a redox cycling mechanism of action, which indirectly inhibits the catalytic activity of proteins by oxidizing accessible cysteines, thereby separating false positives from genuine hits.”

With the growing number of high affinity and selective inhibitors revealed by the DUBprofiler assay, it becomes more pressing to determine the DUB target engagement profile of these molecules within cells to facilitate drug development programs toward clinical use. DUBprofiler-Cell and DUBprofiler-Tissue rely on the use of ubiquitin-containing activity-based probes (ABPs) to engage and enrich active DUBs.

“Our focus at Ubiquigent is to enable the discovery and development of novel DUB modulators as exciting new therapeutics for areas of high unmet medical need. We remain committed to supporting researchers in their endeavors by continuing to develop and expand our unique range of drug discovery services, and in that regard are delighted to be able to offer the DUBprofiler-Cell and DUBprofiler-Tissue assays,” said Frame.

These assays have been developed by Ubiquigent to address several key elements in the DUB-focused drug-discovery workflow. The assays can support target identification and validation, by highlighting which DUBs are expressed and active in different cell or tissue types, thus highlighting those DUBs that may be implicated in disease. The assays also demonstrate target engagement of test compounds against specific DUBs in lysates, live cells, and tissues.⁷

The readout of these assays is target engagement (TE), demonstrated by either Western blotting or mass spectrometry (MS). Typically, binding conditions are determined using Western blotting first to confirm TE to the target DUB. However, a more comprehensive MS analysis allows investigators to interrogate the cellular selectivity of the compounds in cells or tissues, which complements the findings from the in vitro DUBprofiler assay. The binding assays can be performed by incubating the ABPs with untreated lysates incubated with compounds ex vivo or lysates prepared from live cells or tissues which have been exposed to compounds in situ. The parallel analysis of lysates and live cells allows investigators to define a compound’s cellular permeability and measure associated biomarkers where TE has been detected. TE can also be demonstrated in tissues from animals dosed with DUB inhibitors as part of a preclinical efficacy data package, allowing valuable correlations to be made between TE, biomarker readouts, PK (pharmacokinetics), and efficacy.

“DUBprofiler-Cell and DUBprofiler-Tissue are valuable, physiologically relevant assays that allow researchers to determine target engagement and selectivity of their DUB-targeting compounds in cells and tissues. The assays complement our full range of in vitro assays, including DUBprofiler, and contribute greatly to our provision of an end-to-end drug discovery solution for all working in this field. These assays have also been instrumental in the development of our own growing pipeline of DUB-focused programs,” Frame said.

DUB biology and DUB-focused drug discovery have progressed dramatically over the past decade, with increasing numbers of small-molecule DUB inhibitors being explored and refined. With advanced molecules developed by key pharma and biotech companies, and the technologies developed by specialist CROs and academic investigators to underpin their development, we may soon witness the full therapeutic potential of DUBs.

References

1. Harrigan JA, Jacq X, Martin NM, Jackson SP. Deubiquitylating enzymes and drug discovery: emerging opportunities. Nat Rev Drug Discov. 2018;17(1):57-78.
2. Murali R, Wiesner T, Scolyer RA. Tumours associated with BAP1 mutations. Pathology (Phila). 2013;45(2):116-126.
3. Ma ZY, Song ZJ, Chen JH, et al. Recurrent gain-of-function USP8 mutations in Cushing’s disease. Cell Res. 2015;25(3):306-317.
4. Wrigley JD, Eckersley K, Hardern IM, et al. Enzymatic characterisation of USP7 deubiquitinating activity and inhibition. Cell Biochem Biophys. 2011;60(1-2):99-111.
5. Mevissen TET, Komander D. Mechanisms of Deubiquitinase Specificity and Regulation. Annu Rev Biochem. 2017;86:159-192. doi:10.1146/annurev-biochem-061516-044916
6. Sahtoe DD, Sixma TK. Layers of DUB regulation. Trends Biochem Sci. 2015;40(8):456-467.
7. Ubiquigent. Ubiquigent | DUBprofiler^TM. Accessed September 1, 2022.
8. Turnbull AP, Ioannidis S, Krajewski WW, et al. Molecular basis of USP7 inhibition by selective small-molecule inhibitors. Nature. 2017;550(7677):481-486.
9. Ubiquigent. Ubiquigent | Ubiquigent celebrates milestone anniversary of DUBprofiler^TM compound profiling platform. Accessed September 12, 2022.