Active constituents and Molecular Analysis of Psidium guajava Against Multiple Protein of SARS-CoV-2

The severe acute respiratory syndrome COVID-19 declared as a global pandemic by the World Health Organization has become the present wellbeing worry to the whole world. The latest development of COVID-19 spread in Indonesia has reached 1.024.298 cases, with 28.855 patients died, updated on January 28, 2021. Unfortunately, these numbers continue to overgrow, and no drug has yet been approved for effective treatment. There is an emergent need to search for possible medications and explore the potential of Indonesian herbal compounds. Ministry of Health Indonesia stated that Psidium guajava can be use as daily nutritional supplement during COVID-19 pandemic. This study aims to determine the potential active constituents in Psidium guajava as an inhibitor for multiple SARS-CoV-2 proteins using molecular analysis. MPro, Spike and ACE2.


Introduction
The new coronavirus, called SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), was rst identi ed in Wuhan, China, in December 2019 [1]. SARS-CoV-2 belongs to the Coronaviridae family, a single-stranded RNA virus (+ ssRNA) that is widespread among humans and other mammals, causing a wide range of infections from common cold symptoms to fatal illnesses, such as severe respiratory syndrome [2,3]. The latest development of COVID-19 spread in Indonesia has reached 1.024.298 cases, with 28.855 patients died, updated on January 28, 2021 (data taken from https://covid19.go.id). The infected numbers continue to overgrow and reached more than 10.000 new case per day. Unfortunately, there are no drugs approved yet as an effective treatment. Therefore, the need to discover and develop drugs to treat the Coronavirus Disease 2019  is become more urgent.
There are two categories of anti-coronavirus therapy depending on the target, one act on the human immune system or human cells, and the other one is on the coronavirus itself. In terms of the human immune system, the innate immune system response plays an essential role in controlling the replication and infection of coronavirus and to enhance the immune response [4]. Blocking the signalling pathways of human cells required for virus replication may exhibit a speci c antiviral effect. The therapies that work on the coronavirus itself include preventing the synthesis of viral RNA by acting on the genetic material of the virus, inhibiting virus replication through acting on critical enzymes of the virus, and blocking the virus from binding to human cell receptors or inhibiting the viral assembly process by working on several structural proteins [5].
In an attempt to ght against coronavirus, scientists are coming up with different strategies. One of the strategy is exploring natural compounds to nd out their activity against the multiple SARS-CoV-2 protein targets. Especially, in Indonesia, people are more familiar with using herbal to care their health in daily life, we also need to consider developing agents from herbal. Erlina et al.
Computational methods to understand the ligand protein interaction is one of the fastest ways to identify the candidate drug and target. Scientists are screening existing molecules from the database, which might be effective against coronavirus as a strategy [9][10][11]. In case of SARS-CoV-2, inputs from this traditional way were lacking because of unavailability of protein structure. But 3D structures of different proteins of this novel SARS-CoV-2 are constantly being deposited in the database and hence in the present study we decided to do molecular docking analysis of these newly deposited proteins against some repeatedly discussed drugs as a repurposing therapeutic approach. We had selected those proteins for docking which play an important role in propagation of virus.
We selected 3CLpro and Mpro which are the main protease of the virus, spike protein from virus, and Papain like protease (PL pro) [11]. Virus enters the cell via angiotensin receptor converting enzyme 2 therefore blocking this enzyme could be of immense importance [12][13][14]. This enzyme is also selected as one of the target proteins. In this study, some known antiviral drugs like Ritonavir, Lopinavir, and Remdesivir, which are being used as therapeutic agents against COVID-19, are used as candidate ligands for docking. Molecular docking was performed using Autodocktools 1.5.6.

Ligand (Psidium guajava and antiviral) structures
Fourteen Psidium guajava active constituents were prepared as ligand (Table 1). Known antiviral drugs like ritonavir, lopinavir, and remdesivir, which are used to SARS-CoV-2 therapeutic agents, also selected as a candidate ligands for docking against viral proteins.

Molecular docking validation
Molecular docking validation was done using redocking methods by Autodocktools 1.5.6. 3CL Pro (PDB ID: 6LU7) has native ligand namely inhibitor N3 and PL pro (PDB ID: 6WX4) has native ligand namely peptide inhibitor VIR251. Grid box for 6LU7 and 6WX4 develop into three set (40x40x40 Å, 50x50x50 Å and 60x60x60 Å). Run Genetic Algorithm (GA) was set to 100 times. Binding energy (Kcal/mol) and RMSD will evaluate per each docking results.

Molecular docking analysis and visualization
AutoDock software was utilized in all the docking experiments, with the optimized model as the docking target. Ligand and protein optimization were done using Autodocktools 1.5.6. For ligand optimization, the geometry of ligands was cleaned and torsion were set to fewest. For protein optimization, the water was removed, hydrogen polar only were added, hydrogen non polar were merged and Gasteiger charge were added. The docking was performed by using AutoDock4. Run Genetic Algorithm (GA) was set to 10 times. The docking analysis were performed using PyMOL version 2.

Results And Disscussion
Molecular docking validation 6LU7 (PDB ID) was choose as 3CL Pro receptor because it has a resolution value of 2.16 Å. Based on molecular docking validation with a re-docking method between 6LU7 and its native ligand (N3), the optimum grid box is 40x54x40 Å with binding energy value − 8.72 kcal/mol, RMSD value 3.96 Å and inhibition constant value 408.59 nM. The optimum grid center is x= -9.768; y = 11.424; z = 68.935 with 0.375 Å spacing for default setting.
After we got molecular docking results, we further checked the previous studies to nd the biological activities of each compound. So that this research can be useful for the community, we also tried to nd from commodity crops. One of the commodity crops in Indonesia is Guava (Psidium guajava) that can be harvested continuously in one year. In Indonesia, production of guava in the year 2018 is 230,697 tons, with growth rate from the year 2017 to 2018 is 15.06% [21]. Guava is consumed not only as food but also as a traditional medicine in subtropical areas around the world due to its pharmacologic activities. Based on Herbal Regulation as Healthy Supplement for Fighting COVID-19 in Indonesia published by The Indonesian Food and Drug Authority (BPOM) (May, 2020), we can consume Psidium guajava (Guava) 1-4 fruits per day (55-100 gram/fruit) which contain vitamin C 228.3 mg in 100 gram fruit. For the administration, Guava can be eaten directly or processed as juice. There is no case for toxicity for long term consumption, overall this herbal is safe to use as daily nutritional supplement [22]. Phenolic compound from Guajava has been proved as immunomodulator and antioxidant [23].
Guava is well known has several avonoids compounds, i.e. myricetin, quercetin, luteolin, kaempferol, isorhamnetin [6], and Hesperidin [7]. These compounds were also shown in our result, although without the aglycones. Luteolin is known as a furin protein inhibitor [24] which is predicted to be one of the enzymes that break down Coronavirus S (spike) protein as in MERS into units S1 and S2 [25]. In the S1 unit, there is a Receptor Binding Domain (RBD) where the ACE2 peptidase binds so that the virus can bind to the host [25]. Hesperidin / Hesperitin compounds in the in silico study are known to inhibit RBD domain binding of the SARS-COV-2 Spike protein with ACE2 receptors in humans so that it is predicted to inhibit the entry of the SARS-COV-2 potentially [5]. It is also known that luteolin is a neuraminidase inhibitor as well as oseltamivir which is currently one of the drugs used in the CDC protocol. Hesperitin (the form of hesperidin aglycone) and quercetin are known to also act as inhibitors of 3Clpro [26,27]. Other compounds in guava such as myricetin are known to act as SARS coronavirus helicase inhibitors [28]. The kaempferol has the potential to be a non-competitive inhibitor of 3CLPro and PLpro as well as quercetin [29]. Another interesting thing is that kaempferol acts as a modulator of autophagy, which can be utilized in strategies to inhibit SARS-COV-2 virus.

Conclusions
Based on the molecular docking analysis, it was found there are two potential compounds from Psidium guajava that showed higher binding a nity score namely gamma sitosterol and peri-xanthenoxanthene-4,10-dione,2,8-bis (1-methylethyl). Therefore, the extensive work carried out in this study to understand the interaction of multiple viral proteins and different drug ligands which are discussed for treatment in COVID-19. Proteins important in viral replication were selected in the study because inhibiting these proteins might be useful to block the initiation of infection and chain of replication.
Abbreviations Table 1 Due to technical limitations, Table 1 is only available as a download in the supplemental les section