Discovery of a natural product inhibitor targeting protein neddylation by structure-based virtual screening

Biochimie 94 (2012) 2457e2460

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Discovery of a natural product inhibitor targeting protein neddylation by structure-based virtual screening Hai-Jing Zhong a, b,1, Victor Pui-Yan Ma c,1, Zhen Cheng c, Daniel Shiu-Hin Chan c, Hong-Zhang He c, Ka-Ho Leung c, Dik-Lung Ma c, **, Chung-Hang Leung a, b, * a b c

Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China State Key Laboratory of Quality Research in Chinese Medicine, University of Macau, Macao SAR, China Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 December 2011 Accepted 5 June 2012 Available online 16 June 2012

NEDD8-activating enzyme (NAE) controls the specific degradation of proteins regulated by cullin-RING ubiquitin E3 ligase, and has been considered as an attractive molecular target for the development of anti-cancer drugs. We report herein the identification of the dipeptide-conjugated deoxyvasicinone derivative (1) as an inhibitor of NAE by virtual screening of over 90,000 compounds from the ZINC database of natural products. Molecular modelling results suggested that 1 may be a non-covalent competitive inhibitor of NAE by blocking the ATP-binding domain. Compound 1 was able to inhibit NAE activity in both cell-free and cell-based assay with potencies in the micromolar range and selectivity over analogous E1 enzymes UAE and SAE. We envisage that the identification and molecular docking analysis of this bioactive scaffold as an NAE inhibitor would provide the scientific community with useful information in order to generate more potent analogues. Ó 2012 Elsevier Masson SAS. All rights reserved.

Keywords: Drug discovery Virtual screening Ubiquitin-like protein NEDD8-activating enzyme Natural product

1. Introduction The ubiquitin-proteasome system (UPS) is the master regulator of protein degradation in eukaryotic cells [1]. Inhibitors targeting the proteasome, bortezomib [2e4] and salinosporamide A [5], have been shown to be effective anti-cancer agents. However, the general inhibition of proteasome activity may affect the turnerover of thousands of proteins, potentially increasing the toxicity and lowering the therapeutic index of the drugs [6]. The ubiquitinylation of the unwanted proteins is achieved by the coordinated efforts of the E1 activation, E2 conjugation and E3 ligase enzymes [7]. Ubiquitin-like protein (Ubl) conjugation pathways, including NEDD8 and SUMO, have also been identified as key players in the protein degradation process [7,8]. Similar to the ubiquitin pathway, NEDD8 is first activated by the E1-like NEDD8-activating enzyme (NAE) and is then transferred to Ubc12, the NEDD8conjuating enzyme. Finally, the NEDD8 is conjugated to cullin proteins to activate the cullin-RING ubiquitin E3 ligase (CRLs) activity [9,10]. Therefore, NAE could be considered as a more selective upstream target, controlling the specific degradation of proteins * Corresponding author. Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China. Tel.: þ853 8397 8518; fax: þ853 2884 1358. ** Corresponding author. Tel.: þ852 3411 7075; fax: þ852 3411 7348. E-mail addresses: [email protected] (D.-L. Ma), [email protected] (C.-H. Leung). 1 Both authors contributed equally to the work. 0300-9084/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2012.06.004

regulated by CRLs, such as the cancer related substrates p-IkBa and cmyc. The NAE inhibitor MLN4924 [11,12], currently in Phase 1 clinical trials, has been shown to be effective against both solid and haematological human cancers [6]. Recently, our group has shown that the natural product-like 6,600 -biapigenin dose-dependently inhibited NAE activity in cancer cells and suppressed the growth of a human epithelial colorectal adenocarcinoma cell line [13]. Virtual screening (VS) has emerged as a powerful approach to complement the array of existing high-throughput screening technologies [14,15]. Using computer-aided VS, potential leads can be rapidly identified in silico in order to narrow the range of molecules to be tested in vitro and in vivo. The integration of orthogonal approaches to the development of drug leads can greatly reduce the economical investment in chemical synthesis and/or preliminary testing. Meanwhile, natural products have long captured the attention of medicinal chemists due to the diversity of their chemical scaffolds, potentially lower toxicities and bioactive substructures [16e22]. 2. Results and discussion In this study, we expanded the scope of our structure-based drug discovery strategy and screened over 90,000 compounds from the ZINC natural product database for NAE inhibitors in silico. The flexible ligands were docked to a grid representation of the receptor and assigned a score reflecting the quality of the ligandereceptor complex the according to the Internal Coordinate Mechanics

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(ICM) method [ICM-Pro 3.6-1d molecular docking software (Molsoft)] [23]. After visual inspection to eliminate compounds sharing high structural similarity with similar ICM docking scores (0.5), 9 compounds were obtained based on their availability and accessibility from commercial suppliers. The 9 high-scoring compounds were tested in a preliminary E1 NAE activity assay. Four of the compounds showed inhibitory effects against Ubc12-NEDD8 conjugation in vitro at a concentration of 12.5 mM. Their structures, ICM docking scores and potencies against NAE activity are summarized in Table S1. From the preliminary E1 NAE activity assay, the dipeptide-conjugated deoxyvasicinone derivative (1) (CAS number: 951950-33-7) emerged as the top candidate under the tested conditions (Fig. 1A). Pyrrolo[2,1-b]quinazoline alkaloids are known to display a variety of pharmacological activities [24e28]. Deoxyvasicinone, a member of the pyrrolo[2,1-b]quinazoline family and congener of 1, exhibits anti-microbial, anti-depressant and antiinflammatory activities [29]. However, to our knowledge, no biological activity of compound 1 has been reported in the literature. The molecular modelling analysis of the 1-NAE-NEDD8 complex revealed that the top-scoring binding mode of 1 was different from that of 6,600 -biapigenin, but similar to that of ATP or MLN4924 [13]. The deoxyvasicinone ring is predicted to occupy the hydrophobic pocket near Ile148 and Met101 of UBA3 and participate in hydrogen bonding with Gln149 through its pyrimidone nitrogen (Fig. 1B). The pendant dipeptide moiety resides in the relatively hydrophilic region and is predicted to form multiple hydrogen-bonding interactions with NAE in this site, including two H-bonds with the side chain of Lys124 through its amide oxygen atoms, a single H-bond to Asp167 through its carbonyl oxygen atom, and an H-bond to Asp100 through its amide hydrogen atom. Intriguingly, our model predicts that 1 is capable of forming additional H-bonds with both NEDD8 and the APPBP1 subunit through its amide and terminal carboxylate groups of its dipeptide side chain, respectively. The H-bonds formed between 1 and residues Asp100, Lys124, Asp 167 and Gln149 mimic those formed in the NAEeATP complex [30]. (Fig. 1B). The predicted binding coordinates of ATP in the binding pocket are within 2.0 Å RMSD of the reported values in the X-ray crystal structure [30]. The binding score for 1 with NAE was calculated to be 34.2, compared to 30.3 and 30.8 for ATP and MLN4924, respectively [13]. MLN4924 is a mechanism-based inhibitor of NAE, as it forms a tight covalent adduct with the carboxyl terminus of NEDD8 via its sulfamate group. However, since 1 lacks a nucleophilic moiety, it is

unlikely to inhibit NAE via this mechanism. Instead, the strong binding affinity predicted for 1 could be attributed to the ability of the dipeptide side chain to form multiple hydrogen bonding interactions with the charged residues inside the binding site including those of NEDD8 and the APPBP1 subunit. Furthermore, the molecular model revealed that the binding pose of 1 overlapped considerably with that of ATP, suggesting that 1 may act as a reversible competitive inhibitor of NAE by blocking the ATPbinding domain. Additionally, we also performed molecular modelling of 1 to the SAE-SUMO complex. The binding score for 1 to the SAE-SUMO protein complex was calculated to be 20.9, which was significantly lower than that for ATP (32.2). The docking analysis for UAE could not be performed as no suitable X-ray crystal structure was available for molecular modelling. To validate our molecular modelling results, a doseeresponse experiment was performed using a cell-free enzymatic assay to evaluate the ability of 1 to inhibit the activity of NAE and its analogous E1 enzymes for ubiquitin and SUMO. A dose-dependent reduction in the level of the Ubc12eNEDD8 thioester conjugate was observed upon incubation of NAE with 1, with nearly complete inhibition observed at 12.5 mM of 1 (Fig. 2A). Encouragingly, 1 was selective for NAE over the analogous ubiquitin-activating enzyme (UAE) and SUMO-activating enzyme (SAE), with no observable inhibition of the Ubc10eUb or Ubc9eSUMO products, respectively, at the maximum concentration of 1 tested. Densitometry analysis revealed that the IC50 value of 1 against NAE activity in the cell-free system was approximately 0.8 mM (Fig. 2B). The weak in vitro activity of 1 against SAE was consistent with the molecular docking results for 1 with the SAE-SUMO complex described previously. We then tested 7 commercially available analogues of 1 with various side chains for their NAE inhibitory activities in vitro. Interestingly, all analogues showed some degree of inhibition of NAE activity in the cell-free environment, suggesting that the deoxyvasicinone scaffold may be one of the crucial factors in determining inhibitory activity against NAE. The structures, ICM docking scores and potencies of the 7 analogues against NAE activity are summarized in Table S2. We next investigated the ability of 1 to inhibit NAE activity in human cancer cells. Caco-2 (human epithelial colorectal adenocarcinoma) cells were incubated with varying concentrations of 1 for 24 h and analysed for Ubc12eNEDD8 conjugate levels. A dosedependent inhibition of Ubc12eNEDD8 levels was observed upon treatment with 1, with an estimated IC50 value of ca. 6 mM from

Fig. 1. (A) Chemical structures of small molecule NAE inhibitors 1, 6,600 -biapigenin and MLN4924. (B) Low-energy binding conformation of (a) 1, and (b) superposition of the lowenergy binding conformations of 1 and ATP to the NAE heterodimer generated by virtual ligand docking. Proteins APPB1 (blue), UBA3 (purple) and NEDD8 (yellow) are displayed in the ribbon form. Small molecule inhibitor 1 is depicted as a ball-and-stick model showing carbon (yellow), hydrogen (grey), oxygen (red) and nitrogen (blue) atoms. ATP is represented as the red ball-and-stick model. H-bonds are indicated as blue lines. The binding pocket of the NAE is represented as a translucent green surface.

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Fig. 2. (A) Compound 1 inhibits Ubc12eNEDD8 formation in a dose-dependent manner, but has no observable effect on Ubc10eUb or Ubc9eSUMO formation. E1 enzymes charged with Ubc12/NEDD8, Ubc10/Ub, and Ubc9/SUMO were incubated for 60 min with the indicated concentrations of 1. The reaction mixture was fractionated by non-reducing SDS-PAGE and immunoblotted using antibodies for Ubc12, Ubc10 and Ubc9 respectively. (B) Densitometry analysis of the Western blot showing 1-mediated reduction of Ubc12eNEDD8. Approximate IC50 value ¼ ca. 1 mM. (C) Western blot showing selective dose-dependent inhibition of cellular Ubc12eNEDD8 level by 1, while Ubc10eUb and Ubc9eSUMO levels remained unaffected. Caco-2 cells were treated with the indicated concentrations of 1 for 16 h. cell lysates were fractionated by non-reducing SDS-PAGE and immunoblotted using antibodies for Ubc12, Ubc10 and Ubc9 (upper panel). Equal loading was confirmed by GAPDH levels (lower panel). (D) Densitometry analysis of the cell-based Western blot showing 1-mediated reduction of Ubc12eNEDD8 but not the Ubc10-Ub and Ubc9-SUMO. Approximate IC50 value ¼ ca. 6 mM. (E) Caco-2 cells were treated with 1 or MLN4924 for 16 h. cell lysates were immunoblotted to analyse the CRL substrate p27kip1 level using the p27kip1 antibody (upper panel). Equal loading was confirmed by beta-actin level (lower panel).

densometry analysis (Fig. 2C and D). Consistent with the results of the cell-free enzymatic assay, 1 was inactive towards UAE and SAE at the highest dosage of 1 tested, suggesting that 1 is selective towards NAE. Since NAE inhibition should lead to the down-regulation of the CRL activity, we examined the effects of 1 on the levels of CRL substrate p27kip1 in cells by immunoblotting analysis [11]. The results showed that treatment of Caco-2 cells with 1 induced an accumulation of p27kip1 protein in a dose-dependent manner, presumably due to its ability to block the neddylation pathway by interfering with NAE activity (Fig. 2E). We also examined the cytotoxicity of 1 against the growth of cancer cells in vitro using an MTT assay. Our results indicated that 1 was moderately toxic against Caco-2 cells, with an IC50 value of ca. 10 mM after incubation for 48 h (Fig. S2). It is conceivable that the cytotoxicity of 1 against Caco-2 cells could be attributed, at least in part, to the inhibition of NAE activity by 1, and leading to the accumulation of CRL substrates such as tumour suppressor protein p27kip1. 3. Conclusion In conclusion, we have discovered a new small molecule NAE inhibitor from the ZINC natural product database of >90,000

compounds using high-throughput virtual screening. To our knowledge, the dipeptide-conjugated deoxyvasicinone derivative 1 is only the third example of a small molecule inhibitor of NAE. 1 was able to inhibit NAE activity in both cell-free and cell-based assay with potencies in the micromolar range and with selectivity over analogous E1 enzymes UAE and SAE. Furthermore, compound 1 inhibited CRL-mediated substrate degradation and showed moderate cytotoxicity against Caco-2 cells in an MTT assay. Our molecular modelling results suggest that 1 may be non-covalent competitive inhibitor of NAE since it shares a similar binding mode to ATP. We are currently conducting in silico lead optimization of 1 with the aim of generating more potent analogues for in vitro biological testing. Acknowledgements This work is supported by Hong Kong Baptist University (FRG2/1011/008 and FRG2/11-12/009), Environment and Conservation Fund (ECF Project 3/2010), Centre for Cancer and Inflammation Research, School of Chinese Medicine (CCIR-SCM, HKBU), the Research Fund for the Control of Infectious Diseases (RFCID/11101212), the Research Grants Council (HKBU/201811), the University of Macau (Start-up

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Research Grant to C.-H. Leung, MYRG091(Y1-L2)-ICMS12-LCH and MYRG121(Y1-L2)-ICMS12-LCH). Appendix A. Supplementary material Supplementary material associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biochi. 2012.06.004

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