Molecular Docking Studies of Phytoconstituents Identified in Traditional Siddha Polyherbal Formulations Against Possible Targets of SARS-CoV-2

Ever since the outbreak of COVID-19 caused by Severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) in Wuhan, China, the world has witnessed the rapid spread of the pandemic across the world1. World Health Organization (WHO) reported approximately 82,579,768 COVID-19 cases and 1,818,849 deaths as of January 2nd, 2021, with cases reported in more than 222 countries/territories. This novel coronavirus outbreak has posed a severe burden to the global economic, medical, and public health infrastructure2. The COVID-19 is primarily a droplet-spread infection, and patients exhibit various symptoms of which fever, dry cough, and fatigue are predominant3. In some cases, the symptoms had rapidly developed to acute respiratory distress syndrome, metabolic acidosis, septic shock, coagulation dysfunction, eventually leading to multiple organ failure4-6. However, mild or asymptomatic COVID-19 patients can recover shortly after isolation and healthy lifestyle and food habits7. There is no particular treatment available for COVID19 infection except for comprehensive support by the combination of broad-spectrum antibiotics, antiviral and anti-malarial drugs, corticosteroids, and convalescent plasma therapy8. Numerous clinical trials are in progress, including identifying vaccines against SARS-CoV-2. Researchers and health care professionals are in desperate search of an effective Molecular Docking Studies of Phytoconstituents Identified in Traditional Siddha Polyherbal Formulations Against Possible Targets of SARS-CoV-2


INTRODUCTION
Ever since the outbreak of COVID-19 caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Wuhan, China, the world has witnessed the rapid spread of the pandemic across the world 1 . World Health Organization (WHO) reported approximately 82,579,768 COVID-19 cases and 1,818,849 deaths as of January 2 nd , 2021, with cases reported in more than 222 countries/territories. This novel coronavirus outbreak has posed a severe burden to the global economic, medical, and public health infrastructure 2 . The COVID-19 is primarily a droplet-spread infection, and patients exhibit various symptoms of which fever, dry cough, and fatigue are predominant 3 . In some cases, the symptoms had rapidly developed to acute respiratory distress syndrome, metabolic acidosis, septic shock, coagulation dysfunction, eventually leading to multiple organ failure 4-6 . However, mild or asymptomatic COVID-19 patients can recover shortly after isolation and healthy lifestyle and food habits 7 . There is no particular treatment available for COVID-19 infection except for comprehensive support by the combination of broad-spectrum antibiotics, antiviral and anti-malarial drugs, corticosteroids, and convalescent plasma therapy 8 . Numerous clinical trials are in progress, including identifying vaccines against SARS-CoV-2. Researchers and health care professionals are in desperate search of an effective

Molecular Docking Studies of Phytoconstituents Identified in Traditional Siddha Polyherbal Formulations Against Possible Targets of SARS-CoV-2
cure for this pandemic. In the current scenario where the conventional drugs do not prove to be much efficacious, exploring the traditional system of medicine could be a feasible and hopeful strategy 9 . Traditional, complementary, and alternative medicine has a long history of providing primary beneficial health care to the population 10 . India has an unmatched alternative system of medicine in the form of Ayurveda, Yoga, and Naturopathy, Unani, Siddha, Homeopathy, which is now jointly referred to as Ayush, recognized by the Government of India 11 . Siddha Medicine is one of India's oldest (5000 years old) and well-documented medical systems and is practiced mainly in South India, especially in Tamil

Docking protocol Preparation of ligands
The ligand minimization was carried out by the LigPrep module in Maestro 11.8. The 3D ligand structure was generated, and hydrogen atoms were introduced. Salt reduction and ionization (pH 7.0±2.0) were conducted, and the minimization was performed utilizing the OPLS-2005 force field 30,31 .

Preparation of protein
Protein Preparation Wizard was used to prepare protein structures. Bond orders were assigned, and hydrogen atoms were inserted. Within 3 Å of the het groups, the water molecules were removed, and the missing side chains were filled with prime. As a result, hydrogen bonds (H-bond) were optimized and reduced using the OPLS 2005 force field. The cocrystallized ligand binding sites have been identified after elimination. The receptor grid was then created using the "Glide's Receptor Grid Generation" module with a 20 Å radius 30,31 .

Molecular docking and free energy calculation
The molecular docking between receptor binding sites and ligands was conducted using the Glide Module of Maestro 11.8, and the lowest binding pose of each ligand was maintained. Glide docking scores were performed in three high-throughput virtual screening (HTVS), standard precision (SP), and extra precision (XP) modes. Firstly, docking was performed with reference molecules of respective proteins to validate the docking protocol. We used the XP mode for docking. After XP mode docking, compounds were sent to Prime MMGBSA from Maestro 11.8 for free energy calculations.

ADME and toxicity analysis
Out of the 36 compounds, ten compounds were chosen based on the docking performance. The chosen compounds were used in the ADME study using the QikProp module from Maestro 11.8, and the following parameters were determined.

Molecular docking and free energy calculation
Compounds with a docking score of less than -6.0 were deemed possible candidates against SARS-CoV-2, as represented in Table I for a comparative study. Out of 36 molecules, luteolin, chrysoeriol, and cucurbitacin B have been associated with more than two receptor structures. Luteolin displays a docking score less than -6 with M Pro , Nsp15 endoribonuclease, and RdRp, as seen in Figure 1.  Chrysoeriol also displays a docking score less than -6.0 with M Pro , Nsp15 endoribonuclease, and RdRp, as seen in Figure 2. The associations of luteolin and chrysoeriol with various SARS-CoV-2 target forms were comparatively analyzed, in which H-bond and hydrophobic pockets were presented in Tables II and  III. Luteolin shows hydrogen bonding with nearly four amino acids of most of the targets. This finding shows its high binding potency towards the SARS-CoV-2.  In the molecular docking of phytoconstituents with M Pro (5R82), luteolin had a higher affinity with a docking score of -7.408, followed by scutellarein and chrysoeriol with docking scores of -6.807 and -6.473, respectively. These phytoconstituents had a higher affinity to M Pro (5R82) than remdesivir, displaying a docking score of -5.478. Chrysoeriol had a higher affinity with a docking score of -6.394, followed by scutellarein and luteolin with docking scores of -6.081 and -6.036, respectively with the target M Pro (6Y2F). These phytoconstituents had a higher affinity to M Pro (6Y2F) than remdesivir, with a docking score of -5.306. Scutellarein had a greater affinity with a docking score of -7.587, followed by luteolin and chrysoeriol with -7.470 and -7.342, respectively, for molecular docking of phytoconstituents with M Pro (6LU7). These phytoconstituents had a higher affinity than remdesivir, which had a docking score of -7.189. Remdesivir shows greater affinity with a docking score of -7.829, followed by scutellarein and cucurbitacin B with a score of -7.314 and -7.191, respectively, in the docking analysis with Nsp15 endoribonuclease (6W01). With RdRp (6M71), remdesivir had a higher affinity with a docking score of -8.643, followed by pyrethrin and cucurbitacin B with docking scores -6.704 and -6.488, respectively. Hydroxychloroquine had a higher affinity with a docking score of -8.748, followed by remdesivir and cycleanine, which had a docking score of -7.206 and -6.907, respectively, with the target spike protein (6VW1). Most phytoconstituents exhibited similar reference drugs in binding energies and binding pockets, except gallic acid, pyrethrin, chebulagic acid, and cycleanine. Chrysoeriol shows less hydrogen bonding than the luteolin but better than other phytoconstituents. The hydrogen bonding of both luteolin and chrysoeriol could be increased by substitute better chemical groups. The prime MM-GBSA was generally accepted for the re-scoring of docked complexes. Both of the chosen complexes were subjected to prime MM-GBSA measurements after XP Docking 33 . MM-GBSA DGbind scores for all chosen compounds were displayed in Table IV. The negative DG-bind values indicate that the selected compounds associate favorably with the receptor. Ligand binding energies for both substances vary from -40.0 to -100.0 kcal/mol. The binding energies of several of the substances were relatively close to those of the reference drug binding energy. These findings indicate that the selected compounds would inhibit SARS-CoV-2.

ADME analysis
The absorption, distribution, metabolism, and elimination of substances play an essential role in the drug development phase. In silico ADME analysis would save thousands of dollars spent in the drug development phase by producing fewer new compounds 34 . The ADME parameters, such as mol MW, QPlogPo/w, QPlogBB, percent human oral absorption, Rule of Five, and Rule of Three using QikProp showed a better score for the docked compounds 35 . Both of the chosen nine compounds have enhanced ADME properties and drug-likeness according to the spectrum as shown in Table V. All of the nine phytoconstituents have enhanced ADME properties. Cucurbitacin B violates a rule of 1 of 5, which was appropriate. Gallic acid and pyrethrin were in breach of a law of three that was fitting. Luteolin and chrysoeriol display improved drug-likeness and high binding capacity, all of which were essential to the drug candidate.

In silico toxicity study
The oral rat LD50 The endpoint of the oral rat LD50 was the amount of the chemical (chemical mass per rat body weight) that destroys half of the rats when administered orally 36 . The oral rat LD50 was conducted in four methods for all of the chosen compounds, and the findings were comparatively evaluated in Table VI. All substances have been shown to have an acceptable toxicity limit for drug production and preclinical and clinical assessment.

Developmental toxicity
Developmental toxicity includes embryonic and fetal mortality, miscarriage, and other abnormal developmental symptoms such as liver toxicity, lowered body weight, growth, developmental retardation, and physical abnormalities (teratogenic effects) 37 . Developmental toxicity was performed in four approaches with all of the chosen compounds, and the findings were comparatively analyzed in Table VI. A predicted value greater than 0.5 indicates toxicity. Except gallic acid, all other compounds show developmental toxicity.

Ames mutagenicity
In Ames assay, frame-shift mutations or base-pair substitutions could be identified by exposure of histidine-dependent strains of Salmonella typhimurium to the test compound. When these strains were exposed to a mutagen, reversing mutations that restore the functional capacity of the bacteria to synthesize histidine would cause the bacterial colony to develop on a medium histidine deficiency (revertant) 38 . A compound was labeled Ames positive if it significantly induces the development of the reverting colony in at least one of the five strains. If a compound was positive for the Ames test, it could be a possible mutagen 39 . Ames mutagenicity was conducted in four methods for all of the chosen compounds, and the findings were comparatively analyzed in Table VI. A predicted value greater than 0.5 indicates mutagenicity. All the nine phytoconstituents except pyrethrin were not mutagens based on the results on the Ames mutagenicity as predicted by T.E.S.T software. proteolytically cleaves the overlapping pp1a and pp1ab polyproteins to functional proteins, crucial in viral replication. In the viral replication cycle, the M Pro acts as the primary enzyme. Its inhibition could thus interfere with the production of infectious virus particles and reduce disease symptoms 45 . The SARS-CoV-2 spike protein mediates the binding of the virus to its receptor angiotensin-converting enzyme 2 (ACE2) and facilitates the integration of viral and host cell membranes and the entrance of the virus into the host cell. Thus, the Spike protein was vital in neutralizing and T-cell reactions and maintaining immunity during SARS-CoV-2 infection. Given the essential role of the S-protein in viral infection and adaptive immunity, most methods and therapies were based on the S-protein 46 . RNA-dependent RNA Polymerase was an enzyme that replicates RNA from an RNA template. RNA-dependent RNA Polymerase was one of the Nsp (Nsp12) that plays a key role in the coronavirus life cycle 47 .
Nsp15 was responsible for protein interaction with the innate immune response, although other studies suggest that the mechanism was independent of endonuclease activity. In order to conceal it from the host's immune system, there were also reports that Nsp15 degrades viral RNA 48 . Nevertheless, in coronavirus biology, Nsp15 was important. The active site, located in a shallow groove between the two βsheets, carries six key residues conserved among SARS-CoV-2, SARS-CoV, and MERS-CoV proteins: His235, His250, Lys290, Thr341, Tyr343, and Ser294 27 .

CONCLUSION
The present research was planned to classify potential drug candidates exhibiting potential binding affinity to all possible SARS-CoV-2 targets (M Pro , Nsp15 endoribonuclease, RdRp, and spike protein). Based on the findings obtained from molecular docking, free energy measurement, ADME analysis, as well as toxicity analysis, luteolin and chrysoeriol exhibit stronger docking score, binding energy, ADME properties, and lower toxicity than all other compounds.

CONFLICTS OF INTEREST
There are no conflicts of interest to declare.

FUNDING
None.

DATA AVAILABILITY
All data are available from the authors.