In silico studies reveal potential antiviral activity of phytochemicals from medicinal plants for the treatment of COVID-19 infection

The spread of COVID-19 across continents has led to a global health emergency. COVID-19 disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected nearly all the continents with around 1.52 million confirmed cases worldwide. Currently only a few regimes have been suggested to fight the infection and no specific antiviral agent or vaccine is available. Repurposing of the existing drugs or use of natural products are the fastest options available for the treatment. The present study is aimed at employing computational approaches to screen phytochemicals from the medicinal plants targeting the proteins of SARS-CoV2 for identification of antiviral therapeutics. The study focuses on three target proteins important in the life cycle of SARS-CoV-2 namely Spike (S) glycoprotein, main protease (Mpro) and RNA-dependent RNA-polymerase (RdRp). Molecular docking was performed to screen phytochemicals in medicinal plants to determine their feasibility as potential inhibitors of these target viral proteins. Of the 30 plant phytochemicals screened, Silybin, an active constituent found in Silybum marianum exhibited higher binding affinity with targets in SARS-CoV-2 in comparison to currently used repurposed drugs against SARS-CoV-2. Withaferin A from Withania somnifera also showed significant binding to the targets proteins. In addition, phytochemicals from Tinospora cordiofolia and Aloe barbadensis displayed good binding energetics with the target proteins in SARS-CoV-2. These results provide a basis for the use of traditional medicinal plants as alternative lines of treatment for COVID-19 infection.


Introduction
COVID-19 disease caused by the novel coronavirus SARS-CoV-2 has been declared as a global pandemic by WHO. Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2), previously named 2019 n-CoV, first emerged in late 2019 in China [1]. Since December 2019, COVID-19 infection has spread to nearly all the continents. According to the latest situation report released by WHO, globally 1.61 million confirmed cases and around 99690 deaths have been reported due to COVID-19 as on 11 th April, 2020 [2]. India records 6634 confirmed cases and 242 deaths due to  infection (as on 11 th April, 2020) [3]. The virus has high rate of transmissibility and spreads via droplets, physical contact with infected individuals, contaminated surfaces and possibly through oralfecal route [4]. Common symptoms of a person infected with coronavirus include fever, cough, shortness of breath, and dyspnea. In more severe cases, the infection can cause pneumonia, severe acute respiratory syndrome, kidney failure, and even death due to multiple organ failure [5].
SARS-CoV-2 is an enveloped RNA viruse belonging to the Coronaviridae family and genus betacoronavirus and is distant from SARS-CoV with 79% identity. Phylogenetic analysis of the SARS-CoV-2 shows 50% identity with Middle East respiratory syndrome coronavirus MERS-CoV, 88% identity to two bat-derived (SARS)-like coronaviruses bat-SL-CoVZC45 and bat-SL-CoVZXC21 [6] and 96.2% identity to bat CoV RaTG13 [7]. The complete genome of Wuhan-Hu-1 coronavirus (WHCV), a strain of SARS-CoV2 with a size of 29.9 kb was first isolated from a pneumonia patient in Wuhan [8]. The genome has variable number of Open reading frames (around 6-11) [9]. Viral RNA located in the ORF1 translates two polyproteins, pp1a and pp1ab, and encodes 16 non-structural proteins (NSP), while remaining ORF codes for structural proteins. Corona virus has four major structural proteins, namely the Spike (S) protein, envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein [10]. Among these, S glycoprotein of SARS-CoV-2 binds to host cell receptors, angiotensinconverting enzyme 2 (ACE2) that is a critical step for virus entry [11]. Both the structural proteins and NSPs have played important roles from drug design perspectives. The therapies for SARS-CoV-2 can target different pathways-structural proteins that block the entry of virus into the human host cell, critical enzymes involved in viral replication and virus RNA synthesis, proteins that cause virulence or aid virus assembly process and many more. Availability of crystal structures of SARS-CoV-2 proteins in PDB (Table 1) has guided structure-based drug design endeavors for development of antiviral agents.
To date, no specific therapeutic drug or vaccine has been approved for the treatment of coronavirus.
There is an urgent need to discover novel antivirals for the ongoing pandemic situation caused by Severe Respiratory Corona Virus 2 (SARS-Cov-2). Drug discovery for the very infectious COVID-19 is a challenging job owing to frequent mutations [12]. In addition, researchers across the globe are racing to develop potential vaccines [13]. The development of both novel antiviral compounds as well as vaccines presents several challenges and requires significant amounts of effort and time for validation. Therefore, exploring the repurposing of already-approved pharmaceuticals or the use of natural compounds can provide alternatives to the development of novel antiviral drugs. Chloroquine, a repurposed drug known widely for the treatment of malaria is used to treat COVID-19 infection [14].
In vitro studies have shown hydroxychloroquine, a less hepatotoxic derivative of chloroquine, to be foremost effective drug in inhibiting SARS-CoV-2 infection [15]. Few clinical trials have also shown chloroquine phosphate to be effective in COVID-19 associated pneumonia [16]. Other antivirals in combination or alone are also being used to determine their effectiveness against SARS-CoV-2 [17][18]. However, at present the treatment is only symptomatic and more effective antivirals need to be developed to fight the deadly disease.
Medicinal plants are valuable sources of drugs used globally as alternative medicines. India is a rich source of biodiversity with more than 7000 plants species used as medicinal plants [19].
India also has a rich ancient tradition of alternative medicines -Ayurveda, Yoga, Unani and Siddha and Homeopathy system (AYUSH) that is still in use today. It has also been estimated that 70-80 % of people in developing countries are totally dependent on herbal drugs for their primary healthcare   [60]. It has also been reported that consumption of A. vera might be helpful to human immunovirus-infected individuals since it enhances the CD4 count and thereby improves the functioning of the immune system. [60]. Lignans are a class of natural products that possess diverse pharmacological properties and are known to be effective as antitumor, antioxidant, antibacterial and antiviral agents [61]. Milk thistle (Silybum marianum) is another medicinal plant that has been used for thousands of years as a remedy for a variety of ailments [62]. The major component of S.
marianum fruit extract (silymarin) is a flavonoind lignan called silybin that has been used in Indian medicines for liver and gallbladder problems [63][64]. Many studies have also reported that silymarin is an effective antiviral treatment for hepatitis C virus (HCV) [65][66]. Withania somnifera (Ashwagandha) is one of the most important herbs of Ayurvedic system of Indian medicine [67]. Withaferin A (WA) is an active constituent of Withania somnifera that has been shown to have a broad range of medicinal properties including antiviral activity [68][69]. Withonalides are steroidal lactones present in ashwagandha possessing diverse pharmacological activities [70].
Based on the above observations, this work focused on the study of phytochemicals derived from the medicinal plants for their antiviral activity towards SARS-CoV-2. The targets were carefully identified that played a key role in the viral attachment to host cell, viral replication and viral synthesis. A library of 30 natural metabolites in medicinal plants included curcumin and its derivatives, chemical constituents of giloy (Tinospora cordifolia),, Aloe Barbadensis, S. marianum, Withania somnifera and plant lignans were studied by docking to three targets Main protease, Spike S protein and RNA dependent RNA polymerase (RdRp) in SARS-CoV-2. In addition to the library of phyochemicals, we also included the currently used hydroxychloroquine along with other repurposed drugs as ligands for molecular docking.

Ligand Preparation
A library of phytochemicals in medicinal plants was compiled as ligands from the review of literature and their 2D structures were retrieved from PubChem database [75]. For the ligands whose structures were not available, the 2D structures were drawn using MarvinSketch [76]. The protein and ligand structures were prepared using the preparation wizard in Flare (module from CRESSET software) [77][78].

Prediction of Binding site
Binding site of the main protease was analyzed by using the information about the amino acid residues interacting with the known, co-crystallized ligand. The interface residues of the spike receptor binding domain with ACE2 (Angiotensin converting enzyme 2) were selected for grid generation for spike protein. The binding site for RNA-dependent RNA-polymerase (RdRp) was predicted using CASTp web server [79].

Protein-Ligand Molecular Docking
Molecular docking of the ligand library composed of natural compounds and repurposed drugs currently in use for COVID-19 treatment was carried out with target proteins main protease, spike protein (S) and RNA-dependent RNA-polymerase (RdRp) using Flare module provided by CRESSET software. For the docking process, the targets were prepared and minimized, the grid box was  (Table 3) were prepared for docking. The amino acid residues of target proteins considered for binding during molecular docking are enlisted in    The list provides proteins details of SARS-CoV-2 as obtained from GenBank: NC_045512.2