Seminars in Cell and Developmental Biology 132 (2022) 213–229 Contents lists available at ScienceDirect Seminars in Cell and Developmental Biology journal homepage: www.elsevier.com/locate/semcdb Recent advances in the pharmacological targeting of ubiquitin-regulating enzymes in cancer Ana Maria Montagut a, 1, Marc Armengol b, 1, Gema Gorjón de Pablo a, Roger Estrada-Tejedor a, José Ignacio Borrell a, *, Gaël Roué b, * a Grup de Química Farmacèutica, IQS School of Engineering, Universitat Ramon Llull, Barcelona, Spain b Lymphoma Translational Group, UBIRed, Josep Carreras Leukaemia Research Institute, Badalona, Spain A R T I C L E I N F O A B S T R A C T Keywords: As a post-translational modification that has pivotal roles in protein degradation, ubiquitination ensures that Ubiquitin ligase intracellular proteins act in a precise spatial and temporal manner to regulate diversified cellular processes. Deubiquitinase Perturbation of the ubiquitin system contributes directly to the onset and progression of a wide variety of dis­ Cancer eases, including various subtypes of cancer. This highly regulated system has been for years an active research Structure-based drug design area for drug discovery that is exemplified by several approved drugs. In this review, we will provide an update Preclinical screening Clinical trial of the main breakthrough scientific discoveries that have been leading the clinical development of ubiquitin- targeting therapies in the last decade, with a special focus on E1 and E3 modulators. We will further discuss the unique challenges of identifying new potential therapeutic targets within this ubiquitous and highly complex machinery, based on available crystallographic structures, and explore chemical approaches by which these challenges might be met. 1. Introduction: aberrant ubiquitin signaling and cancer in both physiological and malignant settings [8–10]. Structurally, E3s can be divided into ‘really interesting new gene’ The ubiquitin-proteasome system (UPS), which elementary compo­ (RING) class, the ‘homologous to E6-AP carboxy-terminus’ (HECT) class nents are the 26 S proteasome and the small ubiquitin molecule, is a which forms a thioester bond with ubiquitin and then conjugates it to major protein degradation system that regulates a number of cellular the substrate, and the ‘RING-between-RING’ (RBR) class which have a functions that are critical to eukaryote cell homeostasis, such as cell RING1-in-between RING-RING2 motif [11]. cycle [1], apoptosis [2], DNA damage [3] and immune functions [4]. Together with ubiquitin ligase activity, deubiquitination is also a Ubiquitination refers to the enzymatic post-translational modification complex and dynamic process in which enzymes known as deubiquiti­ (PTM) in which the ubiquitin protein is covalently attached to cellular nases (DUBs) are responsible for removing ubiquitin from a substrate. In proteins. This process takes place in three steps, ending with the ubiq­ this process, the isopeptide bond between ubiquitin and its substrate can uitin union to lysine residues on the target protein [5]. The core enzymes be cleaved by the specific DUBs to produce monoubiquitin for recycling regulating this process are the ubiquitin-activating enzyme (UAE or E1), [12]. More than 100 DUBs have been discovered so far, classified into the ubiquitin-conjugating enzyme (UBC or E2), and the ubiquitin ligase five different subfamilies based on the presence of conserved catalytic (E3) [6]. There are two known E1 activating enzyme genes (UBA1 domains: the ubiquitin-specific proteases (USP), ovarian tumor domain (UBE1) and UBA6 (UBE6)), about 50 E2 enzymes, and more than 600 E3 (OTU), Machado–Joseph domain (MJD), ubiquitin-C terminal hydro­ ligases encoded in the human genome [7]. As E3 enzymes ultimately lases (UCH), and the Jab1/MPN (JAMM) metalloproteases [13]. Thanks determine the target of ubiquitination and the specificity of substrate to the balanced action of both ubiquitin ligases and deubiquitinases, recognition, they mediate either proteasomal degradation or cells are able to adapt their proteome in response to a variety of cellular non-degradative signaling and therefore play a critical role in the and environmental factors. functionality of the UPS and in the regulation of key intracellular players Accumulating evidence indicates that cancer cells are highly * Corresponding authors. E-mail addresses: j.i.borrell@iqs.url.edu (J.I. Borrell), groue@carrerasresearch.org (G. Roué). 1 These authors contributed equally https://doi.org/10.1016/j.semcdb.2022.02.007 Received 6 November 2021; Received in revised form 4 February 2022; Accepted 7 February 2022 Available online 17 February 2022 1084-9521/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). A.M. Montagut et al. Seminars in Cell and Developmental Biology 132 (2022) 213–229 Fig. 1. Impact of the ubiquitin system in tumorigenesis. Insulin receptor substrate 1 (IRS-1), Phosphatidylinositol 4,5-bisphosphate (PIP2), Phosphatidylinositol (3,4,5)-trisphosphate (PIP3), Phosphoinositide 3-kinase (PI3K), Phosphatase and tensin homolog (PTEN), Protein kinase B (AKT), Tumor necrosis factor receptor (TNFR), Receptor-interacting protein kinase 1 (RIPK1), cellular inhibitor of apoptosis protein-1/2 (cIAP1/2), TNF Receptor Associated Factor 2/6 (TRAF2/6), NF-κB essential modulator (NEMO), linear ubiquitin chain assembly complex (LUBAC), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IKBα), Skp1-cullin 1-F-box β-Transducin Repeat-Containing Protein (SCFβ-TrCP), X-linked inhibitor of apoptosis protein (XIAP), WW domain-containing protein 1/2 (WWP1/2), Ovarian tumor proteases domain-containing deubiquitinase with linear linkage specificity (OTULIN), Ubiquitin-specific proteases 7/10/36/37 (USP7/ 10/36/37), F-box/WD repeat-containing protein 7 (FBXW7), S-phase kinase associated protein 2 (SKP2), Cereblon (CRBN), G1 To S Phase Transition 1 (GSPT1), Ikaros (IKZF1), Aiolos (IKZF3), Tripartite Motif Containing 24 (TRIM24), Mouse double minute 2 homolog (MDM2), Mitochondrial E3 ubiquitin protein ligase 1 (MULAN), carboxy-terminus of Hsc70 interacting protein (CHIP). [Created with BioRender.com]. dependent on a functional UPS system for tumor initiation, tumor largest group of E3 ubiquitin ligases whose substrates are generally key metabolism, and survival. Indeed, UPS dysregulation and the conse­ players in regulating vital cellular processes such as DNA replication and quent aberrant activation or deactivation of signaling pathways are cell cycle. Dysregulation of this ligase family usually leads to cancer common hallmarks of several cancer subtypes [14]. Interestingly, dys­ [18]. The intracellular accumulation of c-MYC, together with the acti­ regulated ubiquitin ligases and deubiquitinase activities and consequent vation of the mechanistic target of rapamycin (mTOR)-dependent augmentation (gain of function) o impairment (loss of function) activity translation, is also regulated at the early stage of transformation by of target proteins has been associated with the initiation and progression F-box/WD repeat-containing protein 7 (FBW7), another E3 ubiquitin of multiple cancer subtypes [15]. ligase with a key tumor suppressor activity in cancer development and Depending on the substrate specificity and on the affected signaling which mutation-associated loss of function is frequently found in ma­ pathway, ubiquitin ligases and DUBs can act as either tumor promoters lignant cells [19,20]. It is worth noting that FBW7-mediated degrada­ or tumor suppressors (Fig. 1). Regulators of the cell cycle are among the tion of c-MYC is also tightly dependent on the overexpression of the main factors affected by aberrancies in the UPS pathway. For instance, nucleolar DUB USP36, found in a subset of human breast and lung the cyclin-dependent kinase (CDK) inhibitor p27KIP1 is expressed at high cancers and linked to the overexpression of the proto-oncogene [21]. levels in quiescent cells; nevertheless, its expression levels decrease Finally, c-MYC is also regulated at transcriptional level by the tran­ considerably in tumor cells due to its proteasome-mediated degradation scription factors Ikaros and Aiolos, found overexpressed in hyperdiploid [16]. Together with cyclin E/A/CDK2 complexes and the E3 S-phase multiple myeloma (MM), and which proteasome-dependent degradation kinase-associated protein 2 (SKP2), p27KIP1 also constitutes a regulatory is regulated by cereblon (CRBN), the substrate receptor of the CRL4CRBN network engaged in a bidirectional crosstalk with the c-MYC E3 ubiquitin ligase [22]. proto-oncogene, being the stability and the role on cell cycle regulation Besides cell cycle, overexpression of the tumor suppressor p53 - and senescence of this latter dictated by phosphorylation and ubiq­ which protects cells from genomic insults and regulates several target uitylation [17]. SKP2 belongs to the SCF (Skp1/cullin/F-box) family, the genes that take part in cell-cycle control, apoptosis, senescence, and 214 A.M. Montagut et al. Seminars in Cell and Developmental Biology 132 (2022) 213–229 DNA repair [23,24] - is associated with somatic mutations of TP53 gene 2. Therapeutic targeting of the ubiquitin machinery in cancer that occur at rates from 38% to 50% in ovarian, esophageal, colorectal, patients head and neck, larynx, and lung cancers, and detected in about 5% of primary leukemia, sarcoma, testicular cancer, malignant melanoma, and Data obtained during the last decades on the regulation and func­ cervical cancer [25]. These high expression levels are accompanied by tions of the ubiquitin system have accelerated dramatically our knowl­ deregulation of a p53-regulating E3 ubiquitin ligase, murine double edge of the ubiquitin code. Given the multiple alterations regarding the minute 2 (MDM2 or HDM2 for humans), which binds to the trans­ expression and/or activity of several ubiquitin-regulating enzymes in activation domain of p53, impairing its transcriptional activity and numerous malignancies, these latter have attracted extreme attention mediating its transport from the nucleus to the cytoplasm for its for the treatment of cancer in the last decades and increasing number of degradation [26,27]. The DUB that counteracts MDM2-induced p53 inhibitors targeting specific components of the UPS are continuously nuclear export and degradation is USP10, a cytoplasmic being studied and developed [48]. Beside proteasome inhibitors, which ubiquitin-specific protease that is stabilized after DNA damage by an have already shown remarkable usefulness in the clinical management ataxia-telangiectasia mutated (ATM)-dependent process followed by its of different subtypes of cancers, especially lymphoid neoplasms [49], translocation to the nucleus where it activates p53 [28]. Importantly, several inhibitors targeting either E3 ligases or DUBS are currently at USP10 is frequently found under-expressed in renal cell carcinoma [29]. different development stages in clinical studies. Furthermore, tripartite motif containing 24 (TRIM24), the E3 ubiquitin ligase that negatively regulates p53 via a conserved RING domain, is 2.1. Development of E3 ligase and deubiquitinase small molecule depleted in human breast cancer cells where its loss favors inhibitors p53-dependent apoptosis [30]. Another crucial process regulating the cell fate that can be deregu­ Deregulation of E3 enzymes is frequently associated with poor lated in cancer due to UPS improper activity is apoptosis signaling. prognosis in different cancer subtypes, conferring a logical interest in Among the antiapoptotic factors found to be accumulated due to their therapeutic targeting. A vivid example of this interest is exempli­ improper degradation in malignant cells, the BCL-2 family member fied with the preclinical development of MDM2 inhibitors as a novel myeloid cell leukemia 1 (MCL-1) exerts a crucial role in promoting approach to cancer therapy [50], although this process has been rela­ cancer cell survival and resistance to chemotherapy and is regulated by tively slow. Indeed, since the publication of MDM2 crystal structure in the E3s tripartite motif containing 17 (TRIM17), MULE and FBW7 1996, multiple molecules able to interrupt p53–MDM2 binding have [31–33]. The stabilization of BCL-2-like protein 11 (BIM), a been described, but it was only in 2004 that the cis-imidazoline ana­ pro-apoptotic member of the BCL-2 family linking stress-induced signals logues known as Nutlins (Nutlin-1, − 2, and − 3) showed selectivity to the apoptotic executioners and which defective expression can be against p53 and potent anti-cancer activity [51–53]. Among these found for instance in lung cancer and B-cell lymphoma [34,35], is molecules, Nutlin-3 showed a potent antitumoral activity in mouse regulated by the UPS mediated by MAPK/ERK signaling [36]. xenograft models of human cancer, retaining the wild-type (wt) p53 A crucial regulator of malignant cell survival and tumor progression [51]. In addition, it has been shown in vitro that Nutlin-3 disrupts the is protein kinase B, (PKB) also known as AKT, which stability and ac­ interaction between MDM2 and the p53 homologue p73 and activates tivity are negatively regulated by the mitochondrial E3 MULAN (MUL1/ the transcription factor E2F-1, together with the hypoxia-inducible GIDE/MAPL) and by the DUB cylindromatosis (CYLD), respectively factor-1α (HIF-1α), therefore producing consistent p53-independent ef­ [37]. While MULAN expression is lost in head and neck cancer [38], fects, although at higher doses [54–56]. CYLD deficiency has been shown to promote cancer cell proliferation, Supporting the notion that toxicity of conventional chemothera­ survival, glucose uptake, and tumoral growth in mice models of prostate peutics can be reduced by means of non-genotoxic agents able to acti­ tumor [39]. AKT signaling is also affected by the intracellular accumu­ vate p53, ex vivo experiments in patient samples retaining wt p53 show lation of the ubiquitin ligase X-linked inhibitor of apoptosis (XIAP/­ that Nutlin-3 synergizes with doxorubicin and 1-β-Darabinofur­ BIRC4) which ubiquitinates the main negative regulator of the AKT anosylcytosine (Ara-C) in acute myeloid leukemia (AML) and doxoru­ pathway, i.e. phosphatase and tensin homolog deleted on chromosome bicin, chlorambucil, and fludarabine in CLL, amongst others [57]. 10 (PTEN), directing this latter to degradation and modulating the These encouraging results with Nutlins paved the way to the devel­ sensitivity of ovarian cancer cell to platinum [40]. In chronic lympho­ opment of new small molecule MDM2 inhibitors such as MI-219, which cytic leukemia (CLL), the DUB USP7 controls the nuclear pool of PTEN, was shown to disrupt p53-MDM2 binding, leading to p53 activation and both in TP53-wild type and -null environment, and, in this model, PTEN suppression of tumour cell growth in both in vitro and in vivo models of acts as the main tumor suppressive mediator along the USP7-PTEN axis lung cancer [58]. Administration of the compound activated a in a p53-dispensable manner [41]. USP7 is also found overexpressed in p53-dependent response in tumor tissue and in cell lines as indicated by prostate cancer, where it is associated with PTEN nuclear exclusion [42]. p53 accumulation, and led to a strong antitumoral activity in xenograft The linear ubiquitin chain assembly complex (LUBAC) is composed models of human cancer with wt p53, without evidence of toxicity [59, by Heme-oxidized IRP2 ubiquitin ligase-1 L (HOIL-1 L), HOIL-1 L- 60]. interacting protein (HOIP), and Shank-associated RH domain interactor CGM097 is a substituted dihydroisoquinolinone derivative designed (SHARPIN), being the function of HOIP controlled by the DUBs OTULIN to mimic crucial hydrophobic interactions of p53 within the MDM2 and CYLD. While the dysregulation of the LUBAC-mediated linear pocket at Phe19, Trp23, and Leu26 [61]. In vitro, CGM097 is able to ubiquitination pathway has been linked to the development of cancer, block the proliferation of primary AML blasts and AML cell lines with wt the ubiquitination of NF-κB-essential modulator (NEMO) by this system p53, to reduce the growth of AML xenografted tumors, and to improve is critical for genotoxic NF-κB activation and for the protection of tumor the survival in different mouse models of AML patient-derived xeno­ cells from DNA damage-induced cell death [43,44]. grafts [62,63]. Tumor necrosis factor alpha (TNFα) is an inflammatory cytokine that Milademetan is a dispiropyrrolidine-based inhibitor able to reac­ plays significant roles in both immunity and cancer [45] and which tivate p53 signaling in cancer cells with wt p53, and with remarkable signaling mediated by different cell death complexes can be restricted by antitumor effect in in vitro and in vivo models of AML, B-cell non- ubiquitin hydrolases or DUBs, namely A20/TNFAIP3, CYLD, and OTU Hodgkin lymphoma (B-NHL), and other non-hematological cancers deubiquitinase with linear linkage specificity (Otulin) [46,47] (Fig. 1). [64,65]. APG-115 is a spirooxindole-derived inhibitor with improved stability when compared to the HDM2 blocking agent SAR405838, and with demonstrated capacity to block the proliferation of AML malignant cells 215 A.M. Montagut et al. Seminars in Cell and Developmental Biology 132 (2022) 213–229 Table 1 E1, E3, or DUB modulators currently under clinical development. Structure Compound Mechanism of Action Clinical trial stage NCT number References E1 modulators TAK-243 UAE inhibition by Ub-TAK-243 complex Phase 1 in AML, CML, MS NCT03816319 [88,89] formation E3 modulators CC-122 (Avadomide) Increases CRL4CRBN binding to IKZF1/3 Phase 1/2 in NHL NCT03310619 [90] CGM097 MDM2 inhibitor Phase 1 in solid tumors NCT01760525 [91] CC-220 (Iberdomide) Increases CRL4CRBN binding to IKZF1/3 Phase 1/2 in MM NCT04564703 [92] APG-115 MDM2 inhibitor Phase 1/2 AML and CML NCT04358393 [93] Tasisulam degradation of RBM39 Phase 3 in melanoma NCT01006252 [94] Indisulam degradation of RBM39 Phase 2 in colorectal NCT00165867 [95] cancer NSC-339004 degradation of RBM39 Phase 2 in lung cancer NCT00008372 [96] DS-3032 MDM2 inhibitor Phase 1 in AML NCT03671564 [97] (Milademetan) DUB modulators VLX1570 USP14 > UCH37 inhibitor Phase 1/2 in MM NCT02372240 [98] 216 A.M. Montagut et al. Seminars in Cell and Developmental Biology 132 (2022) 213–229 and to impair the spread of AML disease in mice [66]. evaluation with patients with either advanced solid or hematological Regarding the SCF family, since the F-box determines the specificity tumors (Table 1). A first phase 1, open-label dose escalation study of these complexes, most small molecule inhibitors have been developed (NCT02045095) established the maximum tolerated dose (MTD) in 29 against the corresponding residues. Oridonin, a diterpenoid isolated patients with solid cancers. Although the efficacy of the compound was from Rabdosia rubescens [67], promotes the deubiquitination and demonstrated histologically by the detection of decreased ploy- degradation of c-MYC and triggers apoptosis in leukemia and lymphoma ubiquitinated chains, this trial highlighted serious advert events (AEs) cells [68]. Other molecules like ZL25 and compound A directly target in > 30% of the patients. A subsequent phase 2 study is currently SKP2, resulting first in cellular senescence and lastly in p27KIP1-me­ enrolling patients undergoing relapsed or refractory (R/R) AML, mye­ diated cell death and cell cycle arrest [67,69]. Erioflorin is another lodysplastic syndrome (MDS), or chronic myelomonocytic leukemia natural compound that stabilizes the tumor suppressor programmed cell (CMML) (NCT03816319). Beside UBA1 expression and ubiquitylation death 4 (PDCD4) by blocking its degradation, therefore repressing NF-κB status, the development of biomarkers that may allow monitoring pa­ pathway activity, and reducing the proliferation rate of cancer cells tient response to TAK-243 in these clinical trials, is still necessary. [70]. GS143 is a β-TrCP1 ligase inhibitor which also blocks NF-kB signaling by inhibiting the ubiquitination and proteasomal degrada­ 2.2.2. Cereblon (CRBN)- targeting agents tion of the intracellular inhibitor of NF-κB (IκBα) [71]. The Cullin 4-RING ligase (CRL4) complexes are known to exert a Among the different DUBs that are being deeply evaluated as significant role in tumorigenesis through their roles in the control of possible targets for anticancer therapy, counteracting the activity of DNA damage response and DNA repair. CRBN is a substrate recognition USP9X, USP14, UCH37, and USP5 using the inhibitor WP1130 has been subunit of CRL4 that represents a great therapeutic potential, especially shown to trigger apoptosis by promoting p53-dependent signalling and in hematological cancers. IMiD agents, that encompass the antiemetic by deregulating the anti-apoptotic MCL-1 [19]. P53 may also be stabi­ agent thalidomide and its derivative lenalidomide and pomalidomide, lized in vitro by the small molecule inhibitor HBX41108 [72], which are being clinically evaluated and/or approved for the treatment of impairs USP7-mediated deubiquitination, thus promoting the degrada­ different hematological malignancies in the relapse setting. CRBN was tion of the p53 negative regulator, MDM2. In parallel, the selective USP7 identified in 2004 as primary target of thalidomide and as the mediator inhibitor P5091 not only evokes apoptosis but can also overcome bor­ of the reported teratogenic activity of the drug [99]. However, this tezomib resistance in in vitro models of MM and exert a synergistic effect discovery also shed light onto the mechanism of action of IMiDs in MM in vivo when co-administered with the glucocorticoid dexamethasone patients, in which the proteasome-mediated degradation of the tran­ and/or the immunomodulatory drug (IMiD) lenalidomide [73]. Another scription factors Ikaros and Aiolos with a crucial role in the determining interesting player in the stability of p53 is OTU Domain-containing of B-cell fate, has been shown to depend on CRBN E3 ligase activity ubiquitin aldehyde-binding protein 1 (OTUB1) which, similarly to [100–102]. IMiD binding at a conserved pocket near the C-terminus of USP7, stabilizes p53 both in vitro and in vivo by non-canonical inhibi­ CRBN triggers CRL4CRBN-mediated polyubiquitination and tion of MDM2-mediated ubiquitination [74]. Specifically, OTUB1 sup­ proteasome-dependent degradation of its substrates, in a process termed presses UBCH5, the E2 partner of MDM2. The understanding of OTUB1 “molecular glue” [102]. In MM, IMiD-induced degradation of Ikaros and function in the regulation of p53 activity, has opened the door to the Aiolos leads to interferon regulatory factor 4 (IRF4) downregulation and study of possible small molecules that disrupt the interaction between interleukin-2 (IL-2) upregulation, followed by apoptotic cell death [101, MDM2 and UBCH5, inhibiting both its p53-dependent and 103]. In both preclinical and clinical settings, IMiDs are being used p53-independent oncogenic properties [75]. successfully in combination with proteasome inhibitors, steroids, and monoclonal antibodies as anti-myeloma therapy, and as single agents in 2.2. Clinical activity of E1-targeting drugs and E3 modulators other hematological malignancies, including MDS with deletion of chromosome 5q, CLL, and mantle cell lymphoma (MCL) [104]. A novel 2.2.1. UBA1 inhibitors set of thalidomide analogs has been developed recently by Celgene Corp, Prior to the recent elucidation of UBA1 crystal structure, different which includes the cereblon E3 ligase modulators (CELMoDs) CC-122 strategies were carried out to modulate the activity of the E1 enzyme, (avadomide), CC-220 (iberdomide), and CC-885, all being evaluated thanks to the close similarity between human UBA1 and related proteins in active clinical trials. Thanks to the presence of a conserved gluta­ [76–78]. Among the first modulators described, the adenosine sulfamate rimide required for CRBN binding in its structure, CC-122 contains ex­ and first-in-class UBA1 inhibitor, TAK-243 (MLN7243), is the only drug erts pleiotropic immunomodulatory and antitumor activities [105,106]. that has entered into clinical evaluation (Table 1) [77]. This compound, Mechanistically, CC-122 binds CRL4CRBN E3 ligase to induce the designed on the structural basis of the NEDD8-activtating enzyme (NAE) degradation of Ikaros and Aiolos in MM cells, de-repression of interferon inhibitor pevonedistat [79], is active against UBA1, and in lesser extent (IFN)-regulated genes, and co-stimulation of T cells followed by against UBA6 and NAE [77]. In vitro, TAK-243 treatment leads to reduce apoptotic cell death in in vitro and in vivo models of diffuse large B-cell levels of mono- and poly-ubiquitylated proteins and to the accumulation lymphoma (DLBCL) [106,107]. Following these results, CC-122 recently of several ubiquitin-regulated proteins, including p53, c-JUN, c-MYC, entered into clinical trials with patients suffering from B-NHL, MM, and MCL-1, and XIAP, and to apoptotic cell death consequent to DNA CLL (Table 1). In a dose-escalation phase 1 clinical trial (NCT01421524) damage and cell cycle arrest at G1 and/or G2/M phase [77,80–83]. aimed at evaluating the safety, tolerability, pharmacokinetics and pre­ Another important mechanism involved in the antitumor effect of liminary efficacy of CC-122 in patients with relapsed cancers, including TAK-243 in different disease models is the induction of proteotoxic MM, B-NHL and advanced solid tumors, CC-122 monotherapy showed stress at the level of the endoplasmic reticulum (ER), leading to acti­ an acceptable safety profile with a maximum tolerated dose (MTD) of vation of the unfolded protein response (UPR) [77,80,81,83,84]. The 3.0 mg and with manageable AEs reported after a median duration of pleiotropic effects of TAK-243 towards multiple signaling pathways treatment of 58 days (44% fatigue, 29% neutropenia, 15% diarrhea) beside cell cycle and ER stress [80], supported its efficacy and selectivity [46]. Authors reported one complete response (CR) and two partial in a wide range of solid tumors including cutaneous squamous cell response (PR) in B-NHL patients, being the compound able to evoke the carcinoma, small-cell lung cancer, pancreatic cancer, and glioblastoma degradation of Aiolos in circulating B and T cells and to reduce the [77,83–86], but also in several life-threatening hematological condi­ number of B cells in the peripheral blood of the patients [104]. tions, like AML, MM, CLL and a set of indolent or aggressive B-cell CC-220 is another thalidomide derivative developed, among others, lymphoma [77,80–82,87]. for the treatment of relapsed/refractory MM, and with enhanced ca­ Based on these preclinical data and considering the favorable toxi­ pacity to trigger CRBN-mediated degradation of Aiolos [108]. In a cological profile of the molecule, TAK-243 entered into clinical dose-escalation phase 1 study (NCT01733875), no severe AEs were 217 A.M. Montagut et al. Seminars in Cell and Developmental Biology 132 (2022) 213–229 attributed to CC-220 when administrated daily to healthy volunteers. Table 2 The compound was generally well tolerated and was able to evoke the MDM2 ligands reported in literature grouped by scaffold (*) PDB identification degradation of both Ikaros and Aiolos in peripheral blood mononuclear code is used for those ligands without any specific name. cells (PBMCs) [49]. Following preclinical reports of CC-220 synergistic Chemical scaffold Compound PDBID PubChem References interaction with the proteasome inhibitor bortezomib with or without CID the glucocorticoid dexamethasone regarding Ikaros and Aiolos degra­ pyrrolidine RO5313109 4JRG 45139212 [52] dation and anti-myeloma activity, and of efficacy of CC-220/anti-CD38 RO5316533 4JSC 59249286 [52] (daratumumab) combination in vitro [109], a subsequent imidazoline Nutlin-2 1RV1 5288631 [51] IMY* 1TTV 49867154 [123] dose-escalation phase 1b/2a study was launched with relapsed or re­ WK-298 3LBJ 24969086 [124] fractory (R/R) MM patients to evaluate the efficacy and safety of the WK-23 3LBK 44825260 [124] CELMoD when administered either as single agent or in combination BLF* 4DIJ 56951871 [125] with dexamethasone, with or without daratumumab or bortezomib RG-7112 4IPF 57406853 [126] therapies (NCT02773030). Preliminary results showed an overall Nutlin-3a 4HG7 11433190 [127] Nutlin-3a 4J3E 11433190 [128] response rate (ORR) of 29% in these heavily pretreated patients, with no RO0503918 4J74 71816470 [129] MTD reached. Main AEs in patients receiving CC-220 and dexametha­ RO5045331 4J7D 58894234 [129] sone combination were manageable and included grade 3–4 neutropenia RO5524529 4J7E 71763144 [129] (29%), infections (25%), and thrombocytopenia (12%) [110] (Table 1). NUT* 5Z02 11433190 NA NUT* 5ZXF 11433190 NA imidazole 2V8 * 4OQ3 50996417 [130] 2.2.3. MDM2 antagonists 6GG* 5J7F 126963310 [131] In hematologic malignancies in which p53 is predominantly 6GG* 5J7G 126963310 [131] expressed in its wt form, such as AML, the pharmacological targeting of H0W* 6I29 71155423 NA MDM2 has become particularly attractive. HTZ* 6Q9L 138320068 [132] HU8 * 6Q9O 67032088 [132] The Nutlin derivative RG7112 [111] was the first MDM2-P53 dis­ 3-pyrroline-2-one 4NJ* 4ZFI 122177104 [133] ruptor to enter into the clinic, where it efficiently led to p53 stabilization furanone 4NX* 4ZGK 122177105 [133] and to the upregulation of some apoptosis and cell cycle regulators hydantoin RO-2443 3VBG 136683437 [134] (BAX, PUMA, FAS, TNFRSF10B, CDKN1A) in cells from R/R AML and 13Q* 2LZG 56591370 [135] 0R2 * 4ERE 56591324 [136] CLL patients [111]. In AML patients, this agent was active either as AM-8553 4ERF 56965957 [136] single agent or in combination with cytarabine, allowing some cases to 0Y7 * 4HBM 56591282 [135] reach a CR [111,112]. However, given the frequency of hematological 2SW* 4OAS 68000593 [137] AEs (neutropenia, thrombocytopenia and hemorrhage) and gastroin­ piperidinone 2U0 * 4ODE 73386675 [138] testinal toxicity following RG7112 administration, a more potent and 2U5 * 4OGN 73386677 [138] 35 S* 4QO4 58573999 [139] selective Nutlins derivative, i.e. the pyrrolidine RG7388 (RO5503781, 35T* 4QOC 67999919 [140] idasanutlin), was developed [52]. In preclinical models, this AM-7209 4WT2 77108133 [141] second-generation MDM2 inhibitor harbored similar activity as its pre­ morpholinone 1MN* 4JV7 71305070 [142] cursor, but at doses that were significantly lower [52]. In clinical set­ 1MO* 4JV9 71305071 [142] 1MQ* 4JVE 137347970 [142] tings, this compound was well tolerated and showed significant activity 1MY* 4JWR 71305074 [142] with manageable toxicity in R/R AML patients [113]. Different 2TW* 4OBA 71544420 [143] RG7388-based combinations are being evaluated clinically. Among 2TZ* 4OCC 73386674 [138] these, a phase 1b/2 trial (NCT02670044) is currently investigating the 2U1 * 4ODF 73386676 [138] safety, tolerability, and preliminary efficacy of idasanutlin in combina­ 2U6 * 4OGT 73386678 [138] 2U7 * 4OGV 73386679 [138] tion with the BCL-2 antagonist venetoclax in patients with R/R AML who dihydroisoquinolinone NVP- 4ZYF 53240420 [61] are not eligible for chemotherapy. Another ongoing phase 1b/2 trial CGM097 (NCT03850535) is evaluating the safety, efficacy, and pharmacological NVP- 4ZYI 58437867 [61] properties of RG7388 when given alongside cytarabine and daunoru­ CGM097 4SS* 4ZYC 91820721 [144] bicin to newly diagnosed AML patients. isoindolinone TUZ* 7BIR 135282535 [145] CGM097 has been evaluated in monotherapy in a first-in-human TV5 * 7BIT 135282593 [145] dose-escalation phase 1 study (NCT01760525) in patients with TUW* 7BIV 135282862 [145] advanced solid cancers and with wt TP53 gene (Table 1). Results were TVH* 7BJ0 86763270 [145] disappointing with relatively limited activity and notable AEs including TVK* 7BJ6 155883011 [145] U3Z* 7BMG 135281739 [145] grade 3–4 thrombocytopenia and/or neutropenia [114]. indolinone RO5027344 4LWT 117071888 [146] The DS3032b compound is being tested clinically since 2013 and is imidazolopyrrolidinone 6ZT* 5LN2 71565460 [147] now under evaluation in patients with advanced solid tumors or lym­ Siremadlin 5OC8 71678098 [148] phomas, or with other hematological cancers like AML, MDS, MM, acute EYH* 6GGN 71678945 [149] lymphoblastic leukemia (ALL) and chronic myelogenous leukemia HRE* 6Q96 89601515 [132] dihydroimidazothiazole VZV* 3VZV 44608665 [150] (CML) (NCT02319369, NCT01877382, NCT02579824, Table 1). First LTZ* 3W69 58007243 [151] reports from these trials highlighted a general lack of efficacy of benzodiazepinedione DIZ* 1T4E 656933 [152] DS3032b although 77% of patients had stable disease, with main he­ Spiro-oxindole RO5353 4LWV 56649312 [153] matological AEs being thrombocytopenia and neutropenia [115]. In the RO8994 4LWU 53236857 [146] MI-63- 3LBL 49867311 [124] subgroup of patients with R/R AML or high-risk MDS, a decrease in bone analog marrow infiltration by tumoral blasts was detected in 15/38 cases and a 1MT* 4JVR 71305073 [142] CR was observed in two AML patients, although a TP53-mutated clone 6SK* 5LAV 122198471 [154] appeared alongside the treatment in these two cases [116]. 6SJ* 5LAW 118439641 [154] In two phase 1 studies (NCT02319369 and NCT01877382), mila­ 6SS* 5LAY 122198472 [154] BI-0252 5LAZ 118439587 [154] demetan showed promising activity in patients with R/R AML, high-risk SAR405838 5TRF 53476877 [155] MDS, or with solid tumors or lymphomas, with 3 patients presenting a H28 * 6I3S 137349477 [156] CR. Although almost all the cases presented grade 3–4 AEs including 218 A.M. Montagut et al. Seminars in Cell and Developmental Biology 132 (2022) 213–229 Fig. 2. Molecular representation of the MDM2 inhibitors reported in the PDB. nausea, diarrhea, vomiting, fatigue, anemia, thrombocytopenia, neu­ micromolar range of concentrations [120]. Its enhanced activity against tropenia, hypotension, hypokalemia, and hypomagnesemia, the safety DUB activity in MM cells over other tumor types prompted its evaluation profile was considered acceptable [116–118]. Importantly, among 79 in patients with R/R MM. In a phase 1 study aimed at characterizing its patients evaluated with liposarcoma, solid tumors, and lymphomas, 47 safety, tolerability, and pharmacokinetic profile (NCT02372240), a total of them (60%) presented a stable disease [117]. Following these of 15 patients having relapsed after proteasome inhibitor and IMiD encouraging trials, milademetan remains currently under clinical eval­ therapies received either a low dose (0.05–0.60 mg/kg) or a high dose uation in different cancer subtypes. (1.2–2.0 mg/kg) of the compound. In the low-dose groups, although First results of a phase 1 clinical trial testing APG-115 in patients only minimal AEs were observed, including grade 1–2 fatigue, rash, with solid tumors (NCT02935907) showed that dose-limiting toxicities nausea, and anemia, and DLTs were not reported, half of the patients (DTLs), including essentially thrombocytopenia and fatigue, but also were withdrawn from the study due to progressive disease. In the decreased neutrophil and white blood cell counts among others, were high-dose group, patients developed fatal pulmonary toxicity within detected [119]. No information about drug efficacy is available so far. days of receiving two doses of the compound. These disappointing re­ sults led to premature study termination [121]. 2.3. Clinical activity of the DUB modulator VLX1570 3. Inhibitors in the pipeline VLX1570 is a competitive inhibitor of proteasome DUB activity, and As stated previously, four proteins belonging to the UPS, i.e. HOIP, an analogue of the previously described compound b-AP15, that shows MDM2, USP7, and USP14, have been identified as promising targets for selective inhibitory activity for USP14 with an IC50 in the low 219 A.M. Montagut et al. Seminars in Cell and Developmental Biology 132 (2022) 213–229 cancer therapy and they could attract a widespread interest in the near Table 3 future. Having information related to the structure and the binding List of crystallized HOIP structures complexed with a small molecule available in mechanism for these proteins is crucial to make possible the design of the Protein Data Bank grouped according to their chemical scaffold. new inhibitors by applying structure-based drug design techniques. The Core Ligand PDBID PubChem CID References Protein Data Bank (PDB, https://www.rcsb.org/) is the main worldwide compound 5a 6GZY 137349349 [164] repository of 3D structures of macromolecules including proteins, NH O nucleic acids, and their complexes. Over the past few years, the number of crystal structures involving the aforementioned proteins, including complexes with small molecules, has grown dramatically. Having Ligand 3 6SC7 145946065 [165] available structural information allowed the application of structure- Ligand 4 6SC8 145946064 Ligand 2 6SC5 145946063 based methods for designing new inhibitors [122]. HOIPIN-8 6KC6 146035892 [166] 3.1. Structure-based design of E3 ubiquitin-protein ligase inhibitors 6SC9 146018040 [165] HOIPIN-1 6KC5 146035891 [166] 3.1.1. Identification of new potent MDM2-interacting drugs The huge number of entries in the PDB (more than 110 3D structures are available) demonstrates the interest of the scientific community in the MDM2 protein. Interestingly, from the chemical point of view, small Table 4 molecules complexed to MDM2 involve a high variety of chemical List of crystallized USP7 structures complexed with a small molecule available in scaffolds, being most of them heterocyclic compounds (Table 2). The the Protein Data Bank grouped according to their chemical scaffold. molecular name reported in the original reference has been used for Chemical scaffold Ligand PDBID PubChem CID References those molecules without a standard name. compound 7 6VN5 146026046 [167] The binding site of MDM2/HDM2 is mostly hydrophobic [157], and most of the MDM2 inhibitors have been shown to mimic the three key p53 residues Phe19, Trp23, and Leu26, which are part of an α-helix of compound 2 5WHC 135567363 [168] that protein. Different chemical families have been assessed for the in­ hibition of the MDM2-p53 by targeting the aforementioned interactions. The molecular design has played a pivotal role during the develop­ L55 6M1K 155804189 [169] ment of these candidates. Structure-based drug design (SBDD) tech­ FT827 5NGF 137348762 [170] FT671 5NGE 121457027 [170] niques, mainly represented by docking and molecular dynamics compound 5 5N9T 131750088 [171] simulations, have been widely applied not only for gaining insights into MDM2 structural features [158] but also to guide the hit to lead process. The benzodiazepinedione (BDP) family was described for the first time GNE6776 5UQX 122531750 [172] by Grasberger et al. as antagonists of HDM2-p53 interaction [152]. The GNE6640 5UQV 122531786 [172] crystal structure of the BDP-HDM2 complex (PDBID: 1T4F, 1T4E) revealed that this family of compounds is able to interact with the p53 binding site, by reproducing the interaction pattern of this latter (recognizing the Phe19, Trp23, and Leu26 residues) and by mimicking compound 46 6F5H 132990905 [173] compound 1b 6VN4 146026045 [167] its α-helix structure. Thanks to the analysis of the binding site available compound 16 5N9R 131750081 [171] in the PDB, authors were able to enhance the pharmacokinetic proper­ ties of compound 1, leading to the more potent and orally bioavailable compound 19 [159]. More recently, SBDD was used in the discovery of AM-8553, using molecular docking to predict the binding affinities of compound 14 6VN6 146026047 [167] compound 23 6VN3 138610287 piperidinone derivatives [136]. compound 18 6VN2 146672986 Structure-activity relationship (SAR) analysis has been applied on known MDM2 inhibitors to design more potent derivatives with better XL188 5VS6 131953451 [174] drug-like properties. In the case of Nutlins, the application of scaffold 9HS* 5VSK 131953452 hopping approach led to the identification of dihydroimidazothiazole compound 1a 5VSB 71278663 derivatives as potent MDM2 inhibitors [150]. AMG-232, which is currently in clinical phase 2, was identified as a result of a SAR analysis applied on the AM-8553 inhibitor previously described [137]. The identification of the spiro-oxindole SAR405838 was based on the optimization of a previously studied derivatives, designed using a 3,2’-pyrrolidin]− 2(1H)-one derivatives and applied structure-based structure-based approach to target the three key residues involved in leading to BI-0252 inhibitor. Molecular docking was applied to predict p53 interaction [160]. This small molecule has been advanced to phase and rank the binding affinity of candidates [154]. 1 clinical trial and has a Ki of 0.88 nM. The co-crystal structure of the The isoindolinone scaffold was identified as MDM2 inhibitors and SAR405838-MDM2 complex showed not only the direct interaction of explored using SAR studies, showing a characteristic binding mode in the three key amino acid residues of p53 but also additional interactions which isonindolinone scaffold is placed abutting Phe19 and interacts not observed on the p53-MDM2 complex, e.g. Val14 and Thr16, which with Val93 and Tyr67 [161]. To optimize this kind of structures, authors induce the refolding of the short MDM2 N-terminal region. Altogether, made use of quantum mechanics and ligand-based drug design to finally these characteristics give to SAR405838 a high affinity and specificity identify compound 14 with an IC50 = 4 nM [145] (Fig. 2). character for the inhibition of MDM2. Durable tumor regression or complete tumor growth inhibitor is observed when using SAR405838 in 3.1.2. Design of novel HOIP antagonists RS4, acute leukemia, mouse xenograft models of osteosarcoma, colon HOIP sequence is composed of three main domains: zing finger (ZF) cancer, and prostate cancer [155]. domains, NPl4-type zinc finger 1 (NZF1), and NPl4-type zinc finger 2 Inspired by Wang’s work, Gollner et al. designed spiro[3H-indole- (NFZ2). NZF1 domain acts as a ubiquitin-binding site able to interact 220 A.M. Montagut et al. Seminars in Cell and Developmental Biology 132 (2022) 213–229 Table 5 catalytic cysteine Cys885 and promoted enough residence time to form a List of crystallized USP14 structures complexed with a small molecule available covalent bond. One year after, the same group described the interaction in the Protein Data Bank. mechanism of tetrahydroquinolinone derivatives [164]. These com­ Chemical Scaffold Ligand PDBID PubChem CID Reference pounds interact with HOIP protein, establishing H-bonds with His889 IU1 6IIK 675434 [175] and His887 [165], and aromatic interactions with Phe888. Their studies IU1–248 6IIN 135393505 led to the design of HOIPIN-1 and − 8 ligands [166]. Interestingly, A8L 6IIM 34134404 HOIPIN-8 filled a pocket on the opposite surface of Cys885. It had IU1–47 6IIL 675477 different interactions with HOIP, such as a salt bridge between the ligand carboxylate and Arg935 and hydrogen bonds between the ligand pyrazole group and Asp936 [165] (Fig. 3). with NEMO and Lys63 ubiquitin chains. These interactions are necessary for the recruitment of LUBAC in different signaling pathways. HOIP RBR E3 ligase located at the C-terminal of HOIP is formed by a 3.2. Uncovering new DUB modulators RING1, IBR, and RING2 core. RING1 is the one in charge of recruiting the E2-Ub complex. IBR domain contains an allosteric ubiquitin-binding 3.2.1. Discovery of USP7-blocking agents site that can promote the binding of the Ub-E2 complex (allosteric USP7 is structured in seven domains, the N-terminal TRAF-like (TNF activation). The RING2 domain contains the catalytic cysteine (Cys885), receptor-associated factor) domain, the catalytic core domain, and the responsible for the covalent linkage between LUBAC and the donor five C-terminal ubiquitin-like domains, UBL1–5. Residues 208–882 of ubiquitin. Close to this catalytic cysteine, His887 (also called catalytic USP7 contain the catalytic domain (CD) and the first three Ubl domains. histidine) plays a vital role in the final aminolysis reaction [162–164]. USP7 catalytic domain comprises residues 208–560, located between The elongated conformation of the donor ubiquitin is accomplished the TRAF-like and UBL domains. The catalytic site is formed by a triad of by hydrophobic interactions of RING2 (HOIP) with the Ile36 patch and Cys223, His464, and Asp481 residues. These residues are located in a the C-terminus (Ub). cleft between two regions known as Thumb and Palm. The Finger region Interactions between RING2-LDD and the acceptor ubiquitin brings is the one in charge of binding to ubiquitin. The main role of His464, is to the nucleophilic amine from the Met1 residue closer to the catalytic deprotonate the thiol group of the Cys223 and initiate the nucleophilic cysteine (at distance 3.5 Å) [162] linked to the donor ubiquitin by its Gly76 residue via a thioester bond, allowing the aminolysis reaction. Interestingly, all ligands reported in the PDB (Table 3) are non- reversible inhibitors that target Cys885. Most of them are located in the same region, establishing a secondary interaction with His889 via H- bond, and with Phe888 in most of the cases in presence of arene-arene interactions. Only molecule L68 (HOIPIN-8, the ligand included in the PDBID 6SC9) is located in a different region. The crystal structure of the RING2-LDD domain of HOIP in complex with a covalent inhibitor (compound 5a) was firstly described by Johansson et al. in 2019 [164]. The cyclopentyl pyridone scaffold in­ teracts through H-bond with the His889 backbone. The ester chain of 5a lay within an orthogonal pocket to the ledge and interacted with the hydroxyl side chain of Ser899, this interaction led to a conformation of Fig. 4. Binding cavity described for co-crystal structures available in the the small molecule where the Michael acceptor of 5a approached the PDB (green). Fig. 3. Molecular structure of compounds described as HOIP inhibitors in the PDB. 221 A.M. Montagut et al. Seminars in Cell and Developmental Biology 132 (2022) 213–229 Fig. 5. Molecular representation of USP7 inhibitors reported in the PDB. attack of the final isopeptide bond. The role of the Asp481 residue is the After the three-dimensional structures for USP7 became available, restriction of the side-chain rotation of the His464. The active site SBDD approaches were rapidly applied to address the design of new conformation of the isolated USP7 catalytic domain is different from the USP7 inhibitors using molecular docking, molecular dynamics simula­ ones observed in the available structures of other USPs. To an isolated tions [177], or 3D pharmacophore models [178]. Covalent docking was USP7, the USP7 catalytic triad is found in an unproductive conforma­ applied for predicting the binding mechanism of irreversible inhibitors, tion. Ubiquitination of USP7 leads to the activation of the enzyme [176]. e.g. for the most specific USP7 inhibitors available P22077 and P50429 Most of the described USP7 ligands in the PDB correspond to [179]. reversible inhibitors except for FT827, which acts as an irreversible in­ Several covalent and non-covalent inhibitors of USP7 have been hibitor targeting the catalytic Cys223 (Table 4). According to the in­ designed and synthesized in the early last few years. More in detail, the formation available in the PDB, most of them are placed in the same first two crystal structure USP7-small molecule found in the literature region, establishing interactions with Gln297, Phe409, and Val296. are from year 2017. Covalent inhibitor FT827 and a non-covalent in­ Molecules included in 5UQX, 5WHC, and 5UQV are located in a different hibitor FT671 were identified in an ubiquitin-rhodamine screening region (Fig. 4). assay of approximately 500.000 compounds available at FORMA 222 A.M. Montagut et al. Seminars in Cell and Developmental Biology 132 (2022) 213–229 Therapeutics. Their pyrazolo[3,4-d] pyrimidine-4-one-piperidine scaf­ fold allows them to interact with the USP7 thumb-palm domain, which guides the ubiquitin C terminus into the active site of USP7, specifically with Asp295, Val296, and Gln297 of the thumb subdomain and Phe409 and Tyr465 of the palm subdomain. In the case of FT671, the para- fluorophenyl group is extended towards the fingers subdomain and the 3-fluoropyrazole group points towards the catalytic center abutting the catalytic Cys223. For its part, FT827 covalently binds to catalytic Cys223 through its vinylsulfonamide moiety. Both compounds selec­ tively inhibit USP7 in a panel of 38 DUBs from diverse families [170]. Selective USP7 inhibitors GNE-6640 and GNE-6776 were reported in the same year. Crystallographic data show that these compounds show a non-covalently interaction with USP7 at a distance of 12 Å from the catalytic site. Their inhibition mode of action would be explained by the Fig. 6. USP14 structure with ubiquitin bound (adapted from [181]). interaction with USP7 acidic residues involved in recognizing the ubiquitin Lys48 side chain. They have been shown to be selective to USP7 in a panel of 36 other DUBs. The co-crystal structures demonstrate pyrazolopyrimidone group interacts with Gln297, Arg408, and Phe409, that phenol rings of phenol-aminopyridine moieties fit the USP7 pocket, whereas the (4-aminomethyl)phenyl group of L55 establishes an H-bond while the hydroxyl groups are able to stabilize the interaction via H- with Gln351. The pyrazole ring of the ligand also interacted with bond with His403 and the 2-aminopyridine interacts with Asp349. The Phe409 via π-π stacking. Moreover, the hydroxyl group of the inhibitor NH of the pyridine carboxamide moiety of GNE-6776 engaged an also made hydrogen bond interactions with Asp295 and Val296. Com­ interaction with Asp305 while the NH of the indazole moiety of GNE- pound L55 did not inhibit several DUBs, such as UCH-L1, UCH-L5, USP2, 6640 makes van der Waals interaction in the shallow between α5 and USP25, USP28, and USP11 at 20 µM, indicating its excellent inhibitory α6 [172]. selectivity for USP7 [169]. Consecutively in time, several crystal structures were reported Pyridylbenzofuran derivatives have been the latest scaffold involving different chemical families. The oxadiazole scaffold was described as USP7 ligands in the PDB. Compound 41, a highly potent, involved in a face-to-face π-stacking interaction with Tyr348 [168], e.g. selective, and orally bioavailable USP7 inhibitor was obtained through compound 2-USP7 complex revealed several interactions where the SAR studies. Compound 41 shows tumor growth inhibition in both p53 phenol ring locates into the hydrophobic pocket establishing an mutant and p53 wild type cancer cell lines. Thus, USP7 inhibitors can edge-to-face interaction with Phe324 and the hydroxyl group of the ring suppress tumor growth through several different pathways. Neverthe­ interact via H-bond with His403. Quinazolone scaffold can interact with less, in order to design 41, interaction studies of different compounds in the catalytic site. The co-crystal structure of the potent USP7 inhibitor X-ray co-crystal structures were necessary. In that case, compounds 1b compound 1a, led to the development of XL188, a highly potent and and 7 showed to occupy the same allosteric pocket of the palm region selective inhibitor of USP7. The selectivity of XL188 was assessed although their main scaffold was different, 4-hydroxy piperidine and against a panel of 41 purified DUBs. The analysis of the co-crystal benzofuran, respectively. Both compounds interact via H-bonds with structure XL188-USP7 catalytic domain showed that the compound Val296, Asp295, and the phenol moiety of Tyr465. Interestingly, the co- interacted with the S4-S5 pocket of USP7, between the Palm and Thumb crystallization of compound 14 confirmed two more interactions with area, about 5 Å away of the catalytic triad. The quinazolinone ketone USP7, interacting in a reversible way with Arg408 and Gln297. After scaffold interacted with USP7 forming H-bonds with peptide back-bone compound 14 and some more, they designed compound 18 with a suc­ nitrogen atoms of Arg408 and Phe409. The quinazolinone cyclic nitro­ cinimide moiety introduced to the structure, this moiety was able to gen formed an H-bond with the amide side chain of Gln297. The tertiary interact with Phe409 and Gln297 [167] (Fig. 5). hydroxy group not only interacted via H-bond with the carboxylic group of Asp295 but also with the peptide backbone nitrogen of Val296. 3.2.2. Design of USP14 inhibitors Moreover, the phenyl moiety of compounds 1a and XL188, fitted on the The crystal structures of the 45-kDa catalytic domain of USP14 in S4 pocket and interacted by the aromatic rings of Tyr514, His456, isolation and in a complex with ubiquitin aldehyde reveal precise Phe409, and aliphatic chains of Lys420 and Arg408 [174]. Rational and structural features. In the absence of ubiquitin binding, the catalytic structure-guided design has also been useful in the design of USP7 in­ cleft leading to the active site of USP14 is blocked by two surface loops. hibitors. Particularly, SAR analysis was of utmost importance in the When USP14 is bonded by ubiquitin, it induces a significant confor­ design of pyrazolopyrimidine inhibitors and in identifying compound mational change that translocates the two surface loops, as a result, it 2a. Although most of the reported inhibitors target the catalytic domain, allows the access of the ubiquitin C-terminus to the active site. These the co-crystal structure of compound 2a complexed in human DUB structural observations, in conjunction with biochemical characteriza­ revealed an allosteric binding site of USP7. The phenyl ring of com­ tion, identify important regulatory mechanisms for USP14 [180]. pound 2a is fitted with the hydrophobic cavity generated due to a The full-length human USP14 contains 494 amino acids, with a 9- conformational change of Phe 409, not seen in the apo form or in the kDa Ubl domain at its N-terminus followed by a 45-kDa catalytic ubiquitin-conjugated USP7 structures. This ligand was sited in an exosite domain. The catalytic domain of USP14 comprises three main domains: of about 5.5 Å away from the catalytic Cys223. The rational analysis of Fingers, Palm, and Thumb (Fig. 6). The three-domain association creates compounds 2a interactions allow the design of compounds 4 and 5, a pronounced binding surface between the Fingers and the Palm–Thumb which include a benzylic amine group onto the heterocyclic core, where scaffold, which is predicted to bind to ubiquitin. The active site of free the nitrogen atom of the heterocyclic core interacts with Gln351 and the USP14 is already well formed before substrate binding [180]. nitrogen of the pyrazole moiety interacts with Phe409, both via H-bond The Nɗ1 atom in the imidazole ring of the candidate catalytic histi­ [171]. dine (His435) is approximately 3.3 A◦ away from the Sɣ atom in the side Taking compound 2a as a template, scaffold-hopping leads to com­ chain of the catalytic cysteine (Cys 114), in accordance with a hydrogen pound 46, which showed excellent selectivity in a panel of 21 USP at a bond distance. A third residue, Asp451, stabilizes His435 by accepting a fixed concentration of 10 µM [173]. hydrogen bond from its NƐ1 atom. Thus, Cys114, His435, and Asp451 After a gap of one year, the co-crystal of a pyrazolopyrimidone de­ form a catalytic chord in the active site of free USP14 [180]. rivative (L55) and USP7 was reported. The complex shows that the IU1 is a well-known small molecule for being the first one showing 223 A.M. Montagut et al. Seminars in Cell and Developmental Biology 132 (2022) 213–229 Fig. 7. Molecular structure of IU1 and IU1–248 USP14 inhibitors. inhibition and excellent selectivity for USP14 over the other DUBs protein degradation (TPD) exemplified by the proteolysis targeting (Table 5). All ligands described by Wang and co-workers are reversible chimeras (PROTACs) technology has recently emerged as a promising inhibitors, and all are placed in the same region, establishing in­ approach besides general targeting of E3 and DUBs activity, since it teractions mainly with Ser431, Gln197, and Asp199. Wang and co- allows a more fine-tuned control of pathological targets in cancer, workers reported a co-crystal structure of the catalytic domain of among other diseases. Indeed, in contrast to conventional enzyme in­ USP14 interacting with IU1 and three derivatives. Authors showed that hibitors, PROTACs can elicit selective intracellular proteolysis thanks to all the inhibitors likely interacted with a previously unknown steric their specific heterobifunctional structure associating a protein-binding binding site in USP14, so that they could block the access of the C-ter­ moiety capable of engaging an E3 ubiquitin ligase, with another part minus of ubiquitin to the active site of USP14, thus inhibiting USP14 that can selectively bind to the target protein, being these two moieties activity [175]. covalently associated by a linker. Main benefits of the TPD approach Authors then characterized IU1-USP14, His426, Tyr436, and Tyr476 include the possibility to deplete the target proteins very fast (generally were seen to have an important role due to their contacts formed with in minutes), the elimination of protein variants, and its reversibility, that the benzene ring of IU1, via both hydrophobic and π-π stacking in­ globally offer more fine-tuned control. This new approach, already teractions. The orientation of the benzene ring of IU1 was blocked by the under clinical evaluation in different cancer subtypes, will undoubtedly 2 methyl groups of the pyrrole ring, so that, the existence of these methyl improve the field of precision medicine in oncology. groups was essential for retention of the activity of IU1 derivatives, thus the π-π stacking interactions were essential (Fig. 7). Financial support IU1–248 has been shown to be the best compound designed by Wang and co-workers, exhibiting a 10-fold higher potency than IU1. Never­ This work has been co-financed by the European Regional Devel­ theless, additional efforts will be required to achieve the characteriza­ opment Fund (ERDF) through the Interreg V-A Spain-France-Andorra tion of an ideal inhibitor for USP14 [175]. (POCTEFA) program, project PROTEOblood (EFA360/19). G.R. ac­ knowledges supports from Fondo de Investigación Sanitaria PI18/01383 4. Conclusions and perspectives (ERDF) “Una manera de hacer Europa”. This work was carried out under the CERCA Program (Generalitat de Catalunya). Ubiquitin-regulating enzymes are a rich resource of attractive targets for developing small molecules cancer therapeutics due to their essential Conflicts of interest role in a myriad of signaling pathways controlling cell fate. Studies during the last two decades have shown that deregulated E3 ubiquitin G. Roué received research funding from TG Therapeutics. The ligases play a critical role in the development, progression, and response remaining authors have no competing financial interests. of human cancers to conventional chemotherapy, and their targeting by the means of specific modulators has greatly improved the prognosis of a References subset of cancer patients, especially in hematological cancers. In the case of DUBs, although significant progress has been made in our under­ [1] R. Yau, K. Doerner, E. Castellanos, D. Haakonsen, A. Werner, N. Wang, X. Yang, N. Martinez-Martin, M. Matsumoto, V. Dixit, M. Rape, Assembly and function of standing of their biology and mechanisms of action, there is consider­ heterotypic ubiquitin chains in cell-cycle and protein quality control, 918-933. able work to be done in order to move the use of these molecules into the e20, Cell 171 (2017), https://doi.org/10.1016/J.CELL.2017.09.040. clinical practice. 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