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Identification of novel phytochemicals from Hibiscus rosa sinensis flower as a

prospective inhibitor targeting the 3CLpro enzyme of SARS-CoV-2 using
computational approaches.

Preprint · April 2023

DOI: 10.21203/rs.3.rs-2837087/v3

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Identification of novel phytochemicals from Hibiscus rosa sinensis flower as
a prospective inhibitor targeting the 3CLpro enzyme of SARS-CoV-2 using
computational approaches.
Subhadeep Das (  subhadeep.1426@gmail.com )
GIET University
Sagarika Satapathy
GIET University
Diptikanta Acharya
GIET University
Sushil Kumar Sahu
Visva-Bharati University

Research Article

Keywords: SARS-CoV-2, 3CLpro, molecular docking, physiochemical, drug-likeness, ADMET

Posted Date: April 25th, 2023

DOI: https://doi.org/10.21203/rs.3.rs-2837087/v3

License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Page 1/12
Abstract
Hibiscus rosa sinensis has an infinite resource of phytochemicals and has emerged as a solution for different health-related issues such as anti-diabetic, anti-
microbial and wound healing activity as proved in past clinical studies. Focusing on the current situation, an incessant increase in daily COVID-19 cases and
the struggle to discover effective treatment measures for SARS-CoV-2 had led to a global health catastrophe. Upsurge in COVID-19 cases had revealed a
pattern characterised as a first, second, third wave and beyond. This cycle of new SARS-CoV-2 variant transmission needed to be terminated by selecting a
favourable effective target, and the 3CL protease enzyme (3CLpro or Mpro) of SARS-CoV-2 acts as a possible target. The objective of this study is to investigate
the phytochemicals identified in Hibiscus rosa sinensis flowers for their potential anti-SARS-CoV-2 properties virtually, targeting the 3CLpro or Mpro, which
regulates viral pathogenesis. The present research protocol includes molecular docking of 34 phytochemicals identified from the Hibiscus rosa sinensis flower
and targeted against the active site of the 3CLpro enzyme. Computational analysis revealed that the top 3 ligands: cyanidin-3-sophoroside-5-glucoside (-10.9
kcal/mol), 1,2-benzenedicarboxylic acid isodecyl octyl ester (-10.1 kcal/mol) and rutin (-9.3 kcal/mol) had better binding affinity as compared to the control
inhibitor remdesivir (-8 kcal/mol). Further investigation in terms of ligand-protein interaction, physiochemical, ADMET and drug-likeness parameters showed
that cyanidin-3-sophoroside-5-glucoside possessed promising properties and could act as a potentially effective drug candidate. However, our study needs to
be supported by in vitro and in vivo evaluations to determine the precise mechanism of inhibitory action.

1. Introduction
1.1. The emergence of COVID-19:

Coronavirus disease 2019 (COVID-19) is a highly transmittable illness and was reported for the first time in Wuhan, Hubei province, central China in December
2019 [1]. It was characterised mostly based on clinical feature of respiratory distress resembling to the complications that happened during Severe acute
respiratory syndrome-coronavirus (SARS-CoV) and Middle East respiratory syndrome-coronavirus (MERS-CoV) epidemics [2, 3]. COVID-19 associated clinical
symptoms include fever, cough, breathing difficulties and pneumonia leading to progressive respiratory failure [1]. The infection was reported to be caused by
a novel RNA virus from the family Coronaviridae entitled as Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) by the World Health
Organisation [4]. Outspreads of disease have affected many provinces and cities in China, and later, the progressively rampant spread of disease to other
countries resulted in an upsurge in mortality. On 30 January 2020, the first COVID-19 was reported in India [5]. The first case of COVID-19 in the India was
identified in Thrissur district of Kerala state, but due to proper strategy implementation to control the COVID-19 cases, Kerala has shown a high recovery rate,
low mortality and slow advancement of COVID-19 cases during the initial stage [6]. As the COVID-19 began to outspread across the globe with an exponential
infectious rate, on 11 March 2020, the World Health Organisation (WHO) declared the outbreak as a global pandemic [7]. On 8 January 2022, the
epidemiological data revealed that a 1.36% cumulative Case Fatality Rate (CFR) recorded in India, low in comparison to the world’s 1.80% CFR [8, 9].

COVID-19 is an envelope and spherical virus with a positive single-stranded RNA genome, characterised under Coronaviridae family and order Nidovirales,
further categorised into four genera specifically α, β, γ and δ of which COVID-19 belongs to β genus [10]. COVID-19 genome is closely related to SARS-CoV,
thus named as severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Metagenomic analysis based on RNA next-generation sequencing utilised to
interpret the genome length of 29,891 bp (GenBank no. MN908947) which contain 38% G+C content, encodes 9860 amino acids and absence of
hemagglutinin-esterase gene [11, 12]. Sample analysis till January 2022 concluded that SARS-CoV-2 evolved with increasing U-content and lowered genome
size compared to previous SARS-Cov-2 variants [13]. Analogous to other coronaviruses, the positive sense single-stranded RNA genome consists of a 5’-cap
and 3’-poly-A-tail. Further, on genomic sequence analysis of SARS-CoV-2, it was found to be composed of structural and non-structural protein genes. At the 5’-
end, ORF contains a frameshift between ORF1a and ORF1b, which generates two polypeptides that gets proteolytically cleaved to yield 16 non-structural
proteins (nsp1-16). At the 3’-end of the positive sense single-strand RNA of SARS-CoV-2, ORF10 and ORF11 encode four structural proteins includes: spike
protein (S), nucleocapsid protein (N), membrane protein (M) and envelope protein (E), whereas two-thirds of the 5’-end region encodes non-structural proteins
(nsp) includes: RNA-dependent RNA polymerase (nsp12), helicase (nsp13), papain-like protease (nsp3) and 3-chymotrypsin-like protease (nsp5) [12]. Deep
RNA sequencing study using RT-PCR established the abundant presence of RNA-dependent RNA polymerase and the N protein gene in the blood of a severely
infected SARS-CoV-2 patient [14]. Furthermore, 3’-end also encodes eleven accessory proteins includes: ORF3, ORF6, ORF7a, ORF7b, ORF8 and ORF9b factors
involved in host interaction and virulence in coronavirus but shows decreased conservation with SARS-CoV despite the close phylogenetic relationship [12].
SARS-CoV-2 shares 96.3% genetic identity with bat coronavirus RaTG13, although no credible evidence supports RaTG13 as an immediate source of SARS-
CoV-2 [15]. Following genome transcription analysis, beta-coronavirus produces an approximately 800 kDa polypeptide, and the polypeptide chain
is proteolytically cleaved by enzymes to produce various polypeptides. Upon virus entrance into the host cell, two replicases, polypeptide pp1a (~486 kDa) and
pp1ab (~790 kDa) are expeditiously translated [16]. Furthermore, 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro), both encoded by
ORF1a/b, are associated in the proteolytic cleavage of pp1a and pp1ab polypeptides [17], thus 3CLpro and PLpro executes a predominant role in genome
replication.

1.2. 3CLpro as a potential target for antiviral therapeutics:

3-Chymotrypsin like protease (3CLpro or Mpro) commonly referred to as main protease, is a cysteine protease with 33,796.8 Da molecular weight composed of
306 amino acids that directly results in proteolytic cleavage of the polypeptide for proper viral replication and function [18]. Comparative homology modelling
identified 3Clpro SARS-CoV-2 shares closest 99.02% sequence identity with bat SARS-like coronavirus 3CLpro (Bat-CoV/RaTG13) compare to 3CLpro of MERS-
CoV, SARS-CoV, Human-CoV and Bovine-CoV homologs of sequence identity with 87%, 96.08%, 90% and 90% respectively [19]. At 11 different sites, 3CLpro
cleaves the polypeptide to produce different non-structural polypeptides that are crucial for viral duplication [20]. The substrate-binding site of the enzyme
consists of four sub-sites based on the position of the substrate polypeptide namely S1, S2, S4 and S1’ [21]. Catalytically active structure of 3CLpro functions
as a homodimer, three dimensional structure analyses of 3CLpro revealed that two polypeptides (protomer A and B) conjugate to form a dimer, each protomer

Page 2/12
of this single-chain monomeric model contains three domains: I, II and III [18]. Domain I (8-101 residues) and domain II (102-184 residues) both forms
antiparallel β-barrel structure and domain III (201-303 residues) is comprised of five alpha-helices organised into an antiparallel globular cluster which is
further linked by a long loop region (185-200 residues) to domain II. Cleft between domain I and II, Cys145-His41 catalytic dyad is located (Figure 1), which is
predicted to be crucial in proteolytic action [20, 22–24].

Past studies explored in depth analysis of molecular mechanism of proteolytic cleavage by cysteine proteases, proposed that the polarisation and activation
of SH group of cysteine by the imidazole group of histidine forms CysS -/HisH+ion consist strong nucleophilic property to interact with substrate [25]. A
comparable research was conducted by Świderek and Moliner, cleavage of Ac-Val-Lys-Leu-Gln-ACC polypeptide catalysed by 3CLpro SARS-CoV-2. First, a
proton is transported from Cys-145 to His-41 with the nucleophilic action of the peptide bond's carbonyl carbon atom by the sulfur atom of Cys-145, resulting
in a thiohemiketal intermediate, which facilitates the breaking of peptide bonds. The primary outcome of this process is an ACC product, which is discharged
from the active site. Following the release of ACC, an activated water molecule targets the Gln-5 peptide's carbonyl carbon atom, triggering a proton transfer to
His-41. After the covalent link between Cys-145 and the peptide in the thiohemiketal intermediate is disrupted, the second product species is discharged [26].
Catalytic efficiency of 3CLpro SARS-CoV-2 measured at 28,500 M−1 s−1 higher comparative to 3CLpro of SARS-CoV and human rhinovirus catalytic
efficiency 26,500 M−1 s−1 and 920 M−1 s−1 respectively [18, 27, 28]. According to the comparative study on the structure of 3CLpro from SARS-CoV and 3CLpro
from SARS-CoV-2, these two proteases structurally differ by 12 amino acids (Val35Thr, Ser46Ala, Asn65Ser, Val86Leu, Lys88Arg, Ala94Ser, Phe134His,
Asn180Lys, Val202Leu, Ser267Ala, Ser284Ala and Leu286Ala), with α carbon atoms all positioned approximately 1 nm distant from the active site of
3CLpro [19, 29]. Recognition sequence of the active site is Leu-Gln↓(Ser,Ala,Gly) [↓ denotes cleavage site] [30]. However, a similar cleavage site is absent in
human proteases [30]. Also 3CLpro exhibits high tolerance level against mutation [31], which adds to the potency of the 3CLpro enzyme as a possible target
whereby inhibiting them can effectively terminate viral proliferation.

1.3. Hibiscus rosa sinensis based phytochemicals extracted from the flower- a prospective inhibitor for SARS-CoV-2:

Since ancient times, plants and their secondary metabolites remained as the major source of therapeutic formulations. During the period from 1 January 1981
- 30 September 2019, different natural product-based and derived drugs were approved: 247 anticancer drugs, 63 antidiabetic drugs and 53 anti-inflammatory
drugs [32]. Natural products could be an effective, safer and cheaper alternative to orthodox drugs [33]. In 2013, the World Health Organization (WHO) officially
announced the “WHO Traditional Medicine Strategy 2014–2023”, highlighting the significance of integrating traditional and orthodox medicine to improve
global healthcare [34].

India is one of the countries with a rich legacy of traditional medical systems as well as diverse biodiversity to support the herbal demands of these traditional
medical system treatments. India comprises approximately 45,000 plant species, accounting for an estimated 7% of the total worldwide plant species [35].
Various species of the Hibiscus genus plant widely distributed in India. Hibiscus rosa sinensis is one of the species of the Hibiscus genus and belongs to the
Malvaceae family. In India, traditionally, Hibiscus rosa sinensis flower extract is generally used for anti-diabetic, anti-microbial and wound healing activity [36–
38].

“Fail fast, fail early” has become a strategy for the modern drug developing industry to negotiate long-term investment depreciation [39, 40]. Conventional
approaches are expensive, time-consuming, and laborious. Recently, there has been a growing interest in mining natural product repositories for novel
therapeutic agents, and it would be interesting to estimate their potential against the SARS-CoV-2 using computational approaches. Computer-aided drug
discovery approaches rely on predicting the geometrical and energetically favourable fitness of potential lead compounds to the binding pocket of a molecular
target of interest. Moreover, in silico methods are now employed to evaluate the pharmacokinetic and pharmacodynamics properties of drug candidates,
thereby limiting animal trials to a great extent. From the past literature survey, there is no study evaluated the inhibitory property of phytochemicals from
Hibiscus rosa sinensis flower targeting 3CLpro SARS-CoV-2. The present work is aimed at investigating the binding affinity of phytochemicals identified from
Hibiscus rosa sinensis flower as potential 3CLpro inhibitors through an in silico approach.

2. Methodology
2.1. Preparation of 3CLpro structure for docking:

Three dimensional crystal structure of the 3-Chymotrypsin like protease (3CLpro) protease enzyme of SARS-CoV-2 (PDB ID: 6W79) with a resolution of 1.46Å,
was derived from the RCSB Protein Data Bank database (http://www.rcsb.org/pdb) [41]. The 3CLpro protein structure was pre-processed by using AutoDock
tools v1.5.7 [42]. Firstly, by eliminating X77 inhibitor, water molecules and hetero atoms were removed. Missing atoms were checked and repaired, polar
hydrogen and Kollman charges were added to the 3CLpro structure. After completion of pre-processing, the protein structure converted to PDBQT format.

2.2. Ligand Preparation:

From the literature survey, a total of 34 phytochemicals were identified, which were extracted from Hibiscus rosa sinensis flower. The SDF files of each ligand
molecule were retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov) [43]. Remdesivir (PubChem CID: 121304016) is a worldwide studied
drug that targets3CLpro [44, 45],which is considered as control for our in silico study. The SDF ligand files were converted to PDB format using OpenBabel
version 3.1.1 software [46], and ultimately converted to PDBQT format using AutoDock tools v1.5.7 [42].

2.3. Prediction of amino acid residues in the active site:

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PrankWeb (https://prankweb.cz/) online resource was used to estimate the amino acid residues forming the active site of 3CLpro (PDB:6W79 with X77
inhibitor removed) [47]. Based on the best probability score of 0.835, the programme predicted a potential active site formed by amino acid residues: Thr-25,
Thr-26, Leu-27, His-41, Cys-44, Ala-46, Met-49, Phe-140, Leu-141, Asn-142, Gly-143, Cys-145, His-163, His-164, Met-165, Glu-166, Asp-187, Arg-188 and Gln-189.
Manually comparing the amino acid sequence of the active site from our result with N3-bound 3CLpro complex (PDB ID: 6LU7) was done in LigPlot+ [48].
Comparative analyses revealed that the amino acid residues of the active site (PDB ID: 6LU7) identified were majority analogous to the PrankWeb predicted
result for 3CLpro (PDB: 6W79).

2.4. Receptor Grid generation:

Receptor grid was generated enclosing all the amino acid residues of target protein 3CLpro (PDB: 6W79) active site with coordinates centered at x,y,z: 16.910,
-29.376, 21.789 respectively and grid box dimensions of x,y,z: 80Å, 80Å, 80Å respectively. Grid box spacing of 0.375Å and exhaustiveness of 20 were selected
in AutoDock tools v1.5.7 [42].

2.5. Molecular docking of ligand and target protein:

Site-specific molecular docking of ligands against 3CLpro target (PDB: 6W79) with respect to generated receptor grid box was accomplished by utilising the
AutoDock Vina [49]. Docking was proceeding with rigid 3CLpro and flexible ligand structure. The PDBQT output file generated by AutoDock Vina, least binding
affinity (kcal/mol) interaction of the ligand-protein structure was visualised using PyMol (The PyMOL Molecular Graphics System, Version 2.5.2 Schrödinger,
LLC).

2.6. Two-dimensional visualisation of Ligand-Protein interaction:

Top 3 ligand-protein docked interacted structures along with the control remdesivir-protein structure were further analysed at the molecular level using
LigPlot+ [48]. The system generates a 2D ligand-protein interaction diagram that portrays the hydrogen bond interaction and hydrophobic contact pattern
between the ligand and residues of the active site of the target protein.

2.7. Absorption, distribution, metabolism, excretion and toxicity (ADMET) prediction:

ADMET properties of the top 9 docked ligands along with remdesivir were determined by using the ADMETlab 2.0 online server
(https://admetmesh.scbdd.com/) [50]. Physiochemical as well as drug-likeness parameters of the ligands were also calculated to evaluate non-toxic and
effective drug candidates.

3. Results
3.1. Computational molecular docking of bioactive phytochemicals against 3CLpro target:

After completion of the docking experiment for all 34 phytochemicals and control remdesivir by using AutoDock Vina, a vast range of ligand poses were
generated. Ligand pose with the best binding affinity was analysed in further study. Docking experiment was analysed in terms of the binding affinity of the
ligand with the active site of target 3CLpro. The statistical data of the top-ranked ligand obtained from docking is presented in Table 1.

Analysing the docking experiment of the top 9 ligands suggests that the value of binding affinity of phytochemicals ranges from -10.9 kcal/mol to -8.2
kcal/mol (Table 1). Cyanidin-3-sophoroside-5-glucoside (PubChem ID: 44256732) has shown the best binding affinity of -10.9 kcal/mol and quercetin-3-
diglucoside (PubChem ID: 10211337) has ranked ninth with a binding affinity of -8.2 kcal/mol (Table 1). The second ranked ligand, 1,2-benzenedicarboxylic
acid isodecyl octyl ester (PubChem ID: 14902) and third ranked ligand rutin (PubChem ID: 5280805) has shown binding affinity of -10.1 kcal/mol and -9.3
kcal/mol respectively (Table 1).Also, control inhibitor remdesivir (PubChem ID: 121304016) has shown a -8.0 kcal/mol binding affinity (Table 1). Analysing the
result suggests that the listed phytochemicals have a better affinity to 3CLpro target than the known inhibitor remdesivir. The top 3 docked phytochemicals
were further studied for ligand-protein interaction.

S.No PubChem ID Ligand name Binding affinity (kcal/mol)

1 44256732 Cyanidin-3-sophoroside-5-glucoside -10.9
2 14902 1,2-Benzenedicarboxylic acid isodecyl octyl ester -10.1
3 5280805 Rutin -9.3
4 33934 1,2-Benzenedicarboxylic acid, diisooctyl ester -9.1
5 44256718 Cyanidin-3-5,diglucoside -8.9
6 44259992 Hibiscetin-3-glucoside -8.4
7 44256720 Cyaniding 3-sophoroside -8.3
8 10121947 Quercetin-3,7-diglucoside -8.2
9 10211337 Quercetin-3-diglucoside -8.2
Control 121304016 Remdesivir -8.0

Table 1: Docking result of top-ranked ligands identified against 3CLpro active site of SARS-CoV-2 using AutoDock Vina.

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3.2. Two-dimensional visualisation of Ligand-Protein interaction:

For verification of site-specific ligand binding, LigPlot+ was used to visualise the interaction between the ligand and 3CLpro [48]. The primary amino acid
residues responsible for the formation of the active site are predicted, which include: Thr-25, Thr-26, Leu-27, His-41, Cys-44, Ala-46, Met-49, Phe-140, Leu-141,
Asn-142, Gly-143, Cys-145, His-163, His-164, Met-165, Glu-166, Asp-187, Arg-188 and Gln-189. Analysis of two-dimensional non-covalent interactions reveals
the important amino acid residues of the active site involved in interacting with potential phytochemicals. The binding interaction of top-ranked
phytochemicals based on docking results is depicted in Figure 2. Detailed analyses of ligand-protein interactions proved that the top 3 phytochemicals and
remdesivir interact with the catalytic dyad of 3CLpro as depicted in Table 2. The selected ligands interact with either both catalytic dyad residues (His-41 and
Cys-145) or at least one catalytic dyad residue through hydrogen bonding or hydrophobic interaction (Table 2). Cyanidin-3-sophoroside-5-glucoside (PubChem
ID: 44256732) formed hydrogen bond interaction with six amino acid residues of active site including catalytic dyad residue (Thr-25, His-41, Cys-44, Thr-45,
Asn-142, Gly-143,) and four hydrophobic interacting residues (Ala-46, Met-49, His-164, Met-165). Based on past docking studies, the more number of hydrogen
bond interactions, supports the stronger interaction between the ligand and the active site of the protein [51]. Analysing Table 2 concludes that the cyanidin-3-
sophoroside-5-glucoside (PubChem ID: 44256732) has shown most number of hydrogen bond interacting residues with less bond distance as compared to
the other two ligands and control remdesivir. After cyanidin-3-sophoroside-5-glucoside, rutin (PubChem ID: 5280805) formed the second most hydrogen bonds
interactions with five residues of the active site, including His-41 residue of the catalytic dyad (Table 2).

S.no PubChem Hydrogen bond residues Hydrophobic interaction Interaction with

ID Catalytic dyad residue
1 44256732 Thr-45, Cys-44, Thr-25, His- Met-49, Ala-46, His-164, Met-165 Yes
41, Gly-143, Asn-142
2 14902 - Ser-144, Glu-166, Cys-145, His-164, Met-165, Asp-187, Thr-45, Met-49, Cys-44, Thr-25, Yes
Ala-46, His-41, Leu-141, Phe-140, His-163
3 5280805 Cys-44, His-41, Thr-190, Thr-45, Ala-46, Thr-25, Cys-145, Glu-166, Met-165, Asp-187, Gln-189, Arg-188, Met-49 Yes
Tyr-54, Asn-142
Control 121304016 His-41 Thr-26, Thr-45, Gly-143, Met-165, Glu-166, Gln-189, Leu-167, Gln-192, Arg-188, Met-49, Yes
Thr-190, Tyr-54, Asp-187, Ala-46, Cys-44, Thr-25, Cys-145

Table 2- List of interacting residues of 3CLpro with top 3 ranked phytochemicals and control inhibitor remdesivir.

3.3. Physiochemical, Pharmacokinetic and drug-likeness property prediction:

Physiochemical property prediction parameters with an optimal range mentioned in Table 3 are: molecular weight, density, number of hydrogen bond acceptor
and donor, TPSA, Log S (water solubility) and Log P (partition coefficient). Smaller the TPSA value, the greater permeability of the ligand through the cell
membrane. When LogP = 0, the ligand is equally distributed between the lipid and aqueous phases; a negative value of LogP determines the ligand has a
stronger affinity for the aqueous phase (it is more hydrophilic); a positive value of LogP determines the ligand has a greater concentration in the lipid phase
(i.e., the ligand is more lipophilic) [52]. Also, the water solubility of a drug is determined by the Log S value prediction: less than -10 (poorly soluble), less than
-6 (moderately soluble), less than -4 (soluble), less than -2 (very soluble) and less than 0 (highly soluble) [53].

The physiochemical properties of the top 9 docked ligands and control remdesivir were predicted and studied as shown in Table 3. Different physiochemical
parameters along with their optimal range are mentioned in Table 3. Analysing Table 3 revealed that cyanidin-3-sophoroside-5-glucoside (PubChem ID:
44256732) violates five parameters of physiochemical properties: molecular weight (773.21 g/mol), number of hydrogen bond acceptors (21), number of
hydrogen bond donors (14), TPSA (349.9Ų) and Log P (-2.233). The second rank docked ligand, 1,2-benzenedicarboxylic acid isodecyl octyl ester (PubChem
ID: 14902), showed violation of two physiochemical parameters: Log S (-7.225) and Log P (8.534) (Table 3). Rutin (PubChem ID: 5280805) showed four
violations of the physiochemical properties prediction (Table 3).And, control inhibitor remdesivir (PubChem ID: 121304016) violates two parameters of
physiochemical properties: number of hydrogen bond acceptor (14) and TPSA (204.28Ų) (Table 3).

PubChem Ligand name MW (130-725 Density nHB acceptor (0- nHB donor (0- TPSA (0-140 LogS (-4 to 0.5 LogP (0-3)
ID g/mol) 12) 7) Ų) mol/L)

44256732 Cyanidin-3-sophoroside-5- glucoside 773.21 1.116 21 14 349.9 -1.092 -2.233

14902 1,2-Benzenedicarboxylic acid isodecyl octyl 418.31 0.887 4 0 52.6 -7.225 8.534
ester
5280805 Rutin 610.15 1.105 16 10 269.43 -3.742 -0.038
33934 1,2-Benzenedicarboxylic acid, diisooctyl ester 390.28 0.893 4 0 52.6 -7.04 7.494
44256718 Cyanidin-3-5,diglucoside 709.16 1.116 19 10 303.12 -2.144 -0.753
44259992 Hibiscetin-3-glucoside 496.09 1.129 14 10 250.97 -3.659 -0.587
44256720 Cyanidin 3- sophoroside 611.16 1.104 16 11 270.75 -2.107 -1.066
10121947 Quercetin-3,7-diglucoside 626.15 1.116 17 11 289.66 -3.673 -1.521
10211337 Quercetin-3-diglucoside 626.15 1.116 17 11 289.66 -3.303 -1.367
121304016 Remdesivir 602.23 1.055 14 5 204.28 -2.392 1.664

Table 3-Physiochemical descriptors of top-ranked phytochemicals and control remdesivir.

(Abbreviations: MW- Molecular weight, nHB- Number of Hydrogen bond and TPSA- Topological Polar Surface Area.)

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The five major components of pharmacokinetics are absorption, distribution, metabolism, excretion, and toxicity (ADMET). All five factors play a significant
role in the pharmacological efficacy of therapeutic drugs as well as their bioavailability. In this study, Table 4 determines the indicative parameters with an
optimal range of ADMET parameters prediction, includes: Caco-2 and MDCK Permeability (Absorption); Plasma Protein Binding, Volume Distribution and
Blood Brain Penetration (Distribution); CYP1A2, CYP2C19, CYP2C9 and CYP2D6 inhibitor (Metabolism); Clearance and T1/2 (Excretion); hERG blockers,
Human Hepatotoxicity, AMES Toxicity, Skin Sensitization, Carcinogenicity and Respiratory Toxicity (Toxicity). The oral bioavailability (drug-likeness) of a drug
predicted through Lipinski’s rule, a chemical agent is predicted to be a non-orally available drug if two or more Lipinski’s rule are violated [54].

Analysing the ADMET and drug-likeness data from Table 4 revealed that cyanidin-3-sophoroside-5-glucoside (PubChem ID: 44256732) follows the majority of
ADMET parameters of distribution, metabolism and toxicity, except three: low Caco-2 permeability (-6.532), with an intermediate blood brain penetration and
low clearance (1.01 ml/min/kg), also violates Lipinski’s rule. The second ranked ligand, 1,2-benzenedicarboxylic acid isodecyl octyl ester (PubChem ID:
14902), violates three ADMET parameters: high plasma protein binding (98.29%), potential CYP2C19 inhibitor and highly toxic to skin sensitisation (Table 4).
And the third ranked ligand, rutin (PubChem ID: 5280805), violates three ADMET parameters: low Caco-2 permeability (-6.336), low clearance (1.349
ml/min/kg), AMES toxic and three Lipinski’s rule violation (Table 4). Among all the nine ligands, control inhibitor remdesivir (PubChem ID: 121304016) violates
the seven ADMET parameters: low Caco-2 Permeability (-5.996), highly potential CYP2D6 inhibitor, low clearance (3.434 ml/min/kg), intermediate hERG
blockers, high human hepatotoxicity, AMES toxicity and respiratory toxicity (Table 4).

ADMET Parameters Cyanidin-3- 1,2- Rutin 1,2- Cyanidin-3- Hibiscetin-3- Cyanidin 3- Quercetin- Quercetin-3-
sophoroside- Benzenedicarboxylic (PubChem Benzenedicarboxylic 5,diglucoside glucoside sophoroside 3,7- diglucoside
5-glucoside acid isodecyl octyl ID: acid, diisooctyl ester (PubChem (PubChem (PubChem diglucoside (PubChem
(PubChem ester 5280805) (PubChem ID: ID: ID: ID: (PubChem ID:
ID: (PubChem ID: 33934) 44256718) 44259992) 44256720) ID: 10211337)
44256732) 14902) 10121947)

Absorption Caco-2 -6.532 -4.837 -6.336 -4.655 -6.57 -6.426 -6.459 -6.412 -6.412
Permeability
(higher than
-5.1510–6 cm/s)
MDCK 0.00028 0.000015 0.00003 0.000018 0.000073 0.00001 0.000069 0.00011 0.000071
Permeability
(2x10-6 -20x10-
6cm/s)

Distribution Plasma Protein 62.47% 98.29% 83.81% 97.63% 77.07% 85.48% 80.52% 77.14% 81.13%
Binding
(<90%)
Volume 0.458 2.067 0.754 1.445 0.576 0.854 0.702 0.731 0.733
Distribution
(0.04-20 L/Kg)
Blood Brain - --- -- --- -- --- - - --
Penetration
Metabolism CYP1A2 --- -- --- -- --- --- --- --- ---
inhibitor
CYP2C19 --- + --- + --- --- --- --- ---
inhibitor
CYP2C9 --- -- --- - --- --- --- --- ---
inhibitor
CYP2D6 --- -- --- -- --- --- --- --- ---
inhibitor
Excretion Clearance 1.01 7.783 1.349 9.241 1.455 4.395 1.542 1.51 1.444
(High:>15
ml/min/kg,
Low: <5
ml/min/kg)
T1/2 0.494 0.034 0.524 0.044 0.776 0.929 0.696 0.522 0.668
(Probability
value of long
half-life)
Toxicity hERG blockers --- -- --- -- --- --- --- --- ---
Human -- --- --- --- -- -- -- --- --
Hepatotoxicity
AMES Toxicity -- --- ++ --- + ++ + + ++
Skin --- +++ --- +++ --- ++ --- --- ---
Sensitisation
Carcinogenicity --- -- --- - -- --- --- -- ---
Respiratory --- --- --- --- --- --- --- --- ---
Toxicity
Oral drug- No. of 3 1 3 1 3 2 3 3 3
likeness Lipinski’s rule
violation

Page 6/12
Table 4: ADMET properties and drug-likeness prediction of top-ranked phytochemicals and control inhibitor remdesivir using ADMETlab 2.0.

*Note- The predicted probability values are interpreted into six symbols: (---)0-0.1, (--)0.1-0.3, (-)0.3-0.5, (+)0.5-0.7, (++)0.7-0.9, and (+++)0.9-1.0.

Indication of the coloured box (according to ADMETlab 2.0):

Potentially harmful in a particular parameter

Potentially safe in a particular parameter
Potentially intermediately safe in a particular parameter

4. Discussion
Phytochemicals are an incredible resource of potential therapeutic agents that target a wide range of diseases conditions including viral infection.
Identification of potential plant-based products that can target the viral life cycle and virus-host specific interactions would be a promising alternative to
synthetic drug compounds. The current COVID-19 pandemic has urged us to investigate potential therapeutic agents from a wide spectrum of natural plant-
based products. In silico methodologies generate essential preliminary data on binding affinity and thermodynamic stability of the target-ligand complex,
which could be used to optimise experimental methods in future research. In this study, we targeted 3CLpro from SARS-CoV-2 and virtually screened
phytochemicals from Hibiscus rosa sinensis flower as potential COVID-19 drug candidates. Molecular docking, protein-ligand interaction analysis,
physiochemical, ADMET and oral drug-likeness analyses were performed. Amongst the 34 screened phytochemicals from Hibiscus rosa sinensis flower, nine
phytochemicals showed a favourable strength of binding affinity as compared to the control inhibitor remdesivir against 3CLpro (Table 1). Docking result
revealed that cyanidin-3-sophoroside-5-glucoside (PubChem ID: 44256732) shows the strongest binding affinity (-10.9 kcal/mol) against the active site of
3CLpro SARS-CoV-2 in contrast to other ligands and control remdesivir (Table 1). Also, ligand-protein interaction data analysis suggests that cyanidin-3-
sophoroside-5-glucoside formed the most number of hydrogen bond interactions with the amino acid residue of the active site and His-41 residue of the
catalytic dyad (Figure 2 and Table 2). The second rank docked ligand, 1,2-benzenedicarboxylic acid isodecyl octyl ester (PubChem ID: 14902) has favourable
binding affinity -10.1 kcal/mol (Table 1) but ligand-protein interaction revealed that it showed no hydrogen bond interaction with 3CLpro active site although
hydrophobic interaction formed with the catalytic dyad residues (His-41 and Cys-145) along with other 13 amino acid residues (Figure 2 and Table 2). Third
ranked ligand rutin (PubChem ID: 5280805) has shown binding affinity of -9.3 kcal/mol and formed second most number of hydrogen bond interaction with
amino acid residues of 3CLpro active site and His-41 residue of the catalytic dyad, also hydrophobic interaction with Cys-145 residue (catalytic dyad) along
with other nine amino acid residues of the active site (Figure 2 and Table 2). Analysing the above mentioned data proves that cyanidin-3-sophoroside-5-
glucoside efficiently binds with the active site complementary targeting catalytic dyad residue with the strongest binding affinity and formed maximum
hydrogen bond interaction with active site residues.

Predicted data of physiochemical properties revealed that among the top 3 docked ligands, cyanidin-3-sophoroside-5-glucoside (PubChem ID: 44256732)
violates the majority of parameters: molecular weight (773.21 g/mol), number of hydrogen bond acceptors (21), number of hydrogen bond donors (14), TPSA
(349.9 Ų) and Log P (-2.233), suggests that cyanidin-3-sophoroside-5-glucoside possess high hydrophilic nature and low permeability through the cell
membrane (Table 3). ADMET analyses support the physiochemical properties data of cyanidin-3-sophoroside-5-glucoside, which predicts low Caco-2
permeability (-6.532), with intermediate blood-brain penetration and low clearance (1.01 ml/min/kg), also violates Lipinski’s rule but follows other ADMET
parameters (Table 4). Physiochemical prediction revealed that the 1,2-benzenedicarboxylic acid isodecyl octyl ester (PubChem ID: 14902) has poor water
solubility and lipophilic in nature (Table 3). Also, ADMET analysis predicts three violations of 1,2-benzenedicarboxylic acid isodecyl octyl ester: high plasma
protein binding (98.29%), potential CYP2C19 inhibitor and highly toxic to skin sensitisation, but follows Lipinski’s rule (Table 4). And rutin with four
physiochemical properties violations concludes with high hydrophilic nature, low permeability through the cell membrane, high number of hydrogen bond
donors and acceptors (Table 3), also violates ADMET parameters: low Caco-2 permeability(-6.336), low clearance (1.349 ml/min/kg) and AMES toxic, also
violates Lipinski’s rule (Table 4). And, control remdesivir does not follow two physiochemical properties (Table 3) and showed maximum ADMET properties
violation as compared to other ligands (Table 4).

Considering the above mentioned data, rutin has shown less strength of binding affinity as compared to cyanidin-3-sophoroside-5-glucoside and 1,2-
benzenedicarboxylic acid isodecyl octyl ester, which also violates a favourable number of ADMET properties and Lipinski’s rule, so rutin is not considered for
further evaluation. Cyanidin-3-sophoroside-5-glucoside and 1,2-benzenedicarboxylic acid isodecyl octyl ester have good strength of binding affinity, and1,2-
benzenedicarboxylic acid isodecyl octyl ester follows the majority of physiochemical and ADMET properties with no Lipinski’s rule violation as compared to
cyanidin-3-sophoroside-5-glucoside. But hydrogen bonding interaction analysis from the ligand-protein diagram (Figure 2) favours cyanidin-3-sophoroside-5-
glucoside as more efficient and high strength of binding affinity against the 3CLpro active site targeting residue of the catalytic dyad as compared to 1,2-
benzenedicarboxylic acid isodecyl octyl ester with no hydrogen bond interaction with the amino acid residue of the 3CLpro active site. Structural modification
of cyanidin-3-sophoroside-5-glucoside could improve and optimise a few physiochemical and ADMET properties. From past studies identified 3CLpro of SARS-
CoV-2 shows 99.02% sequence homology to 3CLpro of Bat-CoV [19], this signifies that cyanidin-3-sophoroside-5-glucoside identified from our study could also
be an effective phytochemical against Bat-CoV (RaTG13), which can be experimentally examine in future studies. Also, cyanidin-3-sophoroside-5-glucoside
showed Lipinski’s rule violation means the ligand is non-orally bioavailable, it can be administered through different routes such as intravenous, subcutaneous
or intramuscular for better bioavailability. Further investigation of cyanidin-3-sophoroside-5-glucoside in terms of in vitro, in vivo experiment and clinical trials
to assess and optimise the therapeutic action against 3CLpro of SARS-CoV-2.

5. Conclusion

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Our research aimed to perform sequential in silico investigations including molecular docking, ligand-protein interaction, physiochemical, ADMET and drug-
likeness properties prediction to discover potential phytochemical inhibitors against 3CLpro enzyme to terminate the proliferation of SARS-CoV-2. We identified
cyanidin-3-sophoroside-5-glucoside isolated from Hibiscus rosa sinensis flower [55] as a potential drug candidate that targets the His-41 residue of the
catalytic dyad and binds strongly to the active site of 3CLpro with a binding affinity of -10.9 kcal/mol. Following the majority of parameters of physiochemical,
ADMET and drug-likeness properties analysis predicts the stability of cyanidin-3-sophoroside-5-glucoside in the biological system. From this study, we
conclude that the identified novel phytochemical cyanidin-3-sophoroside-5-glucoside has potential inhibitory action against SARS-CoV-2. Our experiment also
underscores the importance of employing computational approaches to mine nature’s repository of plant-based compounds as therapeutic agents against the
pandemic virus.

Declarations
6. ACKNOWLEDGEMENT

The authors are grateful for the support received from the Department of Biotechnology at Gandhi Institute of Engineering and Technology University.

7. DECLARATIONS

Ethical Approval: This article does not contain any studies with human participants or animals performed by any authors.

Competing interests: The authors declare no competing interests.

Authors' contributions: Subhadeep Das conceived the original idea and designed the computational experimental framework. Diptikanta Acharya and Sushil
Kumar Sahu supervised the findings of this work. All the authors contributed to the analysis of experimental data. Subhadeep wrote the manuscript in
consultation with Sagarika Satapathy, Diptikanta Acharya and Sushil Kumar Sahu. All authors have approved the manuscript for publication.

Funding: No funding acquired.

Availability of data and materials: The data used to support the findings of this study are included within the article and also available on request from the
corresponding author.

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Figures

Figure 1

Cartoon representation of SARS-CoV-2 3CLpro [PDB ID- 6W79] with removed X77 inhibitor using PyMol. The residue of catalytic dyad Cys-145 is represented in
an orange-coloured clustered sphere and His-41 is shown in a red-coloured clustered sphere.

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View publication stats

Figure 2

Schematic 2D representation of the ligand-protein interaction with bond length analysis: (A) Cyanidin-3-sophoroside-5-glucoside; (B) 1,2-Benzenedicarboxylic
acid isodecyl octyl ester; (C) Rutin and (D) Remdesivir docked with 3CLpro SARS-CoV-2 (PDB ID: 6W79) using LigPlot+.

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