Phytochemicals As a Potential Inhibitor of COVID-19: An In-Silico Perspective

The current research has centered on the use of pharmacological and binding affinity methods to test the 36 compounds as bioactive constituents’ inhibitors for COVID-19. Six compounds out of 36 phytoconstituents (rutin, quercetin, catechin gallate, rhamnetin, campesterol and stigmasterol) have demonstrated outstanding molecular docking and drug-like properties as HIV inhibitors Lopinavir and Indinavir. Interestingly, the lowest binding energies (LBE) and the inhibition constant (Ki) have showed that these compounds are able to bind to the P-glycoprotein substrate of 3CLpro and Nsp15. Interestingly, rutin has been found to be an excellent potential inhibitor for COVID-19 proteins because it has the best LBE score and Ki value than those of other compounds, and of its ability to form strong H-bonds with COVID-19 proteins. The compounds that come next to the rutin compound are stigmasterol and campesterol. As a result, these compounds are considered possible novel inhibitors of COVID-19. In order to validate the computational results, more in vitro and in vivo investigations are required to support the findings of this research.


INTRODUCTION
Phytochemicals and secondary metabolites from medicinal plants have activity against COVID-19 as many other plants, which have shown promising therapeutic potential activity against SARS-CoV and SARS-CoV-2 [1][2][3][4][5]. Phytochemicals as well as some medicinal plants with approved antiviral, antimicrobial, and antifungal agents have been exploited towards the development of prophylactic and therapeutic agents for COVID-19 [6][7][8]. Until this date, there is no specific treatment for COVID-19; therefore, researchers have been directed to test medicinal herbs and phytochemicals as antiviral and anti-corona agents [9,10]. Currently, antiviral treatments shift toward plant-derived products because they are generally recognized as safe and have less potential for resistance development [11].

PHOTOCHEMISTRY AND MAGNETOCHEMISTRY
The medicinal plants and phytochemicals target multiple proinflammatory and oxidative mediators such as tumor necrosis factor-α (TNF-α) [12]. Among others and because of the involvement of inflammation in the pathogenesis of lung injury, phytochemicals are good candidates as novel compounds in combating corona viruses [13]. In a recent study [10], many compounds isolated from medicinal plants by GC/MS and HPLC such as gallic acid, quercetin, naringin, capsaicin, and psychotrine are important sources for novel antiviral drugs targeting COVID-19. In another study [14], quercetin, kaempferol, myricetin, apigenin, and resveratrol have shown prominent activities against coronaviruses. Alkaloids have also shown antiviral effects against coronaviruses such as Lycorine, which is an indolizidine alkaloid isolated from Lycoris radiata (L'Hér) Herb [8].
Proteins identified in SARS-CoV-2, such as nonstructural protein 15 (Nsp15) [15] and chymotrypsinlike protease (3CL pro ) [16], have been targeted by potential medications. Nsp15 is significant in viral replication as indicated with the investigation of a Human CoV 229E [17]. Nsp15 is an endo-ribonuclease that separates 3' of uridylates through a ribonuclease, and this finding can help increating powerful medications against COVID-19. On the other hand, 3CL pro plays an important role in processing of translated polyproteins. Upon exposure to SARS-CoV-2, several chemical and biological processes occur in the human body [18], and several small molecule medications that counteract corona virus have been surveyed [18].
Nelfinavir has been reported as the best potential compounds against COVID-19 based on docking results [19]. In addition, remdesivir has been reported as one of the 100 inhibitors that inhibit viral replication of SARS-CoV-2 [20,21]. Furthermore, disulfiram and neratinib as putative covalent inhibitors of SARS-CoV-2 virus 3CL pro [22].
The present study aimed to test many phytoconsitutents by docking technique using Autodock software with different computational methods. This study will add new values in the field of finding new promising inhibitors.
Several active compounds of the medicinal plants were obtained via Dr. Duke's Phytochemical and Ethnobotanical Databases [24]. All the compounds were undergone a minimization process using Avogadro software [25] with MMFF94 force field, and were saved in PDB format. Gasteiger charges were assigned for all ligands and inhibitors, and the degree of freedom remained default (the number of active torsional movement for all the ligands were less than 6). Ligand molecules were converted to PDBQT format using AutoDock 4.2.6 software [26,27].

Molecular Docking
This part was achieved by using AutoDock 4.2.6 software, where all rotatable bonds of the compounds were set randomized as completely flexible during the simulation process. Polar hydrogens and Kollman charges were added to 3CL pro and Nsp15 and saved as PDBQT. Grid box size was set to 50 × 50 × 50 points for the active binding sites. The coordinates (x, y, and z, respectively) of the 3CL pro binding site were -10.2439, 17.966, and 66.5084, and of the Nsp15 binding site were -94.65, 19.58, and -28.99 [27,28]. A maximum number of 100 runs were chosen for each independent Lamarckian genetic algorithm. The remaining parameters were kept default. AutoDock 4.2.6 was used to simulate the docking process. The 2D and 3D potential were visualized and analyzed by the Discovery Studio Visualizer 19.

RESULTS
It has been reported that the key amino acids in the active binding site of 3CL pro are HIS41 and CYS145, whereas in Nsp15 is THR341 [29][30][31][32][33]. Accordingly, there is broad consensus among researchers that promising new antiviral activity drugs need to interact with these key amino acids to stop protein activity in viral replication.
Here, the molecular docking analysis has been carried out to evaluate the interaction of medicinal plant components (flavonoids and alkaloids) with the two target SARS-CoV-2 proteins (3CL pro and Nsp15). The binding affinity of the interacting compounds to the active site residues (CYS145 and HIS41) of 3CL pro and THR341 of Nsp15 are shown in Table 1.
All the selected ligands docked in 3CL pro and Nsp15 pockets possess varying scores with the enclosed amino acids according to the determined coordinates. Remarkably, as shown Table 1, rutin, quercetin, catechin gallate, rhamnetin, stigmasterol, campesterol overtake the rest of the drugs by forming a strong interaction with the protease of both enzymes with lowest binding energy (LBE) scores and lowest K i values. Obviously, the affinity of binding of the campesterol has the least value as compared to the values of all selected drugs with 3CL pro , whereas rutin has the least value as compared to the values of all selected drugs with Nsp15.

DISCUSSION
The docking simulations have been carried out for all the selected ligands with two COVID-19 proteins (see Table 1). The findings of this study show that some of phytoconstituent compounds tend to the enzymes more than other compounds. The lowest binding energy (LBE) scores of drugs that are lower than -7.46 kcal/mol with Nsp15 are assumed to show strong interactions and may significantly impair enzymatic activities. For instance, six phytoconstituent compounds (rutin, quercetin, catechin gallate, rhamnetin, stigmasterol and campesterol) have lower LBE scores than -7.46 kcal/mol for Nsp15 and -5.76 kcal/mol for 3CL pro . These compounds are predicted to have the highest potency with strongest interaction in terms of LBE scores and lowest inhibition intensity K i than the other standard compounds. In addition, these compounds reveal a strong binding affinity to both proteins and their LBE scores are lower than the LBE score of vitamin D, which is considered a potential inhibitor [34]. Amazingly, our pharmacokinetic prediction results have shown that rutin, quercetin, catechin gallate, stigmasterol and campesterol have excellent analysis (LBE and K i ), which are better than the LBE and K i of the standard HIV-inhibitors (lopinavir and indinavir).The approved antiparasitic drugs by Food and Drug Administration (FDA), such as, ivermectin, spiramycin and praziquantel show good LBE scores and K i values for Nsp15 and weak interaction with 3CL pro as compared with other phytoconstituents compounds, which are not approved by the FDA for the treatment of any viral infection [35,36]. The docked structures of rutin, quercetin, catechin gallate, rhamnetin, stigmasterol, and control complexes of HIV-inhibitors (lopinavir and indinavir) with main protease 3CL pro and endoribonuclease of SARS-CoV-2 are presented in Figs. 1 and 2, respectively, to recognize key amino acid interactions in the pockets and to evaluate the inhibitory effects in the viral replications.
Strikingly, the findings of this study have exhibited the ability of the rutin, quercetin, catechin gallate, rhamnetin, campesterol and stigmasterol to form strong H-bonds with the key amino acids of the main protease 3CL pro (HIS41 and/or CYS145) from the hydroxy group of each compound in the following order: stigmasterol > campesterol > quercetin > rutin > rhamnetin > catechin gallate. Furthermore, for each potential inhibitor, H-bonds with the key residue THR341 in the Nsp15 are found to be in the following order: rutin > campesterol > stigmasterol > catechin gallate > rhamnetin > quercetin.
Interestingly, compared with standard control, the strongest potential natural compounds for both enzymes are found to be in the following order: rutin > stigmasterol > campesterol. This observation indicates that these compounds may be potent drugs to inhibit the viral replication of COVID-19 by halting the activity of the two essential proteins. CONCLUSIONS LBE scores, K i values, and the interactions of all compounds with the active sites of 3CL pro and Nsp15have been measured to confirm the affinity of these compounds to interact with these proteins, and to classify the potential lead drugs according to their affinity and pharmacological properties. Docking scores have revealed that rutin, quercetin, catechin gallate, rhamnetin, campesterol, and stigmasterol have lower LBE score than -7.46 kcal/mol for Nsp15 and -5.76 kcal/mol for 3CL pro . Moreover, the analysis of the interactions of these compounds with the key amino acids in 3CL pro (HIS41 and/or CYS145) and Nsp15 (THR341) has shown that rutin has the best LBE score and K i value compared with the LBE score and K i value of other compounds, due to it stability to form strong H-bonds with proteins. The compounds that come next to the rutin compound are stigmasterol and campesterol. In order to confirm the computational findings in this study, the results of this study need further in vitro and in vivo investigations. RUSSIAN   Interactions van der Waals π-Alkyl π-σ van der Waals