Ubiquitin-specific Protease 7 Inhibitors Research Articles

USP7 inhibition inhibits proliferation and induces megakaryocytic differentiation in MDS cells by upregulating gelsolin.

Myelodysplastic syndrome (MDS), a largely incurable hematological malignancy, is driven by complex genetic and epigenetic alterations from an aberrant clone of hematopoietic stem/progenitor cells (HSPCs). Ubiquitin-specific protease 7 (USP7) has been demonstrated to have an important oncogenic role in the development of several cancer types, but its role in MDS is unknown. Here, we demonstrate that USP7 expression is elevated in MDS cell lines and patient samples. The USP7-selective small-molecule inhibitors P5091 and P22077 inhibited cell proliferation and induced megakaryocytic differentiation in both cell lines and primary cells. Furthermore, pharmacological inhibition of USP7 markedly suppressed the growth of MDS cell lines in xenograft mouse models. To explore the mechanisms underlying the observed phenotypic changes, we employed RNA-seq to compare the differences in genes after USP7 inhibitor treatment and found that gelsolin (GSN) expression was increased significantly after USP7 inhibitor treatment. Furthermore, knockdown of GSN attenuated the proliferation inhibition, apoptosis induction and megakaryocyte differentiation induced by USP7 inhibitors in MDS cells. Collectively, our findings identify previously unknown roles of USP7 and suggest that the USP7/GSN axis may be a potential therapeutic target in MDS.

USP7 deubiquitinates and stabilizes EZH2 in prostate cancer cells.

Regulation of target proteins by the ubiquitin-proteasome system (UPS) is common in a wide range of cellular events, including transcriptional regulation, cell cycle progression, differentiation, and tumorigenesis. Ubiquitin-specific protease 7 (USP7) has been implicated in tumor development and metastasis in various malignancies through the regulation of target protein stability. In this study, we found that the enhancer of zeste homolog 2 (EZH2), which catalyzes the methylation at lysine 27 of histone H3, is a target of USP7 and is stabilized by USP7-mediated deubiquitination. In prostate cancer cells, the transcriptional repression function of EZH2 was inhibited by USP7-knockdown. Furthermore, ectopic introduction of EZH2 restored the cell migration, invasion, and sphere-forming potential of prostate cancer cells, which had been decreased by USP7-knockdown. Moreover, combined treatment with the USP7-specific inhibitor P5091 and EZH2 inhibitors, such as GSK126, EPZ6438, and DZNep, induced synergistic inhibitory effects on cell migration, invasion, and sphere-forming potential in prostate cancer cells. Collectively, our findings revealed that the promotion of the malignancy-associated characteristics of prostate cancer cells by USP7 was in part due to EZH2 stabilization. Thus, we suggest that simultaneous treatment with a USP7 inhibitor and an EZH2 inhibitor could be a rational strategy for treating EZH2-dependent cancers.

Binding affinity and dissociation pathway predictions for a series of USP7 inhibitors with pyrimidinone scaffold by multiple computational methods

Ubiquitin specific protease 7 (USP7) has attracted increasing attention because of its multifaceted roles in different tumor types. The crystal structures of USP7-inhibitor complexes resolved recently provide reliable models for computational structure-based drug design (SBDD) towards USP7. How to accurately estimate USP7-ligand binding affinity is quite critical to guarantee the reliability of SBDD. In this study, we assessed the reliability of multiple computational methods to the binding affinity prediction for a series of USP7 inhibitors with the pyrimidinone scaffold, including molecular docking scoring, MM/PB(GB)SA, and umbrella sampling (US). It was found that the accuracy of the evaluated computational methods for binding affinity prediction follows the order: US-based method > MM/PB(GB)SA > Glide XP scoring. The calculation results demonstrate that incorporating protein flexibility through induced-fit docking or ensemble docking cannot improve the performance of the Glide scoring based on rigid-receptor docking. For the MM/PB(GB)SA methods, the choice of the protein structure and the calculation procedure has a marked impact on the predictions. More importantly, we discovered for the first time that there are significant differences in the dissociation pathways of strong-binding inhibitors and weak-binding inhibitors of USP7, which may be used as a new criterion to judge whether an inhibitor is a strong binder or not. It is expected that our work can provide valuable guidance on the design and discovery of potent USP7 inhibitors.

Small-molecule inhibitors of ubiquitin-specific protease 7 enhance type-I interferon antiviral efficacy by destabilizing SOCS1.

Type-I interferons (IFN-I) are used as common antiviral drugs for a range of viral diseases in clinic. However, the antiviral efficacy of IFN-I is largely restricted by negative regulators of IFN-I signaling in cells. Therefore, identification of intracellular inhibitors of IFN-I signaling is important for developing novel targets to improve IFN-I antiviral therapy. In this study, we report that the deubiquitinase ubiquitin-specific protease 7 (USP7) negatively regulates IFN-I-mediated antiviral activity. USP7 physically interacts with suppressor of cytokine signaling 1 (SOCS1) and enhances SOCS1 protein stability by deubiquitination effects, which in turn restricts IFN-I-induced activation of Janus kinase-signal transducer and activator of transcription 1 signaling. Interestingly, viral infection up-regulates USP7 and therefore facilitates viral immune evasion. Importantly, the USP7 small-molecule inhibitors P5091 and P22077 inhibit SOCS1 expression and enhance IFN-I antiviral efficacy. Our findings identify a novel regulator of IFN-I antiviral activity and reveal that USP7 inhibitors could be potential enhancement agents for improving IFN-I antiviral therapy.

USP14 Inhibition Regulates Tumorigenesis by Inducing Autophagy in Lung Cancer In Vitro.

The ubiquitin–proteasome system is an essential regulator of several cellular pathways involving oncogenes. Deubiquitination negatively regulates target proteins or substrates linked to both hereditary and sporadic forms of cancer. The deubiquitinating enzyme ubiquitin-specific protease 14 (USP14) is associated with proteasomes where it trims the ubiquitin chain on the substrate. Here, we found that USP14 is highly expressed in patients with lung cancer. We also demonstrated that USP14 inhibitors (IU1-47 and siRNA-USP14) significantly decreased cell proliferation, migration, and invasion in lung cancer. Remarkably, we found that USP14 negatively regulates lung tumorigenesis not only through apoptosis but also through the autophagy pathway. Our findings suggest that USP14 plays a crucial role in lung tumorigenesis and that USP14 inhibitors are potent drugs in lung cancer treatment.

Inhibition of ubiquitin‑specific protease14 promotes connexin32 internalization and counteracts cisplatin cytotoxicity in human ovarian cancer cells.

Although cisplatin is one of the most accepted therapies for ovarian cancer, recurrence and drug resistance remain problematic. Both the ubiquitin‑proteasome system (UPS) and connexin(Cx) are closely related to tumor progression. However, the role of ubiquitin‑specific protease14 (USP14) and Cx in mediating drug resistance remains unclear. In the present study, we aimed to determine whether USP14 is involved in cisplatin resistance and modulates the internalization of connexin32 (Cx32) in ovarian cancer. The results of the deubiquitinase (DUB) trap assay and western blot analysis revealed that the expression and activity levels of USP14 were downregulated in A2780 cisplatin‑resistant cells (A2780‑CDDP) relative to these levels in A2780 cisplatin‑sensitive cells (A2780). CCK‑8 assay results showed that inhibition of USP14 by a specific inhibitor or siRNA decreased cisplatin cytotoxicity in A2780 cells. Additionally, USP14 inhibition increased the expression of Cx32 without changing its mRNA and ubiquitination levels, as showed by Real‑time qPCR and immunoprecipitation assay respectively. Cisplatin resistance induced by USP14 inhibition was counteracted by Cx32 knockdown. Moreover, USP14 inhibition contributed to Cx32 internalization, as determined by western blot analysis and a reduction in gap junction intercellular communication(GJIC), as showed by parachute dye‑coupling assay. Collectively, these data suggest that Cx32 internalization by USP14 inhibition modulates the cisplatin resistance in ovarian cancer cells, thus serving as a potential drug target to challenge chemotherapy failure. In addition, USP14 can also be used as a marker to monitor the development of cisplatin resistance in ovarian cancer treatment.

Inhibition of Ubiquitin-Specific Protease 14 Suppresses Cell Proliferation and Synergizes with Chemotherapeutic Agents in Neuroblastoma.

Neuroblastoma is the most common extracranial malignant solid tumor in children, and drug resistance is a major reason for poor outcomes. Elevated proteasome activity plays an important role in neuroblastoma tumor development and resistance to conventional chemotherapy. Ubiquitin-specific protease 14 (USP14), one of three deubiquitinases associated with the regulatory subunit of the proteasome, is emerging as a potential therapeutic target in multiple tumor types. However, the role of USP14 in neuroblastoma is yet to be elucidated. We found that USP14 inhibition in neuroblastoma via knockdown or a specific inhibitor such as b-AP15 suppressed cell proliferation by inducing cell apoptosis. Furthermore, b-AP15 significantly inhibited neuroblastoma tumor growth in NGP and SH-SY5Y xenograft mouse models. For combination treatment, b-AP15 plus conventional chemotherapeutic agents such as doxorubicin or VP-16 resulted in synergistic antitumor effects on neuroblastoma. Our study demonstrates that USP14 is required for cell viability and is a novel therapeutic target in neuroblastoma. Moreover, USP14 inhibition may add value in combination therapy due to its powerful synergistic effects in treating neuroblastoma.

USP7: Novel Drug Target in Cancer Therapy.

Ubiquitin specific protease 7 (USP7) is one of the deubiquitinating enzymes (DUB) that erases ubiquitin and protects substrate protein from degradation. Full activity of USP7 requires the C-terminal Ub-like domains fold back onto the catalytic domain, allowing the remodeling of the active site to a catalytically competent state by the C-terminal peptide. Until now, numerous proteins have been identified as substrates of USP7, which play a key role in cell cycle, DNA repair, chromatin remodeling, and epigenetic regulation. Aberrant activation or overexpression of USP7 may promote oncogenesis and viral disease, making it a target for therapeutic intervention. Currently, several synthetic small molecules have been identified as inhibitors of USP7, and applied in the treatment of diverse diseases. Hence, USP7 may be a promising therapeutic target for the treatment of cancer.

Ubiquitin-specific protease 7 (USP7) is essential for endometrial stromal cell decidualization in mice.

Ubiquitin-specific protease 7 (USP7), a member of the deubiquitinating (DUB) enzyme family, regulates protein stability and has a well-characterized function in tumorigenesis. Given its critical role in growth and development, it was speculated to be involved in modulating processes in the female reproductive system but its exact role has not been elucidated. Decidualization is one of the key processes in pregnancy and aberrant decidualization is a cause of pregnancy failure. The uterine endometrium layer undergoes significant structural and functional changes during decidualization in preparation for and after embryo implantation. Here, we hypothesized that USP7 could be involved in mediating endometrial stromal cell (ESC) decidualization and set out to determine its function with a primary stromal cell culture. Using in situ hybridization and immunohistochemical techniques, we observed increased USP7 expression during uterine decidualization and found that it was predominantly localized to the decidual zone in the post-implantation uterus. Since the ovarian hormones, progesterone (P4) and estrogen (E2), function in promoting stroma decidualization, we investigated their relationship with USP7 expression and found that they exert minimal influence. Moreover, increased USP7 expression observed during deciduoma development was found to be independent of blastocyst attachment. Using a specific USP7 inhibitor, HBX19818, we demonstrated an additional novel role for USP7 in endometrial stroma decidualization in mice during early pregnancy. Our findings could potentially be applied towards future research and development in female infertility.

USP7 deubiquitinates and stabilizes NOTCH1 in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive leukemia that is primarily caused by aberrant activation of the NOTCH1 signaling pathway. Recent studies have revealed that posttranslational modifications, such as ubiquitination, regulate NOTCH1 stability, activity, and localization. However, the specific deubiquitinase that affects NOTCH1 protein stability remains unestablished. Here, we report that ubiquitin-specific protease 7 (USP7) can stabilize NOTCH1. USP7 deubiquitinated NOTCH1 in vivo and in vitro, whereas knockdown of USP7 increased the ubiquitination of NOTCH1. USP7 interacted with NOTCH1 protein in T-ALL cells, and the MATH and UBL domains of USP7 were responsible for this interaction. Depletion of USP7 significantly suppressed the proliferation of T-ALL cells in vitro and in vivo, accompanied by downregulation of the NOTCH1 protein level. Similarly, pharmacologic inhibition of USP7 led to apoptosis of T-ALL cells. More importantly, we found that USP7 was significantly upregulated in human T-ALL cell lines and patient samples, and a USP7 inhibitor exhibited cell cytotoxicity toward primary T-ALL cells, indicating the clinical relevance of these findings. Overall, our results demonstrate that USP7 is a novel deubiquitinase that stabilizes NOTCH1. Therefore, USP7 may be a promising therapeutic target in the currently incurable T-ALL.

Abstract SY23-03: Development and mechanistic characterization of USP7 deubiquitinase inhibitors

Abstract The ubiquitin system regulates the majority of cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains, and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates, including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumor suppressor and other proteins critical for tumor cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance (NMR)-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumor cell death and enhance cytotoxicity with chemotherapeutics and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 noncovalently target USP7 12Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate H-bond interactions with the ubiquitin Lys-48 side-chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties having free Lys-48 side-chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding via NMR, a study that substantiated our hypothesis. This preferential binding significantly protracted the depolymerization kinetics of Lys-48-linked ubiquitin chains relative to Lys-63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity. [LK, PDL, and LR contributed equally to this work.] Citation Format: Ingrid Wertz, Lorna Kategaya, Paola Di Lello, Lionel Rouge, Richard Pastor, Kevin R. Clark, Jason Drummond, Tracy Kleinheinz, Eva Lin, John-Paul Upton, Sumit Prakash, Johanna Heideker, Mark McCleland, Maria Stella Ritorto, Dario R. Alessi, Matthias Trost, Travis W. Bainbridge, Michael C. Kwok, Taylur P. Ma, Zachary Stiffler, Bradley Brasher, Yinyan Tang, Priya Jaishanker, Brian Hearn, Adam R. Renslo, Michelle R. Arkin, Frederick Cohen, Kebing Yu, Frank Peale, Florian Gnad, Matthew T. Chang, Christiaan Klijn, Elizabeth Blackwood, Scott E. Martin, William F. Forrest, James A. Ernst, Chudi Ndubaku, Xiaojing Wang, Maureen H. Beresini, Vickie Tsui, Carsten Schwerdtfeger, Robert A. Blake, Jeremy Murray, Till Maurer. Development and mechanistic characterization of USP7 deubiquitinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY23-03.

Growth arrest and apoptosis induction in androgen receptor-positive human breast cancer cells by inhibition of USP14-mediated androgen receptor deubiquitination

It has been well known that androgen receptor (AR) is critical to prostate cancer development and progression. It has also been documented that AR is expressed in more than 60% of breast tumors, which promotes the growth of estrogen receptor-negative (ER–)/AR-positive (AR+) breast cancer cells. Thus, AR might be a potential therapeutic target for AR-positive/ER-negative breast cancer patients. Previously we reported that in prostate cancer cells proteasome-associated deubiquitinase ubiquitin-specific protease 14 (USP14) stabilized AR protein level by removing its ubiquitin chain. In the current study, we studied the USP14-AR protein interaction and cell proliferation status after USP14 reduction or inhibition in breast cancer cells, and our results support the conclusion that targeting USP14 is a novel strategy for treating AR-responsive breast cancer. We found that inhibition of USP14 accelerated the K48-ubiquitination and proteasome-mediated degradation of AR protein. Additionally, both genetic and pharmacological inhibition of USP14 significantly suppressed cell proliferation in AR-responsive breast cancer cells by blocking G0/G1 to S phase transition and inducing apoptosis. Moreover, AR overexpression inhibited USP14 inhibition-induced events, suggesting that AR deubiquitination by USP14 is critical for breast cancer growth and USP14 inhibition is a possible strategy to treat AR-positive breast cancer.

USP7: Target Validation and Drug Discovery for Cancer Therapy.

USP7 (ubiquitin specific protease 7, also known as HAUSP) is one of the deubiquitinating enzymes (DUB) that reverses ubiquitination and spares substrate proteins from degradation. After a brief introduction of ubiquitin-proteasome system (UPS) and human DUB, this review focuses on the structural and functional complexity of USP7 in tumor development and progression. Afterwards, physiological regulatory mechanisms and manipulation strategies for USP7 are elaborated. Finally, we discuss the advances and difficulties of USP7 as a novel therapeutic target for cancer. It is mostly concerned that USP7 regulates the dynamics of the p53 and Mdm2 network by deubiquitinating both p53 and its E3 ubiquitin ligase, Mdm2. Recently, USP7 has also been recognized as a regulator of many other tumor associated proteins such as FOXO, PTEN and Claspin, consequently being involved in cell cycle control, DNA damage response, apoptosis and many other cellular processes. Consistently, aberrant USP7 expression and activity have been connected to various types of cancers, which along with lots of validating genetic and functional experiments make this enzyme a compelling target for the treatment of cancer. Currently disclosed inhibitor discovery programs and relevant research have identified several synthetic small molecules, natural compounds, small peptides and one ubiquitin variant that have specific USP7 inhibitory effects and considerable antitumor activities. Taken together, USP7 is a promising therapeutic target and USP7 inhibitors hold promise as a new approach to cancer therapy.

Abstract B193: Characterization of selective active-site targeted covalent inhibitors of usp7

Abstract The ubiquitin-specific protease 7 (USP7) has emerged as an attractive oncology/immune-oncology target owing to its critical roles in several cancer-related signaling pathways as well as its essential role in maintaining functions of Foxp3+ T-regulatory cells (Tregs), the key players in tumor immune evasion. Progenra has developed a series of compounds that inhibit purified USP7 selectively and attenuate USP7 activity in cells and in vivo; these inhibitors exert antitumor activity directly and also facilitate immune-mediated antitumor activity by suppressing Treg functions. However, the precise mechanism of action of these compounds remains unclear. In this study, using a combination of NMR spectroscopy, mass spectrometry, and single amino-acid substitution approaches, we have now demonstrated that our USP7 inhibitors specifically target the catalytic pocket of USP7 and modify its active site cysteine (Cys223) by forming a covalent adduct. Consistent with the covalent binding mechanism, pharmacokinetic studies revealed long-lasting, irreversible USP7 inhibition after a short pulse treatment with inhibitor, accompanied by changes in the level and ubiquitylation of various pharmacodynamic markers, including the Treg lineage-specific transcription factor Foxp3. Detailed knowledge of the mechanism of USP7 inhibition will permit the rational design of improved inhibitors as a new class of anticancer agent. Citation Format: Feng Wang, Jian Wu, Liqing Wang, Ivan Sokirniy, Hui Wang, Lee Chen, Brigid Cunnion, David Sterner, Charles Grove, Thomas Bregnard, Joseph Weinstock, Michael Mattern, Irina Bezsonova, Wayne W. Hancock, Suresh Kumar. Characterization of selective active-site targeted covalent inhibitors of usp7 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B193.

Active site-targeted covalent irreversible inhibitors of USP7 impair the functions of Foxp3+ T-regulatory cells by promoting ubiquitination of Tip60.

Accumulation of Foxp3+ T-regulatory (Treg) cells in the tumor microenvironment is associated with tumor immune evasion and poor patient outcome in the case of many solid tumors. Current therapeutic strategies for blocking Treg functions are not Treg-specific, and display only modest and transient efficacy. Recent studies revealed that ubiquitin-specific protease 7 (USP7) is essential for Treg functions by stabilizing expression of Tip60 and Foxp3, which together are central to the development and maintenance of the Treg cell lineage. Pharmacological inhibition of USP7 is therefore a promising strategy for suppressing Treg functions and promoting anti-tumor immunity. Previously, we reported the P5091 series of small molecule USP7 inhibitors and demonstrated their direct anti-tumor activity in vivo using xenograft models. However, the precise mechanism of action of these compounds was not well defined. In this study, we report the development and characterization of P217564, a second-generation USP7 inhibitor with improved potency and selectivity. P217564 selectively targets the catalytic cleft of USP7 and modifies its active site cysteine (C223) by forming a covalent adduct. Irreversible inhibition of USP7 results in durable downstream biological responses in cells, including down-regulation of Tip60 and consequent impairment of Treg suppressive function. In addition, we demonstrate that both USP7 and various USP7 substrates are subjected to Lys48-mediated ubiquitin modification, consistent with increased proteasomal degradation of these proteins because of USP7 inhibition.

Discovery of Small-Molecule Inhibitors of Ubiquitin Specific Protease 7 (USP7) Using Integrated NMR and in Silico Techniques.

USP7 is a deubiquitinase implicated in destabilizing the tumor suppressor p53, and for this reason it has gained increasing attention as a potential oncology target for small molecule inhibitors. Herein we describe the biophysical, biochemical, and computational approaches that led to the identification of 4-(2-aminopyridin-3-yl)phenol compounds described by Kategaya ( Nature 2017 , 550 , 534 - 538 ) as specific inhibitors of USP7. Fragment based lead discovery (FBLD) by NMR combined with virtual screening and re-mining of biochemical high-throughput screening (HTS) hits led to the discovery of a series of ligands that bind in the "palm" region of the catalytic domain of USP7 and inhibit its catalytic activity. These ligands were then optimized by structure-based design to yield cell-active molecules with reasonable physical properties. This discovery process not only involved multiple techniques working in concert but also illustrated a unique way in which hits from orthogonal screening approaches complemented each other for lead identification.

Discovery and characterization of highly potent and selective allosteric USP7 inhibitors.

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

USP7 small-molecule inhibitors interfere with ubiquitin binding.

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

Chemical Approaches to Intervening in Ubiquitin Specific Protease 7 (USP7) Function for Oncology and Immune Oncology Therapies.

Ubiquitin specific protease 7 (USP7), the most widely studied among the nearly 100 deubiquitinating enzymes, supports cancer by positively affecting tumor growth and negatively affecting the patient's immune response to tumors. Great interest exists, therefore, in developing USP7 inhibitors for clinical evaluation. While the proteasome inhibitor field has enjoyed clinical success, very few clinically appropriate effectors of deubiquitinating (protease) or ubiquitinating (ligase) enzymes have appeared. The ubiquitin protease/ligase field is moving from the initial discovery of potent, selective modulators with cell proof of concept and in vivo activity to the optimization of these molecules to impart drug-like properties or the discovery of new inhibitor scaffolds by improved screening or rational design. This Perspective focuses on the current status of USP7 inhibitors from various organizations active in developing these compounds for the clinic and suggests undertakings that are both achievable and necessary to lead to successful clinical outcomes for USP7 inhibitors in cancer treatment.

Abstract 5336: Characterization of selective covalent inhibitors of USP7

Abstract The ubiquitin-specific protease 7 (USP7) has emerged as an attractive therapeutic target owing to its critical roles in several cancer signaling pathways as well as its essential role in maintaining Foxp3+ T-regulatory cell (Treg) functions. Pharmacological inhibition of USP7 using Progenra’s small molecule inhibitors resulted in direct anti-tumor activity as well as the unleashing of anti-tumor immunity by suppressing Treg functions. However, the precise mechanism of action these compounds was not well characterized. Using a combination of NMR spectroscopy, mass spectrometry, and single amino acid substitution approaches, we have now demonstrated that our USP7 inhibitors specifically target the catalytic cleft of USP7 and covalently modify its active site cysteine (Cys223). Pharmacokinetic studies revealed sustained irreversible USP7 inhibition after short term inhibitor treatment as well as subsequent changes in the level and ubiquitylation of various pharmacodynamic markers, including the Treg lineage specific transcription factor Foxp3. Detailed knowledge of the mechanism of USP7 inhibition has allowed us to incorporate rational design strategies into the lead optimization to obtain improved molecules that will serve as the basis of a new class of anti-cancer immunotherapy agents. Citation Format: Feng Wang, Jian Wu, Liqing Wang, Ivan Sokirniy, Hui Wang, Charles Grove, Phuong Nguyen, Thomas Bregnard, Joseph Weinstock, Michael Mattern, Wayne Hancock, Irina Bezsonova, Suresh Kumar. Characterization of selective covalent inhibitors of USP7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5336. doi:10.1158/1538-7445.AM2017-5336