XL177A

Characterization of naturally occurring pentacyclic triterpenes as novel inhibitors of deubiquitinating protease USP7 with anticancer activity in vitro

Abstract
Deubiquitinating protease USP7 is a promising therapeutic target for cancer treatment, and interest in developing USP7 inhibitors has greatly increased. In the present study, we reported a series of natural pentacyclic triterpenes with USP7 inhibitory activity in vitro.
Among them, both the ursane triterpenes and oleanane triterpenes were more active than the lupine triterpenes, whereas ursolic acid was the most potent with IC50 of 7.0±1.5 μmol/L. Molecular docking studies showed that ursolic acid might occupy the ubiquitin binding pocket of USP7, with the 17-carboxyl group and 3-hydroxyl group playing a vital role in the USP7-ursolic acid interaction. Using the cellular thermal shift assay, we demonstrated that ursolic acid interacted with USP7 in RPMI8226 human myeloma cells. Ursolic acid dose-dependently inhibited the proliferation of the myeloma cells with IC50 of 6.56 µmol/L, accompanied by reductions in USP7 substrates such as MDM2, UHRF1 and DNMT1. Overexpression of USP7 partially, but significantly attenuated ursolic acid-induced cell death as well as downregulation of MDM2, UHRF1 and DNMT1. In conclusion, we demonstrate for the first time that pentacyclic triterpenes represent a novel scaffold for developing USP7 inhibitors and that USP7 inhibition contributes to the anti-cancer effect of ursolic acid.

Introduction
Ubiquitination is a highly dynamic process orchestrated by a series of conjugating enzymes[1]. Deubiquitylating enzymes (DUBs), which include 5 structurally different DUB enzyme families, can deconjugate ubiquitin from polyubiquitylated proteins[2]. Ubiquitin-specific enzymes (USPs) are the largest subfamily of DUBs, with more than 60 members in humans[3,4]. USPs such as USP1, USP2, USP7, USP9x and USP14 have been found to be closely correlated with the development of cancer[3-7]. Among them, USP7 has received special atten- tion. The inhibition of USP7 is predicted to destabilize MDM2 and stabilize p53, which promotes the cell cycle arrest andapoptosis of cancer cells[8]. Moreover, many cancer-related proteases have been reported as additional substrates of USP7, such as UHRF1, DNMT1, claspin, FOXO4 and PTEN[9-13]. The overexpression of USP7 has been observed in prostate cancer, myeloma cells, neuroblastoma, breast cancer, and ovarian can- cer[7, 14-17], while inhibiting USP7 can inhibit proliferation and induce cell death in cancer cells. Therefore, the development of USP7 inhibitors has attracted great attention for the treat- ment of cancer. Although many small-molecule USP7 inhibi- tors have been reported, no natural product has been reported as an inhibitor of USP7 to date[18].Pentacyclic triterpenes and their derivatives occur widely in the plant kingdom and have been reported to have a variety of biological activities, such as antitumor, anti-inflammatory, antiviral and antioxidant activities[19, 20].

For example, ursolic acid has been shown to inhibit the proliferation of a variety of cancers, such as breast, prostate, and liver cancer and leuke-mia. In addition, multiple signaling pathways including the STAT3, NF-κB, PTEN, and TRAIL pathways have been shown to be involved in the anticancer effect of ursolic acid[21, 22]. We previously reported that CDDO-Me has USP7 inhibitory activ- ity. This result prompted us to investigate whether deubiqui- tylating enzymes play a role in the action of pentacyclic triter- penes such as ursolic acid.In the present study, we demonstrated for the first time that a series of naturally occurring pentacyclic triterpenes inhibit USP7 activity by directly interacting with USP7. Among them, ursolic acid (1) shows inhibitory activity against USP7 in vitro and in cells. In addition, the inhibition of USP7 contributes to the apoptosis-inducing effect of ursolic acid in myeloma cells. Our results suggest that natural pentacyclic triterpenes represent a novel scaffold for developing USP7 inhibitors and that USP7 is a novel target involved in the anticancer effect of ursolic acid.Natural pentacyclic triterpenes (compounds 1–13, purity>98%) were purchased from Herbpurify (Chengdu, China). All sol- vents were purified and dried by standard procedures and stored over a suitable drying agent prior to use. Unless oth- erwise noted, all reagents were purchased from commercial sources and used without further purification.For chemical synthesis, the reactions were monitored by thin layer chromatography on plates coated with silica gel. 1H NMR data were acquired with CDCl3 as the solvent on a Bruker DMX 500 or a JEOL ECA-400 spectrometer.

The chemical shifts (δ) were expressed in parts per million (ppm) relative to TMS (tetramethylsilane). Spin-spin coupling con- stants (J) were measured directly from the spectra and given in Hz. Exact mass (HR-MS) spectra were recorded on a Thermo Fisher Scientific LTQ FT Ultra (USA). Purity was based on the integrated UV chromatogram (254 nm), and compounds 14–15 had purity ≥ 95% as verified by HPLC.Human myeloma cells (RPMI8226) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI-1640 (Gibco, Foster City, CA, USA ) supplemented with 10% (w/v) fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco). The cell lines were maintained at 37°C in a humidified atmosphere with 5% CO2.USP7 (5 nmol/L) was incubated with compounds for 10 min at 37 °C in reaction buffer (50 mmol/L Tris-HCl pH 8.0, 20 mmol/L NaCl, 2 mmol/L DTT). Then, GST-UBA52 (3.92 µmol/L, final concentration) was added and incubated for another 45 min at 37 °C. The reaction was terminated by add- ing loading buffer and boiling on a heat block. The proteins were separated by 12% SDS-PAGE and visualized with Coo- massie brilliant blue (G250). The density of the bands wasmeasured with Quantity One software (version 4.69, Bio-Rad, CA, USA), and IC50 was determined using GraphPad software (version Prism 5 Demo, GraphPad Software, Inc, CA, USA). All experiments were repeated three times.The pET28a(+)-UBA52 plasmid was transfected into E coli BL21, and the expression of UBA52 was induced with 0.5 mmol/L isopropyl-D-thiogalactoside (IPTG) at 16 °C for 12 h. Cell pellets were resuspended in lysis buffer (300 mmol/L NaCl, 50 mmol/L PBS pH 8.0, 10 mmol/L imid- azole, 10 mmol/L β-mercaptoacetic ethanol and 10 mmol/L glycerol) and sonicated. His-tagged proteins were purified on Ni-NTA-agarose and eluted with 250 mmol/L imidazole in 300 mmol/L NaCl, 50 mmol/L PBS pH 8.0, 10 mmol/L β-mercaptoacetic ethanol and 10% glycerol.

RPMI8226 cells were harvested and washed with PBS. The cells were diluted in PBS supplemented with a complete pro- tease inhibitor cocktail. The cell suspensions were frozen and thawed three times using liquid nitrogen. The soluble fraction (lysate) was separated from the cell debris by centrifugation at 20 000×g for 20 min at 4°C. The cell lysates were diluted with PBS and divided into two aliquots, one treated with DMSO and the other with diluted ursolic acid. After 30 min of incubation at room temperature, the respective lysates were divided into smaller (50 μL) aliquots and heated individually at different temperatures for 3 min (Veriti Thermal Cycler, Applied Biosystems/Life Technologies) followed by cooling for 3 min at room temperature. The appropriate temperatures were determined in preliminary CETSA experiments (data not shown). The heated lysates were centrifuged at 20 000×g for 20 min at 4°C to separate the soluble fractions from the precipitates. The supernatants were transferred to new micro- tubes and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting analysis.Cell apoptosis was measured with the Annexin V Apoptosis Detection Kit (BD Pharmingen, USA) following the manu- facturer’s instructions. Annexin V-positive and propidium iodide-negative cells were considered to be in the early apop- totic phase, and cells with positive staining for both Annexin V and propidium iodide were considered to be in the stages of late apoptosis or necrosis. All data were collected, stored, and analyzed with the LYSIS II software (BD Biosciences). All experiments were repeated three times.

Cells were washed with PBS and lysed with lysis buffer (50 mmol/L Tris-HCl, pH 6.8, 100 mmol/L DTT, 2% SDS, 10% glycerol). Cell lysates were centrifuged at 20 000 × g for 10 min, and the proteins in the supernatants were quantified. Equal amounts of protein extracts were loaded onto an 8% to12% SDS–polyacrylamide gel, electrophoresed, and trans- ferred to a nitrocellulose membrane (Bio-Rad). The blots were stained with 0.2% Ponceau S red to ensure equal pro- tein loading. After blocking with 5% nonfat milk in PBS, the membranes were probed with antibodies against USP7, MDM2, caspase-3, PARP1, UHRF1, and DNMT1. The sig- nals were detected with a chemiluminescence Phototope- HRP Western Blot Detection Kit (Cell Signaling) according to the manufacturer’s instructions. All experiments were repeated three times with similar results.Molecular docking was carried out using the software AutoDock4.2. The X-ray structure of the USP7 catalytic domain (PDB ID: 4M5W) was retrieved from the Protein Data Bank (http://www.rcsb.org/pdb) for the docking calculations. Water and bromide ions were all removed. To prepare for both the protein and the small molecule, first, all hydrogens were added; then, Gasteiger charges were computed, and non- polar hydrogens were merged. The active site was defined by a grid box of 70×70×70 points with a grid spacing of 0.375 Å using AutoGrid4. The box was centered on the residue Tyr514 in the crystal structure of USP7. The protein was considered rigid for the docking study. The docking parameters were setas follows: ga_pop_size=150 (number of individuals in popu- lation) and ga_run=100 (the number of dockings that were performed). The default values in the software were used for other parameters. Protein-ligand interactions were handled using the Lamarckian genetic algorithm. Finally, the confor- mation was selected according to the predicted binding free energy.The data are presented as the mean±SD of at least three inde- pendent experiments. Student’s t-tests were performed using the GraphPad Prism 5.0 software (GraphPad Software); P<0.05 was considered statistically significant. Results Thirteen natural pentacyclic triterpenes (Figure 1A) were biologically evaluated for inhibitory activity against USP7 in vitro. Based on their structural skeleton, pentacyclic triter- penes can be classified into three major types: (a) the ursane type of triterpenes (compounds 1-4); (b) the oleanane type of triterpenes (compounds 5-11); and (c) the lupane type of trit- erpenes (compounds 12, 13). As shown in table 1, ursolic acid(1)was shown to be the most potent of these analogues, withTo elucidate the structural basis of USP7 inhibition by pen- tacyclic triterpenes, the USP7–ursolic acid complex was modeled based on the crystal structure of the USP7 catalytic domain (PDB ID: 4M5W). Our results showed that ursolic acid occupied the ubiquitin binding pocket of USP7 (Figure 2). The skeleton of ursolic acid hydrophobically interacts with residues Met328, Tyr367, Ala369 and Val393. Two hydro- gen bonds in the complex structure are the most notable: the 17-carboxyl group of ursolic acid could form hydrogen bonds with Glu371, and the 3-hydroxyl group could form a hydrogen bond with Gln351. This computational result gave a reason- able explanation for the lack of activity of compounds 14 and15. Both the theoretical analyses and experimental examina- tion revealed that the 17-carboxyl group and 3-hydroxyl group played vital roles in the USP7–ursolic acid interaction.Ursolic acid interacts with USP7 in myeloma cellsan IC50 value as low as 7.0 μmol/L for USP7; oleanolic acid(5) showed similar activity to that of ursolic acid (1); corosolic acid (2), hederagenin (6), pristimerin (9) and betulinic acid (13) showed mild inhibition of USP7; and the other compounds showed no inhibition. These results revealed that, among the different structural skeletons, both the ursane triterpenes and the oleanane triterpenes were more active than the lupine triterpenes (compounds 1 and 5 vs 13). The number and position of the hydroxyl groups of triterpenes on ring A (for the numbering system of the chemical structures, see Figure 1B) seemed to have an important influence on their potency. Introducing a 2-hydroxyl group and 23-hydroxyl group led to an obvious decrease in the inhibition of USP7 (compound 1 vs 2 and 3). The same tendency was also observed in oleanane triterpenes (compound 5 vs 6 and 7).and the inhibition of USP7 inhibits cell proliferation and induces cell death in myeloma cells[16]. To determine whether the USP7-inhibitory effect of pentacyclic triterpenes observed in vitro existed in cells, we selected ursolic acid (1) for further investigation using RPMI8226 myeloma cells as a cell model. First, we used the cellular thermal shift assay (CETSA) to evaluate drug binding to target proteins in cells[17,18]. Aliquots of RPMI8226 cell lysate incubated with DMSO or ursolic acid were heated to different temperatures. After cooling, the samples were centrifuged to separate the soluble fractions from precipitated proteins. We then quantified the presence of USP7 in the soluble fraction by Western blotting. Com- pared with the DMSO-treated cell lysate, ursolic acid treat- ment led to an obvious increase in the USP7 protein level in the supernatant (Figure 3A–B). Consistent with these results, ursolic acid could increase the thermal stability of USP7 in a dose-dependent manner (Figure 3C–D). To investigate the selectivity of ursolic acid, we examined its effect on the ther- mal stability of several other cysteine proteases, such as USP8,USP18, USP44, USP47, caspase-3 and caspase-8. The results showed that ursolic acid did not affect the thermal stability of these cysteine proteases (Supplementary Figure S1). These data suggest that ursolic acid does interact with other cysteine proteases in cancer cells.Ursolic acid inhibits USP7 activity in myeloma cellsThe inhibition of USP7 can inhibit proliferation and induce apoptosis in myeloma cells. As expected, ursolic acid treat- ment for 24 h inhibited the proliferation of RPMI8226 cells, with an IC50 of 26.56 µmol/L (Figure 4A). The percentage of apoptotic cells was evaluated by Annexin V/PI double stain- ing. As shown in Figure 4B, ursolic acid induced apoptosis in RPMI8226 cells in a dose- and time-dependent manner. Con- sistent with these findings, ursolic acid treatment led to the activation of caspase-3 and cleavage of PARP1, indicating the activation of apoptosis (Figure 4B). Moreover, in parallel with the inhibition of USP7, ursolic acid treatment at 20 μmol/L resulted in a decrease in UHRF1 and DNMT1, two substrates of USP7 (Figure 4B). These results indicate that ursolic acid inhibits USP7 activity and induces apoptosis in myeloma cells.To investigate the role of USP7 in ursolic acid-induced cell death, a USP7 plasmid or control vector was stably transfected into RPMI8226 cells (Figure 4C), and the cells were treated with ursolic acid. As shown in Figure 4D, compared with the control cells, the overexpression of USP7 partially but signifi- cantly inhibited ursolic acid-induced apoptosis (P<0.05). Con- sistent with this result, the cleavage of caspase-3 and PARP1 was also inhibited (Figure 4E). Interestingly, the ursolic acid- induced decreases in MDM2, UHRF1 and DNMT1 were also partially blocked by the overexpression of USP7 (Figure 4E). These data indicate that the inhibition of USP7 contributes toursolic acid-induced apoptosis. Discussion Pentacyclic triterpenes are a broad family of compounds with a variety of activities. However, whether this kind of com- pound exhibits activity against deubiquitinating enzymes was not known. In the present study, we demonstrated for the first time that natural pentacyclic triterpenes are a novel type of scaffold for the development of USP7 inhibitors. Using ursolic acid as a model compound, we demonstrated that targeting USP7 contributes to the anti-cancer effect of ursolic acid.Ursolic acid exhibits wide-ranging anti-inflammatory and anticancer activities. In addition, many signaling pathways such as the Nrf2, NF-κB, STAT3, and AKT pathways can be modulated by ursolic acid[21]. However, the direct target of ursolic acid is not clear. The identification of USP7 as a novel target of ursolic acid may explain some of the activi- ties of ursolic acid. For instance, ursolic acid has been shown to upregulate the expression of p53[22]. This upregulation may result from the USP7 inhibition-induced degradation of MDM2; ursolic acid could reduce the DNA methyltransferase DNMT1, which might be related to the inhibition of USP7[23]. Moreover, as USP7 has other substrates such as PTEN, FOXO4 and UHRF1, it is possible that ursolic acid could regulate these protein-related signaling pathways and show broad activities against cancers with dysregulation of these pathways.Pentacyclic triterpenoids have highly diverse structures[24]. Our data showed that some of the tested pentacyclic triter- penoids had USP7 inhibitory activity. The SAR analysis sug- gested that the number and position of hydroxyl groups of triterpenes in ring A seemed to have an important influence on potency. Consistent with this hypothesis, the conversion of the 17-carboxyl group and 3-hydroxyl group of ursolic acid to ester groups (compounds 14 and 15) resulted in a significant loss of potency (compound 1 vs 14 and 15). These data suggest that the interaction between pentacyclic triterpenoids and USP7 has unique stereospecificity. In addition, this character- istic may allow the development of novel derivatives that are more selective against USP7.Currently, the USP7 inhibitory effect of the tested pentacy- clic triterpenoids is not very potent. Thus, using synthetic pentacyclic triterpenoids to develop novel potent USP7 inhibi- tors is a promising strategy. Crystalline ursolic acid of high purity is easily obtained, and many ursolic acid derivatives have been synthesized[25, 26]. However, most of these synthe- sized compounds are not directed to a clear target. Therefore, whether these ursolic acid derivatives have USP7 inhibitory activities warrants further investigation. In conclusion, to our knowledge, this report is the first to show that natural pentacyclic triterpenes have USP7 inhibitory activity and that pentacyclic triterpenes represent a novel scaf- fold for designing novel USP7 inhibitors. Using ursolic acid as a model compound, we demonstrate that USP7 is a novel tar- get of ursolic acid, which may broaden the XL177A use of ursolic acid in USP7-related cancers.