key: cord-0056397-jdhebxc2
authors: Prasanth, D. S. N. B. K.; Manikanta, Murahari; Chandramohan, Vivek; Bhavya, Gangadharappa; Lakshmana Rao, Atmakuri; Panda, Siva Prasad; Rao, G. S. N. Koteswara; Chakravarthi, Guntupalli; Teja, Nayudu; Suguna Rani, Peddireddy; Ashu, Gummadi; Purnadurganjali, Chittiprolu; Akhil, Puvvala; Vedita Bhavani, Gorriputti; Jaswitha, Tirumalasetti
title: In-silico strategies of some selected phytoconstituents from Melissa officinalis as SARS CoV-2 main protease and spike protein (COVID-19) inhibitors
date: 2021-02-08
journal: nan
DOI: 10.1080/08927022.2021.1880576
sha: 8a65d97bfc0eb8182f7b9e0caaf5db2191dd835a
doc_id: 56397
cord_uid: jdhebxc2

Melissa officinalis (Lamiaceae) was used to treat multiple human afflictions. Literary works demonstrated that it has many biological activities. Today’s research aims to recognise Melissa officinalis phyto-derived anti-viral compounds against main protease and spike protein of COVID-19, to gain insight into the molecular interactions. In the current study, 12 molecules taken from Melissa officinalis were analysed through docking, which is derived from the PubMed database. Docking experiments were conducted with Autodock tool. AdmetSAR and Data warrior servers were eventually used for drug-like prediction. Our research shows that three phytoconstituents from Melissa officinalis, namely, Luteolin-7-glucoside-3′-glucuronide, Melitric acid-A and Quadranoside-III have exhibited better binding affinity and stability with the targets of COVID-19 main protease and spike protein. The identified substances can be further extended for in vitro and in vivo studies to assess their effectiveness against COVID-19.

. Exact origin of the preliminary transmission to human beings is still unidentified. Presently, there are >100 total genome patterns recognised in the NCBI GenBank, coming from over 10 nations [3] . The variant in between these series is much less than 1%. SARS-CoV-2 has been identified as β-coronavirus which causes severe respiratory tract infection in humans by utilising angiotensin-converting enzyme-2 (ACE2) receptors to infect humans [3] . Chinese experts separated SARS-CoV-2 and also sequenced the genome SARS-CoV-2 on 7 January 2020 [4] . The Main protease (Mpro) is an essential protein required for proteolytic maturation of the virus [5] . Thus, targeting Mpro has the potential to provide effective treatment against SARS-CoV-2 by inhibition of the viral polypeptide cleavage. Spike protein of virus binds to the tissue membrane layer with a receptor-mediated communication which enables a way to host cell. Also makes it possible for the application of well-known protein designed to rapidly develop a version for medicine breakthrough on this brandnew SARS-CoV-2 [6] .

The COVID-19 pandemic triggered by SARS CoV-2 has resulted in substantial rates of morbidity and mortality worldwide. The strategy adopted here was to look for in silico potential of phyto-constituents against SARS-COV-2 by computational protocols against spike glycoprotein as well as main protease. On the other hand, plants have been essential to human welfare for their uses as therapeutics since ancient times [7, 8] . A significant amount of antiviral compounds produced from numerous kinds of plants have been used in many studies [9] [10] [11] . Researchers across the globe are screening therapeutic molecules from existing antiviral plant secondary metabolites and are also trying to find novel compounds from medicinal plants to avert this pandemic crisis [12] . In-silico based testing has been confirmed to be a handy tool to overcome the obstacles of drug discovery. These computational strategies conserve information in terms of money and time [13] [14] [15] [16] . Screening from existing plant metabolites, researchers have been trying to identify and optimise novel compounds from medicinal plants to prevent numerous diseases, including COVID-19.

Thus, we have screened a small library of natural compounds against Mpro and spike protein by in silico studies. We have selected M. officinalis for the current research, which is an important source of anti-viral agents [17] [18] [19] . A traditional Indian medication used hundreds of years to treat different lung diseases, including pneumonia, is the Melissa officinalis (Lamiaceae). Several reports have also recently provided convincing and exposing scientific data of its antioxidant, antimicrobial [20] anti-viral [21, 22] , to treat for moderate Alzheimer [23] , neuroprotective [24] , anti-cancer [25, 26] , hepatoprotective [27] , anti-depressant [28] and antidiabetic [29] activities.

The present manuscript therefore attempts to describe in silico potential of Melissa officinalis metabolites that have antiviral properties could be aligned as an alternative for COVID-19. The recent emerging viruses, drug targets and drug development methods have thus become very critical challenges in order to identify specific/efficient therapeutics.

A list of active phytochemicals was acquired from Indian Medicinal Plants, Phytochemistry and Therapeutic Data Base [30, 31] .

Phytochemical components were downloaded from PubChem, and the structures were translated into SDF format and the drug-likeliness of the compounds was evaluated by using Data Warrior program [32] .

The protein's atomic coordinates of COVID-19 main protease (PDB ID: 6LU7) and spike protein (PDB ID: 6LZG) were retrieved from the RCSB PDB site [33] [34] [35] . The charge assignment, solvation parameters and fragmental volumes to the protein were performed using Autodock Tool 4 (ADT) before study or docking. Protein molecule was further processed for molecular docking [36] (Figure 1 ).

The 3D structure of 12 active compounds from M. officinalis includes, Quadranoside-III (Pubchem ID: 10327092), Luteolin-7-glucoside-3 ′ -glucuronide (Pubchem ID: (Pubchem ID:128859), Conhydrinone (Pubchem ID:426124), gamma-Coniceine (Pubchem ID:442632) and 5-Ketooctanal (Pubchem ID: 12538121) were retrieved from PubChem database [37] . Drug likeliness properties of ligands were analysed using Data Warrior tools for the selected active compounds [38] (Figure 2 ).

An important step is a precise prediction of active sites throughout bioinformatics. During this analysis, Active Site of Main Protease and Spike Protein was projected ( Figure 3 ) by Biovia Drug Discovery Studio Visualizer 2020 (PDB ID: 6LU7, 6LZG).

Autodock 4.0 technique has been validated to ensure the simulated screening process using the corresponding co-crystallised ligands of target proteins. Autodock 4.0 reflects a reliable RMSD score with target-receptor binding [34] .

The Autodock wizard was used as docking source to check molecular compound libraries with Autodock software [39] . Ligands were found flexible during the docking process, and protein was kept rigid. Grid parameter configuration file was created for autodock with dimensions of (X = −10.711837, Y = 12.411388, Z = 68.831286 for 6LU7; X = −31.495980, Y = 24.224738, Z = −14.498712 for 6LZG). In this study, amino acids in the active protein site interacting with ligands were identified. Root-mean-square deviation (RMSD) of less than 1.0 Å were deemed optimal and grouped to determine the desirable relation. The best ligand was selected based on the binding affinity at the active pocket with the respective target protein.

Biovia Drug Discovery Studio 2020 was used to examine the docking site visually, and the results were confirmed with Autodock Vina [40] .

ADMET of the ligands is their pharmacokinetic properties that are required to be examined to establish their function inside the body. The ADMET inheritance of ligands was predicted by making use of admetSAR [41, 42] .

The Manosa officinalis prediction for anti-viral behaviour was generated with software support of PASS. PASS is a computerbased system used for estimating various kinds of physiological actions for several phyto-constitutional compounds. The calculated effect of a compound is projected as probable (Pa) activity and possibly (Pi) inactivity. Substances which disclose Pa higher than Pi are the only components deemed possible for a particular medicinal activity [43] [44] [45] .

MD simulation was carried out with GROMACS 2019.4 [46] based on the GROMOS96 force field as reported previously [34, 47] . All MDs were carried out on a GPU enabled workstation. We carried out MD simulations for the following list of complexes: (i) 6LU7-APO, (ii) 6LU7-LUT, (iii) 6LU7-QUA, (iv) 6LGZ-APO, (v) 6LZG-LUT and (vi) 6LZG-MEL. The primary Molecular dynamics was done using the PDB coordinate file for the proteins PDB ID 6LU7 and PDB ID 6LGZ as the starting point. Residues missing in the 6LU7 and 6LGZ of the PDB structure were added using Discovery studio [48] . The system was arranged in a cubic box (size 1.0 nm), solvated with water (SPC) and further neutralised by addition of sodium ions. For the docked complex MD, the ligand topology file for PDB ID 6LU7 and PDB ID 6LGZ were generated using the PRODRG2 server setup [49] Using the steepest descent algorithm, energy minimisation was done at 1000 steps. Position restraints were employed to 6LU7 and 6LZG, after that a temperature balancing (300 K) and pressure balancing (1 bar) was executed at 50,000 ps each. The MD production runs were then conducted for 50 ns, maintaining temperature at 300 K and pressure at 1 bar. Graphs from the MD simulations data were plotted using Xmgrace [50] . MM-PBSA calculations were performed for last 20 ns of respective complexes.

Data Warrior software was used to study the physicochemical properties of selected 12 active compounds. Except for three compounds, remaining all the compounds obeyed Lipinski's rule ( Table 1 ). The basic physicochemical properties of TPSA and AMR mainly include drug intake, distribution and penetration functions [51] .

Molecular docking was conducted on 12 phytoconstituents acquired from M. officinalis to discover a prospective COVID-19 candidate against main protease and spike protein (PDB ID: 6LU7 and 6LZG). Such 12 compounds were bound to the target enzyme COVID-19 and rated based on their dock results. Top three compounds with a dock value of −8.0 kcal/ mol or lower for 6LU7 interactions and −9.0 kcal/mol or lower for 6LZG communications were considered to be a good illustration of COVID-19 control. For a detailed review, refer to Table 2 . A total of 3 compounds were chosen based on 6LU7 and 6LZG binding interactions (Figures 4-10 ).

The rigid docking results were foreseen utilising Discovery Studio for communications assessment. Tables 3 and 4 provided the best binding sites for protein-ligand interaction. The best possible associate with Luteolin-7-glucoside-3 ′ -glucuronide (−10.1 kcal/mol) of the phytochemicals was identified to be spike protein (6LZG) of SARS-CoV-2 ( 

The ADMET attributes of ligands have been calculated using admetSAR. All the substances demonstrated good human intestinal absorption (HIA) and penetration of the bloodbrain barrier (BBB). None of them were found carcinogenic. None of the substances demonstrated negative for AMES. Table 5 lists the tests of HIA, BBB, LD 50 for compounds.

Virtual PASS version collected the biological activity range of previously identified plant constituents. Such projections were flexibly interpreted and given in Table 6 .

The Root Mean Square Deviation (RMSD) is frequently used to measure the steadiness of an enzyme or drug bounded enzyme during the simulation period [1] . We examined RMSD of the simulated system over time by measuring the deviation of 6LZG with its crystal structure (APO). Figure 11 (A) shows that the RMSD of systems reaches equilibration state at 50 ns and the analysis was performed within 10-50 ns frames. The 6LZG-LUT and 6LZG-MEL were found to deviate from crystal conformation giving an indication that the bound state of drugs may persuade the structure of protein.

Average values of RMSD of the systems 6LZG-APO, 6LZG-LUT and 6LZG-MEL were found to be 0.363, 0.238 and 0.221 nm, respectively, confirming the stability of compounds during the simulation time.

We analysed the root mean square fluctuations (RMSF) of 6LZG-APO, 6LZG-LUT and 6LZG-MEL systems to evaluate the lack of stability area wise. RMSF values for 6LZG-APO, 6LZG-LUT and 6LZG-MEL were shown in Figure 11 (B). RMSF plot indicates that residues of alpha helix and beta sheet region are not changing in the 50 ns simulation period. Average values of 6LZG-APO, 6LZG-LUT and 6LZG-MEL were found to be 0.116, 0.118 and 0.128 nm, respectively.

Further, we calculated the radius of gyration (Rg) of protein to understand the influence of compounds on compactness of the protein. Rg values for the protein alone, 6LZG-APO and protein-ligand complexes of 6LZG-LUT and 6LZG-MEL were shown in Figure 11 (C). Mean Rg value of 6LZG-APO, 6LZG-LUT and 6LZG-MEL were found to be 2.530, 2.496 and 2.519 nm, respectively. From Figure 11 (C) it was clear that observable deviations recorded before 50 ns in all the simulations indicating that ligands strongly bind to the active site and maintained the stability of protein structure.

The binding pattern of protein-ligand complex was analysed for its hydrogen-bonding interactions. Hydrogen-bonding interactions of protein-ligand complexes 6LZG-LUT and 6LZG-MEL were shown in Figure 11 (D). The 6LZG-LUT and 6LZG-MEL were found to be 3 and 8, respectively, indicating the strong bonding interactions throughout the 50 ns of MD simulations.

Root Mean Square Deviation (RMSD) is frequently used to measure the steadiness of an enzyme or drug bounded enzyme during the simulation period [1] . We examined the RMSD of the simulated system over time by measuring the deviation of 6LU7 with its crystal structure (APO). Figure 12(A) shows that the RMSD of systems reached the equilibration state at 10 ns and the analysis was performed within 10-50 ns frames. The 6LU7-LUT and 6LU7-QUA were found to deviate from crystal conformation giving an indication that the bound state of drugs may persuade the structure of protein. Average values of RMSD of the systems 6LU7-APO, 6LU7-LUT and 6LU7-QUA were found to be 0.376, 0.275 and 0.345 nm, respectively, confirming the stability of compounds during the simulation time. We analysed the root mean square fluctuations (RMSF) of 6LU7-APO, 6LU7-LUT and 6LU7-QUA systems to evaluate the lack of stability area wise. RMSF values for 6LU7-APO, 6LU7-LUT and 6LU7-QUA were shown in Figure 12 (B). RMSF plot indicates that residues of alpha helix and beta sheet region were not changing in the 50 ns simulation period. The average value of 6LU7-APO, 6LU7-LUT and 6LU7-QUA were found to be 0.156, 0.155 and 0.192 nm, respectively.

Further, we calculated the radius of gyration (Rg) of protein to understand the influence of drugs on compactness of the protein. Rg values for the protein alone, 6LU7-APO and protein-ligand complexes of 6LU7-LUT and 6LU7-QUA were shown in Figure 12 (C). Mean Rg values of 6LU7-APO, 6LU7-LUT and 6LU7-QUA were found to be 2.243, 2.226 and 2.256 nm, respectively. From Figure 12 (C) it was clear that observable deviations recorded before 50 ns in all the simulations indicating that ligands strongly bind to the active site and maintained the stability of protein structure.

The binding pattern of protein-ligand complexes were analysed for their hydrogen-bonding interactions. Hydrogenbonding interactions of protein-ligand complexes 6LU7-LUT and 6LU7-QUA were shown in Figure 12 (D). The 6LU7-LUT and 6LU7-QUA were found to be 2 and 7, respectively, indicating the strong bonding interactions throughout the 50 ns of MD simulations.

The binding affinity of 6LU7 with LUT and QUA was examined using relative binding strength at active centre pocket. Binding energy values were provided in Table 7 . Binding free energy values of 6LU7 with LUT and QUA were observed to be −84.035 ± 16.402 and −82.056 ± 15.906 kJ/mol indicating that LUT has a strong binding affinity with the 6LU7. Whereas, the binding free energy of 6LZG with LUT and MEL was found to be −106.398 ± 25.384 and −137.222 ± 18.027 kJ/mol indicating MEL with better binding affinity with 6LZG. Further, we calculated the active site residues involved in interaction with the drug molecules for 30-50 ns frame. The interactions of 6LU7 with LUT and QUA and 6LZG with LUT and MEL were tabulated (Table 7) .

Further, we calculated the time scale dynamics studies of 6LU7 and 6LZG systems in 0, 100, 200 and 300 ns for 6LU7 systems and 0, 50 and 100 ns for 6LZG systems. The result were depicted in Figure 13 .

Coronaviruses have a long history of human and animal infection involving diseases of the gastrointestinal, urinary, liver and central nervous system [52] . A newly arrived SARS-CoV-2 now poses major threats to human health [53] . Main focus was on clinical management that includes infection prevention, control and help. No specific clinical treatments for SARS-CoV-2-mediated infections are currently available [54] . It is, therefore, necessary to identify and discover potential drug candidates to overcome the loss of health caused by SARS-CoV-2.

This recent discovery of Mpro in COVID-19 provided an excellent opportunity to recognise possible candidates for coronavirus treatment. In this way, natural products in the last few years have gained prominence, as successful anti-viral agents [55, 56] . Because of the urgent need for COVID-19 therapeutics and natural products facilities in medicines development, we have screened M. officinalis phytoconstituents. The current study may help in identification or optimisation of potential hit molecule for further experimental investigation.

Our examination majorly concentrated on exploring in silico potential of phytochemical valuable novel constituents from the herbal plant of M. officinalis against COVID-19. From the docking results, three selected phytoconstituents from M. officinalis were specifically chosen for further molecular dynamics and MM-PBSA studies.

Three compounds, out of 12 candidates, had a higher binding affinity with lower energy binding to the main protease and spike protein. Regarding the main protease, Quadranoside III has a minimum binding energy of −8.6 kcal/mol, which forms three hydrogen bonds with three amino acids have been identified, i.e. LYS A:137 (4.60), ASN A:238 (4.52), GLU A:288 (4.82) and one hydrophobic interaction LEU A:272 (4.46). The bond length for hydrogen bonds is <5 A°, meaning that Our previous study identified tenufolin and Pavetannin C1 from Cinnamon with good binding affinity and stability against same Mpro and Spike protein from docking, molecular dynamics and MM-PBSA calculations. Mpro-tenufolin complex was stabilised with binding energy value of −123.949 ± 16.613 kJ/mol and spike protein-Pavetannin C1 complex with −158.870 ± 30.378 kJ/mol, respectively [34] . In comparison with binding energy values in Table 7 , M. officinalis compounds have exhibited higher binding energy values in GROMACS MM-PBSA calculations. Further experimental investigation will give a better understanding about the efficacy of compounds isolated from Cinnamon or M. officinalis.

Interesting correlation was observed with the docking results and molecular weight of compounds taken in the study. However, a number of compounds taken were very few and does not have a wide range of molecular weight. Three compounds Quadranoside III, Luteolin-7-glucoside-3 ′ -glucuronide and Melitric acid A have a molecular weight of 666.846, 625.51 and 538.46 g/mol, respectively. All three compounds have demonstrated good binding affinity and stability in molecular dynamics integrated with MM-PBSA calculations against both target proteins. The next two compounds were Labiatenic acid and Nepetoidin A with a molecular weight of 360.317 and 314.292 g/mol have exhibited less binding affinity in comparison with the above three compounds. All the other compounds have a molecular weight of less than 200 g/mol and also observed poor docking results. The study indicates that the active pocket of both target proteins has the ability to accommodate large molecules. However, validation with experimental results will give a clear understanding of the relation with molecular weight of compounds. Lipinski's five-rule is a primary standard for assessing drug likeliness. Lipinski's law specifies the molecular properties essential to the pharmacokinetics of medication in the human body, such as ADME. Lipinski's rule of five conditions for optimal medicines. The top three scored compounds failed to obey Lipinski's rule, specifically in the number of H-bond donors and acceptors, as they were derived from natural origin. ADME predictions of selected three compounds have qualified the ADME check screens (Table 3) . From the MD simulations and MM-PBSA calculations, 6LU7-LUT and 6LZG-MEL complexes were found stable and recognised as potential in silico inhibitors for respective target proteins. This preliminary screening of possible molecules (anti-virals) will help to provide rapid input for further experimental analysis against SARS-CoV-2 (COVID-19).

In the research, we used bioinformatics tools, Autodock, GRO-MACS, admetSAR and PASS analysis to recognise the potential Manosa officinalis molecules against COVID-19 main proteases and spike target proteins. Our findings suggest that the two phytoconstituents, Luteolin-7-glucoside-3 ′ -glucuronide and Melitric acid-A, should be extended for in vitro and pre-clinical studies as potential inhibitors to COVID-19 main proteases and spike-proteins.

No potential conflict of interest was reported by the authors. 

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