More than a decade after a Nobel Prize was awarded for the discovery of the ubiquitin-proteasome system and clinical approval of proteasome and ubiquitin E3 ligase inhibitors, first-generation deubiquitylating enzyme (DUB) inhibitors are now approaching clinical trials However, although our knowledge of the physiological and pathophysiological roles of DUBs has evolved tremendously, the clinical development of selective DUB inhibitors has been challenging In this Review, we discuss these issues and highlight recent advances in our understanding of DUB enzymology and biology as well as technological improvements that have contributed to the current interest in DUBs as therapeutic targets in diseases ranging from oncology to neurodegeneration

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Deubiquitylating enzymes and drug discovery: emerging opportunities.

The deubiquitylating enzymes (DUBs, also known as deubiquitylases or deubiquitinases) maintain the dynamic state of the cellular ubiquitome by releasing conjugated ubiquitin from proteins. In light of the many cellular functions of ubiquitin, DUBs occupy key roles in almost all aspects of cell behaviour. Many DUBs show selectivity for particular ubiquitin linkage types or positions within ubiquitin chains. Others show chain-type promiscuity but can select a distinct palette of protein substrates via specific protein–protein interactions established through binding modules outside of the catalytic domain. The ubiquitin chain cleavage mode or chain linkage specificity has been related directly to biological functions. Examples include regulation of protein degradation and ubiquitin recycling by the proteasome, DNA repair pathways and innate immune signalling. DUB cleavage specificity is also being harnessed for analysis of ubiquitin chain architecture that is assembled on specific proteins. The recent development of highly specific DUB inhibitors heralds their emergence as a new class of therapeutic targets for numerous diseases. By opposing protein ubiquitylation, deubiquitylating enzymes (DUBs) regulate various cellular processes, including protein degradation, the DNA damage response, cell signalling and autophagy. Many DUBs show high specificity for ubiquitin chain architecture and/or the protein substrate that they recognize, and have emerged as exciting therapeutic targets within the field of proteostasis.

Breaking the chains: deubiquitylating enzyme specificity begets function.

SARS-CoV-2 is the pathogen responsible for the COVID-19 pandemic. The SARS-CoV-2 papain-like cysteine protease (PLpro) has been implicated in playing important roles in virus maturation, dysregulation of host inflammation, and antiviral immune responses. The multiple functions of PLpro render it a promising drug target. Therefore, we screened a library of approved drugs and also examined available inhibitors against PLpro. Inhibitor GRL0617 showed a promising in vitro IC50 of 2.1 μM and an effective antiviral inhibition in cell-based assays. The co-crystal structure of SARS-CoV-2 PLproC111S in complex with GRL0617 indicates that GRL0617 is a non-covalent inhibitor and it resides in the ubiquitin-specific proteases (USP) domain of PLpro. NMR data indicate that GRL0617 blocks the binding of ISG15 C-terminus to PLpro. Using truncated ISG15 mutants, we show that the C-terminus of ISG15 plays a dominant role in binding PLpro. Structural analysis reveals that the ISG15 C-terminus binding pocket in PLpro contributes a disproportionately large portion of binding energy, thus this pocket is a hot spot for antiviral drug discovery targeting PLpro. The SARS-CoV-2 papain-like protease (PLpro) is of interest as a drug target. Here, the authors identify GRL0617 as a PPI (protein–protein interaction) inhibitor of SARS-CoV-2 PLpro that inhibits its deISGylating activity and present the mechanism of action of the compound through the GRL0617-bound PLpro crystal structure and NMR studies.

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The complex structure of GRL0617 and SARS-CoV-2 PLpro reveals a hot spot for antiviral drug discovery.

p53 is the major tumor suppressor and the most frequently inactivated gene in cancer. p53 could be disabled either by mutations or by upstream negative regulators, including, but not limited to MDM2 and MDMX. p53 activity is required for the prevention as well as for the eradication of cancers. Restoration of p53 activity in mouse models leads to the suppression of established tumors of different origin. These findings provide a strong support to the anti-cancer strategy aimed for p53 reactivation. In this review, we summarize recent progress in the development of small molecules, which restore the tumor suppressor function of wild-type p53 and discuss their clinical advance. We discuss different aspects of p53-mediated response, which contribute to suppression of tumors, including non-canonical p53 activities, such as regulation of immune response. While targeting p53 inhibitors is a very promising approach, there are certain limitations and concerns that the intensive research and clinical evaluation of compounds will hopefully help to overcome.

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Inhibition of p53 inhibitors: progress, challenges and perspectives.

Deubiquitinating enzymes, or DUBs, comprise a family of proteases that regulate ubiquitination dynamics. Since their discovery, genetic and functional studies have nominated DUBs as a promising class for drug discovery across diverse therapeutic areas. Consequent probe and drug discovery efforts over the past 15 years have resulted in over 50 reported inhibitors and advances in DUB structural studies, assay formats, and chemical biology tools. Accumulating knowledge from these studies has enabled several important recent breakthroughs. In this review, we highlight recent successes in solving DUB-ligand co-structures and the development of rigorously characterized potent and selective inhibitors. We posit that these advances in pharmacological targeting of DUBs establish the enzyme family as targetable and provide a framework for other DUBs programs. Accordingly, we envision increasingly rapid progress in the development of potent and selective inhibitors for a wide range of DUBs and advancement of DUB-targeting drugs to the clinic.

Advances in Discovering Deubiquitinating Enzyme (DUB) Inhibitors

Given the importance of ubiquitin-specific protease 7 (USP7) in oncogenic pathways, identification of USP7 inhibitors has attracted considerable interest. Despite substantial efforts, however, the development of validated deubiquitinase (DUB) inhibitors that exhibit drug-like properties and a well-defined mechanism of action has proven particularly challenging. In this article, we describe the identification, optimization and detailed characterization of highly potent (IC50 < 10 nM), selective USP7 inhibitors together with their less active, enantiomeric counterparts. We also disclose, for the first time, co-crystal structures of a human DUB enzyme complexed with small-molecule inhibitors, which reveal a previously undisclosed allosteric binding site. Finally, we report the identification of cancer cell lines hypersensitive to USP7 inhibition (EC50 < 30 nM) and demonstrate equal or superior activity in these cell models compared to clinically relevant MDM2 antagonists. Overall, these findings demonstrate the tractability and druggability of DUBs, and provide important tools for additional target validation studies.

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Discovery and characterization of highly potent and selective allosteric USP7 inhibitors

Ubiquitin specific protease 7 (USP7, HAUSP) has become an attractive target in drug discovery due to the role it plays in modulating Mdm2 levels and consequently p53. Increasing interest in USP7 is emerging due to its potential involvement in oncogenic pathways as well as possible roles in both metabolic and immune disorders in addition to viral infections. Potent, novel, and selective inhibitors of USP7 have been developed using both rational and structure-guided design enabled by high-resolution cocrystallography. Initial hits were identified via fragment-based screening, scaffold-hopping, and hybridization exercises. Two distinct subseries are described along with associated structure–activity relationship trends, as are initial efforts aimed at developing compounds suitable for in vivo experiments. Overall, these discoveries will enable further research into the wider biological role of USP7.

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Identification and Structure-Guided Development of Pyrimidinone Based USP7 Inhibitors

Over the past decade, protein ubiquitination has emerged as an important post-translational modification with roles in a plethora of cellular processes, and dysregulation in the ubiquitin proteasome system pathway (UPS) has been implicated in multiple human disorders including cancer. Ubiquitin specific proteases (USPs) are cysteine proteases that catalyse the de-ubiquitination of numerous protein substrates including tumor suppressors and oncogenes, hence regulating their levels and/or functions. USPs therefore represent a fast growing and attractive target class for pharmacological intervention. USP7 in particular has attracted considerable attention for its implications in multiple key oncogenic pathways including most notably MDM2/p53, PTEN and DNA damage. As part of a focussed effort towards targeting USPs, fragment screening was performed against a panel of family members, including USP7. Hits were identified by surface plasmon resonance and validated using orthogonal biophysical techniques (NMR, thermophoresis). Subsequent hit expansion identified molecules for which high-resolution co-crystal structures have been solved providing unique opportunities for structure-based design. Medicinal chemistry optimisation has yielded a series of novel, reversible and potent USP7 inhibitors (e.g. IC50 In summary, we report the discovery and detailed biochemical and cellular profiling of novel, potent and selective inhibitors of USP7. These molecules have drug-like properties and may provide opportunities for the development of new anticancer therapeutics. Citation Format: Gerald Gavory, Colin O9dowd, Keeva McClelland, Ewa Odrzywol, Alan Brown, Stephanie Burton, Oliver Barker, Frank Burkamp, Matt Helm, Iain James, Jakub Flasz, Elias Arkoudis, Tim Harrison. Discovery and characterization of novel, highly potent and selective USP7 inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-257. doi:10.1158/1538-7445.AM2015-LB-257

Elias Arkoudis

Papers

Discovery and characterization of highly potent and selective allosteric USP7 inhibitors

Identification and Structure-Guided Development of Pyrimidinone Based USP7 Inhibitors

Abstract LB-257: Discovery and characterization of novel, highly potent and selective USP7 inhibitors