to Molecular docking analysis on 16 therapeutic ligands of Ocimum tenuiflorum L. (Tulasi) and their prospects in drug design for COVID-19

The PyRx software and Discovery studio were used in the present molecular docking studies of the 16 ligands of Ocimum tenuiflorum L., selected based on their high therapeutic potentials, viz., (E)-6-hydroxy-4,6-dimethylhept-3-en-2-one, Apigenin, Bieugenol, Cirsilineol, Cirsimaritin, β -Caryophyllene epoxide, Dehydrodieugenol B, Eugenol, Ferulaldehyde, Isothymonin, Isothymusin, Linalool, Luteolin, Ocimarin, Rosmarinic acid, and Thymol. Saquinavir was used as a positive control. The binding affinities of the 16 ligands to the main proteases of COVID-19 6LU7 and 6Y2E (critical for viral replication) and their ability to arrest the virus replication were recorded. The binding affinities of the ligands to 6LU7 and 6Y2E ranged from -4.3 and -4.7 kcal/mol (for (E)-6-hydroxy-4,6-dimethylhept-3-en-2-one) to -7.6 (for Rosmarinic acid to both target proteins). While the corresponding values for the control drug Saquinavir were -7.8 and -7.6 respectively. The Rosmarinic acid, in binding with both the proteases (-7.6 and -7.6 kcal/mol) showed six conventional hydrogen bonds, one carbon hydrogen bond (ASP 153 had one conventional hydrogen bond and one carbon hydrogen bond), one Pi-alkyl bond, one Pi-Pi stacked bond, eight van der waals bonds for 6LU7 protease; it formed three conventional hydrogen bonds, two Pi-alkyl bonds, one unfavourable donor – donor bond and 14 van der waals bonds with 6Y2E protease. The control drug – Saquinavir in binding with 6LU7 protease showed 12 van der waals, one alkyl, one Pi-alkyl, one Pi-cation, one Pi-stacked and four conventional hydrogen bonds, which indicates that it has less affinity when compared with Rosmarinic acid. Similarly, the control drug on binding with 6Y2E protease exhibited ten van der waals, four Pi-alkyl, one cation and three hydrogen bonds. The results are in conformity to similar other studies, and herald a promising scope for Rosmarinic acid as lead molecule in the drug discovery for COVID-19.


INTRODUCTION
The COVID-19, an infectious dreadful virus disease, has been pandemic since December 2019, and the unimaginable volume of human casualties witnessed so far has propelled the scientific community to explore the possibilities of developing safe herbal drugs (Chojnacka et al., 2020), either as preventive or curative (Kiran et al., 2020), against the COVID-19 (Sampangi-Ramaiah et al., 2020). The present investigation on 16 therapeutic ligands of Ocimum tenuiflorum L., (Vernacular name, "Tulasi" in Tamil) (Singh & Chaudhuri, 2018) the latter being the key ingredient in Kandankathiri Legiyam-a Siddha polyherbal formulation used in respiratory diseases, comprise the binding affinities of them to the two main proteases of COVID-19, viz., 6LU7 (Peele et al., 2020) and 6Y2E, and their ability to arrest the virus replication (Sampangi-Ramaiah et al., 2020). The PyRx software and Discovery studio were used in the study to discover lead molecules for the drug design and explore the possibility of repurposing the popular herbal drug in the treatment of COVID-19 (Trott & Olson, 2010). The COVID-19 proteases, 6LU7 (Sisakht et al., 2021) and 6Y2E, are critical for viral replication and the effective binding of the viral proteases will arrest viral replication and the disease (Sampangi-Ramaiah et al., 2020). Hence, the present investigation was taken up towards achieving this purpose.
Modern medicines of the 21 st Century have their origin in traditional herbal medicinal practices. An array of compounds is reported as effective lead molecules in drug design and discovery (Wu et al., 2020). Ocimum tenuiflorum L., is documented extensively in Siddha and Ayurveda literature to cure respiratory diseases (Singh & Chaudhuri, 2018). There are studies on Ocimum tenuiflorum L., to show the presence of several class of phytochemicals (Soni & Sosa, 2013) such as alkaloids, carbohydrates, phenolic compounds, tannins, flavonoids, steroids, and saponins (Siva et al., 2016) such phytochemicals possess antibacterial, antiviral, antifungal, antiprotozoal, antimalarial, anti-helminthic, antidiarrheal, analgesic, antipyretic, anti-inflammatory, antiallergic, antihypertensive activities (Pandey & Sharma, 2010. A total of 41 phytochemicals are reported in Ocimum tenuiflorum L., (Singh & Chaudhuri, 2018) and listed in Table -1, out of these 41 reported phytochemicals, 23 phytochemicals are available in the PUBCHEM database (Kim et al., 2019) with canonical SMILES (Simplified Molecular Input Line Entry System), so these 23 phytochemicals are initially screened using SWISSADME software (Daina et al., 2017) for their drug-likeness (Bhadran et al., 2021).

PyRx Software and Discovery Studio
PyRx is software, it is used for molecular docking studies (Dallakyan & Olson, 2015). PyRx docks the compounds available in "Protein Data Base -(PDB)" format online in the data base against potential drug targets (Trott & Olson 2010). It enables pharmacologists to perform simulations for the drug design and discovery process (Raj, 2021). It comprises a docking wizard with easy-to-use user interface which makes it a valuable tool for computer-aided drug design (Chen, 2014). The rational drug design is made possible by this software because of its chemical spreadsheet-like functionality, and powerful visualization engine (Shaker et al., 2020). The software is unique in docking five ligands with the desired target protein ( Figure 3). The latter feature of PyRx is explored in the present study (Chaudari et al., 2020). Discovery studio is allied software to PyRx for visualizing the protein database files of ligand and target before and after docking by PyRx (Shaker et al., 2020). It is mandatory that the input files must be in PDB format for both ligand and target protein (Yuliana et al., 2013).

Preparation of Target Protein and Molecular Docking
The water molecules and HET atoms of 6LU7 and 6Y2E were eliminated (Figure 2), and the polar hydrogens were added and saved in the system using Disovery studio software. Later, the saved PDB file of the target protein is given as an input file in PyRx software along with the PDB files of the 16 ligands ( Figure 1). The files get converted to pdbqt format and then the docking site was confirmed by a grid box with the dimensions (Angstrom) of X: 63.20, Y: 64.98 and Z: 25.00 for 6LU7 protease (Ounthaisong & Tangyuenyongwatana, 2017) and for 6Y2E protease it is X: 51.21, Y:70.17, Z: 25.00 (Herowati & Widodo, 2014) (Figure 3).
The output of the docking score was obtained in CSV format was saved as MS-EXCEL spread sheet for tabulation. The docked  files with RMSD value with zero alone were saved as PDB file and visualized through discovery studio software to comprehend the ligand-protein interactions. It is pertinent to mention here that the RMSD < 2.0 Å provides a good solution, hence docked position with zero was prioritised and saved as PDB file ( Figure 3) (Ramírez & Caballero, 2018) for visualization through Biovia discovery studio software. The output of PyRx software results were presented in figures 4-9 and Tables 1-3.
The ascending order of binding affinity for the 16 ligands with 6LU7 protease was (E)-6-Hydroxy-4,6-dimethyl-3-heptene-2-one (C 9 H 16 O 2 ) < Linalool ( Table 2). The rest of the ligands did not exhibit such C-H bond and conventional hydrogen bond. It is pertinent to mention here that different types of pi-bonds and van der waals electrostatic attractions are weaker than C-H bond and conventional hydrogen bonds. Thus, it is evident that Rosmarinic acid has a strong binding affinity with 6LU7 than the other 15 ligands (Figure 4-9).
The binding affinity of the control drug Saquinavir were -7.8 and -7.6 for 6LU7 and 6Y2E respectively (Table 1). Whereas, that of Rosmarinic acid, in binding with both the proteases the binding affinity were -7.6 and -7.6 kcal/mol. The Rosmarinic acid showed six conventional hydrogen bonds, one carbon hydrogen

CONCLUSION
As the binding affinity of Saquinavir (Sampangi-Ramaiah et al., 2020), the synthetic control drug, with 6LU7 protease showed 12 van der Waals, one alkyl, one Pi-alkyl, on Pi-cation, one Pistacked, four conventional hydrogen bonds, which indicates that it has less affinity when compared with Rosmarinic acid. Similarly, the synthetic control drug on binding with 6Y2E protease exhibited 10 van der Waals, four Pi-alkyl, one cation, three hydrogen bonds. The results are in conformity to similar other studies and herald a promising scope for Rosmarinic acid as lead molecule in the drug discovery for COVID-19.