Repurposing old drugs as new inhibitors of the ubiquitin-proteasome pathway for cancer treatment

Abstract

The ubiquitin-proteasome system (UPS) plays a central role in the degradation of cellular proteins. Targeting protein degradation has been validated as an effective strategy for cancer therapy since 2003. Several components of the UPS have been validated as potential anticancer targets, including 20S proteasomes, 19S proteasome-associated deubiquitinases (DUBs) and ubiquitin ligases (E3s). 20S proteasome inhibitors (such as bortezomib/BTZ and carfilzomib/CFZ) have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of multiple myeloma (MM) and some other liquid tumors. Although survival of MM patients has been improved by the introduction of BTZ-based therapies, these clinical 20S proteasome inhibitors have several limitations, including emergence of resistance in MM patients, neuro-toxicities, and little efficacy in solid tumors. One of strategies to improve the current status of cancer treatment is to repurpose old drugs with UPS-inhibitory properties as new anticancer agents. Old drug reposition represents an attractive drug discovery approach compared to the traditional de novo drug discovery process which is time-consuming and costly. In this review, we summarize status of repurposed inhibitors of various UPS components, including 20S proteasomes, 19S-associated DUBs, and ubiquitin ligase E3s. The original and new mechanisms of action, molecular targets, and potential anticancer activities of these repurposed UPS inhibitors are reviewed, and their new uses including combinational therapies for cancer treatment are discussed.

1. Introduction

1.1. The ubiquitin-proteasome system (UPS)

The UPS plays an essential role in the degradation of more than 80 % of intracellular proteins. The Nobel Prize in chemistry 2004 was awarded to Avram Hershko, Irwin Rose, and Aaron Ciechanover "for the discovery of ubiquitin-mediated protein degradation". Ubiquitin is a small protein with 76 amino acid residues found universally in eukaryotic cells [1,2]. Ubiquitination, a process of the covalent attachment of ubiquitin to a substrate protein, is highly regulated, involving the sequential action of several enzymes. Initially, ubiquitin activating enzyme (E1) uses energy from ATP hydrolysis to form a thiolester bond between itself and ubiquitin. Following activation, ubiquitin is then transferred to ubiquitin conjugating enzyme (E2) forming an ubiquitin thioester in a similar fashion. In the third step catalyzed by a ubiquitin ligase (E3) an isopeptide is formed between the ubiquitin C-terminus and ε-NH2 group of the substrate protein’s Lys residues [3,4] (Fig. 1).

Fig. 1.

The ubiquitin-proteasome pathway. Ubiquitination, a process of the binding of ubiquitin (Ub) to the substrate, involves the sequential actions of E1, E2 and E3 enzymes in an ATP-dependent manner. Most ubiquitinated substrates are then targeted for degradation by the 26S proteasome, which consists of 20S proteasome and 19S proteasome. The 20S proteasome contains two identical, 7-membered α-rings and two identical, 7-membered β-rings. There are three β-subunits that bear protease active sites. Ubiquitination is a reversible process due to a number of deubiquitinases (DUBs) that catalyze the removal of ubiquitin from target protein.

Once modified by a polyubiquitin chain, most of the target protein is ultimately degraded by 26S proteasome complex (Fig. 1). The 26S proteasome is composed of a core 20S proteasome and one or two regulatory cap 19S proteasomes [5]. The ubiquitinated substrates are recognized by the 19S proteasome, which performs its regulatory functions by binding and removing the polyubiquitin chains. Subsequently, the substrates are unfolded and translocated into the 20S proteasome, where they are degraded into short peptides. The 20S proteasome is a barrel-like complex, which consists of four rings, two identical outer rings being formed by seven α subunits and two identical inner rings formed by seven β-subunits. The proteolytic active sites of 20S proteasome are located on β1-, β2- and β5-subunits, displaying the postglutamyl-peptide-hydrolysis-like (PGPH-L), trypsin-like (T-L) and chymotrypsin-like (CT-L) activities, respectively [5].

Ubiquitination is a dynamic reversible process due to a number of deubiquitinases (DUBs) that catalyze the removal of ubiquitin from the substrate protein (Fig. 1). The human genome encodes approximately one hundred of DUBs which can be divided into six subfamilies [6]. Five of them belong to cysteine DUBs: ubiquitin specifc proteases (USP), ubiquitin C-terminal hydrolase (UCH), Machado-Josephin domain-containing proteases (MJD), ovarian tumor proteases (OTU), and monocyte chemotactic protein-induced protein (MCPIP). The sixth subfamily is the JAB1/MPN/Mov34 metalloenzyme (JAMMs), which are zinc metalloproteases. There are three DUBs associated with the 19S proteasome: USP14/Ubp6, UCHL5/UCH37 and Rpn11/POH1. While Rpn11/POH1 is a permanent 19S proteasomal member, both UCHL5 and USP14 are cysteine DUBs which can physically bind with two 195-base components,ubiquitin receptors Rpn13 and Rpn1, respectivley [7,8].

Targeting protein degradation has been demonstrated as a novel strategy for cancer therapy. Several different components of the UPS have been validated as effective anticancer targets. The first-in-class proteasome inhibitor bortezomib (BTZ) is a dipeptidyl boronic acid that reversibly inhibits the β5 catalytic subunits of the 20S proteasome. BTZ was clinically approved as a therapeutic agent for multiple myeloma (MM) and mantle cell lymphoma (MCL) by the US Food and Drug Administration (FDA) in 2003 and in 2006, respectively [9,10]. In addition, BTZ has shown significant anticancer activity in plasma cell dyscrasias and non-Hodgkin lymphomas, and BTZ has substantial activity in combination with multiple clinically used drugs [11-16]. In addition to BTZ, several second-generation proteasome inhibitors have also been approved by the US FDA, including carfilzomib (CFZ), an irreversible inhibitor, and ixazomib (IXZ), an orally administered reversible inhibitor [17]. The cancer cell selectivity of proteasome inhibition was thought to be due to higher amounts of oncoproteins or aberrant proteins in malignant cells that make the tumor cells more sensitive to the UPS inhibition than normal cells [18,19]. Proteasome inhibition will lead to the buildup of polyubiquitinated proteins in the cancer cells resulting in cell cycle arrest and cell death induction. BTZ-based therapies have become a staple for the treatment of MM; as a result, the survival rate of MM patients has improved significantly since clinical introduction of BTZ and other immunomodulatory drugs. However, BTZ has some shortcomings, such as drug resistance and various toxic side effects [20]. Nevertheless, the clinical success of 20S proteasome inhibitors suggests that the UPS could be considered as a potential target for the development of more specific and potent anticancer agents.

DUBs are an important part of the UPS, and majority of cancer cells have altered expression of DUBs which might act as oncogenes or tumor suppressors or regulators of various essential cancer-associated pathways. Consequently, a number of DUBs are emerging as attractive targets for cancer therapies [21]. Of the three DUBs associated with the 19S proteasome, Rpn11/ POH1 has been suggested as a potential target for drug discovery due to its absolute requirement for cancer cell viability [22]. Overexpression of Rpn11 in mammalian cells can induce the resistance to several cytotoxic drugs [23]. Rpn11 is frequently overexpressed in MM cells and the extent of Rpn11 expression is inversely correlated with the overall patient survival [24]. Knockdown of Rpn11 produces a phenotype similar to that produced by 20S proteasome inhibition, including decreased 20S proteasome activity and buildup of high-molecular-weight ubiquitinated proteins [22]. In addition to Rpn11/POH1, the other two proteasomal DUBs UCHL5 and USP14 have also been shown to be dysregulated in MM cells [25]. UCHL5 can activate the TGF-β signaling through deubiquitinating and stabilizing Smads as well as TGF-β receptor I (TGF-β R1) [26]. UCHL5 was also found to regulate the ubiquitination of an essential RNA splicing factor PRP19, thus promoting the cell migration and invasion of hepatocellular carcinoma (HCC) cells [27]. These observations support the hypothesis that inhibitors of 19S proteasomal DUBs could have potent anticancer properties and potential to treat BTZ-resistant cancer cells [25,28-30].

1.2. Ubiquitin-like systems

Ubiquitin-like proteins (UBLs) are defined as protein families that share structural and evolutionary relationships with ubiquitin as well as the ability to be conjugated to substrates through the concerted action of evolutionarily related E1s, E2s, and E3s. UBLs are conjugated to target proteins or lipids to regulate their activity, stability, subcellular localization, or macromolecular interactions [31].

Several UBLs have been identified, including SUMO, NEDD8, ATG8, ATG12, URM1, UFM1, FAT10, and ISG15, all of which share a similar three-dimensional structure to ubiquitin [32]. Most UBLs are conjugated to substrate proteins via an enzymatic cascade that resembles ubiquitylation, but have their own unique enzyme cascades and specific cellular functions [33,34]. The most well characterized UBLs are SUMO and NEDD8. SUMO can regulate most of the nuclear processes including ribosome maturation, DNA repair, transcription and splicing [35]. Many knockouts of SUMO-activating, -conjugating or -deconjugating enzymes in eukaryotic models gave lethal phenotypes, highlighting the essential role of SUMO signal transduction [36]. NEDD8 controls the stability and function of RING-Cullin E3 ligases, thereby regulating the turnover of a subset of substrates involved in cellular processes associated with cancer cell pathways [37].

1.3. Reposition of old drugs and natural compounds

Drug repositioning represents an attractive drug discovery strategy compared with the traditional de novo drug discovery approach, which is a time-consuming and costly process. Drug repositioning refers to the identification of new indications for already approved drugs and the application of the newly identified drugs to the treatment of a different pathological condition from the drug’s intended disease. For some old drugs, their preclinical data and safety information for original indications are known, which enables them to be developed via a cheaper and faster cycle and more efficiently translated from bench to bedside [38]. Drugs, which target the complex diseases (such as cancer) with multiple targets offer a great opportunity to reposition [39]. Drug combination can also be considered as another form of drug repositioning.

Recently, high-throughput screening (HTS) assays have been widely used to identify old drugs/agents or nature compounds as new candidate UPS inhibitors, another type of drug repurposing. HTS allows the testing of large numbers of compounds for their potential inhibitory activity to the UPS. For example, HTS assays can be applied to measure the catalytic activity in a purified or reconstituted enzyme/complex, cell extracts or intact cells [40]. As a result of HTS application, many tool compounds have been identified that have significantly enhanced basic scientific research and identification of lead compounds to be used as repurposed UPS inhibitors (see below and [41]). Application of these technologies has also provided a unique opportunity to re-establish some natural products as new candidate UPS inhibitors.

2. Repurposed drugs as 20S proteasome inhibitors

2.1. Clioquinol and analogs

Through screening National Cancer Institute (NCI)-Diversity Set Library, NSC-109268, an organic copper (Cu) compound (Table 1), was found able to inhibit the purified 20S and the 26S proteasome activities in human prostate cancer cell model [42]. Based on the chemical structure similarity to NSC-109268, the Cu-binding hydroxyquinoline (HQ) family members 8-hydroxyquinol (8-HQ) and clioquinol (5-chloro-7-iodo-8-HQ, CQ) (Table 1) were identified as novel 20S proteasome inhibitors [42].

Table 1.

Repurposed 20S proteasome inhibitors.

	Compounds	Chemical Structure	Proteasome Inhibitory activity	Ref.
HQ	NSC-109268		Inhibition of the CT-L activity of purified 20S rabbit proteasome (IC50 = 6 μM)	[42]
	Clioquinol (CQ)		CQ/Cu complex: Inhibition of the CT-L activity of purified 20S proteasome (IC50 = 2.5 μM); ~80% inhibition of proteasomal CT-L activity in prostate cancer cells at 20 μM	[49]
	8-Hydroxyquinoline (8-HQ)		8-HQ/Cu complex: 60 % inhibition of the CT-L activity of purified 20S rabbit proteasome at 10 μM; ~67 % inhibition of proteasomal CT-L activity in DCIS cells.	[42,61]
	5-amino-8-hydroxyquinoline (5AHQ)		Inhibition of the CT-L activity of 20S proteasome with IC50 ranging from 0.30 to 0.51 μM, no significant difference with or without Cu; Inhibition of the purified rabbit proteasome activity with a mean Ki of 2.1 μM (95% CI = 2.08 to 2.33 μM)	[63,64]
	2-amino-8-hydroxyquinoline (2AHQ)		Inhibition of the CT-L activity of 20S proteasome activity with IC50 ranging from 0.30 to 0.51 μM, no significant difference with or without Cu.	[63]
	5-aminomethyl-8-hydroquinoline (AMHQ)		Inhibition of the CT-L activity of 20S proteasome activity with IC50 ranging from 0.30 to 0.51 μM, no significant difference with or without Cu	[63]
	5-chloro-8-hydroxyquinoline (ClHQ)		Inhibition of the CT-L activity of 20S proteasome activity with IC50 about 5 μM, enhanced by Cu	[63]
	5-chloromethyl-8-hydroxyquinoline (ClMHQ)		Inhibition of the CT-L activity of 20S proteasome activity with Cu (IC50 =1.3 μM)	[63]
HIV-PIs	Ritonavir		Inhibition on proteasome activity in mouse model; Inhibition of the CT-L activity of the purified 20S proteasome	[68,69]
	Saquinavir		Inhibition of the CT-L and PGPH-L activities of the 26S proteasome and purified 20S proteasome	[71]
	Nelfinavir		Pan-proteasome inhibition in AMO-1 and U266 myeloma cells; 60 % inhibition of the CT-L activity of 26S proteasome at 5 μM; Inhibition the CT-L activity of the 26S proteasome of CD138-selected plasma cells from MM patients	[72,73]
	Indinavir		Inhibition of the CT-L and T-L activities of human 26S proteasome, but not PGPH-L activity of the 26S proteasome and the three peptidase activities of the 20S proteasome	[74]
	Tipranavir		Inhibition of the CT-L activity of 26S proteasome	[75]
Lithium		LiCl	Inhibition of the CT-L and PGPH-L activities of both 20S and 26S proteasome	[91]

CQ has been used for the treatment of neurodegerative disease [43-45]. It acts as a chelator or ionophore of divalent metals, such as Cu and zinc (Zn). X-ray crystallography and X-ray absorption spectroscopy shows that CQ forms complex with Cu through coordination of two CQ molecules to one Cu(II) [46,47]. Further study revealed that CQ could chelate at least some of the Cu from Cu chaperone proteins and oxidizes Cu forming a CQ-Cu(II) complex in the tumors [48].

The complex of CQ and Cu (CQ/Cu) has potent inhibitory effect on the proteasomal activity. It inhibits the purified 20S and the intracellular 26S proteasome activities, leading suppression of cell proliferation and induction of apoptotic cell death in human prostate cancer cells and xenografts [49]. As a Cu chelator, CQ exerts selective anticancer activity toward breast cancer, leukemia, and myeloma with less effect on normal cells [50,51]. Besides acting as the Cu chelator, CQ also works as Zn ionophore [52]. CQ can transport zinc across the plasma membrane [53] and release Zn into the lysosome [54], thereby leading to lysosomal disruption and cell death [53,55]. As cancer cells accumulate more Cu than the normal tissues, it’s reasonable to design a strategy targeting intratumor Cu using CQ to achieve selective anticancer efficacy. However, results from a phase I clinical trial showed that CQ exerted minimal inhibition of the proteasome, and no clinical responses were observed in patients with advanced hematologic malignancies [56]. The possible cause could be the low cellular concentration of CQ in tumor cells. Indeed, one patient with the highest intracellular levels of CQ had inhibition of the proteasome, consistent with the assumption that low cellular CQ was unable to generate biological and clinical effects. Thus it should be taken into consideration, when one plans a future clinical anticancer trial, of improving CQ delivery, increasing CQ cellular levels, and/ or combining CQ with Cu.

8-HQ (Table 1) is an organic compound that is structurally related to CQ. Similar to CQ, 8-HQ and its derivatives can chelate with divalent metal ions, which confers most of their bioactivities [42,57-60]. The anticancer activity of 8-HQ and its derivatives relies on Cu, and they fail to induce cell death if they lose Cu-binding abilities [61]. 8-HQ can inhibit the CT-L activity of purified 20S proteasome in a Cu-dependent manner. The inhibition of CT-L proteasome activity and apoptotic induction by 8-HQ/Cu was not from Cu-mediated oxidative damage to proteins, but from the formation of a proteasome inhibitor inside the tumor cells [42,61]. Based on that finding, several Cu-8-HQ derivatives complexes were developed for cancer treatment, including CuHQTS (Cu 8-HQ-2-carboxaldehyde-thiosemicarbazide) and CuHQDMTS (Cu 8-HQ-2-carboxal dehyde-4,4-dimethyl-3-thiosemicarbazide), two Cu/thiosemicarbazone complexes that showed potent anticancer activity against cisplatin-resistant neuroblastoma cells [62]. In addition, amino-HQ and chloro-HQ derivatives (Table 1) could also inhibit the CT-L activity of the proteasome and induce growth inhibition and apoptosis [63]. Some of the derivatives are FDA-approved drugs, for example, nitroxoline (5-nitro-8-HQ, or 5AHQ; Table 1) is a clinical used urinary antibacterial agent that has a more potent anticancer activity than CQ and 8-HQ [60]. It has been demonstrated that 5AHQ can bind with the α subunits of the 20S proteasome at non-catalytic sites, which results in inhibition of the proteasome and preferable induction of cell death in primary myeloma and leukemia cells over normal hematopoietic cells [64]. Thus, 8-HQ and its derivatives can act as potent 20S proteasome inhibitors with potential anticancer application for various types of cancer.

2.2. Human immunodeficiency virus (HIV)-Protease inhibitors

Since 1996, nine HIV protease inhibitors (HIV-PIs) (ritonavir, saquinavir, nelfinavir, amprenavir, atazanavir, indinavir, tipranavir, darunavir and lopinavir) have been approved by the U.S. FDA and widely used for the treatment of HIV infection [65]. The possible use of HIV-PI as a cancer treatment primarily originated from their success in treating HIV-related Kaposi's sarcoma [66]. As the HIV-I virus coded proteases have similar cleavage activities to the proteasome [67], researchers were interested in knowing whether these HIV-PI could inhibit proteasomes, thus considering to expand their medical implications for the treatment of cancers that are not related to HIV.

Of the nine HIV-PI, five of them are shown to inhibit the proteasome (Table 1). Ritonavir and saquinavir can inhibit the purified 20S proteasome [68-71]. They prefer to inhibit one or two proteasomal proteolytic activities other than the T-L activity [68-71]. The remaining three HIV-PIs can only inhibit the intracellular proteasome. Nelfinavir, is a pan-inhibitor of the 26S proteasome [72], which is a unique property among the HIV-PIs with proteasome inhibitory effects. Also, nelfinavir inhibits the CT-L activity of the 26S proteasome in CD138-selected tumor plasma cells from MM patients [73], while indinavir and tipranavir inhibit CT/T-L and CT-L activities of human 26S proteasome, respectively [74,75].

Similar to specific 20S proteasome inhibitors, inhibition of CT-L activity of proteasome by HIV-PIs leads to accumulation of ubiquinated proteins, inhibition of the NF-κB and induction of ER stress, eventually resulting in growth inhibition and apoptosis in various cancers [70,76-79]. It is well known that upregulation of the unfolded protein response (UPR) by proteasome inhibitors is needed for their anticancer activities and that UPR downregulation is responsible, at least partially for proteasome inhibitor resistance in MM [80]. Ritonavir at clinically relevant concentrations was able to significantly induce UPR; ritonavir-induced UPR can further enhance BTZ’s anticancer effect, resulting in increased levels of cancer cell apoptosis [81,82]. The combination of ritonavir with a second generation proteasome inhibitor such as delanzomib or IXZ also showed synergistical effects [83,84]. These data suggest that HIV-PIs could overcome tumor cell resistance to BTZ or 2nd generation proteasome inhibitors.

The efficacy of HIV-PIs against cancer in preclinical studies has pushed their translation into clinics. Most of the clinical trials are focused on nelfinavir and ritonavir. Nelfinavir can inhibit the intracellular β2 proteasome activity that is not targeted by BTZ or CFZ [72], thus it has been used to overcome 20S proteasome inhibitor resistance in clinical trials. A Phase I trial of nelfinavir-bortezomib-dexamethasone (NeVd) combination from Swiss Group showed promising activity in patients with heavily pretreated, BTZ-lenalidomide doublerefractory MM [85]. Furthermore, NeVd activity was observed in MM refractory to BTZ and more than one immunomodulatory drug in multicenter participated phase II trial, and 65 % objective response rate (ORR) was obtained in this heavily pretreated cohort, similar to that observed with first-line Vd treatment in BTZ-naïive patients [86].

A phase I trial of nelfinavir in combination with chemoradiotherapy for locally advanced pancreatic cancer showed that nelfinavir in combination with radiation and chemotherapeutics (gemcitabine and cisplatin) was well tolerated. Partial chemotherapy responses were observed in 5 of 10 patients who completed chemoradiotherapy, and minor responses were observed in 2 of 10 patients [87], indicating that it can increase the chemoradiotherapy’s effects in pancreatic ductal adenocarcinoma. A phase I trial of nelfinavir with concurrent chemoradiotherapy for unresectable stage IIIA/IIIB non-small cell lung cancer (NSCLC) showed that nelfinavir had a positive effect in NSCLC [88].

Most of the completed trials involving ritonavir were set up for HIV-related Kaposi's sarcoma, but the results are currently not available. In a phase II trial, ritonavir/lopinavir was tested in 19 patients with progressive or recurrent high-grade gliomas. This study did not meet its primary efficacy endpoint [89]. Promising antitumour activity was observed using ritonavir in combination with escalating dose of docetaxel (ModraDoc001/r or ModraDoc006/r) for the treatment of solid tumors [90]. Encouraged by this promising result, the same regimen but with variable doses of docetaxel (ModraDoc001-006/r) is currently under investigation in seven prostate and breast cancer clinical trials.

2.3. Lithium

Lithium (Table 1) has been successfully used for the treatment of depression for over 60 years. It has been reinvestigated as an anticancer agent mainly due to its inhibition on GSK3. It should be noted that lithium also has GSK3-independent activities. In addition, it has been found that lithium chloride (LiCl) specifically inhibits the CT-L and PGPH-L activities of both the 20S and 26S proteasome [91,92]. Inhibition of proteasome activity was also observed in LiCl-treated cells prior or post purification, suggesting that LiCl may affect the proteasome structure irreversibly [91]. It possibly occupied the binding site of magnessium, which is required for the activation of CT-L and PGPH-L activities [92]. The effect of LiCl on proteasome enzymatic activity led to stabilization of proteasome substrates such as the CCAAT/enhancer-binding protein alpha (C/EBPα) and the retinoic acid receptor, and potentiating retinoid-induced cell differentiation [91,93].

It has been found that the inactivation rates for the two proteasomal subunits (β5/CT and β1/PGPH) by LiCl are slow, which limits its application as a cancer cell death inducer like BTZ [92]. Clinical studies indicate that LiCl at doses used for bipolar disorder (BD) and depression did not show any anti-tumor effects in low-grade neuroendocrine tumors [94]. However, it can reduce the overall cancer risk in patients with BD [95]. Results from a randomized phase III study using lithium gamolenate in patients with advanced pancreatic adenocarcinoma showed that a low-dose treatment led to survival times similar to those of other treatments, while a high-dose treatment appeared to have an adverse effect. Thus lithium was not recommended for the treatment of advanced pancreatic cancer [96]. However, the lithium salt of gamma-linolenic acid was shown to prolong survival of inoperable pancreatic cancer patients [97].

Since lithium is not a potent proteasome inhibitor and apoptosis inducer, it can not be used as a solo anticancer agent. Consistently, there are some clinical trials currently to investigate lithium’s combinations with clinically used chemotherapeutics in various cancers.

3. Repurposed drugs as 19S-DUB inhibitors

3.1. Old drugs containing α-β-unsaturated ketone as UCHL5/USP14 inhibitors

3.1.1. WP1130

As a repurposed inhibitor of proteasomal cysteine DUBs, UCHL5 and USP14, WP1130 (Degrasyn, Table 2) has an α,β-unsaturated ketone, which could covalently interact with the cysteine sites of these DUBs through the Michael addition. Indeed, it has been found that WP1130 inhibits multiple cysteine DUBs, including proteasomal DUBs (UCHL5 and USP14), USP5, and USP9x [98]. WP1130 can induce proteasomal degradation of the anti-apoptotic protein Mcl-1, which is a substrate of USP9x [99]. In addition, WP1130 has been reported to regulate the deubiquitination of a subset of kinases, such as Bcr-Abl, Janus-activated kinase 2 (Jak2), and unc-51 like autophagy activating kinase 1 (ULK1), thus regulating the signal transduction pathways of these kinases [100-102]. WP1130 was also shown to enhance cytotoxicity of chemotherapeutic drugs such as BTZ, doxorubicin gemcitabine, cisplatin, and spindle poisons in several types of tumor cells [103-106].

Table 2.

Summary of repurposed 19S-deubiquitinating enzyme inhibitors.

	Compounds	Targets	Chemical Structure	DUB Inhibitory activity	Ref.
UCHL5i/USP14i	WP1130	UCHL5, USP14, USP5, USP9x		WP1130 inhibits the labeling of UCHL5, USP14, USP5 and USP9x in lysates by ≥80 % at 5 μM.	[98]
	b-AP15/ RA-9/ VLX1570	UCHL5, USP14		b-AP15 inhibits the proteasomal DUB activity in vitro at 50 μm. RA-9 inhibits the proteasomal DUB activity in vitro at 20 μM. VLX1570 inhibits proteasomal DUB activity in vitro with IC50 of 10 μM.	[108,113,115]
	Curcumin/ AC17	UCHL5 USP14		AC17 inhibits the proteasomal DUB activity in vitro with IC50 of 4 μM.	[118,123]
	Gambogic acid/dihydrogambogic acid	UCHL5, USP14, 20S		Gambogic acid inhibits the proteasomal DUB activity in vitro at 2 μM and inhibits the CT-like activity of 20 proteasome in vitro with IC50 of 0.5 μM.	[125,126]
	Metal-PT (Cu, Zn, Pt, and Ni)	UCHL5, USP14		Metal-PT inhibits the proteasomal DUB activity in vitro at ~0.5 μM.	[130,136,137,139]
	Gold compound (Auranofin, Au(PPh3)PT)	UCHL5, USP14		Auranofin inhibits the proteasomal DUB activity in vitro at 2 μm. Au(PPh3)PT inhibits the proteasomal DUB activity in vitro at 0.25 μM.	[151, 154]
UCHL5i	RA190	UCHL5		RA190 inhibits deconjugation of K48-linked tetraubiquitin by UCHL5 in vitro at 1 μM.	[157]
	Isothiocyanates	UCHL5, USP9X, USP1		BITC and PEITC inhibit the labeling of UCHL5 in lysates with IC50 of 22 μM and 13 μM, respectively.	[168]
USP14i	IU1/IU1-248	USP14		IU1 and IU1-248 inhibit the USP14 activity in vitro with IC50 of 12.25 and 0.83 μM, respectively.	[170,177]
Rpn11i	Capzimin/8TQ	Rpn11		Capzimin and 8TQ inhibit the Rpn11 activity in vitro with IC50 of 0.34 and 2.4 μM, respectively.	[178]
	Thiolutin	Rpn11		Thiolutin inhibits the Rpn11 activity in vitro with IC50 of 0.53 μm.	[187]
	ETPs	Rpn11		SOP11 inhibits the Rpn11 activity in vitro with IC50 of 1.3 μM.	[195]

3.1.2. b-AP15/RA-9/VLX1570

As another example of drug reposition for 19S-DUB inhibition, b-AP15 (Table 2) has been identified as a specific UCHL5 and USP14 inhibitor through screening a library of potential anticancer drugs that induce the lysosomal membrane permeabilization-mediated apoptosis [107]. b-AP15 induced apoptosis that was independent of p53 and Bcl2 status and displayed antitumor activity in both non-solid and solid tumor models [107-109]. b-AP15 induced accumulation of polyubiquitinated proteins and overcame resistance to 20S proteasome inhibitors in MM cells [109]. Moreover, b-AP15 induced strong apoptotic response through induction of endoplasmic reticulum (ER) stress and production of oxidative stress [110]. b-AP15 also caused atypical cell death by induction of severe mitochondrial damage and proteotoxic stress and inhibition of protein translocation over the ER [111,112].

RA-9 (Table 2), possessing a structure similar to b-AP15, is an inhibitor of UCHL5 and USP14 [113]. However, RA-9 also targets other DUBs such as UCHL1, UCHL3, USP2, USP5, and USP8 [114].

VLX1570 (Table 2) is another chalcone derivative of b-AP15 and prefers to inhibit USP14 over UCHL5. VLX1570 showed increased biological activity and specificity compared to b-AP15, and was advanced to a clinical trial for relapsed MM (now discontinued) [115]. Due to the highly expression of USP14, MM cells were particularly sensitive to VLX1570 treatment [116].

3.1.3. Curcumin/AC17

Curcumin (Table 2) is a natural compound extracted from the golden spice turmeric (Curcuma longa) that has a wide range of beneficial properties, including antitumor, antioxidant, anti-inflammatory, and antiviral [117]. It contains two electrophilic α,β-unsaturated carbonyl groups, which can react with cysteine group of DUBs. Curcumin induces p53-independent cell death partly due to the inhibition of DUBs [118]. Curcumin can also directly inhibit the activity of 20S proteasome, a result from its carbonyl carbons directly interacting with the hydroxyl group of the amino-terminal threonine residue of the 20S proteasomal CT-L subunit [119,120]. Several clinical trials have been carried out to evaluate the beneficial effects of curcumin on multiple types of cancer. It has been confirmed that curcumin can provide symptomatic relief, as well as other parameters of various cancer conditions, including MM, lung, breast, colon, colorectal and prostate cancer [121].

AC17 (Table 2), a 4-arylidene curcumin analog, exhibits improved antitumor activity over curcumin and is initially identified as an inhibitor of IKK (Inhibitory κB Kinase) [122]. AC17 can block the 19S proteasomal DUBs without inhibiting the proteolytic activities of 20S proteasome, resulting in NF-κB inhibition and p53 reactivation. AC17 could be potentially developed into an anticancer agent [123].

3.1.4. Gambogic acid

Gambogic acid (GA) (Table 2) is the principal pigment of gamboge resin of several Garcinia species. The α,β-unsaturated ketone of GA has been found to be essential for growth inhibition and apoptosis induction in cancer cell lines [124]. It was shown that GA and dihydrogambogic acid (Table 2) can inhibit proteasomal DUB activity [125]. GA can also inhibit 20S proteasome function after being metabolized by intracellular CYP2E1, and the C9-C10 double bond of GA is responsible for the proteasome-inhibitory function [126]. Importantly, cancer cell lines have been shown to be more sensitive to GA treatment than normal cell lines [127]. In both animal tumor models and clinical trials, GA shows a favorable safety profile, with minimal side effects [128].

3.2. Metal-based old drugs as UCHL5/USP14 inhibitors

3.2.1. Metal-Pyrithione (M-PT)

Pyrithione (PT) has excellent metal chelating properties that complex with various metals including copper, zinc, platinum, and nickel (Table 2). PT binds with copper which enhances its apoptosis-inducing ability. The pyrithione copper complex, CuPT, has been used as an antifouling ingredient [129]. It was reported that CuPT inhibited the UPS by mainly targeting proteasomal DUBs, UCHL5 and USP14 [130]. Results of silicon docking and active site labeling experiments confirmed that CuPT interacts with these two cysteine DUBs [130]. The pyrithione zinc complex, ZnPT, has been widely used as a booster biocide in antifouling paints and antidandruff shampoos [131-133], and has been shown a significant anticancer potential [134,135]. ZnPT could also target the proteasomal DUBs UCHL5 and USP14, without inhibiting the activities of 20S proteasome [136]. ZnPT efficiently induced apoptosis in cancer cells and significantly suppresses tumor growth in nude mouse xenografts.

Like CuPT and ZnPT, other metal-PT complexes, such as platinum pyrithione (PtPT) and nickel pyrithione (NiPT), can also target the proteasomal DUBs. PtPT dose not bind with DNA, distinct from clinically used platinum-containing drug cisplatin [137]. Cisplatin is highly reactive towards DNA as it has two non-leaving groups (NH3) and two fast-rate leaving groups (Cl) connecting the platinum ion [138]. However, the PT structure in PtPT or NiPT contains two medium-rate leaving groups (S and O) that allows PtPT to react with protein targets such as proteasomal cysteine DUBs over the nuclear DNA. It was found that PtPT and NiPT exhibited better anticancer activities than cisplatin in multiple cancer cell lines and overcome cisplatin resistance in NSCLC cells [137,139]. Both PtPT and NiPT remarkably inhibited tumor growth in nude mice xenografts, without showing any toxic side effects [137,139,140]. Future work should focus on the comprehensive evaluation of the safety of various PT-metal complexes in vivo.

3.2.2. Gold complexes

As early as ancient Chinese days, gold has been used for medicinal purposes towards skin ulcers, infections and inflammations [141]. Moreover, gold complexes were described to exhibit antitubercular activity [142] in 1890 and discovered to be rheumatoid arthritis (RA)-therapeutic in 1929 [143]. Several studies have shown that gold(III) compounds were able to target purified 20S proteasome and 26S proteasome in human breast cancer cells [144-146]. Auranofin (AF, Table 2) is a gold(I)-containing compound approved by the FDA in 1985 for the treatment of rheumatoid arthritis [147]. In recent years, the antitumor activity of AF has been studied by many investigators [148-150], and clinical trials were approved to evaluate the anticancer activity of AF and sirolimus (also called rapamycin, an antifungal agent and an mTOR inhibitor) in ovarian and lung cancer ( NCT03043001; NCT01737502). In 2014, it was reported that AF was a specific inhibitor of the proteasomal DUBs USP14 and UCHL5 [151]. Moreover, in another study, AF showed a potent antitumor activity against imatinib-sensitive and -resistant chronic myeloid leukemia (CML) cells in vitro and in vivo [152]. Notably, DUB inhibition but not ROS induction is required for AF-induced caspase activation and apoptosis [150,152,153]. Recently, it was reported that a new gold(I) complex, Au(PPh3)PT (Table 2), could also suppress tumor growth in vitro and in vivo, and induce apoptosis in cancer cells from acute myeloid leukemia patients [154]. Mechanistically, Au(PPh3)PT impaired UPS function by selectively inhibiting proteasomal DUBs UCHL5 and USP14 as well as other non-proteasomal DUBs, but with minimal effects on the function of 20S proteasome. Importantly, Au(PPh3)PT elicited less cytotoxicity in normal cells than several other metal compounds [154]. In conclusion, repurposing of AF and other gold complexes as effective DUB inhibitors may bring new strategies for cancer treatment.

3.3. UCHL5 inhibitors

3.3.1. RA190

Rpn13 regulates substrate deubiquitinaton by recruiting UCHL5 and activating UCHL5 catalytic activity [155]. Recently, RA190 (Table 2), a bis-benzylidine piperidone compound, was shown to covalently bind to cysteine 88 of Rpn13 in the 19S proteasome, resulting in accumulation of higher-molecular weight polyubiquitylated proteins [156]. The authors also found that the degradation of Ub-AMC by either purified UCHL5 enzyme or purified 19S proteasome was minimally affected by RA190 [156]. More recently, they performed an in vitro assay to directly probe the effects of RA190 on deconjugation of K48-linked tetraubiquitin by UCHL5, and found that RA190 could directly inhibit UCHL5 activity [157]. By using mass spectrometry, RA190 was found to directly react with several cysteines of UCHL5 [157]. Thus, RA190, as a repurposed inhibitor, can inhibit both Rpn13 and UCHL5 through parallel mechanisms. RA190 exerted antitumor effect through induction of caspase-dependent apoptosis and UPR. Moreover, RA190 was found to act synergistically with BTZ, lenalidomide, or pomalidomide against MM [158]. Importantly, RA190 administration retarded growth of MM tumors and ovarian cancer xenografts in mouse models, without affecting body weight or compromising spontaneous antitumor immunity [156]. These findings provide the rationale for novel therapeutics targeting Rpn13 or UCHL5 to treat cancer.

3.3.2. Isothiocyanates (ITCs)

Isothiocyanates (ITCs) (Table 2) are small molecules derived from glucosinolates in edible cruciferous vegetables like broccoli and watercress. The most commonly used ITCs include allyl ITC (AITC), benzyl ITC (BITC), 4-methylthiobutyl-ITC (erucin), sulforaphane (SFN) and phenethyl-ITC (PEITC) [159]. Several clinical trials of ITCs have been approved by the FDA for therapy of malignancies including breast, lung, gastrointestinal tract and prostate cancer [160-162]. Considerable evidence suggests that the possible molecular mechanisms of action by ITCs include: cell cycle arrest, induction of autophagy and apoptosis, and inhibition of inflammatory response [160,161,163-165]. SFN was reported to induce expression of several DUB genes that might be involved in Nrf2-dependent detoxification and antioxidant response [166]. BITC and PEITC have been reported to be strong proteasome inhibitors that suppress the growth of MM cells [167]. Moreover, BITC and PEITC were shown to inhibit DUBs USP9X and UCHL5 through the adduct formation with their active site cysteine residues [168]. USP9X is able to mediate Mcl-1 deubiquitination, and consistently Mcl-1-dependent cancer cells were found to be more sensitive to ITCs than other cells. Using SILAC assisted quantitative mass spectrometry, several other PEITC-targeted DUBs (such as USP1) were identified [169]. PEITC impaired USP1 activity, and thus increased the ubiquitination of PCNA, which functions as an adaptor for recruiting multiple factors to the site of DNA repair [169]. Hence, USP1 inhibition could explain the ability of ITCs to enhance cancer cell sensitivity to the DNA damage inducer cisplatin [169]. Thus, repositioning of ITCs as new potent and selective DUB inhibitors may facilitate their use in clinics.

3.4. USP14 inhibitors

IU1 (Table 2) was identified as a repurposed USP14 inhibitor by HTS from 63,052 compounds using Ub-AMC hydrolysis assay of VS-proteasomes reconstituted with USP14 [170]. IU1 enhanced degradation of proteasomal substrate tau or TDP-43 in murine embryonic fibroblasts (MEFs), and inhibition of USP14 was proposed as a therapeutic strategy for neurodegenerative diseases characterized by tau or TDP-43 aggregation [170]. However, IU1 failed to enhance proteasomal degradation of tau in neurons [171]. In addition, whether IU1 could improve degradation of tau proteins in vivo is still unknown. USP14 inhibition by IU1 induced cell cycle arrest in androgen receptor (AR)-positive prostate and breast cancer cells through inducing the degradation of AR proteins [172,173]. IU1 also enhanced the antiumor effect of enzalutamide through cell growth inhibition and apoptosis induction [174]. USP14 can be phosphorylated and activated by the protein kinase Akt, thus negatively regulating autophagy by removing K63-linked ubiquitination from Beclin 1 [175]. Therefore, USP14 may modulate both UPS and autophagy through regulating K48 and K63 ubiquitination, respectively. The co-crystal structures of USP14 with IU1 showed that IU1 can bind to the thumb-palm cleft region of the USP14 catalytic domain, resulting in steric blockade of the binding of the ubiquitin C-terminus to USP14 [176,177]. Although an IU1 derivative, IU1-248 exhibited a 10-fold higher potency than IU1 under in vitro conditions, there was no in vivo information available about IU1-248 [177].

3.5. Rpn11 inhibitors

3.5.1. Quinoline-8-thiol (8TQ)/Capzimin

Using a fluorescence polarization assay that specifically measures the DUB activity of Rpn11, Li et al. screened hundreds of metal-binding pharmacophores, which yielded quinoline-8-thiol (8TQ) (Table 2) as a Rpn11 inhibitor [178]. They also used HTS assay to screen 330,000 small molecules and identified one compound S-(quinolin-8-yl)-2-bromobenzothioate, a thioester derivative of 8TQ, as a potent Rpn11 inhibitor [178]. These two screenings reveal that 8TQ can serve as a parent scaffold for the further optimization of selective Rpn11 inhibitors. This was consistent with another report in which use of a fragment-based drug discovery approach lead to identification of 8TQ with high affinity for Rpn11 [179]. Inhibition of Rpn11 activity by 8TQ could be through binding to the catalytic Zn²⁺ ion in the active site of Rpn11. Next, structure-activity relationships (SAR) were explored to improve the potency of 8TQ. A novel compound capzimin (Table 2) showed a marked selectivity for Rpn11 [178]. The computational analysis of binding modes and selectivities of capzimin showed that it binds to the active site Zn²⁺ of Rpn11 and this interaction extends to the distal ubiquitin binding site [180]. Given that quinoline scaffold is prevalent in several clinical drugs, capzimin may act as a promising proteasome inhibitor for cancer therapy.

3.5.2. Thiolutin

Dithiolopyrrolones (DTPs) are a class of disulfide-containing natural products, which are synthesized by actinomycetes and proteobacteria [181]. The DTP thiolutin (Table 2) was originally thought to inhibit bacterial and fungal RNA polymerases, but no direct data support this hypothesis [182-184]. Thiolutin is also known to inhibit tumor angiogenesis [185]. Moreover, a quantitative mass spectrometric analysis showed that thiolutins stimulated Hsp27 phosphorylation in endothelial cells [186]. The effect of thiolutin on removal of ubiquitin by the proteasomal Rpn11 was tested and Rpn11 was found to be the direct target of thiolutin [187]. Thiolutin inhibits Rpn11 by binding of Zn²⁺ [187]. Thus thiolutin is a repurposed Rpn11 inhibitor. However, its antitumor potential remains to be further investigated.

3.5.3. Epidithiodiketopiperazines

Epidithiodiketopiperazines (ETPs) (Table 2) are a large group of structurally complex fungal metabolites that exhibit potent antibiotic and antitumor activities [188-190]. Various targets have been proposed to explain the biological activity of ETPs, including histone methyltransferase, HIF-1α/p300 complex, and thioredoxin reductase [191-193]. Previous reports showed that gliotoxin, an ETP, acts as an inhibitor of 20S proteasome depending on its disulfide bond [194]. Using the UbnGST-Wbp2 substrate to monitor 26S proteasome activity, gliotoxin and its core scaffold compound, SOP11 were found to inhibit proteasome function [195]. Furthermore, it was found that SOP11 potently inhibited Rpn11 activity of the proteasome through chelating the catalytic Zn²⁺ ion [195]. However, further research on the pharmacological antitumor properties of the ETPs is required.

4. Repurposed drugs as ubiquitin ligase inhibitors

An alternative strategy to targeting the UPS is to target enzymes upstream (e.g., E1, E2 or E3) of the 20S proteasome. The success in developing inhibitors of NEDD8-activating enzyme (NAE), an E1-like enzyme for cancer treatment supports this thought [196]. Recently, Piperacillin 1, a semi-synthetic, beta-lactam antibiotic was found to inhibit NAE reversibly. It inhibits Ubc12-NEDD8 formation with high selectivity [197]. Piperacillin 1 has been used for the treatment of febrile and neutropenic types of cancer in combination with a beta-lactamase inhibitor [198]. The discovery of a new therapeutic target may expand its application for other types of cancers that have high levels of NAE. Largazole is another repurposed E1 inhibitor which is also acknowledged as a histone deacetylase (HDAC) inhibitor [199]. It can inhibit the formation of the ubiquitin-adenylate complex without affecting thioester bond formation between ubiquitin and E1 [200].

It has been found that UBE2N (also known as Ubc13; an E2 enzyme) and its partners UBE2V1 and UBE2V2 are highly expressed in some types of cancers such as diffuse large B-cell lymphoma and MM [201,202]. Knockdown of UBE2N induces cell death and decreases cancer cell proliferation as well as tumor growth, suggesting that UBE2N may be an attractive therapeutic target for cancer treatment [201,202]. NSC697923, a novel UBE2N inhibitor repurposed from an existing compound library [201] was shown to decrease melanoma xenograft growth [202], demonstrating anti-cancer effects. NSC697923 exploits a binding groove unique to UBE2N, resulting in covalent adduct formation at the UBE2N active site cysteine. Mutation analysis reveals that the cytotoxic effect of NSC697923 is largely due to specific UBE2N inhibition [203]. Inhibition of UBE2N by NSC697923 in neuroblastoma leads to p53 nuclear accumulation and subsequent tumor suppression. It also exerts antitumor efficacy in neuroblastoma orthotopic xenografts [204]. In addition, NSC697923 was shown to be able to overcome radiation resistance., because this compound causes degradation of hypoxia-inducible factor 1 alpha subunit (HIF1α), a key factor in radiation resistance [205].

Although all three enzymes of the ubiquitination cascade (E1, E2 and E3) are potentially highly druggable, targeting E3 ligase is likely to be much more specific. Due to lacking of clinical data of the repurposed inhibitors of E1 and E2, this section will focus on repurposed drugs as E3 inhibitors.

4.1. IMiDs as repurposed inhibitors of CRL4^CRBN E3 Ligase

Thalidomide (Table 3A), an FDA-approved immunomodulatory imid drug (IMiD), was initially developed as a sedative to treat morning sickness in pregnant women, but later repurposed for the treatment of MM, owing to its antiangiogenic, immunomodulatory, and anti-inflammatory properties [206-209]. The use of thalidomide is strictly controlled due to the teratogenicity and adverse side-effects [210]. This led to the development of more potent and less toxic IMiDs, such as lenalidomide (Table 3A) and pomalidomide. All these IMiDs are approved by FDA for the treatment of MM [211]. In addition, lenalidomide has also been approved for the treatment of myelodysplastic syndrome (MDS) with 5q deletion and mantle cell lymphoma (MCL) resistant to BTZ [212,213].

Table 3.

Repurposed E3 inhibitors (3A) and DTC-based proteasome inhibitors (3B).

	Compounds	Targets	Chemical Structure	Inhibitory activity	Ref.
3A	Thalidomide	CRBN		Stabilizes CRBN by inhibiting its autoubiquitination through direct binding, inducing CRBN-mediated ubiquitination and degradation of target proteins	[214]
	Lenalidomide	CRBN		Same as Thalidomide	[217]
	ARV-825	CRBN, BRD4		Promotes the CRBN-dependent degradation of BRD4 through dual interactions with both molecules.	[222]
	Lovastatin	Skp2		Decreases the expression of Skp2 and results in the inhibition of Skp2-mediated ubiquitination and degradation of p27 and p21, leading to cell cycle arrest and apoptosis	[231,232]
	Clomipramine	ITCH		Interferes with the HECT Ub transthiolation activity of ITCH in an irreversible manner	[250]
	JS-K	Mdm2		Inhibits Mdm2 E3 ultimately accumulating p53 concentrations leading to inhibition of the ubiquitin proteasome pathway; Also, able to generate DNA damage and increase p53 concentrations	[264]
	Suramin	CRL		Targets CRL-mediated K48 polyubiquitination and inhibits Cullin-RING E3 ubiquitin ligase	[267]
3B	DSF	20S		DSF-Cu complex: Inhibition of the CT-L activity of the purified 20S proteasome (IC50=~7.5 μM); > 95 % inhibition of proteasomal CT-L activity in MDA-MB-231 cancer cells at 20 μM	[272]
	DDTC	20S		DDTC/Cu complex: 35 % inhibition of the CT-L activity of purified 20S proteasome at 50 μM; > 90 % inhibition of cellular 26S proteasome at 20 μM in intact cells	[275]
		19S DUB		AgDT: inhibition of the DUB activities of UCHL5 and USP14 at 0.08 μM.	[283]
		NPL4		CuET: Inhibition of p97-segregase–dependent protein degradation via interacting with NPL4 and inducing NPL4 immobilization and aggregation.	[290]
	PDTC	20S		Inhibition of the CT-L activity of purified 20S proteasome with IC50 values of 13.8 μM for PDTC/Zn and 5.3 μM for PDTC/Cu	[287]
	Au(DMDT)Br2,	20S		Inhibition of the CT-L activity of the purified 20S proteasome (IC50 = 7.4 μM) and 26S proteasome in intact MDA-MB-231 cancer cells (10–20 μM)	[144]
	AuBr2(ESDT)	20S		Inhibition the CT-L activity of the purified 20S proteasome (IC50 = 1.13 μM) and 26S proteasome in intact MDA-MB-231 cancer cells	[288]
	[Au(ESDT)]2	20S		Inhibition of the CT-L activity of the purified 20S proteasome (IC50 = 17.72 μM) and 26S proteasome in intact MDA-MB-231 cancer cells	[288]

Mechanically, thalidomide targets the substrate receptor protein cereblon (CRBN) [214], a component of CUL4-RBX1-DDB1-CRBN E3-ligase complex (CRL4^CRBN E3 ligase) [215]. As a substrate receptor, CRBN is involved in the ubiquitination of a wide range of substrates, regulating a broad range of essential cellular processes including DNA repair and replication as well as chromatin remodeling [216]. It has been demonstrated that IMiDs binding to CRBN can inhibit auto-ubiquitination of CRBN, but promote CRL4^CRBN-mediated ubiquitination and proteasomal degradation of substrate proteins [214,217]. IMiDs promotes myeloma cell death via CRL4^CRBN-dependent proteasomal degradation of IKZF1 (Ikaros) and IKZF3 (Aiolos), which are key transcription factors involved in B- and T-cell development [218-220].

Recently, IMiDs were exploited for proteolysis targeting chimeras (PROTACs). PROTACs are bifunctional or hybrid molecules that link the IMiD structure to another small molecule that binds a protein of interest (POI). Such IMiD-based PROTACs are capable of guiding the CRL4^CRBN E3 ligase to the POI, resulting in its ubiquitination and degradation [221]. For example, ARV-825 (Table 3A) is a hetero-bifunctional PROTAC that conjugated JQ1, an established chemical inhibitor of bromodomain and extraterminal domain (BET) family members, to a thalidomide analog to promote the CRBN-dependent degradation of Bromodomain-containing protein 4 (BRD4) [222]. BRD4 is a member of the BET family and acts as both a transcriptional/ epigenetic regulator for transcription of oncogenes and apoptosis regulators, such as c-Myc, Bcl-2 and Bcl-xL. Therefore, it has been validated as an important drug target for cancers [223]. It has been demonstrated that ARV-825 has a promising therapeutic potential in various types of cancer, including MM, acute myeloid leukemia, Burkitt's lymphoma, and prostate cancer [222,224-226].

Taken together, the binding of IMiDs or IMiD-based PROTACs to CRBN can induce CRL4^CRBN-mediated ubiquitination and degradation of target proteins, leading to the anticancer effect in MM and other types of cancer.

4.2. Statins as repurposed inhibitors of SCF^Skp2 E3 ligase

Statins (Table 3A) are a class of small fungal metabolites that inhibit the conversion of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) to mevalonate, a rate-limiting step for the cholesterol biosynthesis [227]. Statins are used as cholesterol-lowering drugs approved by the FDA [228]. It has also been demonstrated that statins are modulators of the UPS [229,230]. Recently, studies indicated that statins could induce the degradation of S-phase kinase-associated protein 2 (SCF^Skp2) [231,232]. SCF^Skp2 is the substrate recognition factor of Skp2-Skp1/Cullin-1/F-box-protein complex (SCF^Skp2 E3 ligase). SCF^Skp2 is a promising therapeutic target for cancer treatment since it selectively ubiquitinates and degrades cyclin-dependent kinase inhibitors (CKIs) such as p27 and p21 as well as the G₁-/S-specific cyclin E [233]. Notably, previous studies also reported that statins could lead to the induction of the p27 and p21, resulting in decreased cyclin-dependent kinase activity and subsequent cell cycle arrest in G₁ phase [234-236]. Clinical studies demonstrated an association of statin use with reduced cancer risk and prolonged survival in patients with certain types of cancer [237-240]. Numerous clinical trials have been performed in cancer patients using statins alone or in combination with other agents (https://clinicaltrials.gov/ct2/results?term=statin+AND+cancer&Search=Search). Although statin monotherapy did not give impressive results [241-243], the combination therapy of statins with other agents have shown more promising results [244-248]. Thus, repurposing statins as anticancer agents provides an encouraging strategy for cancer treatment.

4.3. Clomipramine, a repurposed inhibitor of ITCH E3 ligase

Clomipramine (CMI, brand name Anafranil) is an FDA-approved tricyclic antidepressant. It has been widely used for over 40 years in the treatment of patients with psychiatric disorders [249]. By using an ELISA-based HTS assay, CMI was identified as an ITCH E3 ligase inhibitor that interferes with the HECT Ub transthiolation activity of this enzyme in an irreversible manner (Table 3A) [250]. A number of ITCH substrates that are involved in tumorigenesis and chemosensitivity have been identified, such as p63, p73, Notch1, LATS1 (large tumor suppressor 1) and others [251-255]. Recent study indicated that inhibition of ITCH resulted in the suppression of proliferation and induction of apoptosis in lung cancer cells [256]. These results indicated that ITCH may play a relevant role in cancer biology and inhibition of ITCH is a potential strategy for cancer therapy. CMI has been reported to enhance the anticancer activity of several chemotherapeutic agents since 1980s [257,258]. It also has cytostatic properties and can induce apoptotic cell death [259,260]. As an antidepressant drug, CMI is very well-tolerated and can effectively cross the blood–brain barrier, thus it has potential anticancer activity for glioblastoma [261]. In addition, desmethylclomipramine (DCMI), the active metabolite of CMI, has been reported to inhibit growth of lung cancer stem cells (CSCs), decrease their stemness potential and increase the cytotoxic effect of conventional chemotherapeutic drugs [262]. Future studies should further clarify CMI’s anticancer mechanisms and confirm its anticancer effect in clinical settings.

4.4. Repurposing JS-K as an MDM2 inhibitor

First identified as a NO prodrug used to stabilize NO release, JS-K (Table 3A), a member of the diazeniumdiolate family, was used as a nitric oxide donor, releasing NO in a cell sustainably in conjunction with its reaction with glutathione-S-transferase (GST) [263,264]. Recently, JS-K has been shown to have inhibitory effects on p53 ubiquitylation via inhibiting Mdm2 [264]. Mdm2 is a ubiquitin E3 ligase responsible for p53 ubiquitylation [264]. JS-K-mediated Mdm2 inhibition leads to inhibition of p53 degradation, resulting in accumulation of p53 protein. In addition, by generating DNA damage, JS-K is also able to increase p53 expression [264]. Through this mechanism, JS-K has also been shown to regulate stability and activity of another E3 ligase, Siah2, which may explain how JS-K treatment links to growth inhibition of prostate cancer cells [264]. Other studies also support the idea that JS-K can be repurposed as a novel agent for treating various types of cancer [265].

4.5. Suramin as a repurposed inhibitor of Cullin-RING E3 ligase

Suramin (Table 3A) was first discovered as a remedy for African sleeping sickness and river blindness [266]. More recently, suramin has been recognized as a potent inhibitor of Cullin-RING (CRL) E3 ubiquitin ligase [267]. CRL controls various biological processes by directing several protein substrates for proteasomal degradation. Using HTS, suramin was found to target CRL-mediated K48 polyubiquitination [267]. CRL activity is secondary to the recruitment of the E2 ubiquitin-conjugating enzyme Cdc34, and therefore by targeting E3 cullin’s basic canyon region with suramin, CRL substrates accumulate and the E2-E3 interface is inhibited [267]. Although suramin is toxic and causes a fair number of side effects, it at noncytotoxic doses can sensitize tumor cells to chemotherapeutic agents [268,269]. Therefore, combinations of suramin with chemotherapies have been investigated in various cancer clinical trials (https://clinicaltrials.gov/ct2/results?term=Suramin+AND+cancer&Search=Search).

5. Repurposing disulfiram for cancer therapy

5.1. Dithiocarbamate-based compounds targeting the proteasome

Disulfiram (DSF; Table 3B), also known as Antabuse, is a member of the dithiocarbamate (DTC) family that was approved by the FDA in 1951 as the first drug to treat alcohol dependence. Mechanically, DSF targets and inhibits the enzyme activity of aldehyde dehydrogenase (ALDH), which is a critical enzyme in the metabolism of alcohol. Since the 1970s, scientists have noticed the tumor-suppressive effect of DSF [270], and in the past three decades, increasing evidence indicates that DSF has great potential to treat various human cancers in vitro and in vivo [271]. Inhibition of the UPS has been thought to be the primary mechanism accounting for the anti-cancer effects of DSF, because DSF has been identified as a proteasome inhibitor in a cell-based screening assay that monitors the 26S proteasome activity. Several studies have indicated that DSF has strong inhibitory effect on 26S proteasome activity in various cancer cell lines in a Cu-dependent manner, leading to induction of cancer cell death (Table 3B) [272]. However, several reports found that DSF had no inhibition on purified 20S proteasome [273-276], although one study showed that DSF potently inhibited the CT-L activity of purified human 20S proteasome at low micromolar pharmacological concentrations, via a mechanism different from that of BTZ or MG132 that block the active site of the proteasome [277]. Therefore, DSF may act at some other sites on the proteasome as a slow-binding partial noncompetitive inhibitor.

In acidic or Cu-rich environments, such as the stomach, DSF is reduced to diethyldithiocarbamate (DDTC). DDTC is a very strong chelator of transition divalent metal ions and is an effective drug against nickel carbonyl poisoning [278]. Several metal-DDTC complexes have also been reported to possess both proteasome-inhibitory and apoptosis-inducing activities in cancer cells. Although studies indicated that DDTC/Cu complex inhibits the proteasomal CT-L activity and induces apoptosis in breast, prostate and pancreatic cell lines [276,279], effects of Zn(DDTC)₂ and Cu(DDTC)₂ against purified 26S proteasome were much stronger than against purified 20S proteasome (Table 3B), indicated that these metal complexes may target 19S proteasome [275]. Moverover, the analysis of electronic density on atoms and bonds within Cu(DDTC)₂/Zn(DDTC)₂ showed that Cu(DDTC)₂/Zn(DDTC)₂ should be active against the active site of JAMMs [275], suggesting that Cu(II)-DDTC and Zn(II)-DDTC complexes might also inhibit 19S-JAMMs in tumor cells. Silver ions and silver complex have proven to exhibit excellent anticancer activity in vitro [280-282]. Recently, scientists report that the combination of silver ions and DSF can induce enhanced anticancer activity mainly through inhibition of proteasome function [283]. The silver complex AgDT (Table 3B), synthesized from silver and DDTC, efficiently induced cell death in NSCLC cells by targeting proteasomal DUBs USP14 and UCHL5 [283]. AgDT also induced DUB inhibition and antineoplastic effects in the NSCLC cell xenograft mouse models [283].

Other DTCs, such as pyrrolidine dithiocarbamate (PDTC, Table 3B), is a synthetic analog of DTC that has been reported to inhibit transcription factor nuclear factor-kappaB (NF-κB) [284]. When combined with either Zn(II) or Cu(II) chloride, PDTC was shown to inhibit the UPS [50,285,286]. PDTC/Cu and PDTC/Zn complexes were able to inhibit the CT-L activity of a purified 20S proteasome (Table 3B). These complexes were also able to inhibit proteasome activity and induce apoptosis in prostate and breast cancer cells. However, PDTC alone failed to exhibit similar results in cultured cells [287]. In addition, several gold dithiocarbamates, particularly derivatives of N,N-dimethyldithiocarbamate (DMDT) and ethylsarcosinedithiocarbamate (ESDT), such as Au (DMDT)Br₂, AuBr₂(ESDT) and Au(ESDT)₂ were able to inhibit purified 20S proteasome as well as intact 26S proteasome (Table 3B), resulting in accumulation of ubiquitinated proteins and proteasome target proteins, induction of apoptosis, and inhibition of tumor growth [144,288].

Taken together, all these studies suggest that DTC-based compounds are potent proteasome inhibitors in human cancer cells [289]. Since some of the compounds are clinically used for treatment of other diseases with well-established safety profiles and known side effects, repurposing them as proteasome inhibitors for cancer treatment may reduce the time, cost and risk for the development of novel anticancer drugs.

5.2. CuET targeting p97 segregase adaptor NPL4

Recently, Skrott et al. demonstrated that DDTC/Cu complex (CuET) can target p97 segregase adaptor NPL4 (nuclear protein localization 4) at much lower concentration and suggested that DSF inhibits the processing of ubiquitylated proteins by targeting NPL4-dependent segregase p97 (Table 3B) [290]. p97 is a type II AAA (ATPases associated with various cellular activities) ATPase and acts upstream of the proteasome degradation and downstream of substrate ubiquitination [291,292]. p97 has been reported to use energy from ATP to extract (segregate) polyubiquitinated proteins from different macro-complexes and cellular locations, and then present them to proteasome for degradation, thus functioning as a segregase [293,294]. Furthermore, p97 is also involved in degradation of highly folded ubiquitinated soluble proteasome substrates and thus functions as an unfoldase [295]. The functional diversity of p97 is facilitated by association with many different adaptor proteins that act to recruit or modify substrates. Heterodimer of NPL4 and UFD1 (ubiquitin fusion degradation 1) is the best-characterized p97 adaptor [296]. The NPL4-UFD1 complex binds to the N-terminal domain of p97, forming a p97-UFD1-NPL4 complex, which extracts polyubiquitinated proteins, primarily labeled with Lys48-linked ubiquitin chains, from their holding environment, subcellular membrane or multi-protein complex and delivers them to the proteasome for degradation [296-298]. Given the critical function of p97-UFD1-NPL4 complex in the UPS, it has emerged as an attractive cancer therapeutic target and several inhibitors are under development as anticancer drugs, such as DBeQ and Eeyarestatin I for targeting p97 [299,300].

CuET acts on NPL4 through interacting the putative zinc finger-NPL4 (ZF-NPL4) domain, inducing NPL4 immobilization and aggregation, thereby inhibiting p97-segregase-dependent protein degradation. This is followed by triggering heat-shock response (HSR) and endoplasmic reticulum (ER) stress/UPR and eventually cell death [290]. The authors also detected a higher CuET concentration in tumors than the liver and brain tissues from the same animals after treated with DSF [290]. The preferential accumulation of CuET in tumors may explain the previously finding that the DSF/Cu complex is highly cytotoxic to cancer cells but not damage normal cells [272,301].

5.3. Repurposing DSF in combinational therapy to target cancer stem cells

Recently, accumulated evidence also showed that DSF harbors potent cancer stem cell (CSC)/ tumor initiating cell (TIC)-targeting function based on its irreversible inhibitory effect on aldehyde dehydrogenase (ALDH). The canonical function of ALDH is to convert acetaldehyde to acetate in metabolizing alcohol. In 2007, a pioneer study demonstrated that the tumorigenic cell fraction, identified by high ALDH activity, is capable of self-renewal and of generating tumors that recapitulate the heterogeneity of the parental tumor in breast carcinomas [302]. Later studies further showed that ALDH1A1 and ALDH1A3, two of 19 ALDH isoforms expressed in humans, were generally believed to be responsible for the ALDH activity of CSC or TICs [303,304]. It is worth noting that both normal and cancer human mammary epithelial cells with increased ALDH have stem/progenitor properties [302]. Moreover, the results of our recent study, and several others, also showed an enrichment of ALDH⁺ cells under therapeutic conditions including chemotherapies and radiation therapy [305-307]. These findings are consistent with the clinical observation that recurrent tumors frequently show more aggressive progression than primary tumors along with resistance to original therapeutic reagents.

As early as 1987, the effect of ALDH inhibitors, including DSF, was studied on the ex vivo sensitivity of human multipotent and committed hematopoietic progenitor cells and malignant blood cells [308]. However, intensive investigation of the effect of DSF on ALDH⁺ cancer stem cell subpopulation was not started until 2012. So far, significant inhibitory effect of DSF on ALDH⁺ stem cell has been shown in multiple human cancers including glioblastoma [309], brain cancer, breast cancer [305], NSCLC [310], adult B-lineage acute lymphoblastic leukemia [311], acute myeloid leukemia (AML) [312], MM [313], and pancreatic cancer [314]. The results obtained from these studies indicate that DSF treatment not only prevents tumor recurrence and chemotherapy resistance, but also decreases tumor metastasis. However, the heterogeneity and plasticity of cancer stem cell remains a critical factor that needs to be addressed in order to achieve a better therapeutic efficacy.

Using breast cancer as an example, CD44⁺/CD24⁻ is has been characterized as another stem cell subpopulation in addition to ALDH⁺ subpopulation. Both the ALDH⁺ and CD44⁺/CD24⁻ sub-populations have strong self-renewal capability and will regenerate tumor mass when they were implanted in mice [315,316]. Multiple research groups, including ours, showed that ALDH⁺ and CD44⁺/CD24⁻ populations are not identical in terms of their morphological and oncogenic characteristics [307,317]. Cells co-expressing ALDH⁺ and CD44⁺/CD24⁻ may harbor strongest oncogenic and self-renewal capability [318,319]. This notion was confirmed by a transcriptional profiling study based on RNA and single cell sequencing [320]. Aligned with these reports, it has been reproducibly reported that either chemotherapy or radiation therapy could lead to accumulation of CSC cells in vitro and in vivo [321-324]. Due to the heterogeneous and plasticity pattern of CSCs in tumor, it is unlikely that targeting one CSC subpopulation will be therapeutically sufficient to prevent all CSCs’ function. Thus, simultaneously targeting multiple CSC populations or CSC-plasticity related signaling pathways is a more viable alternative.

This speculation was supported by our recent proposed combination therapy, in which DSF was used as an ALDH⁺-targeting reagent along with BKM120, a pan PI3K inhibitor and a CD44⁺/CD24⁻-targeting reagent [307]. Treatment with DSF/Cu or BKM120 induced higher levels of apoptosis in ALDH⁺ or CD44⁺/CD24⁻ sub-populations, respectively, than in bulk tumor cells. Combining DSF/Cu and BKM120 treatment simultaneously decreased the ALDH⁺ and CD44⁺/CD24⁻ CSCs/TICs. Using a TNBC (triple-negative breast cancer) tumor xenograft mouse model, we found that DSF/BKM in combination with Taxol significantly reduced the tumor burden and delayed tumor recurrence compared to Taxol treatment alone [307]. This study is the first of its kind to use two different drugs to abolish two major TIC subpopulations simultaneously and inhibit tumor recurrence. These results lay a foundation for developing a novel therapy that can improve chemotherapeutic efficacy.

5.4. Current clinical trials of DSF for cancer therapy

As an FDA-approved drug, DSF has a well-studied pharmacokinetics and excellent safety profile. The abilities of DSF to target the UPS and CSC further support it as a repurposed, promising anticancer drug. Hence, numerous clinical trials have been performed in patients with different types of cancer to confirm the anticancer effect of DSF either alone or in combination with other chemotherapeutics (https://clinicaltrials.gov/ct2/results?term=disulfiram+AND+cancer&Search=Search).

Since DSF is capable of penetrating blood-brain barrier, almost half of the trials are aimed to evaluate the effectiveness of DSF/Cu in patients with different stages of glioblastoma (GBM) ( NCT01907165, NCT03034135, NCT02715609, NCT03363659, NCT01777919, NCT02678975, NCT03151772, and NCT02770378). A phase I clinical trial ( NCT01907165) showed that DSF in combination with temozolomide (TMZ) had an acceptable safety profile and produced promising progression free survival (PFS) in GBM patients [325]. A recent phase II study ( NCT03034135) from the same group showed that addition of DSF/Cu to TMZ for TMZ-resistant isocitrate dehydrogenase (IDH)-wild type GBM appears well tolerated but has limited activity for unselected population [326]. A prostate cancer trial ( NCT01118741) demonstrates that DSF has no significant benefit on patients’ survival [327]. An ongoing new phase lb study is to investigate intravenous Cu loading with oral DSF’s effect on prostate cancer ( NCT02963051). Currently, no results are available yet from clinical trials investigating DSF’s effects against other types of tumors, such as melanoma ( NCT02101008, NCT00256230, NCT00571116), pancreatic ( NCT03714555, NCT02671890), liver ( NCT00742911) and breast ( NCT03323346) cancer.

Taken together, the available results from clinical trials indicate that DSF is well tolerated in most cancer patients and it can be combined with Cu or chemotherapeutic agents. All these findings provide promising prospects for repurposing this compound as an effective anticancer drug by further larger scale of trials.

6. Conclusions and perspectives

Since 2003, the 20S proteasome has been validated as an anticancer drug target and several 20S proteasome inhibitors (such as BTZ) have been approved by the FDA for the treatment of MM and some other liquid tumors. BTZ-based therapies have become a staple for the treatment of MM at all stages and the survival rate of MM patients has been increased at least 2-3-folds. However, BTZ has several limitations, such as drug resistance and neuro-toxicities.

Overcoming BTZ resistance can definitely improve the current status of cancer treatment. This could be achieved by, at least, developing new proteasome inhibitor drugs and old drug repositioning. The latter represents an attractive drug discovery strategy compared to the time-consuming, costly traditional de novo drug discovery process.

Literature has presented many examples of discovering old drugs as new inhibitors of the UPS. This review has summarized selected repurposed inhibitors of various UPS components, including 20S proteasomes, 19S proteasome-associated DUBs, and E3 enzymes.

Compared to typical drug discovery and development, old drug repurposing has both advantages and disadvantages. The advantages of drug repositioning include: previously well-known pharmacokinetics and pharmacodynamics in patients; fast drug development cycle; economical, efficient translation from bench to bedside; and easy to get clinical approval. The limitations of drug reposition include low target-specificity and off-target effects.

The fact that the UPS regulates degradation of many substrate proteins supports it as one of the most important drug repurposing targets for cancer treatment. Since an old drug was not designed to treat a new disease, there are often more targets that can be hit by the same old drug. However, anticancer drugs with multiple protein targets could represent excellent candidates for reposition. HTP of old drug libraries and natural product collections could also generate novel leads for drug/agent reposition as cancer treatment. Finally, an old drug, when combined with other compounds/drugs/agents could offer new opportunities to its repositioning.

It is exciting to see that rapidly developed modern technologies have made old drug reposition more feasible, precisional, and systematic. For example, better understanding genetic and epigenetic regulations of tumor formation and progression have increased opportunities of discovering more repurposed anticancer therapeutics. Indeed, recent genome-wide association studies (GWASs) have discovered numberous new potential targets for drug repositioning, which, of course, should be further validated by other functional technologies. It is believed that fast development in modern technologies, including computational, informatics, medicinal chemistry, and clinical profiling and analysis, has offered, and will offer us more oppotunites to reuse approved drugs for treating other human diseases such as cancer.