Molecular Docking Studies of SARS-Cov-2 Mainprotease Potential Inhibitors

BACKGROUND : SARS-Cov-2 causes an coronavirus disease 2019 (COVID-19), and the vaccines or drugs of this disease have not been found to inhibit this replication of the virus. Researchers are engaged in all elds of study to discover new potential inhibitors. This study aimed to compute the binding energy (BE) and interactions between the new potential inhibitors and the SARS-Cov-2 Mainprotease (Mpro). METHODS: In this study, we docked between twenty-seven patented drugs and the Mpro receptor (PDB ID: 6W63). The molecular docking calculation was performed using AutoDock Tools 1.5.6. software. Moreover, the information about the biological activity of ligands was calculated using the PASS online server. The result of the calculation was then analyzed and visualized using Biovia Discovery Studio Visualizer. Further calculation, such as the ligand-protein interaction using STITCH database and Lipinski’s rule ve were employed in this research. RESULTS: The molecular docking calculation results showed that nelnavir was strongly bound to Mpro with BE of -9,51 kcal/mol, followed by lopinavir, vitamin D, ritonavir, and dexamethasone. From these ligands, we considered dexamethasone because this ligand works as an anti-inammatory agent. CONCLUSIONS: Following the calculation, nelnavir, lopinavir, and dexamethasone are proposed as a potential inhibitor of the Mpro receptor, but there is a need for further investigation.

receptor glycosylation, so it prevents the SARS-Cov-2 binding the cell target 9 . Other than 3CLpro, the virus replication can also be inhibited by inhibiting Mpro.
The crystal structure of the SARS-Cov-2 Mpro has been identi ed and stored at Protein Data Bank, and one of them is a liganded 6W63 receptor with X77, which is a novel inhibitor. The previous study reported that nel navir ritonavir, and darunavir may interact with this receptor 6 . Because the vaccines or cures for COVID- 19 have not yet been identi ed, patented drugs have been used as a calculation input for this research. The information on the drug was used in this study was collected from the previous research 10 .
Thus we performed molecular docking calculation to identify candidates as potential inhibitors of Mpro.

Molecular Docking Calculation
The molecular docking calculation was conducted using 27 compounds which are Nel na r, Lopinavir, Vitamin D [11][12][13] , Ritonavir, Dexamethasone, Baloxivir Marboxil, Alpha Tocopherol Acetate (Vitamine E), Vitamin A, Umifenovir, Darunavir, Cobicistat 6 , Meropenem 10 , Midazolam, Levo oxacin, Chloroquine Phosphate, Chloroquine 14 , Oseltamivir, Chloroquine Sulfate, Hydroxychloroquine, Cefotaxime, Salbutamol Sulfate, Remdesivir, Acetaminophen, Ribavirin, Favipiravir, Ascorbic acid (Vitamin C), and Acetylcysteine that were retrieved from pubchem.ncbi.nlm.nih.gov, and the receptor is obtained from www.rcsb.org with PDB ID: 6W63. Before molecular docking calculation was performed, the receptor and ligand were prepared using Chimera 1.14 15 . The redocking calculation was conducted between native ligand (X77) and the active site of the 6W63 receptor. The molecular docking calculation was performed utilizing AutodockTools 1.5.6 sized the grid box 40 x 40 x 40 points, spacing 0,375 Ǻ. For searching the parameter, we used the Lamarckian Genetic Algorithm (LGA) in 100 runs 16 . The result of the calculation with the best conformation was visualized using Biovia Discovery Studio Visualizer to observe molecular interactions.

Biological Activity Analysis
Twenty-seven ligands were analyzed using the PASS server online (http://www.pharmaexpert.ru/passonline) to determine their biological activity. This analysis is based on Structure-Activity Relationship (SAR). This step yielded a probability of active (Pa) and the probability of inactive (Pi) with range 0-1. If the Pa value is greater than 0.7, so the compound has a biology activity 17,18 . Lipinski's Rule and STITCH Database Page 4/25 The drug-like properties of the 27 potential inhibitors were calculated by using Drug Likeness Tools (DruLiTo) software based on the Lipinski's rule ve. The potential inhibitors were then further analyzed using a server online Search Tool for Interactions of Chemical (STITCH) (http://stitch.embl.de/) to predict ligand-protein interaction.

Result
In this study, we use the 6W63 receptor liganded to X77. The redocking calculation was conducted for standard data and method validations, and then further calculation was performed on a new potential inhibitor with the same parameter as the redocking process. The result of redocking between receptor and the native ligand is depicted in Figure 1. The result of the molecular docking calculation between the Mpro receptor and 27 drugs is given in Table  Sulfate, Remdesivir, Acetaminophen, Ribavirin, Favipiravir, Ascorbic acid (Vitamin C), and Acetylcysteine 6,10-14 . Following Table 1, there is a ligand with BE comparable or better than native ligand, nel navir.  Table 1 shows that several ligands interact with the same amino acid residues compared to the native ligands through conventional hydrogen bond and other secondary interactions. The potential inhibitors that bind to one amino acid residue are nel navir, vitamin D, ritonavir, vitamin E, vitamin A, umifenovir, darunavir, meropenem, chloroquine, oseltamivir, chloroquine sulfate, cefotaxime, salbutamol sulfate, acetaminophen, and acetylcysteine. There are also other potential inhibitors that bind two amino acid residues, such as baloxivir marboxil, cobicistat, favipiravir, and Vitamin C. Other ligands such as lopinavir, dexamethasone, midazolam, levo oxacin, chloroquine posphate, hydroxychloroquine, remdesivir, and ribavirin do not form the same interaction with native ligand. In addition, Table 1 also shows BE information of its ligands with the active site of Mpro receptor. Nel navir is the lowest BE compared to native ligand, while the other potential inhibitors are higher than native ligand. In addition to the BE calculations, amino acid residues are crucial information to discuss a potential inhibitor of Mpro receptor inhibition. Figure 1 shows that the native ligand binds three amino acid residues via conventional hydrogen bonds such as GLU166, CYS145, and GLY143, even as non-classical hydrogen bonds are LEU141, SN142, and THR26. Native ligand also has secondary interactions such as electrostatic (HIS164) and hydrophobic (MET165, LEU141, CYS145, HIS41, and LEU27). Nel navir attaches the residues of amino acids GLN189, GLU166, PHE140, and HIS164. The interaction with GLU166 amino acid residu shows that nel navir has the same interaction with native ligand. Hydrophobic interaction with MET165 amino acid residue has also occured in this complex. Nel navir also interacts with CYS145 through sulphur interaction (see pic. 1.c. and 1.d.). Referring to Figure 1.d, nel navir binds four amino acid residues such as HIS164, PHE140, GLU166, and GLN189. HIS164 is bound to hydroxyl, PHE140 hydroxyl, and aromatic, GLU166 to a ketone, and GLN189 to an amide. Other potential inhibitors that included on the top seven BE are depicted in Figure 2. Figure 2 shows 2-dimensional visualization of several Mpro receptor inhibitors, namely Lopinavir, vitamin D, ritonavir, dexamethasone, baloxivir marboxil, vitamin E, which have BE of -9.13 to -8.10 kcal/mol. Lopinavir is tied to HIS41 on the Mpro receptor through a conventional hydrogen bond. Vitamin D and ritonavir bind the same amino acid residue, GLU166. Dexamethasone has different interactions with the native ligand. Baloxivir marboxil interacts with GLU166 and GLY143, while vitamin E interacts with GLY143. The Pa analysis, Lipinski's rule, and ligand-protein interactions were performed to explain the potential inhibitors of Mpro. To ensure of molecular docking data, we use the PASS server online to perform a Pa value analysis. The Pa value was obtained from the SMILE of each of the potential inhibitors. The SMILE of each potential inhibitors was used to calculate Pa value. The results of this analysis are Pa and Pi values, which can be considerable discussion of this study. The Pa analysis of inhibitors is used to explore a speci c activity as an antiviral agent based on SAR. Table 2 shows the Pa of antiviral activity in the range 0-1. Based on There is a crucial analysis in drug design, i.e., Lipinski's rule of ve. Table 3 shows that the 27 simulated potential inhibitors do not display more than two violations of Lipinski's rule of ve. Among the potential inhibitors on Table 3, it can be observed that dexamethasone, umifenovir, meropenem, midazolam, levo oxacin, chloroquine, oseltamivir, hydroxychloroquine, salbutamol sulfate, acetaminophen, ribavirin, favipiravir, vitamin C, and acetylcysteine do not violate. At the same time, the remaining shows contravention of 1 namely nel navir, lopinavir, vitamin D, E, A, and cefotaxime. Ritonavir, darunavir, cobicistat, and remdesivir are in violation 2. Clculations for the chemical-protein interaction of four (4) potential inhibitors of Homo sapiens are shown in Figure 3. Drugs are depicted as shaped pills, while the proteins are represented as a sphere. The nodes describe a drug action to protein such as green (activation), red (inhibition), blue (binding), cyan (phenotype), purple (catalysis), black (reaction), pink (posttranslational modi cation), and yellow (transcriptional regulation). Based on the STITCH database, the interaction scores are divided into three (3) clusters, namely medium con dence (0.40-0.70), high con dence (0.70-0.90), and the highest con dence (0.90-1.00). The most important interaction that contributing to this prediction is binding, inhibition, and activation 20 , so the discussions in this article are based on three clusters.

Discussion
The previous studies have shown that nel navir may bind with other Mpro receptors such as 6LU7 and 3M3V. The BE results of these studies were -10.72 and -17.31 kcal/mol for 6LU7 and 3M3V. Although their tendency to interact poses was similar, the interactions of these complexes with each protein were different. In this study, nel navir interacts with the residues of amino acids GLN189, HIS164, PHE140, and GLU166, while it was bound with CYS145, MET49, MET165, GLU166, LEU167, PRO168, GLY170, GLN189, THR190, ALA191 and HIS41 in the active site of the 3M3V receptor 1 . As a result, nel navir is often used as a standard for molecular docking research 6,7 Previous studies have shown that, lopinavir also has a functional inhibition of the Mpro receptor. In this research lopinavir is one of the top four, -9.13 kcal/mol. Although the BE score is higher than native ligand, this drug is reported as an inhibitor of the SARS-Cov-2 on Vero E6 cells with EC 50 below 100 µM 21 . This drug may be used in vitro against SARS-Cov, MERS-Cov, and hCov-229E 7 . Lopinavir is often combined with ritonavir for inhibiting the Mpro receptor. This step causes the uses of lopinavir in a mixture that is not too much 7,22,23 . The previous data showed that twenty-seven COVID-19 patients have treated with lopinavir 400 mg/ritonavir 100 mg 14 . These drugs have also been reported to inhibit HIV protease, a member of β-coronavirus, due to their therapeutic bene ts as antivirals. Lopinavir was shown to have the best therapeutic bene t compared to ritonavir 24 . In addition to nel navir and lopinavir, the results of this study show that vitamin D in potent as an inhibitor of Mpro.
Vitamin D has the lowest BE among vitamins in this study, -9.12 kcal/mol. This vitamin is used in the treatment to improve the immune system of patients and as a preventive measures. Previous studies have reported that the use of vitamin D in therapy may reduce T helper cell type 1 (Th1) cytokines (tumor necrosis factor-α and interferon-γ) and expression of pro-in ammatory [25][26][27] . The use of vitamin D in the Mpro receptor inhibitor reduces the expression of the dipeptidyl peptidase-4 receptor (DPP-4), a molecular virulent that reacts with the SARS-Cov-2 spike glycoprotein 28 . However, there is no rm evidence of the relationship between vitamin D and the death of patients 12,13 . Vitamin A has BE -7.99 kcal/mol and may also increase the immune system in the human body. This vitamin is included in the list of drugs used to treat and to maintain immune system stabilization in patients 29,30 .
Vitamin E and C are also used as immune boosters for treatment with COVID-19 globally. The BE for vitamin E and C is -8.1 dan -4.08 kcal/mol, respectively. Both compounds are antioxidants that can be obtained from fruit and vegetables in our dietary menu. Also these antioxidants may be obtained from supplements. The BE of Vitamin E is lower than Vitamin C, so vitamin E is expected to be more strongly bound to the Mpro receptor. The previous study explained that this antioxidant might increase T-cell subsets, lymphocyte reactions to mitogen, interleukin-2 production, potentiated natural killer cell activity, and in uenza virus response compared with placebo. The use of antioxidants in the therapy process is essential to maintain physical and spiritual conditions during the quarantine period, and the healing process is quickly completed 31,32 .
The anti-in ammatory agent is included in the list of the drugs used for therapy because the immune response of COVID-19 patients has increased such as the number of leukocytes, platelets, lymphocytes, and neutrophils 33,34 . Dexamethasone is one of the corticosteroid groups used to cure a patient of SARS-Cov infection in patients 19,35 . Oxford University recently researched the use of dexamethasone to chronic patients in the United Kingdom (UK), and the WHO has accepted the results of the. Based on the previous research review, the corticosteroid group has been shown to have clinical bene t. Critically, genomic SARS-Cov-2 and SARS-Cov are similar, so a literature review is conducted to gather data and evidence on the e cacy of the drug in the treating of patients. The use of this drug is better than Non-steroidal Anti-In ammation Drugs (NSAIDs) 35,36 . In general, NSAIDs are used as a pain killer for acute to chronic in ammation. Long-term consumption of this drug may cause adverse cardiovascular events in the human body 16 . Ibuprofen is one NSAIDs, and the use of these drugs causes an increase in ACE2 expression. The treatment of patients with co-morbid (diabetic) using NSAIDs may increase the incidence of SARS-Cov-2 infection 37 . Researchers are still discussing COVID-19, followed co-morbid. The previous study reported that co-morbid (cancer) is not directly related to acute in ammation 38 .
In addition to previous ligands, there are a number of other potential inhibitors such as umifenovir, darunavir, cobicistat, meropenem, midazolam, and levo oxacin. The BE of these ligands shall be between -7.83 and -7.0 kcal/mol. Generally, the BE of these potential inhibitors is higher than that of native ligand, but these inhibitors are still used for therapy. Umifenovir's function is to inhibit the interaction of the virus with the host cell membrane 14 . Umifenovir (arbidol) is used in vitro to cure in uenza A infection. Applications of this drug to SAR-Cov-2 therapy still require advanced research to complete the information 39 . Darunavir is also known as an inhibitor of HIV-1 protease, and this drug may prevent the dimerization of HIV-1 protease 40 . In Atlanta, at dose of 800 mg darunavir/Cobicistat 150 mg, darunavir/cobicistat was used for 2 weeks (one daily) of patients 14 . In addition to antioxidants, antibiotics are also used in the treatment of patients. Earlier work described that almost all patients received antibiotics, antiviral, and glucocorticoid. Meropenem and levo oxacin are mostly antibiotics used in the medication of COVID-19 victims [41][42][43][44] .
Chloroquine phosphate, chloroquine, oseltamivir, chloroquine sulfate, hydroxychloroquine, cefotaxime, salbutamol sulfate, remdesivir, acetaminophen, ribavirin, favipiravir, vitamin C, and acetylcysteine have BE -6.55 to -3.51 kcal/mol. The BE of these potential inhibitors is higher than native ligand, so these drugs are expected to interact weakly with the active site of the Mpro receptor. Newly, chloroquine phosphate has an excellent clinical effect on the treatment of this viral virus, while the molecular docking calculation of this study is -6.55 kcal mol -1 . Based on this score, this drug is weakly bound to the Mpro receptor 45 .
Recently, a viral inhibitor has been used to treat patients with chloroquine, an antimalaria agent. The inhibition chloroquine mechanism on SARS-Cov-2 has not yet been invented. The previous study recommended that this drug be developed to treat COVID-19 with diabetes in India. The therapy was designed with a view to minimizing the risk, prolonged use, cost, and source of raw material 14 . The coronavirus seems to be using hemaglutinin-esterase, which binds sialic acid to the surface of the glycoprotein. There is an indication that this virus interacts with ACE2. When this virus binds to ACE2, so this process will continue to replicate the virus. Chloroquine could well interfere with the mechanism of ACE2, and this drug inhibits the reaction with the target cell. Chloroquine may interfere with the biosynthesization of sialic acid. This drug also modulates the acidity of the endosome to prevent the formation of autophagosomes. When mitogen-activated protein (MAP) kinase activation is reduced, virus replication may be managed to stop. This potential inhibitor interfere in the maturation of protein M and the creation of virion before entering the host cell 9 . The e cacy of this drug is to increase the pH of the endosome, a condition for the fusion virus 23 .
Previous study reported that chloroquine demonstrated inhibition of Mpro receptor performance by indirect immuno uorescence assay. Inhibition of infection with this drug has been used to destabilize the terminal glycosylation of the cellular receptor, ACE2 46 . Chloroquine is known to have been cheap and widely used 8,45 . This drug may react with ritonavir/lopinavir in patient therapy 22,47  Besides the potential inhibitor explained above, there are ligands that have to BE higher than native ligands such as oseltamivir, chloroquine sulfate, hydroxychloroquine, cefotaxime, salbutamol sulfate, remdesivir, acetaminophen, ribavirin, favipiravir, vitamin C, and acetylcysteine. Oseltamivir with the trademark Tami u was recognized as an antiviral agent that alters free carboxylate as an active metabolism 22,49 . Apart from oseltamivir, many researchers suggest hydroxychloroquine as opposed to chloroquine because hydroxychloroquine has a low toxin level. In vitro, hydroxychloroquine was the most proposed drug than chloroquine 22,50 . Since then, the USA has claimed that remdesivir can be used in the treatment of COVID-19 patients in their country. Subsequently, the recent study claimed that remdesivir had no clinical bene t from chronic COVID-19 51 . This overview reveals that nding drugs or vaccines for this viral virus still requires a long research in all eld studies.
Based on Table 1, favipiravir has a BE value of -4.14 kcal mol -1 . This value is higher than native ligand, so this compound is estimated to be weakly constrained to the Mpro receptor relative to a native ligand.
China and Japan used this drug to diagnose and treat in uenza 52 . Favipiravir has not shown a binding effect under 100 µM 21 . Recent studies have suggested that favipiravir has been used to treat in uenza infection (A, B, C). This drug was also used to treat H5N1, Ebola (EBOV), human norovirus, and human arenavirus 49,53 . The inhibition of RNA-dependent polymerase is a mechanism of favipiravir in the healing process 14 . The further research in order to obtain more comprehensive data 54,55 . Chloroquine sulfate, cefotaxime, salbutamol sulfate, acetaminophen, and acetylcysteine do not show any results that could be considered from BE results.
According to Table 1, the native ligand is well-posed at the binding site of GLU166, CYS145, and GLY143 due to conventional hydrogen bonds. Subsequently, this novel inhibitor also interacts with residues of amino acids in the hydrophobic regions MET165, LEU141, CYS145, HIS41, and LEU27. In addition to hydrogen bond and hydrophobic interaction, the X77 also forms electrostatic interaction through HIS164 to strengthen its position. The existence of a hydrogen bond is crucial to the inhibition mechanism between drug candidates and Mpro receptors. This nding by the authors is compromised by a previous study 56 . Almost all molecular docking calculations between ligands and Mpro receptor always react with the amino acid residues of GLY143, next CYS145, GLU166, and HIS163, so the amino acid residues are hypothesized as a priority in inhibiting Mpro receptor 57 .
According to BE calculation, nel navir is the most potent potential candidate in this study, but this drug was compared to native ligand only via GLU166 for inhibiting the Mpro receptor. Nel navir is only attached to MET65 in the hydrophobic region and shows sulfur interaction with CYS145. A conventional hydrogen bond is a primary interaction in this discussion, but the hydrophobic also contributes to the stabilization of the drug-receptor complex 55 .
Lopinavir unveils a different interaction with native ligand and interacts with HIS41 via conventional hydrogen bonds. This potential inhibitor binds to MET49, PRO52, ARG188, MET165, and LEU27 in the hydrophobic environment. Sulfur interaction also presents in this complex, CYS145. The previous study showed that this drug was used as an experimental standard. This drug was also successfull in inhibiting HIV, SARS, and MERS treatments 7,36 . Lopinavir is used in combination with ritonavir to cure COVID-19 patients in several countries 51 . Lopinavir is also co-formulated with ribavirin for an inhibitor of SARS-Cov 36 . The routine use of this drug has received a weak recommendation because it does not have su cient evidence in the case of this pandemic 58 . According to the interaction of amino acids, nel navir is still considered an excellent potential inhibitor of the Mpro receptor. Beside lopinavir, ligand from vitamin group is regarded as a new potential inhibitor, vitamin D. Vitamin D, an immune system booster, is used to protect COVID-19 patients. It shows hydrogen bond interaction with GLU166 and is useful in agreement with the native ligand. Although it does not show 100% the same interaction relative with the native ligand, with a vitamin D hydrophobic environment to stabilize the complex, namely MET165, HIS41, CYC145, MET49, LEU27, and CYS44. In order to clarify the explanation, we conducted a Pa analysis, Lipinski's rule, and ligand-protein interaction of potential inhibitors to explore their therapeutic bene ts and drug-like properties.
In Pa calculation, umifenovir has speci c activity an in uenza antiviral. This result is consistent with the previous study that umifenovir was antiviral in uenza in Russia and China 19 . To contribute, the new potential inhibitor is simulated by an online analysis using the PASS server to identify therapeutic candidates as antiviral agents. According to Table 2, umifenovir, darunavir, oseltamivir, remdesivir, and ribavirin have Pa values between 0.1 to 1. Compounds with an antiviral activity value of Pa in a computational or laboratory analysis 17,18 . The Pa value of the PASS analysis results is not speci c to SARS-Cov-2. This can be used as a material for futher research consideration. In this result, Pa value nel navir has a low antiviral activity score, while oseltamivir shows the highest score.
Lipinski's rule of ve gives us an overview of the drug-like inhibitor candidate. Overall, in Table 2, compounds with the violation value of more than one do not meet the criteria for the drug-likeness 59 .
The value of MV is a parameter for the penetration of drugs in the human body's cell membrane. If the MV is more than 500 Da, the drugs hardly enter into the cell membrane. Nel navir, lopinavir, ritonavir, baloxivir marboxil, darunavir, cobicistat, and remdesivir have MV greater than 500 Da, and may interfere with the diffusion of the cell membrane. It was also the case for logP, the H-bond donor, and the acceptor. If the limit of Lipinski's rule of ve is violated by a candidate inhibitor, the candidates may not be suitable for laboratory synthesis 60 . Lipinski's rule of ve still shows that nel navir has good criteria to be a drug candidate for this viral virus.

Conclusion
Calculation of molecular docking using 27 potential inhibitors and the Mpro receptor yields several data.
The BE of these potential inhibitors shows that nel navir is the best Mpro receptor complex supported by amino acid residue interaction. Other potential inhibitors in this study is lopinavir, and vitamin D. Previous study reported that, there was no direct evidence between vitamin D and COVID-19 infection. The analysis of Lipinski's rule of ve shows that nel navir, lopinavir, and vitamin D do not display more than one violation. Dexamethasone as an anti-in ammatory agent may also be considered in this study because there is no violation of the result of Lipinksi's rule of ve. Based on the calculation and analysis data in this study, nel navir, lopinavir, and dexamethasone are recommended as candidates for a new potential Mpro receptor inhibitor. However, further research needs to be conducted to obtained more accurate data.  Interaction of potential inhibitors on the Mpro receptor based on complexes(d)