In Silico Docking Studies of Ag Nanoparticles and Its Derivatives Against NS5B Protein of Hepatitis-C Virus

Nanotechnology refers to the synthesis of nanomaterials (1-100nm) and their applications. Nanoscience can deal with individual atoms and molecules. In recent times, an agreement has started to rise about the informatics foundation expected to accumulate, curate, and share data among every one of the elds in nanotechnology. Nanoinformatics is fullling this demand. It is the science about guring out which data is important to the nanoscale science and after that creating and implementing viable systems for gathering, approving, sharing, analyzing, modeling, and applying that information. Nanoinformatics is essential for ecient production and relative description of nanomaterials. The present study focused on the prediction of interactions of three ligands i.e. silver nanoparticles, tyrosine capped silver nanoparticles, and silver oxide nanoparticles with NS5B protein of HCV. ΑutoDock 4, Discovery Studio, ChemDraw Ultra, OpenBabel, and Chimera software were used. Computational docking helps to evaluate conformations of small ligands attached to macromolecular proteins. NS5B plays a crucial role in HCV replication. It weighs about 66-KDa. It is an RNA-dependent RNA membrane-associated polymerase. The results were obtained from AutoDock 4 and visualized in Discovery Studio and Chimera. Silver nanoparticles showed interactions with LYS81, LYS172, LYS173, TYR176, and ASP177. Tyrosine capped silver nanoparticles formed bonds with SER218, ASP220, GLU357, and LEU362 in the palm region. Silver oxide nanoparticles interacted with LEU260, TYR261, ARG280, and ALA281 in the nger domain. All these three ligands showed promising results to inhibit the NS5B enzyme halting HCV replication.


Introduction
Nanotechnology Nanotechnology refers to the synthesis of nanomaterials (1-100 nm) and their application. Indeed, even well before the beginning of the "nano-era", individuals were subconsciously running over different nanosized objects and the related nanolevel procedures and utilizing them practically. For instance, since the time before BC, people utilize characteristic fabrics such as cotton, silk, wool, ax. They could produce them and process them into items. These textures were having pores of about 1-20 nm. Owing to the presence of nanopores, they were having some good properties like well sweat absorbing, rapid swelling, and drying making them suitable for wearing.
In 1959, rst of all, Richard P. Feynman gave the concept of nanotechnology in his renowned address "There's Plenty of Room at the Bottom." This address is considered as the beginning of the nanotechnology prototype. In 1974, N.Taniguchi was the rst person who introduced the word "Nanotechnology" at the international conference on industrial production in Tοkyο. Metal nanoparticles like gold, silver, and platinum have achieved extensive consideration in few years because of their crucial and mechanical intrigue. Currently, chemical, physical, and most preferably biological synthesis of nanoparticles are being carried out. Nanotechnology plays important role in agriculture, electronics, textiles, pharmaceutics, etc. [1] because of the remarkable properties of nanomaterials. Small particles of different substances had properties different from those of similar substances with the bigger molecule. The characteristics of small particles resemble atomic properties beneath 1 nm. On the other hand, their properties resemble that of material at the macro level when greater than 100 nm [2]. From 1nm to 100 nm, a particle exhibits new and different behavior due to quantum effects. In this era of the pandemic, it is a nanotechnology that is creating hope to defeat COVID-19 because a vaccine against SARS-CoV-2 based on mRNA was designed and delivered through a nano-liposomal entity. This vaccine is in clinical trials right now [3].

Metal Nanoparticles
Metal nanοparticles have been used in different applications. They are gaining interest in the scienti c and commercial elds [4]. They can impressively change biological and physicochemical characteristics as they have high electrical prοperties, increased tolerance to mechanical and thermal pressure, high surface area, and high οptical and magnetic prοperties [5,6]. These unique characteristics have enabled nanοmaterials tο be used in different elds including electrical, magnetic, οptical, and electrοnic devices.
Because οf these unique characteristics, nanοmaterials can be used in different products including electrical, magnetic, οptical, and electrοnic devices. Some nanoparticles are modi ed to increase their e ciency and usage. Silver oxide, titanium oxide, and copper oxide are few prominent examples of engineered nanoparticles. Some nanomaterials are also used in different production industries, for example, the production of sunscreens and stain-repellent clothes. Investigations and diagnosis are also facilitated by the use of simple and engineered nanomaterials in medical equipment and procedures such as diagnοstic kits, imaging, magnetic resοnance imaging (MRI), and drug delivery [7]. The biomedical industry is also blessed with metal nanoparticles. In the light of nanotechnology, the eld of nanomedicine has been a point of intense consideration for e cient and quick diagnosis and creating various methods of therapies utilizing nanoparticles in various diagnostic gadgets [8]. Ag, Pt, and Au nanoparticles are considered noble nanoparticles [6]. These nanoparticles exhibited nontoxic positive effects in biological systems revealing a new dimension of exploration in biological research [9]. Some engineered nanoparticles, such as titanium dioxide (TiO 2 ), zinc oxide (ZnO), ferrous oxide (FeO), cupric oxide (CuO), silver oxide (Ag 2 O), aluminium oxide (Al 2 O 3 ), also have antimicrobial properties and perform noteworthy activities in numerous medical applications. TiO 2, for instance, is used to inhibit the spread of various diseases [10]. In addition, aluminum oxide nanoparticles have many applications and demonstrated antimicrobial characteristics [11]. The zinc-doped titania nanoparticles have uncovered improved pro-angiogenic properties, which may be helpful in various applications [12]. Silver nanoparticles have tremendous antimicrobial activity. While drug delivery mechanism is made e cient by using gold nanoparticles to cure many illnesses such as cancer [13].

Silver Nanoparticles
Because of their exceptional properties, silver nanoparticles have been utilized for many applications counting as anti-viral and anti-bacterial agents. They are being used in healthcare products, beauty care products, the food industry, pharmaceutical industries, and medical and electronic devices [14]. The biοlοgical characteristics of silver nanοparticles depend on various parameters [15]. In systematic and local administratiοn, biοavailability of therapeutic agents get improved because of the physiοchemical properties of the nanοparticles [16]. Silver nanοparticles produce by using the berry extract of Sea Buckthοr, display a broad range of antiοxidant, anti-in ammatory and anticancer activities [17]. AgNPs are proven to be safe antibacterial and antibiο lm compounds against MDR K. pneumοnia [18]. Silver nanοparticles produced by Sphingοbium sp. MAH-11 may act as an intense antimicrobial agent in many treatments [19]. Hence, the synthesis of silver nanoparticles in a controlled manner is useful in several biomedical applications [20].
Currently, medication and immunization advancement for the evacuation of different viral ailments are under critical consideration, various viral strains have been developed that are no more sensitive to drugs and vaccines. So it is imperative to present the multidisciplinary approaches with the established epidemiology, alongside the clinical phases to present a new drug or vaccine which possesses great effectiveness against the resistant strain. Nanotechnology has revolutionized the eld of medicine.
Nanoparticles, especially silver, have antiviral activities against the many viruses that are ruining lives worldwide.

Nanoinformatics
Biological data are being produced at an extraordinary rate [21]. Because of this exponential growth of information, computers have turned out to be irreplaceable for biological research. Such an approach is perfect due to the simplicity with which computers can deal with a huge amount of information [22]. The utilization of computational systems to comprehend and sort out the data related to biological macromolecules is known as bioinformatics. In recent times, an agreement has started to rise about the informatics foundation expected to accumulate, curate, and share data among every one of the partners in nanotechnology [23]. The inconstancy of nanomaterials made risk assessment unrealistic. Easily accessible data and arti cial intelligence approaches are necessary to guarantee consumer wellbeing [24]. A more effective way is required by utilizing nanoinformatics for e cient and broad sharing of data related to nanotechnology. Nanoinformatics is depicted as "the science regarding gure out which data is important to the nanoscale science and after that creating and implementing systems for gathering, approving, sharing, analyzing, modeling and applying that information".

Molecular Docking
Nanoinformatics is an emerging science. It contains databases and tools. Some of them are Nanomaterial Biological Interactions Knowledgebase, InterNano, Nanoparticle Information Library, etc. Nowadays, as nanomedicines are being used and found to have high e ciency, molecular docking of nanomaterials is in trend. In drug discovery, docking is a critical computational technique predicting protein-ligand interactions. The two fundamental characteristics of docking programs are docking precision and scoring reliability [25]. Docking accuracy demonstrates how similar the predicted ligand to the experimental data, whereas scoring reliability positions ligands because of their a nities. Docking accuracy evaluates searching algorithm and scoring reliability assesses scoring functions. In the docking program, the numerous searching algorithms work differently as for randomness, speed, and the area covered. Many searching algorithms show good performance when used against the known structure. Presently, numerous sorts of docking programs are easily accessible, among which, ΑutoDock is frequently used and openly accessible [26]. As protein-nanoparticle interactions are not easy to examine utilizing experimental techniques, molecular docking tools facilitate to ease this di culty.

NS5B Protein
Hepatitis C virus is included in the family Flaviviridae. It has a +RNA single strand (Choo 1989). The HCV genome contains roughly 9,600 nucleotides, which encode for 3,000 amino acid residues for polyprotein precursor. About 170 million people are carriers of HCV around the world. A signi cant number of these people are awaited to be suffered from critical HCV-related liver diseases. NS5B stands for nonstructural 5B protein present in HCV. It weighs about 66-KDa. It is an RNA-dependent RNA membrane-associated polymerase [27]. It takes part in RNA replication, however, the exact molecular mechanism is not completely known yet.
The RNA replication is comprised of two phases. In the rst phase, the formation of a new RNA strand starts at the 3' end of the RNA template. This initiation phase does not need a primer to start, therefore can be called a primer independent or de novo mechanism. Hydroxyl group at 3' position of rst NTP makes a bond with new coming NTPs. In the second phase, elongation occurs by adding more complementary NTPs.
NS5B has three structural domains denoted as ngers (residues 1 to 187 and 228 to 286), palm (residues 188 to 227 and 287 to 370), and thumb (residues 371 to 563). Its catalytic site contains residues from 214 to 332. At the enzymatic molecular surface, there is a site in a pocket speci c for the binding of rGTP molecule. This speci c site is at a distance of 30 Å from the catalytic site. It is situated at the junction of ngers and thumb domains regulating the enzymatic activity allosterically.
The repetitive cases of Hepatitis C virus (HCV) infection causes more than 71 million people to face chronic stage that results in different liver diseases [28]. A deeper understanding of HCV revealed vital proteins that are important for HCV survival and enabled the scientists to target them to make HCV therapy more e cient [28,29]. Antiviral drugs are designed to directly act on 3 important HCV functional proteins i.e. NS5B polymerase, NS5A, and NS3 [30]. The present HCV therapy using ledipasvir, ombitasvir, and sofosbuvir has some adverse effects like anemia, rash, bilirubin, nausea, pruritus, and photosensitivity [31]. The need for reduction in these adverse effects and increment in liver diseases demand improved treatment. This article focuses on the docking of different derivatives of silver nanoparticles to look into alternative, safe, and highly e cient HCV therapy methods.

Retrieval of NS5B 3D Structure from Protein Data Bank
Protein Data Bank (PDB) was built up as the rst freely available repository for biological molecules in 1971. It was a single worldwide library for 3-dimensional structures of bio-molecules and their complexes with other small molecules. Presently, the PDB archive contains ~167518 entries (August 2020). The PDB repository contains data obtained through three techniques: X-ray crystallography, nuclear magnetic resonance spectroscopy (NMR), and electron microscopy [32]. 3D structure of HCV encoded nonstructural 5B (NS5B) protein with 2HWH identity number was taken from PDB website.

Deleting Water Molecules
Water molecules present in NS5B structure were deleted using ΑutoDock tools because many of the water molecules present in protein structure are either loosely bound and easily displaced by a ligand or not necessarily quite in the positions they appear to be-tting water molecules to the residual electron density after the protein structure is tted, is an inexact science. In most cases, water molecules are not involved in the binding. That's why they are preferably removed to ease computations and clear water molecules present in a catalytic pocket so that the pose searching process would not be disturbed. In docking, there is a search for molecules that can create multiple favorable contacts to the protein, water molecules might confound this procedure. As a result, wrong conformation pose is obtained as the ligand forms more solvent assisted salt bridge interactions.

Adding Hydrogen Atoms
Protein-ligand or protein-protein complexes are virtually observed in molecular docking simulation. Macromolecules are present in a charged form with no atom missing in the human/animal body. So, it is necessary and sensible to add charges and missing atoms (hydrogen and in some cases non-hydrogen) to protein before proceeding with a docking experiment.
Most macromolecular structure data do not contain hydrogen atoms in their corresponding PDB les and docking software requires the hydrogen atoms to be in place to compute algorithmic calculations. So, the addition of hydrogen atoms is necessary for docking. The polar hydrogen atoms allow the establishment of hydrogen bonds that may be present between the macromolecule and the ligands tested. Hence, missing hydrogen atoms were added to the NS5B protein.

Compute Gasteiger Charge
To get important and useful outcomes from any electrostatic calculations, designating suitable atomic partial charges to ligand and macromolecule is necessary. Marsili-Gasteiger partial charges are appointed employing a two-phase algorithm. First, each atom in a molecule is designated with seed charges. Then, some amount of these initial charges are transferred from one atom to the other bonded atom. The direction of movement of partial charges depends on the electronegativity difference between two bonded atoms. With each cycle of repeating algorithm, attenuation of charges occurs. Gasteiger partial charges of 17.0062 were added to the NS5B protein in the ΑutoDock tool.

Energy Minimization
Computational chemistry depicts energy minimization as the process of searching a pattern in space where atoms are gathered, the total inter-atomic force on every atom is near to zero and the potential energy surface (PES) is a static point. This searching mechanism during the energy minimization process is based on some computational model of chemical bonding. Energy minimizatiοn is essentially abοut "settling" the mοdel intο a relatively energetically favοrable state. Prοtein structures οften have errοrs οf variοus magnitude such as atοms partially οverlapping, side chains in the wrοng pοsitiοns, etc. Energy minimizatiοn lοοks fοr the pathway that gives the mοst reductiοn in the οverall energy οf the system, relaxing bοnd lengths, angles, nοn-bοnded interactiοns, etc. intο mοre favοrable states.

Retrieval of Ligand 3D Structures
Silver nanoparticles, tyrosine capped silver nanoparticles and silver oxide nanoparticles were used as ligands in molecular docking. The structure of silver, tyrosine capped silver, and silver oxide nanoparticles were drawn in Chemdraw Ultra 12.0 software. Chemdraw Ultra software is used to draw a nearly unlimited variety of biological and chemical drawings.

Preparing Grid Parameter File (GPF)
The Grid box center was assigned on active site cavity in NS5B protein with 1 angstrom spacing of each grid point. While x,y, and z coordinates for grid points were 1.874, -0.603, and 27.509 respectively. In this way, the whole protein molecule was covered within a grid box to allow free rotation of the ligand molecule within the protein.
All the grid-related information was saved as a grid parameter le (gpf). The gpf determines the search space in the receptor.

Running Autogrid4
The autogrid was run by using a grid parameter le (gpf). Autogrid4 gets information about the receptor around which potential is to be computed, map types to be gured out, and the extent and location of those maps from the grid parameter le [33]. The probe atom is employed in 3D space at regular points to pre-calculate the energy in the receptor. These pre-calculated energies get stored as grid maps. Each type of atom contains its grid map. Besides, electrostatic and desolvation maps are also generated. In this way, in ligand molecule, every type of atom is subjected to a rapid evaluation of energy to precalculate its a nity potentials [34].
Preparing Docking Parameter File (DPF) In docking parameters, a genetic algorithm with default settings was selected to be used during the docking process. The docking parameter le reveals ΑutoDock about the utilization of map les, movement of the ligand molecule, center, torsions, beginning of the ligand, movement of the exible residues in the receptor if modeling of the side chain motion is required, type of algorithm to employ and its iteration. It contains le extension as ".dpf" [33].

Running ΑutoDock4
ΑutoDock runs search algorithms using grid maps to determine ligand-protein binding at each point to nd the suitable conformations. Subsequently, numerous docked conformations are acquired. ΑutoDock needs grid maps for each type of ligand atom evaluated by AutoGrid, a ligand PDBQT le, and a docking parameter le which determines the parameters for the docking [34].

Result Analysis
The results obtained from ΑutoDock were observed and analyzed in Chimera and Discovery Studio Visualizer version 17.2.0.16349.

Results & Discussion
NS5B contains three domains named palm, ngers, and thumb ( gure 1). Palm includes the residues from 188 to 227 and 287 to 370, ngers residues from 1 to 187 and 228 to 286, and thumb contains residues from 371 to 563 [35].

Ag NP-NS5B Protein Interaction
Silver nanoparticles interacted with the nger domain of NS5B protein. Five amino acid residues i.e LYS81, LYS172, LYS173, TYR176, and ASP177 showed interaction with Ag nanoparticles. LYS81 is involved in metallic bonding with Ag nanoparticles. LYS81 is one of the amino acids to which RNA binds. Hence Ag nanoparticle can cause hindrance in the attachment of RNA to LYS81. Amino acid residues from 168-183 in the nger domain also help to bind template RNA [35]. Interaction of Ag nanoparticle with LYS172, MET173, TYR176, and ASP177 can inhibit the template RNA to bind with the nger domain.

Tyrosine Capped Ag NP-NS5B Protein Interaction
Tyrosine capped silver nanoparticles were found to have hydrogen bonding with SER218, ASP220, GLU357, and LEU362. The ligand molecule is having three hydrogen bonds (green color) with SER218 with bond lengths of 2.14 Å, 2.36 Å, and 2.51 Å. While ASP220 and GLU357 are interacting with the ligand through a single hydrogen bond with 2.23 Å and 1.80 Å distances respectively. Furthermore, there is a metallic interaction of 3.06 Å with LEU362.
Tyrosine capped silver nanoparticle interacted with the palm region of NS5B protein. The site where the ligand attached with four amino acid residues is a catalytic site. ASP220 and SER218 are part of motif A and GLU357 and LEU362 are included in the motif E region [35]. ASP220 plays a role to coordinate magnesium ions to assist in nucleotide addition during the RNA elongation process. While motif E helps to maintain relative positioning of thumb and palm domains [36]. The ligand molecule can inhibit the ASP220 to bind with magnesium ion to stop the addition of new nucleotides during RNA synthesis. It may also distort the ability of motif A to select the type of nucleic acid which needs to be gone under the polymerization process. Furthermore, it may deform the motif E and hence the spatial arrangement of the thumb and palm domain can be disturbed. As the ligand molecule is present within the catalytic pocket, it may cause hindrance for the RNA molecule that is being synthesized. In this way tyrosine capped silver nanoparticles can act as an inhibitor for NS5B protein to stop the replication of HCV.

Silver Oxide NP-NS5B Protein Interaction
The oxygen atom of the ligand molecule showed three hydrogen bonds with TYR261, ARG280, and ALA281 with distances of 3.15 Å, 3.64 Å, and 1.98 Å respectively. Additionally, one silver atom of the ligand molecule was involved in charge repulsion with ARG280 with 2.33 Å distance and the other silver atom is making metallic interaction with LEU260 with 3.33 Å distance.
NS5B encircles the active site due to the extensive interaction between nger and thumb domains and that's why is not allowed to change their spatial arrangement freely of each other. Because ngers and thumb are associated through two exible nger loops ( 1 and 2), a conformational disturbance in one domain causes the change in the other domain [37]. Subdomains of ngers and thumb determine the shape of the binding channel of the enzyme which binds the nucleic acid [38,39]. At the front and back of the enzyme, ngertips are involved in the formation of template and NTP channels respectively [40]. NS5B protein in complex with GTP has been crystallized. The crystallized structure of NS5b complexed with GTP molecule shows that GTP not only binds to the active site but also to the thumb domain near to the delta-1 loop of ngertip which lies between the thumb and nger domains. Even though this GTP binding site is situated 30 Å far from the active site, it has a regulatory role in dynamic interactions of subdomains of ngers and thumb [38].
Silver oxide nanoparticle Binds with four amino acid residues in the nger domain. It can induce a structural change in the nger region distorting the spatial arrangement of the domain. As the nger domain is linked with the thumb domain so a change can occur in the positioning of the thumb domain as well. Disturbed positioning and structure of thumb and nger domains result in the deforming of the template and nucleic acid binding channel. The attached ligand can also act as a blocker for newly coming rNTPs. Furthermore, the regulatory mechanism of NS5B through the GTP molecule can be breached and damaged by the ligand through dis guring the GTP binding site resulting in allosteric inhibition.
Binding energy is emitted as a result of ligand-target binding causing a decrease in overall complex potential energy. The release in binding energy facilitates the ligand to transform its conformation from its maximum energy state to bound conformation having minimum energy. Hence, the greater the released binding energy, the greater will be the binding a nity of the ligand to the protein. The ligandbinding process will be spontaneous if the binding energy is in a negative value. While the binding process will be nonspontaneous and require energy if the binding energy is in positive value.
The binding energy of silver nanoparticles is -0.17 kcal/mol indicating its low a nity with the protein.
Hydrogen bonding dramatically affects the binding energies. Tyrosine capped Ag NP comparatively showed the highest binding energy i.e. -5.29 Kcal/mol due to the presence of multiple atoms in it forming multiple H-bonds, hence increasing binding energy.

Conclusion
All the results obtained from computational docking of the four ligands individually to NS5B protein show promising effects to inhibit the protein activity. NS5B protein is an RNA polymerase and plays a key role in HCV replication. HCV becomes extinct without this protein molecule. There are three domains of the protein i.e. palm, ngers, and thumb which collectively form the active site. All the ligands were docked near to active site inhibiting the RNA replication process. The most effective ligand was tyrosine capped silver nanoparticles Its relative highest binding energy i.e. -5.29 kcal/mol showed its intensive binding with the protein molecule causing more damage to the integral residues forming the active site.
Declarations Funding Not applicable.

Con icts of interest
There is no con ict of interest among the authors.

Ethics approval
Ethical Review Committee permission was undertaken.

Consent to participate
Consent to participate was taken from the research supervisor.

Consent for publication
Before submission of the study, consent was taken from the research supervisor.
Availability of data and material Data gathered was original, transparent and reliable.