Active Compounds Activity from the Medicinal Plants Against SARS-CoV-2 using in Silico Assay

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), better known as the coronavirus, is a new type of coronavirus that is transmitted to humans. This virus infection is called COVID-19 and was first discovered in the city of Wuhan, China, at the end of December 2019. This virus spread quickly and has spread to other regions in China and several countries, including Indonesia. This disease results in coronavirus pandemic 2019-2020. The objective of this research is to determine the inhibitory ability of several active compounds from natural sources against COVID-19 target protein in silico using molecular docking. In silico research was conducted using autodock 4.2 program by evaluating the binding energy between the active compound with ACE2, TMPRSS2, RdRp, 3CLpro and PLpro as the target proteins. All chemical compounds that evaluated such as asiatic acid, andrographolide, apigenin, brazilein, brazilin, catechin, curcumin, gingerol, hesperidin, hesperetin, kaemferol, luteolin, myricetin, naringenin and quercetin had an affinity to target protein. It reflects that active compounds in medicinal plants can be used as antiviral against COVID-19. Brazilein and brazilin from secang wood (Caesalpinia sappan L.) have a superior bond to ACE2 and lower binding energy value than chloroquine, arbidol, remdesivir, ribavirin and lopinavir. Citrus sp containing hesperidin had an excellent affinity to TMPRSS2. Secang wood and citrus sp. could be developed as an antiSARS-CoV-2 through inhibiting ACE2, TMPRSS2, RdRp and protease (3CLpro and PLpro) that interfered the process of virus infection at the entry, replication and advanced stages, causing worst effect such as pneumonia.

In the last few months, COVID-19 has become a very feared disease caused by a coronavirus. Coronavirus becomes part of a large family of viruses that cause diseases that occur in animals or humans. 1 Humans infected with the virus will show signs of respiratory tract infections ranging from the flu to more serious ones, such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS).2 SARS-COV and MERS COV is belong to a beta-coronavirus. In December 2019 a novel beta-coronavirus that infect human was found in Wuhan, China. It was named as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), while the disease is called Coronavirus Disease-2019 . The SARS-COV-2 not only infected the lower respiratory system induce a severe pneumonia, but also develop other disorders in the digestive system, heart, liver, kidneys, and nervous system, causing multi-organ failure. The elderly and patients with comorbidities such as diabetes, heart, and kidney are very susceptible to COVID-19 disease. [3][4][5] Cases of COVID-19 have spread throughout the world, and WHO states COVID-19 is categorized as a pandemic. The spread of this disease also reached Indonesia, not only in imported cases but also in local transmission. It is necessary to develop the discovery of an antiviral drug that can treat COVID-19 effectively. There are two therapeutic strategies for COVID-19, namely the first, therapy to increase the body's resistance of the host or human cells and secondly, is the restriction for the virus itself. Coronavirus is a virus that has glycoprotein spikes arranged like a crown. Coronavirus infects its host with three stages. The first stage is the virus infects its host by attaching the transmembrane spike glycoprotein to host through angiotensin-converting enzyme 2 (ACE2) in the host so that a complex is formed between S-glycoprotein with ACE2 with the help of transmembrane protease, serine 2 (TMPRSS2) produced by host cells. 6,7 The next stage is the replication stage using RNA-dependent RNA polymerase (RdRp). Coronaviruses are RNA viruses that use host cells to replicate. Coronavirus uses RdRp to make new RNA copies. The last stage is the maturation stage of virus replication in the host cell using proteases such as 3CLpro (3C-like protease) and PLpro (Papain-like protease). 8 Some drugs are known to be able to inhibit these three processes, including arbidol as ACE2 inhibitors; camostat mesylate as TMPRSS2 inhibitors; remdesivir and ribavirin as RdRp inhibitors; Lopinavir and ritonavir as protease inhibitors. Chloroquine, Lopinavir, Ritonavir and Remdesivir are an antimicrobials that declared has a potential activity against SARS-CoV 22,9,10 Indonesia has abundant biodiversity. Many active compounds from Indonesia's biological resources have the potential to be developed as medicines. For this reason, research and evaluation of active compounds will be designed to overcome COVID-19. The Active compounds in natural substances that are explored as candidates for inhibiting and reducing infections from coronavirus cause those compounds boost the immune system and act as antioxidants such as Asiatic acid, andrographolide, apigenin, brazilin, brazilein, catechin, curcumin, gingerol, hesperidin, hesperetin, kaemferol, luteolin, myricetin, naringenin and quercetin. [11][12][13][14][15][16] The active compound is traced its ability to inhibit the infection process of COVID-19 in silico with molecular docking. The target protein, which is the target of inhibition, are ACE2, TMPRSS2, RdRp, 3CLpro and PLpro. The purpose of this study is to evaluate the inhibitory ability of the active compound in the target protein that plays a role in the process of virus entry in host cells, virus replication and the process of virus maturation in host cells by binding affinity analyze using in silico assay. Hopefully, this research can contribute to the discovery of a drug for the COVID-19 antiviral and provide an overview for the public to use natural materials for daily consumption as a preventative measure for COVID-19 disease.

optimization of ligand structure
Preparation and optimization of the three-dimensional structure of the test compounds were carried out on a two-dimensional structure downloaded from https://pubchem.ncbi.nlm. nih.gov/compound/(chosen the name of active compound). The two-dimensional all active compounds files that downloaded were in the sdf format, which will later be converted to pdb format with the Open Babel Gui application and subsequently demonstrated using the HyperChem 8 application.

Preparation of the target Protein
Protein preparation begins by selecting the structure of the target protein ACE2, TMPRSS2, RdRp, 3CLpro and PLpro in the active form that binds to the native ligand. The target protein preparation was carried out using the Chimera 1.11.1 program, beginning with the removal of water molecules (H2O) contained in the target protein. The next step was to eliminate native ligands. 17,18 The results of the protein preparation were stored in the form of a pdb file, which will later be used in the method validation and docking steps of the test compound.

Molecular docking Protocol Validation
Validation of the molecular docking method was done by docking (redocking) the native ligand on the target protein that was previously removed by the native ligand by using the AutoDockTools 1.5.6 application. Validation of molecular docking method aims to ensure the method used has been validated and obtain a suitable method so that it can be used for the next stage of research. The validation parameter of the molecular docking method was the value of RMSD (Root mean square distances) resulting from redocking native ligands with their proteins. docking the active test Compound to the target Protein The optimized test compound was then docking to the prepared target protein (eliminated its native ligand) using a validated method. Docking was done using the AutoDockTools 1.5.6 Application program, equipped with the Autodock and Autogrid programs. The docking process of the active compound on the target protein results in the form of bond energy values and intermolecular interactions (in this case the type of bond formed) that stabilizes the interaction between the test compound and the target protein. 17,19 data analysis Data analysis was performed using descriptive methods. The results obtained from molecular docking are the bond energy and the type of bond between the compound and the target protein. Bond energy values indicate affinity (bond strength) between compounds and target proteins. The binding energy between the active test compound and the target protein is negative, meaning that the test compound has an affinity for the target protein and potentially to inhibit the target protein, whereas if it is positive, it indicates that the active test compound has no affinity for the target protein. The negative bond energy value indicates a spontaneous reaction and indicates a stable system that allows the formation of bonds, while the positive energy value suggests a system that has very little or even no tendency for the reaction to occur so that the bond was not formed. 20,21 results and disCussion the active test Compounds have Been optimized and Prepared using hyperchem 8 The preparation of the structure of the test compound is done by first changing the two-dimensional structure of the compound ( Figure 1) into a three-dimensional structure. Three-dimensional structure optimization of compounds was done by single-point calculations and continued with geometry optimization. Single point calculation is a calculation used to determine the total molecular energy of a structure before the optimization process of the test compound was carried out. Geometry optimization was a process to minimize total energy so that the structure of the most stable test compound was obtained, characterized by a decrease in the overall energy value of the structure of the test compound. [22][23] In geometry optimization results a shift in the structure of compounds into the most stable structure, so that there was a decreasing energy value of the structure of the test compound. Three-dimensional structure of the compound test results of single-point calculations and geometry optimization.
This study used several active compounds of medicinal plants that are known to have high antioxidant content and enhance the immune system. These active compounds may have the potential to inhibit infection from coronavirus. Asiatic acid contained in Centella asiatica; andrographolide from Andrographis paniculata NEES; apigenin, luteolin, quercetin contained in Moringa oleifera; brazilin and brazilein from  Protein preparation aim to separate the native ligand from the target protein, thereby providing pocket or binding sites that will be used during the docking process. Protein preparation was to choose a chain that binds with the appropriate native ligand. The use of one chain aims to make it easier to determine the coordinates of the space (pocket cavity) of the ligand that will be used for docking. Figure 2 demonstrated the results of protein preparation.

Caesalpinia sappan Containing Brazilin; Brazilein and Citrus sp. Containing hesperetin, hesperidin and naringenin exhibit strong Affinity to the Target Protein that Plays a Role in Coronavirus infection
Docking the active test compounds on the five target proteins, namely ACE2, TMPRSS2, RdRp, 3CLpro and PLpro are carried out after the docking method has been validated with a fulfilling RMSD value of = 3 Å. 24 The docking process was done using the AutoDockTools 1.5.6 application, which equipped with Autodock4 and Autogrid4. Docking the test compound was carried out at the binding site of each target protein using the same grid box coordinate settings during the validation process. Using the same grid box coordinates at the time of validation with the docking process can ensure that the interactions that occur between the test compounds are indeed at the binding site of each target protein. Table 1 presented the docking score of natural compounds breaking up the target protein. The overall data gives negative values for binding energy, except Asiatic acid. Asiatic acid has no affinity to ACE2 and RdRp.
In this in silico study, we seen that brazilin in secang (Caesalpinia sappan L.) wood exhibited the most negative value of docking score toward ACE2, means the best affinity was formed. In TMPRSS2 protein, the compound that has the most stable bond is hesperidin in Citrus sp, while quercetin shows the best bond with RdRp. The chemical compound in natural materials that have the potential to bind the most strongly with 3CLpro is andrographolide. Besides that curcumin has the strongest bond compared to other chemical compounds in PLpro. Curcumin and quercetin also were reported as protease inhibitor with PDB ID (6LU7), 25 and these two compounds had an antiaging effect. [26][27][28] Thus this invention will increase the usefulness of active compounds for developing antiviral agents, which are also rich in antioxidants and act as antiaging. The binding energy obtained between the active compounds and the five target proteins was also compared to the bond energy of the drug that acts as ACE2; TMPRSS2; RdRp; 3CLpro and PLpro inhibitors. If the binding energy value of the chemical compound with the five target proteins was more negative than the inhibitors of the five proteins, the ability or affinity of the active compound to bind to the target protein was more exceptional. This shows that more robust and more stable interactions that occur between active compounds and target protein.
Among all the data in table 1, it appears that brazilin and brazilein in secang wood bond to all target proteins and have lower binding energy compared to arbidol, chloroquine, camostat mesylate, remdesivir, ribavirin and lopinavir and have the best bond with ACE2. This shows that it can act as an entry inhibitor of SARS-Cov-2 by inhibiting ACE2 and TMPRSS2. Secang wood also inhibit RdRp which plays a role in RNA replication in the host cell. Proteases was slowed down too, so that the coronavirus maturation process was stopped. Another compound that also has the inhibiting ability in three critical processes of coronavirus infecting its host was Citrus sp. Citrus sp contains hesperidin, which has the highest ability on TMPRSS2 so that the process of forming the S-glycoprotein complex with ACE2 in the host cell was be bothered. Other research also states that Citrus sp can be developed for the discovery of anti-SARS-CoV-2 drugs because it is able to inhibit S-glycoprotein and proteases. 29 The interaction of hydrogen bonds between the target protein with the drug and brazilin, brazilein and hesperidin was shown in figure 3. Further research must be conducted to evaluate more deeply about the potential activities of secang wood and Citrus sp to eradicate SARS-CoV-2. People can use these medicinal plants for prevention and combination therapy with drugs against coronavirus.

ConClusion
Brazilein and brazilin from secang (Caesalpinia sappan L.) wood can be developed as an angiotensin-converting enzym 2 (ACE2) and protease inhibitor. This also occurs in the content of compounds in Citrus sp, especially hesperidin. Secang wood and Citrus sp could be used as a prevention agent and supportive therapy against SARS-CoV-2.