In Silico Screening of Bioactive Compounds from Garcinia mangostana L. Against SARS-CoV-2 via Tetra Inhibitors

DDR


INTRODUCTION
The COVID-19 pandemic is caused by positivesense beta-coronavirus RNA, namely Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). 1 Covid-19 cases were first reported in Wuhan, Hubei China in late December 2019 and have since spread into a global pandemic. 2 Currently, SARS-CoV-2 is responsible for 6 million deaths from SARS-CoV-2. 3 According to genome analysis, SARS-CoV-2 had a 79.5% resemblance to SARS-CoV which had caused an epidemic in 26 countries in 2003. 4 SARS-CoV-2 spreads uncontrollably through aerosols. Furthermore, this virus also causes fever, cough, respiratory failure, septic shock, and organ failure. 5,6 SARS-CoV-2 infection in human cells occurs mediated by receptor binding of the spike domain in the terminal C domain (SARS-CoV-2-CTD) with angiotensin-converting enzyme 2 (ACE-2). 7,8 Viral RNA helicase plays a role in the replication and folding of RNA in the target cell. 9 Furthermore, viral RNA is translated into pp1a and pp1ab proteins then folded by proteolytic enzymes such as Mpro into nonstructural proteins (nsps). 10,11 RdRp binds to genome RNA (negative-sense) to form genome and subgenome RNA (positive sense) via replication and transcription. 12 Several therapeutic strategies of antiviral drugs have been developed today including lopinavir, baricitinib, remdesivir, and favipiravair. 13 In addition, nanotechnology is now widely applied as an anti-SARS-CoV-2. 14 Indonesia is known as megabiodivesity country with numerous medicinal plants. 15 The bioactive of medicinal plants are expected to provide antiviral compounds for SARS-CoV-2. 16 Some Indonesian plants that can be used in medicine are various groups of mangosteen.
Garcinia mangostana L. is a member of the Guttiferae family. 17 The plant grows primarily in tropical Asia including Indonesia and its fruits are widely used in food and traditional medicine. 18,19 Mangosteen peel has been shown to have antimetabolic poperties. 20,21 SARS-CoV-2 inhibitor compounds studies take a long time, but preliminary research to determine the potential of plants as drug candidates can be done with an in silico approach. 22 Therefore, this study aims to determine the potential of G. mangostana L. as inhibitors of RBD spike, helicase, Mpro, and RdRp activity of SARS-CoV-2 in silico.

Antiviral probability prediction
Bioactivity prediction was carried out using the PASS Online (http:// way2drug.com/PASSOnline/) web. The category of predictions sought was antiviral. The potential activity standards used are Pa > 0.3 and Pa > Pi. The score is preferred as computational evidence in molecular docking. 26

Virtual screening
Docking simulations were carried out to calculate the binding energy. Molecular docking is performed with PyRx due to its high accuracy. 21 The purpose of molecular docking was to identify the binding energy of bioactive compounds to target proteins as anti-SARS-CoV-2. 27

Interaction and visualization
The goal of visualization is to provide more detailed description of the docking results. The most negative affinity compounds were displayed. Visualization of the target ligand-protein complex and its interaction was performed using Discovery Studio. The result of PyMol visualization is a complex structure of ligand-targeted proteins. 28,29

RESULT AND DISCUSSION
Lipinski rule of five is important for determining drug candidate compounds by being treated as drug-like compounds. 30 Based on the drug similarity analysis, it was determined that 6 bioactive compounds meet the Lipinski rule of five so that they can be interpreted as drug-like compounds. Meanwhile, an antiviral probability analysis performed using PASS Online revealed that mangostanol, mangiferin, mangostenol, and epicatechin had Pa > Pi and Pa > 0.3 26 (Table 1). The score indicates that the bioactive compounds have higher activation potential than the inactivation as antivirals. As a result, these compounds are preferred in bioinformatics studies. 31 However, the results of this prediction still require in vitro and in vivo studies. Compounds docked against the next four targeted SARS-CoV-2 proteins satisfy Lipinski rule of 5 and have high antiviral probability.
The molecular docking results are used to determine the stability of the interaction between the ligand and targeted protein complexes. The highest level of stability interaction is indicated by the low binding affinity, indicating the inhibitory activities of the ligands to the targeted protein are greater. 6 Table 2 shows that all bioactive compounds have binding activity against targeted proteins. However, the compound to be visualized is that has the lowest binding affinity, mangiferin.
SARS-CoV-2 RBD spike has more extensive mutation than SARS-CoV RBD spike. This results in the formation of vdw (van der Waals) interactions and hydrogen bonds when binding to ACE2 is greater, increasing the potential for SARS-CoV-2 transmission. 7 Gln, Cys, His, and Asp are some conservative residues in RBD spike that can be used in molecular docking. 32 Mangiferin forms chemical bonds with conservative residues such as hydrogen bonds to Asp350 and vdw bonds to His378, Asp382, and His401. Hydrogen bonds cause smaller binding energy so that the ligand-targeted protein complexes interactions are stable. 33 Furthermore, the presence of hydrophobic interactions such as pi-pi T-shaped and vdw bonds aids the interaction stability and ligand turnover to the targeted protein in cellular processes. 34 SARS-CoV-2 helicase is involved in RNA replication and folding. 9 Some helicase segments of SARS-CoV-2 variants show similarities to NSP13 5' RNA site, suggesting that it has great potential in the in silico anti-SARS-CoV-2 development in silico. 35 Mangiferin forms various bonds to helicase, including hydrogen, pi-alkyl, and vdw bonds. The pi-alkyl bond is formed from the interaction of the aromatic cloud from the aromatic group with the electron cloud from the alkyl group. 36 The presence of weak bonds such as pi-alkyl and vdw bonds affect ligand activity against targeted proteins in viral entry inhibition, maturation, and protein folding. 37 In addition, there is an unfavorable acceptoracceptor bond to Leu235. This bond is a less preferred inhibitor in docking due to several factors such as the presence of induced fit bonds, irrelevant binding modes, critical cell activity, and unpredictable bonding models at other binding sites.
Mpro is a cysteine protease responsible for viral maturation and protein folding. 10,11 Various SARS-CoV-2 variants are so conserved that their mutation rates are low. 38 Some of these conserved sites are catalytic residues include Asn, Gln, and Pro. 39 This position can be used as a molecular docking position in the development of anti-SARS-CoV-2.
Mangiferin indicates the presences of hydrogen bonds, pi-alkyl, vdw, donors are disliked, and acceptors are not liked. The existence of an unfavorable donor-donor bond causes the binding energy to be more positive. More in vitro and in vivo research is required to determine the significance of these bonds. 40 RdRp is an essential component of coronavirus replication and transcription because it plays a role in the formation of genomic and non-genome RNA. 41,12 In the role, RdRp is assisted by other proteins such as nsp7 and nsp8. 1 RdRp contains a large number of conservative catalytic residues, the majority of which are Ser and Gly. 12 Molecular docking results show that mangiferin has a hydrogen bond to Ser255. The presence of hydrogen bonds in catalytic residues aids stabilize ligand complex bonds with targeted proteins resulting in the desired interaction conformation. 42 There are also pi-cation, pi-alkyl, pi-stacked amide, vdw, and unfavorable donor-donor bonds that form a relatively negative binding affinity. [43][44][45][46][47][48][49][50] CONCLUSION Mangosteen (Garcinia mangostana L.) has an anti-SARS-CoV-2 potential. The bioactive compound mangiferin from mangosteen is known to have the lowest binding energy to RDB spike, helicase, Mpro, and RdRp of SARS-CoV-2. However, more research is needed to support the results of this study.