The Study of Potential Antiviral Compounds from Indonesian Medicinal Plants as Anti-COVID-19 with Molecular Docking Approach

Corona Virus Disease 2019 (COVID-19) is a disease caused by the outbreak of SARS-CoV-2 infection, a new coronavirus strain1. Food and Drug Administration of America (US FDA) stated that there is still no specific drug to inhibit the virus until this paper was written. Only broad-spectrum antiviral such as remdesivir are used to treat the infection2. Remdesivir is a prodrug and nucleoside analog, as a prodrug remdesivir has two active metabolites such as remdesivir nucleoside (GS-441524) and remdesivir triphosphate (GS-443902)3,4. The rapid transmission of COVID-19 from human to human forces researchers to find a potent drug, including using natural sources as an alternative5. However, natural drug development has its limitation, including it is time-consuming. This limitation could be addressed by using the molecular docking approach6,7. Molecular docking is one of the in silico approaches used to design or select compounds that can be used as an inhibitor or an activator of a target protein, and it also assesses the mechanism of action in the molecular state8. The Study of Potential Antiviral Compounds from Indonesian Medicinal Plants as Anti-COVID-19 with Molecular Docking Approach


INTRODUCTION
Corona Virus Disease 2019 (COVID-19) is a disease caused by the outbreak of SARS-CoV-2 infection, a new coronavirus strain 1 . Food and Drug Administration of America (US FDA) stated that there is still no specific drug to inhibit the virus until this paper was written. Only broad-spectrum antiviral such as remdesivir are used to treat the infection 2 .
The rapid transmission of COVID-19 from human to human forces researchers to find a potent drug, including using natural sources as an alternative 5 . However, natural drug development has its limitation, including it is time-consuming. This limitation could be addressed by using the molecular docking approach 6,7 . Molecular docking is one of the in silico approaches used to design or select compounds that can be used as an inhibitor or an activator of a target protein, and it also assesses the mechanism of action in the molecular state 8 .
As mentioned by the Indonesian Food and Drug Administration (BPOM Indonesia), several medicinal plants from Indonesia with antiviral activity can be used to treat COVID-19 patients, such as turmeric (Curcuma longa), curcuma (Curcuma xanthorrhiza), gale of the wind/meniran (Phyllanthus niruri), green chiretta/sambiloto (Andrographis paniculata), and guava (Psidium guajava) 9 . Some research has been done to prove the plants have potential secondary metabolite as an antiviral to support this statement. Curcumin, demethoxycurcumin, and bisdemethoxycurcumin in C. longa or C. xanthorrhiza have been shown to have an activity to inhibit 3C-like protease (3CL pro ) and spike protein of SARS-CoV-2 in the in silico study. At the same time, another study also reported that curcumin affect 3CL pro of SARS-CoV-2 in the in vitro research 10, 11 . Based on in vitro study, quercetin in P. guajava was shown to inhibit SARS-CoV-2 papain-like protease (PL pro ) with an IC50 of 8.6 µM 12 . Luteolin, apigenin, quercetin, and kaempferol in P. guajava also shown potency to inhibit 3CL pro of SARS-CoV-2 based on in silico study 13 . Based on another in silico study with AutoDock Vina, quercitrin in P. niruri, as well as andrographolide and neoandrographolide from A. paniculata, show the best potential as a 3CL pro SARS-CoV-2 inhibitor based on free energy acquisition 14 .
In the life cycle of SARS-Cov-2, two non-structural proteins have a crucial role. First, 3CL pro plays a role in replicating polypeptides into functional proteins to multiply viruses 15 . The 3CL pro plays a role in synthesizing replicas through proteolysis mechanisms, and viruses will use these replicas to construct structural proteins to multiply themself 16 . Meanwhile, PL pro serves as an essential virulence factor for the virus. The PL pro works by untying ISG15 from IRF3, thus inhibiting Interferon (I and III) formation, which serves to signal the immune system 17 . This protein has the potential to be used as a target for treatment; in addition to genetic similarities between SARS-CoV (96%) and SARS-CoV-2, 3CL pro and PL pro also has not undergone mutations such as D614G in viral spike proteins 18 . Based on this background, this study aims to assess the potency of compounds from five medicinal plants from Indonesia, as mentioned before, as a potential inhibitor of PL pro and 3CL pro from SARS-CoV-2, using a molecular docking approach.

Docking protocol
Overall, the docking protocol used in this study was based on the docking protocol used by Purnomo et al 20 .

Preparation of target protein and native ligand
The target protein and native ligand preparation were carried out using YASARA View. The protein was obtained by deleting its native ligand in the PDB file (Figure 1). In contrast, the native ligand was obtained by deleting the protein from the PDB file (Figure 2). In

Docking protocol validation
Docking protocol validation aims to obtain a root median square deviation (RMSD) value. The RMSD value was asses using YASARA View by re-docking the native ligand to its protein. Docking protocols stated as a valid protocol if the RMSD value <2 Å 21 .

Test ligand docking
Docking was done using PLANTS by typing the commands in cmd.exe. The optimized test ligand was docked to the active site of both PL pro and 3CL pro . PLANTS will read the command that has been set before to obtain the best docking score of each test ligand. The final result of the docking score would be compared with the best score of the native ligand.

Assessment
The assessment was performed using a descriptive approach. The results from the docking process were the docking score of each interaction between the test ligand and target protein. The docking score indicates affinity between the ligand and target protein. The docking score was described as a negative value, meaning the test ligand had a good affinity and potential as the inhibitor of the target protein 22 . The test ligand was stated to had potential as the target protein inhibitor if the docking score was more negative than the docking score of remdesivir triphosphate.

Curcuma longa and Curcuma xanthorrhiza
The docking score between curcumin, demethoxycurcumin, and bisdemethoxycurcumin with both PL pro and 3CL pro was shown in Figure 4. The interaction shows that curcumin had the best potency as a PL pro inhibitor because the docking score (-103.609) was almost the same as remdesivir triphosphate (-103.827). Another in silico research using AutoDock Vina claimed curcumin acted best as PL pro inhibitor among four other targets like angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2), RNAdependent RNA polymerase (RdRp), and 3CL pro , with the free binding energy (ΔG) of -8.45 kcal/mol, which was less than those ACE2 (-7.99 kcal/mol), TMPRSS2 (-7.19 kcal/mol), RdRp (-5.3 kcal/mol), and 3CL pro (-7.24 kcal/mol) 23 . Interaction with 3CL pro shows demethoxycurcumin was the most potent inhibitor of 3CL pro . The docking score of demethoxycurcumin (-87.1625) was less than remdesivir triphosphates (-86.1811). Sharma et al. 24 reported that demethoxycurcumin was one of the best inhibitors of 3CL pro with ΔG of -7.02 kcal/mol. This result was based on in silico study using AutoDock 4 to assess some medicinal plants metabolites as 3CL pro inhibitor. Curcuminoids in both plants have been shown to have antiviral activity, in which curcumin shows antiviral activity in HIV as a protease inhibitor in HIV-1 and HIV-2 based on in vitro and in vivo studies 25 . Other studies suggest that curcumin shows the activity as a SARS-CoV-2 3CL pro and spike protein inhibitor based on molecular docking study 10,11 . Curcumin also reported inhibiting 3CL pro in SARS-CoV with an IC50 of 3.3 to 10 µM 26 .

Psidium guajava
The docking score between luteolin, apigenin, quercetin, and kaempferol with both PL pro and 3CL pro was shown in Figure 5. It turns out that the ligands were not potent enough as a PL pro inhibitor. However, among the four ligands, quercetin had the lowest docking score of -80.286. Interaction with 3CL pro shows that luteolin (-73.4345), apigenin (-70.3368), quercetin (-71.5539), and kaempferol (-68.4321) had potency as 3CL pro inhibitor, although the docking score was still higher than remdesivir triphosphate (-86.1811). Previous in silico research has shown that based on the free binding energy data, apigenin, luteolin, and quercetin had more potential as 3CL pro inhibitors (-7.4; -7.12; and -6.83 kcal/mol, respectively) than as PL pro inhibitor (-6.6; -6.9; and -6.6 kcal/mol) 23 .

Phyllanthus niruri
The docking score of formononetin-7-O-glucuronide with PL pro and 3CL pro was presented in Figure 6. The docking score data shows that formononetin-7-Oglucuronide might not have potency as a PL pro inhibitor but had potency as an inhibitor of 3CL pro because the docking score (-72.0154) was less than native ligand (-64.0074) but higher than remdesivir triphosphate (-86.1811). Docking between formononetin-7-O-glucuronide and the receptor for SARS-CoV-2 has not been previously reported, whereas another similar study using a metabolite of P. niruri chose nirurin as the test ligand 27 .

Andrographis paniculata
Andrographis paniculata was an Indonesian plant with the main metabolite in lactone terpenoids such as andrographolide and neoandrographolide 28 . The docking score of andrographolide and neoandrographolide with PL pro and 3CL pro was shown in Figure 7. Based on the results, both andrographolide (-79.1989) and neoandrographolide (-97.2452) had no potency as PL pro inhibitors. On the contrary, the interaction between neoandrographolide with 3CL pro shows the docking score (-93.7746) less than remdesivir triphosphate (-86.1811) and could act as the most potent ligand for 3CL pro inhibitor. Murugan et al. 14 reported that in the in silico research using AutoDock Vina, neoandrographolide had ΔG as 3CL pro (-31.4 kcal/mol), PL pro (-28.5 kcal/mol), RdRp (-17.1 kcal/mol), and spike protein (-23.9 kcal/mol) inhibitors, in which they were best for 3CL pro inhibitor. In addition, andrographolide was the main antiviral compound that was often found in A. paniculata 29 . This compound could inhibit the dengue virus in HeLa (EC50 22.739 µM) and HepG2 (EC50 21.304 µM) cells 30 .     Based on the docking score of test ligands with PL pro , curcumin was the most potent inhibitor of PL pro with the lowest docking score of -103.609, while apigenin was the ligand with the highest docking score, as shown in Table I. This result indicates that curcumin had a good affinity when the interaction occurred between the ligand and PL pro . Based on the docking score, neoandrographolide was the ligand with the lowest docking score of -93.7746, while kaempferol was the highest with -68.4321. The docking score of neoandrographolide indicated a good affinity with 3CL pro and potency to acted as the most potent ligand to inhibit 3CL pro . Curcumin and neoandrographolide were stated as the most potent candidate for PL pro and 3CL pro inhibitors, respectively. The prediction from this research could be used to develop an anti-COVID-19 drug from herbal compounds such as combination with extract or isolate that contains curcumin and neoandrographolide as the main compounds.
Moreover, combining these two compounds could enhance the inhibitor effect in PL pro or 3CL pro of SARS-CoV-2. However, further in vitro and in vivo research was required to confirm this finding.

CONCLUSION
Based on the docking score, it can be concluded that curcumin has the most potential as a PL pro inhibitor, while neoandrogapholide has the most potential as a 3CL pro inhibitor.

CONFLICTS OF INTEREST
The authors have no conflicts of interest to declare that are relevant to the content of this article.

FUNDING
None.

DATA AVAILABILITY
All data are available from the authors.