Deubiquitinating enzymes (DUBs), also known as deubiquitinating peptidases or ubiquitin proteases, ubiquitin hydrolases, represent a large group of proteases that cleave monoubiquitin and polyubiquitin chains from proteins. They have attracted great attention in the drug discovery world due to their significant roles in various cellular processes [1].
At Life Chemicals, we have developed two deubiquitinase-focused screening sets of over 20,000 drug-like screening compounds selected with both ligand-based and structure-based approaches that will surely boost DUB drug discovery campaigns:
The compound selection can be customized based on your requirements, cherry picking is available.
Please, contact us at orders@lifechemicals.com for any additional information and price quotations.
For a pre plated set based on this Screening Library, please explore our Pre-plated Focused Libraries.
Background Information
Deubiquitinating enzyme family comprises approximately 100 proteins and can be subdivided into two primary classes: cysteine proteases and metalloproteases. DUBs have been implicated in numerous human diseases, including cancer, chronic inflammation, autoimmune disorders, and neurodegenerative diseases [2].
The conjugation of ubiquitin to a target protein is a multistep process involving the sequential action of a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin protein-ligase (E3). Post-translational modifications of proteins by the ubiquitination machinery include the addition of a single ubiquitin protein or chains of ubiquitin to lysine residues of substrate proteins, which can activate and inactivate proteins, regulate protein degradation via the proteasome and lysosome, and modulate protein-protein interactions [3].
Ubiquitin proteases play an essential role in the ubiquitin pathway by reversing these effects by removing post-translational modifications. DUBs' catalytic activity includes thiol-dependent hydrolysis of ester, thioester, amide, peptide, and isopeptide bonds formed by the C-terminal Gly of ubiquitin [1, 4]. The well-defined active site of DUBs, which contains a catalytic cysteine, makes them attractive targets for small-molecule drug discovery.
Deubiquitinase Focused Library
A reference set of over 25,500 bioactive compounds with reported DUB inhibitor activity was prepared based on the data from the ChEMBL database. Then the Life Chemicals HTS Compound Collection was screened against it using a 2D fingerprint similarity search (85 % Tanimoto similarity cut-off). The PAINS filter together with our in-house developed toxicophore and undesired functionalities filters were also applied.
As a result, a screening set of over 15,300 structurally-diverse molecules with potential deubiquitinase inhibitory activity against different DUB-related targets, focusing primarily on two deubiquitinating enzymes, ubiquitin carboxyl-terminal hydrolases UCH1 and UCH2, has been obtained.
In addition, a Diversity Screening Set of more than 3,900 structurally-diverse deubiquitinase-targeting screening compounds was added as a convenient starting point for DUB-focused drug discovery.
Representative compounds from the Deubiquitinase-focused Diversity Screening Set
Deubiquitinase Targeted Library
Our Deubiquitinase-targeted Screening Library consisting of more than 4,900 structurally diverse molecules for biological screening has been developed using both structure-based and ligand-based approaches. To design this Set, our cheminformatics team has selectively focused on the following deubiquitinase-related drug targets:
- OTU protease of Crimean Congo Hemorrhagic Fever Virus
- OTU domain-containing protein 4 (OTUD4)
- Ubiquitin Specific Peptidases USP1/USP2
- Ubiquitin Like Modifier Activating Enzyme 5 (UBA5)
Along this way they utilized a proprietary in silico screening platform to predict novel bioactive compounds. All supporting data were obtained from the ChEMBL DB and RCSB Protein Data Bank. Applying the Glide module from the Schrödinger Suite, the entire Life Chemicals HTS Compound Collection has been analyzed in search for docking occurring in the active site of the corresponding proteins. To enhance the accuracy and precision of our docking results, we referred to a set of constraints.
OTU protease of Crimean Congo Hemorrhagic Fever Virus
The Ovarian Tumor domain (OTU domain) is a conserved protein domain found in the RNA-directed RNA polymerases (RdRps) of various viruses [5], including the Crimean-Congo Hemorrhagic Fever Virus (CCHFV). The OTU domain enables the cleavage of ubiquitin and interferon-stimulated gene 15 (ISG15) from host cellular proteins, which allows the virus to avoid the host immune response and facilitates viral replication [6]. It should be pointed out that the CCHFV is a highly dangerous virus with a mortality rate of up to 30%, with the OTU domain in the structure of its RdRps being crucial for its virulence [7]. Inhibition of the OTU domain inhibits CCHFV replication in cell culture [8], and organisms infected by the virus lacking the OTU domain in the structure of RdRps generally have a reduced viral load and lower levels of proinflammatory cytokines [9]. Thus, the OTU domain in the CCHFV RdRp L protein is essential for the virus's ability to evade the host immune response and replicate in infected cells. Understanding the structure and function of the OTU domain provides insight into the pathogenesis of the CCHFV and may lead to the development of new antiviral drugs that will have an inhibitory effect on this domain.
Having previously determined the binding site of the OTU protease of Crimean Congo Hemorrhagic Fever Virus, docking of the HTS Compound Collection was carried out to result in 1,070 drug-like screening compounds.
Key features:
- Method: glide ligand docking (standard precision)
- X-Ray data used: 5V5H (site3)
- Constraints: no
- Filters used: QikProp properties and descriptors
- Number of compounds selected: 1,070
Figure 1. OTU (PDB ID: 5V5H) binding sites prediction. We selected site3 (inhibitor binding site in the PDB structure).
Figure 2. Spatial structure binding site of the complex of OTU (PDB ID: 5V5H) with lead docking molecule F0192-0243.
OTU domain-containing protein 4 (OTUD4)
OTU domain-containing protein 4 (OTUD4) is a deubiquitinating enzyme that plays a crucial role in regulating various cellular processes, including DNA repair, immune response, and cell proliferation. The OTUD4 protein contains a catalytic domain called the ovarian tumor (OTU) domain, which enables it to cleave ubiquitin chains from substrate proteins [10]. OTUD4 interacts with the DNA damage response protein MDC1 and stabilizes it, thereby promoting the repair of double-stranded DNA breaks [11]. In addition, OTUD4 is known to regulate the innate immune response by deubiquitinating the STING (stimulator of interferon genes) signaling molecule [12]; OTUD4 can promote cell proliferation by regulating the stability of the oncogenic protein c-Myc, and OTUD4 expression is upregulated in various types of cancer, suggesting its potential as a therapeutic target [13].
The emerging data suggest that OTUD4, a deubiquitinating enzyme, is a key player in maintaining cellular homeostasis and is implicated in the pathogenesis of several diseases, including cancer. However, further investigation is required to unravel the precise molecular mechanisms underlying OTUD4's functions and its potential as a therapeutic target. In this regard, the Life Chemicals team makes its contribution by introducing a docking subset for OTUD4 into deubiquitinase screening libraries.
Reconstruction of the spatial structure of OTU domain-containing protein 4 and prediction of binding sites was carried out. As a result of docking the HTS Compound Collection and subsequent filtering by bioactivity and toxicity, around 600 compounds were selected.
Key features:
- Method: glide ligand docking (standard precision)
- Docked in the sites1 and the sites2 of I-TASSER models (1 and 2)
- Constraints: His148 (H-bond)
- Docking resulting compounds were compared between the two models, separately for the site1 and the site2.
- Filters used: QikProp properties and descriptors
- Number of compounds selected: 599
Figure 3. The process of innate antiviral signaling involves the OTUD protein. RNA-containing viruses trigger a signaling cascade that activates NF-κB and IRF3/7, followed by the induction of type I IFN and proinflammatory cytokines. The illustration shows representative compounds from the Life Chemicals library that can inhibit OTUD protein and E1 protein, which is involved in recognizing viral genome replication.
Figure 4. I-TASSER MODELING OF OTUD4 (UniProt: Q01804).
Figure 5. OTUD4 binding sites prediction.
Figure 6. Spatial structure binding site of the complex of OTUD4 with lead docking molecule F6727-2153.
Ubiquitin Specific Peptidases USP1/USP2
USP1 and USP2 are ubiquitin specific peptidases that regulate various cellular processes, including DNA damage response, cell cycle progression, and protein degradation. Dysregulation of USP1 and USP2 has been implicated in various diseases, including cancer, making them potential therapeutic targets. Modulators of USP1 and USP2 activity could potentially lead to the development of novel therapeutics for cancer and other diseases.
Pharmacophore modeling was applied to search for DUB-specific screening compounds with the aid of both Cresset and Schrödinger modules. As a result, over 1,100 potential small-molecule inhibitors of ubiquitin-specific proteases (USP1,2) were identified for the Deubiquitinase USP1,2 Focused Screening Set.
Figure 7. Signaling cascade involving ubiquitin-specific protease 1 (USP1) and its intracellular functions.
Ubiquitin Like Modifier Activating Enzyme 5 (UBA5)
UBA5 is a member of the E1 class of enzymes that activate ubiquitin-like modifiers, which play important roles in various cellular processes, including protein degradation, DNA repair, and signal transduction. Dysregulation of UBA5 has been implicated in various diseases, including cancer and neurodegenerative disorders. UBA5 represents a potential therapeutic target for drug discovery, and modulators of UBA5 activity could lead to the development of novel therapeutics for these diseases.
In-silico virtual screening provided 2,200 in-stock drug-like screening compounds with potential E1 enzyme activity for the Deubiquitinase E1 Targeted Screeening Set.
References:
- Lopez-Castejon G, Edelmann MJ. Deubiquitinases: Novel Therapeutic Targets in Immune Surveillance. Mediators Inflamm. 2016:3481371. doi:10.1155/2016/3481371.
- Harrigan, J., Jacq, X., Martin, N. et al. Deubiquitylating enzymes and drug discovery: emerging opportunities. Nat Rev Drug Discov 17, 57–78 (2018). https://doi.org/10.1038/nrd.2017.152
- Song L, Luo ZQ. Post-translational regulation of ubiquitin signaling. J Cell Biol. 2019;218(6):1776-1786. doi:10.1083/jcb.201902074.
- Callis J. The ubiquitination machinery of the ubiquitin system. Arabidopsis Book. 2014;12:e0174. Published 2014 Oct 6. doi:10.1199/tab.0174.
- Du J, Fu L, Sui Y, Zhang L. The function and regulation of OTU deubiquitinases. Front Med. 2020;14(5):542-563. doi:10.1007/s11684-019-0734-4
- Scholte FEM, Hua BL, Spengler JR, et al. Stable Occupancy of the Crimean-Congo Hemorrhagic Fever Virus-Encoded Deubiquitinase Blocks Viral Infection. mBio. 2019;10(4):e01065-19. Published 2019 Jul 23. doi:10.1128/mBio.01065-19
- Shahhosseini N, Wong G, Babuadze G, et al. Crimean-Congo Hemorrhagic Fever Virus in Asia, Africa and Europe. Microorganisms. 2021;9(9):1907. Published 2021 Sep 9. doi:10.3390/microorganisms9091907
- Scholte FEM, Zivcec M, Dzimianski JV, et al. Crimean-Congo Hemorrhagic Fever Virus Suppresses Innate Immune Responses via a Ubiquitin and ISG15 Specific Protease. Cell Rep. 2017;20(10):2396-2407. doi:10.1016/j.celrep.2017.08.040
- Zhang W, Bailey-Elkin BA, Knaap RCM, et al. Potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants. PLoS Pathog. 2017;13(5):e1006372. Published 2017 May 18. doi:10.1371/journal.ppat.1006372
- Zhao Y, Mudge MC, Soll JM, et al. OTUD4 Is a Phospho-Activated K63 Deubiquitinase that Regulates MyD88-Dependent Signaling. Mol Cell. 2018;69(3):505-516.e5. doi:10.1016/j.molcel.2018.01.009
- Ye H, Chu X, Cao Z, et al. A Novel Targeted Therapy System for Cervical Cancer: Co-Delivery System of Antisense LncRNA of MDC1 and Oxaliplatin Magnetic Thermosensitive Cationic Liposome Drug Carrier. Int J Nanomedicine. 2021;16:1051-1066. Published 2021 Feb 11. doi:10.2147/IJN.S258316
- Jiang M, Chen P, Wang L, et al. cGAS-STING, an important pathway in cancer immunotherapy. J Hematol Oncol. 2020;13(1):81. Published 2020 Jun 22. doi:10.1186/s13045-020-00916-z
- Zhao X, Su X, Cao L, et al. OTUD4: A Potential Prognosis Biomarker for Multiple Human Cancers. Cancer Manag Res. 2020;12:1503-1512. Published 2020 Feb 28. doi:10.2147/CMAR.S233028