Accepted Article
Received Date : 18-Nov-2013 Revised Date
: 11-Jan-2014
Accepted Date : 14-Feb-2014 Article type
: Research Article
Discovery of Novel Secretory Phospholipase A2 Inhibitors Using Virtual Screen
Shunchen Qiu1, Fangjin Chen2, Ying Liu1, 2, Luhua Lai1,2,*
1
BNLMS, State Key Laboratory of Structural Chemistry for Unstable and Stable
Species, College of Chemistry and Molecular Engineering, 2
Center for Quantitative Biology, Peking University, Beijing 100871, China
*To whom correspondence should be addressed:
Luhua Lai
College of Chemistry and Molecular Engineering
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12307 This article is protected by copyright. All rights reserved.
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Peking University, Beijing 100871, China
E-mail: [email protected]
Phone: 86-10-62757486
Fax: 86-10-62751725
Key words: secretory phospholipase A2, virtual screen, drug design, novel inhibitor, continuous fluorescence assay.
Running title: sPLA2 inhibitor virtual screen
Abstract Human non-pancreatic secretory phospholipase A2 (hnpsPLA2) was reported to be associated with inflammatory diseases and considered as a potential drug target for inflammation and other related disease treatment. Though many hnpsPLA2 inhibitors were reported, few entered into the drug development stage due to various problems. In the present study, we discovered seven novel hnpsPLA2 inhibitors using virtual screen. Of the 99 compounds tested by continuous fluorescence assay, seven are potent hnpsPLA2 inhibitors with micromolar IC50 values. Typical molecules include 9-Fluorenylmethoxycarbonyl protected α-phenylalanine derivatives and azo This article is protected by copyright. All rights reserved.
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compounds, which may serve as novel scaffold for developing potent hnpsPLA2 inhibitors. These compounds bind to hnpsPLA2 by interacting with the catalytic calcium ion and the hydrophobic regions in the substrate binding pocket.
Introduction PLA2 hydrolyzes the sn-2 position of cellular phospholipids, liberating arachidonic acid (AA) and leading to the biosynthesis of eicosanoid products including PGs, TXs and LTs (1, 2). Secreted phospholipases A2 (sPLA2), a growing family of PLA2 enzymes, are small, water-soluble, calcium-dependent enzymes with molecular weights of 13–20 kDa(3). In particular, because the sPLA2s are accessible in the circulation and have high levels of systemic enzyme activity, they are attractive therapeutic targets (4, 5). Human non-pancreatic secretory phospholipase A2 (hnpsPLA2) is an isoform of sPLA2 family which has been isolated from human synovial fluid as well as other human cells (6). HnpsPLA2 are expressed abnormally in many inflammation-related pathological conditions, such as rheumatoid arthritis, atherosclerosis and many other chronic disorders like asthma, psoriasis, Crohn’s disease and ulcerative colitis (7-9). In several inflammation-related pathological conditions, high levels of hnpsPLA2 have been observed (10-13). Inhibition of this enzyme will help deplete the sources of arachidonic acid and controls the inflammatory processes.
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Various PLA2 inhibitors have been reported. In 1980s, Gelb et al. synthesized phospholipid analogues with micromole level inhibition (14). Subsequently, marine natural products that had lower cytotoxicity and higher biocompatibility than chemical small molecules were discovered (15, 16). The inactivating of sPLA2 by marine natural products were found to be irreversible, as they modified multiple lysines on the surface of many different sPLA2s (17, 18). Several crystal structures of hnpsPLA2 and ligand complexes were reported (19-21). The hydrophobic region laying on the N-terminal helix formed by the aliphatic and aromatic residues Leu2, Phe5, Ile9, Ala17, Ala18, Tyr21 and Phe98 were found to bind the fatty acid tails of the substrate. Based on the structure of hnpsPLA2, rational design of inhibitors have been reported (22-25).
Though hnpsPLA2 inhibitors were mostly developed as potential treatment for rheumatoid arthritis (26), these inhibitors were also reported to reduce the level of oxidized low density lipoprotein and C-reactive protein (CRP) in patients with coronary heart disease (27). Thus highly potent and selective hnpsPLA2 inhibitors will be important for the discovery of new anti-inflammatory drugs and effective therapies for cardiovascular events or cancer (28, 29). In the present study, we have carried a structure-based virtual screen to discover novel hnpsPLA2 inhibitors. Several potent inhibitors with novel chemical structures were identified.
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Results and discussion Virtual Screen
The crystal structure of sPLA2 complexed with indole 8 (PDB code: 1DB4) was used to identify potential inhibitors of sPLA2 (19). The substrate binding pocket was used for the docking studies. The SPECS compound database (November 2009 version for 10 mg, 201 007 compounds) and a home-made DCSD compound database (6456 compounds) were used for the virtual screen. Figure 1 shows the three-step virtual screen scheme that has been successfully used to identify inhibitors in other enzyme systems like 5-lipoxygenase (5-LOX) (30) and mPGES-1 (31). Rigid body docking was done using DOCK 6.1 (32) followed by semi-flexible docking Autodock 4 (33). Then the top 837 compounds with predicted Kd lower than 15nM were evaluated manually according to the following criteria: (1) forming more coordination bond to the calcium ion CA198; (2) forming good hydrophobic interactions with the substrate binding sites; (3) containing aromatic ring structure at the center region which forms π−π stacking or hydrophobic interaction with Phe5. After this step, 99 compounds were selected and purchased for experimental testing. The calcium ion CA198 is required by the catalytic reaction, which coordinatied with the carbonyl oxygen on the substrate in the process of the catalytic reaction to stable the tetrahedral intermediate. CA198 also plays an important role in stabilizing the binding poses of the inhibitors by coordinating with carbonyl oxygen or nitro oxygen in the inhibitors. Thus, coordination with CA198 was considered to be important in This article is protected by copyright. All rights reserved.
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inhibitor selection. Phe5 formed a hydrophobic region in substrate combination pocket and was often used to interact with ligand by hydrophobic effect or π−π stacking (34). Hence interaction with Phe5 was used a one feature for selecting possible inhibitors.
Inhibition Testing
The inhibitory activities of the 99 selected compounds were tested in vitro against hnpsPLA2 based on a continuous fluorescence assay using 1, 8-anilinonaphthalene sulfonate (ANS) as an interfacial probe (35). In the initial screen, all compounds were tested at the concentration of 50 μM. 2-(1-Benzyl-5-methoxy-2-methyl-1H-indol-3-yl) acetamide (NI101), one reported hnps-PLA2 inhibitor (36), was used as the positive control and DMSO (5%, v/v) was used as vehicle control. Seven of the 99 compounds showed significant inhibition of hnpsPLA2 activity. The IC50 values of these 7 compounds were then determined (Table 1). All the compounds inhibited the enzyme at micromol concentration. The seven compounds can be separated into two groups according to their chemical structures. Group I contains compounds 1 to 5 from the DCSD database, which were all 9-Fluorenylmethoxycarbonyl (Fmoc) protected amino acids. Group II contains two azo compounds (No. 6 and 7) from the SPECS database.
Binding modes of the group I inhibitors and SAR analysis
The binding poses of the active compounds were predicted using Autodock (33).
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Compounds 1-4 in group I were all Fmoc protected phenylalanine derivatives. Compound 5 was also an alanine derivative with a longer hydrophobic chain. Figure 2 shows the binding of compound 1 and 2 in the enzyme substrate pocket. The carboxyl group of the L-phenylalanine in compound 1 interacts with the calcium ion (NHCHCOO-…Ca2+ 2.0Å). It also forms hydrogen bonds with the main chain of Gly29 and Gly31. The fluorene group interacts with the hydrophobic site and forms aromatic/aromatic and aromatic/aliphatic interactions with residues Phe5, Ile9, Tyr21 and Phe98. And the phenyl ring on the phenylalanine is in a suitable orientation to interact with Tyr51.
In compound 2, a naphthalene ring replaces the phenyl ring in compound 1. Compound 2 showed stronger inhibition activity than compound 1, probably because the naphthalene ring can form better hydrophobic interactions than the phenyl ring.
Compound 3 (Figure S2A) is Fmoc-p-nitro-D-phenylalanine. Because the amino acid in compound 3 is D-phenylalanine which has different orientation from compound 1 or compound 2. Beside the hydrophobic interaction between the fluorene group and hydrophobic region of hnpsPLA2, the hydrophobic interaction between the phenyl group and Tyr 51 is weaker. However, as the nitro group can form hydrogen bonds with the side chain of Lys52 (O…H-N 1.8Å, O…N 2.8Å, O…H-N 1.9Å, O…N 2.9Å), compound 3 shows a little bit better activity than 1.
Compound 4 is Fmoc-3-(4-hydroxyl-3-nitrophenyl)-D-alanine, and Figure S2B shows the interaction between compound 4 and hnpsPLA2. Similar with compound 3, the
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hydroxyl and the nitryl froms hydrogen bonds with Lys52 (O…H-N 2.3Å, O…N 3.3Å and N-O…H-N, 1.8Å O…N 2.8Å). Compound 5 (Figure S2C) has a longer hydrophobic chain between D-alanine and phenyl group. The hydrophobic chain increases the hydrophobic interaction between compound 5 and the side chain of Tyr51 and Lys62.
We have shown here that Fmoc-phenylalanine and derivatives can inhibit hnpsPLA2. This type of compounds have been used in self-assembling studies (37). Recently 2-oxoamide compounds based on α-amino acids were found to inhibit GIIAPLA2 with IC50 of micromole level, in which the aliphatic chains attached to the amino group in amino acids form hydrophobic interactions with the binding site (23). As a comparison, in Fmoc-phenylalanine derivatives, the fluorene forms similar interactions as the aliphatic chains.
Binding modes of the group II inhibitors Compound 6 and compound 7 all contain azo group (Figure 3 and Figure S2D). Analogues of compound 6 were reported to influence the lifespan of eukaryotic organism and analogues of compound 7 were reported to be able to inhibit HCV NS3-4A serine protease(38). In compound 6, the nitryl on the phenyl ring near azo forms a hydrogen bond with the main chain NH of Ala17 (N-O…H-N 2.1Å, O…N 3.1Å). Another nitryl forms hydrogen bond with side chain of Asp48 (N-O…H-O 1.7Å, O…O 2.7Å). C-O-C group and NH of Gly29 also form a hydrogen bond (C-O …H-N 2.1Å, O…N 3.1Å). The carbonyl interacts with the calcium ion (C=O…Ca2+ This article is protected by copyright. All rights reserved.
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3.5Å). The benzenes form hydrophobic interaction with residues Leu2, Phe5, His6, Ile9, Tyr21, Tyr51 and Phe98. For compound 7, the carbonyl interacts with the calcium ion (C=O…Ca2+ 2.5Å). The nitryls form hydrogen bond with Lys52 (N-O… H-N 1.8Å, O…N 2.8Å) and Lys62 (N-O…H-N 1.6Å, O…N 2.6Å). NH on the main chain of Gly29 forms hydrogen bond with carbonyl group (C=O…H-N 2.1Å, O…N 3.1Å) and C=N group (C=N…H-N 2.4Å, N…N 3.4Å). The phenyl rings form hydrophobic interaction with residues Leu2, Phe5, Ile9, Tyr21 and Tyr51. Indole compounds are well studied sPLA2 inhibitors (39). Many indole derivative sPLA2 inhibitors have been reported. Recently, long chain 2-oxamides based on α-, δand γ-amino acids were studied as sPLA2 inhibitors (23). Here, we found that Fmoc-protected α-phenylalanine and derivatives can inhibit hnpsPLA2. In addition, azo compounds were also found to inhibit hnpsPLA2. Conclusively, through structure-based virtual screen, two new types of hnpsPLA2 inhibitors have been identified with micromole inhibition activities. These two types of compounds can be used as scaffolds to develop new types of sPLA2 inhibitors.
Experimental Procedures Materials.
1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was from Avanti Polar Lipids. The selected compounds were from the SPECS, DCSD with purity of more
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than 90% and for most compounds greater than 95% (confirmed by the supplier, using NMR, LS-MS, or both; data are available through the Web site). Other reagents were from Sigma Aldrich unless indicated otherwise.
Protein expression and purification. Protein expression and purification were performed as previously described. In short, a synthesized gene was used to express the enzyme. The protein was harvested as inclusion bodies, refolded, and purified by gel filtration (35). The concentration of eluted wild type enzyme was approximately 3.6 mg/ml.
Inhibition
Inhibitory activity assay of hnps-PLA2 was performed as follows. Reaction buffer (160 μl
50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 μg/ml bovine serum albumin
(BSA), 5μM CaCl2 and 10 mM ANS), 20 μl of substrate stock solution (2 mM DMPC) and 2 mM of sodium deoxycholate in water, sonicated for 5 min, and 10 μl of inhibitor stock solution (dissolved in DMSO) were incubated at 28℃ for 10 min. Reactions were started by adding10 μl of hnps-PLA2 stock solution (0.1mg/ml) and monitored by excitation at 377 nm and emission at 470 nm using a multiwall fluorometer (SYNERGY4, BioTek). The initial reaction rates at different inhibitor concentrations were used for calculating the IC50 value. Molecular Docking.
The SPECS compound database (November 2009 version for 10 mg, 201 007 compounds) and DCSD compound database (6456 compounds) was used for the This article is protected by copyright. All rights reserved.
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virtual screen. The 3D structures of the compounds were built with the LigPrep (40) program of the Schrödinger software in Maestro 9.0.211 (using default settings). A 3D structure library containing 203,667 compounds was established (generation of 3D structure failed for 3796 compounds).For the DOCK step in virtual screen, rigid body docking was performed with default parameters using the program DOCK 6.1 (32). For the Autodock step in virtual screen, flexible ligands and rigid receptor docking was performed using the program Autodock 4 (33) with following parameters: empirical free-energy function and the GALS algorithm (genetic algorithm with local search); number of runs, 50; number of individuals, 300; maximum number of energy evaluations, 25 000 000; maximum number of generations, 270 000; grid box, 61 × 61 × 61 points. The final docking result was selected as the structure with the lowest energy in the largest cluster.
Acknowledgements
This work was supported in part by the Ministry of Science and Technology of China and the National Natural Science Foundation of China. Virtual screen work was carried out at the National Supercomputer Center in Tianjin and the calculations were performed on TianHe-1 (A).
Conflice of Interest
The authors declare that there are no financial and commmercial conflicts of interest.
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Supporting Information
Appendix S1. Ranks of Identified Hits (Active in Cell-free Assay) in Virtual Screen.
Appendix S2.LC-MS spectrums of the identified hits.
Appendix S3. NMR spectrums of the identified hits.
Appendix S4. Hnps-PLA2 inhibitory activity of screened inhibitors in cell-free systems. Appendix S5. Interaction between hnps-PLA2 and compounds 3 (A), 4 (B), 5 (C) and compound 7 (D). Cyan: compound; Origane: Phe5; Green: Calcium ion-CA198, black dotted line: interaction with CA 198; red dotted line: hydrogen bond.
Figure Legends
Fig. 1 Virtual screen scheme to identify hnps-PLA2 inhibitors. This article is protected by copyright. All rights reserved.
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Fig. 2 Interaction between hnps-PLA2 and compounds 1-2. Cyan: compound 1; Magenta: compound 2; Orange: Phe5; Green: Calcium ion-CA198. Fig. 3 Interaction between hnps-PLA2 and compounds 6. Cyan: compound 6; Orange: Phe5; Green: Calcium ion-CA198.
Table 1. Inhibition of hnpsPLA2 by the Active Compounds in Cell-Free.
Compounds
Chemical structure
IC50 (μM)
1 (DCSD 002855)
47.5±3.9
2 (DCSD 002847)
8.6±0.4
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3 (DCSD 001360)
21.4±3.5
4 (DCSD 002267)
41.0±6.8
5 (DCSD 001850)
34.5±8.8
6 (SPECS
9.0±1.1
AG-690/40696769)
7 (SPECS AG-690/40108589)
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12.2±1.2
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Received Date : 18-Nov-2013 Revised Date
: 11-Jan-2014
Accepted Date : 14-Feb-2014 Article type
: Research Article
Discovery of Novel Secretory Phospholipase A2 Inhibitors Using Virtual Screen
Shunchen Qiu1, Fangjin Chen2, Ying Liu1, 2, Luhua Lai1,2,*
1
BNLMS, State Key Laboratory of Structural Chemistry for Unstable and Stable
Species, College of Chemistry and Molecular Engineering, 2
Center for Quantitative Biology, Peking University, Beijing 100871, China
*To whom correspondence should be addressed:
Luhua Lai
College of Chemistry and Molecular Engineering
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12307 This article is protected by copyright. All rights reserved.
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Peking University, Beijing 100871, China
E-mail: [email protected]
Phone: 86-10-62757486
Fax: 86-10-62751725
Key words: secretory phospholipase A2, virtual screen, drug design, novel inhibitor, continuous fluorescence assay.
Running title: sPLA2 inhibitor virtual screen
Abstract Human non-pancreatic secretory phospholipase A2 (hnpsPLA2) was reported to be associated with inflammatory diseases and considered as a potential drug target for inflammation and other related disease treatment. Though many hnpsPLA2 inhibitors were reported, few entered into the drug development stage due to various problems. In the present study, we discovered seven novel hnpsPLA2 inhibitors using virtual screen. Of the 99 compounds tested by continuous fluorescence assay, seven are potent hnpsPLA2 inhibitors with micromolar IC50 values. Typical molecules include 9-Fluorenylmethoxycarbonyl protected α-phenylalanine derivatives and azo This article is protected by copyright. All rights reserved.
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compounds, which may serve as novel scaffold for developing potent hnpsPLA2 inhibitors. These compounds bind to hnpsPLA2 by interacting with the catalytic calcium ion and the hydrophobic regions in the substrate binding pocket.
Introduction PLA2 hydrolyzes the sn-2 position of cellular phospholipids, liberating arachidonic acid (AA) and leading to the biosynthesis of eicosanoid products including PGs, TXs and LTs (1, 2). Secreted phospholipases A2 (sPLA2), a growing family of PLA2 enzymes, are small, water-soluble, calcium-dependent enzymes with molecular weights of 13–20 kDa(3). In particular, because the sPLA2s are accessible in the circulation and have high levels of systemic enzyme activity, they are attractive therapeutic targets (4, 5). Human non-pancreatic secretory phospholipase A2 (hnpsPLA2) is an isoform of sPLA2 family which has been isolated from human synovial fluid as well as other human cells (6). HnpsPLA2 are expressed abnormally in many inflammation-related pathological conditions, such as rheumatoid arthritis, atherosclerosis and many other chronic disorders like asthma, psoriasis, Crohn’s disease and ulcerative colitis (7-9). In several inflammation-related pathological conditions, high levels of hnpsPLA2 have been observed (10-13). Inhibition of this enzyme will help deplete the sources of arachidonic acid and controls the inflammatory processes.
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Various PLA2 inhibitors have been reported. In 1980s, Gelb et al. synthesized phospholipid analogues with micromole level inhibition (14). Subsequently, marine natural products that had lower cytotoxicity and higher biocompatibility than chemical small molecules were discovered (15, 16). The inactivating of sPLA2 by marine natural products were found to be irreversible, as they modified multiple lysines on the surface of many different sPLA2s (17, 18). Several crystal structures of hnpsPLA2 and ligand complexes were reported (19-21). The hydrophobic region laying on the N-terminal helix formed by the aliphatic and aromatic residues Leu2, Phe5, Ile9, Ala17, Ala18, Tyr21 and Phe98 were found to bind the fatty acid tails of the substrate. Based on the structure of hnpsPLA2, rational design of inhibitors have been reported (22-25).
Though hnpsPLA2 inhibitors were mostly developed as potential treatment for rheumatoid arthritis (26), these inhibitors were also reported to reduce the level of oxidized low density lipoprotein and C-reactive protein (CRP) in patients with coronary heart disease (27). Thus highly potent and selective hnpsPLA2 inhibitors will be important for the discovery of new anti-inflammatory drugs and effective therapies for cardiovascular events or cancer (28, 29). In the present study, we have carried a structure-based virtual screen to discover novel hnpsPLA2 inhibitors. Several potent inhibitors with novel chemical structures were identified.
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Results and discussion Virtual Screen
The crystal structure of sPLA2 complexed with indole 8 (PDB code: 1DB4) was used to identify potential inhibitors of sPLA2 (19). The substrate binding pocket was used for the docking studies. The SPECS compound database (November 2009 version for 10 mg, 201 007 compounds) and a home-made DCSD compound database (6456 compounds) were used for the virtual screen. Figure 1 shows the three-step virtual screen scheme that has been successfully used to identify inhibitors in other enzyme systems like 5-lipoxygenase (5-LOX) (30) and mPGES-1 (31). Rigid body docking was done using DOCK 6.1 (32) followed by semi-flexible docking Autodock 4 (33). Then the top 837 compounds with predicted Kd lower than 15nM were evaluated manually according to the following criteria: (1) forming more coordination bond to the calcium ion CA198; (2) forming good hydrophobic interactions with the substrate binding sites; (3) containing aromatic ring structure at the center region which forms π−π stacking or hydrophobic interaction with Phe5. After this step, 99 compounds were selected and purchased for experimental testing. The calcium ion CA198 is required by the catalytic reaction, which coordinatied with the carbonyl oxygen on the substrate in the process of the catalytic reaction to stable the tetrahedral intermediate. CA198 also plays an important role in stabilizing the binding poses of the inhibitors by coordinating with carbonyl oxygen or nitro oxygen in the inhibitors. Thus, coordination with CA198 was considered to be important in This article is protected by copyright. All rights reserved.
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inhibitor selection. Phe5 formed a hydrophobic region in substrate combination pocket and was often used to interact with ligand by hydrophobic effect or π−π stacking (34). Hence interaction with Phe5 was used a one feature for selecting possible inhibitors.
Inhibition Testing
The inhibitory activities of the 99 selected compounds were tested in vitro against hnpsPLA2 based on a continuous fluorescence assay using 1, 8-anilinonaphthalene sulfonate (ANS) as an interfacial probe (35). In the initial screen, all compounds were tested at the concentration of 50 μM. 2-(1-Benzyl-5-methoxy-2-methyl-1H-indol-3-yl) acetamide (NI101), one reported hnps-PLA2 inhibitor (36), was used as the positive control and DMSO (5%, v/v) was used as vehicle control. Seven of the 99 compounds showed significant inhibition of hnpsPLA2 activity. The IC50 values of these 7 compounds were then determined (Table 1). All the compounds inhibited the enzyme at micromol concentration. The seven compounds can be separated into two groups according to their chemical structures. Group I contains compounds 1 to 5 from the DCSD database, which were all 9-Fluorenylmethoxycarbonyl (Fmoc) protected amino acids. Group II contains two azo compounds (No. 6 and 7) from the SPECS database.
Binding modes of the group I inhibitors and SAR analysis
The binding poses of the active compounds were predicted using Autodock (33).
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Compounds 1-4 in group I were all Fmoc protected phenylalanine derivatives. Compound 5 was also an alanine derivative with a longer hydrophobic chain. Figure 2 shows the binding of compound 1 and 2 in the enzyme substrate pocket. The carboxyl group of the L-phenylalanine in compound 1 interacts with the calcium ion (NHCHCOO-…Ca2+ 2.0Å). It also forms hydrogen bonds with the main chain of Gly29 and Gly31. The fluorene group interacts with the hydrophobic site and forms aromatic/aromatic and aromatic/aliphatic interactions with residues Phe5, Ile9, Tyr21 and Phe98. And the phenyl ring on the phenylalanine is in a suitable orientation to interact with Tyr51.
In compound 2, a naphthalene ring replaces the phenyl ring in compound 1. Compound 2 showed stronger inhibition activity than compound 1, probably because the naphthalene ring can form better hydrophobic interactions than the phenyl ring.
Compound 3 (Figure S2A) is Fmoc-p-nitro-D-phenylalanine. Because the amino acid in compound 3 is D-phenylalanine which has different orientation from compound 1 or compound 2. Beside the hydrophobic interaction between the fluorene group and hydrophobic region of hnpsPLA2, the hydrophobic interaction between the phenyl group and Tyr 51 is weaker. However, as the nitro group can form hydrogen bonds with the side chain of Lys52 (O…H-N 1.8Å, O…N 2.8Å, O…H-N 1.9Å, O…N 2.9Å), compound 3 shows a little bit better activity than 1.
Compound 4 is Fmoc-3-(4-hydroxyl-3-nitrophenyl)-D-alanine, and Figure S2B shows the interaction between compound 4 and hnpsPLA2. Similar with compound 3, the
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hydroxyl and the nitryl froms hydrogen bonds with Lys52 (O…H-N 2.3Å, O…N 3.3Å and N-O…H-N, 1.8Å O…N 2.8Å). Compound 5 (Figure S2C) has a longer hydrophobic chain between D-alanine and phenyl group. The hydrophobic chain increases the hydrophobic interaction between compound 5 and the side chain of Tyr51 and Lys62.
We have shown here that Fmoc-phenylalanine and derivatives can inhibit hnpsPLA2. This type of compounds have been used in self-assembling studies (37). Recently 2-oxoamide compounds based on α-amino acids were found to inhibit GIIAPLA2 with IC50 of micromole level, in which the aliphatic chains attached to the amino group in amino acids form hydrophobic interactions with the binding site (23). As a comparison, in Fmoc-phenylalanine derivatives, the fluorene forms similar interactions as the aliphatic chains.
Binding modes of the group II inhibitors Compound 6 and compound 7 all contain azo group (Figure 3 and Figure S2D). Analogues of compound 6 were reported to influence the lifespan of eukaryotic organism and analogues of compound 7 were reported to be able to inhibit HCV NS3-4A serine protease(38). In compound 6, the nitryl on the phenyl ring near azo forms a hydrogen bond with the main chain NH of Ala17 (N-O…H-N 2.1Å, O…N 3.1Å). Another nitryl forms hydrogen bond with side chain of Asp48 (N-O…H-O 1.7Å, O…O 2.7Å). C-O-C group and NH of Gly29 also form a hydrogen bond (C-O …H-N 2.1Å, O…N 3.1Å). The carbonyl interacts with the calcium ion (C=O…Ca2+ This article is protected by copyright. All rights reserved.
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3.5Å). The benzenes form hydrophobic interaction with residues Leu2, Phe5, His6, Ile9, Tyr21, Tyr51 and Phe98. For compound 7, the carbonyl interacts with the calcium ion (C=O…Ca2+ 2.5Å). The nitryls form hydrogen bond with Lys52 (N-O… H-N 1.8Å, O…N 2.8Å) and Lys62 (N-O…H-N 1.6Å, O…N 2.6Å). NH on the main chain of Gly29 forms hydrogen bond with carbonyl group (C=O…H-N 2.1Å, O…N 3.1Å) and C=N group (C=N…H-N 2.4Å, N…N 3.4Å). The phenyl rings form hydrophobic interaction with residues Leu2, Phe5, Ile9, Tyr21 and Tyr51. Indole compounds are well studied sPLA2 inhibitors (39). Many indole derivative sPLA2 inhibitors have been reported. Recently, long chain 2-oxamides based on α-, δand γ-amino acids were studied as sPLA2 inhibitors (23). Here, we found that Fmoc-protected α-phenylalanine and derivatives can inhibit hnpsPLA2. In addition, azo compounds were also found to inhibit hnpsPLA2. Conclusively, through structure-based virtual screen, two new types of hnpsPLA2 inhibitors have been identified with micromole inhibition activities. These two types of compounds can be used as scaffolds to develop new types of sPLA2 inhibitors.
Experimental Procedures Materials.
1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was from Avanti Polar Lipids. The selected compounds were from the SPECS, DCSD with purity of more
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than 90% and for most compounds greater than 95% (confirmed by the supplier, using NMR, LS-MS, or both; data are available through the Web site). Other reagents were from Sigma Aldrich unless indicated otherwise.
Protein expression and purification. Protein expression and purification were performed as previously described. In short, a synthesized gene was used to express the enzyme. The protein was harvested as inclusion bodies, refolded, and purified by gel filtration (35). The concentration of eluted wild type enzyme was approximately 3.6 mg/ml.
Inhibition
Inhibitory activity assay of hnps-PLA2 was performed as follows. Reaction buffer (160 μl
50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 μg/ml bovine serum albumin
(BSA), 5μM CaCl2 and 10 mM ANS), 20 μl of substrate stock solution (2 mM DMPC) and 2 mM of sodium deoxycholate in water, sonicated for 5 min, and 10 μl of inhibitor stock solution (dissolved in DMSO) were incubated at 28℃ for 10 min. Reactions were started by adding10 μl of hnps-PLA2 stock solution (0.1mg/ml) and monitored by excitation at 377 nm and emission at 470 nm using a multiwall fluorometer (SYNERGY4, BioTek). The initial reaction rates at different inhibitor concentrations were used for calculating the IC50 value. Molecular Docking.
The SPECS compound database (November 2009 version for 10 mg, 201 007 compounds) and DCSD compound database (6456 compounds) was used for the This article is protected by copyright. All rights reserved.
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virtual screen. The 3D structures of the compounds were built with the LigPrep (40) program of the Schrödinger software in Maestro 9.0.211 (using default settings). A 3D structure library containing 203,667 compounds was established (generation of 3D structure failed for 3796 compounds).For the DOCK step in virtual screen, rigid body docking was performed with default parameters using the program DOCK 6.1 (32). For the Autodock step in virtual screen, flexible ligands and rigid receptor docking was performed using the program Autodock 4 (33) with following parameters: empirical free-energy function and the GALS algorithm (genetic algorithm with local search); number of runs, 50; number of individuals, 300; maximum number of energy evaluations, 25 000 000; maximum number of generations, 270 000; grid box, 61 × 61 × 61 points. The final docking result was selected as the structure with the lowest energy in the largest cluster.
Acknowledgements
This work was supported in part by the Ministry of Science and Technology of China and the National Natural Science Foundation of China. Virtual screen work was carried out at the National Supercomputer Center in Tianjin and the calculations were performed on TianHe-1 (A).
Conflice of Interest
The authors declare that there are no financial and commmercial conflicts of interest.
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Supporting Information
Appendix S1. Ranks of Identified Hits (Active in Cell-free Assay) in Virtual Screen.
Appendix S2.LC-MS spectrums of the identified hits.
Appendix S3. NMR spectrums of the identified hits.
Appendix S4. Hnps-PLA2 inhibitory activity of screened inhibitors in cell-free systems. Appendix S5. Interaction between hnps-PLA2 and compounds 3 (A), 4 (B), 5 (C) and compound 7 (D). Cyan: compound; Origane: Phe5; Green: Calcium ion-CA198, black dotted line: interaction with CA 198; red dotted line: hydrogen bond.
Figure Legends
Fig. 1 Virtual screen scheme to identify hnps-PLA2 inhibitors. This article is protected by copyright. All rights reserved.
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Fig. 2 Interaction between hnps-PLA2 and compounds 1-2. Cyan: compound 1; Magenta: compound 2; Orange: Phe5; Green: Calcium ion-CA198. Fig. 3 Interaction between hnps-PLA2 and compounds 6. Cyan: compound 6; Orange: Phe5; Green: Calcium ion-CA198.
Table 1. Inhibition of hnpsPLA2 by the Active Compounds in Cell-Free.
Compounds
Chemical structure
IC50 (μM)
1 (DCSD 002855)
47.5±3.9
2 (DCSD 002847)
8.6±0.4
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3 (DCSD 001360)
21.4±3.5
4 (DCSD 002267)
41.0±6.8
5 (DCSD 001850)
34.5±8.8
6 (SPECS
9.0±1.1
AG-690/40696769)
7 (SPECS AG-690/40108589)
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12.2±1.2
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