Natural Products as Anti-COVID-19 Agents : An In Silico Study 1

DOI: 10.2174/2666796701999201116124851 Abstract: Background: The coronavirus disease 2019 (COVID-19) is a life-threatening viral infection caused by a positive-strand RNA virus belonging to the Coronaviridae family called severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2). This virus has infected millions of peoples and caused hundreds of thousands of deaths around the world. Unfortunately, to date, there is no specific cure for SARS-CoV-2 infection, although researchers are working tirelessly to come up with a drug against this virus. Recently, the main viral protease has been discovered and is regarded as an appropriate target for antiviral agents in the search for the treatment of SARS-CoV-2 infection due to its role in polyproteins processing coronavirus replication.


INTRODUCTION
Since December 2019, an outbreak of pneumonia of initially unknown causes was detected in Wuhan (Hubei, China), and was quickly determined to be caused by a novel beta-coronavirus, named novel coronavirus 2019 (also called the severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2)). This virus, belonging to the family Coronaviridae and the order Nidovirales, has led to a severe epidemic in China and more than 200 other countries, resulting in a worldwide concern. It is transmitted through direct contact with respiratory droplets of an infected person (generated through coughing and sneezing), fecal-oral, body fluid routes, or touching surfaces contaminated with the virus [1][2][3]. Coronaviruses (CoVs) are cross-species viruses containing an enveloped positive-stranded RNA genome (Length: 26-32 kb) like pleomorphic particles with crown-like spikes of glycoproteins projecting from their viral envelopes. These envelopes exhibit a corona or halo-like appearance. CoVs include four common cold human coronaviruses [229E (al-*Address correspondence to these authors at the Department of Pharmacy, Life Science Faculty, Bangabandhu Sheikh Mujibur Rahman Science and Technology University, Gopalganj 8100, Bangladesh; E-mail: dmt.islam@bsmrstu.edu.bd and Department of Chemistry, The University of Jordan, Amman 11942, Jordan; E-mail: mmubarak@ju.edu.jo pha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), and HKU1 (beta coronavirus)], and two other types [severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome coronavirus (MERS-CoV)], which cause severe infections [2,4,5].
In late December 2019, this unidentified disease was later named coronavirus disease 2019 (COVID-19), where 'CO' stands for corona, 'VI' for the virus, and 'D' for the disease. Patients with COVID-19 reveal insidious onset with fever, cough, and myalgia-with or without diarrhea or shortness of breath, or both [6]. As of Aug 4, 2020, the number of confirmed cases was 18,142,718 and 691,013 deaths around the world [7]. All types of human coronaviruses (hCoVs) encode an enzyme chymotrypsin-like protease (3CL pro ), which is also named the main protease (M pro ); thanks to playing a pivotal role in the proteolytic process during the virus multiplication [8]. Proteolytic processing (processing of replicase polyproteins) is one of the crucial steps in the life cycle of many positive-stranded RNA viruses, including coronaviruses. The non-structural protein M pro generally cuts two replicase polyproteins and also causes some matured proteins that are essential for viral replication and transcription [9]. Thus, the M pro of SARS-CoV-2 has been considered as an important molecular target for anti-SARS-CoV-2 drug discovery and development. Sarkar et al.

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Natural products and their derivatives have been used for years in folk medicine to treat several diseases, including viral infections [10], and the scope of herbal medicines in the context of the nutraceuticals market is vast [11]. In addition, the acceptability of plant-based drugs is increasing on a daily basis. Along this line, Nigella sativa demonstrated its inhibitory activity against the hepatitis C virus [12]. Furthermore, some natural products exhibit antiviral activity through the inhibition of viral replication [13,14]. On the other hand, several marine natural products [15], and biotechnologically produced compounds [16] have also been shown to exert antiviral effects against different viruses. There is a vast library of compounds derived from natural sources that could turn into drug leads for the treatment of various ailments, including viral diseases [17]. However, there is a lack of adequate research on the development of anti-CoV agents from such natural products. Such agents are not only important to combat CoV but also play an important role in preventing viral attack. In addition, despite the ongoing research on the development of specific therapies or vaccines against protease of SARS-CoV-2, there is currently no effective prophylaxis or therapy for SARS-CoV-2, which hinders the treatment or control of the viral infection. In this study, we designed an in silico study to determine the inhibitory activities of selected natural anti-viral compounds against the protease of SARS-CoV-2 in an attempt to identify the stronger binding affinities. Results of this study could highlight the importance of some natural products as potential drugs for SARS-CoV-2.

Molecular Docking Study
With the aid of Auto Dock vina (version 4; The Scripps Research Institute, La Jolla, CA, USA) in the PyRx platform, molecular docking was performed to elucidate the binding mode of ligands and target structures. For this reason, the atomic coordinate of the protein (PDB ID: 6LU7) was downloaded from Protein Data Bank (PDB). Using Discovery studio visualizer (version 16.1.0.15350; Biovia, San Diego, CA, USA), visualization of the respective protein-ligand complexes, along with non-covalent interactions, were performed. We introduced an updated server called Computed Atlas of Surface Topography of proteins (CASTp 3.0) for the purpose of detecting and characterizing cavities, pockets, and channels of this protein structure.

Ligand Preparation
After reviewing the literature on different anti-viral drugs, we identified 16 natural bioactive compounds that act on several viral infections in order to predict their inhibitory activities against the protease of SARS-CoV-2 (6LU7) to eventually describe candidate drugs that may exhibit antiviral activity against SARS-CoV-2. We downloaded the 3D structure of each compound in Structure-data file (SDF) format with the aid of the PubChem database.

Protein Preparation
All information about the proteins has been collected from the Uniprot (http://www.uniprot.org/).The crystal struc-ture of the SARS-CoV-2 main protease in complex with a peptidomimetic inhibitor (PDB ID: 6LU7) was downloaded from the RCSB protein data bank (http://www.rcsb.org) [18]. We used PYMOL (version 1.7.4.5), a magnificent software, to visualize the protein and remove all water molecules from the protein structure (Fig. 1).Void atomic spaces and crystallographic disturbances were corrected through energy minimization using the Swiss PDB viewer v4.1.0. As a final point, optimized protein structure was saved in ".pdb" format [19].

Binding Pockets Identification of Protein Structure (PDB ID: 6LU7)
Geometric and topological features such as pockets, cavities, and channels were shown with the help of the CASTp 3.0 server thorough the representation of surface atoms participating in their formation (e.g. the stick model shown in Fig. 2 and the sequence panels shown in Fig. 3). In the beginning, the protein structure in the ".pdb" format was uploaded in the CASTp server and a probe radius as input for topographic computation. A default probe radius of 1.4 ˚A, which is considered as the standard value for computing solvent accessible surface area for obtaining pre-computed results, was used. Finally, all surface pockets or amino acid residues in a protein structure were identified and provided a detailed delineation of all atoms participating in their formation. The final output file was directly downloaded from the CASTp server, which is visualized using the PyMOL plugin.

Molecular Docking and Binding Site Prediction
In an in silico molecular docking study, the appropriate binding orientations and conformations of the ligands with the targeted protein and the preferred orientations of the ligand with maximum binding affinities for the active sites of the protein associated with structural pockets were performed using the AutoDock vina in PyRx platform. Shown in Table 1 is a visualization of the binding of compounds 3 with specific amino acid residues, performed by using BIO-VIA Discovery studio visualizer v16.1.0.15350 (Fig. 4), including binding energies (kcal/mol) acquired from PyRx for selected compound-protein complexes.  Table 1 are the results obtained for the determination of the ligand-protein binding affinity and binding pockets. These results indicate that all compounds exhibit typical docking scores with protein and interacting residues against SARS-CoV-2's M pro (PDB code 6LU7). The binding pockets of SARS-CoV-2's M pro were identified with the following amino acid residues: THR24, THR25, THR26,  LEU27, HIS41, THR45, SER46, MET49, PHE140,  LEU141, ASN142, GLY143, SER144, CYS145, HIS163,  MET165, GLU166, and HIS172 as shown in (Fig. 1) and Table 1.

DISCUSSION
The ability of a virus to engross its cellular receptor, enter the cell, and replicate is a complex process that affords many opportunities for the development of antiviral strate-   Table 1. Comparative docking scores of compounds with protein and interacting residues of selected compounds against 6LU7.
CASTp predicted amino acid residues of protein structure (PDB ID: 6LU7)   THR24, THR25, THR26, LEU27, HIS41, THR45, SER46, MET49, PHE140,   LEU141, ASN142, GLY143, SER144, CYS145, HIS163, MET165, GLU166 gies. The first human cases of SARS-CoV-2 were identified in the Chinese city of Wuhan in December 2019, and spread progressively to more than 200 countries outside China [20]. Worldwide, the number of people who have been infected with the coronavirus is more than 53.7 million [7]. Since the outbreak began in December, more than 1.3 million have died in some 220 locations including China [7]. At present, there are no approved treatments for diseases caused by coronaviruses; however, there are drugs and compounds used to treat HIV and other different types of viruses that are being rapidly tested against the new coronavirus. In this study, we used 16 natural compounds from 60 related articles and analyzed their inhibitory activities through an in silico study against the crystal structure of SARS-CoV-2 main protease (PDB ID: 6LU7), which is obtained from the RCSB protein data bank (http://www.rcsb.org) [18].
Depending on the proteolytic processing events on the polyproteins, maturation of CoVs is performed by a threedomain (I, II, and III) chymotrypsin-fold proteinase, called M pro or 3CL pro [21]. The structure of the main protease of novel human coronavirus (HCoV) shows two M pro molecules form an active homodimer. This homodimer plays a significant role in the proteolytic activity when positioned at the interface between domains I and II, where the 2 conserved residues His41 and Cys145 form the catalytic dyad of M pro [22]. M pro has recognized as an applicable target for viral inhibitor development toward SARS-CoV-2 treatment due to its pivotal role in virus maturation [23]. According to the chemical structures, the significant inhibitors of M pro can be classified into two classes -one makes a covalent bond with Cys145 amino acid residue of the catalytic site of that enzyme [24], and the other prevents a substrate entrance 5 (Fig. 4) contd…. Fig. (4). Nonbonding interaction of selected compounds with M pro of SARS-CoV-2 through Discovery studio visualizer v16.1.0.15350. Here, we only presented nonbonding interaction for comparatively high binding scored ligand-protein complexes (≥ -6.9 kcal/mol). (A higher resolution / colour version of this figure is available in the electronic copy of the article).
into the active site cavity through bindings to that enzyme [25].

CONCLUSION
The complete methodology described in this study highlights the prediction of ligand-protein binding affinity and its binding pockets. In agreement with the results from in silico docking of the constituents against the diverse receptor (PDB code 6LU7), findings showed that several natural products exhibit significant antiviral activity, particularly apigenin, alpha-hederin, and asiatic acid. These compounds could be promising leads in the development of antiviral drugs. However, much more work is required that could involve animal models and perhaps human subjects. In short, information obtained from this investigation could be valuable for future vaccine and drug development.

ETHICS APPROVAL AND CONSENT TO PARTICI-PATE
Not applicable.

HUMAN AND ANIMAL RIGHTS
No Animals/Humans were used for studies that are base of this research.

CONSENT FOR PUBLICATION
Not applicable.

AVAILABILITY OF DATA AND MATERIALS
The data supporting the findings of the study is available within the article.

FUNDING
None.