Ubiquitin-specific Protease 7 Inhibitors Research Articles

USP14 inhibition promotes DNA damage repair and represses ovarian granulosa cell senescence in premature ovarian insufficiency

BackgroundPremature ovarian insufficiency (POI) is a condition characterized by a substantial decline or loss of ovarian function in women before the age of 40. However, the pathogenesis of POI remains to be further elucidated, and specific targeted drugs which could delay or reverse ovarian reserve decline are urgently needed. Abnormal DNA damage repair (DDR) and cell senescence in granulosa cells are pathogenic mechanisms of POI. Ubiquitin-specific protease 14 (USP14) is a key enzyme that regulates the deubiquitylation of DDR-related proteins, but whether USP14 participates in the pathogenesis of POI remains unclear.MethodsWe measured USP14 mRNA expression in granulosa cells from biochemical POI (bPOI) patients. In KGN cells, we used IU1 and siRNA-USP14 to specifically inhibit USP14 and constructed a cell line stably overexpressing USP14 to examine its effects on DDR function and cellular senescence in granulosa cells. Next, we explored the therapeutic potential of IU1 in POI mouse models induced by D-galactose.ResultsUSP14 expression in the granulosa cells of bPOI patients was significantly upregulated. In KGN cells, IU1 treatment and siUSP14 transfection decreased etoposide-induced DNA damage levels, promoted DDR function, and inhibited cell senescence. USP14 overexpression increased DNA damage, impaired DDR function, and promoted cell senescence. Moreover, IU1 treatment and siUSP14 transfection increased nonhomologous end joining (NHEJ), upregulated RNF168, Ku70, and DDB1, and increased ubiquitinated DDB1 levels in KGN cells. Conversely, USP14 overexpression had the opposite effects. Intraperitoneal IU1 injection alleviated etoposide-induced DNA damage in granulosa cells, ameliorated the D-galactose-induced POI phenotype, promoted DDR, and inhibited cell senescence in ovarian granulosa cells in vivo.ConclusionsUpregulated USP14 in ovarian granulosa cells may play a role in POI pathogenesis, and targeting USP14 may be a potential POI treatment strategy. Our study provides new insights into the pathogenesis of POI and a novel POI treatment strategy.

Gardenin B, A Natural Inhibitor for USP7: In vitro Evaluation and In silico Identification

Background: Ubiquitin-specific protease 7 (USP7) is one of the most widely studied deubiquitin enzymes (DUBs). The protein level of USP7 is highly expressed in a variety of malignant cancers, which suggests that it is a prognostic marker of cancers and a potential drug target for oncotherapy. Objective: The aim of this study was to identify natural and effective USP7 inhibitors, in order to understand the activation of the USP7/p53 pathway by natural inhibitors. Methods: In this study, USP7 enzyme activity screening assay system and p53 luciferase reporter assay system have been applied for the discovery of natural USP7 inhibitors targeting the catalytic active site. Molecular docking and molecular dynamics (MD) simulation revealed the combined mechanism between USP7 with gardenin B. Results: The gardenin B was screened from our home-lab natural products (160 flavonoids) and had cytotoxicity in HCT116 cells (IC50 = 46.28 ± 2.16 μM). Preliminary in vitro studies disclosed its antiproliferative activity and activated p53 signaling pathway in HCT116 cells. We found that the complex formed by gardenin B and 5vsk (Ledock score = -6.86, MM/GBSA score = -53.35) had the optimal binding conformation. Moreover, the MD simulation showed that the π-π interactions between gardenin B with His461 and Phe409, and the hydrogen bonds interaction between gardenin B with Leu406 played an important role in maintaining the close binding of the complexes. Conclusion: In conclusion, gardenin B could be used as a natural product inhibitor of USP7 for further optimized design and development potential.

Random Forests Regression and Rescoring Strategy for Identifying Inhibitors of Ubiquitin Specific Protease-7

Ubiquitin-specific protease-7 (USP7) is a potential target for cancer therapy. In this work, the evaluation and design of USP7 inhibitors are conducted employing a comprehensive computational approach. Initially, a large database of compounds undergoes preliminary screening via similarity and pharmacophore analyses. Subsequently, a secondary selection process is executed based on both quantitative structure–activity relationship modeling and molecular docking. The final selection is refined through rescoring treatments via consensus scores and MM/GBSA binding free energy. A total of 138 experimental USP7 inhibitors were collected for the construction of random forest regression models using four different feature selection methods. 17 top experimentally active inhibitors were used as models in similarity searching, and eight representative USP7-ligand binding models were used in pharmacophore screening, performed over a database of 14 million compounds. The rescoring approach applied in this study offers an alternative and beneficial method for the ranking and selection of desired molecules. Based on the molecular scaffolds of the highly active inhibitors, some new derivatives have been designed and received high predicted scores for inhibition.

EPEN-07. USP7 IS AN INTERACTION PARTNER OF EZHIP AND POTENTIAL DRUGGABLE TARGET IN PFA EPENDYMOMAS

Abstract BACKGROUND Ependymomas (EPN) arise in the supratentorial brain (ST-EPN), posterior fossa (PF-EPN), or the spinal cord (SP-EPN), in children and adults. Among the three molecular PF-EPN groups, PFAs are characterized by young median age at diagnosis, a balanced genome and poor outcome. Recently, enhancer of zeste inhibiting protein (EZHIP) has been identified as potential driver of PFAs. By inhibiting EZH2, EZHIP prevents the distribution of the epigenetic repressor mark H3K27me3. Without known enzymatic functions though, EZHIP isn´t the urgently needed drug target. We therefore focused on essential and potentially druggable interaction partners. METHODS We investigated protein-protein interactions using co-immunofluorescence, co- immunoprecipitation and proximity-based assays. We utilized CRIPSR/Cas9 and shRNAs-based methods, mutagenesis and PROTAC treatments followed by Western Blot or mass spectrometry for functional analyses. Viability and apoptosis after drug treatments was assessed in vitro by metabolic readouts, synergy was determined using matrix layouts. RESULTS We identified ubiquitin-specific protease 7 (USP7), a known cancer regulator with multiple inhibitors available as direct interaction partner of EZHIP in PFA. We confirmed their interaction in PFA cells and showed that the EZHIP-USP7 interaction is independent of EZH2, a separate interactor of both. Functionally, we show that USP7 de-ubiquitinates EZHIP, preventing its degradation and thus stabilizing it, mediated via the six lysine residues present in EZHIP. We reveal a vulnerability of PFA cells to genetically engineered losses of EZHIP and USP7. Moreover, USP7 inhibitors highly affected the survival and induced apoptosis in different PFA cell lines at a low micromolar IC50 in vitro. Mid-throughput drug library screens additionally identified BET inhibitors as synergistic partners. In vivo treatments with the combination of USP7 and clinically achievable doses of BET inhibitors in PFA PDX models are ongoing. CONCLUSIONS USP7 de-ubiquitinates and thus stabilizes EZHIP, which qualifies it as a candidate therapeutic target in pediatric PFA ependymoma.

Blocking Ubiquitin-Specific Protease 7 Induces Ferroptosis in Gastric Cancer via Targeting Stearoyl-CoA Desaturase.

Gastric cancer (GC) presents a formidable global health challenge, and conventional therapies face efficacy limitations. Ubiquitin-specific protease 7 (USP7) plays pivotal roles in GC development, immune response, and chemo-resistance, making it a promising target. Various USP7 inhibitors have shown selectivity and efficacy in preclinical studies. However, the mechanistic role of USP7 has not been fully elucidated, and currently, no USP7 inhibitors have been approved for clinical use. In this study, DHPO is identified as a potent USP7 inhibitor for GC treatment through in silico screening. DHPO demonstrates significant anti-tumor activity in vitro, inhibiting cell viability and clonogenic ability, and preventing tumor migration and invasion. In vivo studies using orthotopic gastric tumor mouse models validate DHPO's efficacy in suppressing tumor growth and metastasis without significant toxicity. Mechanistically, DHPO inhibition triggers ferroptosis, evidenced by mitochondrial alterations, lipid Reactive Oxygen Species (ROS), Malondialdehyde (MDA) accumulation, and iron overload. Further investigations unveil USP7's regulation of Stearoyl-CoA Desaturase (SCD) through deubiquitination, linking USP7 inhibition to SCD degradation and ferroptosis induction. Overall, this study identifies USP7 as a key player in ferroptosis of GC, elucidates DHPO's inhibitory mechanisms, and highlights its potential for GC treatment by inducing ferroptosis through SCD regulation.

ER Stress-Activated HSF1 Governs Cancer Cell Resistance to USP7 Inhibitor-Based Chemotherapy through the PERK Pathway.

Ubiquitin-specific protease 7 inhibitors (USP7i) are considered a novel class of anticancer drugs. Cancer cells occasionally become insensitive to anticancer drugs, known as chemoresistance, by acquiring multidrug resistance, resulting in poor clinical outcomes in patients with cancer. However, the chemoresistance of cancer cells to USP7i (P22077 and P5091) and mechanisms to overcome it have not yet been investigated. In the present study, we generated human cancer cells with acquired resistance to USP7i-induced cell death. Gene expression profiling showed that heat stress response (HSR)- and unfolded protein response (UPR)-related genes were largely upregulated in USP7i-resistant cancer cells. Biochemical studies showed that USP7i induced the phosphorylation and activation of heat shock transcription factor 1 (HSF1), mediated by the endoplasmic reticulum (ER) stress protein kinase R-like ER kinase (PERK) signaling pathway. Inhibition of HSF1 and PERK significantly sensitized cancer cells to USP7i-induced cytotoxicity. Our study demonstrated that the ER stress-PERK axis is responsible for chemoresistance to USP7i, and inhibiting PERK is a potential strategy for improving the anticancer efficacy of USP7i.

Identification of YCH2823 as a novel USP7 inhibitor for cancer therapy

Inhibition of the human ubiquitin-specific protease 7 (USP7), the key deubiquitylating enzyme in regulating p53 protein levels, has been considered an attractive anticancer strategy. In order to enhance the cellular activity of FT671, scaffold hopping strategy was employed. This endeavor resulted in the discovery of YCH2823, a novel and potent USP7 inhibitor.YCH2823 demonstrated remarkable efficacy in inhibiting the growth of a specific subset of TP53 wild-type, -mutant, and MYCN-amplified cell lines, surpassing the potency of FT671 by approximately 5-fold. The mechanism of action of YCH2823 involves direct interaction with the catalytic domain of USP7, thereby impeding the cleavage of ubiquitinated substrates. An increase in the expression of p53 and p21, accompanied by G1 phase arrest and apoptosis, was observed upon treatment with YCH2823. Subsequently, the knockdown of p53 or p21 in CHP-212 cells exhibited a substantial reduction in sensitivity to YCH2823, as evidenced by a considerable increase in IC50 values up to 690-fold. Furthermore, YCH2823 treatment specifically enhanced the transcriptional and protein levels of BCL6 in sensitive cells. Moreover, a synergistic effect between USP7 inhibitors and mTOR inhibitors was observed, suggesting the possibility of novel therapeutic strategies for cancer treatment. In conclusion, YCH2823 exhibits potential as an anticancer agent for the treatment of both TP53 wild-type and -mutant tumors.

Virtual screening of natural products targeting ubiquitin-specific protease 7

Ubiquitin-specific protease 7 (USP7) is a promising prognostic and druggable target for cancer therapy. Inhibition of USP7 can activate the MDM2-P53 signaling pathway, thereby promoting cancer cell apoptosis. This study based on watvina molecular docking of virtual screening method and biological evaluation found the new USP7 inhibitors targeting catalytic active site. Three hits were screened from 3760 natural products and validated as USP7 inhibitors by enzymatic and kinetic assays. The IC50 values of scutellarein (Scu), semethylzeylastera (DML) and salvianolic acid C (SAC) were 3.017, 6.865 and 8.495 μM, respectively. Further, we reported that the hits could downregulate MDM2 and activate p53 signal pathway in HCT116 cells. Molecular dynamics simulation was used to investigate the binding mechanism of USP7 to Scu, the compound with the best performance, which formed stable contact with Val296, Gln297, Phe409, Tyr465 and Tyr514. These interactions are essential for maintaining the biological activity of Scu. Three natural products are suitable as lead compounds for the development of novel USP7 inhibitors, especially anti-colon cancer drugs. Communicated by Ramaswamy H. Sarma

Abstract PR04: USP7 inhibitors prevent triple negative breast cancer metastasis by inducing UHRF1 protein degradation

Abstract Functional inactivation of RB1 occurs frequently in triple negative breast cancer (TNBC), the most aggressive breast cancer subtype with poor clinical prognosis when tumors metastasize. Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1) is a key epigenetic regulator that is controlled by the RB-E2F pathway and is frequently overexpressed in many types of cancers, including TNBC. Using TCGA and METABRIC databases, we found that UHRF1 overexpression is negatively associated with breast cancer patient survival, including TNBC. Genetic ablation of UHRF1 using CRISPR/Cas9 in TNBC cell lines resulted in a robust decrease in tumorigenicity, including decreased cell clonogenicity (soft agar colony assay), migration (scratch wound healing assay), and invasion (Transwell assay) in vitro and decreased tumor growth and metastasis using orthotopic xenograft in vivo models. Despite strong evidence showing UHRF1 as a promising therapeutic target in several cancers, there is a current lack of UHRF1-specific inhibitors. To overcome this limitation, we explored targeting Ubiquitin-Specific Protease 7 (USP7), a deubiquitinase that prevents UHRF1 degradation, as a therapeutic strategy to trigger UHRF1 degradation. Here, we demonstrate that genetic inactivation or pharmacological inhibition of USP7 decreases TNBC tumor growth and metastasis in a UHRF1-degradation dependent manner. Phenocopying the effects of knocking out UHRF1, genetic ablation of USP7 using CRISPR/Cas9 in TNBC cell lines also resulted in significant decreases in cell clonogenicity, migration, and invasion in vitro. Intriguingly, mice bearing USP7 knockout tumors resulted in a far more robust decrease in tumor growth and absolute absence of metastatic disease. We established that many of the effects driven by USP7 ablation were mediated by the targeted degradation of UHRF1 by comparing vector control cell lines with UHRF1 knockout cells treated with the highly selective USP7 inhibitors, XL177A or FT671. In the absence of UHRF1, USP7 inhibitors showed no effects in cell migration and invasion in the three TNBC cell lines used in this study. Of promise, NSG mice bearing MDA-MB-231 tumors were recruited into vehicle or 50 mg/kg FT671 treatment groups and dosed daily via oral gavage for 20 days. For mice treated with FT671, we observed a significant decrease in tumor growth, with decreased UHRF1 levels in tumors, with minimal systemic toxicities. This study suggests that USP7 is a target with potential clinical relevance in suppressing tumor growth and metastasis in TNBC, and possibly other TP53-null cancers. These are promising results that present a novel opportunity for future clinical interventions for TNBC patients. Citation Format: Ahhyun Kim, Claudia Benavente. USP7 inhibitors prevent triple negative breast cancer metastasis by inducing UHRF1 protein degradation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr PR04.

USP7 Promotes TGF-β1 Signaling by De-Ubiquitinating Smad2/Smad3 in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a long-term, progressive, and irreversible pulmonary interstitial disease. The activation of Smad family member 2 (Smad2) and Smad3 transcription factors by transforming growth factor β-1 (TGF-β1) is a critical event in the pathogenesis of IPF. However, there is still a lack of understanding regarding the molecular mechanisms governing Smad2 and Smad3 proteins. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a vital role in regulating protein stability within cells. However, its regulation of the TGF-β signaling pathway and its significance in IPF remain undiscovered. This study aims to clarify the function of USP7 in the TGF-β signaling pathway, while simultaneously exploring the specific molecular mechanisms involved. Additionally, this study seeks to evaluate the therapeutic potential of targeted USP7 inhibitors in IPF, thereby providing novel insights for the diagnosis and management of IPF. We first detected the expression of USP7 in lung tissues of mice with Bleomycin (BLM)-induced pulmonary fibrosis and in Beas-2B cells treated with or without TGF-β1 through Western blot analysis. Subsequently, we explored the influence of USP7 on fibrotic processes and the TGF-β1 signaling pathway, utilizing in vitro and in vivo studies. Finally, we assessed the effectiveness of USP7-specific inhibitors in an IPF murine model. In the present study, USP7 was found to de-ubiquitinate Smad2 and Smad3, consequently increasing their stability and promoting the TGF-β1-induced production of profibrotic proteins including α-smooth muscle actin (α-SMA) and fibronectin 1 (FN-1). Inhibition or knockdown of USP7 resulted in decreased levels of Smad2 and Smad3 proteins, leading to reduced expression of FN-1, Collagen Type I Alpha 1 Chain (Col1A1), and α-SMA induced by TGF-β1 in human pulmonary epithelial cells. These findings demonstrate that overexpression of USP7 reduces Smad2/3 ubiquitination, whereas inhibition or knockdown of USP7 enhances their ubiquitination. USP7 is abundantly expressed in IPF lungs. The expressions of USP7, Smad2, and Smad3 were upregulated in bleomycin-induced lung injury. The USP7 inhibitor P22077 reduced the expression of FN-1 and type I collagen as well as Smad2/3 and collagen deposition in lung tissue in a model of pulmonary fibrosis induced by bleomycin. This study demonstrates that USP7 promotes TGF-β1 signaling by stabilizing Smad2 and Smad3. The contribution of USP7 to the progression of IPF indicates it may be a viable treatment target.

USP7 Inhibition Suppresses Neuroblastoma Growth via Induction of p53-Mediated Apoptosis and EZH2 and N-Myc Downregulation.

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.

Abstract 6157: USP7 is a promising thersapeutic target for neuroblastoma

Abstract Background: The ubiquitin-proteasome system (UPS) plays an essential role in post-translational modification of proteins to maintain proteostasis. Ubiquitination of target protein is coordinated by different enzymes, which add and link ubiquitin to each other in specific ways that determine the fate of the protein for to be degraded or to be included into protein complex to induce signaling pathways. Ubiquitination is further regulated by deubiquitinases, which remove ubiquitin and rescue target protein from being degraded or diverted from it signaling role. UPS dysregulation has been linked to many human diseases, including cancer. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a critical role in immune response, tumor suppression and DNA repair. USP7 overexpression has been associated with tumor aggressiveness in a variety of tumors, including in neuroblastoma (NB). Therefore, USP7 is a potential therapeutic target for NB. Some new specific USP7 inhibitors are highly potent and selective and have demonstrated significant antitumor activity in preclinical models of adult cancer. We hypothesize that inhibition of USP7 alone or in combination with epigenetic drugs like HDAC inhibitors will be more effective against NB tumor growth. To evaluate the efficacy of USP7 inhibition with specific USP7 inhibitors alone or with combination with epigenetic drugs like HDAC inhibitors (Saha) or chemotherapy against NB tumor growth, NB cell lines were treated with increasing concentrations of USP7 inhibitors alone or in combination with chemotherapy or with HDAC inhibitors. Cell proliferation was measured using continuous live cell imaging and cell viability using AlamarBlue assay. Apoptosis was measured by caspase cleavage using Caspase 3/7 apoptosis assay as well as by PARP cleavage by Western Blotting (WB). Changes in various proteins were measured by WB and co-immunoprecipitation were performed to analyze the ubiquitin status of specific protein targets regulated by USP7. Pharmacologic inhibition of USP7 resulted in significant decreases in both cell proliferation and cell viability in vitro only in p53 wild-type NB cell lines. There was no correlation seen between MYCN amplification status and treatment response. USP7 inhibition induced apoptosis as well as necroptosis with dose-dependent increase. Treatment with USP7 inhibitors led to decrease p53 ubiquitination leading increased of p53 protein expression in all sensitive cell lines tested and decreased MYCN protein level in MYCN-amplified cell lines. EZH2, which is a predominant target of USP7 is significantly downregulated with increasing dose of USP7 inhibitors. In addition, the combination of USP7 inhibitors with chemotherapy commonly used in NB or with combination with HDAC inhibitors showed enhanced efficacy. Our data suggests that USP7 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed neuroblastoma. Citation Format: Christophe Le Clorennec, Karen Lee, Peter Zage. USP7 is a promising thersapeutic target for neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6157.

Development of a robust HTRF assay with USP7 full length protein expressed in E. coli prokaryotic system for the identification of USP7 inhibitors

Deubiquitinating enzyme ubiquitin-specific protease 7 (USP7) is a promising therapeutic target. Several USP7 inhibitors accommodated in the catalytic triad of USP7 have been reported with the aid of high-throughput screening (HTS) methods using USP7 catalytic domain truncation. However, the drawbacks of previously reported biochemical cleavage assays, including poor stability, fluorescence interference, time-consuming, expensive, more importantly the selectivity issue, have challenged the USP7-targeted drug discovery. In this work, we demonstrated the functional heterogeneity and essential role of different structural elements in the USP7 full activation, highlighting the necessity of USP7 full length in drug discovery. Apart from reported two pockets in the catalytic triad, five additional ligandable pockets were predicted based on the proposed USP7 full length models by AlphaFold and homology modelling. A reliable homogeneous time-resolved fluorescence (HTRF) HTS method was established based on the cleavage mechanism of USP7 towards the ubiquitin precursor UBA10. The USP7 full length protein was successfully expressed in the relatively cost-effective E. coli prokaryotic system and used to simulate the auto-activated USP7 in nature. Via screening our in-house library (∼ 1500 compounds), 19 hit compounds with >20% of inhibition rate were identified for further optimization. This assay will enrich the toolbox for the identification of highly potent and selective USP7 inhibitors for clinical use.

Engineered Microparticles for Treatment of Murine Brain Metastasis by Reprograming Tumor Microenvironment and Inhibiting MAPK Pathway.

Brain metastases (BRM) are common in advanced lung cancer. However, their treatment is challenging due to the blood-brain barrier (BBB) and the immunosuppressive tumor microenvironment (ITME). Microparticles (MPs), a type of extracellular vesicle, can serve as biocompatible drug delivery vehicles that can be further modulated with genetic engineering techniques. MPs prepared from cells induced with different insults are compared and it is found that radiation-treated cell-released microparticles (RMPs) achieve optimal targeting and macrophage activation. The enzyme ubiquitin-specific protease 7 (USP7), which simultaneously regulates tumor growth and reprograms M2 macrophages (M2Φ), is found to be expressed in BRM. Engineered RMPs are then constructed that comprise: 1) the RMP carrier that targets and reprograms M2Φ; 2) a genetically expressed SR-B1-targeting peptide for improved BBB permeability; and 3) a USP7 inhibitor to kill tumor cells and reprogram M2Φ. These RMPs successfully cross the BBB and target M2Φ in vitro and in vivo in mice, effectively reprogramming M2Φ and improving survival in a murine BRM model. Therapeutic effects are further augmented when combined with immune checkpoint blockade. This study provides proof-of-concept for the use of genetically engineered MPs for the treatment of BRM.

USP7 Inhibitors in Cancer Immunotherapy: Current Status and Perspective.

Ubiquitin-specific protease 7 (USP7) regulates the stability of a plethora of intracellular proteins involved in the suppression of anti-tumor immune responses and its overexpression is associated with poor survival in many cancers. USP7 impairs the balance of the p53/MDM2 axis resulting in the proteasomal degradation of the p53 tumor suppressor, a process that can be reversed by small-molecule inhibitors of USP7. USP7 was shown to regulate the anti-tumor immune responses in several cases. Its inhibition impedes the function of regulatory T cells, promotes polarization of tumor-associated macrophages, and reduces programmed death-ligand 1 (PD-L1) expression in tumor cells. The efficacy of small-molecule USP7 inhibitors was demonstrated in vivo. The synergistic effect of combining USP7 inhibition with cancer immunotherapy is a promising therapeutic approach, though its clinical efficacy is yet to be proven. In this review, we focus on the recent developments in understanding the intrinsic role of USP7, its interplay with other molecular pathways, and the therapeutic potential of targeting USP7 functions.

Administration of USP7 inhibitor P22077 inhibited cardiac hypertrophy and remodeling in Ang II-induced hypertensive mice

Hypertension is one of the common causes of pathological cardiac hypertrophy and a major risk for morbidity and mortality of cardiovascular diseases worldwide. Ubiquitin-Specific Protease 7 (USP7), the first identified deubiquitinating enzymes, participated in a variety of biological processes, such as cell proliferation, DNA damage response, tumourigenesis, and apoptosis. However, its role and mechanism in cardiac remodeling remain unclear. Here, our data indicated that USP7 expression was increased during Ang II-induced cardiac hypertrophy and remodeling in mice and humans with heart failure, while the administration of its inhibitor p22077 attenuated cardiac hypertrophy, cardiac fibrosis, inflammation, and oxidase stress. Mechanistically, the administration of p22077 inhibited the multiple signaling pathways, including AKT/ERK, TGF-β/SMAD2/Collagen I/Collagen III, NF-κB/NLRP3, and NAPDH oxidases (NOX2 and NOX4). Taken together, these findings demonstrate that USP7 may be a new therapeutic target for hypertrophic remodeling and HF.

USP7 Induces Chemoresistance in Triple-Negative Breast Cancer via Deubiquitination and Stabilization of ABCB1.

Triple-negative breast cancer (TNBC) accounts for 15–20% of all breast cancer. TNBC does not express the estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2. Cytotoxic chemotherapy and surgery are the current therapeutic strategies for TNBC patients, but the chemoresistance of TNBC limits the efficiency of this strategy and shortens the lifespan of patients. The exploration of targeted therapy is ongoing in TNBC research. The aim of the present study was to identify the mechanism underlying acquired resistance in TNBC through the exploration of the relationship between the expression of USP7 and of ABCB1. We found that ubiquitin specific protease 7 (USP7) is a potential therapeutic target for overcoming the chemoresistance of TNBC. USP7 overexpression increased the chemoresistance of TNBC, while the knockdown of USP7 effectively increased the chemosensitivity of chemoresistant TNBC. A USP7 inhibitor effectively induced apoptosis and suppressed metastasis in chemoresistant TNBC. We further clarified that USP7 is a specific deubiquitinating enzyme for ABCB1 that plays an essential role in drug resistance. USP7 directly interacted with ABCB1 and regulated its stability. We concluded that USP7 promotes the chemoresistance of TNBC by stabilizing the ABCB1 protein.

Highlights in USP7 inhibitors for cancer treatment.

Ubiquitin-specific protease 7 (USP7) is a member of one of the most largely studied families of deubiquitylating enzymes. It plays a key role modulating the levels of multiple proteins, including tumor suppressors, transcription factors, epigenetic modulators, DNA repair proteins, and regulators of the immune response. The abnormal expression of USP7 is found in various malignant tumors and a high expression signature generally indicates poor tumor prognosis. This suggests USP7 as a promising prognostic and druggable target for cancer therapy. Nonetheless, no approved drugs targeting USP7 have already entered clinical trials. Therefore, the development of potent and selective USP7 inhibitors still requires intensive research and development efforts before the pre-clinical benefits translate into the clinic. This mini review systematically summarizes the role of USP7 as a drug target for cancer therapeutics, as well as the scaffolds, activities, and binding modes of some of the most representative small molecule USP7 inhibitors reported in the scientific literature. To wind up, development challenges and potential combination therapies using USP7 inhibitors for less tractable tumors are also disclosed.

Recent insights into USP7: Construct, pathophysiology, and inhibitors

The ubiquitin-proteasome pathway (UPP) is essential for proteostasis and cellular homeostasis. Most of the human proteins are degraded through the UPP in which proteins should be tagged with a specific polyubiquitin chain in a sequential cascade of E1 ubiquitin (Ub)-activating enzymes, namely, E2 Ub-conjugating enzymes and E3 Ub ligases. Meanwhile, the ubiquitination process can be reversed by deubiquitinating enzymes (DUBs), which protect the target proteins from ubiquitination, and so far, around 100 DUBs have been reported to present in human cells. Ubiquitin-specific protease 7 (USP7) is a member of the DUBs family, which has been reported to play crucial role in the development of human tumors and diseases; however, the molecular mechanisms of disease and malignant tumor progression mediated by USP7 has not been fully elucidated. In addition, the therapeutic potential of USP7 in cancer treatment remains to be further explored. Therefore, this review begins with a review of the structure and function of USP7, and then focuses on the development of USP7 inhibitors and their potential applications in various human diseases.

Characterizing (un)binding mechanism of USP7 inhibitors to unravel the cause of enhanced binding potencies at allosteric checkpoint.

The ability to predict the intricate mechanistic behavior of ligands and associated structural determinants during protein-ligand (un)binding is of great practical importance in drug discovery. Ubiquitin specific protease-7 (USP7) is a newly emerging attractive cancer therapeutic target with bound allosteric inhibitors. However, none of the inhibitors have reached clinical trials, allowing opportunities to examine every aspect of allosteric modulation. The crystallographic insights reveal that these inhibitors have common properties such as chemical scaffolds, binding site and interaction fingerprinting. However, they still possess a broader range of binding potencies, ranging from 22 nM to 1,300 nM. Hence, it becomes more critical to decipher the structural determinants guiding the enhanced binding potency of the inhibitors. In this regard, we elucidated the atomic-level insights from both interacting partners, that is, protein-ligand perspective, and established the structure-activity link between USP7 inhibitors by using classical and advanced molecular dynamics simulations combined with linear interaction energy and molecular mechanics-Poisson Boltzmann surface area. We revealed the inhibitor potency differences by examining the contributions of chemical moieties and USP7 residues, the involvement of water-mediated interactions, and the thermodynamic landscape alterations. Additionally, the dissociation profiles aided in the establishment of a correlation between experimental potencies and structural determinants. Our study demonstrates the critical role of blocking loop 1 in allosteric inhibition and enhanced binding affinity. Comprehensively, our findings provide a constructive expansion of experimental outcomes and show the basis for varying binding potency using in-silico approaches. We expect this atomistic approach to be useful for effective drug design.