Phytochemical compounds of Morus alba as anti-aging agent towards in silico binding to matrix metalloproteinase proteins

Nội dung text: Phytochemical compounds of Morus alba as anti-aging agent towards in silico binding to matrix metalloproteinase proteins

1. MedPharmRes, 2021, Vol. 5, No. 3 1 MedPharmRes Journal of University of Medicine and Pharmacy at Ho Chi Minh City homepage: and Original article Phytochemical compounds of Morus alba as anti-aging agent towards in silico binding to matrix metalloproteinase proteins Phuong Thuy Viet Nguyena*, Thi Khanh Taa, Giang Le Tra Nguyena aFaculty of Pharmacy, University of Medicine and Pharmacy at Ho Chi Minh City, Ho Chi Minh City, 700000, Vietnam. Received December 07, 2020: Revised March 17, 2021: Accepted March 22, 2021 Abstract: Skin aging is a natural phenomenon which is related to progressive loss of skin structural integrity and physiological function and affects aesthetics which has been of highly interest. Inhibition of matrix metalloproteinases (MMPs) such as MMP-1, MMP-3, MMP-9 is one of the potential approaches for anti- aging treatment as these targets are involved in molecular pathology to skin aging process from sunlight. The aim of the study was to investigate the binding affinity of 9 phytochemical compounds extracted from Morus alba Moraceae into the MMP enzymes leading to potential anti-aging activity by using in silico approaches including molecular docking and molecular dynamics simulations. All the compounds showed binding abilities into the targets. In particular, mulberrofuran H obtained the best docking results on the three MMPs. Molecular dynamics simulations of the complex of mulberrofuran H and MMP-9 showed that this complex was stable. Combination of molecular docking and molecular dynamics simulations results, there was an important hydrophobic interaction between mulberrofuran H and His401 at the active site of the MMP-9, which determined the MMP-9 inhibitory potential of mulberrofuran H. The ligand mulberrofuran H was also stabilized into the MMP-9 protein by hydrogen bonds with Pro421 with the high occupancy of 77.67%. These results demonstrated the good binding of mulberrofuran H on the protein MMP-9 which highlighted its anti- aging potency. Keywords: anti-aging; phytochemicals; Morus alba; molecular docking; molecular dynamics simulations. 1. INTRODUCTION of collagen, elastin, melanine, etc through the inhibition and/ or activation of some enzymes such as hyaluronidase, Skin aging is a complicated phenomenon which is a elastase, matrix metalloproteinase in the extracellular domain combination of natural aging and photo-aging [1]. It is of the skin [1]. related to progressive loss of skin structural integrity and physiological function leading to visible wrinkles and skin Matrix metalloproteinases (MMP) are a endopeptidase sagging on the skin’s surface affecting aesthetics [1]. This family or matrixins that are zinc (II)-dependent proteases [2]. process could be due to the change of various internal There are 23 MMPs (MMP-1 to MMP-23) belonging to five factors, for example hormone, cellular metabolism, genetics main groups including collagenase, gelatinase, stromelysins, and/ or external factors such as the exposure of sunlight (i.e., matrilysins, membrane MMPs (MT-MMP), and other MMPs photo-aging), pollution, and chemical toxins [1]. Of which, [2]. Among these enzymes, MMP-1 (collagenase), MMP-3 UV radiation exposure could cause the production of (stromelysin-1) and MMP-9 (gelatinase) are three major reactive oxygen species (ROS) which is a dangerous agent to enzymes responsible for degradation of various extracellular induce the skin aging prematurely by changing of production protein components of skin such as degrading collagen in the *Address correspondence to Phuong Thuy Viet Nguyen at the Faculty of Pharmacy, University of Medicine and Pharmacy at Ho Chi Minh City, Ho Chi Minh City, Vietnam; Tel: +084-919520708; E-mail: ntvphuong@ump.edu.vn © 2021 MedPharmRes
2. 2 MedPharmRes, 2021, Vol. 5, No. 3 Nguyen et al. epidermis of skin surface [2]. Thus, MMP-1, MMP-3 and Arg424, making it difficult for the ligand to attach with long MMP-9 are considered potential targets for anti-aging drug branches. The MMP-3 has the widest S1 'pocket, as an open discovery. channel, suitable for bulky and highly hydrophobic ligands [2]. MMP family have some common structures [2]. Typically, the structures of MMPs include a propeptide Until now, there are some researches about natural region (77-87 amino acids), a catalytic metalloproteinase compounds such as alkaloids, flavonoids, phenolic domain (about 170 amino acids), a linker peptide (hinge compounds for anti-aging activity, especially for MMPs region) and a hemopexin domain (200 amino acids) [2]. The inhibition. For example, aloin A, B of Aloe vera [3]; catalytic zone contains two Zn2+ ions for structural and xanthorrhizol in the extract of Curcuma xanthorrhiza [4], catalytic and three calcium ions (Ca2+); in which the zinc ion diosgenin of Dioscorea villosa [5], oil extract from Camellia for catalytic binding with three histidine and 3 Ca2+ ions [2]. japonica [6] and fruit extract from Emblica officinalis [7] In the catalytic zone there are active zones, which interact inhibited MMPs enzymes. Morus alba Moraceae has been with substrates and inhibitors. These parts include S1, S2, used as traditional medicine with some bioactive compounds S3, Sn on the left side of Zn2+ ion and S1', S2', S3', Sn' for anti-inflammatory, antioxidant and neuroprotective on the right of Zn2+ ion [2]. In particular, the pockets on the activities [8]. The in vitro effects of Morus alba extract were right are considered to be the places of binding inhibitors as also reported on the MMP-1, MMP-9 [9] and MMP-13 [10] they provide better inhibitory effects and the pockets on the for the treatment of osteoarthritis and periodontitis. left are less interested because of a relatively modest Unfortunately, there is no information about biological inhibitory capacity. The S1' pocket was known to be activity of Morus alba phytochemicals as potential anti- important in determining the MMPs selectivity due to the aging agent. Therefore, the present study investigated the difference in size, hydrophobicity and flexibility among anti-aging potency of natural compounds in the Morus alba MMPs [2]. extract through their binding interactions with the MMP-1, For the MMP-1, this S1' pocket is quite shallow and less MMP-3 and MMP-9 enzymes related to skin aging process by a combination of molecular docking and molecular hydrophobic. However, the amino acid Arg214 at the bottom dynamics simulations approaches. of this pocket is flexible, which can be displaced during ligand binding to create a larger bound pocket. For the 2. MATERIALS AND METHOD MMP-9, the S1' pocket is wider and more hydrophobic, with the bottom of this pocket being the inflexible amino acid 2.1. Molecular docking Table 1. Three structures of MMP-1 (PDB: 966C), MMP-3 (PDB: 1G4K) and MMP-9 (PDB: 2OW0) as targets for anti-aging drug discovery Protein PDB id Resolution Structure type Selected chain for Co-crystallized ligand Reference docking MMP-1 966C 1.9 Å Monomer A Monomer A N-hydroxy-2-[4-(4-phenoxy- [11] benzenesulfonyl)-tetrahydro- pyran-4-yl]-acetamide MMP-3 1G4K 2.0 Å Trimer A, B, C Chain B 5-methyl-5-(4-phenoxy- [12] phenyl)-pyrimidine-2,4,6- trione MMP-9 2OW0 2,.0 Å Dimer A, B Chain A N-[(4'-iodobiphenyl-4- [13] yl)sulfonyl]-d-tryptophan Molecular docking between three receptor targets, files protein. PDBqt and AD4_parameters.dat including for namely MMP-1 (PDB id: 966C [11]), MMP-3 (PDB: 1G4K Zn2+: Rii = 0,87 Å (Rii was the sum of van der Waals radius [12]) và MMP-9 (PDB: 2OW0 [13]) and 9 phytochemical of two similar atoms), epsii ε = 0,35 kcal.mol-1 (ε was the compounds found in the Morus alba extract was conducted depth of van der Waals) and volume of atomic solvation vol in AutoDock 4.0 [14] to explore the binding affinity, binding = ( ) = 0,3446 [16]. pose as well as binding interactions of the targets with respective ligands. Briefly, three targets were retrieved from The nine phytochemicals in the Morus alba extract the protein data bank ( The crystal included 5 prenyl flavonoids (Kuwanon E, Kuwanon S, structures of MMP-1 (PDB: 966C), MMP-3 (PDB: 1G4K) Quercetin, Morusinol and Cudraflavon B), 2 benzofurans and MMP-9 (PDB: 2OW0) were downloaded in (Mulberrofuran Y and Mulberrofuran H), Moracin C and format .PDB file (Table 1), and then removed waters and Mulberroside C. These compounds were selected for the retracted the co-crystallized ligands from the experimental study based on the similar structures to the co-crystallised 2+ structures and kept the ion Zn in the active sites of the ligands in the MMP enzymes. The 2D structures of protein targets. The targets were minimized by using compounds (Figure 1) were downloaded from PubChem CHARMM 36 force field in Discovery Studio 2016 [15], and database ( in the save the monomers in .PDB file. These protein targets were format .sdf file and minimized energy by using ChemDraw subsequently added polar hydrogens, Gasteiger charges by 3D 16.0 and save in the format .PDB. Following, the targets using Autodock Tool 1.5.6 [14] and save in the .PDBqt file. and ligands were prepared using Autodock Tools 1.5.6 under The optimization parameters for docking were updated in the setting parameters of grid boxes (Table 2) and saved
3. Potential anti-aging agent of Morus alba to matrix metalloproteinase proteins MedPharmRes, 2021, Vol. 5, No. 3 3 in .gpf. Autogrid 4.0 converted .gpf to .glg file. Finally, using Discovery Studio 2016 và MOE 2015.10 including molecular docking was carried out using AutoDock 4.0 with binding affinities, different conformational ligands and the the Lamarckian genetic algorithm and input files .glg binding interactions of ligands and protein targets. and .dpf. The docking results (in files .dlg) were analysed Figure 1. Structures of nine compounds in the extract of Morus alba Moraceae Table 2. Parameters of grid boxes for molecular docking of three targets, MMP-1 (PDB: 966C), MMP-3 (PDB: 1G4K) and MMP-9 (PDB: 2OW0), respectively Protein Size (Å)3 Spacing (Å) Coordinates (Å) MMP-1 22 x 22 x 22 1.0 X = 9.166, Y = 6.703, Z = 36.278 MMP-3 20 x 20 x 20 1.0 X = 16.496, Y = 49.347, Z = 68.602 MMP-9 22 x 22 x 22 1.0 X = 26.068, Y = 7.540, Z = 47.575 2.2. Molecular dynamics simulations systems. The production was for 20 ns and the result was taken every 0.01 ns and evaluated based on the values of Molecular dynamics simulations (MDs) were used to RMSD (Root-mean-square deviation), RMSF (Root-mean- investigate stability and flexibility of protein-ligand complex under the influence of temperature, pressure, and solvent. square fluctuation), Rg (Radius of gyration), percentage of hydrogen bond occupancy and hydrophobic interactions and MDs were conducted for protein without ligand (apo protein) SASA (Solvent-accessible surface area). The parameters and protein-ligand complex for 20 ns by using GROMACS 2019 software. From the docking results, choosing the best were calculated by GROMACS, and the results were analysed using VMD version 1.9.3. conformation of the best binding compound and then adding hydrogens to it by Avogadro ver 1.2.0 software were performed. The topology of the ligand was generated by 3. RESULTS CGENFF with CHARMM 36 force field. The complex was placed in a simulation box (12 surface polyhedrons) and 3.1. Molecular docking contained the water solvent (TIP3P model) and a distance of Molecular docking was carried out for the co-crystallised 1.0 nm from the box edge. The sodium cation and chloride ligands in the three experimental structures to evaluate the ion were added to neutralise the system. The complex was reproducibility of Autodock 4. The results showed that these energy minimized to eliminate the negative interactions and native ligands obtained good binding affinities of -7.71; - the system was balanced under the "isothermal-isobaric" 8.19 and -8.98 kcal.mol-1 into the three targets, MMP-1, -3 NVT and NPT conditions (N: number of particles, V: and -9, respectively. The root-mean-square-deviations of ○ volume, T: temperature 300 K, and P: pressure 1 bar). conformations of ligand after docking and the native ones Berendsen thermostat and Parrinello-Rahman barostat were were about 0.2 to 0.6 Å. The active sites of the targets were used for maintaining the temperature and pressure. The confirmed including the co-crystallised ligands, ion Zn2+ at equilibration was run during 1000 ps for each NVT and NPT the center with 3 histidines along with the key residues
4. 4 MedPharmRes, 2021, Vol. 5, No. 3 Nguyen et al. Leu181, Ala182 và Glu219 of MMP-1; Leu164, Ala165, interactions of the experimental structures were also Ala167 and Glu202 of MMP-3; and Leu188, Ala189, reproduced after docking (Table 3). Gln402 of MMP-9, respectively (Figure 2). The binding Figure 2. Docking results of protein targets (a) MMP-1 (PDB: 966C), (b) MMP-3 (PDB: 1G4K) and (c) MMP-9 (PDB: 2OW0) with the co-crystallized ligands in their active sites; superimposition of the co-crystallized ligands in the experimental structures before docking and after docking; and 2D binding interactions of proteins and ligands. Table 3. Binding interactions of MMP-1 (PDB: 966C), MMP-3 (PDB: 1G4K) and MMP-9 (PDB: 2OW0) and the co- crystallized ligands in the experimental structures (before docking) and after molecular docking Ligand Experimental structure After docking Protein MMP-1 Hydroxamic-Ala182 Hydroxamic-Ala182 (PDB: 966C) Hydroxamic-Glu219 Hydroxamic-Glu219 Hydroxamic-Zn2+ Hydroxamic-Zn2+ Sulfone-Leu181 Sulfone-Leu181 MMP-3 Pyrimidine (O2)-Leu164 Pyrimidine (O4)-Leu164 (PDB: 1G4K) Pyrimidine (N1)-Ala165 Pyrimidine (N3)-Ala165 Pyrimidine-H2O-Glu202 Pyrimidine-His211 Pyrimidine-H2O-Ala167 Phenol-His224 Pyrimidine (N2)-Zn2+ Pyrimidine (O2)-Zn2+ Phenoxy-His201 MMP-9 Carboxyl-Zn2+ Carboxyl-Zn2+ (PDB: 2OW0) Carboxyl-His411 Carboxyl-Gln402 Carboxyl-Gln402 Sulfone-Ala189 Sulfone-Ala189 Sulfone-Leu188 Sulfone-Leu188
5. Potential anti-aging agent of Morus alba to matrix metalloproteinase proteins MedPharmRes, 2021, Vol. 5, No. 3 5 Molecular docking of 9 compounds, kuwanon E, kuwanon S docked well on all MMP-1, MMP-3, MMP-9 kuwanon S, quercetin, morusinol, cudraflavon B, proteins with the binding affinities lower than -7.0 kcal.mol- mulberrofuran Y, mulberrofuran H, moracin C and 1. Among the investigated compounds, mulberrofuran H was mulberroside C into three targets, MMP-1 (PDB: 966C), selected as the hit compound with the best binding affinities MMP-3 (PDB: 1G4K) and MMP-9 (PDB: 2OW0) was on three protein targets: -8.23 kcal.mol-1 for MMP-1; -8.87 carried out using Autodock 4.0. The docking results kcal.mol-1 for MMP-3 and -8.34 kcal.mol-1 for MMP-9. demonstrated that all nine compounds were fitted well into Some top compounds with their interactions with three the active sites of all proteins with the good binding affinities proteins were listed in Table 5. (Figure 3 and Table 4). Mulberrofuran H, moracin C and Figure 3. Nine compounds in the extract of Morus alba Moraceae into the three protein targets MMP-1 (PDB: 966C), MMP-3 (PDB: 1G4K) and MMP-9 (PDB: 2OW0), respectively after molecular docking Table 4. Binding affinities (kcal.mol-1) of 9 compounds in the extract of Morus alba Moraceae after docking into the three protein targets MMP-1 (PDB: 966C), MMP-3 (PDB: 1G4K) and MMP-9 (PDB: 2OW0), respectively. No Compounds MMP-1 MMP-3 MMP-9 1 Mulberrofuran H -8.23 -8.87 -8.34 2 Mulberroside C -8.01 -5.92 -8.27 3 Kuwanon S -7.52 -7.64 -6.54 4 Moracin C -7.17 -7.50 -7.50 5 Quercetin -6.27 -6.08 -5.81 6 Kuwanon E -6.17 -6.35 -4.62 7 Mulberrofuran Y -6.14 -7.92 -6.24 8 Cudraflavone B -6.05 -7.44 -6.20 9 Morusinol -5.45 -7.02 -6.04
6. 6 MedPharmRes, 2021, Vol. 5, No. 3 Nguyen et al. Table 5. Interactions of top ligands and the binding pocket of the three protein targets MMP-1 (PDB: 966C), MMP-3 (PDB: 1G4K) and MMP-9 (PDB: 2OW0) Target Compounds Hydrogen bonds Hydrophobic interactions MMP-1 Mulberrofuran H Glu219, Pro238, Tyr237 Leu181, Arg214, Val215, His218, Leu235, Thr241 Mulberroside C Leu181, Ala182, Glu219, Arg214, Val215, His218, Leu235, Tyr240 Thr241 Kuwanon S Ala182, Arg214, Ala234, Tyr210, Tyr240, Thr241 Tyr237 Moracin C Glu219, Ala234, Tyr237 Leu181, Arg214, Val215, His218, His228 MMP-9 Mulberrofuran H Val398, Pro421 Leu188, Leu397, His401, His405, His411, Leu418, Tyr423 Mulberroside C Gly186, Ala189, Gln402, Leu188, Val398, His401 Pro421, Thr426 Kuwanon S Ala189, Tyr420, Met422 His401, His411 Moracin C Ala417 His401, Tyr423 MMP-3 Mulberrofuran H Glu202, Pro221 Leu164, Leu197, Val198, His201, His205, Leu218, His224 Mulberrofuran Y Glu202 Asn162, Val163, Ala165, Leu164, Val198, His201, His211, Kuwanon S Pro221 Leu164, His201, Leu218, Tyr223, His224 Moracin C Glu202, Tyr220 Leu164, His201, His211, Leu218 Cudraflavone B Leu164, Ala165, Glu202, Leu164, Val198, His201, Tyr223 Tyr223 Morusinol Ala165, Leu164, Glu202, Leu197, Val198, His201, His205, His211, Tyr223 Leu218, Tyr223 3.2. Molecular dynamics simulations The RMSD values of protein-ligand complexes reached stable state after 1 ns while the apo protein gained equilibrium The best complex of MMP-9 and mulberrofuran H was state about nearly 6 ns, respectively (Figure 4). The variation of chosen after docking for 20 ns MDs to give insight into the MMP-9 in the complex was quite large with the RMSDs from binding stability and the flexibility of the complex using 2.0-3.0 Å compared to the first position (Figure 4). The RMSD Gromacs 2019. The apo protein (MMP-9 structure without values of ligand were found to be less than 1.0 Å after 6 ns ligand) was also used as the reference one. The MDs were (Figure 4). Therefore, because of ligand binding, the MMP-9 in evaluated by the values of RMSD, RMSF, Rg, percentage of the complex was more stable in compared with the MMP-9 apo hydrogen bond occupancy and hydrophobic interactions and protein. SASA Figure 4. The values of RMSD (Root-mean-square deviation) of the complex MMP-9 with (a) mulberrofuran H and (b) the MMP-9 apoprotein (PDB: 966C)
7. Potential anti-aging agent of Morus alba to matrix metalloproteinase proteins MedPharmRes, 2021, Vol. 5, No. 3 7 The RMSF values showed the fluctuation of each atom Percentage of hydrogen bonds occupancy between protein throughout the simulation, especially calculated for carbon Cα of MMP-9 (2OW0) and ligand mulberrofuran H were calculated at amino acids of protein. As the lack of residues from 215 to 391 the different times 0, 5, 10, 15 and 20 ns (Table 6). Of the in the crystal structure was the reason for the straight light observed hydrogen bonds, mulberrofuran H showed the highest (Figure 5). The RMSF values confirmed that the residues of the presence of important hydrogen bonds with amino acids His401 binding site were less fluctuable. The average RMSF values (83.07%), Pro421 (77.67%), followed by Leu188 (36.16%) and were 1.0 Å. However, residues located near the binding site Gln402 (27.32%), similar to the interactions of the experimental (residues from 180 to 200) showed more fluctuation after ligand complex. In addition, mulberrofuran H also formed the binding. hydrogen bonds with Met422, Tyr420, Ala417 with high occupancy of more than 65%, showing the good interactions with S1' pocket. Through 20 ns simulation of molecular dynamics, the hydrophobic interactions between the benzopyran backbone of mulberrofuran H with His401 was very stable. This is an important hydrophobic interaction in the experimental complex, which determined the MMP-9 inhibitory potential of mulberrofuran H. In addition, there were also interactions between benzopyran scaffold with Leu418, Arg424 and hydrophobic interactions of the tricyclo substituent group and the amino acids Leu187 and Leu188. All the interactions were quite stable, helping the complex to bind firmly (Table 7). Figure 5. RMSF plot of backbone atoms of MMP-9 Figure 7 showed that there was not much difference in (PDB: 966C) in the apoprotein MMP-9 (black) and SASA values of the apo protein and the protein in the complex. bound state (red) – the complex of MMP-9 and SASA values of MMP-9 protein fluctuated in the range of 90- 2 mulberrofuran H over 20 ns simulation 95 nm while the MMP-9 in the complex was in the range of 95-100 nm2. This suggested that the binding of mulberrofuran H As can be seen from Figure 6, the Rg value of MMP-9 was to MMP-9 did not affect the SASA values much as the folding nearly constant throughout 20 ns, and close to 1.5 nm in both structure of the MMP-9 protein could keep the protein in a dynamic models. The results demonstrated that MMP-9 was stable state. There was also a fluctuation of SASA values in the stable after binding to mulberrofuran H, and the ligand did not initial 15 ns before reaching a stable state until the end of change the radius gyration of the protein. simulations. Figure 6. Radius of gyration (Rg) plot of MMP-9 (black) compared to Figure 7. SASA (Solvent-accessible complexed state (red) for 20 ns simulations surface area) values of mulberrofuran H in complexed with MMP-9 protein (red) with respect to the apoprotein (black)
8. 8 MedPharmRes, 2021, Vol. 5, No. 3 Nguyen et al. Table 6. The percentage of hydrogen bonds occupancy between the protein MMP-9 (PDB: 2OW0) and mulberrofuran H during the simulation period of 20 ns Donor Acceptor Occupancy Percentage (%) His401 Ligand 83.07% Ligand Pro421 77.67% Ligand Met422 75.27% Ligand Tyr420 70.03% Ligand His401 66.53% Ligand Ala417 65.13% Ligand Leu418 61.29% Leu188 Ligand 36.16% Complex MMP-9 and Mulberrofuran H Ligand Gly186 32.27% Ligand Leu188 31.72% Ligand Glu416 31.37% Ligand Arg424 31.07% Arg424 Ligand 30.87% Tyr423 Ligand 29.32% Gln402 Ligand 27.32% Ligand Leu397 21.28% Table 7. The hydrophobic interactions between the MMP-9 (PDB: 2OW0) and mulberrofuran H during the 20 ns simulations Time Hydrophobic interactions 0 ns Benzopyran – His401 Benzopyran – Leu418 Benzopyran – Leu397 Benzopyran – Tyr423 Tricyclo – Leu188 5 ns Benzopyran – Leu397 Benzopyran – His401 Benzopyran – Arg424 Benzopyran – Pro430 Tricyclo – Leu188 Tricyclo – Ala189 Complex MMP-9 (PDB: 2OW0) and Tricyclo – Val398 Mulberrofuran H 10 ns Benzopyran – His401 Benzopyran – Tyr423 Tricyclo – Leu187 15 ns Benzopyran – His401 Benzopyran – Met422 Benzopyran – Tyr423 Tricyclo – Leu187 20 ns Benzopyran – Leu418 Benzopyran – His401 Benzopyran – Arg424 Benzopyran – Tyr423 Tricyclo – Leu187 4. DISCUSSION moracin C exhibited better binding on MMP-9 than MMP-1. This could be explained by the fact that the S1 'pocket in the 4.1. Molecular docking MMP-9 was deeper, so the ligand could go deeper and interact with the protein better. In addition, the S1 'pocket in MMP-9 Docking results on MMP-1 and MMP-9 was narrower than MMP-1, leading to a weaker binding result The docking results on MMP-1 and MMP-9 shared many for bulky structures, such as kuwanon S. similarities with the same 4 top candidates, mulberrofuran H, On the other hand, mulberrofuran H, mulberroside C and mulberroside C, kuwanon S and moracin C. The common kuwanon S have a curved V-like configuration, having a feature in the structures of these ligands was the heterocyclic substituted group as an anchor, which was well compatible with groups: benzofuran (mulberrofuran H, moracin C), the shape of the binding cavity for a more stable attachment. furochromane (mulberroside C) and chromon groups (kuwanon Specifically, mulberrofuran H, with a structure of 5-benzofuran S) which could easy get into the S1 'pocket of MMP-1 and 1,3-diol was well-fitted into the pocket. However, moracin C MMP-9 and interact with the pockets. The heterocyclics are not with the isobutylene group could not push the heterocyclic too cumbersome, so the S1 pockets of MMP-1 and MMP-9 group into the binding pocket, so the interactions of this were accessible to them, demonstrating their inhibitory compound and proteins MMP-1 and MMP-9 were weaker. potential. However, mulberrofuran H, mulberroside C and
9. Potential anti-aging agent of Morus alba to matrix metalloproteinase proteins MedPharmRes, 2021, Vol. 5, No. 3 9 The remaining 5 compounds, morusinol, quercitin, kuwanon the dimethylocta branching fitting wellto S1’ site, which could E, mulberrofuran Y and cudraflavone B did not have good also be observed in kuwanon S (-7.64 kcal.mol-1). Moracin C (- binding affinities due to their bulky heterocyclic structure, along 7.50 kcal.mol-1) was different from the others because of the π-π with the formation of endo-molecular hydrogen bonds, making interaction between the 5-benzopyran, 1,3-diol heterocyclic them difficult to access to the S1 'pocket. For example, the -OH backbone with His201 (similar to the co-crystalized ligand of substituents on the furochromane ring of quercitin, kuwanon E, MMP-3 protein). Cudraflavone B (-7.44 kcal.mol-1) is a morusinol and cudraflavone B created the endolytic hydrogen polyphenol structure with short hydrocarbon substituents, so the bonds with cetone oxygen, increasing the cumbersome effect. compound only had few hydrophobic interactions with residues Mulberrofuran Y contains the hydrocarbon dimethylocta in binding pocket such as Leu164, Val198, His201 and Tyr223. substituent on the benzofuran group and the bulky 1,3-diol Morusinol (-7.02 kcal.mol-1) with chromane heterocyclic benzene group at the end of the molecule which was found not structure created many π-π interactions with His201 and Tyr223 able to get into the S1‘ pocket even though the structure is and many other π-alkyl interactions, the hydrocarbon substituent similar to mulberrofuran H. branch created hydrophobic interactions with the trio His201, In terms of interactions between investigated ligands and His205 and His211. Particularly with mulberroside C, a very potential ligand on two MMP-1 and MMP-9 proteins, did not MMP-1 protein, most of the potential compounds mimicked the yield good binding affinity on MMP-3. This could be explained co-crystalized ligands to form the hydrogen bond with the key residue Glu219 (Table 5). Mulberrofuran H, mulberroside C by the fact that the van der Waals interaction of the ligand with MMP-3 was not as strong as with the MMP-1 and MMP-9 and moracin C all belonged to 5-benzofuran 1,3-diol benzyl proteins. backbone, so they shared some hydrophobic interactions in the S1’ binding pocket. The chromane scaffold of the kuwanon S In general, combinations of binding affinities and binding also created a hydrophobic bond with this cavity. The ligand interactions with three targets, MMP-1, MMP-3 and MMP-9 mulberrofuran H generated a hydrogen bond with Pro238 and proteins, the complex of mulberrofuran H and MMP-9 was selected two hydrophobic interactions between its cyclohexan ring and for molecular dynamics simulations (MDs) to give further insight the key residues Leu181 and His218. The mulberroside C into the binding of the compound and the MMP-9. formed hydrogen interactions with Leu181 and Ala182 based on some OH substituents of the pyranose ring. Comparing with 4.2. Molecular dynamics simulations the ligand moracin C, a hydrophobic interaction with the residue After molecular docking, the ligand mulberrofuran H was His228 of S1 pocket was generated by this ligand’s select as the best binding ligand with the good binding affinities hydrocarbon methyl but-2-en substituent. The kuwanon S with into three targets, MMP-1, MMP-3 and MMP-9. Through the two -OH phenol groups on the chromane ring created two values of RMSD, RMSF, Rg, percentage of hydrogen bond hydrogen bonds in S1' pocket with Ala182 and Tyr237. In occupancy, hydrophobic interactions and SASA, the MMP-9 in addition, there were also many hydrophobic interactions the complex was more stable in compared with the MMP-9 between the dimethylocta substituent of kuwanon S and apoprotein. Thereby, the interactions between MMP-9 with Leu181, Tyr210, Tyr240 (Table 5). mulberrofuran H can be found to be beneficial, helping the As for MMP-9 (PDB: 2OW0), there were not many complex to be stable. hydrogen bonds but mainly hydrophobic interactions between Combination of molecular docking and molecular dynamics top ligands and residues in the binding site (Table 5). This might simulations results, there was an important hydrophobic be because the S1' pocket of MMP-9 protein was possibly more interaction between mulberrofuran H and His401 at the active hydrophobic than that of the MMP-1 protein. The ligand site of the MMP-9, which determined the MMP-9 inhibitory mulberrofuran H was stabilized by the π-contacts with Leu188, potential of mulberrofuran H. The ligand mulberrofuran H was His401, His405 and His411 while the mulberroside C fitted the also stabilized into the MMP-9 protein by hydrogen bond with protein through the hydrogen bonds with Arg424 and Thr426 Pro421 (high occupancy of 77.67%). In addition, there were by the active -OH on cyclohexan, with Gly186 and Pro421 by - also interactions between the benzopyran scaffold with Leu418, OH substituent on pynanoside ring, with Leu188 and Ala189 by Arg424 and hydrophobic interactions of the tricyclo substituent -OH phenol substituent of the backbone, respectively. group and the amino acids Leu187 and Leu188. All the Interestingly, the best docking conformation of moracin C interactions contributed to the stability of the complex. The generated a special interaction between -OH of the benzyl-1,3 results demonstrated the anti-aging potency of mulberrofurane diol substituent with Zn2+ ion. Apart from many hydrophibic H through the in silico study of inhibition of MMP-9 protein. and hydrophobic interactions with S1' cavity, the chromane ring Mulberrofurane H was also reported by the inhibitory activity of of the kuwanon S also presented an important electrostatical tyrosinase activity of Mulberrofuran H [17]. The in vitro and in interaction with the positive charge of Zn2+ ion (Table 5). vivo biological tests are required for the confirmation. Docking results on MMP-3 There were some differences of top compounds with good 5. CONCLUSION binding affinities on MMP-3 as the protein cavity was larger Through molecular docking, nine selected natural and deeper which was suitable for bulky structures (Table 5). compounds from Morus alba Moraceae were investigated Mulberrofurane H had the benzopyrane scaffold to be able to the prospective anti-aging agents by binding into three enter deeply insight into S1' site, conducting π-alkyl interactions targets, MMP-1, MMP-3 and MMP-9. All the phytochemical with Leu197, Val198, His224, and Leu218, so this ligand compounds showed binding abilities into the targets. Of -1 obtained the good binding affinity (-8.87 kcal.mol ). which, mulberrofuran H showed the best binding affinity on -1 Mulberrofuran Y (-7.92 kcal.mol ) generated many alkyl all three proteins and the complex of this compound with interactions with Leu164, Ala165, Val198 and His201 due to MMP-9 was selected for MD simulations. The values of
10. 10 MedPharmRes, 2021, Vol. 5, No. 3 Nguyen et al. RMSD, RMSF, hydrogen bond occupancy, hydrophobic I procollagen in ultraviolet-irradiated human skin fibroblasts. interactions, Rg and SASA demonstrated that the Phytotherapy research : PTR 23 (9):1299-1302. doi:10.1002/ptr.2768 5. De Canha MN, Steyn A, Blom van Staden A, Fibrich BD, Lambrechts mulberrofuran H and MMP-9 complex was highly stable IA, Denga LL, Lall N (2020). Book Review: Herbal Principles in during the period of 20 ns. An important hydrophobic Cosmetics: Properties and Mechanisms of Action. Front Pharmacol interaction between mulberrofuran H and His401 was 10:1513. doi:10.3389/fphar.2019.01513 identified, which determined the MMP-9 inhibitory potential 6. Jung E, Lee J, Baek J, Jung K, Lee J, Huh S, Kim S, Koh J, Park D (2007). Effect of Camellia japonica oil on human type I procollagen of mulberrofuran H. The ligand mulberrofuran H was also production and skin barrier function. Journal of ethnopharmacology 112 stabilized into the MMP-9 protein by hydrogen bonds with (1):127-131. doi:10.1016/j.jep.2007.02.012 Pro421 with the high occupancy of 77.67%. These results 7. Fujii T, Wakaizumi M, Ikami T, Saito M (2008). Amla (Emblica demonstrated the inhibitory potential of mulberrofuran H on officinalis Gaertn.) extract promotes procollagen production and inhibits matrix metalloproteinase-1 in human skin fibroblasts. Journal of the protein MMP-9 which could be further tested for anti- ethnopharmacology 119 (1):53-57. aging activity. doi: 8. Rodrigues EL, Marcelino G, Silva GT, Figueiredo PS, Garcez WS, CONFLICT OF INTEREST Corsino J, Guimarães RdCA, Freitas KdC (2019). Nutraceutical and Medicinal Potential of the Morus Species in Metabolic Dysfunctions. The authors declare no conflict of interest. Int J Mol Sci 20 (2):301. doi:10.3390/ijms20020301. 9. Yiemwattana I, Kaomongkolgit R, Wirojchanasak S and Chaisomboon N (2019). Morus alba stem extract suppress matrix metalloproteinases FUNDING (MMP)-1, MMP-9, and tissue inhibitors of metalloproteinase (TIMP)-1 expression via inhibition of lκBα degradation induced by The authors would like to thank University of Medicine Porphyromonas gingivalis LPS signal in THP-1 cells. European Journal and Pharmacy at Ho Chi Minh City for financial support. of Dentistry 13 (2):229-234. doi: 10.1055/s-0039-1694314. 10. Wongwat T, Srihaphon K, Pitaksutheepong C, Boonyo W and ACKNOWLEDGEMENTS Pitaksuteepong T (2020). Suppression of inflammatory mediators and matrix metalloproteinase (MMP)-13 by Morus alba stem extract and The authors would like to thank University of Medicine oxyresveratrol in RAW 264.7 cells and C28/I2 human chondrocytes. and Pharmacy at Ho Chi Minh City for their support and Journal of traditional and complementary medicine 10 (2):132-140. 10.1016/j.jtcme.2019.03.006. funding of conducting this research. 11. Lovejoy B, Welch AR, Carr S, Luong C, Broka C, Hendricks RT, Campbell JA, Walker KAM, Martin R, Van Wart H, Browner MF AUTHORS' CONTRIBUTIONS (1999). Crystal structures of MMP-1 and -13 reveal the structural basis for selectivity of collagenase inhibitors. Nature Structural Biology 6 The authors have completed the work including TKT for (3):217-221. doi:10.1038/6657 carrying out molecular docking, molecular dynamics 12. Dunten P, Kammlott U, Crowther R, Levin W, Foley LH, Wang P, Palermo R (2001). X-ray structure of a novel matrix metalloproteinase simulations; GLTN for checking the research data, molecular inhibitor complexed to stromelysin. Protein Sci 10 (5):923-926. dynamics simulations analysis; and PTVN for preparing, doi:10.1110/ps.48401 editing and proofreading manuscript. 13. Tochowicz A, Maskos K, Huber R, Oltenfreiter R, Dive V, Yiotakis A, Zanda M, Pourmotabbed T, Bode W, Goettig P (2007). Crystal structures of MMP-9 complexes with five inhibitors: contribution of the ORCID ID flexible Arg424 side-chain to selectivity. Journal of molecular biology 371 (4):989-1006. doi:10.1016/j.jmb.2007.05.068 Phuong Thuy Viet Nguyen 14. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell Giang Le Tra Nguyen DS, Olson AJ (2009). AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. Journal of computational REFERENCES chemistry 30 (16):2785-2791. doi:10.1002/jcc.21256 15. Systemes D (2015). BIOVIA, Discovery Studio Modelling 1. Tobin DJ (2017). Introduction to skin aging. Journal of Tissue Viability Environment. Release 4.5 edn., Dassault Systemes: San Diego, CA. 26 (1):37-46. doi: 16. Hu X, Shelver WH (2003). Docking studies of matrix metalloproteinase 2. Cui N, Hu M, Khalil RA (2017). Biochemical and Biological Attributes inhibitors: zinc parameter optimization to improve the binding free of Matrix Metalloproteinases. Prog Mol Biol Transl Sci 147:1-73. energy prediction. Journal of molecular graphics & modelling 22 doi:10.1016/bs.pmbts.2017.02.005 (2):115-126. doi:10.1016/s1093-3263(03)00153-0 3. Barrantes E, Guinea M (2003). Inhibition of collagenase and 17. Paudel P, Seong SH, Wagle A, Min BS, Jung HA, Choi JS (2020). metalloproteinases by aloins and aloe gel. Life sciences 72 (7):843-850. Antioxidant and anti-browning property of 2-arylbenzofuran derivatives doi:10.1016/s0024-3205(02)02308-1 from Morus alba Linn root bark. Food Chemistry 309:125739. 4. Oh HI, Shim JS, Gwon SH, Kwon HJ, Hwang JK (2009). The effect of doi: xanthorrhizol on the expression of matrix metalloproteinase-1 and type-