Comparative Study of Drug Likeness and Pharmacokinetic Properties of Synthetic Antiviral Drugs to that of Remdesivir: In-silico Approach

a Division of Biochemistry, School of Life Sciences, JSS Academy of Higher Education and Research, Mysuru, Karnataka 570015, India. b Department of Studies in Food Technology, Davangere University, Davangere, 577007, India. c Department of Biotechnology and Bioinformatics, School of Life Sciences, JSS Academy of Higher Education and Research, Mysuru, Karnataka 570015, India. d Department of Microbiology and Tissue Culture, School of Life Sciences, JSS Academy of Higher Education & Research, Mysuru570015, India e Department of Sciences, Amrita School of Arts and Sciences, Amrita Vishwa Vidyapeetham, Mysuru Campus, Mysuru, Karnataka 570 026, India. f Department of Studies in Biotechnology, Davangere University, Davangere-577007, Karnataka, India. g ICARNational Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Yelahanka, Bengaluru-560064, India. h Department of Chemistry, Sri Jayachamarajendra College of Engineering, Manasagangotri, Mysore, 570 006, India. i Department of Studies and Research in Biotechnology, Sahyadri Science College, Kuvempu University, Shimoga -577203, Karnataka, India.


INTRODUCTION
At the turn of the twenty-first century At the beginning of the twenty-first century, millions of individuals were infected with the newly discovered coronavirus infection 2019 (COVID-19) [1], it was first detected in China's Hubei province Wuhan, the metropolitan region. It has been widely spread from China to many other countries, and it has been drawing enormous attention all around the globe [2]. According to the WHO, this viral disease is considered very pandemic [3].
In the closing 20 years, numerous viral epidemics, including the severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 to 2003 in Guangdong province, China, and H1N1 influenza in 2009, have been recorded. In 2002 SARS appeared in Southern China and spread to twenty-eight other countries. By July 2003, almost 8,000 individuals had been inflamed, with 774 of them dying. Only four cases were observed in the small outburst in 2004 [4]. Ten years after SARS, another highly infectious pathogenic coronaviruses, MER-CoV, emerged in Middle Eastern countries [5]. It was started in 2012 in Saudi Arabia. More than 1621 cases were infected; 584 people died in 2012 due to middle-east respiratory syndrome coronavirus. Compared to SARS, it was less contagious [6,7]. SARS-CoVs and MERS-CoVs are Zoonotic viruses with bat/cat and dromedary camel as hosts, respectively [7,8]. After SARS-CoV and MERS-CoV,the COVID-19 is said to be the third zoonotic coronavirus [9]. Commonly reported symptoms of a person infected with COVID-19 are runny nose, cough, fever, headache, and shortness of breath. After infection with COVID-19, depending on a person's age and weak immunity, the symptoms appear within 2 to 14 days [10]. Patients with mild symptoms will cure within a week, while in severe conditions, the respiratory system will fail due to the alveolar damage triggered by the virus. This may lead to the demise of patients [11].
Coronaviruses are a broad family of viruses that may infect people and a wide variety of animals. Coronavirus belongs to the subfamily Coronaviridae within the own family of Coronaviridae the order Nidovirales. CoVs are divided into four genera: Alphacoronavirus, Betacoronavirus, Gamma Coronavirus, and Deltacoronavirus based on their genomic and phylogenetic relationship [12]. Generally, mammals are infected by the genera Alphacoronavirus and Betacoronavirus. The Gammaoronavirus and Deltacoronavirus can infect birds, but some of them can also infect mammals [5]. On January 12, 2020, WHO named the novel coronavirus as 2019 novel coronavirus or '2019-nCoV', then on February 11 2020 the ICTV(International Virus Classification Commission) labelled 2019-nCoV as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) according to the guidelines of nomenclature of virus because 2019-nCoV is homologous with SARS-CoV. At the same time, 2019-nCoV-related diseases were designated as COVID-19 by WHO [1].
Coronavirus is a single-stranded RNA virus (+ssRNA), which is about 120nm in diameter. It poses the giant genomes ~26kb to ~32kb among all the known RNA viruses, and it contains a 3' poly-A tail and a 5' cap structure that allows it to act as mRNA to translate polyprotein [13]. The first CoVs were identified in the 1960s by a group of virologists. Depending upon their morphological features, they represent spherical virion with a center shell. The spike, which is protein present on the virus's surface, resembles a crown; the virus has termed the corona; corona in ancient Latin language means crown based on their shape [1]. The positive single-stranded RNA genome of CoVs encodes the 2 significant genes, i.e., OPF1a and ORF1b, which encodes 16nsp (non-structural protein). ORF1a encodes form nsp1-nsp11 and ORF1b encode for nsp12-nsp16 [14,15].
The CoVs structural genes encode the four structural proteins, Spike (S) protein, Membrane protein (M), Nucleocapsid protein (N), and the Envelope protein (P). Some of the beta coronaviruses also have an additional layer of the short spike, consisting of the hemagglutinin esterase protein (HE). These are all required to complete the structure of viral particles [16]. Several functional CoVs proteins are involved in translation, modification of RNA, synthesis, transcription, virus replication, and infections. Among these, PLpro (papain-like protease), 3CLpro (3-chymotrypsin-like protease), RdRp (RNA dependent RNA polymerase), and helicase are the most critical targets for the development of inhibitors [17]. RNA-dependent RNA polymerase plays a vital role in SARS-CoV-2, which acts as a replica that catalyzes the synthesis of a complementary RNA strand using an RNA template for replication and transcription. Thus it can be used as a potent drug target [18,19].
In this study, we archiveda molecular docking to test the several synthetic and photochemical antiviral drugs against the RdRp (RNA dependent RNA polymerase) protein of coronaviruses. In the present study we used the human immune virus (HIV) compounds, antimalarial compounds, and also the earlier reported anti-SARS-CoV and anti-MERS-CoV synthetic compounds and some of the antiviral phytochemicals chosen randomly. The obtain results provides a new repurposed drugs against SARS-CoV-2 using in-silico approach.

Protein Preparation
Coronavirus (COVID-19) related articles were referred to identify the target proteins, after the identification of target protein the 3D structure of RdRp from COVID-19 (PDB code: 6NUR) with the resolution 3.10 Å. The target protein 6NUR was downloaded from the PDB in the PDB file format. Then the saved PDB format of protein was opened in PyMOL. In PyMOL all the ligands, ions, and nsp12 protein was cleaned by removing the water molecules. Then the protein structure was saved in the PDB format [21]. Then the protein 6NUR was opened in AutoDock 4.2.6 to see the docking results.

Ligand Preparation
The 3D structure of each compound is downloaded in SDF format from the PubChem and then converted into PDB format using Open Babel free software. The ligand should be in the PDB format. Finally, the PDB formats of ligands are opened in AutoDock4.2.6. The ligands selected for molecular docking of RdRp are anti-HIV, anti-malarial, and also some of photochemical against anti-viral. The high similarities between the main protein of SARS-CoVs or MERS-CoVs and the recent COVID-19 we also selected the ligand of anti-SARS-CoVs and anti-MERS-CoVs[21,3].

Grid Box Generation
Blind-docking with a search method in AutoDock 4.2.6 was used to calculate the binding site. Primarily, binding site was determined from reading from various Articles related to COVID-19 and docked in the previously ligand binding site of protein was used to dock the selected ligand molecules. Using AutoDock software 4.2.6, a grid box was created around the ligand binding site by changing the grid settings for the X, Y, and Z coordinates [22].The grid center of the RdRp (RNA dependent RNA polymerase) enzyme has the highest affinity binding site at coordinates of X=150.009; Y=147.554; and Z=157.012 with a 3D grid box of 30 x 30 x 30 [17]. The best binding energy conformation of the ligand molecule which had a lower calculated binding energy (negative energy) as compared to the reference molecule was selected for further analysis and considers having a higher binding affinity with the target NSP12 molecule [3].

ADME Properties
ADMET full form Absorption, Distribution, metabolism, Excretion and Toxicity. The toxicity of the bio reactive compounds is determined with the help ofPreADMET. In drug design, and drug discovery process ADMET play an important role because 60% failure of all the drugs in the preclinical phaseis due to these properties. Nowadays ADEM is applied at the early stage of the drug development, and discovery process.
Four main partsofPreADMET are as follows: Molecular descriptor calculation, drug-likeness prediction, ADME prediction, and toxicity prediction [2]. The drug likeness prediction of certain compounds that have a certain pharmacological activity was calculated using Lipinski's rule of 5. The rules states that 1) Not more than 5 H-bond donors(OH and NH) 2) Not more than 15 rotatable bonds 3) Not more than 10 H-bond acceptors 4) Amolecular weight of compound should be less than 500g/mol 5) A partition coefficient(log P) should not be less than 5 [24,25].

Docking Visualization and Analysis
Visualization and analysis of the docking site was performed by using PyMOLandAutoDock was used for result validation. The interaction between the protein and ligand was checked usingPyMOL along with bond length and the amino acid residue to which the ligand binds to the protein[2] [3]. The visual analysis of twodimensional molecular interactions between the protein-ligand complex structures was performed using BIOVIA Discovery studio visualizer software. It also used to depict the hydrogen bonds, hydrophobic bonds of each molecular docking poses in the form of graphical representation.

Selection of Protein and Ligand for Docking Analysis
Coronaviruses are grouped under the viruses which can infect humans. Lakhs of people were killed all around the world. COVID-19 pandemic is the most damaging pandemic in recent human history. Our research points to the RNA dependent RNA polymerase protein in coronavirus (RdRp/nsp12) as a possible therapeutic target for the development of efficient antiviral medicines in the context of coronavirus infection (PDB ID: 6NUR). 6NUR is one of the main proteins in the coronavirus. RdRp plays a vital role inproliferation in the life cycle of coronavirus by RNA replication. RdRp consist of 3 unique chains, i.e., nsp12, nsp8 and nsp7, it also consists of 2 Zinc atoms (Fig. 2). The nsp12 bound to the nsp7 and nsp8, the resolution is 3.10 Å, total structural weight is 162.52kDa and it as 1087 amino acid residues. Therefore, RNA dependent RNA polymerase is ideal as therapeutic targets to identify the inhibitors for coronavirus, by this we can stop the spreading of virus in the human body. Therefore, a basic preliminary screening of ligands was carried out by using Autodock4.2.6, selected50 synthetic ligands, were studied against the target protein RNA dependent RNA polymerase of COVID-19 (Fig. 3)

Molecular Docking Analysis
To search potential inhibitor for SARS-CoV-2, total fifty synthetic ligands were selected for docking analysis using the Autodock 4.2.6 computational screening against the important protein RdRp. Considering the biological activity like anti-MERS, Anti-SARS, Anti-HIV, antimalarial and antiviral agents we have selected the ligands. The majority of the inhibitors exhibited greater than -6.48 kcal/mol in the docking findings of fifty ligands against the RdRp target, and the results were comparable to the conventional medication Remdesivir, which pretended to be a transitory therapeutic treatment. As the core part of all the ligands were similar, ten ligands which showed the maximum docking score against the protein RdRp were considered for further in-silico ADMET and drug likeness study. 2D chemical structureof the selected lead compounds are represented in Fig.  4.A graphical representation of top 10 ligands shows the average binding affinity of ligands which inhibit the protein RdRp are represented in Fig. 5.

ADME Properties and Analysis
The selected compounds were analysed after molecular docking with the protein RNA dependent RNA polymerase as a target of COVID-19. The top 10 ligands selected on the basis of their higher docking scored were screened further ADMET and drug-likeness properties are interpreted based on marginal values compared with the resultant value.
We analysed the pharmacokinetics like blood brain barrier, plasma protein binding and human intestinal absorption. The computational BBB value corresponds to the weather the compound passes across the blood brain barrier.

Protein Ligand Integration and Visualization
There are more than 10 synthetic ligands which showed good activity against RNA dependent RNA polymerase functional sties. Depending on the higher docking score, ADMET and drug likeness prediction top 5 ligands were selected nelfinavir, NSC335985, NSC29007, NSC159375, AZD7986 were having lower binding scores than other ligands, which reveals promising binding affinity towards the active site of coronavirus RdRp . Fig 6shows the

CONCLUSIONS
Coronavirus (CoVs) have induced a main outbreak of human fatal pneumonia due to the fact that the start of the twenty-first century. There is no specific antiviral drug or treatment till date. In summary, we performed the molecular docking studies of synthetic drugs chosen by literature survey against anti-MERS, Anti-HIV, Anti-malarial to inhibit the protein RNAdependent RNA polymerase and compared the docking score with the standard drug Remdesivirpretended to be a temporary therapeutic drug for COVID-19. Our study revealed all the tested synthetic drugs showed highest docking score compared to Standard ligand Remdesivir. Out of fifty compounds docked, the ten compounds with high binding energies were selected for further drug-likeness and pharmacokinetic prediction. Out of ten compounds top five Compounds were selected which showed best binding energy, ADMET and drug likeness properties nelfinavir>NSC335985 > NSC29007> NSC159375> AZD7986. Among them Nelfinavir has the highest binding affinity (-10.06 kcal/mol) to COVID-19 RNA dependent RNA polymerase with good drug likeness and pharmacokinetics properties. Further research is urgently required to investigate the potential antiviral drug uses of these drugs for designing and developing an effective medicine against COVID-19.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.