Search for therapeutics against COVID 19 targeting SARS-CoV-2 papain-like protease: an in silico study


 Background: The global pandemic of novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped, positive-sense, single-stranded RNA betacoronavirus of the family Coronaviridae. Papain-like protease (PLpro) of SARS CoV-2 is an important target of COVID-19 because it is a multifunctional cysteine protease essential for coronaviral replication. Large numbers of phytochemicals with varied chemical structures isolated from medicinal plants have been shown to possess antiviral activity. Some of these phytochemicals have been chosen on the basis of literature survey for this study. Reported inhibitors of the papain-like protease are taken as control and for QSAR study.Methods: Three dimensional structure of target was downloaded from Protein Data Bank and docked with phytochemicals & inhibitors by using software FlexX. Inhibitors of the papain-like protease were taken from binding database and QSAR analysis was performed by using EasyQSAR software.Results: Six phytochemicals: Baicalin, Rutin, Biopterin, Licoleafol, Luteolin and Quercetin shows stable bonding pattern with the target in compare to known inhibitors as it shows least score in docking, forms maximum number of hydrogen bonds with the active residues of the receptor. The predicted IC50 values of the phytochemicals are also better than the known inhibitors.Conclusion: Based on present observation of docking score of both phytochemicals and known inhibitors, IC50 value of known inhibitors and predicted IC50 of phytochemicals, we suggests above mentioned six phytochemicals may be the Papain-like protease (PLpro) targeted potent drug leads against Covid-19.

In human being COVID 19 experience as a mild to moderate respiratory problems and can be improved without any particular cure but senior peoples with diabetes, chronic respiratory diseases, cancer and cardiovascular disease are prone to high risk in this infection. The clinical practitioners report says patients with COVID-19 showed sign of sore throat, cough, fever, muscle pain, tiredness and viral pneumonia. This virus spread from diseased person to other through coughing and sneezing and can be avoided by keeping a proper distance with others and sanitizing hands with alcohol regularly. So practicing personal sanitation and public distancing is the only means to prevent from this deadly pandemic (2,3). Several countries have enforced lockdown which is helping in con ning the spread of the disease, however it has not been totally successful. In addition to loss of human lives, COVID-19 is causing rigorous economic losses to both developed and developing nations. According to WHO report as of May 31, 2020, the virus has infected 5934936 people in more than 215 countries including a shocking 367166 deaths (2).
HCoVs generally are positive-sense single-stranded RNA (30kb) viruses. HCoVs are characterize by two groups of protein; structural such as Spike (S), Nucleocapsid (N) Matrix (M) and Envelope (E), and nonstructural proteins such as RNA dependent RNA polymerase (RdRp) (nsp12) the Papain-like protease (PLpro) and 3C-like protease (3CLpro). PLpro is a crucial enzyme in the life cycle of RNA viruses, comprising coronaviruses. PLpro is a multifunctional cysteine protease that processes the viral polyprotein and host cell proteins by hydrolysing the peptide and isopeptide bonds in viral and cellular substrates leading to the virus replication. It is responsible for the cleavages of N-terminus of the replicase poly-protein to release Nsp1, Nsp2 and Nsp3, which is essential for correcting virus replication.
PLpro also antagonize the host's innate immunity. As a vital enzyme in the process of coronavirus replication and infection of the host, PLpro is an accepted target for coronavirus inhibitors. It is very important for targeting PLpro to treat coronavirus infections (6,7). Stripping ubiquitin and ISG15 from host-cell proteins to assist coronaviruses in their evasion of the host innate immune responses is an added function of PLpro. Inhibiting viral replication and inhibiting the dysregulation of signaling cascades in infected cells leading to cell death in surrounding and uninfected cells may be achieved by targeting PLpro (8). Therefore, the papain-like protease (PLpro) is an important target for antiviral drug design (9).
At present, there is no evidence from randomized clinical trials (RCTs) that any possible therapy improves outcomes in patients with either suspected or con rmed COVID-19, therefore, there is an urgent need for effective drugs (10).
Plants have naturally developed over the years in diverse weather conditions on earth and have been bestowed with rich composite of secondary metabolites/phytochemicals with wide pharmacokinetic spectrum. Around 2500 medicinal plant species have been recognized worldwide to treat a myriad of in ictions and ailments (11,12). A large number of compounds of varied chemical structures isolated from medicinal plants possess antiviral activity (13)(14)(15)(16)(17)(18)(19) (Table 1).
Experimental approaches for the study of interactions between drug compounds and target proteins are expensive and time consuming. In silico approaches propose techniques to examine hypotheses of new putative drugs by reducing the cost and shortening the time.
Therefore, the present study was conducted to identify potential inhibitors of SARS-CoV-2 papain-like protease from natural compounds using in silico approaches. Reported inhibitors of the Papain-like protease are taken as control and for QSAR study.

Materials And Methods
The Ligands Antiviral phytochemicals were taken based on literature survey and known inhibitors of the papain-like protease of SARS CoV-2 were taken from the Binding Database (20). The structure of these phytochemicals and known inhibitors of the Papain-like protease were retrieved from PubChem Compound and by drawing using ChemO ce tools. The three dimensional structure of these compounds in sdf format were generated using OpenBabel software (21).

The receptor
The crystal structure of the papain-like protease of SARS Active site identi cation The active sites of the receptor were identi ed by the FlexX software during receptor preparation process.

Protein -Ligand interaction using FlexX
Docking is a term used for computational schemes that attempt to nd the best matching between two molecules: a receptor and ligand (22). The receptor was docked with known inhibitors of the Papain-like protease and phytochemicals using software FlexX (23

Results
Interaction energies between ligand and receptor play the most crucial role in drug designing. In this work, the papain-like protease of SARS CoV-2 (PDB ID: 6W9C) was selected as drug target and the interactions of the compounds were studied using FlexX software. The docking results of phytochemicals with target are described in table 2 and the docking results of papain-like protease inhibitors with target are described in table 3. The docking poses of best docked phytochemicals and inhibitors are shown in Figures (Figure 1 -Figure 9). Phytochemicals: Baicalin, Quercetin, Licoleafol, Biopterin, Luteolin and Rutin show much more binding a nity with the target in comparison to the reported inhibitors of the papain-like protease.

Discussion
The least score in docking was preferred for considering better ligand as it indicates more stability in binding (22). The interactions of phytochemicals and the Papain-like protease inhibitors with target were screened based on hydrogen bonding based prediction (25 Luteolin an antiviral avone against herpes and poliomelytis viruses isolated from Matricaria inodora L. plant (14) has binding e cacy with the target with a docking score of -25.9438 Kcal/mol and forms eight hydrogen bonds with the receptor.
Quercetin exhibit remarkable activities against picornaviruses and vesicular stomatitis virus (14) a potent antioxidant avonoid found mostly in onions, grapes, berries, cherries, broccoli, and citrus fruits shows good binding a nity with the target, forms eight hydrogen bonds with a docking score of -24.9869 Kcal/mol.
The predicted IC50 values of above mentioned phytochemicals were much less than the most of the inhibitors ( Table 4).
The Papain-like protease (PLpro) is a multifunctional cysteine protease that processes the viral polyprotein and host cell proteins by hydrolysing the peptide and isopeptide bonds in viral and cellular substrates leading to the virus replication. Targeting PLpro with antiviral drugs may have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signaling cascades in infected cells that may lead to cell death in surrounding, uninfected cells (6, 7 and 8).
Six phytochemicals: Baicalin, Rutin, Biopterin, Licoleafol, Luteolin and Quercetin shows stable bonding pattern with the target in compare to known inhibitors as it shows least score in docking, forms maximum number of hydrogen bonds with the active residues of the receptor. The predicted IC50 values of the phytochemicals are also better than the known inhibitors. Therefore, these six phytochemicals have more potentiality to inhibit the Papain-like protease.

Conclusion
Based on present observation of docking score of both phytochemicals and known inhibitors, IC50 value of known inhibitors and predicted IC50 of phytochemicals, we suggests six phytochemicals: Baicalin, Rutin, Biopterin, Licoleafol, Luteolin and Quercetin may be the Papain-like protease (PLpro) targeted potent drug leads against Covid-19. However, further studies are required to validate the same in vivo or in vitro.