Medicinal Plants Against Coronavirus (SARS-COV-2) in Morocco Via Computational Virtual Screening Approach

With the spread of the coronavirus in all countries of the world and in the absence of a vaccine or effective treatment to inhibit the infection. Several medicinal plants are used by the Moroccan population either to treat or prevent infection. The frequent use of its plants without documentation of their effectiveness on the Covid-19 is justified by cultural and economic reasons and their pharmacological activity scientifically proven. The objective of this study is to evaluate the inhibitory effect of the main polyphenols and flavonoids of Syzygium aromaticum and citrus limon as well as the main organosulfur compounds of garlic against coronavirus 6lu7 protease and 6y2e protease using in-silico molecular docking analysis. The COVID-19 3CLpro/Mpro (PDB ID: 6LU7) and free enzyme of the SARS-CoV-2 (2019- nCoV) main protease (PBD ID: 6Y2E) structures were obtained from the https://www.rcsb.org/ website in .PDB format. The COVID-19 and main protease ReaxFF force field. In this force field, the general energy function We have docked 34 natural products belonging to the three medicinal plants. In 6LU7 protease, 24 compounds exhibited a binding affinity greater than or equal to -6 Kcal/mol. While, in 6Y2E protease, 6 compounds exhibited a binding affinity greater than or equal to -6 Kcal/mol. We found that ellagic acid, narirutin, neoeriocitrin and neohesperidin are highly suggested as an inhibitor of SARS-COV-2.


Introduction
COVID-19 is the infectious disease caused by the last coronavirus that was discovered. This new virus and disease were unknown before the outbreak occurred in Wuhan, China in December 2019.
COVID-19 is now pandemic and affects many countries around the world with more than 31,000,000 infections and more than 970,000 death.
In the absence of an effective vaccine against this virus, several studies have been done to examine the inhibitory effect of natural bioactive molecules on papain-like protease (PLpro) and 3chymotrypsin-like protease (3CL pro ) using molecular docking analysis to arrive at binding affinity (Aanouz et al., 2020;Khaerunnisa, Kurniawan, Awaluddin, Suhartatiand Soetjipto, 2020;ul Qamar, Alqahtani, Alamriand Chen, 2020). 3CL pro plays an important role in the replication of viral particles, it is a potential target for anti-coronaviruses inhibitors screening (ul Qamar et al., 2020). While, Pl pro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread (Shin et al., 2020).
In Morocco, more than 100.000 cases of coronavirus confirmed and more than 1,800 cases of death.
In front of this pandemic situation, several medicinal plants are used by the Moroccan population either to treat or prevent infection. The frequent use of its plants without documentation of their effectiveness on the Covid-19 is justified by cultural and economic reasons and their pharmacological activity scientifically proven.
We carried out a survey on the medicinal plants used by the Moroccan population to treat or prevent covid19 and we found the massive use of three plants (garlic, citrus limon and clove). Garlic is widely used for the treatment of viral infections due to the presence of several bioactive molecules such as allicin, diallyl trisulfide and ajoene. The antiviral effect of garlic extract has been shown against rhinovirus, HIV, herpes simplex virus 1 (Tsai et al., 1985), against herpes simplex virus 2 (Weber et al., 1992), influenza A and B (Fenwick and Hanley, 1985), cytomegalovirus, viral pneumonia, and rotavirus (Bayan, Koulivandand Gorji, 2014). Citrus limon another plant used by the Moroccan population to prevent the covid-19 pandemic. This plant rich in flavonoids such as diosmin, eriocitrin and hesperidin. some authors suggest that these flavonoids have several biological activities like antiviral activities (Del Rıo et al., 2004).
The antiviral activity of eugeniin extracted from clove has been reported against herpes virus and it was deducted that one of the major targets of eugeniin is the viral DNA synthesis by the inhibition of the viral DNA polymerase (Cortés-Rojas, de Souzaand Oliveira, 2014). Moreover, eugenol is one major constituent of cloves has shown antiviral activity against human herpes simplex (Aboubakr et al., 2016).
The objective of this study is to evaluate the inhibitory effect of the main polyphenols and flavonoids of Syzygium aromaticum and citrus limon as well as the main organosulfur compounds of garlic against covid-19 6lu7 protease and 6y2e protease using in-silico molecular docking analysis.
The 3D structures of the selected ligands were obtained from the https://pubchem.ncbi.nlm.nih.gov/ website in the .SDF format. Then they optimized by ucsf chimera 1.14 software from https://www.cgl.ucsf.edu/chimera/ and saved in .MOL2 format. Table 1   At every MD (Molecular Dynamics) step, this force field updates the bond orders and provides a pathway for bonds to form and break during the simulation. ReaxFF can reproduce with acuity all relevant quantum mechanical data, as well as provide atomistic descriptions of several complex chemical reactions.
The simulations were done using the target ligands close to the protein structure, maintained at a constant temperature of 298 K. In MD simulations, temperature is set based on the displacement velocities of each atom/molecule. ReaxFF forcefield was applied with a timestep of 0.1 fs, so every interaction and bond could be observed during the simulation.
Reactions involving protein + ligand systems usually require a high amount of time. The total number of iteractions used in these simulations were 25M, thus representing a timeframe of 2.5 ns. Although it may seem small, common methods of MD applied in these systems (e.g. CHARMM forcefield) use timesteps of 2 fs, 8 times higher than the one used in ReaxFF, so the timeframe of both methods can be equated. The calculation time of each simulation was around 700 hours.
ReaxFF is not commonly used in these systems, as it was originally built for analyzing fast reactions or complex mechanisms. However, due to the usage of bond orders for determining the energies, it can be an interesting methodology for analyzing interactions between large structures and ligands. Physical interactions or chemical bonds can be observed and thus the behavior of the ligand and the overall binding energy/stabilization of the system can be predicted.
In 6Y2E protease, 6 compounds exhibited a binding affinity greater than or equal to -6 Kcal/mol. In addition to oleanolic acid, diosmin, eriocitrin hesperidin and neohesperidin which are active on the 6lu7 site are also active on the 6y2e site with different energy between the two proteases.
The presence of hydrogen bonds in the complex explains the good interaction between the molecule and the protease. Knowing that hydrogen bonds are considered first level interactions, while other types of interactions such interactions between p systems and cation -p interactions and hydrophobic contacts and non-specific Van der Waals interactions are considered second and third level interactions respectively (Aanouz et al., 2020).
Despite the known antiviral activity of garlic, their organosulfur compounds studied in our research such as allicin, S-allyl-cysteine sulfoxide, diallyl disulfide, diallyl sulfide, diallyl trisulfide and S-allyl-cysteine present a weak band affinity which does not exceed -4.3 kcal / mol for 6lu7 and 6y2e protease (Table 2).

MD simulation
Simulations using the ReaxFF force field require a small timestep, so every bond break or formation is accurately observed. The aim of the simulations was to observe whether the ligand would bind to the protein, and which would be the energy variation of this process. Therefore, the behavior could be analyzed. Unit cells of the systems were built with the protein plus the ligand, with a minimum distance from each other of 20 Å, so after minimization, both would be kept in approximately the same place.
In all 4 cases, during production phase, the ligand succeeded in binding with the protein. The total energy of the system decreased significantly, showing a higher stabilization of the system with both structures bond together.
During the approach, the ligand structure suffered rotation, possibly due to the branches of the protein.
Dipole/electrostatic interactions are constantly being evaluated during the simulations, so it is expected that the ligand (smaller structure) adapts to the protein surface for the most favorable bonding.
Figure 4 below presents the system (6lu7 + hesperidin) from the beginning of the simulation to the complete binding of the ligand. In this specific case, the binding occurred at 80 ps after the beginning, but was finished at approximately 120 ps from start.
As previously stated, hesperidin rotates before reaching the protein surface, and the approach becomes faster when the intermolecular interactions become stronger. The variation observed in the total energy of the system was approximately ∆E = -11695.5 kJ/mol. Figure 3 presents the energy variation over time for this case.
Highest energy variation occurs in the first 25 ps. As the system was minimized at 5K, and then kept at 200 K during the equilibration phase, there is a rearrangement of the structures when applying 298 K in the production phase, thus stabilizing the system with this new parameter. This behavior is expected and observed in all 4 cases simulated. Hesperidin in contact with 6y2e (case 2) was much slower than in case 1. After 120 ps from the beginning of the production phase, the ligand was still approaching the protein structure. The binding process started at approximately 200 ps after the start, but took almost 300 ps to complete.
One could say that hesperidin has more affinity to 6lu7 than to 6y2e due to the difference in velocities.
However, the total energy variation (Fig. 5) of this simulation was ∆E = -20495.7 kJ/mol, almost double of the one observed in case 1.
Therefore, besides the kinetics may be slower, the interaction between hesperidin and 6y2e is much stronger than with 6lu7, which may result in a better performance of the ligand in deactivating the active site of the protein. Figure 6 presents the frames of the system in different times after the start of the production phase. Cases 3 and 4 evaluate the behavior of Diosmin when in contact with 6lu7 and 6y2e, respectively. In case 3, the ligand took almost 100 ps to approach and interact with the protein structure. After the initial interaction, only after 400 ps from the beginning of the production phase, the ligand was completely bound to the protein surface (Fig. 7).
An energy evaluation shows a difference of ∆E = -12787.5 kJ/mol from the beginning. The system becomes more stable, indicating a strong binding between protein and ligand (Fig. 8).
In comparison to case 1, 6lu7 shows a stronger binding with Hesperidin than with Diosmin, but as the difference is small (~10%), perhaps the kinetics presents a more important figure in this scenario. The complete bond of the ligand in case 3 is 3 times slower than in case 1, so possibly Hesperidin could be a more suitable ligand for protein 6lu7. The last system evaluated was Diosmin + 6y2e (case 4). Among all system, this was the one with highest kinetics i.e., the approach of the ligand to the protein surface was the fastest. As observed in Fig. 9, after 35 ps from the beginning of the production phase, the ligand was remarkably close to the protein 6y2e, showing a probable high interaction (dipole, Van der Waals etc.) between the structures. Some rearrangements (rotation) of Diosmin was observed, but after 200 ps, the ligand was completely bound to the protein.
When comparing this system with case 2 (6y2e + Hesperidin), it is seen that the overall kinetics and energy ( Fig. 10) of case 4 favors Diosmin as a more suitable ligand for protein 6y2e, with total energy variation of ∆E = -21695 kJ/mol. Therefore, a faster coupling and a highest energy variation make this ligand the most favorable option for this protein. properties of SARS-COV-2. In our studies, we found that ellagic acid, narirutin, neoeriocitrin and neohesperidin are highly suggested as an inhibitor of SARS-COV-2.

Conflicts of interest:
The authors declare that there are no conflicts of interest.

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