2.1 Structure of DUBs

DUBs generally consist of one or more binding domains for substrate recognition, such as zinc-finger (ZnF) motifs, UBD, ubiquitin-binding domains (UBD), ubiquitin-like domains (UBL), coiled-coil domains, and ubiquitin interaction motifs (UIM), which work in conjunction with indispensable catalytic domains to precisely control hydrolysis. The catalytic domains contain the classical catalytic triad necessary for the hydrolysis of ubiquitin chains, including cysteine (Cys), histidine (His), and aspartic acid (Asp) or glutamic acid residues.¹⁵ Other domains work in concert to catalyze and precisely regulate hydrolysis while influencing cellular localization and functional diversity.^16-18 Most identified DUB families belong to Cys proteases, except for the JAMM family, which is classified as zinc metalloproteases. DUBs with Cys protease activity typically exhibit a catalytic pocket composed of Cys, His, and acidic residues, while JAMM family DUBs are characterized by their zinc-dependent metalloprotease activity, coordinating zinc ions with aspartate, His, and serine residues to facilitate water molecule recruitment and initiate isopeptide bond hydrolysis (Figure 2).

Due to their conserved structure and complex functional mechanisms, USP family members are widely present in eukaryotes, exhibiting a high degree of evolutionary conservation. The core of USP family members is the USP domain, characterized by a highly conserved catalytic core, which typically consists of nine tandem ubiquitin-like folds (Ubs) and two large insertion sequences. The three-dimensional structure of the USP domain exhibits a unique α/β fold, which includes multiple β sheets and α helices, providing a stable framework that allows USP family members to efficiently recognize and hydrolyze ubiquitin or ubiquitin chains. USP1 consists of 785 amino acids and features a conserved USP domain typical of the DUB family, which includes the catalytic residues (Cys90, His593, and Asp751).¹⁹ USP5 consists of a ZnF domain and a USP domain with a catalytic core composed of a Cys box and a His box and two ubiquitin-associated motifs.¹⁹ USP7 is a well-studied USP protein among nearly 100 DUBs that is characterized by a structure consisting of 1102 amino acids, including a catalytic core domain flanked by an N-terminal TRAF-like domain and five C-terminal UBL domains.²⁰ The catalytic core domain, formed by residues Cys223, His464, and Asp481, is primarily responsible for cleaving the isopeptide bond between ubiquitin and substrate proteins.²¹ The UBL domains of USP7 contribute to interactions with various proteins, particularly in substrate recognition and specificity, while the unique TRAF-like domain aids in substrate binding and initiating conformational changes necessary for catalytic activity.²² USP14 is composed of 494 amino acids and features two distinct domains: an N-terminal UBL domain that regulates proteasome activity and a C-terminal USP domain responsible for its DUB activity.²³ USP22 encodes a peptide with 525 amino acids and has a molecular weight of approximately 60 kDa. Its structure comprises an N-terminal ZnF domain and a C-terminal catalytic domain. Unlike other DUBs, the ZnF domain in USP22 does not directly bind to ubiquitin but instead interacts with other proteins to form a tightly locked deubiquitinase module, which deubiquitinates target proteins to alter their expression.²⁴

The OTU domain is the core of OTU family members, characterized by a conserved protein structure consisting of five beta strands flanked by alpha helical domains of varying sizes. This structural arrangement provides a stable framework for OTU family members to efficiently recognize and cleave ubiquitin or ubiquitin chains.^{25, 26} The catalytic mechanism of OTUs involves the initial recognition and binding to ubiquitin substrates or ubiquitin chains. The sulfur atom of the Cys residue at the catalytic center attacks the carbonyl carbon atom of the isopeptide bond between ubiquitin and substrate proteins or within the ubiquitin chain. The adjacent His residue regulates the pK_a of the catalytic Cys, while Asp or asparagine stabilizes and polarizes the catalytic histidine, forming a catalytic intermediate. This intermediate is subsequently dissociated through the participation of water molecules, leading to the hydrolysis of bonds between ubiquitin and substrate protein or within the ubiquitin chain. The cleaved ubiquitin and substrate protein (or the remaining part of the ubiquitin chain) are then dissociated from the enzyme, completing the deubiquitination process.^{9, 27, 28} The unique crystal structure of OTUB1 provides it with two distinct ubiquitin-binding sites—a distal site and a proximal site containing approximately 45 N-terminal residues of OTUB1.²⁹ Studies have shown that the activity of the OTUB1 enzyme is closely related to the regulation of E2 enzymes. OTUB1 not only exhibits classical DUB activity by directly removing ubiquitin molecules attached to substrates, but also possesses nonclassical DUB activity by binding to E2 enzymes to inhibit the ubiquitination process.^{30, 31} Unlike other DUBs, A20 exhibits both DUB activity and E3 ubiquitin ligase activity. The N-terminal OTU domain endows A20 with DUB activity, while the C-terminal ZnF domain confers E3 ligase activity.³² OTUD5 consists of 571 amino acids and contains two conserved structural domains: the OTU domain and the UIM domain.²⁴

The MPN (Mpr1/Pad1 N-terminal) domain is a notable feature of the JAMM family, characterized by a crystal structure that includes an eight-stranded beta sheet and two alpha helices. MPN domain proteins can be further classified into two subfamilies: the MPN+ family, which exhibits heteropeptidase activity, and the MPN– family, which lacks catalytic activity. The MPN+ family contains a zinc-coordinated JAMM motif, while the MPN– family often functions as a scaffold in various JAMM multisubunit complexes.³³ All MJD family members share a common Josephin domain that consists of approximately 180 amino acids. Ataxin-3 (ATXN3) and ATXN3-like protein (ATXN3L) features an N-terminal Josephin domain and a flexible C-terminal domain with UIMs. Although JOSD1 and JOSD2 contain only a single Josephin domain, this domain is highly conserved across all eukaryotes, indicating the essential role of MJD family members despite their largely unexplored biological functions.³⁴ The conserved UCH catalytic domain with classical α-β-α folding is a characteristic shared by all UCH family members. UCHL1 is composed of 223 amino acids and encompasses a UCH catalytic domain with short N-terminal and C-terminal extensions.³⁵ BAP1, consisting of 729 amino acids, includes the UCH catalytic domain and the C-terminal nucleosome binding domain.^{35, 36}

In summary, the conserved catalytic triad and additional functional domains endow DUB family members with precise substrate specificity and regulatory capabilities, underscoring their multifaceted roles in cellular processes. This foundational understanding of DUB domains sets the stage for exploring their broader biological implications and therapeutic potential.

2.2 Catalytic mechanisms of DUBs in recognition and cleavage

The catalytic domains of DUBs recognize and bind modifications through at least one ubiquitin-binding site, namely, the S1 site.³⁷ DUBs can either directly bind the substrate to remove the ubiquitin attached to it, or recognize and cleave the ubiquitin chain itself. Cys protease DUBs typically contain a catalytic triad composed of Cys, His, and an acidic residue. The catalytic process involves the deprotonated Cys residue attacking the isopeptide bond to form a tetrahedral intermediate, releasing the proximal ubiquitin, followed by hydrolysis to form a second tetrahedral intermediate, ultimately releasing the distal ubiquitin and regenerating the apo-enzyme. Zinc-dependent metalloprotease DUBs (JAMM family) have an active site comprising a zinc atom coordinated by conserved catalytic residues and a water molecule. The catalytic process involves a nucleophilic attack by the activated water molecule, forming a tetrahedral intermediate, cleaving the isopeptide bond, and simultaneously releasing the two ubiquitin parts. The arrangement and nature of ubiquitin binding sites in DUBs determine their ability to cleave polyubiquitin chains either exo- (distal or proximal) or endo-cleavage.

Most USP and UCH subfamily members exhibit nonspecific interactions with substrates, while other subfamilies such as OTU, JAMM, Josephine, and MINDY demonstrate chain specificity, particularly the OTU family.³⁸ For instance, OTUB1 demonstrates a preference for Lys48-linked chains, which typically tag proteins for degradation via the 26S proteasome. In contrast, OTUD1 preferentially cleaves Lys63-linked chains, which are associated with nondegradative functions such as DNA repair, signal transduction, and endocytosis.¹⁷ Other OTU family members, such as OTUD7B, show specificity for Lys11-linked chains.^{39, 40} OTULIN, on the other hand, specifically targets Met1-linked linear chains, which play a critical role in the activation of the NF-κB signaling pathway.⁴¹ TRABID exhibits specificity for Lys29- and Lys33-linked chains, which may have distinct functions in regulating specific cellular signaling processes.⁴²The OTU family exhibits linkage specificity or debranching characteristics by binding ubiquitin chains at specific S1′ and S2′ sites. The study of DUB cleavage mechanisms indicates that OTUs cleave ubiquitin chains by interacting with the distal and proximal parts of the ubiquitin chain at the S1 and S1′ sites, respectively. The S1′ site of OTUs binds to the proximal ubiquitin, allowing only one connection point of the proximal ubiquitin to enter the catalytic center. Therefore, the S1′ site is the determining factor for chain-specific cleavage. Many S1′ sites are located on the catalytic domain of OTUs. Additional Ub binding sites such as S2, S2′, or auxiliary UBDs enhance the functional capacity of DUBs.⁹ For example, OTUD2 can specifically recognize and cleave longer Lys11-linked chains, a specificity enhanced by the S2 site within the OTU domain.⁹

In summary, the arrangement of ubiquitin binding sites (S1, S1′, S2, S2′) determines their ability to perform either exo- or endo-cleavage, contributing to their substrate specificity and catalytic efficiency. Further research on catalytic mechanisms of DUBs is needed to understand their intracellular functions and may provide new targets for drug development, with significant scientific and clinical implications.

2.3 Regulation of DUB enzymatic activity

The activity of DUBs influenced not only by their inherent structural features but also by various intracellular signaling and regulatory mechanisms. These include PTMs, protein–protein interactions, conformational changes, and cellular environmental factors. Such regulatory mechanisms ensure that DUB function at the appropriate time and location, thereby preventing potential cellular damage due to uncontrolled activity. Dysregulation of DUB activity is closely associated with the pathogenesis of various diseases, particularly cancer, neurodegenerative diseases, and inflammatory conditions.

3.1 NF-κB signaling pathway

The NF-κB signaling pathway is fundamental for regulating immune responses and inflammation. Maintaining its precise control is crucial to ensure appropriate cellular responses and to prevent diseases such as chronic inflammation and cancer.⁶² DUBs are critical modulators within this pathway, targeting various key proteins to fine-tune the signaling processes. Here, we will delve into the comprehensive and intricate regulation of the NF-κB pathway by different DUBs, focusing on their specific roles and mechanisms of action (Figure 3A).

The NF-κB family includes transcription factors such as p105/p50, p100/p52, p65 (RelA), RelB, and c-Rel, activated primarily through the canonical and noncanonical pathways. The classical pathway responds to proinflammatory signals like TNF-α and LPS, activating the IκB kinase (IKK) complex, composed of IKKα, IKKβ, and NEMO. This activation leads to phosphorylation, ubiquitination, and proteasomal degradation of IκB proteins, releasing NF-κB dimers to translocate to the nucleus and initiate transcription of target genes involved in inflammation and immune response.^{63, 64}

3.2 MAPK signaling pathway

The MAPK signaling pathway is essential for transmitting signals from extracellular stimuli, impacting cancer cell behavior, including proliferation, survival, and drug resistance. This pathway involves a cascade of phosphorylation events triggered by membrane receptor engagement, leading to MAPKKK and MAPKK activation, and culminating in MAPK regulation of gene expression. This complex signaling network is modulated by DUBs, which influence various MAPK pathway branches, including ERK, JNK, and p38 MAPK⁸² (Figure 3A).

The ERK pathway is central to promoting cell proliferation and survival in response to growth factors. This pathway is tightly regulated by various DUBs to ensure proper signal transduction. For instance, USP21 is crucial for maintaining the stability of MAPK/ERK kinase (MEK) 2, a core component of the ERK pathway. By deubiquitinating MEK2, USP21 ensures sustained activation of ERK, which is vital for the progression of hepatocellular carcinoma (HCC).⁸³ This stabilization mechanism underscores the role of DUBs in prolonging the active state of pivotal signaling proteins.

Another key regulator is USP4, which stabilizes transforming growth factor-beta (TGF-β)-activated kinase 1 (TAK1), thus influencing the ERK pathway indirectly. TAK1 activation leads to downstream signaling cascades including the ERK pathway, highlighting the interconnectedness of MAPK pathways and the broad regulatory impact of DUBs like USP4.⁸⁴

Moreover, USP8 and OTUD7B (Cezanne-1) modulate the EGFR signaling axis, a significant upstream activator of the ERK pathway.^{85, 86} By deubiquitinating and stabilizing EGFR, these DUBs amplify the transmission of proliferative signals through the ERK cascade, showcasing their role in enhancing growth factor-mediated signaling.

The JNK pathway responds primarily to stress stimuli and is pivotal in controlling apoptosis and cellular differentiation. OTUD1 emerges as a critical player in this pathway by stabilizing apoptosis signal-regulating kinase 1 (ASK1), an upstream MAPK kinase kinase.⁸⁷ OTUD1's action ensures ASK1's activity, thereby promoting the activation of JNK signaling in response to stress signals. Interestingly, OTUD1 achieves this stabilization independently of its deubiquitinase function, suggesting a unique regulatory mechanism that extends beyond conventional deubiquitination.

Furthermore, OTUD5 exhibits a broad influence by inhibiting the phosphorylation of ERK1/2, p38, and JNK, thereby providing a counterbalance within the MAPK signaling network.⁸⁸ This inhibition is crucial for finely tuning cellular responses to avoid excessive apoptosis or uncontrolled cell proliferation that could result from aberrant JNK activation.

The p38 MAPK pathway is activated in response to stress and inflammatory cytokines, playing a critical role in apoptosis, cell differentiation, and immune responses. OTUB1 acts as a key modulator in this pathway by inhibiting the sustained activation of LPS-induced p38 MAPK signaling.⁸⁹ This action serves as a preventive measure against chronic inflammation and associated pathologies, highlighting OTUB1's role in maintaining cellular homeostasis under stress conditions.

USP11 contributes to the regulation of the p38 pathway in a PPP1CA-dependent manner. PPP1CA, one of the catalytic subunits of protein phosphatase 1, influences MAPK signaling and is protected from proteasome-mediated degradation by USP11. This protection allows for enhanced activity of the p38 pathway, particularly in tumorigenesis processes seen in CRC cells. By controlling PPP1CA levels, USP11 indirectly modulates p38 MAPK signaling, underscoring the complex interplay between DUBs and MAPK pathways.⁹⁰

The regulation of MAPK pathways by DUBs is crucial for normal cellular function. Dysregulation can lead to conditions such as cancer, neurodegenerative diseases, and chronic inflammation. The complexity of DUB regulation is highlighted by their overlapping roles and interactions within MAPK signaling, converging on key molecules and pathways. This intricate network ensures properly scaled and timed cellular responses, preventing aberrant signaling. Understanding the specific roles of DUBs in MAPK pathways could reveal new therapeutic strategies for treating diseases linked to dysregulated MAPK signaling.

3.3 PI3K/Akt/mTOR signaling pathway

The PI3K/Akt/mTOR signaling pathway is crucial for regulating cell growth, proliferation, and survival. Dysregulation of this pathway is often implicated in various cancers and other diseases.⁹¹ DUBs play significant roles in modulating this pathway by influencing the stability and activity of key signaling proteins. This section explores the complex regulation of the PI3K/Akt/mTOR pathway by DUBs, focusing on their mechanisms and impact on critical components (Figure 3B).

The PI3K pathway is activated when extracellular growth factors bind to receptor tyrosine kinases (RTKs) on cell surfaces. This binding activates PI3K, which converts PIP2 into PIP3. PIP3 recruits Akt (protein kinase B) to the cell membrane, where Akt is activated by phosphoinositide-dependent kinase-1 (PDK1). Activated Akt phosphorylates various downstream targets, including the mechanistic target of rapamycin (mTOR), a central regulator of cell growth and metabolism. mTOR functions in two distinct complexes, mTORC1 and mTORC2, each playing unique roles in cellular metabolism and growth.⁹²

Central to the regulation of the PI3K/Akt pathway is PTEN, which dephosphorylates PIP3 back to PIP2, antagonizing PI3K signaling. PTEN's tumor suppressor function is tightly regulated by ubiquitination, with several DUBs influencing its stability and activity. USP22-mediated upregulation of SIRT1 leads to PTEN deacetylation, thus activating the PI3K/Akt/mTOR pathway, an effect that can be reversed by SIRT1 knockdown.⁹³

Additional interactions include USP13, which directly binds and deubiquitinates PTEN, stabilizing it and suppressing tumorigenesis in PTEN-positive cancer cells. The loss of USP13 results in enhanced Akt phosphorylation and tumor growth.⁹⁴ Similarly, members of the Josephin family, including ATXN3, ATXN3L, and JOSD1, increase the expression of PTEN and its competing endogenous RNA PTENP1, indicating a transcriptional regulatory mechanism.⁹⁵

Akt is a central node in the PI3K/Akt/mTOR pathway, and its activity is tightly controlled by phosphorylation and ubiquitination status. CYLD, a well-characterized DUB, removes ubiquitin moieties from Akt, thereby preventing its activation in the absence of growth factors. Upon stimulation, CYLD dissociates from Akt, allowing E3 ligases to ubiquitinate and activate Akt.⁹⁶

Similarly, OTUD1 and OTUD7B remove K63-linked ubiquitination from Akt under different conditions, promoting its membrane recruitment and phosphorylation, thereby activating the pathway.^{97, 98} Interestingly, USP1 also interacts with Akt but primarily inhibits its activity by suppressing phosphorylation at T308, while complete Akt inhibition involves the complex formation with PHLPP1, which dephosphorylates S473.⁹⁹ This coordination ensures precise control over Akt signaling during nutrient deprivation.

mTOR functions in two distinct complexes, mTORC1 and mTORC2, each regulating different aspects of cell physiology. A critical interaction involving OTUB1 shows that it stabilizes RACK1, thus preventing abnormal activation of the PI3K/Akt pathway. OTUD5 stabilizes RNF186, which promotes the degradation of the mTORC1 feedback inhibitor sestrin2, thereby activating mTOR signaling. Additionally, OTUD5 modulates the protein abundance of DEPTOR, an inhibitory subunit of both mTORC1 and mTORC2.¹⁰⁰

Another intriguing regulator is USP32, whose knockout results in hyperubiquitination of the Ragulator complex subunit LAMTOR1. This modification impairs its interaction with vacuolar H-ATPase, reducing Ragulator function and ultimately limiting mTORC1 recruitment. Consequently, mTOR kinase localization to lysosomes diminishes, leading to decreased mTORC1 activity and increased autophagy.¹⁰¹

The complexity of DUBs in regulating the PI3K/Akt/mTOR pathway highlights their potential as therapeutic targets. In summary, DUBs exert multifaceted regulatory effects on the PI3K/Akt/mTOR signaling pathway. Their interactions with key components like PTEN, Akt, and mTOR complexes crucially influence the pathway's activity, adding layers of control in cellular signaling networks. Understanding these intricate dynamics provides insights into potential therapeutic interventions for diseases characterized by aberrant PI3K/Akt/mTOR signaling.

3.4 Wnt/β-catenin signaling pathway

The Wnt/β-catenin signaling pathway is crucial for embryonic development and the maintenance of adult tissue homeostasis. Dysregulation of this pathway is implicated in numerous cancers.¹⁰² The pathway is initiated when Wnt proteins bind to the Frizzled receptor and the LRP5/6 coreceptor on the cell surface. This interaction inhibits the destruction complex, which is composed of Axin, Adenomatous Polyposis Coli (APC), Glycogen Synthase Kinase-3β (GSK-3β), and Casein Kinase 1 (CK1). The inhibition of this complex leads to the stabilization and accumulation of β-catenin in the cytoplasm. Subsequently, β-catenin translocates to the nucleus, where it forms a complex with T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors, driving the expression of target genes that promote cell proliferation and survival^{103, 104} (Figure 3B).

Central to the regulation of the Wnt/β-catenin pathway is the control of β-catenin levels and activity. This regulation is achieved through a balance between ubiquitination and deubiquitination processes, where DUBs play pivotal roles. One of the primary regulatory nodes of the Wnt/β-catenin pathway is the stabilization of β-catenin. In the absence of Wnt signaling, β-catenin is targeted for ubiquitination and proteasomal degradation by the destruction complex. Upon Wnt stimulation, this complex is inhibited, allowing β-catenin to accumulate in the cytoplasm and subsequently translocate to the nucleus. DUBs such as USP5 and USP9X directly interact with β-catenin to remove inhibitory ubiquitin chains, thereby stabilizing β-catenin. USP5 deubiquitinates β-catenin, preventing its degradation and facilitating its nuclear accumulation. Elevated expression of USP5 is associated with larger tumor sizes and poorer differentiation in non-small cell lung cancer (NSCLC), underscoring its role in tumor progression.¹⁰⁵ Similarly, USP9X stabilizes β-catenin by removing K48-linked polyubiquitin chains, promoting β-catenin's nuclear translocation and activation of downstream oncogenic targets such as c-Myc and cyclin D1. Higher levels of USP9X correlate with worse prognosis in high-grade gliomas.^{106, 107}

The destruction complex itself is another critical target for DUB regulation. Components of this complex, such as Axin and APC, are essential for β-catenin degradation. USP15, for instance, targets β-catenin through the APC complex, stabilizing the destruction complex and facilitating the degradation of β-catenin.¹⁰⁸ This action highlights USP15's role as a tumor suppressor by preventing the overactivation of Wnt signaling and subsequent tumor formation.

In addition, cellular stress conditions, such as DNA damage, can hijack the normal regulation of β-catenin. OTULIN, for example, is phosphorylated at Tyr56 following DNA damage, enhancing its association with β-catenin.¹⁰⁹ This stabilized binding prevents β-catenin degradation and enhances its nuclear accumulation to maintain cell survival pathways under stress conditions. Moreover, OTUD7B exemplifies a nonenzymatic regulatory mechanism in the Wnt/β-catenin pathway. Instead of deubiquitinating β-catenin directly, OTUD7B interacts with LEF1, promoting its nuclear localization and enhancing its interaction with β-catenin.¹¹⁰ This interaction underscores the importance of spatial dynamics in the regulation of β-catenin's transcriptional activity.

Conversely, certain DUBs act to suppress Wnt/β-catenin signaling. A20 functions in this capacity by modulating the ubiquitination state of RIPK4, a kinase involved in Wnt signaling.¹¹¹ By destabilizing RIPK4, A20 diminishes downstream signaling, providing a negative feedback mechanism to prevent excessive activation of the Wnt pathway.

Given the centrality of β-catenin regulation in the Wnt pathway, DUBs represent potent therapeutic targets. Targeting specific DUBs could provide a means to modulate β-catenin levels and activity, offering a strategy to intervene in cancers driven by aberrant Wnt signaling. In sum, the regulation of the Wnt/β-catenin signaling pathway by DUBs is multifaceted and context-dependent. DUBs influence various aspects of the pathway, from stabilizing β-catenin to modulating the activity of destruction complex components and interacting with key transcription factors. Understanding these intricate regulatory mechanisms provides valuable insights into potential therapeutic targets for diseases characterized by dysregulated Wnt signaling, particularly cancer. Future research focused on the development of DUB inhibitors or modulators holds promise for more effective cancer treatments.

3.5 Hippo signaling pathway

The Hippo signaling pathway is a critical regulator of cell proliferation, apoptosis, and organ size, with its core components, including the MST1/2 kinases, LATS1/2 kinases, and the transcription coactivators YAP/TAZ. DUBs are essential modulators within this pathway, influencing the stability and activity of its key components¹¹² (Figure 3B).

Central to Hippo signaling is the regulation of YAP/TAZ, which activate genes linked to cell growth and survival upon nuclear translocation. DUBs like OTUB1 and OTUB2 prevent YAP degradation, thus sustaining its activity. OTUB1 specifically removes K48-linked ubiquitin chains from YAP, facilitating its nuclear accumulation.¹¹³ Additionally, poly-SUMOylated OTUB2 enhances YAP/TAZ signaling, highlighting the importance of posttranslational modifications.¹¹⁴

USP1 and USP19 further exemplify the regulation of YAP/TAZ stability. USP1 focuses on suppressing K11-linked polyubiquitination of TAZ, linked to the progression of HCC, while USP19 targets K48- and K11-linked chains on YAP, maintaining its activity in cancer.^{115, 116}

The activity of LATS1/2 kinases, pivotal for phosphorylating and inhibiting YAP/TAZ, is also modulated by DUBs. YOD1 indirectly enhances YAP/TAZ activity by stabilizing ITCH, an E3 ligase, consequently reducing LATS-mediated phosphorylation of YAP/TAZ.¹¹⁷ This demonstrates how DUBs can regulate upstream components to control pathway outputs. USP9X plays a dual role by interacting with both YAP/TAZ and LATS2. Its absence leads to heightened YAP activity and oncogenesis, highlighting its complex involvement in both promoting and inhibiting Hippo pathway activity depending on context.^{118, 119}

Beyond direct protein stabilization, DUBs like USP47 influence wider cellular processes by modulating proteins such as NLRP3, impacting inflammation and immunity.¹²⁰ This action potentially affects Hippo pathway signaling indirectly, demonstrating the extensive reach of DUB activity across cell regulatory networks.

The dynamic interplay between DUBs and the Hippo signaling pathway is crucial for maintaining cellular homeostasis. By precision-tuning ubiquitination processes, DUBs offer potential pathways for targeted therapeutic strategies. Continued investigation into DUB mechanisms within Hippo signaling will further illuminate their broader implications in health and disease management.

3.6 TGF-β signaling pathway

The TGF-β signaling pathway plays a crucial role in regulating a wide array of cellular processes, including proliferation, differentiation, apoptosis, and extracellular matrix production. Dysregulation of this pathway is implicated in various pathologies, particularly cancer.¹²¹ Central to the TGF-β signaling cascade is the activation of receptor serine/threonine kinases, which then phosphorylate receptor-associated Smads (R-Smads). These R-Smads form complexes with Co-Smads and translocate to the nucleus to regulate the transcription of target genes. DUBs are central to maintaining the balance of this pathway by modulating the ubiquitination status of key signaling components¹²² (Figure 3B).

At the heart of TGF-β signaling are the type I and type II receptors (TGF-βRI and TGF-βRII), which are activated upon ligand binding. The stability and availability of these receptors are crucial for proper signal transduction. UCHL1 has been shown to protect TGF-βRI from degradation, enhancing the overall signaling strength and downstream impact. By preventing the ubiquitination of TGF-βRI, UCHL1 facilitates sustained receptor presence on the cell surface, promoting continuous signaling cascades that are vital for processes such as tissue repair and immune response.¹²³

Similarly, USP15 plays a significant role in stabilizing TGF-βRI by deubiquitinating it within the SMAD7–SMURF2 complex. This action prevents receptor degradation and ensures ongoing signaling.¹²⁴ This stabilization is crucial for numerous physiological responses, including cellular proliferation and differentiation. The involvement of USP15 highlights how DUBs can maintain receptor integrity, thereby modulating signal intensity and duration.

Central to TGF-β signaling are the SMAD proteins, which translocate to the nucleus to regulate gene expression once activated by receptor-mediated phosphorylation. DUBs exert significant control over these proteins by modulating their ubiquitination status, thus influencing signaling efficiency. OTUD1 has a dual regulatory effect by modulating the ubiquitination of both SMAD3 and SMAD7. It enhances the formation of the SMAD3/SMAD4 complex by removing ubiquitin chains from SMAD3, facilitating its nuclear translocation and transcriptional activity.¹²⁵ Concurrently, OTUD1 stabilizes SMAD7, a negative regulator of the pathway, by cleaving its ubiquitin chains.¹²⁶ This dual action enables OTUD1 to fine-tune the pathway, promoting or inhibiting signal propagation as needed, thus contributing to cellular homeostasis.

USP26 also impacts SMAD7's stability. By limiting its degradation, USP26 contributes to the attenuation of TGF-β signaling, providing a check against excessive pathway activation.¹²⁷ This regulatory feedback loop involving USP26 ensures that cells can swiftly respond to changes in signaling demands, maintaining equilibrium in cellular responses.

The influence of DUBs extends beyond immediate pathway components to affect broader developmental and pathological outcomes. USP9X, for example, deubiquitinates SMAD4, a critical mediator in the TGF-β pathway.¹²⁸ This regulation is essential during embryonic development, particularly in mesoderm formation. The dysfunction of USP9X leads to impaired neurodevelopment and associated disorders, emphasizing its role in balancing signaling during critical development phases.¹²⁹

Understanding how DUBs influence the TGF-β pathway provides insights into their potential as therapeutic targets. Given their role in fine-tuning critical pathway components, targeting specific DUBs offers a strategy for modulating TGF-β signaling in diseases characterized by its dysregulation. The dual roles of DUBs in promoting and inhibiting signaling suggest that selective modulation can restore balance in disorders such as cancer and fibrosis.

These pathways significantly influence various biological processes such as cell proliferation, differentiation, and apoptosis, further revealing the complex role and potential therapeutic value of the DUB family in tumor biology. Therefore, studying the specific functions and regulatory mechanisms of the DUB family in these pathways can enhance our understanding of cancer pathogenesis and may provide a theoretical basis for developing new anticancer strategies.

4.1 DUBs and tumor development

Increasing evidence suggests that DUBs play a significant role in tumor initiation and progression, especially in common cancers such as breast cancer, liver cancer, lung cancer, and CRC. The impact of DUBs is not singular or fixed; rather, they influence key processes such as tumor cell growth, proliferation, migration, and invasion through their deubiquitination activity. This effect is primarily achieved by regulating the stability of key proteins and their interactions with known ubiquitinases (Figure 4A).

4.2 DUBs and tumor cell death

In exploring cancer therapies, the regulation of cell death mechanisms is particularly crucial. The DUBs plays a central role in regulating various cell death pathways. Although apoptosis is the primary mechanism of tumor cell death, increasing research indicates that nonapoptotic forms of cell death, such as ferroptosis, autophagy, and necroptosis, also represent emerging regulatory pathways that inhibit tumor progression. In-depth studies of these mechanisms not only help understand the survival strategies of tumor cells but may also reveal new therapeutic target (Figure 4B).

4.3 DUBs in DNA damage repair and treatment resistance

DNA damage repair is an essential cellular mechanism that ensures genomic integrity and stability. Dysregulation of these repair pathways, especially those involved in addressing DNA double-strand breaks (DSBs), is a hallmark of cancer and implicates multiple proteins, heavily relying on ubiquitination processes.³⁴⁸ DUBs are pivotal in regulating protein ubiquitination and managing DNA damage repair, thus impacting tumor growth and progression. Furthermore, resistance to drug therapy, radiotherapy, and chemotherapy presents a significant challenge in cancer treatment. This section will explore DNA damage repair mechanisms and their complex relationship with treatment resistance (Figure 4B).

USP1 is critical in the Fanconi anemia (FA) pathway for repairing DNA crosslinks. It deubiquitinates FANCD2 and FANCI, ensuring their function in the S phase of the cell cycle for effective DNA repair.^{135, 349} USP1 also deubiquitinates proliferating cell nuclear antigen, influencing the translesion DNA synthesis repair pathway, and regulates checkpoint kinase 1 (CHK1) activity, impacting DDR and cellular differentiation.^{133, 350}

OTUB2 is pivotal in DSB repair, affecting the choice between homologous recombination (HR) and nonhomologous end joining (NHEJ). By inhibiting RNF8-mediated ubiquitination, OTUB2 limits NHEJ and promotes HR through the activation of YAP/TAZ, maintaining genomic stability during DSB repair.²⁶³ USP22 also plays a role in DSB repair by deubiquitinating H2B-K120, necessary for chromatin remodeling. The absence of USP22 impairs H2AX phosphorylation and reduces NHEJ efficiency, underscoring its importance in genomic stability.²¹⁰

In mismatch repair (MMR) and nucleotide excision repair (NER), OTUB1 stabilizes CHK1 through deubiquitination, enhancing repair and radiation resistance.²⁵³ Additionally, it inhibits the ubiquitination of MMR protein MSH2, regulating DNA damage signaling and resistance to genotoxic agents.³⁵¹ OTUB1 also stabilizes and activates p53, further supporting DDR by promoting DNA repair pathways.³⁵¹

USP22 and A20 are crucial for chromatin remodeling and DDR protein recruitment. USP22 deubiquitinates H2B-K120, facilitating the recruitment of repair proteins.²¹⁰ In contrast, A20 binds and inhibits RNF168, disrupting its interaction with H2A and preventing the accumulation of repair proteins like 53BP1, impacting DDR and enhancing cancer cell resistance to treatments.³⁵²

In conclusion, DUBs are integral to the regulation of diverse DNA damage repair pathways. USP1, OTUB1, OTUB2, USP22, and A20 exhibit varied mechanisms through which they influence DNA repair processes. Understanding these context-specific roles emphasizes the therapeutic potential of targeting DUBs in cancer treatment and underscores the need for ongoing research to reveal their precise regulatory functions and interactions within DDR pathways.

4.4 DUBs and tumor cell metabolism

The metabolic characteristics of tumor cells often differ significantly from those of normal cells, with the most notable phenomenon being the Warburg effect. This effect describes how tumor cells preferentially generate energy through glycolysis rather than the more efficient oxidative phosphorylation, even under sufficient oxygen conditions.³⁵³ Additionally, tumor cells regulate lipid and amino acid metabolism to meet their needs for energy, biosynthetic precursors, and maintenance of redox balance. By exploring the diverse roles of DUB family proteins in tumor cell energy metabolism, we can better understand their complex roles in tumor development and provide a scientific basis for developing new therapeutic strategies (Figure 4B).

DUBs significantly impact the glycolytic pathway, a primary metabolic route in tumor cells, especially under the Warburg effect where cells favor glycolysis over oxidative phosphorylation even in the presence of oxygen. USP37, for instance, has been shown to regulate glycolysis by targeting c-Myc. In H1299 cells, USP37 knockdown leads to decreased glucose consumption and lactate production, which can be reversed by re-expressing c-Myc. This indicates that USP37 enhances the Warburg effect through c-Myc stabilization.²³⁷ Similarly, OTUB1 promotes aerobic glycolysis by preventing MYC degradation, thereby facilitating breast cancer cell proliferation via increased expression of hexokinase 2 (HK2).³⁵⁴ USP14 also supports glycolytic metabolism in OSCC by deubiquitinating and stabilizing phosphofructokinase-1 liver type (PFKL), which boosts tumor cell proliferation and migration.¹⁸⁴ In contrast, A20 reduces PFKL stability, thereby inhibiting glycolysis and liver cancer cell growth.³⁰⁸

In terms of lipid metabolism, USP22 plays a crucial role by directly interacting with and deubiquitinating peroxisome proliferator-activated receptor gamma (PPARγ). This deubiquitination stabilizes PPARγ, which is vital for de novo fatty acid synthesis. High levels of USP22 expression in HCC correlate with the expression of PPARγ, ATP-citrate lyase (ACLY), and acetyl-CoA carboxylase, and are associated with poor prognosis, underscoring its role in lipid metabolism and tumor progression.²¹⁴

Hypoxia, a common feature of the TME, induces specific metabolic adaptations mediated by hypoxia-inducible factor 1-alpha (HIF-1α). DUBs such as USP25 and USP11 regulate HIF-1α stability, thereby influencing hypoxia-adapted metabolic pathways. USP25 enhances HIF-1α transcriptional activity by deubiquitinating it under hypoxic conditions, promoting glycolysis and enabling the survival of pancreatic ductal adenocarcinoma (PDAC) cells in hypoxic regions.²¹⁶ Similarly, USP11 stabilizes HIF-1α, driving glycolytic pathways involving PDK1 and lactate dehydrogenase A, contributing to HCC proliferation and metastasis.¹⁶⁹

DUBs play multifaceted roles in the metabolic reprogramming of tumor cells by stabilizing critical metabolic regulators. This supports tumor cell proliferation, migration, and survival, making DUBs potential targets for cancer therapy. Understanding these regulatory mechanisms offers insights into novel therapeutic strategies aimed at disrupting tumor metabolism to hinder cancer growth and progression.

4.5 DUBs and tumor immunity

Tumor cells develop a multitude of complex mechanisms to escape immune surveillance and suppress antitumor immune responses, thereby fostering an environment favorable for their growth and survival. Programmed death-ligand 1 (PD-L1) is a critical immune checkpoint protein that, when overexpressed on tumor cells, binds to PD-1 receptors on T cells, resulting in immune evasion.³⁵⁵ Achieving a detailed understanding of how DUBs regulate PD-L1 is essential for the development of effective cancer immunotherapies (Figure 4B).

PD-L1 stability is intricately regulated by several DUBs, which directly influence its expression levels on tumor cells. For instance, USP5 stabilizes PD-L1 by removing its ubiquitin tags, thereby preventing its degradation. Elevated levels of USP5 in NSCLC are associated with increased PD-L1 expression and poor patient outcomes. This highlights USP5's role in promoting immune evasion by stabilizing PD-L1, which inhibits T cell activity.¹⁴⁷

Similarly, USP22 is another DUB that stabilizes PD-L1 by direct deubiquitination. USP22 operates through the USP22–CSN5–PD-L1 axis, enhancing PD-L1 levels and hindering T cell cytotoxicity, which in turn promotes tumorigenesis.²¹³ Additionally, USP7 has been shown to enhance PD-L1 stability directly. The inhibition of USP7 disrupts the PD-L1/PD-1 interaction, increasing the susceptibility of cancer cells to T cell-mediated killing both in vitro and in vivo.¹⁵³

USP2 also facilitates immune evasion by stabilizing PD-L1. Depletion of USP2 induces PD-L1 degradation, thereby enhancing antitumor immunity. Combining USP2 depletion with anti-PD-1 therapy exhibits a synergistic antitumor effect, underscoring USP2's role in immune escape and its potential as a therapeutic target.¹³⁷

In lung cancer, the deletion of A20 activates the TBK1–STAT1–PD-L1 axis, promoting tumor cell evasion from CD8+ T cell-mediated surveillance.³⁵⁶ In melanoma, increased A20 expression enhances STAT3 activity and PD-L1 expression, thereby impairing CD8+ T cell functions.³⁵⁷

Beyond PD-L1, OTUD1 displays dual roles in tumor immunity. It enhances antitumor immunity by regulating iron transport through the OTUD1–IREB2–TFRC axis, yet it also contributes to CRC immune escape via the TAM/TNFα–OTUD1–FGL1 pathway within the liver microenvironment.^{271, 279} Additionally, OTUD5 deubiquitinates and stabilizes STING, playing a role in radiation-mediated antitumor immunity.²⁹⁸

In conclusion, DUBs significantly modulate tumor immunity by regulating the stability of key immune regulatory proteins like PD-L1. These interactions can either promote immune evasion or enhance immune surveillance, indicating the potential of targeting DUBs in cancer immunotherapy. Understanding the precise mechanisms through which DUBs influence PD-L1 and other immune regulators will be crucial for advancing effective cancer treatments.

4.6 DUBs and TME

The TME plays a critical role in promoting tumor growth and immune evasion. This complex milieu consists of various cell types, including immune cells, fibroblasts, and endothelial cells, along with cytokines and other chemical signals.³⁵³ DUBs within the TME modulate the immune responses by regulating the stability and function of numerous proteins, directly impacting tumorigenesis and immune evasion mechanisms (Figure 4B).

USP7 is crucial for macrophage homeostasis, particularly in balancing M1 and M2 phenotypes. Its inhibition reprograms M2 macrophages, leading to increased CD8+ T cell proliferation and improved antitumor responses. In Lewis lung carcinoma models, USP7 inhibitor treatment reduced tumor growth and enhanced M1 macrophage and CD8+ T cell infiltration via the p38 MAPK pathway.¹⁵⁴

In PDAC, USP22 deletion reshapes the TME by reducing myeloid cell infiltration while promoting T and NK cell infiltration, thereby enhancing the response to combination immunotherapy and suppressing metastasis in a T cell-dependent manner.³⁵⁸ Similarly, USP14 stabilizes IDO1, promoting immune suppression in CRC. USP14 inhibition decreases IDO1 levels, increases CD8+ T cell infiltration, reverses immune tolerance, and sensitizes CRC cells to anti-PD-1 therapy.³⁵⁹

OTUD4 contributes to tumor immune escape by stabilizing CD73, thus inhibiting CD8+ T cell function and IFN-γ production.²⁹⁰ Conversely, increased USP18 expression in tumor cells inhibits carcinogenesis, while decreased USP18 expression fosters tumor growth by reducing IFN-γ synthesis and CTL survival in the TME.³⁶⁰

OTUB1 enhances antitumor immunity by modulating IL-15 ubiquitination, which activates CD8+ T cells and matures NK cells.³⁶¹ Additionally, OTUD6B expression in tumors correlates with macrophage infiltration and T cell activity, shaping the tumor immune microenvironment.³⁶²

TME is a highly complex and dynamic system involving various cell types and signaling pathways. DUBs play significant roles in key immune processes and cell types, thereby modulating immune responses and the infiltrative capacity of immune cells. Targeting DUBs presents a promising therapeutic approach, and continued research in this area will be instrumental in developing novel strategies to enhance antitumor immune responses and improve the efficacy of existing treatments.

5.1 Role of DUBs in neurodegenerative disorders

DUBs play crucial roles in neurodegenerative disorders maintaining neuronal health and are increasingly recognized for their involvement in neurodegenerative disorders.^{363, 364} Neurodegenerative diseases are characterized by the progressive loss of specific neuron populations due the formation of neurotoxic aggregates in the brain, including PD, Alzheimer's disease (AD), and polyglutamine diseases.³⁶⁵ Recent evidence highlights the significant roles of DUBs in neurodegenerative disorders, making them potential therapeutic targets.

A polymorphic USP8 allele (USP8D442G) have been significantly found in Chinese PD patients, which could enhance interaction with α-synuclein, leading to its accumulation and altered localization, highlighting this polymorphism as a potential therapeutic and diagnostic target for PD.³⁶⁶ HD, driven by polyglutamine-expanded mutant huntingtin (mHTT), is ameliorated by the deubiquitinase Usp12, which protects against mHTT toxicity and enhances autophagy in neurons. Notably, Usp12's neuroprotective and autophagy-inducing effects do not depend on its catalytic activity, highlighting its unique role in regulating neuronal proteostasis.³⁶⁷ USP14 is a DUB enzyme crucial for maintaining the nervous system's health and function by regulating the cellular pool of monomeric ubiquitin. In USP14-deficient mice, developmental deficits are observed in neuromuscular junctions and motor neuron endplates, accompanied by activation of pathways involving mixed lineage kinase 3 and downstream targets like c-jun N-terminal kinase (JNK). USP14 plays a vital role in synaptic transmission and neuronal development, especially at the neuromuscular junctions.³⁶⁸ USP14 influences the clearance of ubiquitinated toxic proteins by inhibiting their proteasome-mediated degradation, exemplified by the enhanced degradation of tau and ATXN3 upon inhibition of its DUB activity.³⁶⁹ USP14 inhibitor have been developed as a potential therapeutic agent to protect cells from neurodegenerative stressors cell by enhancing proteasome activity and promoting the degradation of proteotoxic proteins.^{370, 371} Furthermore, a broader role of USP14 in memory formation and neuroprotection against ischemic injury, highlighting it as a promising therapeutic target in neurodegenerative disorders.^{372, 373} A recent study reports that loss of USP30 enhances mitophagy and reduces neurodegeneration by counteracting α-synuclein's effects, and a USP30 inhibitor MTX115325 shows potential as a disease-modifying therapy for PD.³⁷⁴

ATXN3 plays significant roles in neurodegenerative disorders, particularly in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease. This disease is triggered by CAG repeat expansions in the ATXN3 gene, leading to a malformed ATXN3 protein with an extended polyglutamine tract,³⁷⁵ which results in toxic protein aggregates. Emerging therapies, including RNA-based silencers and CRISPR/Cas9, target mutant ATXN3 expression, showing promise in reducing oxidative stress and mitochondrial dysfunction in SCA3 models.^376-378 These results suggest that targeting mutated ATXN3 could offer a promising therapeutic strategy for SCA3, highlighting the need to develop effective ATXN3 inhibitors.

UCHL1, a key DUB enzyme abundant in the brain, plays a vital role in maintaining neuronal health by regulating ubiquitin homeostasis at synapses.³⁷⁹ Mutations and modifications in UCHL1 have been associated with human neurodegenerative disorders such as PD and AD, as well as brain injuries.³⁸⁰ UCHL1 acts as both a deubiquitinase and a ligase—its ligase activity, which extends Lys63–polyubiquitin chains on α-synuclein, is enhanced by oligomerization but can be disrupted by disease-associated mutations.³⁸¹ Structural studies reveal that UCHL1 variants like I93 M increase PD's risk by disrupting activity, while S18Y is protective by reducing dimerization and ligase activity. Beyond α-synuclein regulation, UCHL1 interacts with and stabilizes TrkB, a neurotrophin receptor critical for neuronal survival and synaptic plasticity, highlighting a mechanism where UCHL1 modulates BDNF–TrkB signaling by preventing TrkB degradation.³⁸² These findings underscore the multifaceted role of UCHL1 in neuroprotection and its potential as a therapeutic target in neurodegenerative disorders.

In conclusion, DUBs are fundamental to preserving neuronal function through their regulation of protein homeostasis and mitigation of aggregate-prone proteins via proteasome and autophagy pathways. Their contribution to neurodegenerative conditions underscores their potential as therapeutic targets, warranting further exploration in neuronal models to advance treatment strategies for these debilitating disorders.

5.2 Role of DUBs in cardiovascular diseases

DUBs are crucial in cardiovascular diseases as they prevent degradation of substrate proteins by the ubiquitin–proteasome system, thereby regulating autophagy, metabolism, and oxidative stress.³⁸³ They play significant roles in cardiac hypertrophy, MI, and diabetes-related cardiac conditions, influencing disease onset and progression.

DUBs play pivotal roles in the development and regulation of cardiac hypertrophy through their involvement in various cellular signaling pathways. These enzymes influence key processes such as inflammation, apoptosis, autophagy, and calcium homeostasis, all of which contribute to the cardiac hypertrophic response. DUBs such as USP19, USP2, and USP4 have been identified as potential biomarkers and regulators in cardiac hypertrophy.^384-386 USP4, for instance, exhibits an antimyocardial hypertrophy effect by inhibiting the TAK1–(JNK1/2)/p38 signaling pathway, while USP22 exacerbates hypertrophy by activating the same pathway.³⁸⁷ Moreover, USP14 fosters cardiac hypertrophy by enhancing GSK-3β activation, suggesting its role as a modulator of hypertrophic signaling.³⁸⁸ The stabilization of calcium homeostasis by DUBs like USP25 and JOSD2 further underscores their protective roles against hypertrophy, as they regulate SERCA2a levels.³⁸⁹ USP10 regulates hypertrophic pathways via enhancing the stability and function of SIRT6, leading to downregulation of the Akt signaling pathway and attenuation of maladaptive cardiac hypertrophy.³⁹⁰ These insights highlight the potential of targeting DUBs for therapeutic interventions to treat cardiac hypertrophy and related cardiovascular diseases.

DUBs can be involved in the pathogenesis and progression of MI by modulating various cellular processes such as apoptosis, ferroptosis, and pyroptosis. USP47 is upregulated in MI, enhancing NF-κB activity and promoting cardiomyocyte apoptosis during ischemia/reperfusion injury, thus its knockdown may serve as a therapeutic target by repressing MI progression.³⁹¹ Conversely, USP49 activates DUSP1–JNK1/2 signaling to reduce apoptosis of cardiomyocytes, offering cardio-protective effects against ischemia–reperfusion injury.³⁹² Ferroptosis, another form of programmed cell death, is regulated by USP7 and OTUD5 during MI, with USP7 knockdown reducing ferroptosis via the p53 pathway, while OTUD5 prevents ferroptosis by stabilizing GPX4, thus both alleviating myocardial injury.^{13, 393} Moreover, USP11 and USP47 has been shown to worsen ischemia–reperfusion injury by enhancing pyroptosis through aggravating immune inflammation.^{394, 395} These findings underscore DUBs as potential therapeutic targets in MI management.

In diabetic heart disease, DUBs play a significant role in regulating mitochondrial function and cardiac health. Specifically, in cardiac dysfunction amelioration in type 2 diabetes mellitus (T2DM), the S-sulfhydration of USP8 enhances its deubiquitination of parkin, which facilitates parkin's translocation into mitochondria and mitigates oxidative stress and mitochondrial apoptosis.³⁹⁶ Additionally, the USP10/Notch1 axis mediates the cardiac protective effects of follicle-like protein 1 in T2DM with MI,³⁹⁷ highlighting USP10 as a potential therapeutic target for managing diabetic heart disease.

Taken together, many DUBs are crucial regulators of cardiovascular diseases, influencing key cellular processes such as inflammation, apoptosis, autophagy, and calcium homeostasis. These findings highlight the therapeutic potential of targeting DUBs to treat cardiovascular diseases, warranting further research into their specific roles and mechanisms.

5.3 Role of DUBs in inflammatory disorders

DUBs play crucial roles in inflammatory disorders by modulating ubiquitin signaling pathways, significantly impacting central and peripheral immune responses often disrupted in conditions such as RA, systemic lupus erythematosus (SLE), IBD, psoriasis, and MS.³⁹⁸ These enzymes regulate the balance between proinflammatory and anti-inflammatory signals, and their dysregulation contributes to the progression of various inflammatory disorders, highlighting them as potential therapeutic targets for treatment.

RA is classified as a systemic poly-articular chronic autoimmune joint disease that primarily affects the hands and feet, with pathological features including immune cell infiltration, hyperplasia of the synovial lining, pannus formation, and destruction of articular cartilage and bone.³⁹⁹ A20 plays a crucial role in negatively regulating NF-κB signaling in response to inflammatory stimuli and has been implicated as a susceptibility gene for RA. Myeloid-specific A20-deficient mice spontaneously developed severe destructive polyarthritis, characterized by increased inflammatory cytokines, sustained NF-κB activation, and enhanced osteoclastogenesis, indicating A20's critical, cell-specific role in the disease.⁴⁰⁰ CYLD, another DUB, also impacts NF-κB and is involved in the pathogenesis of synovial inflammation in RA.⁴⁰¹ Moreover, USP4 interacts with RORγT in Th17 cells, enhancing IL-17A production, making it a promising target for developing new treatments for T-helper 17 (Th17)-mediated autoimmune diseases like RA.⁴⁰² Together, these DUBs highlight novel therapeutic targets and pathways in RA management.

SLE is a complex chronic autoimmune disease characterized by heightened B and T cell responses and a loss of tolerance to self-antigens, leading to widespread organ damage.⁴⁰³ The regulation of autoimmune-related genes through ubiquitination and deubiquitination processes is critical in SLE pathogenesis. In SLE patients, the DUB USP7 is a crucial regulator of the protein levels of the human IFNα-2 receptor (IFNAR1). It stabilizes IFNAR1 by dismantling IFNAR1-dependent polyubiquitin chains, thereby activating the IFNα pathway. USP7 is significantly overexpressed in SLE patients and shows a positive correlation with IFN scores, SLE disease activity index scores, and anti-double-stranded DNA, suggesting that USP7 may be associated with SLE disease activity through the stabilization of IFNAR1.⁴⁰⁴ Furthermore, it is reported that genetic variations in the A20 OTU domain are associated with an increased risk of SLE.⁴⁰⁵ Although A20 typically regulates NF-κB, mutations in its OTU domain do not lead to enhanced NF-κB signaling.⁴⁰⁶ Instead, they cause an upregulation of the PADI4 enzyme, which promotes protein citrullination and neutrophil extracellular trap (NET) formation, enhancing autoimmune responses. Individuals with A20 DUB polymorphisms in SLE patients exhibit increased NETs and autoantibodies to citrullinated epitopes, indicating that these genetic alterations increase susceptibility to SLE through these mechanisms.⁴⁰⁷ These insights suggest that modulating DUB can offer novel therapeutic strategies for managing SLE, aiming to restore immune balance and mitigate disease progression.

DUBs are integral to the pathogenesis and progression of IBD by modulating ubiquitin signaling pathways that regulate inflammatory processes. Prominent among these is A20, which tempers NF-κB signaling—a key pathway in IBD development—by removing K63-linked polyubiquitin chains from NF-κB signaling factors like NEMO, RIPK1, and TRAF6, thus maintaining immune homeostasis.³² Mice lacking A20 exhibit severe intestinal inflammation, underlining its role in intestinal health.⁴⁰⁸ Similarly, CYLD, another DUB, suppresses NF-κB activation by deubiquitinating TRAF2 and TRAF6, further preventing excessive inflammation.⁷⁶

Some DUBs regulate the pathogenesis of psoriasis, a chronic immune-mediated disorder characterized by skin, nail, and joint inflammation, which involves dysregulated cytokine signaling, activating transcription factors like STAT3, STAT1, and NF-κB.⁴⁰⁹ For instance, USP4 and USP17 enhance Th17-mediated gene expression and IL-17 secretion by deubiquitinating RORγT, contributing to inflammatory pathways.⁴⁰² USP4 also regulates TNFα-induced NF-κB activation by deubiquitinating TAK1,⁴¹⁰ highlighting its potential as a therapeutic target. These findings underscore the critical involvement of DUBs in managing inflammatory pathways in psoriasis.

MS is a chronic autoimmune disease that causes inflammation, leading to demyelination and immune cell infiltration in the central nervous system, affecting the protective covering around nerves in the brain and spinal cord.⁴¹¹ DUBs regulate key signaling pathways such as NF-κB, JAK–STAT, and MAPK to influence T cell differentiation, activation, and survival, thus affecting inflammation in MS.⁴¹² For instance, OTUD7B enhances T cell receptor (TCR) signaling and exacerbates experimental autoimmune encephalomyelitis (EAE) in a MS mouse model by deubiquitinating Zap70 and preventing Sts1/Sts2 binding, whereas its knockout diminishes T cell activation and Th1 cell differentiation by reducing TCR–CD28 signaling.⁴¹³ USP18 interacts with TAK1 to reduce its ubiquitination and, by decreasing NF-κB and NFAT activation, impairs IL-2 production, thus inhibiting Th17 polarization and EAE pathogenesis, with USP18 deficiency leading to reduced Th17 differentiation.⁴¹⁴ These insights underscore DUBs’ significant roles in MS pathophysiology, offering targets for therapeutic intervention.

In summary, DUBs are vital regulators of the inflammatory process, influencing immune signaling, cytokine production, and the pathogenesis of inflammatory disorders. Continued exploration of their roles could lead to breakthroughs in the treatment of inflammatory disorders, providing more targeted and effective therapies.

5.4 Role of DUBs in developmental diseases

Notably, emerging evidence points to a critical involvement of DUBs in developmental diseases, often resulting from mutations that disrupt their normal function. These mutations typically lead to severe developmental disorders, marked by early-onset neurological deficits, highlighting the significance of DUBs in neurodevelopmental processes. Such disorders are frequently attributed to loss-of-function mutations, underscoring a crucial dependency on DUB activity for normal development.

Hao-Fountain syndrome, a developmental disorder characterized by seizures, behavioral abnormalities, hypogonadism, and hypotonia, is caused by heterozygous mutations in the USP7 gene.⁴¹⁵ The pathogenesis of the syndrome is associated with USP7's roles in cellular protein trafficking, specifically through the regulation of the MAGE–L2–TRIM27 complex, which plays a critical part in retromer-dependent endosomal recycling.⁴¹⁶ This insight aligns with similar phenotypic features observed in related syndromes, indicating that aberrant endosomal sorting underlies the disorder. Mutations in the X-linked deubiquitinase USP9X are associated with both syndromic and nonsyndromic intellectual disabilities.^{417, 418} Specifically, USP9X regulates TGF-β signaling via SMAD4 deubiquitination, stabilizes the centriole replication factor STIL, and regulates cilia assembly, all of which have overlapping phenotypes with diseases like microcephaly and ciliopathies.⁴¹⁹ Additionally, USP9X influences dendritic spine development through the stabilization of ankyrin-G, with variants in ankyrin-G linked to neurodevelopmental disorders. Notably, TGF-β supports cortical spine development via USP9X-dependent stabilization of ankyrin-G, suggesting interconnected pathways in USP9X-related neurodevelopmental coordination, warranting further investigation into these interactions and their gender-specific manifestations.⁴²⁰

OTUD7A, a K11-specific OTU deubiquitinase located in the 15q13.3 locus, is implicated in a range of neurodevelopmental and psychiatric disorders when deleted.^{421, 422} Studies indicate OTUD7A's crucial role in neurodevelopment, evidenced by its control over dendritic branching and its knockout leading to neurodevelopmental deficits and abnormal EEGs in mice.^{423, 424} Moreover, biallelic OTUD7A missense variants in individuals with epilepsy support its association with the condition.⁴²⁵ Despite these findings, the detailed molecular mechanisms, including its interaction with E3 ligases and substrates, remain to be elucidated, which could provide insights into distinct epilepsy forms. Moreover, bi-allelic loss-of-function mutations in OTUD6B result in global developmental delay, feeding difficulties, structural brain abnormalities, and congenital heart disease.⁴²⁶ While OTUD6B is linked to protein translation and proteasome stability, its precise mechanistic role remains unclear, necessitating further research to understand its impact on differentiation processes.⁴²⁷ Hemizygous missense and deletion variants in OTUD5, an X-linked OTU deubiquitinase that cleaves K48- and K63-linked ubiquitin chains, cause a male-specific congenital disorder characterized by central nervous system, craniofacial, cardiac, skeletal, and genitourinary anomalies.⁴²⁸ In mice, Otud5 knockout results in embryonic lethality, and OTUD5-depleted human ESCs show neuroectodermal differentiation defects that can be rescued by wild-type OTUD5.⁴²⁸ A patient variant affecting only K48-linked ubiquitin cleavage fails to rescue differentiation, indicating the disorder stems from loss of OTUD5's activity on degradative K48-chains. OTUD5 prevents the degradation of chromatin remodelers like ARID1A/B, HDAC2, and HCF1, essential for enhancer activation during differentiation.⁴²⁷ These findings highlight the critical role of K48-ubiquitin chain cleavage in coordinating chromatin remodeling in early development, with further research needed to explore its broader embryogenic regulation.

Overall, DUBs are vital in modulating ubiquitin signaling necessary for embryonic and postnatal development, with their dysregulation associated with congenital disorders. Despite advancements, questions about their substrate specificity, regulatory mechanisms, and full physiological roles remain, often due to their study relying on incomplete protein variants. Advances in genomic sequencing now enable the identification of key DUBs as potential disease-causing genes, suggesting that prioritizing these for research could reveal new developmental pathways and improve disease diagnosis and treatment, influencing advances in precision medicine.

6.1 Targeting USPs

As research on the critical roles of the USP family in diseases increases, the discovery of small molecule drugs targeting the USP family has also been burgeoning. Existing studies have provided detailed reviews of the various small molecule inhibitors currently targeting the USP family.^{24, 464, 465} To provide the latest updates, this section will summarize some of the newly emerged USP inhibitors in the past 3 years, focusing on their development strategies and pharmacological activities (Table 2).

The development strategy for USP1 inhibitors involved designing and synthesizing a series of pyrido[2,3-d]pyrimidin-7(8H)-one derivatives based on the structure of known inhibitors, with systematic exploration of their structure-activity relationships. Compound 1m showed strong antiproliferative effects, arrested cancer cells in the S phase, acted as reversible and noncompetitive inhibitors, and enhanced the efficacy of the PARP inhibitor olaparib, demonstrating excellent oral pharmacokinetic properties in mice.⁴²⁹ ML323 is a USP1 inhibitor that shows specifically antagonizing the USP1-UAF1 complex without affecting other DUBs. Recently, ML323 effectively suppressed the proliferation of HCC and ESCC cells, inhibited TGF-β induced EMT in TNBC cells, and induced apoptosis and DNA damage in ESCC cells, demonstrating its potential as a viable anticancer treatment.⁴³⁰

Through a scaffold hopping strategy to enhance the cellular activity of FT671 (known USP7 inhibitor), resulting in the discovery of YCH2823 as a novel and potent USP7 inhibitors. YCH2823 demonstrated potent inhibition of USP7, inhibiting cancer cell growth, promoting G1 phase arrest and apoptosis, increasing p53 and p21 levels, and showing synergistic effects with mTOR inhibitors, making it a promising anticancer agent for both TP53 wild-type and mutant tumors.⁴³² Recently, a series of pyrrole[2,3-d]pyrimidin-4-one derivatives were designed for USP7 inhibition, with the representative compound Z33 (YCH3124) showing high potency and selectivity. YCH3124 exhibited strong antiproliferative activity across various cancer cell lines, inhibited the USP7 pathway, led to the accumulation of p53 and p21, disrupted cell cycle progression at G1 phase, and induced significant apoptosis, highlighting its potential as a potent anticancer agent.⁴³³ Moreover, a series of 2-aminopyridine derivatives were designed and synthesized based on structural modifications of the lead USP7 inhibitor, GNE6640. Compounds 7 showed the most potential, with IC₅₀ values of 7.6 µM, promoting MDM2 degradation, p53 stabilization, and p21 expression, though they displayed moderate antiproliferative activities against HCT116 cells.⁴³⁴ Furthermore, the proteolysis targeting chimera (PROTAC) strategy was employed to discovery small molecule to degrade USP7 for inhibiting its function. To design PROTAC targeting USP7, known USP7 ligand molecule FT671 and XL188 were optimized by modifying the right-hand side tail groups to enhance target binding affinity and lipophilicity. Among the synthesized compounds, PROTAC 17 exhibited the most potent USP7 inhibition with an IC₅₀ value of 1.7 µM, aligning with docking results and cell viability profiles, indicating its potential efficacy.⁴³⁵

A novel compound D1 with quinolin-4(1H)-one scaffold was identified as a lead USP10 inhibitor due to its high potency and selectivity. D1 significantly inhibited HCC cell proliferation and clone formation by targeting the ubiquitin pathway, promoting YAP degradation, downregulating p53 and p21, arresting cells in S-phase, and inducing apoptosis, demonstrating its potential for USP10-positive HCC treatment.⁴³⁶ Spautin-1 has been reported as an inhibitor of USP10 and USP13, significantly reducing the proliferation and migration of glioblastoma cells. Mechanistically, Spautin-1 disrupts the RAF–ERK pathway and glycolysis, and decreases the proto-oncogene SKP2, highlighting its potential to counteract key mechanisms involved in glioblastoma progression.⁴³⁸

Though high-throughput screening and structure-based optimization, BAY-805 was developed as a highly potent and selective noncovalent USP21 inhibitor. BAY-805 shows the low nanomolar affinity and robust target engagement, leading to strong NF-κB activation, representing a valuable chemical probe for exploring USP21 biology.⁴³⁹ Utilizing predictive models to screen and identify potential compounds from the United States Food and Drug Administration (US FDA)-approved drug library, nifuroxazide was identified as the most potent USP21 inhibitor with an IC₅₀ of 14.9 µM. Nifuroxazide demonstrated efficacy in inhibiting USP21, modulating its substrate ACLY, increasing p-AMPKα levels, and elevating miR-4458, both in HepG2 cells and in vivo, highlighting its potential for treating metabolic disorders and cancer proliferation.⁴⁴⁰ Based on computer-aided drug design methodologies, several potential ligands were designed to target the catalytic domain of USP21, as a result, MOLHYB-0436 showed the best binding affinity and stability. MOLHYB-0436 can disrupt the Wnt signaling pathway, which is activated by USP21 in PDAC, displaying promising pharmacokinetic and pharmacodynamic profiles, good bioavailability, and low toxicity, indicating its potential as a treatment for pancreatic cancer.⁴⁴¹ Recently, identifying a traditional Chinese secoiridoid compound, gentiopicroside, was identified as a USP22 inhibitor by stably binding to the USP22 catalytic pocket. Gentiopicroside decreased Foxp3 expression and Treg immune suppressive activity, increased histone 2B monoubiquitination, and decreased PD-L1 expression in cancer cells, enhancing antitumor immunity. In vivo studies showed that gentiopicroside significantly inhibited the growth of lung adenocarcinoma in mice and increased CD8+ T cell production of IFN-γ and granzyme B.⁴⁴²

USP25 and USP28, which are highly homologous and associated with diseases like cancer and neurodegenerative disorders, have recently become focal points for the development of therapeutic inhibitors.^{466, 467} Through screening a synthetic compound library and further structure optimization, CT1113 was synthesized and developed as a promising USP inhibitor with potent activity against USP28 and USP25. CT1113 can reduce c-MYC levels and inhibit USP28 and USP25 in various cancer cell lines, resulting in decreased expression of MYC–MAX target genes and increased ubiquitination of substrates like c-MYC and Tankyrase, thus exhibiting a broad-spectrum anticancer activity.⁴⁴³ Moreover, Vismodegib was identified as a lead structure for USP28 inhibition by screening a drug library, and further structure-based optimization led to the discovery of a more potent derivative 9p. Compound 9p showed high selectivity and potency in inhibiting USP28, causing cytotoxicity in colorectal and lung cancer cells, and enhancing CRC cell sensitivity to Regorafenib. Compound 9p also downregulated c-MYC via the ubiquitin–proteasome system, highlighting its potential anticancer effects through USP28 inhibition.⁴⁴⁴

Given the similar conserved catalytic domain structures in the USP family, different USP targeted inhibitors can also serve as a starting point for ligand design. For example, USP7 and USP47 are closely related in systematics and functionally perform some similar biological functions. The study of the biochemical and structural characteristics of USP47 reveals why the USP7 inhibitor derivative P50429 has targeting and inhibitory abilities toward USP47, and provides new insights for further design of USP47 specific inhibitors.⁴⁴⁵ To develop USP48 inhibitors, 14 commercially available USP inhibitors were screened and ML364 (known as a USP2 inhibitor) was identified as a candidate for further optimization. Using a ligand-based approach, a series of ML364 analogs were synthesized, with compound 17e showing an IC₅₀ of 12.6 µM in inhibiting USP48 activity. Further structural refinements are necessary to enhance the inhibition activity and specificity of these compounds.⁴⁴⁶

6.2 Targeting OTUs

Recent advancements in OTU deubiquitinase inhibitors have opened new avenues for targeted cancer therapy by modulating deubiquitination processes to suppress tumor growth. Studies have reported that various small-molecule compounds, such as SJB3-019A, OTUB2-IN-1, and OTUDin3, exhibit significant inhibitory effects on deubiquitinases along with notable antitumor activities. This section primarily explores the screening processes, structural optimization, and application potential of several representative OTU deubiquitinase inhibitors across different tumor models, underscoring the importance of further studies to enhance drug selectivity and safety.

Screening was conducted on 20 reported databases of DUB or DUB-like protease inhibitors, and two USP1-like inhibitors, SJB2-043 and SJB3-019A, were found to exhibit significant inhibitory effects on OTUB1 and USP8 at a concentration of 2.5 µM. To further explore the potential of these inhibitors, in-depth structure-activity relationship studies were conducted. Four analogues, compounds 60, 61, 62, and 63, were developed by modifying the 2-phenyl ring of SJB2-043 with single substitution, double substitution, and phenyl ring modification. Subsequently, protein thermal stability and enzyme activity experiments of compound 61 on OTUB1, as well as molecular docking mechanism analysis, showed that compound 61 has high selectivity and strong inhibitory effects on OTUB1 and USP8. This compound not only demonstrated strong inhibitory effects on OTUB1 and USP8 in vitro but also showed significant antiproliferative effects in H1975 and A549 NSCLC cell lines. Additionally, compound 61 effectively inhibited tumor growth in the H1975 xenograft mouse model after low-dose intraperitoneal administration. However, despite the significant antitumor activity exhibited by compound 61, studies also found that the OTUB1/USP8 protein exhibits instability when bound to compound 61. This discovery suggests that further validation of the binding mode of inhibitors is needed in the future, and their efficacy and safety should be evaluated in other animal models. In particular, in-depth studies of the pharmacokinetic and pharmacological properties of compound 61, as well as its mechanisms of action in different tumor types, will help optimize its structure, improving its stability and selectivity.⁴⁴⁷

As the first effective OTUB2 inhibitor, OTUB2-IN-1 forms hydrogen bonds between its two carboxyl groups and four amino acid residues (Thr45, Gly49, Ser223, and His224) in the OTUB2 catalytic pocket, significantly inhibiting the hydrolysis activity of the recombinant GST-OTUB2 protein toward substrate Ub-R110. In the mouse LL/2 tumor model, OTUB2-IN-1 treatment significantly inhibited tumor growth, prolonged mouse survival, and exhibited no significant toxicity. Subsequent animal model experiments and immunohistochemical analysis showed that OTUB2-IN-1 also demonstrated therapeutic potential in mouse B16-F10 and KLN205 tumor models. Additionally, tumor infiltration of CD8 and GZMB CTLs was significantly increased in OTUB2-IN-1-treated mouse tumors, while the expression of PD-L1 was significantly reduced. This result indicates that OTUB2-IN-1 exerts its antitumor immune effect by degrading PD-L1. In summary, OTUB2-IN-1 exhibits significant antitumor potential by precisely targeting DUBs to regulate tumor immune escape mechanisms.⁴⁴⁸

In the study of OTUD3, computer-aided virtual screening and biological experiments identified two OTUD3 inhibitors, OTUDin3 and Rolapitant, which inhibit the deubiquitinase activity of OTUD3. Molecular docking analysis shows that OTUDin3 forms various interactions with OTUD3, including hydrophobic interactions at different sites, hydrogen bonding, and π–π stacking interactions. In vitro experiments demonstrated that OTUDin3 can inhibit the growth, migration, and invasion of H1299 cell lines and induce cell apoptosis by targeting OTUD3. Rolapitant significantly inhibited the proliferation of NSCLC cell lines such as A549, H1299, and H460. In the in vivo validation experiment, OTUDin3 significantly inhibited tumor growth in a nude mouse model carrying H1299 tumors, with no significant health issues or weight loss observed. Rolapitant showed significant effects in inhibiting tumor growth in nude mouse models carrying A549 tumors, especially when used in combination with targeted drugs. These research findings further confirm the effectiveness of strategies to influence tumor growth and cell death by regulating specific DUBs.^{449, 450}

Additionally, effective inhibitors 7Ai and 7Bi targeting OTUD7A and OTUD7B were discovered using AtomNet virtual screening technology. First, a homologous model of the OTUD7A-OTU domain was generated based on the available crystal structures of closely related OTUD7B-OTU domains. Using the generated structures, 4 million compounds were screened, and based on their ability to reduce the abundance of EWS-FLI1 protein in A673 and SK-N-MC cells, it was found that 7Ai can effectively block OTUD7A-mediated deubiquitination of EWS-FLI1 without affecting the binding of OTUD7A to EWS-FLI1. Preliminary studies have shown that 7Ai treatment can significantly reduce the proliferation and migration ability of A673 cells. Although this study lacks formal in vivo experiments, this discovery provides more possibilities for exploring the combination therapy of 7Ai and active chemotherapy drugs in the future. Similarly, 10 candidate compounds for OTUD7B inhibitors were screened through AtomNet technology and biological experiments. Subsequently, the inhibitory effects of these compounds on Akt pS473 signaling in various NSCLC cell lines were investigated, and OTUD7B deubiquitinase activity was measured in vitro using K11-linked dimeric ubiquitin (di-ub) as a substrate. Finally, the study found that compound 7Bi specifically inhibits OTUD7B and inhibits the growth of various NSCLC cells, including A549, in vitro. The discovery of 7Ai and 7Bi inhibitors indicates that AtomNet has significant potential in discovering different inhibitors of closely related deubiquitinase members.^{451, 452}

Although there is limited research on the role of TRABID in tumors, computer screening of crystal structures based on the A20 OTU domain, searching for compounds that may bind to TRABID catalytic sites, combined with preliminary biological experiments, identified NSC112200 and NSC267309 as TRABID inhibitors with IC₅₀ values of approximately 3 µM. At a concentration of 20 µM, these two compounds not only inhibited the growth of colorectal tumor cell lines HCT-116 and SW480 but also inhibited the growth of two abnormal cell lines, NIH3T3 and HEK293.⁴⁵³ Subsequently, Zhang et al.⁴⁵⁴ conducted a more in-depth study on the mechanism of action of the inhibitor NSC112200. The results showed that NSC112200 could inhibit the deubiquitinase activity of ZRANB1, promote the degradation of EZH2 protein, and effectively kill TNBC cells at micromolar concentrations. However, the inhibitor exhibits certain toxicity in mice, so its structure needs further optimization.⁴⁵⁴ These studies indicate that NSC112200 and NSC267309 have significant antitumor potential as TRABID inhibitors. Further optimizing the structure of these inhibitors not only helps to improve their selectivity and safety but also provides important theoretical and experimental bases for the development of new-generation TRABID inhibitors.

To accelerate the discovery of OTU family inhibitors, an efficient screening platform was established by designing a chemical library based on the chemical types of various DUB inhibitors and combining it with activity-based protein mass spectrometry analysis. Through this platform, the inhibitor of VCPIP1, the fluoroquinolone compound CAS-12290-201, was successfully screened. This compound exhibits high selectivity and strong inhibitory effect on VCPIP1, with an enzymatic IC₅₀ value of 70 nM. Additionally, cell experiments have shown that CAS-12290-201 has low toxicity and good target binding ability. This study demonstrates the feasibility of developing DUB inhibitors through structure-guided methods. Specifically, the successful application of this virtual platform demonstrates that activity-based protein mass spectrometry analysis and well-designed chemical libraries can effectively screen and optimize DUB inhibitors. This method not only improves screening efficiency but also significantly enhances the specificity and efficacy of inhibitors, providing important references and tools for future drugdevelopment.⁴⁵⁵

In addition to developing inhibitors that directly interact with catalytic site domains, a novel strategy for creating heterobifunctional chimeric molecules targeting DUBs has recently been proposed, known as DUBTAC technology. This technology aims to form chimeric molecules that target specific proteins for degradation in a ubiquitin-dependent manner, thereby improving selectivity and intracellular efficiency. For instance, a recently developed covalent small molecule ligand for OTUB1, acrylamide EN523, can specifically target the noncatalytic allosteric Cys C23 on OTUB1 and effectively bind to OTUB1 within cells. This binding method does not inhibit the deubiquitination activity of OTUB1; instead, it interacts with OTUB1 through a novel mechanism, highlighting the potential role and application prospects of DUBTAC technology in the future development of OTU-targeted drugs.⁴⁶⁸ The advancement of this technology provides a new strategy for treating diseases such as cancer and is expected to play a significant role in the field of precision medicine.

6.3 Targeting other DUBs

Significant advancements in the development of DUB inhibitors have expanded the scope beyond the well-studied USP and OTU families, leading to the discovery of potent and selective inhibitors targeting other DUB family members. This section highlights key compounds such as GK13S, a selective inhibitor of UCHL1, VLX1570 targeting USP14 and UCHL5, and the Rpn11 inhibitor capzimin. These inhibitors showcase high potency and specificity, serving as essential tools for biochemical research and potential therapeutic applications.

One notable example is the UCHL1-targeted inhibitor GK13S. This 3-carboxy-N-cyanopyrrolidine probe exhibits an IC₅₀ value of 50 nM, demonstrating high potency. The cocrystal structure reveals that GK13S binds to UCHL1 in a hybrid conformation, partaking in both the apo and ubiquitin-bound states. This inhibitor covalently attaches to the C90 residue via its cyanamide warhead and occupies the enzyme's catalytic cleft. Specificity is conferred by the positioning of the pyrrolidine moiety in a unique pocket exclusive to UCHL1's apo conformation. Additionally, although GK13S primarily targets UCHL1, it also shows binding affinity for PARK7 and its family member C21orf33. Remarkably, GK13S effectively inhibits UCHL1 activity in cellular contexts without compromising the viability of HEK293 and U-87 MG cells.⁴⁵⁶

Furthermore, VLX1570, a dual inhibitor of USP14 and UCHL5, was developed from b-AP15. Exhibiting greater drug-like properties, VLX1570 has enhanced potency and solubility in aqueous solutions.⁴⁵⁷ Specifically, VLX1570 shows superior activity in hydrolysis assays using Ub-AMC as a substrate, yielding an IC₅₀ of 13.0 µM compared with b-AP15's 16.8 µM.⁴⁵⁸ In Waldenstrom macroglobulinemia (WM) cells, targeting USP14 and UCHL5 with VLX1570 induces apoptosis and leads to the accumulation of high molecular weight polyubiquitinated protein conjugates.⁴⁵⁹ Additionally, in vivo studies report a significant reduction in tumor burden and extended survival in WM-xenografted mice following a 21-day VLX1570 treatment regimen. Although VLX1570 is the first DUB inhibitor to enter clinical trials, the study was terminated due to dose-limiting toxicities observed at higher doses.⁴⁶⁰

Another significant discovery is iBAP, a selective inhibitor of BAP1, with an IC₅₀ value of 253 nM. This inhibitor exhibits high specificity for BAP1 over other DUBs. Research shows that leukemia cells harboring truncated ASXL1 gain-of-function mutations, such as K562 cells and THP1 cells, are particularly sensitive to iBAP treatment. RNA-seq analysis indicates that iBAP downregulates leukemia-associated genes including HMGN5, STAT5A, HOXA11, TWIST1, and MBD2. In vivo studies corroborate iBAP's efficacy, showing delayed progression in leukemia and improved survival in mice models bearing ASXL1 mutations.⁴⁶¹

Rpn11 inhibition has also made strides with the identification of 8-thioquinoline (8TQ) and its optimized derivative, capzimin. 8TQ initially exhibited strong Rpn11 inhibition through chelating the Zn²⁺ ion at the enzyme's active site, with an IC₅₀ of 2.5 µM. However, its lack of specificity prompted further optimization, resulting in capzimin, which shows potent inhibition of Rpn11 with an IC₅₀ of 0.34 µM and high selectivity over other JAMMs. Capzimin effectively inhibits cell proliferation and induces apoptosis in various cancer cell lines, highlighting its potential as a therapeutic agent.

Targeting CSN5 with siRNA has been shown to halt cell cycle progression and induce apoptosis in HCC cells, making CSN5 a compelling therapeutic target. Utilizing a specialized high-throughput screening platform, CSN5i-3 emerged as a highly potent inhibitor. CSN5i-3 inhibits CSN-mediated CRL deneddylation with an exceptionally low IC₅₀ of 0.0058 µM and exhibits favorable pharmacokinetic properties. Its selectivity is noteworthy, with minimal inhibition observed for other JAMMs such as AMSH-LP (IC₅₀ > 100 µM) and RPN11 (IC₅₀ = 53 µM). In tests on 500 diverse cancer cell lines, CSN5i-3 showed a range of inhibitory activities and significantly suppressed the growth of human xenografts in mouse models. These findings position CSN5i-3 as a promising candidate for treating cancers driven by CSN5 overexpression.⁴⁶³

Recent progress in developing inhibitors for various DUBs beyond the USP and OTU families underscores significant advancements in this field. Compounds such as GK13S, VLX1570, iBAP, capzimin, and CSN5i-3 demonstrate high potency and specificity, providing valuable tools for biochemical research and potential therapeutic applications. These inhibitors not only enhance our understanding of DUB-related cellular mechanisms but also offer promising prospects for treating cancers and other diseases associated with dysregulated ubiquitination pathways. Continued research in this area is likely to yield even more potent and specific inhibitors with broad therapeutic potential.

Although significant progress has been achieved in developing inhibitors for the entire DUB family, substantial challenges persist in this area of research. A major obstacle is the identification of potent compounds that selectively target the catalytic pocket of DUBs. Furthermore, the dynamic alternation between active and inactive conformations of DUBs complicates the design of predictive biochemical assays and the development of drug-like compounds. Additionally, the ubiquitin transfer mediated by reactive thiols in DUBs presents difficulties in the screening of inhibitors. These challenges have limited the advancement of DUB inhibitors, with only a few making preliminary progress in clinical trials for cancer treatment.

Mitoxantrone, approved by the US FDA as a USP11 inhibitor, is undergoing numerous phase I/II trials targeting conditions including neoplasms, acute myeloid leukemia, MS, and breast cancer.⁴⁶⁹ A notable 2014 study (Clinical Trial ID: NCT02043756) assessed its pharmacokinetics and safety in 20 solid tumor patients, with doses up to 18 mg/m² shown to be effective and well-tolerated. CSPC ZhongQi Pharmaceutical Technology is conducting a phase II trial (Clinical Trial ID: NCT03776279) to evaluate its efficacy in 106 patients with refractory T-cell lymphomas. Additionally, the University of Pittsburgh initiated a study (Clinical Trial ID: NCT03839446) in 2019 to assess Mitoxantrone combined with etoposide and gemtuzumab ozogamicin for acute myeloid leukemia.

VLX1570 demonstrated promising antimyeloma effects at doses of 0.6 mg/kg or higher. However, its development faced setbacks due to severe respiratory complications at elevated doses, leading to the study's discontinuation, with questions remaining about the role of its formulation in these adverse effects.⁴⁶⁰

Pimozide is being evaluated in phase II trials at the University of Calgary for amyotrophic lateral sclerosis (ALS). The first trial (Clinical Trial ID: NCT02463825) focuses on 25 patients with neuromuscular junction dysfunction, while a second placebo-controlled trial (Clinical Trial ID: NCT03272503) involves 100 participants to explore its potential in slowing ALS progression.

To advance DUB inhibitors toward clinical application, future research should pivot toward gaining deeper biological insights. While traditional methods involve screening known libraries and identifying hit compounds, a shift is needed toward confirming target engagement in cellular and in vivo models through biomarker studies, gene knockout phenotypes, and competitive binding assays. Chemical proteomics is indispensable in uncovering novel DUB functions and substrate interactions, capitalizing on the unique adaptability, dose responsiveness, and selectivity of small molecules. Beyond simple validation, attention should be given to exploring new biological roles of DUBs using quantitative proteomics, transcriptomics, and phenotypic analyses in various cell types.