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Interaction of human chymase with ginkgolides, terpene trilactones of Ginkgo biloba investigated by molecular docking simulations
Biochemical and Biophysical Research Communications xxx (2016) 1e6
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Interaction of human chymase with ginkgolides, terpene trilactones of Ginkgo biloba investigated by molecular docking simulations Amit Dubey a, b, Anna Marabotti a, c, Pramod W. Ramteke b, Angelo Facchiano a, * a
Istituto di Scienze dell'Alimentazione e CNR, Via Roma 64, Avellino, 83100, Italy Jacob School of Biotechnology and Bioengineering, Sam Higginbottom Institute of Agriculture, Technology and Sciences, Allahabad, 211007, India c degli Studi di Salerno, Via Giovanni Paolo II 132, Fisciano, 84084, SA, Italy Dipartimento di Chimica e Biologia "Adolfo Zambelli", Universita b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 February 2016 Accepted 8 March 2016 Available online xxx
The search for natural chymase inhibitors has a good potential to provide a novel therapeutic approach against the cardiovascular diseases and other heart ailments. We selected from literature 20 promising Ginkgo biloba compounds, and tested them for their potential ability to bind chymase enzyme using docking and a deep analysis of surface pocket features. Docking results indicated that the compounds may interact with the active site of human chymase, with favorable distinct interactions with important residues Lys40, His57, Lys192, Phe191, Val146, Ser218, Gly216, and Ser195. In particular, proanthocyanidin is the one with the best-predicted binding energy, with seven hydrogen bonds. Interestingly, all active G. biloba compounds have formed the hydrogen bond interactions with the positively charged Lys192 residue at the active site, involved in the mechanism of pH enhancement for the cleavage of angiotensin I site. Ginkgolic acid and proanthocyanidin have better predicted binding energy towards chymase than other serine proteases, i.e kallikrein, tryptase and elastase, suggesting specificity for chymase inhibition. Our study suggests these G. biloba compounds are a promising starting point for developing chymase inhibitors for the potential development of future drugs. © 2016 Elsevier Inc. All rights reserved.
Keywords: Ginkgo biloba Chymase Herbal nutraceutical Screening Molecular docking Inhibitor Cardiovascular diseases
1. Introduction Cardiovascular disease is becoming a global phenomenon all over the world, and one particular manifestation, heart failure, is perilously increasing in regularity. The quantity of cardiac mast cells is remarkably increased in patients with heart failures, and a strong connection between heart failure and chymase (EC 3.4.21.39), an important enzyme present in abundance in secretory granules of mast cells, has been already proved [2]. Chymase is accumulated in mast cells in an inactive form and it is activated suddenly after its release into the interstitial tissues at pH 7.4, in particular when mast cells are stimulated by inflammation or injury. This enzyme is the extensive extravascular source of vasoactive angiotensin II, which is formed very rapidly through the hydrolysis of the Phe8eHis9 bond of angiotensin I [8]. For these
Abbreviations: GB, Ginkgo biloba; OHH, 2-[3-{methyl [1-(2-naphthoyl) piperidin-4-yl] amino} carbonyl)-2-naphthyl]-1-(1-naphthyl)-2-oxoethylphosphonic acid; PDB, Protein Data Bank. * Corresponding author. E-mail address: [email protected] (A. Facchiano).
reasons, there is a strong interest to develop specific chymase inhibitors [3], as a new therapeutic treatment for the disease as well as a potential therapeutic modality for atherosclerotic plaque stabilization. Ginkgo biloba (Ginkgoaceae) (GB), also known as ‘maidenhair tree’, is the best-selling herbal remedy in the USA [31]. GB extracts have two major fractions of compounds: flavonoids and terpenes. The chemical structure of flavonoids comprises an aromatic ring and a double bond that seem to react preferentially with hydroxyl radicals [35]. Terpenes comprise the ginkgolides A, B, C, J and M, and bilobalide [27]. Terpenes were found to strongly diminish platelet activation and collection by antagonizing plateletactivating factor [28,20], whereas bilobalide, a sesquiterpenetrilactone, was shown to reduce cerebral edema, cortical infarct volume and ischemic damage in patients who have suffered a stroke [9]. Moreover, an extract of GB was proven to reduce the atherosclerotic nanoplaque formation after a 2-months therapy. This effect was attributed to an up-regulation of radical scavenging enzymes and the inhibition of the risk factors, oxidized Low Density Lipoprotein/Low Density Lipoprotein and Lipoprotein [26]. Furthermore, in vitro experiments on a rat model of obese type 2
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Please cite this article in press as: A. Dubey, et al., Interaction of human chymase with ginkgolides, terpene trilactones of Ginkgo biloba investigated by molecular docking simulations, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.03.028
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diabetes show that GB extract has beneficial effects on blood circulation and hyperglycemia, and provides protective effect in patients with diabetes and atherosclerosis [17]. In our in silico study, we tested components of GB extracts for their ability to bind to human chymase enzyme. Since chymase is also involved in atherosclerosis [7], we would like to challenge the hypothesis of a correlation between them. 2. Methods 2.1. Selection of compounds Twenty natural compounds from GB were selected from literature [6,15,23,24,29,32] (see Fig. S1 for their chemical structures). PubChem [14] was used to retrieve all ligand molecules in the SDF format. Their 3D structures were then converted to Protein Data Bank (PDB) format using Chimera program [22]. PDB [4] includes 18 human chymase structures, and they were compared and analyzed for resolution, reliability and Ramachandran plot with PDB Redo [13]. The crystal structure of chymase complexed with the ligand JNJ-10311795 (2-[3-{methyl [1-(2naphthoyl) piperidin-4-yl] amino} carbonyl)-2-naphthyl]-1-(1naphthyl)-2-oxoethylphosphonic acid) (OHH) (PDB code 1T31) [10] has been selected as the best one in terms of completeness and quality. Two known natural chymase inhibitors, i.e. keto-betaboswellic acid and beta-boswellic acid [30], were downloaded from PubChem database and used as control for molecular docking simulations. 2.2. Molecular docking simulation of GB compounds Proteineligand docking simulations were performed using AutoDock version 4.2 and ADT Suite 1.5.6 software [19]. The structure of the crystallographic inhibitor OHH bound to chymase was used as a control to perform a self-docking prediction, to check for correctness of the parameters as well as for estimating its binding energy. Furthermore, in order to check for selectivity of the GB compounds towards chymase, other proteins sharing the same enzymatic activity were selected, namely kallikrein (PDB code: 1LO6) [5], tryptase (PDB code: 2FPZ) [18], elastase (PDB code: 5ABW) [33], and docking simulations were performed. Polar hydrogens were added to the protein and ligands, and charges were assigned according to Gasteiger [12]. A box of 76 60 74 points was used for all docking simulations towards chymase, with a spacing of 0.353 Å, targeting the pool of amino acids Lys40, His57, Asp102, Phe191, Lys192, Gly193, Ser195, Tyr215, Gly216, Arg217, Ala226 identified in the PDB file as the ones involved in the interaction with the ligand. For docking towards kallikrein, a box of 64 66 72 points was used, with a spacing of 0.353 Å, centered on the pool of amino acids involved in the interaction with the ligand (as reported in the 1LO6.pdb file), i.e. His57, Asp189, Ser190, Cys191, Gln192, Gly193, Asp194, Ser195, Val213, Ser214, Tyr215, Gly216, Asn217, Ile218, Cys220, Ser221, Pro225, Gly226, Tyr228. For tryptase, a box of 76 68 66 points was used, with a spacing of 0.353 Å, centered on the pool of amino acids involved in the interaction with the ligand (as reported in the 2FPZ.pdb file), i.e. His57, Tyr172, Ser190, Cys191, Gln192, Gly193, Asp194, Ser195, Val213, Tyr215, Gly216, Glu217, Gly219, Cys220, Ala221, Arg224, Pro225, Gly226, Ile227, Tyr228. For elastase, a box of 70 68 74 points was used, with a spacing of 0.353 Å, centered on the pool of amino acids involved in the interaction with the ligand (as reported in the 5ABW.pdb file), i.e. His57, Tyr94, Val97, Asn98, Leu99, Arg177, Val190, Cys191, Phe192, Gln192, Gly193, Asp194, Ser195, Gly196, Ala213, Ser214, Phe215, Val216, Arg217, Gly218, Cys220. Before docking, water molecules and the ligands present in the
crystallographic structure were removed. For each GB compound, 100 docking runs were performed using the AutoDock Lamarckian genetic algorithm, considering the protein as rigid and the ligand as flexible. The others parameters were set to default. RMSD value of 2.0 Å was taken for clustering docking poses. The Discovery Studio mes BIOVIA) was used for superimposing software (Dassault Syste co-crystallized inhibitor of chymase and GB compounds to compare the position of binding. The conformations illustrative of the best energetic and the most populated cluster of poses were selected, saved in .pdb format and investigated for their H-bonds and hydrophobic interactions with the enzyme by using the tools available in AutoDock Tools and Discovery Studio. 2.3. Chymase surface pocket analysis CASTp server [16] was used with default parameters to identify the possible ligand binding pockets within the active site of the 3D structures of chymase. 3. Results 3.1. Molecular docking of G. biloba compounds with chymase By molecular docking simulations, we found that all the 20 compounds derived from GB derivatives are predicted to bind to the active site of chymase, since their predicted binding energies are negative (see Table 1). In particular, on the basis of lowest predicted binding energy, the detailed study of the best four different GB compounds (highlighted in Table 1) predicted to bind to chymase showed that all of them interact with at least one of the key amino acids constituting the active site of human chymase, which are Lys40, His57, Asp102, Phe191, Lys192, Gly193, Ser195, Tyr215, Gly216, Arg217, Ala226 (Fig. 1). Their detailed interactions are reported in Supplementary Table 1. In order to compare the results of the selected GB compounds with known natural chymase inhibitors, we performed molecular docking simulations with beta-boswellic acid and keto-betaboswellic acid [30]. Moreover, we performed a re-docking procedure with OHH compound, co-crystallyzed with chymase in PDB structure 1T31. Results are shown in Table 2. While the re-docking of OHH indicates very low binding energy, the values of predicted binding energy for the other two known chymase inhibitors are very similar to those obtained with ginkgolic acid, proanthocyanidins, quercetrin and rutin, supporting the hypothesis that the GB-derived compounds can indeed bind chymase. From this analysis, it appears that these best four GB compounds are predicted to interact with chymase key residues Lys40, His57, Phe191, Lys192, Ser195 and Gly216. We performed further docking simulations to verify the possibility of interactions of the four GB compounds with other serine proteases, i.e, kallikrein, tryptase, elastase (Table 3). Ginkgolic acid and proanthocyanidin have better predicted binding energy towards chymase than towards kallikrein, tryptase and elastase. Therefore, these two compounds appear rather selective for chymase. On the contrary, quercitrin shows similar predicted binding energy for all proteins and therefore appears to be not selective. Finally the predicted binding energies for rutin indicate that this molecule appears to bind preferentially to kallikrein than to chymase, but less preferentially to tryptase and elastase. 3.2. Chymase surface analysis and comparison to other serine proteases The chymase structure was analyzed by CASTp program to
Please cite this article in press as: A. Dubey, et al., Interaction of human chymase with ginkgolides, terpene trilactones of Ginkgo biloba investigated by molecular docking simulations, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.03.028
A. Dubey et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6
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Table 1 GB compounds interactions with Chymase, showing residues exhibiting hydrogen bonds and hydrophobic bonds with the different compounds. Name of compound (PUBCHEM ID)
Predicted binding No. of poses in Hydrogen energy (kcal/mol) the cluster bonds
Hydrophobic interactions
Ginkogolic acid (5281858) Proanthocyanidin (108065) Quercetrin (5280459) Rutin (5280805) Ginstein (5280961) Diosmetin (5281612) Genkwanin (5281617) Luteolin (5280445) Ginkgolide M (71450923) Procyanidins (107876) Kaempferol (5280863) Isorhamnetin (5281654) Quercetin (5280343) Apigenin (5280443) Myricetin (5081672) Ginkgolide J (163776) Bilobalide (73581) Ginkgolide A (6419993) Ginkgolide B (6324617) Ginkgolide C (161120)
¡8.45 ¡8.43 ¡8.36 ¡8.13 7.73 7.72 7.65 7.6 7.53 7.53 7.5 7.36 7.36 7.32 7.04 6.55 6.5 6.37 6.28 6.15
LYS40, PHE41, PHE191, LYS192, ALA226 HIS57, PHE191, LYS192, TYR215 PHE191, LYS192, TYR215, ALA226 PHE191, LYS192, TYR215, GLY216, ALA226 PHE191, LYS192, TYR215, ALA226 PHE191, LYS192, ALA226 PHE191, LYS192, ALA226 PHE191, LYS192, VAL213
1 14 66 39 100 62 72 79 100 33 49 71 74 64 86 44 100 50 85 98
LYS192 LYS40, HIS57, PHE191, LYS192, GLY193, GLY216 LYS40, HIS57, SER189, ALA190, PHE191, LYS192, SER195 SER189, SER195, SER214, ARG217 LYS40, HIS57, SER189, SER195 SER189, LYS192, SER218 SER189, LYS192 ALA190, LYS192, SER195, SER218 LYS192, SER195, GLY216 ALA190, LYS192, TYR215, GLY216 SER189, LYS192, SER195, GLY216 ARG143, SER189, PHE191, LYS192 ALA190, LYS192, SER195, SER218 SER189, PHE191, LYS192 SER189, LYS192, SER195, SER218 LYS192, GLY193, ASP194, SER195 SER214,ARG217 LYS192, GLY193, SER195, GLY216 LYS40, LYS192, SER195, GLY216 LYS192, SER195 GLY216 LYS192, GLY193, SER195, GLY216
ALA190, PHE191, LYS192, VAL213, ALA226 PHE191, LYS192, ALA226 PHE191, LYS192, ALA226 PHE191, LYS192, VAL213 PHE191, LYS192, VAL213, ALA226 PHE191, LYS192, ALA226 LYS192 PHE191, LYS192, TYR215 LEU99, PHE191, TYR215 LEU99, PHE191, TYR215 VAL213
Compounds shown in bold are those with the better predicted binding energy.
identify accessible surface pockets. The analysis identified three distinct major cavities, named here as A, B and C: the first one of 162.2 Å3; the second one of 149.2 Å3; the third one of 19.5 Å3. Pocket A is made by the following residues: Ser189, Ala190, Phe191,
Lys192, Ser195, Val213, Tyr215, Gly216, Arg217, Ala220, Lys221, Pro224, Ala226. Pocket 2 is made by residues Pro38, Ser39, Lys40, Phe41, Cys42, Arg143, Lys192, Gly193, Ser195. Finally, pocket C includes residues His57, Leu99, Asp102, Ser214, Tyr215. Their position
Fig. 1. Interaction of selected active site residues of chymase with the best four GB compounds, Ginkgolic acid (panel A), Proanthocyanidin (panel B), Quercetrin (panel C) and Rutin (panel D).
Please cite this article in press as: A. Dubey, et al., Interaction of human chymase with ginkgolides, terpene trilactones of Ginkgo biloba investigated by molecular docking simulations, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.03.028
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Table 2 Predicted binding energies and number of poses of the known chymase inhibitors docked to chymase. Known natural inhibitors
Focused docking results Lowest binding energy
Keto-beta-boswellic acid Beta-boswellic acid OHHa a
Table 4 Occupation of the different cavities of chymase by GB compounds.
9,6 8,76 14.84
No. of poses in the cluster 100 100 90
OHH is the co-crystallyzed ligand in the PDB structure of chymase 1T31.
are very close and, all together, they constitute the active site of the chymase. The comparison of the binding sites of GB compounds with CASTp results (Table 4) suggests that rutin, ginstein, procyanidins, ginkgolide A, B and J, and bilobalide occupy all the pockets identified, whereas the others GB compounds interact preferentially with A and B pockets only. All the best four GB compounds found by molecular docking were compared with OHH, the inhibitor co-crystallyzed in the chymase structure used (Fig. 2). Based on similarity of residues shared and CASTp analysis, we show that the co-crystallized inhibitor occupies mainly the pocket C and a small part of pocket A, whereas ginkgolic acid (Fig. 2 (top left)) and quercetrin (Fig. 2 (bottom left)) are shifted towards the catalytic pocket B and some moieties interact with the nearby centered pocket region A. The binding pocket of proanthocyanidin (Fig. 2 (top right)) and rutin (Fig. 2 (bottom right)) is instead more similar to that of the crystallographic inhibitor.
Name of compound
CASTP pocket
Ginkogolic acid Proanthocyanidin Quercetrin Rutin Ginstein Diosmetin Genkwanin Luteolin Ginkgolide M Procyanidins Kaempferol Isorhamnetin Quercetin Apigenin Myricetin Ginkgolide J Bilobalide Ginkgolide A Ginkgolide B Ginkgolide C
A, B A,B A, B A,B,C A,B,C A,B A,B A,B A, B A,B,C A,B A,B A,B A,B A,B A, B, C A,B,C A,B,C A,B,C A, B
in the active site region. This enhanced interaction may produce more efficient cleavage of angiotensin I [1]. In addition, the charged amino acids Lys40, Arg143 and Lys192 in the chymase are located in positions (Pocket A and B) where they possibly can interact with GB compounds.
4.1. The extended GB compounds-binding site on human chymase 4. Discussion The chymase inhibition is thought to play a crucial role in the therapeutic action against cardiovascular diseases, fibrotic disorders and various other allergic inflammations. Based on literature search, we selected 20 most active GB compounds with the aim of evaluating their potential ability to bind chymase enzyme. Molecular docking investigation studies on the crystal structure of the human chymase have revealed that all the GB compounds were docked into the active site of human chymase and show favorable distinct interactions with the key residues Lys40, His57, Val146, Phe191, Lys192, Ser195, Gly216, and Ser218. The assessment of hydrogen bond network has determined that almost all the active GB compounds can potentially form hydrogen bond interactions with the positively charged Lys192 residue at the active site. The catalytic activity of chymase is dependent on the basicity of His57 of the catalytic triad positions. The identification of hydrogen bonding at Lysl92 residue of human chymase suggests a possible mechanism for the pH enhancement. Lys residues are positively charged at neutral pH and become neutral over the alkaline pH range; neutralization of the amino group of Lys192 by high pH would reduce its need to be solvated by water, thereby enhancing its ability to interact with groups of the substrate buried
The extended GB compounds-binding site is the region of the chymase that interacts with residues inclining the scissile bond. Chymase is capable of interacting with important residues of a protein by assuming the shape of a cleft. As shown in the molecular docking results (Table 1), while analyzing the best four GB compounds, almost all show interactions with Lys40 and Lys192. In chymase, quercetrin binds into the binding pocket B (Fig. 2 (bottom left)), occupied by the group of Ser218 and by the positively charged residue Arg217, whereas proanthocyanidin (Fig. 2 (top right)) and rutin (Fig. 2 (bottom right)) fit inside the large binding pocket A and B and can bind Tyr215 residue very easily. The residues Phe191, Lys192, Tyr215, Gly216, Ser218 and Ala220 located in the binding pockets A and B interact mostly with hydroxyl and ring aromatic groups of GB compounds. These residues are considered to execute important role in the natural substrate acceptance and hydrolysis process in chymase. Therefore, the establishment of strong interactions at the catalytic residue Lys40 plays a very diverse role in active inhibition. All the best four GB compounds share interactions with residues Ser189, Ala190, Gly216, and Ala226, involved in important interaction for the substrate selectivity and hydrolysis [11,21,34]. Alanine is also more hydrophobic with respect to Serine, and may enhance hydrophobic interactions with substrate lysine side chains, hence improving the binding affinity [25]. Interestingly,
Table 3 Comparison of the predicted binding energies of the best GB compounds docked to chymase with respect to those obtained for kallikrein, tryptase and elastase. Name of compound
Chymase Lowest binding energy
Ginkgolic acid Proanthocyanidins Quercitrin Rutin
8.45 8.43 8.36 8.13
Kallikrien No, of clusters 1 14 66 39
Lowest binding energy 7.23 7.64 9.47 9.7
Tryptase No, of clusters 14 38 56 11
Lowest binding energy 6.15 7.6 8.09 7.28
Elastase No, of clusters 11 62 42 11
Lowest binding energy 7.24 6.14 8.23 6.25
No, of clusters 22 19 37 10
Please cite this article in press as: A. Dubey, et al., Interaction of human chymase with ginkgolides, terpene trilactones of Ginkgo biloba investigated by molecular docking simulations, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.03.028
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Fig. 2. Comparison of the position of the structure of chymase co-crystallized ligand (green) with best four Ginkgo biloba compounds, (top left) Ginkgolic acid (Yellow); (top right) Proanthocyanidin (Black); (bottom left) Quercetrin (Orange); (bottom right) Rutin (Cyan). The three pockets identified by CASTp analysis are shown as colored spheres and labeled A, B, and C. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
proanthocyanidin is the compound with the higher predicted number of hydrogen bond formation, with Lys40, Phe191, Lys192, Gly193, Ser195, Gly216 and Ser218. Moreover, some of these compounds, in particular ginkgolic acid and proanthocyanidin, seem more selective for chymase than to other serine proteases like kallikrein, tryptase and elastase. We found GB compounds with predicted favorable binding energy against chymase enzyme, thus becoming a potential starting point for testing this hypothesis experimentally, over in vitro and in vivo models. Therefore, these natural molecules, with wide therapeutic mechanism, can become novel candidates for therapeutic action, and may enhance the possibility for the potential development of future drugs. Acknowledgments This work has been partially supported by the Flagship InterOmics Project (PB.P05, funded and supported by the Italian Ministry of Education, University and Research and Italian National Research Council organizations). A. D. is supported by ICGEB Smart Fellowship program. Appendix A. Supplementary data Supplementary data related to this article can be found at http://
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Interaction of human chymase with ginkgolides, terpene trilactones of Ginkgo biloba investigated by molecular docking simulations Amit Dubey a, b, Anna Marabotti a, c, Pramod W. Ramteke b, Angelo Facchiano a, * a
Istituto di Scienze dell'Alimentazione e CNR, Via Roma 64, Avellino, 83100, Italy Jacob School of Biotechnology and Bioengineering, Sam Higginbottom Institute of Agriculture, Technology and Sciences, Allahabad, 211007, India c degli Studi di Salerno, Via Giovanni Paolo II 132, Fisciano, 84084, SA, Italy Dipartimento di Chimica e Biologia "Adolfo Zambelli", Universita b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 February 2016 Accepted 8 March 2016 Available online xxx
The search for natural chymase inhibitors has a good potential to provide a novel therapeutic approach against the cardiovascular diseases and other heart ailments. We selected from literature 20 promising Ginkgo biloba compounds, and tested them for their potential ability to bind chymase enzyme using docking and a deep analysis of surface pocket features. Docking results indicated that the compounds may interact with the active site of human chymase, with favorable distinct interactions with important residues Lys40, His57, Lys192, Phe191, Val146, Ser218, Gly216, and Ser195. In particular, proanthocyanidin is the one with the best-predicted binding energy, with seven hydrogen bonds. Interestingly, all active G. biloba compounds have formed the hydrogen bond interactions with the positively charged Lys192 residue at the active site, involved in the mechanism of pH enhancement for the cleavage of angiotensin I site. Ginkgolic acid and proanthocyanidin have better predicted binding energy towards chymase than other serine proteases, i.e kallikrein, tryptase and elastase, suggesting specificity for chymase inhibition. Our study suggests these G. biloba compounds are a promising starting point for developing chymase inhibitors for the potential development of future drugs. © 2016 Elsevier Inc. All rights reserved.
Keywords: Ginkgo biloba Chymase Herbal nutraceutical Screening Molecular docking Inhibitor Cardiovascular diseases
1. Introduction Cardiovascular disease is becoming a global phenomenon all over the world, and one particular manifestation, heart failure, is perilously increasing in regularity. The quantity of cardiac mast cells is remarkably increased in patients with heart failures, and a strong connection between heart failure and chymase (EC 3.4.21.39), an important enzyme present in abundance in secretory granules of mast cells, has been already proved [2]. Chymase is accumulated in mast cells in an inactive form and it is activated suddenly after its release into the interstitial tissues at pH 7.4, in particular when mast cells are stimulated by inflammation or injury. This enzyme is the extensive extravascular source of vasoactive angiotensin II, which is formed very rapidly through the hydrolysis of the Phe8eHis9 bond of angiotensin I [8]. For these
Abbreviations: GB, Ginkgo biloba; OHH, 2-[3-{methyl [1-(2-naphthoyl) piperidin-4-yl] amino} carbonyl)-2-naphthyl]-1-(1-naphthyl)-2-oxoethylphosphonic acid; PDB, Protein Data Bank. * Corresponding author. E-mail address: [email protected] (A. Facchiano).
reasons, there is a strong interest to develop specific chymase inhibitors [3], as a new therapeutic treatment for the disease as well as a potential therapeutic modality for atherosclerotic plaque stabilization. Ginkgo biloba (Ginkgoaceae) (GB), also known as ‘maidenhair tree’, is the best-selling herbal remedy in the USA [31]. GB extracts have two major fractions of compounds: flavonoids and terpenes. The chemical structure of flavonoids comprises an aromatic ring and a double bond that seem to react preferentially with hydroxyl radicals [35]. Terpenes comprise the ginkgolides A, B, C, J and M, and bilobalide [27]. Terpenes were found to strongly diminish platelet activation and collection by antagonizing plateletactivating factor [28,20], whereas bilobalide, a sesquiterpenetrilactone, was shown to reduce cerebral edema, cortical infarct volume and ischemic damage in patients who have suffered a stroke [9]. Moreover, an extract of GB was proven to reduce the atherosclerotic nanoplaque formation after a 2-months therapy. This effect was attributed to an up-regulation of radical scavenging enzymes and the inhibition of the risk factors, oxidized Low Density Lipoprotein/Low Density Lipoprotein and Lipoprotein [26]. Furthermore, in vitro experiments on a rat model of obese type 2
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Please cite this article in press as: A. Dubey, et al., Interaction of human chymase with ginkgolides, terpene trilactones of Ginkgo biloba investigated by molecular docking simulations, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.03.028
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diabetes show that GB extract has beneficial effects on blood circulation and hyperglycemia, and provides protective effect in patients with diabetes and atherosclerosis [17]. In our in silico study, we tested components of GB extracts for their ability to bind to human chymase enzyme. Since chymase is also involved in atherosclerosis [7], we would like to challenge the hypothesis of a correlation between them. 2. Methods 2.1. Selection of compounds Twenty natural compounds from GB were selected from literature [6,15,23,24,29,32] (see Fig. S1 for their chemical structures). PubChem [14] was used to retrieve all ligand molecules in the SDF format. Their 3D structures were then converted to Protein Data Bank (PDB) format using Chimera program [22]. PDB [4] includes 18 human chymase structures, and they were compared and analyzed for resolution, reliability and Ramachandran plot with PDB Redo [13]. The crystal structure of chymase complexed with the ligand JNJ-10311795 (2-[3-{methyl [1-(2naphthoyl) piperidin-4-yl] amino} carbonyl)-2-naphthyl]-1-(1naphthyl)-2-oxoethylphosphonic acid) (OHH) (PDB code 1T31) [10] has been selected as the best one in terms of completeness and quality. Two known natural chymase inhibitors, i.e. keto-betaboswellic acid and beta-boswellic acid [30], were downloaded from PubChem database and used as control for molecular docking simulations. 2.2. Molecular docking simulation of GB compounds Proteineligand docking simulations were performed using AutoDock version 4.2 and ADT Suite 1.5.6 software [19]. The structure of the crystallographic inhibitor OHH bound to chymase was used as a control to perform a self-docking prediction, to check for correctness of the parameters as well as for estimating its binding energy. Furthermore, in order to check for selectivity of the GB compounds towards chymase, other proteins sharing the same enzymatic activity were selected, namely kallikrein (PDB code: 1LO6) [5], tryptase (PDB code: 2FPZ) [18], elastase (PDB code: 5ABW) [33], and docking simulations were performed. Polar hydrogens were added to the protein and ligands, and charges were assigned according to Gasteiger [12]. A box of 76 60 74 points was used for all docking simulations towards chymase, with a spacing of 0.353 Å, targeting the pool of amino acids Lys40, His57, Asp102, Phe191, Lys192, Gly193, Ser195, Tyr215, Gly216, Arg217, Ala226 identified in the PDB file as the ones involved in the interaction with the ligand. For docking towards kallikrein, a box of 64 66 72 points was used, with a spacing of 0.353 Å, centered on the pool of amino acids involved in the interaction with the ligand (as reported in the 1LO6.pdb file), i.e. His57, Asp189, Ser190, Cys191, Gln192, Gly193, Asp194, Ser195, Val213, Ser214, Tyr215, Gly216, Asn217, Ile218, Cys220, Ser221, Pro225, Gly226, Tyr228. For tryptase, a box of 76 68 66 points was used, with a spacing of 0.353 Å, centered on the pool of amino acids involved in the interaction with the ligand (as reported in the 2FPZ.pdb file), i.e. His57, Tyr172, Ser190, Cys191, Gln192, Gly193, Asp194, Ser195, Val213, Tyr215, Gly216, Glu217, Gly219, Cys220, Ala221, Arg224, Pro225, Gly226, Ile227, Tyr228. For elastase, a box of 70 68 74 points was used, with a spacing of 0.353 Å, centered on the pool of amino acids involved in the interaction with the ligand (as reported in the 5ABW.pdb file), i.e. His57, Tyr94, Val97, Asn98, Leu99, Arg177, Val190, Cys191, Phe192, Gln192, Gly193, Asp194, Ser195, Gly196, Ala213, Ser214, Phe215, Val216, Arg217, Gly218, Cys220. Before docking, water molecules and the ligands present in the
crystallographic structure were removed. For each GB compound, 100 docking runs were performed using the AutoDock Lamarckian genetic algorithm, considering the protein as rigid and the ligand as flexible. The others parameters were set to default. RMSD value of 2.0 Å was taken for clustering docking poses. The Discovery Studio mes BIOVIA) was used for superimposing software (Dassault Syste co-crystallized inhibitor of chymase and GB compounds to compare the position of binding. The conformations illustrative of the best energetic and the most populated cluster of poses were selected, saved in .pdb format and investigated for their H-bonds and hydrophobic interactions with the enzyme by using the tools available in AutoDock Tools and Discovery Studio. 2.3. Chymase surface pocket analysis CASTp server [16] was used with default parameters to identify the possible ligand binding pockets within the active site of the 3D structures of chymase. 3. Results 3.1. Molecular docking of G. biloba compounds with chymase By molecular docking simulations, we found that all the 20 compounds derived from GB derivatives are predicted to bind to the active site of chymase, since their predicted binding energies are negative (see Table 1). In particular, on the basis of lowest predicted binding energy, the detailed study of the best four different GB compounds (highlighted in Table 1) predicted to bind to chymase showed that all of them interact with at least one of the key amino acids constituting the active site of human chymase, which are Lys40, His57, Asp102, Phe191, Lys192, Gly193, Ser195, Tyr215, Gly216, Arg217, Ala226 (Fig. 1). Their detailed interactions are reported in Supplementary Table 1. In order to compare the results of the selected GB compounds with known natural chymase inhibitors, we performed molecular docking simulations with beta-boswellic acid and keto-betaboswellic acid [30]. Moreover, we performed a re-docking procedure with OHH compound, co-crystallyzed with chymase in PDB structure 1T31. Results are shown in Table 2. While the re-docking of OHH indicates very low binding energy, the values of predicted binding energy for the other two known chymase inhibitors are very similar to those obtained with ginkgolic acid, proanthocyanidins, quercetrin and rutin, supporting the hypothesis that the GB-derived compounds can indeed bind chymase. From this analysis, it appears that these best four GB compounds are predicted to interact with chymase key residues Lys40, His57, Phe191, Lys192, Ser195 and Gly216. We performed further docking simulations to verify the possibility of interactions of the four GB compounds with other serine proteases, i.e, kallikrein, tryptase, elastase (Table 3). Ginkgolic acid and proanthocyanidin have better predicted binding energy towards chymase than towards kallikrein, tryptase and elastase. Therefore, these two compounds appear rather selective for chymase. On the contrary, quercitrin shows similar predicted binding energy for all proteins and therefore appears to be not selective. Finally the predicted binding energies for rutin indicate that this molecule appears to bind preferentially to kallikrein than to chymase, but less preferentially to tryptase and elastase. 3.2. Chymase surface analysis and comparison to other serine proteases The chymase structure was analyzed by CASTp program to
Please cite this article in press as: A. Dubey, et al., Interaction of human chymase with ginkgolides, terpene trilactones of Ginkgo biloba investigated by molecular docking simulations, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.03.028
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Table 1 GB compounds interactions with Chymase, showing residues exhibiting hydrogen bonds and hydrophobic bonds with the different compounds. Name of compound (PUBCHEM ID)
Predicted binding No. of poses in Hydrogen energy (kcal/mol) the cluster bonds
Hydrophobic interactions
Ginkogolic acid (5281858) Proanthocyanidin (108065) Quercetrin (5280459) Rutin (5280805) Ginstein (5280961) Diosmetin (5281612) Genkwanin (5281617) Luteolin (5280445) Ginkgolide M (71450923) Procyanidins (107876) Kaempferol (5280863) Isorhamnetin (5281654) Quercetin (5280343) Apigenin (5280443) Myricetin (5081672) Ginkgolide J (163776) Bilobalide (73581) Ginkgolide A (6419993) Ginkgolide B (6324617) Ginkgolide C (161120)
¡8.45 ¡8.43 ¡8.36 ¡8.13 7.73 7.72 7.65 7.6 7.53 7.53 7.5 7.36 7.36 7.32 7.04 6.55 6.5 6.37 6.28 6.15
LYS40, PHE41, PHE191, LYS192, ALA226 HIS57, PHE191, LYS192, TYR215 PHE191, LYS192, TYR215, ALA226 PHE191, LYS192, TYR215, GLY216, ALA226 PHE191, LYS192, TYR215, ALA226 PHE191, LYS192, ALA226 PHE191, LYS192, ALA226 PHE191, LYS192, VAL213
1 14 66 39 100 62 72 79 100 33 49 71 74 64 86 44 100 50 85 98
LYS192 LYS40, HIS57, PHE191, LYS192, GLY193, GLY216 LYS40, HIS57, SER189, ALA190, PHE191, LYS192, SER195 SER189, SER195, SER214, ARG217 LYS40, HIS57, SER189, SER195 SER189, LYS192, SER218 SER189, LYS192 ALA190, LYS192, SER195, SER218 LYS192, SER195, GLY216 ALA190, LYS192, TYR215, GLY216 SER189, LYS192, SER195, GLY216 ARG143, SER189, PHE191, LYS192 ALA190, LYS192, SER195, SER218 SER189, PHE191, LYS192 SER189, LYS192, SER195, SER218 LYS192, GLY193, ASP194, SER195 SER214,ARG217 LYS192, GLY193, SER195, GLY216 LYS40, LYS192, SER195, GLY216 LYS192, SER195 GLY216 LYS192, GLY193, SER195, GLY216
ALA190, PHE191, LYS192, VAL213, ALA226 PHE191, LYS192, ALA226 PHE191, LYS192, ALA226 PHE191, LYS192, VAL213 PHE191, LYS192, VAL213, ALA226 PHE191, LYS192, ALA226 LYS192 PHE191, LYS192, TYR215 LEU99, PHE191, TYR215 LEU99, PHE191, TYR215 VAL213
Compounds shown in bold are those with the better predicted binding energy.
identify accessible surface pockets. The analysis identified three distinct major cavities, named here as A, B and C: the first one of 162.2 Å3; the second one of 149.2 Å3; the third one of 19.5 Å3. Pocket A is made by the following residues: Ser189, Ala190, Phe191,
Lys192, Ser195, Val213, Tyr215, Gly216, Arg217, Ala220, Lys221, Pro224, Ala226. Pocket 2 is made by residues Pro38, Ser39, Lys40, Phe41, Cys42, Arg143, Lys192, Gly193, Ser195. Finally, pocket C includes residues His57, Leu99, Asp102, Ser214, Tyr215. Their position
Fig. 1. Interaction of selected active site residues of chymase with the best four GB compounds, Ginkgolic acid (panel A), Proanthocyanidin (panel B), Quercetrin (panel C) and Rutin (panel D).
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Table 2 Predicted binding energies and number of poses of the known chymase inhibitors docked to chymase. Known natural inhibitors
Focused docking results Lowest binding energy
Keto-beta-boswellic acid Beta-boswellic acid OHHa a
Table 4 Occupation of the different cavities of chymase by GB compounds.
9,6 8,76 14.84
No. of poses in the cluster 100 100 90
OHH is the co-crystallyzed ligand in the PDB structure of chymase 1T31.
are very close and, all together, they constitute the active site of the chymase. The comparison of the binding sites of GB compounds with CASTp results (Table 4) suggests that rutin, ginstein, procyanidins, ginkgolide A, B and J, and bilobalide occupy all the pockets identified, whereas the others GB compounds interact preferentially with A and B pockets only. All the best four GB compounds found by molecular docking were compared with OHH, the inhibitor co-crystallyzed in the chymase structure used (Fig. 2). Based on similarity of residues shared and CASTp analysis, we show that the co-crystallized inhibitor occupies mainly the pocket C and a small part of pocket A, whereas ginkgolic acid (Fig. 2 (top left)) and quercetrin (Fig. 2 (bottom left)) are shifted towards the catalytic pocket B and some moieties interact with the nearby centered pocket region A. The binding pocket of proanthocyanidin (Fig. 2 (top right)) and rutin (Fig. 2 (bottom right)) is instead more similar to that of the crystallographic inhibitor.
Name of compound
CASTP pocket
Ginkogolic acid Proanthocyanidin Quercetrin Rutin Ginstein Diosmetin Genkwanin Luteolin Ginkgolide M Procyanidins Kaempferol Isorhamnetin Quercetin Apigenin Myricetin Ginkgolide J Bilobalide Ginkgolide A Ginkgolide B Ginkgolide C
A, B A,B A, B A,B,C A,B,C A,B A,B A,B A, B A,B,C A,B A,B A,B A,B A,B A, B, C A,B,C A,B,C A,B,C A, B
in the active site region. This enhanced interaction may produce more efficient cleavage of angiotensin I [1]. In addition, the charged amino acids Lys40, Arg143 and Lys192 in the chymase are located in positions (Pocket A and B) where they possibly can interact with GB compounds.
4.1. The extended GB compounds-binding site on human chymase 4. Discussion The chymase inhibition is thought to play a crucial role in the therapeutic action against cardiovascular diseases, fibrotic disorders and various other allergic inflammations. Based on literature search, we selected 20 most active GB compounds with the aim of evaluating their potential ability to bind chymase enzyme. Molecular docking investigation studies on the crystal structure of the human chymase have revealed that all the GB compounds were docked into the active site of human chymase and show favorable distinct interactions with the key residues Lys40, His57, Val146, Phe191, Lys192, Ser195, Gly216, and Ser218. The assessment of hydrogen bond network has determined that almost all the active GB compounds can potentially form hydrogen bond interactions with the positively charged Lys192 residue at the active site. The catalytic activity of chymase is dependent on the basicity of His57 of the catalytic triad positions. The identification of hydrogen bonding at Lysl92 residue of human chymase suggests a possible mechanism for the pH enhancement. Lys residues are positively charged at neutral pH and become neutral over the alkaline pH range; neutralization of the amino group of Lys192 by high pH would reduce its need to be solvated by water, thereby enhancing its ability to interact with groups of the substrate buried
The extended GB compounds-binding site is the region of the chymase that interacts with residues inclining the scissile bond. Chymase is capable of interacting with important residues of a protein by assuming the shape of a cleft. As shown in the molecular docking results (Table 1), while analyzing the best four GB compounds, almost all show interactions with Lys40 and Lys192. In chymase, quercetrin binds into the binding pocket B (Fig. 2 (bottom left)), occupied by the group of Ser218 and by the positively charged residue Arg217, whereas proanthocyanidin (Fig. 2 (top right)) and rutin (Fig. 2 (bottom right)) fit inside the large binding pocket A and B and can bind Tyr215 residue very easily. The residues Phe191, Lys192, Tyr215, Gly216, Ser218 and Ala220 located in the binding pockets A and B interact mostly with hydroxyl and ring aromatic groups of GB compounds. These residues are considered to execute important role in the natural substrate acceptance and hydrolysis process in chymase. Therefore, the establishment of strong interactions at the catalytic residue Lys40 plays a very diverse role in active inhibition. All the best four GB compounds share interactions with residues Ser189, Ala190, Gly216, and Ala226, involved in important interaction for the substrate selectivity and hydrolysis [11,21,34]. Alanine is also more hydrophobic with respect to Serine, and may enhance hydrophobic interactions with substrate lysine side chains, hence improving the binding affinity [25]. Interestingly,
Table 3 Comparison of the predicted binding energies of the best GB compounds docked to chymase with respect to those obtained for kallikrein, tryptase and elastase. Name of compound
Chymase Lowest binding energy
Ginkgolic acid Proanthocyanidins Quercitrin Rutin
8.45 8.43 8.36 8.13
Kallikrien No, of clusters 1 14 66 39
Lowest binding energy 7.23 7.64 9.47 9.7
Tryptase No, of clusters 14 38 56 11
Lowest binding energy 6.15 7.6 8.09 7.28
Elastase No, of clusters 11 62 42 11
Lowest binding energy 7.24 6.14 8.23 6.25
No, of clusters 22 19 37 10
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Fig. 2. Comparison of the position of the structure of chymase co-crystallized ligand (green) with best four Ginkgo biloba compounds, (top left) Ginkgolic acid (Yellow); (top right) Proanthocyanidin (Black); (bottom left) Quercetrin (Orange); (bottom right) Rutin (Cyan). The three pockets identified by CASTp analysis are shown as colored spheres and labeled A, B, and C. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
proanthocyanidin is the compound with the higher predicted number of hydrogen bond formation, with Lys40, Phe191, Lys192, Gly193, Ser195, Gly216 and Ser218. Moreover, some of these compounds, in particular ginkgolic acid and proanthocyanidin, seem more selective for chymase than to other serine proteases like kallikrein, tryptase and elastase. We found GB compounds with predicted favorable binding energy against chymase enzyme, thus becoming a potential starting point for testing this hypothesis experimentally, over in vitro and in vivo models. Therefore, these natural molecules, with wide therapeutic mechanism, can become novel candidates for therapeutic action, and may enhance the possibility for the potential development of future drugs. Acknowledgments This work has been partially supported by the Flagship InterOmics Project (PB.P05, funded and supported by the Italian Ministry of Education, University and Research and Italian National Research Council organizations). A. D. is supported by ICGEB Smart Fellowship program. Appendix A. Supplementary data Supplementary data related to this article can be found at http://
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Please cite this article in press as: A. Dubey, et al., Interaction of human chymase with ginkgolides, terpene trilactones of Ginkgo biloba investigated by molecular docking simulations, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.03.028