|Year : 2019 | Volume
| Issue : 1 | Page : 17-24
Molecular docking study for evaluation of neuroprotective potential of sericin against cerebral stroke and exploring its biomaterial properties
Khushboo Maurya1, Anand Kumar Pandey2
1 Department of Pharmaceutical Sciences and Technology, Institute of Chemical Engineering, Mumbai, Maharashtra; Department of Biotechnology Engineering, Institute of Engineering and Technology, Bundelkhand University, Jhansi, Uttar Pradesh, India
2 Department of Biotechnology Engineering, Institute of Engineering and Technology, Bundelkhand University, Jhansi, Uttar Pradesh, India
|Date of Web Publication||24-Apr-2019|
Dr. Anand Kumar Pandey
Department of Biotechnology Engineering, Institute of Engineering and Technology, Bundelkhand University, Jhansi, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Cerebral stroke, the third leading cause of death worldwide results from the improper blood supply to the brain due to occlusions in the brain arteries. This leads to production of free radicals contributed by cyclo-oxygenases (COX), acid sensing ion channels (ASIC) and matrix metalloproteinases (MMPs) causing adverse conditions of inflammation, oxidative stress, and acidosis leading to neuronal death thereby proving these enzymes as potent targets. Sericin, a 38 amino acid long protein found in silk fiber is known for its anti-inflammatory and anti-oxidant property. Aim and Objectives: Inhibition of the above-mentioned targets by silk protein sericin to reduce the pathological features by structural interactions as well as reducing inflammation and oxidative stress due to the natural properties of compound. Methodology: In the present study we studied structural inhibition of effective targets by sericin through molecular docking analysis. Also, the semi crystalline nature of sericin was deduced through in silico XRD spectral analysis. Result: Structural inhibition through molecular docking analysis proved highly efficient inhibition. Also, the in silico XRD spectral analysis proved sericin to be a potential biomaterial for scaffold development. Conclusion: Sericin can not only act as an effective drug against cerebral ischemia but can also be used to develop scaffold to repair damaged brain.
Keywords: Acid-sensing ion channel, cerebral stroke, cyclooxygenase, matrix metalloproteinases, sericin
|How to cite this article:|
Maurya K, Pandey AK. Molecular docking study for evaluation of neuroprotective potential of sericin against cerebral stroke and exploring its biomaterial properties. Biomed Res J 2019;6:17-24
|How to cite this URL:|
Maurya K, Pandey AK. Molecular docking study for evaluation of neuroprotective potential of sericin against cerebral stroke and exploring its biomaterial properties. Biomed Res J [serial online] 2019 [cited 2020 Apr 6];6:17-24. Available from: http://www.brjnmims.org/text.asp?2019/6/1/17/257036
| Introduction|| |
Cerebral stroke victimizes about 6.2 million lives around the world and is ranked as the third leading cause of death. Cerebral stroke/ischemia occurs due to the occlusion and reperfusion in the cerebral artery resulting in improper blood supply to different parts of the brain. Such improper supply of blood results in scarcity of oxygen and glucose concentrations in concerned parts, directing it toward the anaerobic pathway of glycolytic cycle producing lactic acid and a high amount of H+ ion, and their reversal to the mitochondrial matrix, leading to the decrease in cytoplasmic pH resulting in acidosis and formation of free radicals. This condition becomes adverse with time and finally develops high oxidative stress. The mechanistic participators which play a vital role in the development of such adverse conditions are cyclooxygenase-2 (COX-2), acid-sensing ion channel (ASIC), and matrix metalloproteinases (MMPs) [Figure 1]. In the diseased state, these enzymes are overexpressed to intolerable levels and lead to inflammation and oxidative stress causing excessive neuronal death., Arachidonic acid metabolism by COX-2 and its overexpression due to AMPA receptor overactivation in ischemic condition is a leading cause of generation of reactive oxygen species (ROS) and glutamate-mediated excitotoxicity and thus finally results in neuronal insult. Past researches made it evident that at low pH in ischemic condition, ASIC-1a is overactivated and plays a leading role in intraneuronal Ca2+ accumulation resulting in neuronal loss mediated by acidosis. Many in vitro and in vivo researches have been done to enumerate the role of ASIC-1a in ischemic brain pathologies. MMPs are Zn-dependent proteinases involved in extracellular matrix remodeling. In ischemic conditions, these proteases play a vital role in blood–brain barrier (BBB) disruption by digestion of extracellular matrix and basal lamina in the capillaries. Such disruptions lead to conditions of edema and hemorrhage and hence neuronal death. In the present study, we have implicated a comparison between reported compounds and our considered natural compound sericin by molecular docking analysis for evaluating the structural inhibitory potential of sericin against the potent targets. Sericin, a glue-like protein, produced by Bombyx mori (silkworm) is composed 18 types of amino acids majorly serein, aspartic acid, and glycine. Its partial structure is made up of 38 amino acids, but many polymeric repeats containing structures are reported. It sticks the silk fibers together. It is a hydrophilic glycoprotein having different types depending on the solubility in warm water, that is, sericin A, B, and C, and based on molecular weight, these are sericin A (250 KDa), sericin M (400 KDa), and sericin P (150 KDa). Large amount of sericin obtained from textile industries is discarded as waste. For centuries, sericin is discarded as waste by textile industries which may require a high oxygen demand for its degradation. Reusing this waste may resolve the environmental problems and serve as a cheap and freely available material to be used for the sake of humans as well as may have high economical and social demands once used. Although it has not yet been employed for many applications, it is well known for its highly significant anti-inflammatory, antitumor, and antioxidant properties along with several other medicinal properties., Being biocompatible in nature and pH responsive, it can easily bind with other molecules and can be used for fabrication with small materials and it acts as a vehicle for drug delivery. It forms cross-links with gelatin, genipin, alginates, collagen, Polyvinly alcohol (PVA)-forming scaffolds, and hydrogels for suitable drug delivery. Furthermore, the chitosan/sericin microspheres are nontoxic as well as degradable in nature. Past researches have also reported that sericin has high cell regeneration, attachment, and proliferation potentials to be used in tissue regeneration. It also possesses high elasticity and porosity to be used as hydrogel in drug delivery systems. Looking at the underestimated potential and usage and its exceptionally good medicinal properties, we hypothesize that sericin has great potential to treat cerebral ischemia as it can combat oxidative stress and inflammation, the most common and dreaded pathologies of the disease. To provide strong additional support to our hypothesis along with considering the medicinal properties, we evaluated the structural inhibition potential of sericin against the potential targets of cerebral ischemia. Hence, the present study deals with the physiochemical analysis of sericin protein along with structural inhibition potential analysis by molecular docking. In addition, in silico analysis of crystalline structure of sericin was performed to reveal its potential to be used as a scaffold for repairing damaged brain parts.
|Figure 1: Pathways showing mechanism of action of cyclooxygenases, acid-sensing ion channel-1a, matrix metalloproteinase-2, and matrix metalloproteinase-9 initiating the neuronal death. ( a) Interrelation of free radicals, acid-sensing ion channel-1a, and matrix metalloproteinases, (b) cyclooxygenase activation and its effects, (c) abnormal opening of acid-sensing ion channel-1a and its effects|
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| Methodology|| |
Preparation of protein
The structures of COX-2, ASIC-1a, MMP-2, and MMP-9 (Protein Data Bank identification [PDB ID]: 5KIR, 4NYK, 1QIB, and 1L6J, respectively) were obtained from the Research Collaboratory for Structural Bioinformatics PDB. The sequence of sericin of B. mori (38 amino acids) was procured from the National Centre for Biotechnology Information. The three-dimensional (3D) structure of sericin was predicted by ModWeb (protein structure modeling software), and the template used was PDB ID 3ULT with 40% sequence identity. The structure validation was done by Verify3D, WHATCHECK, and ProSA-web.
Preparation of ligands
The smile notation of ibuprofen and quercetin was taken from the PubChem, and PDB files were generated using online smile translator.
Active sites of ASIC-1a, MMP-2, and MMP-9 (4nky, 1qib, and 1l6j, respectively) were detected using metaPocket 2.0. It is a meta-server developed at Technical University of Dresden and Zhejiang University jointly, for ID of ligand-binding sites on protein. It employs eight methods, i.e., LIGSITECS, PASS, Q-SiteFinder, Fpocket, SURFNET, GHECOM, ConCavity, and POCASA for evaluating the binding sites and improves the prediction success rate.
Molecular docking of COX-2 with quercetin and ibuprofen was done using SwissDock online server developed by the Molecular Modeling Group of the Swiss Institute of Bioinformatics (Switzerland).
Molecular docking of COX-2 with sericin was done using pyDock online server developed by the Barcelona Supercomputing Center (Spain), which analyzes protein–protein interactions based on Fast Fourier Transform (FTT)-based algorithm. The configuration, electrostatic interaction desolvation energy, van der Waals interaction, total energy, and rank were calculated of every docking configuration for evaluation and comparison. The highest-ranking configuration was reported.
Docking of 1qib and 1l6j with sericin was done using AutoDock Vina. It was developed by Dr. Oleg Trott in the Molecular Graphics Laboratory at The Scripps Research Institute. It uses iterated local search optimizer. Blind docking was carried out using grid sizes 82, 110, and 104 along the X, Y, and Z axes with spacing 0.708 angstrom. The grid center was set to 36.695, 38.779, and 33.924 nm for x, y, and z coordinates, respectively.
Chemical property calculations
A ChemAxon online tool product called Chemicalize was used for the evaluation of chemical properties of sericin including elemental analysis, names and identifiers, pKa, and log P/log D.
Crystal maker software suite
This suite serves for number of software such as CrystalMaker, CrystalDiffract, SingleCrystal, and CrystalViewer. CrystalMaker software was used for advanced chemical and material structure modeling. It transcends crystallographic tools and energy-modeling tools to design new structure and predict their vibrational properties and much more. CrystalDiffract stimulates multiphase X-ray and neutron powder diffraction analysis/simulation patterns using featuring real-time parameter control, etc., Using these two software, the crystal structure of sericin and its X-ray diffraction (XRD) spectra were generated.
| Results and Discussion|| |
Selection of unknown natural compound against ischemic stroke
Sericin is a glue-like protein obtained from B. mori that holds the silk fibers (fibroins) together forming the cocoon. It constitutes about 25%–30% of silk protein, composed of 38 amino acids (TGSSSNTDSNSNSVGSSTSGGSS TYGYSSNSRDGS-36 amino acid sequence was selected for this study based on BLAST analysis performed for sericin structure modeling through ModWeb tool) containing 18 different types of it with maximum concentration of serine (33.4%) and aspartic acid (16.7%)., Furthermore, the physiochemical properties of sericin can be seen in [Table 1]. Validation of structure was done using Verify3D, WHATCHECK, and ProSA-web web server. All the validation tool results proved the validity of structure. The modeled structure passed in Verify3D as more that 80% residues scored ≥0.2 in 3D-1D profile. In WHATCHECK, the modeled structure passed 31 tests out of 41, which proves the overall accuracy of structure. ProSA-web gave the Z score of −1.02 proving similarity with Nuclear Magnetic Resonance (NMR) structure. Furthermore, majority of residues were found to have low energy, hence proving the stability of structure.
|Table 1: Comparison between different molecular properties of ibuprofen, quercetin, and sericin|
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Sericin has many other medically relevant properties such as antioxidant, antitumor, inhibiting cell apoptosis, anticoagulase, antibacterial, moisturizing, wound healing property, biocompatibility, chemoprotector, ultraviolet resistant, antityrosinase, low digestibility which collectively promotes its application in tissue engineering, drug delivery, as well as food and cosmetic industries. It is also reported that sericin shows negligible allergenicity and low immunogenicity, which makes it possible to be easily used as a medicine. It can be used as both media supplement and cryoprotectant in cell culture. Sericin can also be given in diet as it shows resistance to proteases and increases in Zn, Fe, Mg, and Ca concentrations. It can also be given as prebiotics as it promotes colon health. It also enhances the attachment of fibroblast and its proliferation playing an important role in wound healing. The presence of certain polyphenols such as quercetin and kaempferol glycosides in cocoon provides sericin the antioxidant property and anti-inflammatory effect, thereby reducing apoptosis. It reduces the level of tumor necrosis factor-alpha, suppresses free radicals and ROS, and most importantly reduces the expression of COX-2 by chelation with Cu and Fe and hence reduces acidosis which causes oxidative stress. It also reduces proto-oncogenes (c-fos, c-myc, and COX-2) expression and lowers cholesterol, triglycerides, free fatty acids, etc., and thus is anticarcinogenic and antihyperlipidemic, respectively.,. Sulfated sericin shows anticoagulant property and serves as a substitute for heparin. Furthermore, it is evident that sericin/insulin bioconjugates with glutaraldehyde cross-linking are pharmacologically 4 times more active with 2.7 times high half-life than bovine serum albumin/insulin conjugates and intact insulin proving it to be a better substitute.
Evaluation of sericin as therapeutic for cerebral ischemia
Cyclooxygenase-2 as targets
Molecular docking was performed for analysis of structural inhibition of COX-2 by the two reported compounds, i.e., ibuprofen and quercetin and our considered natural silk protein sericin. Our results proved that sericin is highly potent and binds to COX-2 with a very high negative binding energy (compared to the reported ones) of − 30.037 Kcal/mol [Figure 2], [Figure 3], [Figure 4], [Table 2] and [Table 3]. Although sericin is hydrophilic and could not cross BBB, in ischemic conditions, the BBB is ruptured and hence sericin can easily reach and bind its target.
|Table 2: Binding energy value (ΔG, Kcal/mol) of different inhibitors against cyclooxygenase-2 (Kcal/mol)|
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|Table 3: Parameters of different energies while binding of sericin with cyclooxygenase-2 (Kcal/mol)|
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The secondary structure of COX is a tetramer, each containing three domains. The first domain is the epidermal growth factor domain (33–72 residues) containing two antiparallel beta-sheets responsible for signal transduction, the second is membrane domain (73–116 residues) containing four alpha helices for membrane interaction through amphipathic helical segment, and the third is helix D (117–583 residues) extending the catalytic domain 12 through hydrophobic tunnel containing active site. This catalytic domain consists of helix bundle to facilitate heme-binding sites, peroxidase active sites, and COX active sites.,, In our results, we found that sericin binds at the catalytic site of COX-2 and have great potential to inhibit oxidation of arachidonic acid by COX and thus production of prostaglandins, thereby curing inflammation and providing a high potential treatment for cerebral ischemia.
Acid-sensing ion channel-1a as target
The present study showed that molecular docking of sericin with ASIC-1a resulted in binding energy of −5.5 Kcal/mol [Figure 5]. Binding site prediction of ASIC-1a (4nyk) using metaPocket 2.0 shows the most probable active site [Figure 6]. The green dot signifies the metaPocket, and the area surrounding the metaPocket shows the cavity wall made of significant residues.
|Figure 6: (a) Ligand-binding sites of acid-sensing ion channel-1a (green dot: metaPocket and white surface: cavity wall made of residues). (b) Zoomed view of metaPocket|
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Pandey et al. reported the same site as an active site for binding of quercetin, and our compound sericin was also found to attach at the same site proving its efficiency as a drug for the purpose.
Matrix metalloproteinase-2 and matrix metalloproteinase-9 as targets
Docking studies show that sericin inhibits MMP-2 and MMP-9 with an efficient binding energy of − 5.4 and − 6.5 Kcal/mol, respectively [Figure 7]. Pandey et al. reported that MMP-9 has a pocket-like S1' cavity with a floorboard and MMP-2 has a channel-like S1' cavity. The interaction with Leu 164 and Ala 165 is a very important characteristic of broad range of MMP-2 inhibitors. In addition, H-bonding with Ala 217 and Ala 220 provides high binding affinity with MMP-2. Active site of MMP-9 comprises catalytic Zn ion and is separated into large “upper” and small “lower” subdomain.
|Figure 7: Sericin docked with ( a) matrix metalloproteinase-2 and (b) matrix metalloproteinase-9|
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metaPocket 2.0 results showed the probable active sites in the above-mentioned regions for both MMP-2 and MMP-9 [Figure 8]. The sites for previously reported compound quercetin also resembled the ones predicted in our analysis. Sericin docking result showed that the interacting sites in both MMP-2 and MMP-9 were same as that of quercetin proving the high potential of our compound with comparable binding energies. The combined results of docking analysis and the previously reported molecules are present in [Table 4] and the target molecule's pathways effected by sericin inhibition are presented in [Figure 9].
|Figure 8: (a) Ligand-binding sites of matrix metalloproteinase-2 (green dot: metaPocket and white surface: cavity wall made of residues). (b) Zoomed view of matrix metalloproteinase-2 metaPocket. (c) Ligand-binding sites of matrix metalloproteinase-9 (green dot: metaPocket and white surface: cavity wall made of residues). (d) Zoomed view of matrix metalloproteinase-9 metaPocket|
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|Table 4: Comparative binding energies of cyclooxygenase2, acidsensing ion channel1a, matrix metalloproteinase2, and matrix metalloproteinase9 with ibuprofen, quercetin, and sericin (Kcal/mol)|
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|Figure 9: Schematic representation showing inhibitory effects of sericin against considered targets|
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In silico evaluation and analysis of crystal structure of sericin and its comparative study
The in silico evaluation and analysis were done to predict the crystal structure of sericin. The crystal structure was generated using CrystalMaker software [Figure 10]d. The following three-dimensional structural arrangement constitutes the primitive unit cell with all the axial angles, i.e., α, β, and γ = 90°. In silico XRD graph was also generated between intensity and angular position using CrystalDiffract to analyze the crystalline nature of sericin.
|Figure 10: X-ray diffraction spectra of different natural biomaterials: (a) collagen, (b) fibrinogen, (c) sericin, (d) crystal structure of sericin|
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As per the previous studies, the biomaterials to be used for scaffold should have high porosity. Amorphous solids are more porous than crystalline solids. Glass which is often used for bone scaffold is an amorphous solid but somewhere lacks in mechanical strength which is a necessary feature for scaffold preparation. Hence, there is a need of materials which have high porosity (as small interconnected pores support cell adhesion with a better exchange of gases and physiological fluids inside the scaffold) along with mechanical strength which are the characteristics of semi-crystalline solids., The low level of peaks in the XRD graphs describes a reduction in crystalline nature, also signifying the increase in porosity. In our analysis, we found that the XRD peaks of sericin [Figure 10]c are lowly arranged except some peaks at the initials, depicting that sericin has both the features, i.e., crystalline and noncrystalline features. Hence, sericin can be considered to have semi-crystalline nature, which is essential to fulfill the requirements of both porosity and mechanical strength needed for an efficient scaffold development. The XRD graphs of other materials (such as collagen and fibrinogen), used in scaffold development, have comparable peak fashion to that of sericin providing strong support to our in silico prediction [Figure 10]a and [Figure 10]b.,, Thus, on the basis of above mentioned, it can be proposed that sericin has the potential to be used as an active natural biomaterial for scaffold designing due to its semi-crystalline nature and could also be conjugated and cross-linked with other materials. Furthermore, the other properties of sericin such as biocompatibility, low digestibility, promoting cell differentiation, and being a natural biomolecule (glycoprotein) support the idea of using it as a biomaterial for scaffold preparation.
| Conclusion|| |
The present study suggests that sericin has a high binding potential for COX-2, ASIC-1a, MMP-2, and MMP-9 with extremely high binding energy and proves its neuroprotective potential effects against cerebral ischemia. Apart from this, it also has high antioxidant property and is naturally extracted so poses no or less side effects. Hence, application of sericin through dietary supplements as well as intravenously through drug delivery systems can have great therapeutic potential to treat cerebral stroke and associated oxidative stress-mediated damage. The effective scaffold biomaterial property of sericin proves its efficient use in the field of tissue engineering and regenerative medicines for organ development.
The outstanding properties of sericin for the treatment of cerebral ischemia and the structural inhibition of the effective targets of the disease make it an efficient option as drug for further research. The multi varied properties of sericin which can be used in tissue engineering, regenerative medicines and, drug delivery open wide research doors for future. In concerns to scaffold designing, conjugation with other material to enhance its properties for the purpose can lead to outstanding results. Furthermore, its excellent adhesive property opens a new path to be used in the development of BioGlue which would be helpful to heal wounds and seal incisions after surgery.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
[Table 1], [Table 2], [Table 3], [Table 4]