Search Results (43)

Crimean–Congo hemorrhagic fever virus (CCHFV) remains a major public health threat due to its high mortality rates and the absence of approved antiviral therapies. The viral ovarian tumor (OTU) protease is a critical virulence factor that suppresses host innate immunity through its deubiquitinase activity, making it an attractive therapeutic target. In this study, we employed a structure-guided approach to identify and validate novel small-molecule inhibitors targeting the non-catalytic Y89-W99 pocket of the OTU protease. Recombinant OTU protease was successfully expressed, purified, and refolded, yielding a soluble and enzymatically active protein. Cellular assays confirmed that the enzyme retains robust deubiquitinase activity, significantly reducing global ubiquitin conjugates in mammalian cells. In silico analysis of a putative DUB inhibitor library identified several candidate inhibitors with favorable binding interactions within the Y89-W99 pocket. Biochemical validation using a fluorometric Ub-AMC assay revealed that multiple small molecules strongly inhibit OTU activity, including OTUi-10 (~93% inhibition), OTUi-13 (~87%), OTUi-1 (~85%), OTUi-4 and OTUi-11 (~81%), and OTUi-9 (~76%). Additional moderate inhibitors included OTUi-12 (~67%), OTUi-19 and OTUi-21 (~66%), and OTUi-5 (~57%). In silico drug-likeness and toxicity profiling filtered the library to four fully compliant candidates, OTUi-4, OTUi-10, OTUi-11, and OTUi-12, all free of predicted toxicity alerts. These findings suggest that the Y89–W99 pocket may be a pharmacologically relevant site worthy of further investigation and identify OTUi-10, OTUi-4, and OTUi-11 as promising preliminary hit compounds. The results also provide initial insights that may guide future optimization and mechanistic studies of OTU protease inhibitors targeting CCHFV. Full article

Ubiquitin-specific protease 22 (USP22) regulates epigenetic gene expression by deubiquitinating histone H2B (H2Bub1) and upregulating oncogenic proteins and pathways, while antagonizing p53-mediated tumor suppression. USP22 is frequently overexpressed in cancers and associated with therapy resistance and poor prognosis yet remains largely untargeted pharmacologically. Here, using a fluorescence-based USP22 deubiquitinase assay to screen the LOPAC^®1280 library, we identified β-Lapachone, a natural ortho-naphthoquinone with strong anticancer activities, as a potent USP22 inhibitor. β-Lapachone potently inhibited USP22 enzymatic activity, with a half-maximal inhibitory concentration (IC₅₀) of ~0.75 μM, and molecular docking revealed its occupation of the catalytic pocket adjacent to the USP22 active-site triad, supporting a potential binding mode. Functionally, β-Lapachone suppressed proliferation and induced apoptosis in A549 and H1299 RAS-mutant lung adenocarcinoma (LUAD) cells, while USP22 knockout conferred marked resistance, indicating partial USP22 dependence. In patient-derived LUAD models, β-Lapachone inhibited sphere formation and reduced CD133⁺ cancer stem cell populations. Notably, it synergized with cisplatin to enhance DNA damage and apoptosis. In vivo, β-Lapachone significantly suppressed tumor growth in a syngeneic KRAS-mutant/p53-Null mouse lung cancer model and further potentiated cisplatin-induced antitumor effects. Collectively, these findings identify β-Lapachone as a potent inhibitor of USP22 and validate USP22 inhibition as a key mechanism underlying its anticancer activity in LUAD cells, both in vitro and in vivo. Full article

Ubiquitination, a crucial cellular protein regulation process, is linked to various diseases, including cancer. Deubiquitinases (DUBs) can reverse ubiquitination, offering a therapeutic strategy. USP7, a DUB, is a key target in oncology due to its role in destabilizing p53, and small-molecule inhibitors could restore p53 activity and combat tumor growth. In this study, we integrated a machine learning (ML)-based screening approach with molecular docking and molecular dynamics (MD) simulations in order to identify potential small-molecule inhibitors of USP7. ML-based screening identified 22 active molecules from a library of 2301 natural compounds. Among the 22 active compounds, only fifteen compounds fulfilled the drug-likeness criteria. Subsequently, molecular docking found three compounds, PubChem 162957515, 114917, and 442879 as potential inhibitors based on binding affinity and interactions. Further, MD simulations and MM-PBSA analyses were performed to evaluate the stability and dynamic behavior of the complexes. Binding energy calculations Molecular Mechanics Poisson–Boltzmann Surface Area (MM-PBSA) revealed that compounds PubChem 114917 and 162957515 exhibited strong binding affinities of −20.98 kcal/mol and −18.68 kcal/mol, respectively, implying that these compounds could serve as promising inhibitors for the development of anticancer therapeutics. Full article

Neospora caninum is a major apicomplexan pathogen responsible for significant reproductive losses in livestock, yet lacks effective therapeutics. Here, we identify and functionally characterize a previously unstudied OTU-like deubiquitinase (ncOTU; XP_003886403) as a key modulator of host–pathogen interactions. Sequence and structural analyses revealed conservation of the catalytic triad (D257, C260, H362) and a Y305-W315-G316 inhibition pocket analogous to viral OTU proteases. Recombinant ncOTU exhibited robust deubiquitinase activity and significantly reduced global ubiquitination levels in mammalian cells, preferentially targeting mono-ubiquitinated and low-molecular-weight substrates. Transcriptomic analysis demonstrated that ncOTU expression correlates with suppressed NF-κB signaling, type I interferon responses, and downstream antiviral effectors, while partially uncoupling upstream nucleic acid sensing pathways. Structure-based virtual screening and biochemical validation identified multiple small-molecule inhibitors targeting the conserved inhibition pocket. Dose-response analysis revealed submicromolar potency for ncOTUi-9 (IC₅₀ = 0.1 μM), ncOTUi-8 (0.2 μM), and ncOTUi-19 (0.3 μM), whereas ncOTUi-3 showed lower activity (7.1 μM). Interaction analyses confirmed stable binding within the inhibition pocket, with more extensive contact networks correlating with increased potency. Collectively, these findings establish ncOTU as a functional deubiquitinase that contributes to evasion and highlight it as a promising therapeutic target for neosporosis. Full article

Deubiquitinases, or DUBs, have emerged as pivotal regulators of cellular homeostasis, coordinating the delicate balance between protein ubiquitination and deubiquitination. Their versatile roles span from controlling protein turnover to modulating signal transduction pathways, thereby influencing diverse cellular processes, including DNA damage repair, apoptosis, and immune responses. This review comprehensively explores the current understanding of DUBs, elucidating their structural diversity, catalytic mechanisms, physiological functions, and implications in human diseases. Moreover, we discuss the therapeutic potential of targeting DUBs in various pathological conditions, highlighting recent advancements and challenges in developing DUB-specific inhibitors. Full article

Background/Objectives: HNSCC is a highly aggressive malignancy marked by the dysregulation of the cell cycle. In HPV⁻ HNSCC, mutations in the CDKN2A gene frequently result in the loss of the p16 protein, a key inhibitor of the cyclin D1/CDK4/6 complex. This loss results in unchecked G1/S phase progression. The CDK4/6 inhibitor palbociclib has shown therapeutic potential in HPV⁻ HNSCC by inducing G1 phase arrest and reducing cell viability. In this study, we investigated the molecular mechanisms by which palbociclib affects cell viability in HPV⁻ HNSCC. Methods: Four HPV⁻ HNSCC cell lines were treated with palbociclib, and RNA sequencing was performed to assess changes in gene expression. Cell viability was measured using the MTT assay. To further investigate protein localization, interactions, and function, we used immunofluorescence staining, co-immunoprecipitation, small molecule inhibitors, and siRNA-mediated knockdown. Results: We demonstrate that palbociclib downregulates survivin, a protein that plays dual roles in mitosis and apoptosis, thereby inhibiting cell proliferation. We also found that survivin is overexpressed in HPV⁻ HNSCC. Inhibiting survivin dimerization using the compound LQZ-7i significantly reduces cell viability and promotes its export from the nucleus to the cytoplasm. Additionally, we identified USP1, a deubiquitinase, as both a downstream target of CDK4/6 and a key regulator of survivin stability. Inhibiting USP1 activity or silencing its expression significantly reduces survivin levels. Conclusions: Our findings highlight survivin as a critical mediator of cell proliferation in HPV⁻ HNSCC and suggest that targeting the CDK4/6-USP1-survivin axis may offer a promising therapeutic strategy. Full article

Ubiquitin-specific protease 7 (USP7), a deubiquitinase enzyme responsible for removing ubiquitin (Ub) from target proteins, plays a crucial role in oncogenic pathways and has been implicated in various human diseases. X-ray crystallography has revealed distinct conformations of USP7, including apo (ligand-free), allosteric inhibitor-, and Ub-bound states. However, the dynamic mechanisms underlying the allosteric inhibition of USP7 remain unclear. This study investigates the effect of allosteric inhibitor binding on the dynamics of USP7 through multiple replica molecular dynamics simulations. Our results demonstrate that Ub binding stabilizes the USP7 conformation, while allosteric inhibitor binding increases flexibility and variability in the fingers and palm domains of USP7. Furthermore, our analysis of USP7 local regions reveals that allosteric inhibitor binding not only restrains the dynamics of the C-terminal Ub binding site, thereby impeding the accessibility of Ub to USP7, but also disrupts the proper alignment of the catalytic triad (Cys223-His464-Asp481) in USP7. Additionally, community network analysis indicates that intra-domain communications within the fingers domain in USP7 are significantly enhanced upon allosteric inhibitor binding. This study reveals that the binding of an allosteric inhibitor induces a dynamic shift in enzyme’s conformational equilibrium, effectively disrupting its catalytic activity through allosteric modulation. Full article

E3 ubiquitin ligases and deubiquitinating enzymes (DUBs) are pivotal regulators of bone homeostasis, orchestrating osteoblast differentiation, proliferation, and osteoclast activity by controlling protein degradation and stability. This review delineates the roles of key E3 ligases (e.g., Smurf1, Smurf2, TRIM family) and DUBs (e.g., USP family) in bone formation and resorption. E3 ligases such as Smurf1/2 inhibit osteogenesis by degrading BMP/Smad signaling components, while TRIM proteins and HERC ligases promote osteoblast differentiation. Conversely, DUBs like USP2 and USP34 stabilize β-catenin and Smad1/RUNX2, enhancing osteogenic pathways, whereas USP10 and USP12 suppress differentiation. Dysregulation of these enzymes contributes to osteoporosis, fracture non-union, and other bone disorders. The interplay between ubiquitination and deubiquitination, alongside the regulatory role of miRNA and environmental factors, underscores their therapeutic potential. Future research should focus on developing therapies targeting E3 ubiquitin ligases, deubiquitinases, miRNA regulators, and small-molecule inhibitors to restore bone homeostasis in osteoporosis and fracture healing disorders. Full article

Targeted protein degradation has emerged as an alternative therapy against cancer, offering several advantages over traditional inhibitors. The new degrader drugs provide different therapeutic strategies: they could cross the phospholipid bilayer membrane by the addition of specific moieties to extracellular proteins. On the other hand, they could efficiently improve the degradation process by the generation of a ternary complex structure of an E3 ligase. Herein, we review the current trends in the use of TAC-based technologies (TACnologies), such as PROteolysis TArgeting Chimeras (PROTAC), PHOtochemically TArgeting Chimeras (PHOTAC), CLIck-formed Proteolysis TArgeting Chimeras (CLIPTAC), AUtophagy TArgeting Chimeras (AUTAC), AuTophagosome TEthering Compounds (ATTEC), LYsosome-TArgeting Chimeras (LYTAC), and DeUBiquitinase TArgeting Chimeras (DUBTAC), in experimental development and their progress towards clinical applications. Full article

Neuroblastoma (NB) is a pediatric malignancy originating from neural crest cells of the sympathetic nervous system that accounts for 15% of all pediatric cancer deaths. Despite advances in treatment, high-risk NB remains difficult to cure, highlighting the need for novel therapeutic approaches. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a critical role in tumor suppression and DNA repair, and USP7 overexpression has been associated with tumor aggressiveness in a variety of tumors, including NB. Therefore, USP7 is a potential therapeutic target for NB. The tumor suppressor p53 is a known target of USP7, and therefore reactivation of the p53 pathway may be an effective therapeutic strategy for NB treatment. We hypothesized that inhibition of USP7 would be effective against NB tumor growth. Using a novel USP7 inhibitor, Almac4, we have demonstrated significant antitumor activity, with significant decreases in both cell proliferation and cell viability in TP53 wild-type NB cell lines. USP7 inhibition in NB cells activated the p53 pathway via USP7 and MDM2 degradation, leading to reduced p53 ubiquitination and increased p53 expression in all sensitive NB cells. In addition, USP7 inhibition led to decreased N-myc protein levels in both MYCN-amplified and -nonamplified NB cell lines, but no correlation was observed between MYCN amplification and treatment response. USP7 inhibition induced apoptosis in all TP53 wild-type NB cell lines. USP7 inhibition also induced EZH2 ubiquitination and degradation. Lastly, the combination of USP7 and MDM2 inhibition showed enhanced efficacy. Our data suggests that USP7 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed NB. Full article

Placenta accreta is a high-risk condition causing obstetric crisis and hemorrhage; however, its pathogenesis remains unknown. We aimed to identify the factors contributing to trophoblast invasiveness and angiogenic potential, which in turn drive the pathogenesis of placenta accreta. We focused on the transforming growth factor (TGF)-β1-Smad pathway and investigated the intrinsic relationship between the time- and dose-dependent inhibition of the ubiquitinating enzyme UCHL5 using bAP15, a deubiquitinase inhibitor, after TGF-β1 stimulation and the invasive and angiogenic potential of two cell lines, gestational choriocarcinoma cell line JEG-3 and trophoblast cell line HTR-8/SVneo. UCHL5 inhibition negatively regulated TGF-β1-induced Smad2 activation, decreasing extravillous trophoblast invasiveness. Smad1/5/9 and extracellular signal-regulated kinase (ERK) were simultaneously activated, and vascular endothelial growth factor was secreted into the trophoblast medium. However, extravillous trophoblast culture supernatant severely impaired the vasculogenic potential of human umbilical vein endothelial cells. These results suggest that the downstream ERK pathway and Smad1/5/9 potentially regulate the TGF-β1-Smad pathway in extravillous trophoblasts, whereas Smad2 contributes to their invasiveness. The abnormal invasive and angiogenic capacities of extravillous cells, likely driven by the interaction between TGF-β1-Smad and ERK pathways, underlie the pathogenesis of placenta accreta. Full article

Ubiquitin-specific protease 2 (USP2) is a deubiquitinase belonging to the USPs subfamily. USP2 has been known to display various biological effects including tumorigenesis and inflammation. Therefore, we aimed to examine the sensitization effect of USP2 in TRAIL-mediated apoptosis. The pharmacological inhibitor (ML364) and siRNA targeting USP2 enhanced TNF-related apoptosis-inducing ligand (TRAIL)-induced cancer cell death, but not normal cells. Mechanistically, USP2 interacted with survivin, and ML364 degraded survivin protein expression by increasing the ubiquitination of survivin. Overexpression of survivin or USP2 significantly prevented apoptosis through cotreatment with ML364 and TRAIL, whereas a knockdown of USP2 increased sensitivity to TRAIL. Taken together, our data suggested that ML364 ubiquitylates and degrades survivin, thereby increasing the reactivity to TRAIL-mediated apoptosis in cancer cells. Full article

The use of specific inhibitors towards mutant BRAF (BRAFi) and MEK (MEKi) in BRAF-mutated patients has significantly improved progression-free and overall survival of metastatic melanoma patients. Nevertheless, half of the patients still develop resistance within the first year of therapy. Therefore, understanding the mechanisms of BRAFi/MEKi-acquired resistance has become a priority for researchers. Among others, oxidative stress-related mechanisms have emerged as a major force. The aim of this study was to evaluate the contribution of Nrf2, the master regulator of the cytoprotective and antioxidant response, in the BRAFi/MEKi acquired resistance of melanoma. Moreover, we investigated the mechanisms of its activity regulation and the possible cooperation with the oncogene YAP, which is also involved in chemoresistance. Taking advantage of established in vitro melanoma models resistant to BRAFi, MEKi, or dual resistance to BRAFi/MEKi, we demonstrated that Nrf2 was upregulated in melanoma cells resistant to targeted therapy at the post-translational level and that the deubiquitinase DUB3 participated in the control of the Nrf2 protein stability. Furthermore, we found that Nrf2 controlled the expression of YAP. Importantly, the inhibition of Nrf2, directly or through inhibition of DUB3, reverted the resistance to targeted therapies. Full article

The deubiquitinase BAP1 (BRCA1-associated protein 1) is associated with BAP1 tumor predisposition syndrome (TPDS). BAP1 is a tumor suppressor gene whose alterations in cancer are commonly caused by gene mutations leading to protein loss of function. By CRISPR-Cas, we have generated mutations in ubh-4, the BAP1 ortholog in Caenorhabditis elegans, to model the functional impact of BAP1 mutations. We have found that a mimicked BAP1 cancer missense mutation (UBH-4 A87D; BAP1 A95D) resembles the phenotypes of ubh-4 deletion mutants. Despite ubh-4 being ubiquitously expressed, the gene is not essential for viability and its deletion causes only mild phenotypes without affecting 20S proteasome levels. Such viability facilitated an RNAi screen for ubh-4 genetic interactors that identified rpn-9, the ortholog of human PSMD13, a gene encoding subunit of the regulatory particle of the 26S proteasome. ubh-4[A87D], similarly to ubh-4 deletion, cause a synthetic genetic interaction with rpn-9 inactivation affecting body size, lifespan, and the development of germ cells. Finally, we show how ubh-4 inactivation sensitizes animals to the chemotherapeutic agent Bortezomib, which is a proteasome inhibitor. Thus, we have established a model to study BAP1 cancer-related mutations in C. elegans, and our data points toward vulnerabilities that should be studied to explore therapeutic opportunities within the complexity of BAP1 tumors. Full article

Ubiquitination and deubiquitination processes are widely involved in modulating the function, activity, localization, and stability of multiple cellular proteins regulating almost every aspect of cellular function. Several virus families have been shown to exploit the cellular ubiquitin-conjugating system to achieve a productive infection: enter the cell, promote genome replication, or assemble and release viral progeny. In this study, we analyzed the role of deubiquitinating enzymes (DUBs) during chikungunya virus (CHIKV) infection. HEK293T, Vero-E6, and Huh-7 cells were treated with two DUB inhibitors (PR619 or WP1130). Then, infected cells were evaluated by flow cytometry, and viral progeny was quantified using the plaque assay method. The changes in viral proteins and viral RNA were analyzed using Western blotting and RT-qPCR, respectively. Results indicate that treatment with DUB inhibitors impairs CHIKV replication due to significant protein and viral RNA synthesis deregulation. Therefore, DUB activity may be a pharmacological target for blocking CHIKV infection. Full article