Discovery of a novel NEDD8 Activating Enzyme Inhibitor with Piperidin

May 2, 2016 - NEDD8 activating enzyme (NAE) plays an important role in regulating intracellular proteins with key parts in a broad array of cellular f...
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Discovery of a novel NEDD8 Activating Enzyme Inhibitor with Piperidin-4-amine Scaffold by Structure-Based Virtual Screening Peng Lu,† Xiaoxin Liu,† Xinrui Yuan,† Minfang He,‡ Yubin Wang,*,† Qi Zhang,† and Ping-kai Ouyang‡ †

School of Pharmaceutical Sciences, Nanjing Tech University, No. 5 Xinmofan Road, Nanjing 210009, People’s Republic of China College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 5 Xinmofan Road, Nanjing 210009, People’s Republic of China

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S Supporting Information *

ABSTRACT: NEDD8 activating enzyme (NAE) plays an important role in regulating intracellular proteins with key parts in a broad array of cellular functions. Here, we report a structure-based virtual screening of a compound library containing 50 000 small molecular entities against the active site of NAE. Computational docking and scoring followed by biochemical screening and target validation lead to the identification of 1-benzyl-N-(2,4-dichlorophenethyl) piperidin4-amine (M22) as a selective NAE inhibitor. M22 is reversible for NAE, inhibits multiple cancer cell lines with GI50 values in the low micromolar range, and induces apoptosis in A549 cells. Furthermore, it produces tumor inhibition in AGS xenografts in nude mice and low acute toxicity in a zebrafish model. M22, a novel NAE inhibitor, represents a promising lead structure for the development of new antitumor agents.

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(NEDD8 pathway is shown in Figure 1). In contrast to the ubiquition pathway, neddylation acts on only a few substrates and regulates activities of target proteins. Therefore, inhibition of NAE can modulate the rate of ubiquitination and subsequent degradation of proteins which are regulated by CRLs, such as IκBα,9 p27,10 NRF2,11,12 and c-Myc,13 leading to cancer cell death. Besides CRL substrates, NEDD8 also directly regulates MDM2,14 p53,14,15 and other cancer related proteins.16 Targeting a specific class of UPS E3s by inhibition of NAE in NEDD8 pathway will have the potential to selectively stabilize cellular proteins, thereby leading to reduced toxicity and an improved therapeutic index compared with inhibition of the proteasome. As overactivated neddylation has been observed in human lung cancer and intrahepatic cholangiocarcinoma, NAE has recently emerged as a new target for the treatment of cancer.17−19 MLN4924 (1), an AMP analogue, was developed as an efficient mechanism-based NAE inhibitor by Millennium Pharmaceuticals, whose phase I clinical trial for the treatment of both solid tumors and hematological malignancies was initiated in 2008.20 The X-ray crystal structures revealed that MLN4924 formed an NEDD8-MLN4924 in situ and the product was tightly bound to NAE by mimicking NEDD8-AMP.21 Treating cancer cells with MLN4924 led to varieties of biological effects such as checkpoint activation, DNA rereplication, and apoptosis in multiple cancer cell lines.22 However, treatment-emergent

he ubiquitin-proteasome system (UPS) is responsible for regulated degradation of intracellular proteins with important roles in a broad array of cellular functions.1 Proteasome inhibitor bortezomib (Velcade) which targets the proteasome is approved for the treatment of multiple myeloma and mantle cell lymphoma.2,3 The successful clinical application of bortezomib raises the question whether inhibitors of other enzymes that modulate UPS activity can lead to the development of new anticancer drugs. In UPS, the covalent modification of the cullin-RING ubiquitin E3 ligases (CRLs) by neural precursor cell-expressed developmentally downregulated protein 8 (NEDD8), a member of the ubiquitin-like protein family, is known to be essential for the CRL-mediated ubiquitination of downstream targets.4−6 The covalent attachment of NEDD8, termed neddylation, is a reversible and multistep process analogous to ubiquitination. Mature NEDD8 is covalently linked to target protein via a cascade reaction catalyzed by the APPBP1/Uba3 heterodimer complex as E1 (NEDD8 activating enzyme, NAE), Ubc12 or UBE2F as E2 (NEDD8 conjugating enzyme),7 and Roc1/Rbx1 as E3 (NEDD8 ligase).8 First, NEDD8 and Mg2+/ ATP bind to NAE and form an acyl adenylate intermediate, NEDD8-AMP, followed by a transthiolation reaction to form NAE-NEDD8. A second NEDD8-AMP is formed by a second round of NEDD8 and Mg2+/ATP binding, which yields a ternary complex. Subsequently, NEDD8 from the ternary complex is transferred to NEDD8-specific E2s (UBC12 or UBE2F) through transthiolation reaction to form E2-NEDD8. In the presence of E3, NEDD8 from E2-NEDD8 covalently binds to the specific substrates via an isopeptide bond at last © 2016 American Chemical Society

Received: February 19, 2016 Accepted: May 2, 2016 Published: May 2, 2016 1901

DOI: 10.1021/acschembio.6b00159 ACS Chem. Biol. 2016, 11, 1901−1907

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ACS Chemical Biology

Figure 2. Virtual screening protocol and structure of M22.

RMSD of heavy atoms between the top-ranked poses and the crystallographic counterpart. According to results shown in Supporting Information Table S1, among the top-ranked poses, Libscore and estimated binding energy could reproduce the crystallographic pose of ATP within an acceptable tolerance (RMSD < 2.0 Å). Therefore, Libscore and estimated binding energy were selected for further evaluation. A decoy set consisting of 27 NAE inhibitors from published patents32−34 and 1260 drug-like decoys (properties of decoys were shown in Supporting Information Table S2) was used to evaluate sensitivities and specificities of the two docking scores. The decoy set was built in accordance with the method reported by Mysinger and co-workers.35,36 The receiver operating characteristic curve (ROC) was generated to reveal the overall screening performance of two docking scores. According to the result (shown in Supporting Information Figure S2), the AUC value of estimated binding energy was 0.914, which was similar to that of Libscore (0.911). Considering that the calculation speed of LibDock was much faster, we used LibDock to conduct preliminary virtual screening, and AutoDock 4 was used for further screening. In our studies, 50 000 compounds from the ChemBridge database were filtered according to Lipinski’s rule and prepared using the prepare ligands protocol in DS 2.1. In this protocol, a set of ligands were handled as follows: (1) standardizing charges for common groups, (2) retaining only the largest fragment and adding hydrogens, (3) generating tautomers and isomers, and (4) removing duplicates.37 A total of 48 367 retained ligands were docked into a pretreated receptor (PDB ID: 1R4N) and scored by the LibDock protocol in DS 2.1. As the score of ATP was 152, the value of 140 was selected as a cutoff. A total of 1482 compounds whose scores were higher than 140 were docked into the receptor using AutoDock 4, and 100 top-ranked compounds were saved. Tanimoto coefficient (Tc) values of chemical similarity were calculated for each pair of compounds among the 100 candidates using SciTegic extended-connectivity fingerprints (ECFP_4) in DS 2.1. ECFP_4 is an atom type-based method, and the neighbor atoms within a diameter of four bonds are considered when calculating the features for each atom.38 On the basis of the Tanimoto similarity metric, 100 compounds were clustered into 10 groups using the maximum dissimilarity method of DS 2.1. After visual inspection by considering the docking poses, structural diversity, and novelty, 23 compounds from the 100 compounds (structures are shown in Supporting Information Figures S3 and S4) were purchased and tested in a preliminary activity assay in vitro. Biology Screening in Vitro and Target Validation. Compounds were assessed in a CCK-8 assay for cancer cell

Figure 1. NEDD8 pathway and structure of MLN4924.

heterozygous mutations of NAE and resistance to MLN4924 have already been identified in preclinical studies.23−25 Natural product-like compounds 2 and 3, metal complex 4, and enerhodanine analogue 5 (structures are shown in Supporting Information Figure S1) were reported as moderate NAE inhibitors, while there still were some shortages about potency and druglike physical properties in these compounds.26−29 Although the potential benefits for the inhibition of NAE have been recognized for nearly 17 years, there are currently limited classes of effective small-molecule inhibitors, and no NAE inhibitor is approved for the treatment of cancer. Thus, it is highly desirable to discover novel chemotypes that possess high NAE inhibitory potency and selectivity. Herein, we report the discovery of M22 with a piperidin-4-amine scaffold as a novel reversible inhibitor of NAE through high-throughput virtual screening, which shows potent anticancer activity both in vitro and in vivo with low acute toxicity. More importantly, M22 is druglike and served as a lead compound, identified by its low molecular weight, convenient synthesis, and multiple diversifiable positions.

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RESULTS AND DISSCUSSION Virtual Screening. Until now, limited classes of NAE inhibitors were reported. Therefore, we attempted to search for a new chemical scaffold which could effectively inhibit NAE activity. In our study, virtual screening was conducted following the strategy shown in Figure 2. In this case, the LibDock protocol in Discovery Studio 2.1 (DS 2.1)30 and AutoDock 431 were adopted to carry out the docking-based screening. To validate the reliability of the proposed docking application, capabilities of pose prediction and actives discrimination were explored in both native docking and decoy database screening scenarios using the X-ray crystal of NAE with ATP (PDB ID: 1R4N). In the native docking experiment, six types of docking score, Libscore, Ligscore-1, Plp-1, and Jain, together with PMF in DS 2.1 and estimated binding energy in AutoDock 4 were investigated for their abilities to identify correct binding poses. The docking accuracy was determined by calculating the 1902

DOI: 10.1021/acschembio.6b00159 ACS Chem. Biol. 2016, 11, 1901−1907

Articles

ACS Chemical Biology

Figure 3. M22 inhibited neddylation in enzyme-based assay and cell-based assay. (A) Survival curve of M1, M7, M22, and MLN4924 against A549 (n = 3). (B) M22 inhibited the Ubc12-NEDD8 formation in an enzyme assay. (C) ATP-competitive NAE inhibition assay. (D) M22 blocked the neddylation pathway selectively in A549 cells. (E) M22 suppressed degradation of CRL substrates in A549 cells.

followed by the addition of ATP ranging from 125 nM to 1 mM. Inhibition of Ubc12-NEDD8 was reduced when ATP concentration was higher than 10 μM (Figure 3C), suggesting that M22 was a reversible NAE inhibitor and NAE activity recovered at high ATP concentration. Therefore, M22 was considered as a potential reversible NAE inhibitor. In cell-based assays, A549 cells that were treated with M22 under 0−90 μM for 24 h were lysed, and protein extracts were analyzed by Western blot. According to results, formations of Uba3-NEDD8 and Ubc12-NEDD8 were dose-dependently inhibited (Figure 3D), indicating that M22 suppressed NEDD8 activation and the transfer of NEDD8 to Ubc12 (E2) catalyzed by NAE. However, the six negative compounds could not inhibit NAE activity in A549 cells (data shown in Supporting Information Figure S6). The addition of DTT led to the disappearance of the Ubc12-NEDD8 band (Supporting Information Figure S7), indicating that the thioester bond of Ubc12-NEDD8 was reduced by DTT. Therefore, M22 was considered as an NAE inhibitor to prevent NEDD8 activation inside cells. NAE inhibition resulted in a corresponding decrease in the abundance of Cullins-NEDD8 (Figure 3D), which suggested that the neddylation pathway was blocked by M22. Blocking neddylation resulted in the diminished activity of CRLs and subsequently led to the accumulation of CRLtarget proteins. In this assay, degradations of p27 and CDT1, which were known as CRL substrates, were decreased (Figure 3E),21 suggesting that M22 could stabilize CRL substrates through suppressing neddylation. Besides that, NAE inhibition could prevent p53 from degradation in cells leading to an accumulation of p53 as recently reported (Figure 3E).39 Neddylation could be inhibited by M22, not only in A549 cells but also in AGS cells (shown in Supporting Information Figure S7). From those data, M22 was considered to suppress NAE activity and to downregulate neddylation in vitro. As NAE was homologous to ubiquitin (Ub) and other ubiquitin-like protein (UBL) E1s, selectivity of M22 for NAE was also evaluated in a cell-based assay. In this paper, activities of M22 against ubiquitin activating enzyme, SUMO activating enzyme, and FAT10 activating enzyme were tested through detecting levels of Ubc10-Ub, Ubc9-SUMO, and USE1-Ub thioester products inside A549 cells. Surprisingly, formation of those Ubc10-Ub, Ubc9-SUMO, and USE1-Ub were almost not

growth inhibition. A549, an NAE upregulated and neddylation overactivated cell line,17 was chosen for antiproliferation assay first. Among those 23 compounds, M1, M3, and M22 could inhibit A549 cell proliferation in the micromolar range (structures of M1 and M3 were shown in Supporting Information Figure S3). Furthermore, M22 with a piperidin4-amine scaffold could inhibit A549 cell proliferation completely at 30 μM (GI50 = 5.5 μM and GI90 = 19.3 μM, shown in Figure 3A). In order to investigate whether proliferation suppression was induced by general toxicity of M22 or not, noncancer cell line GES-1 (gastric mucosal cell line) was selected as a negative control. In this assay, the GI50 of M22 against GES-1 was 43.0 μM (shown in Supporting Information Figure S5), which was much higher than that against A549. Considering that NAE was widely expressed in noncancer cells at a low level, NAE inhibitors would have an effect on noncancer cells at high concentrations. The difference between the antiproliferation activities of M22 against GES-1 and A549 suggested that proliferation inhibition of M22 against A549 cells was induced by NAE inhibition rather than general toxicity of M22. Besides that, another six piperidin-4-amine based compounds were also measured as negative controls (structures are shown in Supporting Information Figure S6). All six of the compounds showed no proliferation inhibition even at a concentration of 90 μM, suggesting that the growth inhibition was structure-specific and was not induced by general toxicity of piperidin-4-anmine. Therefore, M22 was selected to explore its enzymatic activity on NAE. A dose−response experiment was performed using an enzymatic assay which measured the level of NAE-mediated formation of the Ubc12-NEDD8 thioester product. A decreased level of Ubc12-NEDD8 conjugation indicates the inhibition of NAE. Recombinant human NAE was incubated with NEDD8, Ubc12, and ATP in the presence of M22 for 1 h. Then, levels of Ubc12-NEDD8 thioester product were detected by Western blot (Figure 3B). A concentration-dependent reduction of the intensity of Ubc12-NEDD8 bands was observed, and formation of Ubc12-NEDD8 was significantly inhibited at 3.33 μM. To validate the assumption in our screening strategy that M22 was an ATP-competitive inhibitor, an ATP concentration-dependent assay was conducted. NAE, NEDD8, and Ubc12 were incubated with 3.33 μM M22, 1903

DOI: 10.1021/acschembio.6b00159 ACS Chem. Biol. 2016, 11, 1901−1907

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ACS Chemical Biology

Figure 4. Evaluation of apoptotic/necrotic processes induced by M22. (A) M22 induced apoptosis on A549 with observation at low magnification of a fluorescence microscope using Hoechst/PI staining. (B) M22 induced apoptosis on A549 with observation at high magnification of a fluorescence microscope using Hoechst/PI staining. (C) Evaluation of apoptosis on A549 by Annexin V/PI staining and flow cytometry detecting.

ally, 2,4-dichlorophenyl group was estimated to have cation−π interactions with Arg90 and Arg15. Further Antiproliferation Study of M22. Antiproliferation activities of M22 against K562 (leukemia, MLN4924 sensitive40), Caco-2 (colon cancer, MLN4924 sensitive26), SKOV-3 (ovarian cancer, MLN4924 sensitive41), BXPC-3 (carcinoma of pancreas), and AGS (gastric cancer) were tested. M22 was able to inhibit proliferation of six cell lines in the micromolar range (Figure 6A), and cell growth of K562, SK-OV-3, and BXPC-3 could be inhibited completely by M22. The GI90 values of M22 against Caco-2, K562, SK-OV-3, BXPC-3, and AGS were 12.7 μM, 8.98 μM, 13.2 μM, 26.6 μM, and 14.9 μM, respectively, indicating that M22 could effectively inhibit proliferation of cancer cells from different tissues. Because the NEDD8 pathway locates on the upstream of UPS, M22 may have synergistic effects with bortezomib, which is a selective inhibitor of the 26S proteasome on the downstream of UPS. To verify the effect of a combination of M22 and bortezomib on A549, the Jin equation (the modified Burgi formula), whose concept was universally accepted in pharmacology, was employed. In the Jin equation, the q values presenting 2 suggest antagonism, simple addition, synergism, and apparent synergism, respectively.42 The 48-h-exposure inhibition rates of M22 (5 μM), bortezomib (12.5 nM), and combination treatment (5 μM M22 + 12.5 nM bortezomib) were 42.2%, 33.5%, and 82.1%, respectively (Figure 6B). As the q value was 1.33, M22 was considered having a synergistic effect with bortezomib on A549 growth inhibition. Besides that, the inhibition rate of combination treatment was significantly higher than that of a double dose of bortezomib (P < 0.05) or M22 (P < 0.05). However, neither adriacin nor 5-fluorouracil had synergism with M22 (Supporting Information Figure S8), suggesting that the synergistic effect between M22 and bortezomib was mechanism-based. Tumor Xenograft Growth Inhibition by M22. To evaluate the antitumor activity of M22 in vivo, we administered M22 to nude mice bearing human tumor cell xenografts and monitored tumor growth rate. M22 was administered by intraperitoneal injection once daily to mice bearing AGS xenografts at 60 mg kg−1. Bortezomib was selected as positive control and administered by intraperitoneal injection at 1 mg kg−1. After a two-week treatment, significant inhibition of tumor growth was observed (Figure 7A) and the T/C (average

suppressed at different concentrations of M22 (Figure 3D), which suggested that M22 was a selective NAE inhibitor. M22 Promotes Apoptosis in A549 Cell Line. In order to investigate whether M22 induced apoptotic cell death, A549 cells treated with M22 for 36 h were stained with Hoechst/PI, followed by examination for apoptosis and necrosis under a fluorescence microscopy. Hoechst+ nuclei were detected, and typical morphological features of apoptosis, such as karyopyknosis (marked by red arrows) and apoptotic body (marked by yellow arrow), were visible. Meanwhile, PI+ nuclei were not observed (Figure 4A,B). These results were also confirmed on A549 cells using Annexin V−PI staining followed by flow cytometry. The amount of PI+ M22-treated cells was always below 10%, while annexin V+/PI− cells ranged from 1.30% to 38.93%, depending on the M22 dose (Figure 4C). Taken together, these results suggested that treatment of M22 promoted cell apoptosis in A549. Binding Mode Analysis. In AutoDock 4, molecular modeling results showed that the binding conformation of M22 in the NAE-NEDD8 complex was similar to that of ATP (shown in Figure 5). The binding mode of M22 was different

Figure 5. Molecular modeling results. (A) Low-energy binding conformations of M22 bound to NAE heterodimer generated by AutoDock 4. (B) Merging between top-ranked M22 pose (shown in blue) and ATP conformation (shown in red) from NAE crystal (PDB ID: 1R4N).

from that of MLN4924 (PDB ID: 3GZN). The 2,4-dichloro-1phenethyl group was predicted to map onto the phosphate groups of ATP, and the benzyl group of M22 was forecasted to be located in the pocket where the adenine of ATP occupied. M22 was estimated to form one H-bond between the carboxyl group of Gly176 and the secondary amine of M22. Addition1904

DOI: 10.1021/acschembio.6b00159 ACS Chem. Biol. 2016, 11, 1901−1907

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ACS Chemical Biology

Figure 6. Further antiproliferation study: (A) M22 inhibited proliferation of multiple cell lines in micromole range (n = 3). (B) Synergistic effect between M22 and Bortezomib (n = 6).

Figure 7. Antitumor activity and acute toxicity of M22 in vivo: (A) M22 produced tumor growth inhibition of AGS xenografts in nude mice (n = 6). (B) LC50 of M22 compared with bortezomib in zebrefish model. (C) Morphogenesis of zebrafish embryos treated with M22 at 90 μM and Bortezomib at 10 μM.

be located in the pocket where the adenine of ATP occupied, it may increase ligand−receptor interactions that the benzyl group is replaced by analogous groups of adenine. (3) With a comparison to the X-ray crystal structure of NAE and MLN4924, another group will be introduced to locate the pocket where the indanyl group occupies to enhance binding. The binding mode between M22 and NAE will help to raise our knowledge and to provide an alternative strategy for the design of new NAE inhibitors.

treated tumor volume/average control tumor volume) was 0.284 (P < 0.01), suggesting that M22 treatment inhibited the growth of human tumor xenografts. Acute Toxicity in Vivo. As a convenient and predictive animal model, the zebrafish model organism is increasingly used for assessing drug toxicity.43 Numerous studies confirmed that mammalian and zebrafish toxicity profiles are strikingly similar, and zebrafish usually can serve as an intermediate step between cell-based evaluation and conventional animal testing.44 In this paper, the zebrafish model was used to investigate acute toxicity of M22. Concentrations of M22 from 0.36 μM to 90 μM were investigated, and no zebrafish death was observed even at the highest dose, meanwhile the LC50 of bortezomib was 5.47 μM (Figure 7B,C). From these data, we considered that M22 has low acute toxicity in vivo. Conclusion. In conclusion, M22, a potential NAE inhibitor, has been identified by structure-based virtual screening. M22 effectively inhibited proliferation of the neddylation upregulated cell line as well as MLN4924 sensitive cell lines. It selectively exhibited NAE inhibitory activity in enzyme-based and cell-based assays, with potency in the micromolar range. The treatment of M22 blocked neddylation and reduced the degradation of related substrates, resulting in cell apoptosis. In the in vivo assay, M22 not only prevented tumor growth in the nude mice xenograft model but also presented low acute toxicity in the zebrafish model. The structure of M22 revealed its low molecular weight, convenient synthesis, and multiple diversifiable positions. Furthermore, piperidine is the most commonly used nitrogen heterocycle among U.S. FDA approved pharmaceuticals.45 Taken together, these findings support the feasibility that M22 is an ideal lead compound for developing novel NAE inhibitors. According to the binding model of M22, there are three parts of M22 that can be modified in the future to increase activity: (1) New H-bonds can be made to Arg90 or Arg15, if the 2,4-dichlorophenyl group is replaced by heterocyclic groups or substituent groups were introduced. (2) Since the benzyl group of M22 is predicted to

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METHODS

Full details for all experimental methods are provided in the Supporting Information.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.6b00159.

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Tables S1 and S2 and Figures S1−S8 (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 25 83172108. Fax: +86 25 83172105. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acschembio.6b00159 ACS Chem. Biol. 2016, 11, 1901−1907