Chemical Approaches to Intervening in Ubiquitin Specific Protease 7

Perspective pubs.acs.org/jmc

Chemical Approaches to Intervening in Ubiquitin Specific Protease 7 (USP7) Function for Oncology and Immune Oncology Therapies Jian Wu, Suresh Kumar, Feng Wang, Hui Wang, Lijia Chen, Patrick Arsenault, Michael Mattern,* and Joseph Weinstock Progenra, Inc., 277 Great Valley Parkway, Malvern, Pennsylvania 19355, United States S Supporting Information *

ABSTRACT: 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. proteins.6,7 Attempts to mine the ubiquitin proteasome pathway for new drug targets continue, and in this Perspective, the development of inhibitors of one ubiquitin deconjugating enzyme, USP7, a promising anticancer target, will be examined to see how it may yet be possible to make a safe and efficacious drug starting with a small molecule ubiquitin protease inhibitor. Critical considerations for the development of effective USP7 inhibitors will be discussed, including the pro-oncogenic and immune modulatory activities of USP7, identification of tools and technologies for discovery and development of USP7 inhibitors, and medicinal chemistry considerations in the progression from lead to preclinical development of those inhibitors. 1.1. Ubiquitin Pathway: Mechanisms. The ubiquitin signaling system has emerged as a fundamental regulatory component of various cellular processes.8,9 Through a cascade of enzymatic reactions, proteins are selectively modified with

1. INTRODUCTION In 2004, three pioneers of the ubiquitin pathway (Drs. Aaron Ciechanover, Irwin Rose, and Avram Hershko) were awarded the Nobel Prize in Chemistry;1 in the previous year, the USFDA had approved bortezomib/Velcade (1, Table 1), a proteasome inhibitor, for treatment of refractory relapsed multiple myeloma.2 It seemed that the next few years would see a barrage of new drugs to treat all classes of disease by acting on the ubiquitin pathway’s conjugating and deconjugating enzymes, as well as the proteasome. Today, nearly a decade and a half later, only four additional ubiquitin pathway associated drugs have been approved, despite significant discovery efforts by pharmaceutical companies and others. Two of these drugs [carfilzomib/Kyprolis (3) and ixazomib/ Ninlaro (5)] (Table 1) are proteasome inhibitors, and two [lenalidomide/Revlimid (2) and pomalidomide/Pomalyst (4)] (Table 1) are derivatives of thalidomide.3−5 The first immunomodulatory drug (IMiD), 2, was used clinically before it was known that the cellular pharmacology of this drug class entailed binding to cereblon, a component of certain ubiquitin E3 ligases (ubiquitin conjugating enzymes), thereby interfering with the ability of the ligases to recognize and bind their target © 2017 American Chemical Society

Special Issue: Inducing Protein Degradation as a Therapeutic Strategy Received: March 30, 2017 Published: August 2, 2017 422

DOI: 10.1021/acs.jmedchem.7b00498 J. Med. Chem. 2018, 61, 422−443

Journal of Medicinal Chemistry

Perspective

Table 1. Ubiquitin Proteasome Pathway: USFDA Approved Drugsa

a

www.fda.gov.

or deconjugated from the ε-amino groups of lysines of specific target proteins (see above), resulting in modulations of cellular half-life, activity, and compartmentalization of target proteins. The large numbers of E3 ligases (several hundred) and DUBs (approximately one hundred) present unique opportunities for selectively targeting the ubiquitin pathway.8,13,14 The ubiquitin pathway is characterized by levels of complexity beyond that of multiple enzymes; a single ubiquitin may be conjugated to a target protein or to another ubiquitin, the latter resulting in polyubiquitin chains, which can be linear or branched and attached to one of several ubiquitin lysines.15−18 Thus, modulation of a target molecule by a ubiquitin protease or ligase occurs as part of a complex regulatory environment. The proteasome itself constitutes a target class apart from ubiquitin ligases and proteases; this multiprotein structure captures polyubiquitinated proteins, removes ubiquitin, and degrades the target protein through various constituent protease activities. In recent years, ubiquitin-based anticancer drug development has focused on all five molecular target classes (E1, E2, E3, DUBs

ubiquitin, which then provides a multitude of signals downstream that ultimately determine the fate of the modified protein (Figure 1). Protein ubiquitination is dynamic, and ubiquitin is removed from proteins by enzymes called deubiquitinases (DUBs). There are approximately 100 DUBs in the human genome. Ubiquitin specific proteases (USPs), which represent the largest family of DUBs, are isopeptidases that belong to the cysteine protease family, whose members share a conserved catalytic triad. Among DUBs, ubiquitin specific protease 7 (USP7), also known as herpes-associated ubiquitin specific protease (HAUSP), is the most widely studied DUB. 1.2. Approved Drugs from the Ubiquitin Proteasome Pathway. Many molecularly targeted drugs in current use alter cellular physiology by acting on enzymes that catalyze posttranslational modification of their substrates, the most obvious example being kinase inhibitors currently in the clinic.10−12 Another example comes from the ubiquitin-proteasome pathway, in which the protein tag ubiquitin is conjugated to 423



Perspective

Figure 1. Ubiquitin pathway and inhibitors: Ubiquitin is activated by E1 in an ATP dependent adenylation reaction forming E1−Ub thioester. Activated Ub is transferred to the active site cysteine of an E2 enzyme in a transthiolation reaction to generate E2−Ub thioester, which is subsequently transferred in an E3 ligase dependent manner to the ε-amino group of lysine in a substrate. Substrates modified with lysine 48-linked polyubiquitin chains are degraded by the proteasome. Alternately, ubiquitin can be removed from the substrate by deubiquitinating enzymes called DUBs. Representative examples of small molecule inhibitors that perturb E2, E3, and DUBs are indicated.

substrate protein (p53) (RG7388/idasanutlin, 7).30,31 MLN 4924/pevonedistat (6), an inhibitor of the E1 activating enzyme of the ubiquitin-like protein Nedd8, has undergone numerous Phase I/II trials.32,33 The most recent (2015) ubiquitin pathway entrant to clinical trials, VLX1570 (10), is a competitive inhibitor of proteasome-associated deubiquitinase activities (USP14 and UCHL-5).34,35 It is clear, then, that compounds acting on deubiquitinases and E3 ligases have lagged behind, relative to proteasome inhibitors, in clinical development. An overview of various inhibitors targeting E1, E2, E3, and DUBs has been published recently.36

and proteasomes). To the surprise of many, it was the proteasome inhibitor 1 that became the first marketed ubiquitin pathway drug; it was approved by the USFDA in 2003 for treatment of cancer (Table 1).2 Approvals of proteasome inhibitors have been limited to multiple myeloma, which appears to be particularly sensitive to proteasome inhibitors, and certain types of lymphoma.2,19−21 In early 2017, there were three proteasome inhibitors on the market in the USA, the newest (5) having an oral formulation.22 Compounds 1 and 5 are covalent drugs with boronic acid groups, and 3 is a reactive epoxide based on a peptide-derived natural product. Additional proteasome inhibitors have been and are being evaluated in clinical trials as both single agents and components of combination protocols against multiple myeloma as well as other types of cancer including refractory solid tumors [clinicaltrials.gov].23,24 The only other marketed drugs with a connection to the ubiquitin pathway are derivatives of thalidomide (see above). Compound 2 was approved originally in 2005 in combination with dexamethasone for patients who had received at least one prior therapy.4,25 In 2013, a second thalidomide derivative, 4, was approved for patients with multiple myeloma whose disease had progressed following treatment with other drugs.5 1.3. Ubiquitin Pathway Experimental Drugs Other than Proteasome Inhibitors in Clinical Trials. While numerous trials with FDA-approved as well as experimental proteasome inhibitors are ongoing (combinations, oral administration, extension to cancers other than multiple myelomas and certain leukemias; please see clinicaltrials.gov), comparatively few trials are underway for E3 ligases and deubiquitinases. Moreover, as can be seen in Table 2, two of the E3 ligase compounds that entered clinical trials appear not to be currently under active study. Nevertheless, at least one small molecule inhibitor of each enzyme class of the ubiquitin pathway, with the exception of E2, has advanced from the discovery stage to clinical development (Table 2). Three of these experimental drugs are either E3 ligase inhibitors [(JNJ26854165/serdemetan (8), GDC-0152 (9)] (Table 2)26−29 or antagonists of the binding of an E3 ligase (MDM2) to its

2. USP7: A MAJOR PROCANCER ENZYME 2.1. Substrates and Functions. USP7 is a cysteine protease that was originally identified as a binding partner of ICP0 (Infected Cell Polypeptide 0), an immediate-early gene of the Herpes simplex virus 1 (HSV-1).37 Since then, numerous proteins have been identified as potential substrates or binding partners of USP7.38 USP7 is present in complexes with binding partners including several E3 ligases39 and appears to be essential for normal physiological functions. Conventional germ line knockout of USP7 in mice results in embryonic lethality between days E6.5 and E7.5, underlying the essential role of USP7 in growth and development.40 Conditional deletion of USP7 in neural progenitors inhibits neuronal cell development, decreases cortex thickness, and causes perinatal lethality.41 In addition, both heterozygous deletion (or mutation) and genomic duplication of USP7 have been associated with neurological and behavioral phenotypes, indicating that the role of USP7 is complex and context-dependent.42,43 USP7 also plays an essential role in DNA replication.44 Many of the well characterized substrates of USP7 play critical roles in tumor suppression or progression, cell signaling, epigenetic control, DNA damage repair, and immune responses. USP7-dependent dysregulation of its substrates’ stability and functions has been reported to be essential for the initiation, progression, or recurrence of a series of tumors, including but not limited to lung cancer, colorectal cancer, breast cancer, neuroblastoma, medulloblastoma, and prostate cancer.45−50 424



Perspective

Table 2. Ubiquitin Pathway Enzyme Antagonists in Clinical Triala

a

www.clinicaltrials.gov.

(KMT1A), both epigenetic modulators of gene transcription. USP7 also stabilizes E3 ligase UHRF1 (Ubiquitin-like, containing PHD and RING finger domains, 1) and Dnmt1 (DNA methyl transferase 1), critical regulators of DNA methylation and epigenetic gene silencing. In addition to the p53 pathway, USP7 regulates the functions of tumor suppressors such as PTEN and FOXO proteins (FOXO1/4), both of which require monoubiquitination for their proper localization and function.55,56 By removing ubiquitin from monoubiquitinated PTEN and FOXO1/4, USP7 antagonizes their tumor suppressor functions.56,57 The protein p14ARF acts as a tumor suppressor by inhibiting ribosome biogenesis or initiating p53-dependent cell cycle arrest and apoptosis. By deubiquitinating and stabilizing Trip12, an E3 ligase for p14ARF, USP7 enables downregulation of p14ARF, thereby attenuating its tumor suppressor activity.58 In

p53 is a critical tumor suppressor, and mutations in p53 are found in more than 50% of human cancers. USP7 plays an important role in antagonizing p53 functions through multiple mechanisms. HDM2 is one of the major E3 ubiquitin ligases that downregulate p53 via the ubiquitin proteasome pathway. HDMX, which is a binding partner of HDM2, also inhibits the function of p53 through tight binding to the N-terminal transcription activation domain of p53.51 Both HDM2 and HDMX are deubiquitylated and stabilized by USP7.52,53 Therefore, knockout or knock-down of USP7 protein or inhibition of USP7 enzymatic activity results in downregulation of HDM2/HDMX, leading to stabilization of p53 protein and subsequent activation of the p53 pathway. In addition, USP7 inhibits the expression of p53 as well as the expression of its downstream transcriptional targets by stabilizing lysine specific demethylase 1 (LSD1)54 and histone methyltransferase 1A 425



Perspective

Figure 2. Multimodal antitumor activities of USP7 inhibitors. USP7 inhibitors exhibit intrinsic antitumor activities by downregulating protumorigenic proteins and extrinsic immune mediated antitumor activities by attenuating Treg functions.

pathways by stabilizing their transcription factors Gli,46,62 βcatenin,50 and androgen receptor,63−65 respectively. Tumor cells typically produce higher than normal levels of reactive oxygen species, increasing the extent of DNA damage.63 Accordingly, functional DNA repair pathways are beneficial for tumor cell survival. In addition, DNA damage responses are associated with the generation of resistance to chemotherapy.64 USP7 regulates multiple DNA repair pathways, and loss of USP7 reduces the tolerance of cells to genotoxic agents.65 For example, the antitumor activity of certain alkylating chemotherapy drugs is largely attenuated by the overexpression of alkylation repair proteins such as ALKBH2 and ALKBH3 in cancer cells. USP7 is reported to deubiquitinate and stabilize ALKBH2/3, and the loss of USP7 in tumor cells greatly sensitizes them to alkylating agents, suggesting that USP7 inhibitors could be explored as sensitizers in alkylating agent-based chemotherapy protocols.65 Thus, USP7 is a critical node in various cancer signaling pathways, and USP7 inhibitors are predicted to have intrinsic antitumor activity (Figure 2). In addition to its tumor supportive role, USP7 promotes the evasion of tumors from immune surveillance and killing by stabilizing proteins that are essential for regulatory T cells (Treg) to suppress tumoricidal effector T cells (Teff) in the tumor microenvironment. This immune regulatory role of USP7 is discussed in detail in the following section (Figure 2). 2.2. Therapeutic Application of USP7 Inhibitors in Neoplastic Disease. Part of the renewed and increased interest in developing USP7 inhibitors for cancer treatment stems from the recent demonstration that, in addition to protecting tumor cells directly from apoptosis-associated tumoricidal effects, USP7 plays a role in the capacity of tumors to evade detection and killing by the host immune system. Several chemical biology studies have been conducted in an

addition, USP7 regulates the stability of MEL18, BMI1, and UbE2E1, three protein components of the polycomb repressive complex 1 (PRC1), which are responsible for histone H2A monoubiquitination and gene silencing of p16INK4α, a tumor suppressor protein.59 In its role as a cancer-promoting enzyme, USP7 not only antagonizes tumor repressors but also positively regulates the stability and functions of several oncoproteins, including NMYC, which is dysregulated in a number of advanced-stage human tumors and has been reported as a driver of neuroblastoma (NB) tumorigenesis.47 USP7 has been identified as a regulator of N-MYC, and USP7 expression induces deubiquitination and stabilization of N-MYC.47 Hypoxiainducible factor 1α (HIF-1α) plays a major role in cancer biology, specifically in angiogenesis, tumor cell survival, metastasis, and generation of resistance to chemotherapy and radiotherapy.60 A recent study discovered that hypoxia-induced K63 polyubiquitination of USP7 by the E3 ligase HectH9 enhanced the ability of USP7 to deubiquitinate and stabilize HIF-1α.61 REST is a critical transcription factor in regulating neural progenitor cell self-renewal; neural tumors, such as medulloblastomas and neuroblastomas, normally express high levels of REST, and forced expression of REST can induce malignant transformation of neural progenitors. USP7 has been demonstrated to regulate and stabilize this oncoprotein.49 Abnormally altered cell signaling pathways often drive tumorigenesis. For example, hyperactivation of the Sonic hedgehog (Shh) pathway drives development of medulloblastoma, dysregulation of the Wnt/β-catenin pathway is associated with colorectal tumor growth, and malfunctioning of the androgen receptor signaling pathway is a common cause of prostate cancer initiation and progression. USP7 has been reported to positively regulate the activity of these three 426



Perspective

weight loss, lethargy, or microscopic tissue damage.47 These studies strongly suggest that USP7 inhibitors may be effective in the treatment of human NB by suppressing N-Myc activities irrespective of cellular p53 wild-type or mutant status. Most recently, An et al. demonstrated that USP7 positively regulates Wnt signaling in colorectal cancers.50 USP7 knockdown inhibits proliferation of colorectal cancer cell lines with differing p53 status. USP7 inhibition by treatment with 17 attenuates Wnt signaling by promoting the ubiquitination and subsequent degradation of β-catenin. Treatment with 17 inhibits proliferation and induces apoptosis in colorectal cancer cell lines in vitro. Treatment of HCT116 xenografts in nude mice using ip administration of 17 at 15 and 25 mg/kg, inhibited tumor growth. Further investigation demonstrated reduction in levels of β-catenin and Wnt target genes (Axin, cMyc and Survivin). These results indicated inhibition of Wnt signaling in vivo under these conditions. In addition to USP7, its closest homologue, USP47, also stabilizes β-catenin. Interestingly, 17 was demonstrated to inhibit USP47 in vitro with a potency similar to that of USP7. Therefore, concomitant inhibition of USP47 by 17 in the in vivo experiment above could also have contributed to the downregulation of β-catenin. USP7 regulates the stability and function of PHF8, a histone demethylase necessary for DNA damage response (DDR).71 Acting through PHF8 and RNF168, USP7 regulates both homologous DNA repair (HR) and nonhomologous end joining (NHEJ) repair processes. USP7 mediated stabilization of PHF8 leads to upregulation of cyclin A2, a critical mediator of cell growth and proliferation. USP7 promotes breast carcinogenesis through the USP7/PHF8/cyclin A2 axis. Knockdown of USP7 in the breast cancer cell line MCF7 results in changes in the expression of 1477 genes and significantly abrogates tumor growth in athymic mice.71 Thus, USP7 inhibitors could also be pursued for therapeutic intervention in breast cancer alone or in combination with chemo- or radiotherapies. USP7 overexpression in glioblastoma is associated with disease progression and poor prognosis.54 Yi et al. identified lysine specific demethylase 1 (LSD1) as a USP7 substrate in glioblastoma cells.54 Knockdown of USP7 inhibits the growth of glioblastoma cells in vitro by modulating p53 pathways. This study supports the notion that USP7 inhibitors could be efficacious against glioblastoma. USP7 is expressed at elevated levels in several ovarian cancer cell lines compared to nontumorigenic immortalized ovarian surface epithelial cells.69 In addition, ovarian cancer tissues express significantly higher levels of USP7 than do nontumor tissues, and knockdown of USP7 inhibits the proliferation of ovarian cancer cells. Targeting USP7 may thus represent a novel strategy for treating ovarian cancer. Hyperactivation of the Shh pathway is a hallmark of medulloblastoma (MB), the most common pediatric brain tumor.73 Although Shh pathway inhibitors are being evaluated for treating MB, central nervous system and skeletal muscle developmental toxicities due to Shh inhibition could pose serious challenges, particularly in younger infants.73 Recently Zhan et al. demonstrated that knockdown of USP7 inhibits the growth and migration of medulloblastoma (MB) cells.46 Further, USP7 knockout using the CRISPR/Cas9 system in MB cells suppresses their growth and metastasis. USP7 targeting inhibits the Shh pathway and decreases gliomaassociated oncogene homologue (Gli) protein levels without affecting the p53 pathway. More importantly, treatment with

attempt to validate USP7 as a target of both growth and survival of tumors and their ability to evade surveillance and killing by the host immune system.66,67 It is important to note, however, that the various chemical tools described in the literature to date lack the selectivity, potency, and pharmacokinetic properties required to validate the role of USP7 in tumor biology with complete certainty. Nonetheless, the data generated thus far are intriguing and warrant continued efforts to develop drug-like molecules with improved potency and selectivity that can be employed with confidence to confirm the roles of USP7 in tumor biology suggested by the initial studies. 2.2.1. USP7 Inhibitors in Direct Regulation of Tumors. Many cancers, including multiple myeloma, neuroblastoma, squamous cell carcinoma, hepatocellular carcinoma, lung cancer, glioblastoma, ovarian cancer, breast cancer, and colorectal cancer, overexpress USP7; this overexpression has been correlated with poor prognosis in neuroblastoma and in lung and colorectal cancers.45,50,54,58,66,68−72 Chauhan et al. obtained the first evidence of in vivo antitumor activity of USP7 inhibitors when they demonstrated that P5091 (17, Figure 9) inhibits multiple myeloma growth in MM1.S xenograft models.66 Inhibition of USP7 in multiple myeloma cells results in destabilization of HDM2 and upregulation of p53 and p21 tumor suppressors, ultimately leading to apoptosis.66 In addition, treatment with 17 overcomes multiple myeloma’s resistance to the proteasome inhibitor bortezomib and also synergizes with DNA damaging agents such as etoposide and doxorubicin in killing multiple myeloma cells in vitro.66 Most recently, Das et al. showed that 17 inhibits multiple myeloma activity in synergy with RRx-001, a novel hypoxia selective epigenetic agent, by downregulating Dnmt1.24 Thus, there is strong rationale for clinical evaluation of USP7 inhibitors alone or in combination with existing multiple myeloma therapies to further increase therapeutic efficacy. In 2012, Fan et al. reported that P22077 (19, Figure 9), a close analog of 17, induces apoptosis in neuroblastoma (NB) cells in a p53 dependent manner.68 USP7 inhibition by 19 promotes downregulation of HDM2 and upregulation of p53 in p53 wild-type NB cells.68 Compound 19 significantly augments the cytotoxic effects of doxorubicin and etoposide in NB cells with an intact USP7−HDM2−p53 axis.68 Moreover, treatment with 19 sensitizes chemoresistant NB cells to chemotherapy. In an orthotopic NB mouse model, 19 was shown to significantly inhibit xenograft tumor growth of three different NB cell lines.68 In 2016, Tavana et al. described USP7 as a critical regulator of the proto-oncogene N-Myc, which is amplified in several cancers including neuroblastoma (NB).47 RNAi mediated knockdown in cancer cell lines or genetic ablation of USP7 in mouse brain was shown to destabilize N-Myc, leading to inhibition of N-Myc function. More importantly, treatment with the USP7 inhibitors 17 or 19 promoted ubiquitination and downregulation of N-myc protein levels in MYC-N gene amplified NB cells without significantly altering the MYC-N mRNA levels. Treatment with 19 also promotes apoptosis in p53 proficient NB cells and markedly suppresses the growth of MYC-N amplified human neuroblastoma cell lines in xenograft models. Remarkably, intraperitoneal (ip) administration of 19 at 20 mg/kg daily for 15 days was well tolerated. No weight loss or other health problems were observed during treatment, and no abnormalities were found after full necropsy at the end of treatment. Similarly, wild-type mice treated with 20 mg/kg daily dosage for a 30-day period were found to have no signs of 427



Perspective

level, suggesting that Tip60 plays a dominant role in regulating the function of Treg cells, potentially by promoting the acetylation, multimerization and function of Foxp3. Thus, pharmacologic inhibition of USP7 impairs Treg cell function, likely through Tip60 and Foxp3 dependent mechanisms. USP7 protein levels and overall activity are similar in Treg and Teff. The role of USP7 in Teff, however, is unclear. To address the effect of USP7 inhibitors on Teff, Wang et al. injected mice with P217564 and isolated Treg and Teff, which were evaluated using ex vivo functional assays. Most interestingly, USP7 inhibitor treated Teff exhibited resistance to suppression by Treg.67 Although the precise mechanism behind this resistance remains to be established, these data indicate that treatment with USP7 inhibitors in vivo does not impair the Teff functions, which are critical for their antitumor immune response. Using two distinct syngeneic tumor models, Wang et al. demonstrated antitumor activity of USP7 inhibitors in vivo. In the Treg dependent syngeneic TC1 lung tumor and AE17 mesothelioma models, USP7 inhibitor were shown to exert statistically significant antitumor activity and, more importantly, to promote the accumulation of tumor-antigen specific CD8+ T cells in the tumor microenvironment.67 These tumor specific CD8+ T cells produced IFNγ, potentially helping to break immune tolerance. This study also demonstrated that USP7 inhibitor treatment decreases intratumoral accumulation of Foxp3+ Treg cells. The same tumors, grown in syngeneic immunodeficient mice, were seen to be refractory to USP7 inhibitors given at the same dose, suggesting that under the conditions employed, USP7 inhibitors exerted activity against TC1 and AE17 predominantly by inhibiting Treg functions, promoting an antitumor immune response. Notably, both TC1 and AE17 cancer cells have reduced levels of USP7, MDM2, and p53 compared to the multiple myeloma cells (MM1.S) that are responsive to USP7 inhibitor treatment in immunodeficient animals.67 Thus, those tumors that overexpress USP7 and its substrates may be more sensitive to the direct antitumor activity of USP7 inhibitors, whereas tumors with relatively high levels of suppressive Treg in their tumor microenvironment will also be responsive to the immune mediated antitumor activity of USP7 inhibitors. Moreover, USP7 inhibitors were shown to confer significant therapeutic benefit when used in combination with biologicals commonly used in immune oncology protocols (vaccines as well as anti-PD1 monoclonal antibodies) in mice bearing AE17 tumors and TC1 tumors, respectively.67 Dnmt1 and UHRF1 are two additional critical modulators of the function and development of Treg. In 2013, Wang et al. demonstrated that deletion of Dnmt1, but not Dnmt3a, decreases the numbers and function of Treg and that, conditional deletion of Dnmt1 in mouse Treg leads to lethal autoimmunity by 3 to 4 weeks of age.88 This phenotype is strikingly similar to that of mice with a USP7−/− deletion in their Treg. Conditional deletion of Dnmt1 downregulates global DNA methylation and causes alterations in Treg-specific expression of several hundred genes. This study established that Dnmt1, whose stability is regulated by USP7, is also essential for the maintenance of Treg development and function. Obata et al. demonstrated that deletion of UHRF1 results in a failure of colonic Treg development.89 Although the precise role of UHRF1 in Treg functions remains to be established, this study linked yet another USP7 substrate to immune regulation. USP7 is expressed in a wide variety of tissues, and the emergence of several potential substrates could pose concerns regarding on-target toxicity in clinical applications of USP7

USP7 inhibitors 17 or 19 blocks the proliferation and migration of MB cells in vitro. Thus, selective USP7 inhibition has potential as a treatment option for MB; it remains to determine, however, whether these promising in vitro results translate to the clinic. Zhang et al. identified Ki-67, the known proliferation marker of malignant tumors, as a USP7 substrate in NSCLC.45 Expression levels of both Ki-67 and USP7 are positively correlated with degree of carcinoma malignancy. USP7 interacts with Ki-67 in non-small-cell lung cancer (NSCLC), leading to deubiquitination and stabilization of Ki-67, and inhibition of USP7 by 19 results in accumulation of ubiquitinated Ki-67 and its subsequent downregulation. Moreover, knockdown of USP7 in NSCLC cells suppresses tumor growth in nude mice66 in addition to sensitizing NSCLC cells to gemcitabine and docetaxel.11 Collectively, this study suggests that USP7/Ki-67 is an attractive therapeutic target in NSCLC cells in addition to being a potential prognostic marker. USP7 inhibitors again could be efficacious as single agents or in combination with radio- or chemotherapies. 2.2.2. USP7 in Immune Regulation. Cytotoxic T cells and natural killer cells are capable of killing tumors. Within the tumor microenvironment, Foxp3 + T reg can be highly immunosuppressive; limiting the antitumor responses of Teff such as CD8+ T cells, thereby promoting tumor growth and progression.74−78 Thus, a promising new cancer immunotherapy avenue is the attenuation of Treg suppressive functions.79−82 The Treg lineage and its immunosuppressive functions are determined primarily by the lineage specific transcription factor Foxp3, which is subject to several post-translational modifications, including acetylation and ubiquitination.83−85 Tip60, a histone acetyl transferase, exerts a dominant role in promoting acetylation, multimerization, and functioning of Foxp3 in Treg.86 The role of USP7 in immune regulation became apparent when van Loosdregt et al. showed that USP7 interacts with Foxp3 in Treg; knockdown of USP7 impaired Treg functions in an ex vivo Teff suppression assay.87 Notably, adoptive transfer of wild-type Treg prevented colitis development in mice while transfer of Treg with their USP7 knocked down failed to suppress Teff -mediated colitis development. In these seminal experiments it was shown that USP7 deubiquitinates Foxp3, thereby preventing its proteasomal degradation. The critical role of USP7 in maintaining Treg suppressive functions became evident when Wang et al. provided genetic evidence that developmental deletion of USP7 in Treg cells (USP7−/− Treg) induces lethal systemic autoimmunity in mice.67 USP7 deletion alters the Treg gene expression signature and dampens the suppressive activity of Treg cells. Interestingly, USP7−/− Treg cells proliferate actively, indicating the dominance of p53 independent pathways in these cells. In stark contrast to the wild-type Treg, USP7−/− Treg express more IFNγ and IL-2. All of these data suggest that inhibition of USP7 impairs Treg cell function and potentially promotes antitumor immunity. Pharmacologic blockade of USP7 may therefore provide an avenue for effective cancer immunotherapy. Notably, incubation with the USP7 inhibitor 17 and its more potent analog P217564 (IC50 = 0.58 μM, structure not disclosed) have been shown to abrogate the suppressive activity of Treg.67 While USP7 inhibitor treatment promotes ubiquitination of both Foxp3 and Tip60 and inhibits formation of Foxp3 multimers, USP7 inhibitor treatment or USP7 deletion in Treg leads to a dramatic decrease in Tip60 protein 428



Perspective

Figure 3. (A) Cocrystal structure (PDB ID 4WPI) of ICP0 peptide (green) with USP7 C-terminal UBL-123 domain (gray); (B) key interactions between ICP0 peptide (green) and USP7 UBL-2 domain (gray).

Figure 4. (A) Superimposed cocrystal structures (PDB ID 2FOO and 2FOP) of p53 peptide (green), MDM2 peptide (orange), and USP7 Nterminal TRAF domain (gray); (B) key interactions between substrate peptides (green and orange) and USP7 TRAF pocket (gray).

inhibitors.38 As discussed earlier, germ line deletion of USP7 results in early embryonic lethality in mice and mutations resulting in haploinsufficiency of USP7 leads to a spectrum of neurodevelopmental and behavioral phenotypes in humans.40,42 In addition, USP7 is essential for DNA replication, and USP7 inhibitors strongly inhibit replication origin firing in a p53 independent manner.44 Therapeutic blockade of USP7 using small molecule inhibitors could potentially impact normal neuronal development as well as DNA replication, genomic integrity and proliferation of normal cells. Thus, careful evaluation of toxicities associated with small molecule USP7 inhibitors is critical to establish the therapeutic window. Apart from Treg cells, USP7 inhibitors might affect other cell populations that express Tip60 and other USP7 substrates. Several preclinical in vivo studies have demonstrated that USP7 inhibitors such as 17, 19, and P217564 are well tolerated at doses that reduce tumor growth in mice and exert antitumor activities against a variety of cancers including multiple myeloma, neuroblastoma, lung tumors, and colon can-

cer.47,50,66−68 More important, Treg are relatively small in number (∼5−10% of total CD4+ T cell population)90 and appear to be hypersensitive to USP7 inhibitors.67 Thus, USP7 inhibitors could preferentially attenuate Foxp3+ Treg functions while sparing the host effector CD8+ T cells. Pharmacologic modulation of Treg cells using USP7 inhibitors offers a new way to break immune tolerance in the tumor microenvironment. Suppression of Treg functions by inhibiting USP7 could result in potential autoimmunity. Treatment of mice with P217564 at its therapeutically efficacious dose did not result in autoimmunity, indicating a safe therapeutic window. However, more studies are needed to determine clinical relevance of these observations, and strategies to harness the immune system to fight cancer must take potential autoimmunity and its therapeutic management into consideration. Collectively, these studies show that USP7 inhibitors confer significant benefit when used either as monotherapy in syngeneic tumor models or in combination with other forms of immunotherapy to promote antitumor activity. It will be 429



Perspective

Figure 5. (A) Overlay of Apo-USP7 (PDB ID 4M5X) with Ub-bound USP7 (PDB ID 5JTV). Ub-bound USP7 catalytic domain flexible loops are colored in green, Apo-USP7 catalytic domain flexible loops are colored in red, and ubiquitin is colored in cyan. (B) Shift of catalytic trial residues (C223, H464, and D481) upon the binding of ubiquitin. Inactive conformation is shown in gray, while the active conformation is shown in green.

Figure 6. Catalytic mechanism of USP7.

terminal UBL domains (PDB ID 4WPI) shows that the peptide binds onto a shallow surface area at UBL-2 (Figure 3).92 The key residues involved in the protein−protein interactions (PPIs) are K620 and K624 of ICP0, which form salt bridges with D762 and D764 of USP7, respectively. The key consensus binding motif was confirmed as KXXXK, which was also found in GMPS and UHRF1 to enable their binding with UBL-2. Additionally, upon the binding of GMPS, UBL-4 and -5 have been shown to participate in the allosteric activation of USP7 through a GMPS mediated mechanism, which regulates p53 level.92,93 The N-terminal TRAF domain of USP7 has been reported to bind to substrates such as p53 and MDM2 in a mutually exclusive manner, illustrated by the superimposed cocrystal

interesting to see whether their potential dual antitumor activity will translate to the clinical setting.

3. USP7: CATALYTIC MECHANISM AND 3-D CRYSTAL STRUCTURE The USP7 protein contains a single catalytic domain (residues 208−560), an N-terminal tumor necrosis factor-receptor (TRAF)-like domain (residues 53−208), and five C-terminal ubiquitin-like (Ubl) domains (residues 560−1102). Each domain exhibits unique characters and functions. The five ubiquitin-like domains (UBL-1−5) can interact with USP7 partners such as ICP0, GMPS (guanosine monophosphate synthetase) and UHRF1.91 A crystal structure of ICP0 peptide 617-GPRKCARKTRH-627 with the USP7 C430



Perspective

Figure 7. Various DUB substrates: (A) C-terminal fusion of ubiquitin with fluorophore resulting in Ub-AMC and Ub-Rh110 substrates. (B) Ubiquitin fusion to the amino-terminus of reporter enzymes PLA2 and EKL to generate coupled reporter substrates. (C) Internally quenched fluorescent diubiquitins. (D) Fluorescently labeled substrate derived peptide containing isopeptide linked ubiquitin as substrate for fluorescence polarization assays.

catalytic cysteine C223 is located far away from H464 and D481, autoinhibiting the catalytic activity of USP7 (Figure 5). Upon the binding of ubiquitin (shown in cyan), the catalytic domain is activated, represented by the shift of three switching loops (residues including K281-Q296, F409−N418, and G458−H464 highlighted in green) surrounding the ubiquitin tail (V70−G76), as well as by the formation of the activated catalytic triad (highlighted in green) (Figure 5). The catalytic mechanism is illustrated in Figure 6. When the catalytic triad assumes its active conformation, the hydrogen bond between Asp481 and His464 locks His464 into a favorable location toward the vicinity of Cys223, which in turn results in the deprotonation of the thiol of Cys223 to reveal the nucleophile. The thiol anion then attacks the carbonyl group of the isopeptide bond between the substrate N-terminal lysine and the ubiquitin C-terminal glycine to form a tetrahedral intermediate, which collapses and ejects the free lysine from the substrate, while ubiquitin is attached to the Cys223 of USP7 through a thioester linkage. Next, a molecule of water protonates His464 and hydrolyzes the thioester bond to release Cys223 and the ubiquitin C-terminal glycine.

structures of p53 (358−363)−TRAF (PDB ID 2FOO) and MDM2 (142−147)−TRAF (PDB ID 2FOP) (Figure 4).94,95 Both peptides adopted similar conformations as well as interactions with the TRAF pocket residues including F118, I154, D164, W165, G166, and F167. The key recognition motif can be summarized as PXXS, which accounted for 359-PGGS362 in p53, and 144-PSSS-147 in MDM2. Interestingly, another consensus motif AXXS in both p53 and MDM2 also showed up as a TRAF binder, represented by the p53 site 364-AHSS-367 and 225-DAGVS-229.94,95 Shi and co-workers also observed that the MDM2 peptide was a stronger binder compared to the p53 peptide.95 The selectivity between p53 and MDM2 suggests that inhibition of the TRAF domain of USP7 can potentially destabilize MDM2 more than p53, which can be an attractive target for designing small molecule inhibitors. Although multiple crystal structures showing the interactions between USP7 domains and various peptide substrates have been reported in the literature, very little information is available on the binding between full length USP7 and its substrates. In fact, in addition to the key motif interactions mentioned above, unknown protein−protein interactions, including p53−UBL45 and HDM2−UBL45, have been suggested as contributing to the binding of these substrates.96 Increased structural understanding of these interactions is necessary to identify potential “hot spots” for the binding of small molecule inhibitors with USP7. The catalytic domain of USP7 has been reviewed in the literature.97,98 As a cysteine isopeptidase, USP7 has a catalytic domain whose conformation shows significant structural flexibility.99 Interestingly, this conformational change is unique compared to other USPs including USP8, USP14, and USP4.93,100 In its nonactive conformation (shown in red), the

4. ASSAYS AVAILABLE TO IDENTIFY USP7 INHIBITORS Robust and sensitive biochemical assays are necessary to characterize the enzymatic functions of DUBs in vitro as well as to conduct high throughput screening to identify potential inhibitors. In cells, DUBs catalyze the cleavage of isopeptide linkages between ubiquitin and a substrate protein or between two ubiquitin molecules. Preparation of specific ubiquitinated substrates in quantities sufficient to develop robust high 431



Perspective

Figure 8. Chemical structures of reported USP7 inhibitors from Hybrigenics.

wavelengths of 475 and 550 nm, respectively. Like the UbAMC and Ub-Rh110 based assays, this coupled assay employs cleavage of a linear amide bond, not the physiological isopeptide bond; it does, however, feature cleavage of two polypeptide entities rather than a small molecule fused to a polypeptide. Ubiquitin fused to the amino-terminus of reporter enzyme enterokinase light chain (EKL) is a second-generation DUB substrate for a coupled assay.105 Cleavage of ubiquitin by DUBs releases active EKL whose activity can be monitored using internally quenched FRET peptide substrates such as QXL520DDDDKGSK-FAM (excitation 490 nm/emission 520 nm) and QXL570-DDDDKGSK-TAMRA (excitation 540 nm/emission 590 nm). The Ub-EKL assay offers increased sensitivity and diminished interference from autofluorescence of test compounds. In addition, this reporter-based assay can be used in a multiplex format to monitor activity of more than one isopeptidase at the same time. Several recently developed assays could be used to screen for USP7 inhibitors. For example, a monoubiquitinated peptide from the USP7 substrate PTEN (amino acids 5−21) containing a fluorescent label (TAMRA) at the amino-terminus has been developed as a USP7 specific substrate.106 Cleavage of the ubiquitin by USP7 releases the TAMRA (5-carboxytetramethylrhodamine)-labeled peptide whose fluorescence polarization can be monitored using a plate reader. This assay has the advantage of employing the native isopeptide linkage in its DUB substrate. Diubiquitin substrates are another relatively new class of substrate allowing for continuous measurement of true isopeptidase activity. The C-terminus of wild-type ubiquitin is conjugated via an isopeptide bond to lysine 48 (K48) or lysine 63 (K63) of a second ubiquitin molecule to form an internally quenched fluorescent FRET pair (IQF). Each ubiquitin is labeled with single molecule of either a fluorescent reporter (for example, TAMRA) or a quenching fluorophore (for example, QXL). DUB mediated cleavage of the IQF DiUb leads to the release of the quencher and resultant increase in fluorescence.107 Recently, Ritorto et al. developed a sensitive assay to quantify DUB enzyme activity using matrix assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry.108 In this assay, various diubiquitin isomers are cleaved by DUBs and the monoubiquitin released is quantified using an 15Nubiquitin internal standard. This assay allows determination of

throughput screening assays remains a challenge, and several nonphysiological substrates have been developed as alternatives to monitor activities of DUBs such as USP7 (Figure 7). One of the earliest substrates to be developed was ubiquitin-7-amido-4methylcoumarin (Ub-AMC), where the carboxy-terminal glycine of ubiquitin is fused with the AMC fluorophore using a transpeptidation method.101 Upon incubation with DUBs such as USP7, the ubiquitin moiety is cleaved and the AMC fluorophore is released allowing its emission at 460 nm (λex = 380 nm), the assay readout. The simplicity and robustness of the Ub-AMC based assay led to its widespread use and commercial adaptation into high throughput screening platforms. It was recognized, however, that the excitation wavelength of AMC fluorophore in the UV range (380 nm) constituted a major limitation, as compounds that fluoresce or absorb at this wavelength could interfere with the assay. Moreover, AMC is conjugated to the ubiquitin through an aromatic amide bond rather than the physiological isopeptide linkage cleaved by DUBs. Later screening assays addressed some of the issues associated with the Ub-AMC assay. Fusion of the fluorophore rhodamine 110 to the carboxy terminus of ubiquitin generated the Ub-Rh110 substrate (Ub-Rh110).102 Upon cleavage by DUBs, the released rhodamine-glycine emits intense fluorescence at 485 nM when excited, improving sensitivity compared to Ub-AMC. Ub-Rh110 has been utilized in high throughput screening campaigns to discover DUB inhibitors. Ubiquitin carboxy-terminus fusion with aminoluciferin (UbAML) generated a highly sensitive substrate.103 Cleavage of ubiquitin from Ub-AML releases aminoluciferin, which is subsequently used by luciferase to generate a chemiluminescent signal. Ub-AML based assays are also suitable for high throughput screening. Ubiquitin-reporter enzyme fusion substrates have also been developed to measure DUB activity. In the first generation assay, ubiquitin was fused to the aminoterminus of reporter enzyme phospholipase A2 (PLA2).104 Since the catalytic activity of PLA2 requires a free aminoterminus, ubiquitin fusion inactivates the PLA2 enzyme. However, upon cleavage of the ubiquitin by the DUB, the active PLA2 is released, and it subsequently converts 2-(6-(7nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD C6-HPC) to the fluorophore 7-nitrobenz-2-oxa-1,3-diazole (NBD), whose fluorescence can be monitored using excitation and emission 432



Perspective

Figure 9. Chemical structures of USP7 inhibitors from Progenra.

USP7 inhibitor with an IC50 of 28 μM from a high throughput screen of 36 000 compounds using Ub-AMC as the substrate. Subsequent optimization led to the more potent compound 15, with an IC50 of 8.2 μM. These compounds showed good selectivity for USP7 when tested against other human USPs and other DUBs. An interesting property of the compounds is their covalent irreversible mode of action in inhibiting the USP7 target enzyme. In support of this property, pretreatment of USP7 with the compounds prevents the active site from reacting with an active site-specific probe, Ub-vinyl sulfone. An aromatic chlorine activated by the tetrahydroacridine system may render this mechanism possible. Molecular docking of the inhibitor positioned the Cl substituent in the proximity of the active site Cys, in agreement with the proposed mechanism, while a required positively charged amine substituent interacted with negatively charged residues. In cellular models, these active-site targeting inhibitors were shown to bind directly and specifically to USP7 and modulate the steady-state levels of several USP7 substrates. In 2014, a new family of quinazolin-4-one based compounds were claimed as selective USP7 inhibitors.116,117 The patent application describes 30 examples of compounds that were synthesized, and their inhibition of USP7 was characterized using recombinant and biochemical assays. These compounds can block labeling of USP7, but not of USP8, USP5, USP10, CYLD, or UCH-L3, with Ub-VME. Their IC50 values for USP7 inhibition in cell lysates are in the range of 25−50 μM. A series of experiments was designed to understand better the mechanism of inhibition. The compounds appear to have the characteristics of reversible inhibitors, as judged by measuring enzyme activity recovery using gel filtration, large dilution assays, and native electrospray ionization mass spectrometry. Compound 16 (Figure 8) is the most potent inhibitor (IC50 = 15 μM) of the USP7 activity in this series.114 Hybrigenics researchers were among the first to describe a small molecule, 11, as having USP7 inhibitory activities, and since then 11 has been extensively used as a tool compound. Compound 11 inhibits cell growth and induces apoptosis in HCT116 (human colon carcinoma) cells by activating p53.111 Notably, 11 has been used to demonstrate the cellular role of USP7 in regulating TNF-α induced apoptosis, nucleotide excision repair, DNA damage response, circadian rhythm,

potency and specificity of DUB inhibitors and is configurable for high throughput screening to discover novel DUB inhibitors. In addition to monitoring activity of purified USP7, it is critical to develop cell based assays to characterize USP7 inhibitors. Activity-based probes such as ubiquitin-vinyl methyl ester (Ub-VME) can be used to measure the activity of cellular DUBs, including USP7.109 Incubation of cell lysates with UbVME results in covalent modification of the active site cysteine of DUBs, increasing the molecular weight of the free DUBs by 8 kDa, which can subsequently be detected by Western blotting. In addition, target engagement can be determined using cellular thermal shift assays (CETSA), in which the interaction of a DUB, for example, USP7, with a compound results in its thermal stabilization or destabilization.110

5. DEVELOPMENT OF SELECTIVE USP7 INHIBITORS 5.1. USP7 Inhibitors from Hybrigenics. Hybrigenics conducted a Ub-AMC based high throughput screen of 65 092 compounds for novel USP7 inhibitors, which resulted in the identification of the cyano-indenopyrazine derivative HBX 41,108 (11, Figure 8) with an IC50 of 0.42 μM.111 While 11 is selective against many families of proteases, it inhibits caspase 3 and multiple DUBs. Compound 11 was disclosed as a reversible uncompetitive inhibitor. The chloro-substituent is required for USP7 inhibition with reasonable potency, and the corresponding hydroxyl analog (12) is completely inactive. Molecular docking indicated that the compound may interact specifically with the active, ubiquitin-bound conformation of the core. In a later publication from the original group,112 it was shown that 11 was more potent against USP8 (IC50 = 96 nM) than against USP7 (420 nM, Figure 8). In 2010, Lopez et al. published a patent describing a series of pyrrole compounds as USP7 selective inhibitors.113 The most potent compound disclosed in the patent is compound 13 with an IC50 of 1.6 μM (Ub-AMC assay). No binding mechanism is proposed, but 13 exhibits good selectivity over the small panel of other enzymes tested (USP8, USP5, UCH-L1, UCH-L3, caspase 3, IC50s > 200 μM). In 2012, 9-chloro derivatives of amidotetrahydroacridine were reported by Reverdy et al. as a new family of USP7 inhibitors.114,115 HBX 19,818 (14, Figure 8) was identified as a 433



Perspective

prepared. The data showed that nitro groups in the thiophene series play an important but nonexclusive role in achieving USP7 inhibitory activity. The thiophene-4-nitro group was replaced by a cyano group in certain compounds with little or no loss in USP7 inhibition potency. Replacement of the nitro group in the thiophene series with other electron-withdrawing groups drastically reduces inhibition of purified USP7. It was also found that carboxyamides at C2 provide an opportunity to prepare compounds with enhanced aqueous solubility while maintaining activity against USP7. One of the most potent analogs 21 (USP7 IC50 = 0.42 μM, Figure 9) demonstrates enhanced stability, as illustrated by reduced GSH reactivity (80% recovery) and excellent plasma stability (97% recovery). Consistent with other compounds in this series, compound 21 is a selective USP7 inhibitor and does not inhibit representative USPs (USP2, USP5, USP8, USP21, and USP28; IC50 > 32 μM) or other proteases such as calpain-1, caspase 3, and 20S proteasome.125 Another important finding was the identification of 3,5-dichloro-4-pyridylsulfide as a replacement for the substituted phenyl sulfides at the 5-position of the thiophene. This group increased the solubility and USP7 inhibition potency. Compound 17 and its analogs robustly suppress Treg functions, thereby restoring the proliferation and activity of Teff ex vivo. In a recent publication, 17 and P217564 were reported to have potent and long-lasting inhibitory effects on murine Treg function in vitro.67 For instance, 2 h of preincubation of P217564 with Treg almost completely abrogated their ability to suppress Teff cell proliferation ex vivo. These sustained inhibitory effects on Treg function are remarkable given the brief preincubation and are indicative of essentially irreversible USP7 inhibition. In studies using full length USP7 or just its catalytic domain, preincubation with P217564 was seen to prevent its interaction with an active sitedirected probe, Ub-VME. Most importantly, P217564 treatment at 6.0 mg/(kg·day) was shown to decrease the growth of TC1 lung tumors significantly in immunocompetent mice, while having no effect on tumor growth when tested at the same dosing in immunodeficient mice. The lack of activity in immunodeficient mice could be attributed to the relatively lower level of USP7 present in the TC1 tumor cells, which likely made their growth less sensitive to USP7 inhibition. Recently, a new series of thiophene-based USP7 inhibitors have been described as covalent irreversible inhibitors of USP7 for cancer treatment through immune modulation.126 In this new series, some compounds have been designed with the sulfone incorporated into a small ring, which may restrict the motion of the oxygen and coordinate better with the enzyme. Benzothiophene-1,1-dioxide is shown as one example, and it results in 22 (Figure 9), which was demonstrated to have an IC50 of 100 nM. However, the 2−3 double bond of the thiophene ring may be reactive toward Michael additions and thus a potential source of off-target reactivity. Compound 23, the 2,3-dihydrobenzothiophene-1,1-dioxide analog, does not have a vinyl sulfone moiety but inhibits USP7 inhibition with an IC50 of 130 nM, similar to that of 22. Another way of decreasing the Michael acceptor potential of the double bond is to introduce a 3-methyl group. Compound 24 has an IC50 of 170 nM, while 25, the 3-methyl-2,3-dihydro analog, has an IC50 of 75 nM. Compound 26 increases the solubility of 25 by introducing a solubilizing moiety, giving rise to a reduced cLogP (3.7), and can form salts to further increase solubility. Using an LC-MS based analytical method, it was found that

histone methylation, and lipid and glucose metabolism, as well as the activity of the PRC1 complex. In these studies, USP7 knock-down was employed to further corroborate the results obtained using a USP7 inhibitor.71,118−124 However, the nonselective nature of this compound limits its utility as a USP7 inhibitor. The relatively more selective compound 14 is one of the first irreversible USP7 inhibitors reported, and irreversibility may play an important role in the physiological mechanisms of USP7 inhibitors. To date, in vivo antitumor activity has not been described for this compound. 5.2. USP7 Inhibitors from Progenra. Progenra identified 17 (USP7 IC50 = 4.2 μM, Figure 9) from screening a diversitybased library of small molecules for USP7 inhibitors using the ubiquitin-phospholipase A2 enzyme (Ub-PLA2) reporter assay.66,125 Compound 17 is highly selective and exhibits little or no inhibition activity against numerous USP variants (USP2, USP5, USP8, USP15, USP20, USP21, USP28) or other protease classes, such as caspase, cathepsins, calpain, metalloproteases, and serine proteases.66 Of the proteases tested, only USP47, the DUB most closely related to USP7, was found to be as susceptible as USP7 to inhibition by 17. Comparison of the halogen substituents of the 5-arylsulfanyl moiety of the 2acetyl-4-nitro-5-arylsulfanylthiophenes demonstrates that the unsubstituted phenyl analog P22074 (18) is not active as a USP7 antagonist, whereas all of the 5-mono and dihalo phenylsulfanylthiophenes exhibit USP7 inhibitory activity.66 In addition, the dichloro analogs and difluoro analog 19 are more potent than the monochloro analogs. In the cellular context, 17 induces apoptosis in multiple myeloma cells that are resistant to bortezomib therapy,66 is well tolerated, inhibits tumor growth, and prolongs survival in human multiple myeloma and B cell leukemia xenograft models. Further, combinations of 17 with lenalidomide, SAHA (an HDAC inhibitor), or dexamethasone trigger synergistic antitumor activity in multiple myeloma in vitro. These preclinical data demonstrate the efficacy of USP7 inhibitors in multiple myeloma disease models and validate USP7 as a therapeutic target, providing a basis for future clinical studies on USP7 inhibitors in patients resistant to proteasome inhibitors.66 Characterization of 17 in biological matrices was limited, as it produces a very low MS signal at biologically relevant concentrations. Activity-based chemical proteomics was utilized to determine the potency and selectivity of USP7 inhibitors in cultured cell models. The selectivity of 19 in inhibiting USP7 in cells was demonstrated by activity-based quantitative mass spectrometry.109 In contrast, PR-619 (20), a nonselective DUB inhibitor, has been confirmed to be a broad inhibitor of DUB activity.109 Further, 19 inhibits neuroblastoma growth in xenograft models, as demonstrated in two independent laboratories. In the more recent study reported by Tavana et al, 19, administered by ip injection at 20 mg/kg daily for 15 days in an orthotopic neuroblastoma mouse model, showed significant inhibition of xenograft growth.47 The lack of apparent toxicities indicated that the compound was well tolerated in mice. Compound 19 is detectable at relevant concentrations, but it exhibits low recovery from glutathione (GSH) solutions and poor plasma stability in vitro.125 To address issues regarding potency and developability, analogs of 17 were designed and synthesized, leading to improvements in potency, solubility, and metabolic reactivity profile.125 Given that the nitro group could contribute to reactivity by way of thiol displacement, a series of potentially less reactive cyano compounds was 434



Perspective

Figure 10. Chemical structures of USP7 inhibitors from FORMA.

an NMR based fragment screening of 4862 compounds against the USP7 catalytic domain. The primary hits were confirmed to bind to the enzyme active site by X-ray crystallography. Using shape-based virtual screening combined with NMR sitemapping, the expanded hits were found to bind to a unique site on the palm portion of USP7 within a 0.5 nm distance from the catalytic triad. One of their optimization efforts was focused on converting the early stage compound 32 (Figure 11) with two phenol groups into a more “druglike” and potent USP7 inhibitor. A range of phenol isosteres were incorporated to overcome possible pharmacokinetic and toxicological limitations. A substantial loss in USP7 inhibition potency was observed when using nonclassical bioisosteres such as phenyl acetamide, phenyl carbamate, and phenyl methane sulfonamide. A significant finding from this exercise is that one of the two phenol groups could be successfully replaced by either benzamide or 1H indazole with a resultant increase in USP7 potency. Evidence for the involvement of hydrogen bond formation in the success of this bioisosteric replacement with 1H indazole was provided by the lack of activity observed with the N-methylpyrrolo analog. The most potent analog disclosed in the patent is compound 33, which inhibits USP7 with an IC50 of 0.98 μM (Ub-Rho 110 assay) and little or no inhibition of USP5 and USP47. Regarding biological effects, 34 was seen to cause an increase in ubiquityl-MDM2 in SJSA-1 cells at low serum conditions (IC50 = 2 μM). Genentech has pioneered the structure-based approach in discovering novel USP7 inhibitors and has demonstrated their cellular activities. To date, no ADME or in vivo efficacy data have been reported for these compounds. Structure-based approaches have a reasonable potential to yield a new generation of USP7 inhibitors with improved selectivity and, possibly, alternative modes of inhibition, for example, allosteric inhibitors. 5.5. USP7 Inhibitors from China Pharmaceutical University. Considering thiazole as a bioisosteric structure of thiophene with decreased electron density, Chen and colleagues recently focused their attention on converting 17

preincubation of the catalytic core domain from wild-type USP7 enzyme (USP7WT) with active compounds resulted in a covalent addition of part of the inhibitor to the USP7 enzyme at a ratio of 1:1. This covalent modification of the enzyme is dependent on the presence of active site cysteine C223, as mutation of cysteine 223 to alanine (USP7C223A) results in complete elimination of adduct formation. 5.3. USP7 Inhibitors from FORMA Therapeutics. FORMA Therapeutics recently claimed a new family of 4hydroxy piperidines as noncovalent USP7 inhibitors (27−31, Figure 10).127−131 Optimization showed that R could be a wide variety of ring systems (pyrrolopyrimidines, pyrazolopyrimidines, pyrrololotriazinones, imidazotriazinones, quinazolinones, azaquinazolinones, thieno-pyrimidinones, isothiazolopyrimidinones, pyrazolopyrimidinones, and pyrrolopyrimidinones). These R groups were found to be capable of increasing USP7 inhibition potency. The 4-hydroxy piperidine group, which was retained in a majority of the potent compounds, may be an important pharmacophore for USP7. One of FORMA’s most potent compounds, 29, has an IC50 for USP7 < 200 nM (UbRh110 assay). No detailed selectivity or cell data have been reported. 5.4. USP7 Inhibitors from Genentech. In 2016, Genentech Inc. published a patent describing 2-aminopyridine compounds as USP7 inhibitors and claimed their potential value for the treatment of cancer and immune disorders such as inflammation (Figure 11).132 The hit identification started with

Figure 11. Chemical structures of USP7 inhibitors from Genentech.

Figure 12. Chemical structures of USP7 inhibitors from China Pharmaceutical University. 435



Perspective

Figure 13. Chemical structures of USP7 inhibitors from natural sources.

research group from Kumamoto University reported the first natural product USP7 inhibitor, spongiacidin C (39, Figure 13), obtained from screening the extracts of 700 marine invertebrates using the Ub-Rh110 assay.135 Compound 39, a pyrrole alkaloid isolated from the marine sponge Stylissa massa, showed USP7 inhibition activity (IC50 = 3.8 μM). The close structural analogs debromohymenialdisine (40) and hymenialdisine (41) have much weaker inhibitory activities (20% inhibition at 20 μM for both compounds). This result indicates that the presence of a hydantoin ring in 39, rather than an aminoimidazolinone ring in 40 and 41, is essential to USP7 inhibition. A small panel of cysteine proteases were assayed to evaluate selectivity. Compound 39 was found to inhibit USP21 (IC50 = 17 μM) in addition to USP7, but not to inhibit USP2, USP8, or SENP1 over the tested dose range (Cmax = 32 μM). However, 39 did not inhibit growth of HCT116 cells in vitro and its effect on USP7 activity in cells, if any, remains to be determined. Notably, 40 and 41 have been described as nonselective kinase inhibitors.136 Xestoquinone (42, Figure 13), a pentacyclic quinone isolated from the marine sponge Petrosia alfiani, was identified as a potent USP7 inhibitor, with an IC50 of 0.13 μM.137 Together with 42, its structural dimer 43 and trimer 44 were isolated from the same species. Both 43 and 44 showed potent inhibitory activities against USP7 with IC50s of 0.49 and 0.75 μM, respectively. From the point of view of chemical structure, compound 42 possesses two electrophilic reactivity centers, C-1 and either C-14 or C-15, which could be attracted to the cysteine residue of USP7. However, although the C-14 and C15 electrophilic centers were blocked in trimer 44, this compound still showed USP7 inhibitory activity. Wang et al. reported on a screening campaign conducted with pentacyclic triterpenoid natural products to find USP7 inhibitors, using the Ub-AMC assay.138 Six compounds were found to inhibit USP7 in the screen. Among them, ursolic acid (45, Figure 13) was the most potent inhibitor (IC50 = 7.0 μM); this compound also inhibited USP47, but with reduced potency as compared with USP7 (IC50 = 26 μM). Compound 45 was

and 19 (Figure 9) into a new class of thiazole derivative.133 Preliminary SAR studies revealed that the 4-nitro group in the thiazole series seemed to be critical for USP7 potency. Replacement of nitro in compound 35 (Figure 12) with hydrogen made the compound nearly inactive. No other nitro replacement was discussed. Compound 36 (USP7 IC50 = 6.1 μM), the thiazole analog of 19, was shown to be more potent than the parent 19 (USP7 IC50 = 19 μM, Ub-Rho 110 assay). In bioactivity studies, however, it was found that 36 was a less potent inhibitor of HCT-116 cell growth than 19. No biochemical selectivity data were reported. Consistent with findings reported by Progenra, the 3,5-dichloro-4-pyridyl group is retained in the most potent analog 37 (IC50 = 0.67 μM). Results of docking studies indicated that 3,5-dichloro-4-pyridyl of 37 matched in a hydrophobic groove of the catalytic domain of USP7, which consists of series of aromatic amino acid residues. Based on these docking results, it was hypothesized that 19 and 37 bind to the ubiquitin binding pocket to competitively inhibit the binding of ubiquitin to USP7. In a patent application, the inventors from the China Pharmaceutical University group disclosed thiazole derivatives to be useful as antitumor agents.134 In addition to the thiazole compounds discussed in the application, a new series of thiazole compounds were synthesized and tested for their ability to inhibit in vitro proliferation of the human multiple myeloma cell line RPMI-8226. Compound 38 (Figure 12) is a representative example lacking a nitro group. Compared to 37, 38 has a weaker IC50 for growth inhibition (22 μM), which further suggests that electron-withdrawing substituents on the thiazole group are beneficial for anti-USP7 activity. In the same assay, 17 had been shown to be more potent (12 μM) than 38. The thiazole groups of USP7 inhibitors are predicted to function as irreversible inhibitors similar to the nitrothiophene series. No selectivity data and in vivo data have been reported for the thiazole series. 5.6. USP7 Inhibitors from Natural Sources. Recent efforts in the areas of natural product isolation, synthesis, and screening have identified a variety of USP7 inhibitory compounds derived from natural sources. The Tsukamoto 436



Perspective

studies also inhibit USP47 with similar potency. Under such circumstances, it becomes difficult to establish the relative contribution arising from inhibiting individual USPs to the observed antitumor efficacy of a given compound. In addition, potential off-target activity of these two structurally similar USP7 inhibitors against unrelated targets could contribute to antitumor efficacy. On the other hand, while entirely selective USP7 inhibitors may be desirable to limit side effects, inhibiting more than one USP might be beneficial in inhibiting tumor growth. Precedent for such multitarget inhibition can be found in the kinase field, where dual and multiple kinase inhibitors have shown remarkable anticancer properties in the clinic.140,141 Moreover, the only USP inhibitor that has advanced to the Phase I clinical stage is a dual-specific inhibitor of UCHL5 and USP14. While multiple target therapy may be an attractive avenue, establishing a therapeutic window will be critical for the development of multi-USP inhibitors. Structurally guided design and development will be crucial to generating maximally selective USP7 inhibitors. In this regard, knowledge of the physiological form of the active site of USP7 bound to ubiquitin, its substrate (e.g., Tip60 in the case of Treg suppression), and any other proteins with which it may be complexed in cells will be helpful. In addition, as has been pointed out by others in recent summaries of ubiquitin-based therapeutics, fine-tuning of assays and introduction of novel assays with increased physiological relevance will help to identify high quality inhibitors.36,97,142 Both NMR based screening for USP7 selective binders and high throughput screening to identify compounds that selectively inhibit USP7 interaction with specific substrates have the potential to offer valuable starting points for future development of selective USP7 inhibitors. Although USP7 inhibitors have shown in vivo efficacy against a variety of tumors over a range of doses, very limited in vitro ADME and no in vivo pharmacokinetics data have been presented in the literature for these inhibitors. This gap in development was pointed out in a recent review,97 and in the case of USP7, it still remains a challenge, to judge from what is publicly disclosed. The availability of USP7 inhibitors with drug-like and well-defined pharmacokinetics properties will be essential for making progress in the therapeutic arena. These properties, however, should be considered in the context of the mechanism of inhibition of USP7. As pointed out above, both reversible and irreversible inhibitors of the enzyme have been identified. Recently, molecular targeted drugs that are covalent inhibitors of other enzymes, for example, kinases, have been approved by the FDA, signaling a new and expanded way of looking at the developability profile of an experimental drug and putative clinical candidate.143−145 In comparison with reversible inhibitors, selective covalent irreversible USP7 inhibitors offer potential advantages. For example, a relatively short duration of treatment could result in sustained USP7 inhibition and prolonged biological effects, leading to improved therapeutic efficacy.146 Moreover, development of covalent USP7 inhibitors may be governed by pharmacokinetics parameters that differ from those for reversible inhibitors, particularly with respect to half-life and clearance. Given the tissue-wide expression and functional importance of USP7 in normal cells, selective irreversible inhibitors with fast metabolic clearance could help in mitigating potential on-target as well as off-target toxicities. It should be kept in mind, however, that potential immunogenicity of protein adducts resulting from

also shown to bind to USP7 in cells by the use of a cellular thermal shift assay. The same group published results of screening 350 drugs or drug-like compounds for USP7 inhibition, employing the UbAMC protease assay; they identified the synthetic triterpenoid C-28 methyl ester of 2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid (CDDO-Me, 46, Figure 13), also known as bardoxolone methyl, which was found to inhibit USP7 with an IC50 = 14 μM.69 Compound 46 contains an electrophilic Michael acceptor in ring A (Figure 13). Michael addition of proteins having reactive cysteine residues to the reactive α,β-unsaturated double bond of 46 would be expected to form reversible or irreversible covalent bonds and trigger biological activity. However, the corresponding analog with the reduced double bond in ring A inhibits USP7 with similar potency (IC50 = 25 μM). The authors in fact have described 46 as a reversible inhibitor of USP7 whose activity is unrelated to the Michael acceptor of ring A. Selectivity studies showed that 46 also inhibits USP2 (IC50 = 22 μM), but not cathepsin B or cathepsin D (no inhibition at concentrations of 100 μM in both cases). In HO8910 and SKOV3 xenograft model studies, 46 was reported to inhibit USP7 in vivo and to retard tumor growth.69 The diverse molecular targets identified for 46 include Kelch-like ECH-associated protein 1 (Keap1), IκB kinase β (IKKβ), and peroxisome-activator receptor γ (PPARγ). Although 46 was evaluated in the clinic for kidney disease (diabetic nephropathy), the phase III trial was terminated owing to an increased rate of adverse cardiovascular events with 46 over placebo.139 Thus, any clinical development of 46 as a USP7 inhibitor for anticancer applications would be challenging and require mitigation of the adverse cardiac effects. While the data have shown that the specific moieties or functional groups of these natural products affect their USP7 inhibition activities, it is not very clear how these organic molecules interact with the cysteine protease. Thus, detailed SAR studies, computational chemistry studies, and X-ray cocrystal studies would be helpful to explain their mechanism of USP7 inhibition. In addition, since there is some indication that the compounds are exerting predicted USP7-associated activities in cells, the series may be a candidate for further study in terms of PK/ADME properties if potency and selectivity can be improved.

6. PERSPECTIVE Compelling data accumulated over the past decade have established USP7 as an oncology target. In addition to genetic data obtained by knockouts, conditional deletion, and RNAi mediated knockdowns, chemical biology data employing small molecule inhibitors of USP7 have played a major role in validating USP7 as a preclinical therapeutic target in cancers. Whereas USP7 deletions and knockdowns result in either complete or partial removal of USP7 protein, small molecule inhibitors allow fine-tuning of USP7 activity while potentially maintaining its interaction with other proteins. In moving from the stage of developing excellent tool compounds for translational research to that of developing anticancer drugs, however, one of the major challenges lies in developing potent and selective USP7 inhibitors with drug-like properties. To judge from the available literature, this challenge remains. To date, all of the USP7 inhibitors described appear to inhibit one or more related USPs with similar potencies. For example, although they are among the most selective USP7 inhibitors described, compounds 17 and 19 used in several in vivo efficacy 437



■

covalent inhibitors could generate allergic responses or drug hypersensitivity reactions.146 USP7 inhibitors have shown remarkable antitumor efficacy as single agents against a variety of tumor models. As in the case of other protumorigenic proteins, USP7 overexpressed in a tumor establishes a dependence on its presence for the tumor’s growth and, thus, increased susceptibility of the tumor to USP7 inhibitors. A case in point is multiple myeloma, which is characterized by a tremendous level of USP7 overexpression compared to normal cells and exquisite sensitivity to USP7 inhibitor treatment.66 USP7 expression in tumors could serve as a predictive biomarker in future clinical studies. The observation that USP7 inhibitor synergizes with approved cytotoxic chemotherapy agents in vitro in addition to overcoming resistance against bortezomib opens up avenues for exploring the in vivo efficacy of USP7 inhibitors in combination with chemotherapy agents.66 In particular, combinations with radiation, DNA damaging agents,66 kinase inhibitors, or epigenetic modulators24 are likely to yield improved therapies. In addition to its established role in tumor progression, the essential role of USP7 in maintaining Treg cell functions makes it a highly valuable immune-oncology target. Treg are remarkably sensitive to USP7 inhibition due to their unique dependence on USP7 substrates to maintain their suppressive functions. Most important, functions of CD8+ Teff, the critical players in mounting an effective antitumor immune response, remain unaffected by USP7 inhibitor treatment.67 A protein becomes the true substrate of USP7 only when it is ubiquitinated. Therefore, the sensitivity of a particular cell or tissue to USP7 inhibition is likely to depend on the level of USP7 as well as the presence and dynamics of substrate ubiquitination machinery and any compensatory mechanisms. Clearly, additional studies are required to unravel the precise mechanisms behind the differences in sensitivity of various immune effector cell types to USP7 inhibition. From a therapeutic perspective, selective attenuation of Treg functions within the tumor microenvironment would greatly improve antitumor responses elicited by immunotherapies. As such, combination studies with immune checkpoint blockades such as anti-PD1 antibody, anti-CTLA4 antibodies, and vaccines will provide valuable insights into the therapeutic utility of USP7 inhibitors. In summary, the biology, particularly the immune oncology mechanisms recently elucidated, but also the more classical tumoricidal mechanisms, indicates that USP7 inhibitors would be useful drugs. Numerous small molecules representing diverse pharmacophores have been found to have USP7 inhibition properties. The biochemical and cellular characterization as well as in vivo efficacy of lead compounds described in publications and patents must now be complemented by extensive pharmacokinetics, pharmacodynamic, and toxicology studies. Although more challenging, identification and development of substrate-specific and allosteric USP7 inhibitors is likely to yield more specific and targeted therapies.

■

Perspective

AUTHOR INFORMATION

Corresponding Author

*Phone: 484-328-1724. E-mail: [email protected]. ORCID

Michael Mattern: 0000-0002-8595-000X Notes

The authors declare no competing financial interest. Biographies Jian Wu is an Associate Director, R&D, at Progenra, Inc. Dr. Wu has a B.S. degree in Medicinal Chemistry from the China Pharmaceutical University in Nanjing and a Ph.D. degree in Organic Chemistry from Wayne State University, Detroit, MI. After postdoctoral research at the University of Pennsylvania, Dr. Wu joined Progenra in 2009 and has led medicinal chemistry team in the company’s various ubiquitin ligase and deubiquitinase drug discovery projects targeting cancer and other diseases. Suresh Kumar, Senior Director, R&D at Progenra, Inc., has a B.S. degree in Chemistry, Physics, and Mathematics and an M.S. degree in Biochemistry from the University of Kerala, India, and a Ph.D. in Molecular Biology from the Indian Institute of Science, Bangalore. After postdoctoral research at the University of Pennsylvania, Dr. Kumar joined Progenra in 2007 and has been in charge of validating Progenra’s therapeutic targets and providing cell proof of concept for the company’s preclinical compounds. He now heads drug discovery efforts at Progenra. Feng Wang, Assistant Director, R&D, Progenra, Inc., has a B.S. degree in Biology and an M.S. in Biochemistry from Xiamen University, China, and a Ph.D. degree in Microbiology from the University of Texas at Austin, TX. Dr. Wang joined Progenra at the beginning of 2015 and has been working on various projects in cancer- and neurodegenerative disease-based drug discovery. His studies, one of which defined the irreversible mechanism of inhibition of a class of Progenra’s small molecule inhibitors of the deubiquitinating enzyme USP7, are directed at determining mechanism of action and establishing proof of concept for Progenra compounds. Hui Wang, Senior Scientist, Chemistry Department, Progenra, Inc., has a B.S. degree in Applied Chemistry and an M.S. degree in Organic Chemistry from Lanzhou University, China, and a Ph.D. degree in Organic Chemistry from Dartmouth College, New Hampshire. Dr. Wang practiced medicinal chemistry as an Associate Director at PharmaBlock R&D Co. Ltd., in China and at Scripps Research Institute before joining Progenra in 2016 as a principal medicinal chemist. He has been instrumental in synthesizing SAR compounds for Progenra’s USP7 inhibitor program. Lijia Chen, Senior Scientist, Chemistry Department, Progenra Inc., has a B.S. degree in Pharmaceutical Sciences from the China Pharmaceutical University, Nanjing, and a Ph.D. degree in Pharmaceutical Sciences from the University of Maryland School of Pharmacy. In his academic career, Dr. Chen gained experience in medicinal chemistry and structural biology; his dissertation research topic was the synthesis and evaluation of Mcl-1 inhibitors. Having joined Progenra in 2016, Dr. Chen currently works on USP7 inhibitor chemical optimization and rational design.

ASSOCIATED CONTENT

Patrick Arsenault, Assistant Director, R&D, Progenra Inc., has a B.S. degree in Biology from the University of Massachusetts, Amherst, and a Ph.D. degree in Plant Biochemistry and Molecular Biology from the Worcester Polytechnic Institute, Massachusetts. In Dr. Arsenault’s postdoctoral studies at the University of Pennsylvania, he gained considerable experience in drug discovery and preclinical drug development. At Progenra, he leads efforts in assay development

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00498. Molecular formula strings (CSV) 438



Perspective

(10) Cohen, P.; Goedert, M. GSK3 inhibitors: development and therapeutic potential. Nat. Rev. Drug Discovery 2004, 3, 479−487. (11) Isaacs, C.; Herbolsheimer, P.; Liu, M. C.; Wilkinson, M.; Ottaviano, Y.; Chung, G. G.; Warren, R.; Eng-Wong, J.; Cohen, P.; Smith, K. L.; Creswell, K.; Novielli, A.; Slack, R. Phase I/II study of sorafenib with anastrozole in patients with hormone receptor positive aromatase inhibitor resistant metastatic breast cancer. Breast Cancer Res. Treat. 2011, 125, 137−143. (12) Cohen, P.; Tcherpakov, M. Will the ubiquitin system furnish as many drug targets as protein kinases? Cell 2010, 143, 686−693. (13) Ciechanover, A. The ubiquitin-proteasome pathway: on protein death and cell life. EMBO J. 1998, 17, 7151−7160. (14) Hochstrasser, M. Ubiquitin-dependent protein degradation. Annu. Rev. Genet. 1996, 30, 405−439. (15) Yamao, F. Ubiquitin system: selectivity and timing of protein destruction. J. Biochem. 1999, 125, 223−229. (16) Pickart, C. M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 2001, 70, 503−533. (17) Komander, D.; Reyes-Turcu, F.; Licchesi, J. D.; Odenwaelder, P.; Wilkinson, K. D.; Barford, D. Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains. EMBO Rep. 2009, 10, 466−473. (18) Heideker, J.; Wertz, I. E. DUBs, the regulation of cell identity and disease. Biochem. J. 2015, 467, 191. (19) Adams, J. The development of proteasome inhibitors as anticancer drugs. Cancer Cell 2004, 5, 417−421. (20) Meister, S.; Schubert, U.; Neubert, K.; Herrmann, K.; Burger, R.; Gramatzki, M.; Hahn, S.; Schreiber, S.; Wilhelm, S.; Herrmann, M.; Jack, H. M.; Voll, R. E. Extensive immunoglobulin production sensitizes myeloma cells for proteasome inhibition. Cancer Res. 2007, 67, 1783−1792. (21) Cenci, S.; Oliva, L.; Cerruti, F.; Milan, E.; Bianchi, G.; Raule, M.; Mezghrani, A.; Pasqualetto, E.; Sitia, R.; Cascio, P. Pivotal advance: protein synthesis modulates responsiveness of differentiating and malignant plasma cells to proteasome inhibitors. J. Leukocyte Biol. 2012, 92, 921−931. (22) Ratner, M. FDA approves three different multiple myeloma drugs in one month. Nat. Biotechnol. 2016, 34, 126. (23) Anderson, K. C. The 39th David A. Karnofsky Lecture: benchto-bedside translation of targeted therapies in multiple myeloma. J. Clin. Oncol. 2012, 30, 445−452. (24) Das, D. S.; Ray, A.; Das, A.; Song, Y.; Tian, Z.; Oronsky, B.; Richardson, P.; Scicinski, J.; Chauhan, D.; Anderson, K. C. A novel hypoxia-selective epigenetic agent RRx-001 triggers apoptosis and overcomes drug resistance in multiple myeloma cells. Leukemia 2016, 30, 2187−2197. (25) Richardson, P. G.; Mitsiades, C.; Hideshima, T.; Anderson, K. C. Lenalidomide in multiple myeloma. Expert Rev. Anticancer Ther. 2006, 6, 1165−1173. (26) Kojima, K.; Burks, J. K.; Arts, J.; Andreeff, M. The novel tryptamine derivative JNJ-26854165 induces wild-type p53- and E2F1mediated apoptosis in acute myeloid and lymphoid leukemias. Mol. Cancer Ther. 2010, 9, 2545−2557. (27) Tabernero, J.; Dirix, L.; Schoffski, P.; Cervantes, A.; LopezMartin, J. A.; Capdevila, J.; van Beijsterveldt, L.; Platero, S.; Hall, B.; Yuan, Z.; Knoblauch, R.; Zhuang, S. H. A phase I first-in-human pharmacokinetic and pharmacodynamic study of serdemetan in patients with advanced solid tumors. Clin. Cancer Res. 2011, 17, 6313−6321. (28) You, L.; Liu, H.; Huang, J.; Xie, W.; Wei, J.; Ye, X.; Qian, W. The novel anticancer agent JNJ-26854165 is active in chronic myeloid leukemic cells with unmutated BCR/ABL and T315I mutant BCR/ ABL through promoting proteosomal degradation of BCR/ABL proteins. Oncotarget 2017, 8, 7777−7790. (29) Flygare, J. A.; Beresini, M.; Budha, N.; Chan, H.; Chan, I. T.; Cheeti, S.; Cohen, F.; Deshayes, K.; Doerner, K.; Eckhardt, S. G.; Elliott, L. O.; Feng, B.; Franklin, M. C.; Reisner, S. F.; Gazzard, L.; Halladay, J.; Hymowitz, S. G.; La, H.; LoRusso, P.; Maurer, B.; Murray, L.; Plise, E.; Quan, C.; Stephan, J. P.; Young, S. G.; Tom, J.; Tsui, V.;

and high throughput screening for effectors of ubiquitin pathway enzymes for preclinical development. Michael Mattern, Vice President, Progenra, Inc., has a B.S. degree in Chemistry from Muhlenberg College, Pennsylvania, and a Ph.D. degree from Princeton University in Biochemical Sciences and Molecular Biology. Dr. Mattern worked for several years at the Laboratory of Molecular Pharmacology at the National Cancer Institute/NIH, Bethesda MD, in experimental cancer therapeutics and at GSK Pharmaceuticals in Oncology drug discovery before cofounding Progenra, Inc. Joseph Weinstock, Senior Director, Progenra, Inc., is the head of Medicinal Chemistry at Progenra. Dr. Weinstock has a B.S. degree in Chemistry from Rutgers University, NJ, and a Ph.D. in Organic Chemistry from the University of Rochester, NY. He was a Director in the Medicinal Chemistry department of GSK (and legacy organization) R&D for many years and was responsible for the synthesis of several marketed drugs, including the antihypertensive agent Dyazide, the angiotensin II antagonist Tevetan, and the renal vasodilator Fenoldopam.

■

ACKNOWLEDGMENTS This work has been funded in part by Grant Number CA 174037, awarded to Progenra, Inc., by the National Cancer Institute, National Institutes of Health. We thank Dr. David Newman for a critical reading of the manuscript.

■

ABBREVIATIONS USED DUB, deubiquinating enzyme; IC50, concentration required to inhibit 50% of growth or enzyme activity; HAUSP, herpesassociated ubiquitin specific protease; IMiD, immunomodulatory drug; MB, medulloblastoma; MYCN, N-myc gene; NB, neuroblastoma; NSCLC, non-small-cell lung cancer; Shh, sonic hedgehog; Treg, regulatory T cells; Teff, effector T cells; USP7, ubiquitin specific protease 7

■

REFERENCES

(1) Giles, J. Chemistry Nobel for trio who revealed molecular deathtag. Nature 2004, 431, 729. (2) Richardson, P. G.; Anderson, K. C. Bortezomib: a novel therapy approved for multiple myeloma. Clin. Adv. Hematol. Oncol. 2003, 1, 596−600. (3) Cejalvo, M. J.; de la Rubia, J. Which therapies will move to the front line for multiple myeloma? Expert Rev. Hematol. 2017, 10, 383− 392. (4) List, A. F.; Baker, A. F.; Green, S.; Bellamy, W. Lenalidomide: targeted anemia therapy for myelodysplastic syndromes. Cancer Control 2006, 13 (Suppl), 4−11. (5) Gras, J. Pomalidomide for patients with multiple myeloma. Drugs Today 2013, 49, 555−562. (6) Lu, G.; Middleton, R. E.; Sun, H.; Naniong, M.; Ott, C. J.; Mitsiades, C. S.; Wong, K. K.; Bradner, J. E.; Kaelin, W. G., Jr. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science 2014, 343, 305−309. (7) Gandhi, A. K.; Kang, J.; Havens, C. G.; Conklin, T.; Ning, Y.; Wu, L.; Ito, T.; Ando, H.; Waldman, M. F.; Thakurta, A.; Klippel, A.; Handa, H.; Daniel, T. O.; Schafer, P. H.; Chopra, R. Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN.). Br. J. Haematol. 2014, 164, 811−821. (8) Hershko, A.; Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 1998, 67, 425−479. (9) Ciechanover, A. The unravelling of the ubiquitin system. Nat. Rev. Mol. Cell Biol. 2015, 16, 322−324. 439



Perspective

Um, J.; Varfolomeev, E.; Vucic, D.; Wagner, A. J.; Wallweber, H. J.; Wang, L.; Ware, J.; Wen, Z.; Wong, H.; Wong, J. M.; Wong, M.; Wong, S.; Yu, R.; Zobel, K.; Fairbrother, W. J. Discovery of a potent small-molecule antagonist of inhibitor of apoptosis (IAP) proteins and clinical candidate for the treatment of cancer (GDC-0152). J. Med. Chem. 2012, 55, 4101−4113. (30) Andreeff, M.; Kelly, K. R.; Yee, K.; Assouline, S.; Strair, R.; Popplewell, L.; Bowen, D.; Martinelli, G.; Drummond, M. W.; Vyas, P.; Kirschbaum, M.; Iyer, S. P.; Ruvolo, V.; Gonzalez, G. M.; Huang, X.; Chen, G.; Graves, B.; Blotner, S.; Bridge, P.; Jukofsky, L.; Middleton, S.; Reckner, M.; Rueger, R.; Zhi, J.; Nichols, G.; Kojima, K. Results of the Phase I trial of RG7112, a small-molecule MDM2 antagonist in leukemia. Clin. Cancer Res. 2016, 22, 868−876. (31) Patnaik, A.; Tolcher, A.; Beeram, M.; Nemunaitis, J.; Weiss, G. J.; Bhalla, K.; Agrawal, M.; Nichols, G.; Middleton, S.; Beryozkina, A.; Sarapa, N.; Peck, R.; Zhi, J. Clinical pharmacology characterization of RG7112, an MDM2 antagonist, in patients with advanced solid tumors. Cancer Chemother. Pharmacol. 2015, 76, 587−595. (32) Wong, K. M.; Micel, L. N.; Selby, H. M.; Tan, A. C.; Pitts, T. M.; Bagby, S. M.; Spreafico, A.; Klauck, P. J.; Blakemore, S. J.; Smith, P. F.; McDonald, A.; Berger, A.; Tentler, J. J.; Eckhardt, S. G. Targeting the protein ubiquitination machinery in melanoma by the NEDD8-activating enzyme inhibitor pevonedistat (MLN4924). Invest. New Drugs 2017, 35, 11−25. (33) Shah, J. J.; Jakubowiak, A. J.; O’Connor, O. A.; Orlowski, R. Z.; Harvey, R. D.; Smith, M. R.; Lebovic, D.; Diefenbach, C.; Kelly, K.; Hua, Z.; Berger, A. J.; Mulligan, G.; Faessel, H. M.; Tirrell, S.; Dezube, B. J.; Lonial, S. Phase I study of the novel investigational NEDD8activating enzyme inhibitor pevonedistat (MLN4924) in patients with relapsed/refractory multiple myeloma or lymphoma. Clin. Cancer Res. 2016, 22, 34−43. (34) Wang, X.; Mazurkiewicz, M.; Hillert, E. K.; Olofsson, M. H.; Pierrou, S.; Hillertz, P.; Gullbo, J.; Selvaraju, K.; Paulus, A.; Akhtar, S.; Bossler, F.; Khan, A. C.; Linder, S.; D’Arcy, P. The proteasome deubiquitinase inhibitor VLX1570 shows selectivity for ubiquitinspecific protease-14 and induces apoptosis of multiple myeloma cells. Sci. Rep. 2016, 6, 26979. (35) Wang, X.; D’Arcy, P.; Caulfield, T. R.; Paulus, A.; Chitta, K.; Mohanty, C.; Gullbo, J.; Chanan-Khan, A.; Linder, S. Synthesis and evaluation of derivatives of the proteasome deubiquitinase inhibitor bAP15. Chem. Biol. Drug Des. 2015, 86, 1036−1048. (36) Paiva, S. L.; da Silva, S. R.; de Araujo, E. D.; Gunning, P. T. Regulating the master regulator: controlling ubiquitination by thinking outside the active site. J. Med. Chem. 2017, DOI: 10.1021/ acs.jmedchem.6b01346. (37) Everett, R. D.; Meredith, M.; Orr, A.; Cross, A.; Kathoria, M.; Parkinson, J. A novel ubiquitin-specific protease is dynamically associated with the PML nuclear domain and binds to a herpesvirus regulatory protein. EMBO J. 1997, 16, 1519−1530. (38) Nicholson, B.; Suresh Kumar, K. G. The multifaceted roles of USP7: new therapeutic opportunities. Cell Biochem. Biophys. 2011, 60, 61−68. (39) Kim, R. Q.; Sixma, T. K. Regulation of USP7: a high incidence of E3 complexes. J. Mol. Biol. 2017, DOI: 10.1016/j.jmb.2017.05.028. (40) Kon, N.; Kobayashi, Y.; Li, M.; Brooks, C. L.; Ludwig, T.; Gu, W. Inactivation of HAUSP in vivo modulates p53 function. Oncogene 2010, 29, 1270−1279. (41) Kon, N.; Zhong, J.; Kobayashi, Y.; Li, M.; Szabolcs, M.; Ludwig, T.; Canoll, P. D.; Gu, W. Roles of HAUSP-mediated p53 regulation in central nervous system development. Cell Death Differ. 2011, 18, 1366−1375. (42) Hao, Y. H.; Fountain, M. D., Jr.; Fon Tacer, K.; Xia, F.; Bi, W.; Kang, S. H.; Patel, A.; Rosenfeld, J. A.; Le Caignec, C.; Isidor, B.; Krantz, I. D.; Noon, S. E.; Pfotenhauer, J. P.; Morgan, T. M.; Moran, R.; Pedersen, R. C.; Saenz, M. S.; Schaaf, C. P.; Potts, P. R. USP7 acts as a molecular rheostat to promote WASH-dependent endosomal protein recycling and is mutated in a human neurodevelopmental disorder. Mol. Cell 2015, 59, 956−969.

(43) Sanders, S. J.; Ercan-Sencicek, A. G.; Hus, V.; Luo, R.; Murtha, M. T.; Moreno-De-Luca, D.; Chu, S. H.; Moreau, M. P.; Gupta, A. R.; Thomson, S. A.; Mason, C. E.; Bilguvar, K.; Celestino-Soper, P. B.; Choi, M.; Crawford, E. L.; Davis, L.; Wright, N. R.; Dhodapkar, R. M.; DiCola, M.; DiLullo, N. M.; Fernandez, T. V.; Fielding-Singh, V.; Fishman, D. O.; Frahm, S.; Garagaloyan, R.; Goh, G. S.; Kammela, S.; Klei, L.; Lowe, J. K.; Lund, S. C.; McGrew, A. D.; Meyer, K. A.; Moffat, W. J.; Murdoch, J. D.; O’Roak, B. J.; Ober, G. T.; Pottenger, R. S.; Raubeson, M. J.; Song, Y.; Wang, Q.; Yaspan, B. L.; Yu, T. W.; Yurkiewicz, I. R.; Beaudet, A. L.; Cantor, R. M.; Curland, M.; Grice, D. E.; Gunel, M.; Lifton, R. P.; Mane, S. M.; Martin, D. M.; Shaw, C. A.; Sheldon, M.; Tischfield, J. A.; Walsh, C. A.; Morrow, E. M.; Ledbetter, D. H.; Fombonne, E.; Lord, C.; Martin, C. L.; Brooks, A. I.; Sutcliffe, J. S.; Cook, E. H., Jr.; Geschwind, D.; Roeder, K.; Devlin, B.; State, M. W. Multiple recurrent de novo CNVs, including duplications of the 7q11.23 Williams Syndrome region, are strongly associated with autism. Neuron 2011, 70, 863−885. (44) Lecona, E.; Rodriguez-Acebes, S.; Specks, J.; Lopez-Contreras, A. J.; Ruppen, I.; Murga, M.; Munoz, J.; Mendez, J.; FernandezCapetillo, O. USP7 is a SUMO deubiquitinase essential for DNA replication. Nat. Struct. Mol. Biol. 2016, 23, 270−277. (45) Zhang, C.; Lu, J.; Zhang, Q. W.; Zhao, W.; Guo, J. H.; Liu, S. L.; Wu, Y. L.; Jiang, B.; Gao, F. H. USP7 promotes cell proliferation through the stabilization of Ki-67 protein in non-small cell lung cancer cells. Int. J. Biochem. Cell Biol. 2016, 79, 209−221. (46) Zhan, M.; Sun, X.; Liu, J.; Li, Y.; Li, Y.; He, X.; Zhou, Z.; Lu, L. Usp7 promotes medulloblastoma cell survival and metastasis by activating Shh pathway. Biochem. Biophys. Res. Commun. 2017, 484, 429−434. (47) Tavana, O.; Li, D.; Dai, C.; Lopez, G.; Banerjee, D.; Kon, N.; Chen, C.; Califano, A.; Yamashiro, D. J.; Sun, H.; Gu, W. HAUSP deubiquitinates and stabilizes N-Myc in neuroblastoma. Nat. Med. 2016, 22, 1180−1186. (48) Cheng, J.; Yang, H.; Fang, J.; Ma, L.; Gong, R.; Wang, P.; Li, Z.; Xu, Y. Molecular mechanism for USP7-mediated DNMT1 stabilization by acetylation. Nat. Commun. 2015, 6, 7023. (49) Huang, Z.; Wu, Q.; Guryanova, O. A.; Cheng, L.; Shou, W.; Rich, J. N.; Bao, S. Deubiquitylase HAUSP stabilizes REST and promotes maintenance of neural progenitor cells. Nat. Cell Biol. 2011, 13, 142−152. (50) An, T.; Gong, Y.; Li, X.; Kong, L.; Ma, P.; Gong, L.; Zhu, H.; Yu, C.; Liu, J.; Zhou, H.; Mao, B.; Li, Y. USP7 inhibitor P5091 inhibits Wnt signaling and colorectal tumor growth. Biochem. Pharmacol. 2017, 131, 29−39. (51) Wade, M.; Li, Y. C.; Wahl, G. M. MDM2, MDMX and p53 in oncogenesis and cancer therapy. Nat. Rev. Cancer 2013, 13, 83−96. (52) Li, M.; Brooks, C. L.; Kon, N.; Gu, W. A dynamic role of HAUSP in the p53-Mdm2 pathway. Mol. Cell 2004, 13, 879−886. (53) Meulmeester, E.; Maurice, M. M.; Boutell, C.; Teunisse, A. F.; Ovaa, H.; Abraham, T. E.; Dirks, R. W.; Jochemsen, A. G. Loss of HAUSP-mediated deubiquitination contributes to DNA damageinduced destabilization of Hdmx and Hdm2. Mol. Cell 2005, 18, 565−576. (54) Yi, L.; Cui, Y.; Xu, Q.; Jiang, Y. Stabilization of LSD1 by deubiquitinating enzyme USP7 promotes glioblastoma cell tumorigenesis and metastasis through suppression of the p53 signaling pathway. Oncol. Rep. 2016, 36, 2935−2945. (55) Trotman, L. C.; Wang, X.; Alimonti, A.; Chen, Z.; TeruyaFeldstein, J.; Yang, H.; Pavletich, N. P.; Carver, B. S.; Cordon-Cardo, C.; Erdjument-Bromage, H.; Tempst, P.; Chi, S. G.; Kim, H. J.; Misteli, T.; Jiang, X.; Pandolfi, P. P. Ubiquitination regulates PTEN nuclear import and tumor suppression. Cell 2007, 128, 141−156. (56) van der Horst, A.; de Vries-Smits, A. M.; Brenkman, A. B.; van Triest, M. H.; van den Broek, N.; Colland, F.; Maurice, M. M.; Burgering, B. M. FOXO4 transcriptional activity is regulated by monoubiquitination and USP7/HAUSP. Nat. Cell Biol. 2006, 8, 1064−1073. (57) Song, M. S.; Salmena, L.; Carracedo, A.; Egia, A.; Lo-Coco, F.; Teruya-Feldstein, J.; Pandolfi, P. P. The deubiquitinylation and 440



Perspective

localization of PTEN are regulated by a HAUSP-PML network. Nature 2008, 455, 813−817. (58) Cai, J. B.; Shi, G. M.; Dong, Z. R.; Ke, A. W.; Ma, H. H.; Gao, Q.; Shen, Z. Z.; Huang, X. Y.; Chen, H.; Yu, D. D.; Liu, L. X.; Zhang, P. F.; Zhang, C.; Hu, M. Y.; Yang, L. X.; Shi, Y. H.; Wang, X. Y.; Ding, Z. B.; Qiu, S. J.; Sun, H. C.; Zhou, J.; Shi, Y. G.; Fan, J. Ubiquitinspecific protease 7 accelerates p14(ARF) degradation by deubiquitinating thyroid hormone receptor-interacting protein 12 and promotes hepatocellular carcinoma progression. Hepatology 2015, 61, 1603− 1614. (59) Gil, J.; Peters, G. Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all. Nat. Rev. Mol. Cell Biol. 2006, 7, 667−677. (60) Semenza, G. L. Targeting HIF-1 for cancer therapy. Nat. Rev. Cancer 2003, 3, 721−732. (61) Wu, H. T.; Kuo, Y. C.; Hung, J. J.; Huang, C. H.; Chen, W. Y.; Chou, T. Y.; Chen, Y.; Chen, Y. J.; Chen, Y. J.; Cheng, W. C.; Teng, S. C.; Wu, K. J. K63-polyubiquitinated HAUSP deubiquitinates HIF1alpha and dictates H3K56 acetylation promoting hypoxia-induced tumour progression. Nat. Commun. 2016, 7, 13644. (62) Zhou, Z.; Yao, X.; Li, S.; Xiong, Y.; Dong, X.; Zhao, Y.; Jiang, J.; Zhang, Q. Deubiquitination of Ci/Gli by Usp7/HAUSP regulates hedgehog signaling. Dev. Cell 2015, 34, 58−72. (63) Liou, G. Y.; Storz, P. Reactive oxygen species in cancer. Free Radical Res. 2010, 44, 479−496. (64) Bouwman, P.; Jonkers, J. The effects of deregulated DNA damage signalling on cancer chemotherapy response and resistance. Nat. Rev. Cancer 2012, 12, 587−598. (65) Zhao, Y.; Majid, M. C.; Soll, J. M.; Brickner, J. R.; Dango, S.; Mosammaparast, N. Noncanonical regulation of alkylation damage resistance by the OTUD4 deubiquitinase. EMBO J. 2015, 34, 1687− 1703. (66) Chauhan, D.; Tian, Z.; Nicholson, B.; Kumar, K. G.; Zhou, B.; Carrasco, R.; McDermott, J. L.; Leach, C. A.; Fulcinniti, M.; Kodrasov, M. P.; Weinstock, J.; Kingsbury, W. D.; Hideshima, T.; Shah, P. K.; Minvielle, S.; Altun, M.; Kessler, B. M.; Orlowski, R.; Richardson, P.; Munshi, N.; Anderson, K. C. A small molecule inhibitor of ubiquitinspecific protease-7 induces apoptosis in multiple myeloma cells and overcomes bortezomib resistance. Cancer Cell 2012, 22, 345−358. (67) Wang, L.; Kumar, S.; Dahiya, S.; Wang, F.; Wu, J.; Newick, K.; Han, R.; Samanta, A.; Beier, U. H.; Akimova, T.; Bhatti, T. R.; Nicholson, B.; Kodrasov, M. P.; Agarwal, S.; Sterner, D. E.; Gu, W.; Weinstock, J.; Butt, T. R.; Albelda, S. M.; Hancock, W. W. Ubiquitinspecific Protease-7 inhibition impairs Tip60-dependent Foxp3+ Tregulatory cell function and promotes antitumor immunity. EBioMedicine 2016, 13, 99−112. (68) Fan, Y. H.; Cheng, J.; Vasudevan, S. A.; Dou, J.; Zhang, H.; Patel, R. H.; Ma, I. T.; Rojas, Y.; Zhao, Y.; Yu, Y.; Zhang, H.; Shohet, J. M.; Nuchtern, J. G.; Kim, E. S.; Yang, J. USP7 inhibitor P22077 inhibits neuroblastoma growth via inducing p53-mediated apoptosis. Cell Death Dis. 2013, 4, e867. (69) Qin, D.; Wang, W.; Lei, H.; Luo, H.; Cai, H.; Tang, C.; Wu, Y.; Wang, Y.; Jin, J.; Xiao, W.; Wang, T.; Ma, C.; Xu, H.; Zhang, J.; Gao, F.; Wu, Y. L. CDDO-Me reveals USP7 as a novel target in ovarian cancer cells. Oncotarget 2016, 7, 77096−77109. (70) Zhang, L.; Wang, H.; Tian, L.; Li, H. Expression of USP7 and MARCH7 is correlated with poor prognosis in epithelial ovarian cancer. Tohoku J. Exp. Med. 2016, 239, 165−175. (71) Wang, Q.; Ma, S.; Song, N.; Li, X.; Liu, L.; Yang, S.; Ding, X.; Shan, L.; Zhou, X.; Su, D.; Wang, Y.; Zhang, Q.; Liu, X.; Yu, N.; Zhang, K.; Shang, Y.; Yao, Z.; Shi, L. Stabilization of histone demethylase PHF8 by USP7 promotes breast carcinogenesis. J. Clin. Invest. 2016, 126, 2205−2220. (72) Zhao, G. Y.; Lin, Z. W.; Lu, C. L.; Gu, J.; Yuan, Y. F.; Xu, F. K.; Liu, R. H.; Ge, D.; Ding, J. Y. USP7 overexpression predicts a poor prognosis in lung squamous cell carcinoma and large cell carcinoma. Tumor Biol. 2015, 36, 1721−1729.

(73) Samkari, A.; White, J.; Packer, R. Shh inhibitors for the treatment of medulloblastoma. Expert Rev. Neurother. 2015, 15, 763− 770. (74) Woo, E. Y.; Chu, C. S.; Goletz, T. J.; Schlienger, K.; Yeh, H.; Coukos, G.; Rubin, S. C.; Kaiser, L. R.; June, C. H. Regulatory CD4(+)CD25(+) T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer. Cancer Res. 2001, 61, 4766−4772. (75) Petersen, R. P.; Campa, M. J.; Sperlazza, J.; Conlon, D.; Joshi, M. B.; Harpole, D. H., Jr.; Patz, E. F., Jr. Tumor infiltrating Foxp3+ regulatory T-cells are associated with recurrence in pathologic stage I NSCLC patients. Cancer 2006, 107, 2866−2872. (76) O’Callaghan, D. S.; Rexhepaj, E.; Gately, K.; Coate, L.; Delaney, D.; O’Donnell, D. M.; Kay, E.; O’Connell, F.; Gallagher, W. M.; O’Byrne, K. J. Tumour islet Foxp3+ T-cell infiltration predicts poor outcome in nonsmall cell lung cancer. Eur. Respir. J. 2015, 46, 1762− 1772. (77) Huang, Y.; Wang, F. M.; Wang, T.; Wang, Y. J.; Zhu, Z. Y.; Gao, Y. T.; Du, Z. Tumor-infiltrating FoxP3+ Tregs and CD8+ T cells affect the prognosis of hepatocellular carcinoma patients. Digestion 2012, 86, 329−337. (78) Shou, J.; Zhang, Z.; Lai, Y.; Chen, Z.; Huang, J. Worse outcome in breast cancer with higher tumor-infiltrating FOXP3+ Tregs: a systematic review and meta-analysis. BMC Cancer 2016, 16, 687. (79) Golovina, T. N.; Vonderheide, R. H. Regulatory T cells: overcoming suppression of T-cell immunity. Cancer J. 2010, 16, 342− 347. (80) Ireland, D. J.; Kissick, H. T.; Beilharz, M. W. The role of regulatory T Cells in mesothelioma. Cancer Microenviron. 2012, 5, 165−172. (81) Schabowsky, R. H.; Madireddi, S.; Sharma, R.; Yolcu, E. S.; Shirwan, H. Targeting CD4+CD25+FoxP3+ regulatory T-cells for the augmentation of cancer immunotherapy. Curr. Opin. Invest. Drugs 2007, 8, 1002−1008. (82) Wang, R. F. Functional control of regulatory T cells and cancer immunotherapy. Semin. Cancer Biol. 2006, 16, 106−114. (83) Williams, L. M.; Rudensky, A. Y. Maintenance of the Foxp3dependent developmental program in mature regulatory T cells requires continued expression of Foxp3. Nat. Immunol. 2007, 8, 277− 284. (84) Fontenot, J. D.; Gavin, M. A.; Rudensky, A. Y. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 2003, 4, 330−336. (85) Chatila, T. A.; Blaeser, F.; Ho, N.; Lederman, H. M.; Voulgaropoulos, C.; Helms, C.; Bowcock, A. M. JM2, encoding a fork head-related protein, is mutated in X-linked autoimmunity-allergic disregulation syndrome. J. Clin. Invest. 2000, 106, R75−81. (86) Xiao, Y.; Nagai, Y.; Deng, G.; Ohtani, T.; Zhu, Z.; Zhou, Z.; Zhang, H.; Ji, M. Q.; Lough, J. W.; Samanta, A.; Hancock, W. W.; Greene, M. I. Dynamic interactions between TIP60 and p300 regulate FOXP3 function through a structural switch defined by a single lysine on TIP60. Cell Rep. 2014, 7, 1471−1480. (87) van Loosdregt, J.; Fleskens, V.; Fu, J.; Brenkman, A. B.; Bekker, C. P.; Pals, C. E.; Meerding, J.; Berkers, C. R.; Barbi, J.; Grone, A.; Sijts, A. J.; Maurice, M. M.; Kalkhoven, E.; Prakken, B. J.; Ovaa, H.; Pan, F.; Zaiss, D. M.; Coffer, P. J. Stabilization of the transcription factor Foxp3 by the deubiquitinase USP7 increases Treg-cellsuppressive capacity. Immunity 2013, 39, 259−271. (88) Wang, L.; Liu, Y.; Beier, U. H.; Han, R.; Bhatti, T. R.; Akimova, T.; Hancock, W. W. Foxp3+ T-regulatory cells require DNA methyltransferase 1 expression to prevent development of lethal autoimmunity. Blood 2013, 121, 3631−3639. (89) Obata, Y.; Furusawa, Y.; Endo, T. A.; Sharif, J.; Takahashi, D.; Atarashi, K.; Nakayama, M.; Onawa, S.; Fujimura, Y.; Takahashi, M.; Ikawa, T.; Otsubo, T.; Kawamura, Y. I.; Dohi, T.; Tajima, S.; Masumoto, H.; Ohara, O.; Honda, K.; Hori, S.; Ohno, H.; Koseki, H.; Hase, K. The epigenetic regulator Uhrf1 facilitates the proliferation and maturation of colonic regulatory T cells. Nat. Immunol. 2014, 15, 571−579. 441



Perspective

(90) Pacholczyk, R.; Kern, J. The T-cell receptor repertoire of regulatory T cells. Immunology 2008, 125, 450−458. (91) Pfoh, R.; Lacdao, I. K.; Georges, A. A.; Capar, A.; Zheng, H.; Frappier, L.; Saridakis, V. Crystal structure of USP7 ubiquitin-like domains with an ICP0 peptide reveals a novel mechanism used by viral and cellular proteins to target USP7. PLoS Pathog. 2015, 11, e1004950. (92) Reddy, B. A.; van der Knaap, J. A.; Bot, A. G.; Mohd-Sarip, A.; Dekkers, D. H.; Timmermans, M. A.; Martens, J. W.; Demmers, J. A.; Verrijzer, C. P. Nucleotide biosynthetic enzyme GMP synthase is a TRIM21-controlled relay of p53 stabilization. Mol. Cell 2014, 53, 458− 470. (93) Faesen, A. C.; Dirac, A. M.; Shanmugham, A.; Ovaa, H.; Perrakis, A.; Sixma, T. K. Mechanism of USP7/HAUSP activation by its C-terminal ubiquitin-like domain and allosteric regulation by GMPsynthetase. Mol. Cell 2011, 44, 147−159. (94) Sheng, Y.; Saridakis, V.; Sarkari, F.; Duan, S.; Wu, T.; Arrowsmith, C. H.; Frappier, L. Molecular recognition of p53 and MDM2 by USP7/HAUSP. Nat. Struct. Mol. Biol. 2006, 13, 285−291. (95) Hu, M.; Gu, L.; Li, M.; Jeffrey, P. D.; Gu, W.; Shi, Y. Structural basis of competitive recognition of p53 and MDM2 by HAUSP/USP7: implications for the regulation of the p53-MDM2 pathway. PLoS Biol. 2006, 4, e27. (96) Ma, J.; Martin, J. D.; Xue, Y.; Lor, L. A.; Kennedy-Wilson, K. M.; Sinnamon, R. H.; Ho, T. F.; Zhang, G.; Schwartz, B.; Tummino, P. J.; Lai, Z. C-terminal region of USP7/HAUSP is critical for deubiquitination activity and contains a second mdm2/p53 binding site. Arch. Biochem. Biophys. 2010, 503, 207−212. (97) Ndubaku, C.; Tsui, V. Inhibiting the deubiquitinating enzymes (DUBs). J. Med. Chem. 2015, 58, 1581−1595. (98) Kemp, M. Recent advances in the discovery of deubiquitinating enzyme inhibitors. Prog. Med. Chem. 2016, 55, 149−192. (99) Hu, M.; Li, P.; Li, M.; Li, W.; Yao, T.; Wu, J. W.; Gu, W.; Cohen, R. E.; Shi, Y. Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde. Cell 2002, 111, 1041−1054. (100) Komander, D.; Clague, M. J.; Urbe, S. Breaking the chains: structure and function of the deubiquitinases. Nat. Rev. Mol. Cell Biol. 2009, 10, 550−563. (101) Dang, L. C.; Melandri, F. D.; Stein, R. L. Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido4-methylcoumarin by deubiquitinating enzymes. Biochemistry 1998, 37, 1868−1879. (102) Hassiepen, U.; Eidhoff, U.; Meder, G.; Bulber, J. F.; Hein, A.; Bodendorf, U.; Lorthiois, E.; Martoglio, B. A sensitive fluorescence intensity assay for deubiquitinating proteases using ubiquitin-rhodamine110-glycine as substrate. Anal. Biochem. 2007, 371, 201−207. (103) Orcutt, S. J.; Wu, J.; Eddins, M. J.; Leach, C. A.; Strickler, J. E. Bioluminescence assay platform for selective and sensitive detection of Ub/Ubl proteases. Biochim. Biophys. Acta, Mol. Cell Res. 2012, 1823, 2079−2086. (104) Nicholson, B.; Leach, C. A.; Goldenberg, S. J.; Francis, D. M.; Kodrasov, M. P.; Tian, X.; Shanks, J.; Sterner, D. E.; Bernal, A.; Mattern, M. R.; Wilkinson, K. D.; Butt, T. R. Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities. Protein Sci. 2008, 17, 1035−1043. (105) Tian, X.; Isamiddinova, N. S.; Peroutka, R. J.; Goldenberg, S. J.; Mattern, M. R.; Nicholson, B.; Leach, C. Characterization of selective ubiquitin and ubiquitin-like protease inhibitors using a fluorescencebased multiplex assay format. Assay Drug Dev. Technol. 2011, 9, 165− 173. (106) Geurink, P. P.; El Oualid, F.; Jonker, A.; Hameed, D. S.; Ovaa, H. A general chemical ligation approach towards isopeptide-linked ubiquitin and ubiquitin-like assay reagents. ChemBioChem 2012, 13, 293−297. (107) Bozza, W. P.; Liang, Q.; Gong, P.; Zhuang, Z. Transient kinetic analysis of USP2-catalyzed deubiquitination reveals a conformational rearrangement in the K48-linked diubiquitin substrate. Biochemistry 2012, 51, 10075−10086.

(108) Ritorto, M. S.; Ewan, R.; Perez-Oliva, A. B.; Knebel, A.; Buhrlage, S. J.; Wightman, M.; Kelly, S. M.; Wood, N. T.; Virdee, S.; Gray, N. S.; Morrice, N. A.; Alessi, D. R.; Trost, M. Screening of DUB activity and specificity by MALDI-TOF mass spectrometry. Nat. Commun. 2014, 5, 4763. (109) Altun, M.; Kramer, H. B.; Willems, L. I.; McDermott, J. L.; Leach, C. A.; Goldenberg, S. J.; Kumar, K. G.; Konietzny, R.; Fischer, R.; Kogan, E.; Mackeen, M. M.; McGouran, J.; Khoronenkova, S. V.; Parsons, J. L.; Dianov, G. L.; Nicholson, B.; Kessler, B. M. Activitybased chemical proteomics accelerates inhibitor development for deubiquitylating enzymes. Chem. Biol. 2011, 18, 1401−1412. (110) Jafari, R.; Almqvist, H.; Axelsson, H.; Ignatushchenko, M.; Lundback, T.; Nordlund, P.; Martinez Molina, D. The cellular thermal shift assay for evaluating drug target interactions in cells. Nat. Protoc. 2014, 9, 2100−2122. (111) Colland, F.; Formstecher, E.; Jacq, X.; Reverdy, C.; Planquette, C.; Conrath, S.; Trouplin, V.; Bianchi, J.; Aushev, V. N.; Camonis, J.; Calabrese, A.; Borg-Capra, C.; Sippl, W.; Collura, V.; Boissy, G.; Rain, J. C.; Guedat, P.; Delansorne, R.; Daviet, L. Small-molecule inhibitor of USP7/HAUSP ubiquitin protease stabilizes and activates p53 in cells. Mol. Cancer Ther. 2009, 8, 2286−2295. (112) Colombo, M.; Vallese, S.; Peretto, I.; Jacq, X.; Rain, J. C.; Colland, F.; Guedat, P. Synthesis and biological evaluation of 9-oxo9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile analogues as potential inhibitors of deubiquitinating enzymes. ChemMedChem 2010, 5, 552−558. (113) Lopez, R.; Collura, V.; Wolfgang, S.; Colland, F. Novel Specific Inhibitors of Ubiquitin Specific Protease 7, the Pharmaceutical Compositions thereof and their Therapeutic Applications. International Patent WO2010081783, 2010. (114) Reverdy, C.; Conrath, S.; Lopez, R.; Planquette, C.; Atmanene, C.; Collura, V.; Harpon, J.; Battaglia, V.; Vivat, V.; Sippl, W.; Colland, F. Discovery of specific inhibitors of human USP7/HAUSP deubiquitinating enzyme. Chem. Biol. 2012, 19, 467−477. (115) Menard, R.; Sulea, T. Selective inhibition of USP7. Chem. Biol. 2012, 19, 437−438. (116) Colland, F.; Gourdel, M.-E. Selective and Reversible Inhibitors of Ubiquitin Specific Protease 7. International Patent WO2013030218, 2013. (117) Kessler, B. M. Selective and reversible inhibitors of ubiquitinspecific protease 7: a patent evaluation (WO2013030218). Expert Opin. Ther. Pat. 2014, 24, 597−602. (118) Zaman, M. M.; Nomura, T.; Takagi, T.; Okamura, T.; Jin, W.; Shinagawa, T.; Tanaka, Y.; Ishii, S. Ubiquitination-deubiquitination by the TRIM27-USP7 complex regulates tumor necrosis factor alphainduced apoptosis. Mol. Cell. Biol. 2013, 33, 4971−4984. (119) He, J.; Zhu, Q.; Wani, G.; Sharma, N.; Han, C.; Qian, J.; Pentz, K.; Wang, Q. E.; Wani, A. A. Ubiquitin-specific protease 7 regulates nucleotide excision repair through deubiquitinating XPC protein and preventing XPC protein from undergoing ultraviolet light-induced and VCP/p97 protein-regulated proteolysis. J. Biol. Chem. 2014, 289, 27278−27289. (120) Qian, J.; Pentz, K.; Zhu, Q.; Wang, Q.; He, J.; Srivastava, A. K.; Wani, A. A. USP7 modulates UV-induced PCNA monoubiquitination by regulating DNA polymerase eta stability. Oncogene 2015, 34, 4791− 4796. (121) Zhu, Q.; Sharma, N.; He, J.; Wani, G.; Wani, A. A. USP7 deubiquitinase promotes ubiquitin-dependent DNA damage signaling by stabilizing RNF168. Cell Cycle 2015, 14, 1413−1425. (122) Hirano, A.; Nakagawa, T.; Yoshitane, H.; Oyama, M.; KozukaHata, H.; Lanjakornsiripan, D.; Fukada, Y. USP7 and TDP-43: pleiotropic regulation of cryptochrome protein stability paces the oscillation of the mammalian circadian clock. PLoS One 2016, 11, e0154263. (123) Lee, K. W.; Cho, J. G.; Kim, C. M.; Kang, A. Y.; Kim, M.; Ahn, B. Y.; Chung, S. S.; Lim, K. H.; Baek, K. H.; Sung, J. H.; Park, K. S.; Park, S. G. Herpesvirus-associated ubiquitin-specific protease (HAUSP) modulates peroxisome proliferator-activated receptor 442



Perspective

gamma (PPARgamma) stability through its deubiquitinating activity. J. Biol. Chem. 2013, 288, 32886−32896. (124) Lecona, E.; Narendra, V.; Reinberg, D. USP7 cooperates with SCML2 to regulate the activity of PRC1. Mol. Cell. Biol. 2015, 35, 1157−1168. (125) Weinstock, J.; Wu, J.; Cao, P.; Kingsbury, W. D.; McDermott, J. L.; Kodrasov, M. P.; McKelvey, D. M.; Suresh Kumar, K. G.; Goldenberg, S. J.; Mattern, M. R.; Nicholson, B. Selective dual inhibitors of the cancer-related deubiquitylating proteases USP7 and USP47. ACS Med. Chem. Lett. 2012, 3, 789−792. (126) Weinstock, J.; Wu, J.; Kumar, K. G. S.; Wang, F.; Kodrasov, M. P.; Agarwal, S. Covalent Irreversible Inhibitors of USP7 as Anti-Cancer Agents. International Patent WO2017023684, 2017. (127) Ioannidis, S.; Talbot, A. C.; Follows, B.; Buckmelter, A. J.; Wang, M.; Campbell, A.-M.; Schmidt, D. R.; Guerin, D. J.; Caravella, J. A.; Diebold, R. B.; Ericsson, A.; Lancia, D., Jr. Preparation of Pyrrolo and Pyrazolopyrimidines as Ubiquitin-Specific Protease 7 Inhibitors. International Patent WO2016109515, 2016. (128) Ioannidis, S.; Talbot, A. C.; Follows, B.; Buckmelter, A. J.; Wang, M.; Campbell, A.-M.; Schmidt, D. R.; Guerin, D. J.; Caravella, J. A.; Diebold, R. B.; Ericsson, A.; Lancia, D., Jr. Preparation of Pyrrolotriazinones and Imidazotriazinones as Ubiquitin-Specific Protease 7 Inhibitors. International Patent WO2016109480, 2016. (129) Ioannidis, S.; Talbot, A. C.; Follows, B.; Buckmelter, A. J.; Wang, M.; Campbell, A.-M.; Lancia, D., Jr. Preparation of (Aza)quinazolinones as Ubiquitin-Specific Protease 7 Inhibitors. International Patent WO2016126926, 2016. (130) Ioannidis, S.; Talbot, A. C.; Follows, B.; Buckmelter, A. J.; Wang, M.; Campbell, A.-M.; Lancia, D., Jr. Preparation of Thienopyrimidinones as Ubiquitin-Specific Protease 7 Inhibitors. International Patent WO2016126929, 2016. (131) Ioannidis, S.; Talbot, A. C.; Follows, B.; Buckmelter, A. J.; Wang, M.; Campbell, A.-M. Isothiazolopyrimidinones, Pyrazolopyrimidinones, and Pyrrolopyrimidinones as Ubiquitin-Specific Protease 7 Inhibitors. International Patent WO2016126935, 2016. (132) Blake, R.; Di, L. P.; Drummond, J.; Heideker, C. J.; Kategaya, L.; Maurer, T.; Murray, J. M.; Ndubaku, C.; Pastor, R.; Rouge, L.; Tsui, V.; Wertz, I. E.; Yu, K. Arylpyridinamines as USP7 Inhibitor Compounds and their Preparation. U.S. Patent US20160272588, 2016. (133) Chen, C.; Song, J.; Wang, J.; Xu, C.; Chen, C.; Gu, W.; Sun, H.; Wen, X. Synthesis and biological evaluation of thiazole derivatives as novel USP7 inhibitors. Bioorg. Med. Chem. Lett. 2017, 27, 845−849. (134) Sun, H.; Song, J.; Chen, C.; Wang, J.; Xu, C.; Wen, X.; Gu, W. Thiazole Compounds as Antitumor Agents and their Preparation, Pharmaceutical Compositions and Use in the Treatment of Cancer. International Patent WO2015127797, 2015. (135) Yamaguchi, M.; Miyazaki, M.; Kodrasov, M. P.; Rotinsulu, H.; Losung, F.; Mangindaan, R. E.; de Voogd, N. J.; Yokosawa, H.; Nicholson, B.; Tsukamoto, S. Spongiacidin C, a pyrrole alkaloid from the marine sponge Stylissa massa, functions as a USP7 inhibitor. Bioorg. Med. Chem. Lett. 2013, 23, 3884−3886. (136) Meijer, L.; Thunnissen, A. M.; White, A. W.; Garnier, M.; Nikolic, M.; Tsai, L. H.; Walter, J.; Cleverley, K. E.; Salinas, P. C.; Wu, Y. Z.; Biernat, J.; Mandelkow, E. M.; Kim, S. H.; Pettit, G. R. Inhibition of cyclin-dependent kinases, GSK-3beta and CK1 by hymenialdisine, a marine sponge constituent. Chem. Biol. 2000, 7, 51−63. (137) Tanokashira, N.; Kukita, S.; Kato, H.; Nehira, T.; Angkouw, E. D.; Mangindaan, R. E. P.; de Voogd, N. J.; Tsukamoto, S. Petroquinones: trimeric and dimeric Xestoquinone derivatives isolated from the marine sponge Petrosia alfiani. Tetrahedron 2016, 72, 5530− 5540. (138) Wang, W.; Wu, Y.; Liu, M.; Jing, B. Application of Pentacyclic Triterpenoid in Preparation of USP7 Inhibitor. China Patent CN105534991A, 2016. (139) Wang, Y. Y.; Yang, Y. X.; Zhe, H.; He, Z. X.; Zhou, S. F. Bardoxolone methyl (CDDO-Me) as a therapeutic agent: an update on its pharmacokinetic and pharmacodynamic properties. Drug Des., Dev. Ther. 2014, 8, 2075−2088.

(140) Wilhelm, S.; Carter, C.; Lynch, M.; Lowinger, T.; Dumas, J.; Smith, R. A.; Schwartz, B.; Simantov, R.; Kelley, S. Discovery and development of sorafenib: a multikinase inhibitor for treating cancer. Nat. Rev. Drug Discovery 2006, 5, 835−844. (141) Broekman, F.; Giovannetti, E.; Peters, G. J. Tyrosine kinase inhibitors: multi-targeted or single-targeted? World J. Clin. Oncol. 2011, 2, 80−93. (142) Goldenberg, S. J.; McDermott, J. L.; Butt, T. R.; Mattern, M. R.; Nicholson, B. Strategies for the identification of novel inhibitors of deubiquitinating enzymes. Biochem. Soc. Trans. 2008, 36, 828−832. (143) Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The resurgence of covalent drugs. Nat. Rev. Drug Discovery 2011, 10, 307−317. (144) Burger, J. A.; Tedeschi, A.; Barr, P. M.; Robak, T.; Owen, C.; Ghia, P.; Bairey, O.; Hillmen, P.; Bartlett, N. L.; Li, J.; Simpson, D.; Grosicki, S.; Devereux, S.; McCarthy, H.; Coutre, S.; Quach, H.; Gaidano, G.; Maslyak, Z.; Stevens, D. A.; Janssens, A.; Offner, F.; Mayer, J.; O’Dwyer, M.; Hellmann, A.; Schuh, A.; Siddiqi, T.; Polliack, A.; Tam, C. S.; Suri, D.; Cheng, M.; Clow, F.; Styles, L.; James, D. F.; Kipps, T. J. Ibrutinib as initial therapy for patients with chronic lymphocytic leukemia. N. Engl. J. Med. 2015, 373, 2425−2437. (145) Roskoski, R., Jr. Classification of small molecule protein kinase inhibitors based upon the structures of their drug-enzyme complexes. Pharmacol. Res. 2016, 103, 26−48. (146) Johnson, D. S.; Weerapana, E.; Cravatt, B. F. Strategies for discovering and derisking covalent, irreversible enzyme inhibitors. Future Med. Chem. 2010, 2, 949−964.

443


Chemical Approaches to Intervening in Ubiquitin Specific Protease 7

Recommend Documents