Disulfiram and 6-Thioguanine synergistically inhibit the enzymatic activities of USP2 and USP21

Hsin-Cheng Lin, Ying Kuan, Hsu-Feng Chu, Shu-Chun Cheng, Heng-Chih Pan, Wei-Yi Chen, Chiao-Yin Sun, Ta-Hsien Lin
a Basic Research Division, Medical Research Department, Taipei Veterans General Hospital, Taipei 112, Taiwan
b Department of Life Sciences and Institute of Genome Sciences, National Yang Ming Chiao Tung University, Taipei 112, Taiwan
c Program in Molecular Medicine, National Yang Ming Chiao Tung University and Acedemia Sinica, Taipei 112, Taiwan
d Institute of Biochemistry and Molecular Biology, National Yang Ming Chiao Tung University, Taipei 112, Taiwan
e Biomedical Industry Ph.D. Program, National Yang Ming Chiao Tung University, Taipei 112, Taiwan
f Department of Nephrology, Chang Gung Memorial Hospital, Keelung 204, Taiwan

a b s t r a c t
Disulfiram is a promising repurposed drug that, combining with radiation and chemotherapy, exhibits effective anticancer activities in several preclinical models. The cellular metabolites of disulfiram have been established, however, the intracellular targets of disulfiram remain largely unexplored. We have previously reported that di- sulfiram suppresses the coronaviral papain-like proteases through attacking their zinc-finger domains, suggest- ing an inhibitory function potentially on other proteases with similar catalytic structures. Ubiquitin-specific proteases (USPs) share a highly-conserved zinc-finger subdomain that structurally similar to the papain-like pro- teases and are attractive anticancer targets as upregulated USPs levels are found in a variety of tumors. Here, we report that disulfiram functions as a competitive inhibitor for both USP2 and USP21, two tumor-related deubiquitinases. In addition, we also observed a synergistic inhibition of USP2 and USP21 by disulfiram and 6- Thioguanine (6TG), a clinical drug for acute myeloid leukemia. Kinetic analyses revealed that both drugs exhib- ited a slow-binding mechanism, moderate inhibitory parameters, and a synergistically inhibitory effect on USP2 and USP21, suggesting the potential combinatory use of these two drugs for USPs-related tumors. Taken together, our study provides biochemical evidence for repurposing disulfiram and 6TG as a combinatory treatment in clin- ical applications.

1. Introduction
Cancers are rapidly growing threats to the increased population and extended lifespan of humans. The increment of diagnosed cancers is expecting to hit a new high annually and costs massive medical ex- penses globally. The necessity of anti-cancer treatments promoted the investigation and development of new medicines, but this investment has been hampered by high cost, high failure rate, time-consuming, and lacking molecular mechanisms. Alternatively, repurposing drugs are good candidates for anti-cancer therapeutics, since they have been clinically evaluated for safety and tolerance in human patients. One ofthe promising anti-cancer drugs in the Repurposing Drugs in Oncology (ReDO) project [1] is disulfiram, an FDA-approved alcohol-aversive compound, that inhibits aldehyde dehydrogenase led to the accumula- tion of acetaldehyde in the circulation [2,3] and exhibits anti-cancer ac- tivities in several preclinical models [4–6]. Previous studies have shown that disulfiram is capable of altering the intracellular levels of heavy metal ions and induces oxidative stress response that represses the pro- liferation of multiple types of cancer cells [7]. Furthermore, a nation- wide epidemiological study indicated that disulfiram administration to patients significantly lowers the risk of cancer-associated death [8], suggesting that disulfiram is a promising candidate for drug repurposing. In addition, our group has also reported a synergistic inhi- bition of coronaviral papain-like proteases by disulfiram and 6- Thioguanine [9], which is also an FDA-approved drug used for treating acute myeloid leukemia [10].
Although the disulfiram-associated metabolites and potential targeting pathways have been reported [8], the interactomes that account for the anti-cancer activity of disulfiram are still largely unex- plored. Furthermore, the disulfiram has been shown to displaybroad-spectrum inhibitory characteristics on multiple enzymes, includ- ing aldehyde dehydrogenase [11], urease [12], and methyltransferase [13], by reacting with the cysteine residues in the catalytic domains. In addition, we previously reported that disulfiram shows an intermediate inhibitory function on the papain-like proteases through attacking the zinc-finger domain that induces the ejection of zinc ion and destabilizes the secondary structure of these proteases [9]. Based on these studies, we reason that enzymes with similar zinc-finger domain structures, such as deubiquitinases in the family of Ubiquitin Specific Protease (USP), may also serve as intracellular targets of disulfiram in related cancer cells.
USP is the largest subfamily of deubiquitinating enzymes (DUBs) su- perfamily that catalyzes the removal of conjugated ubiquitin from tar- get proteins [14,15] and involved in multiple cellular pathways include DNA repair, cell cycle regulation, cell growth and apoptosis [16]. Particularly, the dysregulated expressions and activated enzymatic activities of USPs have been reported in a variety of cancers, suggesting that USPs inhibition may be a novel therapeutic strategy for cancer treatment [17–21]. Despite many new compounds have been shown to inhibit the activities of several USPs [22], however, there is no one succeed in the clinical trials for cancer treatment [19,23]. Thus, identifi- cation of potential USP inhibitors from repurposing drugs may serve as a feasible solution.
USPs are cysteine proteases sharing a conserved catalytic domain that composed by palm, thumb, and finger subdomains [15]. Notably, several human USPs contains a zinc-binding motif which plays a crucial role in their enzymatic activities [24]. Among the 56 members of human USP subfamily [25], USP2 and USP21 are phylogenetically categorized to the same subcluster [24] and display high structural similarity, sharing~50% identity in amino acid sequence and significant structural overlap of catalytic domain with a root-mean-square deviation smaller than 1 Å. Besides, USP2 and USP21 contain a functional zinc-binding motif that structurally similar to the zinc-figure domain of the papain-like prote- ases. Furthermore, these two USPs are also implicated, dependent on their DUB activities [26,27], in multiple cancers, including breast cancers [28,29], prostate cancers [30], and hepatocellular carcinomas [31], indi- cating they are potential targets for treating cancers.
Here, we have evaluated the inhibition of enzymatic activities of USP2 and USP21 by disulfiram and/or 6TG. Our results demonstrate that disulfiram was also a moderate competitive inhibitor to tested USP2 and USP21. Mechanistically, disulfiram may target USP2 and USP21 through binding to their catalytic cysteines on the ubiquitin- binding interface. Furthermore, disulfiram treatment destabilized the secondary structures of USP2 and USP21, suggesting that disulfiram may also target the zinc-finger domain and induce the ejection of the zinc ions, which is similar to the effect observed in papain-like prote- ases. We also showed that disulfiram and 6TG can synergistically inhibit the activities of USP2 and USP21, suggesting a repurposing application of disulfiram and 6TG in treating USP-related tumors.

2. Materials and methods
2.1. Expression constructs for recombinant USPs
The expression vector for the USP2 catalytic domain was previously described [20]. The expression vector for the catalytic domain (196–565) of USP21 was amplified from Flag-HA-USP21 (Addgene, # 22574) by PCR with primers 5′-AAACATATGGATGACAAGATGGCTCA TCAC-3′ and 5′-AAAGTCGACCAGGCACCGGGGTGGCTC-3′ andsubcloned into the Nde1-SalI sites of pET29a (Novagen). The USP21 C398S mutant was generated by QuickChange Lightning Site-Directed Mutagenesis Kit using primers 5′-CCACGACCTTCGAGGTTTTTTCTGAC CTGTCCCTGCCCATCCC-3′ and 5′-GGGATGGGCAGGGACAGGTCAGAAAAAACCTCGAAGGTCGTGG-3′ and according to manufacturer’s protocol. All constructs were confirmed by direct DNA sequencing.

2.2. Expression and purification of human USP2 and USP21 catalytic domains
Protein expression of USP2 catalytic domain was carried out as pre- viously described [20]. Briefly, the expression vector for the USP2 cata- lytic domain was transformed into E. coli Rosetta (DE3) cells (Novagen). Transformed cells were inoculated in 0.8 L LB with 25 μg/mL kanamycin and 17 μg/mL chloramphenicol at 37 °C for 4 h (until ~0.8 OD600), 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added for protein induction at 20 °C for 20 h. To express the USP21 catalytic domain, the expression vectors for USP21 and pTf16 (Takara) were co-transformed into E. coli BL21 (DE3) cells and 4 mM L-arabinose was added, to induce the expression of chaperone tig, prior induction of USP21 with IPTG as described above. The induced cells were pellet at 4 °C for 10 min and sonicated in lysis buffer (20 mM Tris, pH 8.5, 250 mM NaCl, 5% glycerol, 0.2% Triton X-100, 2 mM β- mercaptoethanol (βME)). The crude lysates were cleared by 9000 rpm centrifugation and soluble fractions were incubated with 1 mL pre- equilibrated Ni-NTA agarose (Qiagen) at 4 °C for 1 h. The resin was then washed with washing buffer (20 mM Tris, pH 8.5, 250 mM NaCl, 8 ml imidazole, 2 mM βME) and bound proteins were eluted in elution buffer (20 mM Tris, pH 8.5, 30 mM NaCl, 150 mM imidazole, 2 mM βME). Eluted proteins were further fractionated in an S-100 gel- filtration column (GE Healthcare) pre-equilibrated with running buffer (20 mM Tris pH 8.5, 100 mM NaCl, 2 mM dithiothreitol). The purity of recombinant proteins was examined by SDS-PAGE and Coomassie blue staining. Protein samples were concentrated using Amicon Ultra- 4 30 kDa filters (Millipore), supplemented with 5% glycerol, flash freez- ing, and stored at −80 °C.

2.3. IC50 determination
DUB assays were performed in a Luminescence Spectrometer LS50B (Perkin Elmer) as previously described [9]. The fluorogenic substrate Ub-7-amino-4-trifluoromethyl coumarin (Ub-AFC) (Boston Biochem) was used to measure the enzymatic activities of USP2 and USP21. For the inhibition assays, 0.25 μM Ub-AFC and indicated inhibitors were homogenously mixed in 0.5 mL phosphate buffer (20 mM, pH 6.5) followed by the addition of indicated enzymes. The continuous fluores- cent signals, excitation at 350 nm and emission at 485 nm, were imme- diately determined at 30 °C. The concentrations of USP2 and USP21 were 0.08 μM and 0.05 μM, respectively. The data were best fitted to ob- tain the IC50 for each enzyme as described previously [9].

2.4. Steady-state kinetic analysis
The steady-state kinetic assays were performed as previously de- scribed [9]. In brief, the concentrations of USP2 and USP21 were0.08 μM and 0.05 μM, respectively. The substrate Ub-AFC was ranged in 0.25–1.5 μM. Data were analyzed and plotted using SigmaPlot 12.5 (Systat Software Inc., USA).

2.5. Multiple inhibition assay
The assays were carried out as previously described with modifica- tions [9]. Briefly, the DUB activity of USP2 was measured without or with 20 μM disulfiram in the presence of 0–60 μM 6TG, whereas the DUB activity of USP21 was measured without or with 2 μM disulfiram in the presence of 0–40 μM 6TG.

2.6. Thermostability assays
For circular dichroism analysis, the protein concentration was
0.2 mg/mL in 20 mM phosphate buffer (pH 6.5) and measurements were performed with ellipticity at 222 nm and the temperatures ranged from 25 °C to 85 °C in a Circular Dichroism Spectrophotometer Model410 (AVIV Biomedical). To access the effect of disulfiram, indicated measurements were performed in the presence of 0.5 μM disulfiram.

2.7. Recoverability assay
Recombinant USP2 or USP21 was incubated without or with 200 μM disulfiram at 4 °C for 1 h and pass the Sephadex G-25 (Pharmacia Bio- tech) column to remove unbound disulfiram. The DUB activities of treated USP2 and USP21 were then determined as described above in the absence or presence of 5 mM βME.

2.8. Inactivation mechanism
To determine the inactivation mechanism, the enzyme kinetics were performed as described above in the presence of 0–40 μM of disulfiram and 0.2–4 μM Ub-AFC and monitored for 3 min. Results are processed and presented as previously described [9].

3. Results
3.1. The inhibition of USP2 and USP21 by disulfiram
Our group has previously reported that disulfiram displays an inhib- itory effect on the coronaviral papain-like proteases by targeting the cysteine residues of the zinc-finger domain for covalent modification [9]. USPs share a conserved catalytic triad with the catalytic cysteine and zinc-finger subdomains that structurally similar to the zinc-finger domain of papain-like proteases [15]. Additionally, it is proposed that modifications of the catalytic cysteine of USPs interfere with their DUB activities [32]. We, thus, reasoned that disulfiram may be also capable of inhibiting the activities of USPs. Notably, our kinetic assays revealed that the IC50 values of disulfiram on USP2 and USP21 were 25 ± 1.2 μM and 3.7 ± 0.4 μM, respectively (Fig. 1 and Table 1).
Notably, addition of a detergent, Triton X-100, showed no effect to the inhibitory function of disulfiram (Fig. S1), indicating the specificity of disulfiram-mediated inhibition.
Next, to characterize the disulfiram-induced inhibitions of USP2 and USP21, kinetic measurements were carried out with a range of concen- trations of inhibitor, and raw data were fitted to available inhibitory models. Consistent with the previous study in papain-like proteases, we found that the disulfiram-induced inhibitions of USP2 and USP21 were best fitted to the competitive inhibition model (Fig. 2).
Interestingly, our results also revealed that the inhibition constant (Kis) of disulfiram for USP2 was 12.2 ± 1.0 μM which is higher than that for USP21 (4.6 ± 0.7 μM) (Table 2). To clarify the differencebetween IC50 and Kis of disulfiram on USP2 and USP21, we carried out the structural alignment based on Cα coordinates (Fig. 3a).
We found that the Cys398 is located in the ubiquitin-binding core of USP21, whereas a Trp439 was found at a similar position of USP2 (Fig. 3b). We reasoned that Cys398 is an ideal target for disulfiram mod- ification and the distance between Cys398 and the Gly47 of ubiquitin is only 3.4 Å. To test this possibility, we mutated Cys398 of USP21 to Ser- ine, a non-conjugatable residue for disulfiram, and found that the IC50 of disulfiram on USP21 was significantly increased to 27.5 ± 2.5 μM, which is similar to that of USP2 (Fig. 3c and Table 1). This result suggests that cysteine residue on the surface of the USP catalytic domain may be targeted by disulfiram.

3.2. Time-dependent inhibition of USP2 and USP21 by disulfiram
Disulfiram is a thiol-reactive agent that covalently modifies cysteine residues and results in a diethyldithiocarbamate (DDC) moiety on the target protein [33]. Our previous study has shown that disulfiram- induced inhibition of the papain-like proteases of SARS coronavirus could be partially reversed by the reductant βΜΕ treatment [9]. We wondered whether the same cysteine-conjugation mechanism is also applied to disulfiram-induced inhibition of USPs. To test this, we first determined the DUB activities of USP2 and USP21 pre-treated with di- sulfiram followed by passing a Sephadex G-25 column to remove the unbound disulfiram. The results showed that pre-treatment of disulfi- ram diminished nearly 96% and 84% DUB activities of USP2 and USP21, respectively (Fig. 4).
The graph showing the relative DUB activities of disulfiram-treated USP2 (left) and USP21 (right) in the absence or presence of 5 mM βME. The activities of untreated USPs (control) are set to 1.
In addition, the inclusion of βΜΕ in the reaction rescues 27% and 42% DUB activities of USP2 and USP21, respectively (Fig. 4). Furthermore,and in agreement with a previous study on papain-like protease, the ki- netic assays revealed a dose- and time-dependent inhibition of USP2 and USP21 activities by disulfiram (Figs. 5 and S2), suggesting a similar slow-binding inhibitory mechanism on these USPs. These results sug- gest that the cysteine-conjugation may also involve in the by disulfiram-induced inhibition of USPs.

3.3. 6TG inhibits USP2 and USP21 through different inhibitory mechanism
Our previous study also determined the inhibitory effect and unraveled the binding site of 6TG on USP2 [20]. Crystal structure (PDB: 5XU8) further suggests that 6TG covalently binds to catalytic cys- teine of USP2. Enlighten by this observation and the structural similarity of the 6TG-binding pocket of USP2 to that of the USP21 (Fig. S3), we thus reasoned that 6TG may also be a potential inhibitor for USP21. The DUB assays revealed that the IC50 of 6TG on USP21 was 22.7 μM (Fig. 6a).
Interestingly, kinetic assay revealed that 6TG was a competitive in- hibitor for USP21 (Fig. 6b), whereas 6TG showed a noncompetitive mode on USP2 [20]. This observation suggests that the 6TG may interact with the catalytic site of USP21, however, further structural analyses are required to clarify the mechanistic effect of 6TG on USP21. Notably, and in agreement with our previous observation on USP2 [20], the 6TG also displayed a slow-binding manner and moderate inhibitory parameters on USP21 (Fig. S4 and Table 2).

3.4. Disulfiram and 6TG synergistically inhibit USP2 and USP21
Apart from cysteine residues within the catalytic domain and ubiquitin-binding surface of USP, the zinc-finger domain also representsan ideal attacking site for disulfiram. The zinc-finger domains of USP2 and USP21 contain four cysteine residues that bind to a zinc ion, which is similar to the zinc-finger domain of coronavirus papain-like proteases. Our previous study has demonstrated that disulfiram targets cysteine residues in the zinc-finger domain and ejects the bound zinc ions that destabilize the secondary structures of coronavirus papain- like proteases [9]. Given that the structural similarity of the zinc-finger domain, we propose that disulfiram may also interfere with the zinc co- ordinated structures of USP2 and USP21. In addition, we have previ- ously reported that 6TG covalently conjugated with the cysteine in the catalytic domain rather than that of the zinc-finger domain (PDB: 5XU8), suggesting a potential synergistic inhibition by disulfiram and 6TG. Kinetic assays validated this speculation that the two drugs are able to synergistically inhibit the DUB activities of both USPs (Fig. 7).
In the presence of disulfiram, the IC50 values of 6TG on USP2 andUSP21 were decreased to 0.7-fold and 0.4-fold, respectively, of 6TG alone (Table 1). The results also indicate that these two drugs may mod- ify the cysteine residues in different regions of USPs. In the case of USP2, 6TG was reported to target the cysteine residues in the catalytic site [20], the synergistic effect of 6TG, and disulfiram verifies the idea that disulfiram may conjugate the cysteine residues on other regions of USP2. However, the structural analyses are required to uncover the synergistic effect of disulfiram and 6TG on USP21. To test whether disul- firam modulates the structure of and destabilize USPs, the thermo- stability of USPs were determined without or with 0.5 μM disulfiram (Fig. S5). Despite at low concentration of disulfiram, the melting tem- peratures of USP2 and USP21 still showed 2 °C and 3.5 °C decrements, respectively, confirming that disulfiram targets the zinc finger domain and causing the ejection of zinc ion that leads to destabilization of structure.

4. Discussion
4.1. Disulfiram interferes the zinc-coordinated structure to inhibit enzymes
A functional zinc-binding site is contained in 45 out of 56 human USPs [24], suggesting a crucial role for zinc ion in coordinating the pro- tein folding and enzymatic activities of USPs. Our result indicated that disulfiram may target the cysteine residues on the zinc-finger domain of USP2 and USP21 that might lead to the release of zinc ion and desta- bilize the protein structure. In addition, we found that the presence ofCys398 on the ubiquitin-interacting interface of USP21 enhances the in- hibitory function of disulfiram, indicating that other cysteine residues outside of the zinc-finger domain may be also targeted by disulfiram.
Moreover, disulfiram has been reported to exhibit anticancer activities in both cell models and preclinical trials. One potential mechanism for this activity is involving the formation of disulfiram-chelated complexes with heavy metal atoms, such as copper and zinc [7]. Thus, disulfiram could effect on cellular functions by sequestering the intracellular metal ions to block the activities of metal ion-dependent enzymes. For example, disulfiram could diminish the activity of superoxide dismutase to upregulate oxidative stress and matrix metalloproteinases to de- creased cell invasiveness of cancer cells [34]. Our result suggested that the USPs may also serve as alternative targets of disulfiram, by zinc se- questration and/or reacting to cysteine residues on USPs, that may be also implicated in repressing the growth of cancer cells. This idea is sup- ported by the accumulation of poly-Ub proteins in disulfiram-treated cells [8]. Furthermore, disulfiram was reported to enhance the efficacy of auranofin in repressing the proliferation of ovarian cancer cells [35].

4.2. USPs are synergistically inhibited by disulfiram and 6TG that may use- ful for cancer treatment
Upregulated USPs are highly correlated to various cancers. For in- stance, both USP2 and USP21 were reported to promote the tumor pro- gression of Triple-negative breast cancer (TNBC) [36,37]. Moreover, USP2 and USP21 regulate TNBC progression through two different sig- naling pathways, matrix metalloproteinase-2, and Nucleotide oligomer- ization domain-like receptor pathways, respectively. A recent study showed that a USP2 inhibitor ML364 enhances the efficacy ofchemotherapy treatment in TNBC [38]. Interestingly, disulfiram was also reported to suppress the proliferation of TNBC [39,40]. These obser- vations, together with our findings, suggest that USPs may serve as po- tential therapeutic targets of TNBC.
Since disulfiram and 6TG are both FDA-approved drugs with high safety and well-tolerance in humans. Our biochemical results may fur- ther suggest a combinatory application in treating USP-dysregulated tu- mors [7].

5. Conclusions
Our current study has shown that disulfiram is a competitive inhib- itor for tested USP proteins, USP2, and USP21, with moderate kinetic pa- rameters. We further identified 6TG is a competitive inhibitor for USP21, whereas it functions as a non-competitive inhibitor for USP2. In addi- tion, the time-course, recoverability, and thermostability assays suggest that disulfiram may covalently modify cysteine residues and disrupt the ternary structure that leads to the irreversible inhibition of USPs. Impor- tantly, co-treatment of disulfiram and 6TG displayed a synergistic effect of inhibition on both USPs, implying that disulfiram may alternatively target the zinc finger domain as 6TG directly binds to catalytic cysteines. Together, our study provides biochemical evidence for the evaluation of combination treatment of disulfiram and 6TG for USP2 or USP21 dysreg- ulated diseases.

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