Potential activity of a selected natural compounds on SARS-CoV-2 RNA-dependent-RNA polymerase, and binding affinity of the receptor-binding domain (RBD)

doi:10.21203/rs.3.rs-32971/v1

Coronavirus disease (COVID-19) is caused by SARS-CoV-2 and represents the causative agent of a potentially lethal disease. COVID-19 has been described as a significant global public health pandemic by the World Health Organization due to its high mortality rate, rapid spread, and the lack of drugs and vaccines for it. Active antiviral drugs are desperately needed to combat the potential return of severe acute respiratory syndrome (SARS).

In this study, we selected 39 natural compounds present in plants, algae, and sponges with antiviral activity. Molecular docking was used to screen the compounds’ activity on SARS- CoV-2 RNA-dependent-RNA polymerase, receptor-binding domain (RBD), and the human ACE2 receptor. Compounds with binding energy ≤ -6.5 kcal/mol enter pre-clinical testing using in silco ADME/Tox (absorption, distribution, metabolism, excretion, and toxicity).

We found eight potential SARS-CoV-2 inhibitors: (glycyrrhizin, rutin, baicalin, 1, 6-di-O- galloyl-beta-D-glucose, pyropheophorbide A, pheophorbide A, beta-Sitosterol, and vitexin). These outcomes indicate that these compounds could be potential candidates to be utilized in lead optimization for the design and production of the anti-SARS-CoV-2 drug.

Bioinformatics

Drug Discovery, Design, & Development

Coronavirus

SARS-COV2

In silco

Molecular Docking

Natural compounds

Antiviral

In December 2019, a new coronavirus (2019-nCoV) infection was reported in hospitalized patients with pneumonia of unknown cause in Wuhan, Hubei Province, China ¹. On January 30, 2020, the World Health Organization (WHO) officially declared the COVID- 19 epidemic a global health emergency ².

COVID–19 is the name given to this novel coronavirus (CoV), spread throughout the world. Coronaviruses are enveloped RNA that belongs to the subfamily Coronavirinae of the family Coronaviridae; they broadly commonly spread by humans, birds, and other mammals, causing respiratory and intestinal infections in both animals and humans ³. Six species of coronavirus are known to cause human disease ⁴. Two of them are highly pathogenic and have previously caused extreme acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and 2003 in Guangdong Province, China ⁵^,⁶, and Middle East respiratory syndrome coronavirus (MERS-CoV) in in the Middle East in 2012 ⁷. These two viruses have been linked in some cases to fatal illness (Cui et al., 2019). The other four species (HCoV–229E, HCoV-OC43, HCoV- NL63, and HCoV-HKU1) are prevalent and typically induce only mild upper respiratory diseases in immunocompetent hosts ⁴.

COVID–19 is a β-coronavirus (CoV) that causes self-limiting upper infections in immunocompetent hosts. Severe symptoms such as breathing difficulty and pneumonia occur in immunocompromised and elderly persons and those with chronic underlying diseases. Moreover, the touching of contaminated objects seems to characterize the propagation of COVID–19 ⁸.

The angiotensin-converting enzyme 2 (ACE2) is an enzyme that transforms angiotensin II into angiotensin (1–7) ⁹^,¹⁰. Angiotensins are peptides involved in maintaining blood pressure control and arterial hypertension by increasing the secretion of aldosterone ¹¹ and promoting sodium retention by the kidneys ¹². ACE2 receptors are attached to the surfaces of the lungs, heart, and kidney cell membranes ¹³^,¹⁴.

The spike (S) glycoprotein of SARS-CoV–2 contains the receptor-binding domain (RBD) that recognize specific cell receptors for its attachment. Angiotensin is peptides involved in membrane fusion and entry process through endocytosis ¹⁵. It has been reported that ACE2 is the receptor of the SARS-CoV–2 S glycoprotein ¹⁶^,¹⁷ due to the presence of affinity domains ¹⁸..

The interaction of this spike protein with ACE2 could be responsible for lung damage ¹⁹. Therefore, the RBD of spike glycoprotein could be a candidate for drug targets for inhibiting the initiation process of virus infection ²⁰.

In the absence of antiviral treatment, infected patients receive oxygen therapy and immunoglobulin G for critical cases ²¹. Antiviral drugs were previously tested in clinical practice, including nucleoside analogs acting as polymerase inhibitors, such as ribavirin (hepatitis C virus), acyclovir (herpes virus), ganciclovir (cytomegalovirus), and favipiravir (Ebola and influenza A virus); protease inhibitors, such as lopinavir and ritonavir (human immunodeficiency virus, SARS, and MERS), and nafamostat (influenza virus and Ebola); and neuraminidase inhibitors, such as oseltamivir (influenza virus A). However, all of the tests using these molecules were invalid for COVID–19. By contrast, the best results were obtained with the non-antiviral drug chloroquine, which has been used since the 1950s to treat malaria ²². Nevertheless, its antiviral mechanism is not well understood; however, we suppose that this compound inhibits pH-dependent steps of the virus replication ²³ or interferes with the glycosylation of cellular receptors ²⁴. In vitro studies also demonstrated that chloroquine acts at both entry and post-entry stages of the COVID–19 infection on Vero cells ²⁵. Remdesivir, which is an adenosine nucleotide analog prodrug, has been reported to exhibit activity against several RNA viruses ²⁶ by inhibiting RNA polymerase activity ²⁷.

This study aimed to in silico evaluate the effect of 39 natural compounds, which have been reported to exhibiting in vitro antiviral activity, on the SARS-CoV–2 RNA-dependent-RNA polymerase activity as well as on their interaction with RBD and ACE2. The compounds, which displayed a binding energy more than 6.5 Å, were tested for absorption, plasma clearance, tissue distribution, metabolic effects, toxicity, and drug-likeness using Pre ADMET profiling.

2.1. Natural products and active molecules with antiviral activity

Thirty-nine natural compounds isolated from plants, algae, and sponges (Table 1) and three antiviral drugs (Velpatasvir, IDX–184, and Oseltamivir) have been tested by in silico for their inhibitory activity on SARS-CoV–2 RNA-dependent-RNA polymerase activity, as well as RBD and ACE2 interaction.

Table1: Selected natural products displayed antiviral activities

Origin	Natural compound	Targeted Virus	References
Acacia nilotica	6-di-O-galloyl-beta-	-Anti Hepatitis C	²⁸^,²⁹
(Plant)	D-glucose	virus (HCV), Anti
	Melacacidin	HIV-1
	Digallic acid
	Ellagic acid
	Ethyl gallate
	Gallic acid
	Methyl gallate
Nigella sativa	Beta-Sitosterol	AIV (H9N2)	³⁰
(Plant)	Dithymoquinone
	Thymohydroquinone
	Thymol
	Thymoquinone
Commiphora	Guggulsterone E	HSV-2	³¹
gileadensis (Plant)		RSV-B
Opuntia ficus	Pheophorbide A	HSV-2	³¹
indica	Pyropheophorbide A	IIFV-A
(Plant)
Salvadora persica	Benzylisothiocyanate	HSV-1	³²
L.,
(Plant)
Peganum harmala	Harmine	HSV-2	³³
(Plant)
Lycoris radiata	Lycorine	SARS-CoV	³⁴
(Plant)	Glycyrrhizin
Eugenia	Eugenol	HSV	³⁵
caryophyllus
(Plant)
Flos Lonicerae	Chlorogenic acid	SARS-CoV	³⁶
(Plant)

Punica granatum (Plant)	Punicalagin	HEV-71	³⁷
Scutellaria baicalensis (Plant)	Baicalin	SARS-CoV	³⁶
Brassica oleracea (Plant)	Sulforaphane	IFV-A	³⁸
Cladosiphon okamuranus (Brown algae)	Fucoidan	NDV	³⁹
Pseudodistoma antinboja (Sponge)	Butenolides	IFV-A (H1N1)	⁴⁰
Honey (Bee product)	Rutin	VZV IFV	⁴¹^,⁴²
Honey (Bee product)	Acacetin	VZV IFV	⁴¹^,⁴²
	Apigenin
	Catechin
	Chrysin
	Hispidulin
	Kaempferol
	Luteolin
	Myricetin
	Naringetol
	Quercetin
	Tricetin
	Vitexin

AIV: Avian influenza virus; HCV: hepatitis C virus; HSV: herpes simplex virus; HEV: human enterovirus; IFV: influenza virus; NDV: Newcastle disease virus; RSV: respiratory syncytial virus; VZV: varicella-zoster virus

2.1 Homology modeling

The structures of SARS-CoV–2 RNA- dependent RNA polymerase (RdRp) (7BV2), spike protein model (6VSB) and human ACE2 receptor (6VW1) in complex with RBD of SARS-CoV–2, were obtained from the RCSB PDB database ⁴³.

2.2 Preparation of proteins and inhibitors

The structures of RdRp, ACE2, and the SARS-CoV–2 spike protein were prepared for docking by protonation, water molecules removal, atom fixation, RMSD gradients refinement, and energy minimization by the drug discovery platform Molecular Operating Environment (MOE) suite (demo version 2019; Chemical Computing Group Inc: Montreal, QC, Canada). Thirty- nine natural products compounds (Table 1) and three antiviral references (Velpatasvir, IDX- 184, and Oseltamivir) structures were obtained from the PubChem database ⁴⁴.

2.3 Molecular docking

The default docking parameters in MOE were used to study the best stable conformational binding (poses with the lowest energy level) between ligands and protein receptors. Docking with root-mean-square deviation (RMSD) less than 2 Å, was considered a success ⁴⁵. Known and predicted active sites and interacting residues were used for ligand docking ¹⁷.

2.4 Protein-Ligand interaction analysis

The binding affinities, visualization, and interactions of ligand-receptor complexes were examined by the Protein-Ligand Interaction Profiler (PLIP) web server (Salentin et al., 2015), and Discovery Studio Visualizer 2020 (BIOVIA, 2017). Compounds with the most favorable binding energy (≤ –6.5 kcal/mol), were selected for further analysis ⁴⁶.

2.5 ADMET Profiling

To assess the potential pharmacokinetic properties of the top eight compounds (that scored the lowest energy ≤ –6.5), we conducted a preliminary computational screening using PreADMET profiling to estimate their absorption rate, plasma clearance, tissue distribution, metabolic effects, toxicity, and drug likeness ^47–49.

3.1.1 Target proteins active site prediction

MOE software site finder specified the following residues in the active site of the SARS-CoV–2 RdRp: ASP161, TYR163, ASP164, PHE165, GLU167, LYS551, ARG553, GLY616, TRP617, ASP618, TYR619, PRO620, LYS621, ASP760, ASP761, ALA762, VAL763, VAL792, PHE793, MET794, SER795, ALA797, LYS798, CYS799, TRP800, HIS810, GLU811, and PHE812. The active sites of SARS-CoV–2 spike protein RBD and ACE2 receptor were determined according to previously published data ⁴³^,⁵⁰.

3.1 Docking and interaction analysis

From 39 natural compounds virtually screened for their activities on RdRp, ACE2, and RBD proteins, only eight hits scored binding energy ≤ –6.5 kcal/mol (Table 2). Then the promoting eight compounds were selected for further interaction analysis and PreADMET profiling.

Table 2: Molecular Docking results and PubChem ID of the selected compounds and some antiviral agents

			RdRp		ACE2		RBD-SARS- CoV-2
No	Compounds	PubChem ID	S	RMS D	S	RMS D	S	RMS D
1	Glycyrrhizin	14982	-7.9	2.0	-7.8	1.8	-7.3	2.3
2	Rutin	5280805	-7.8	2.0	-6.9	2.9	-6.9	1.5
3	Baicalin	64982	-7.6	1.3	-6	2.4	-6.4	1.0
4	1,6-di-O-galloyl- beta-D-glucose	440221	-7.5	2.9	-6.3	1.7	-6.2	1.7
5	Pheophorbide A	253193	-7.3	1.0	-6.5	3.5	-6.6	2.0
6	Pyropheophorbide A	161456	-7.3	1.4	-6.2	2.0	-6.4	1.5
7	Beta-Sitosterol	222284	-6.9	1.5	-6	3.9	-5.9	1.8
8	Vitexin	5280441	-6.5	2.4	-5.6	1.5	-5.6	2.7

9	Chlorogenic acid	1794427	-6.4	1.5	-5.7	1.8	-5.9	1.9
10	Dithymoquinone	398941	-6.2	1.4	-4.7	2.4	-5.2	0.9
11	Hispidulin	5281628	-6	1.1	-5.2	1.5	-5.4	3.1
12	Melacacidin	169996	-5.8	1.3	-5	2.5	-5.1	1.5
13	Catechin	9064	-5.8	2.4	-5.1	1.4	-5.1	1.9
14	Fucoidan	92023653	-5.8	1.5	-5.2	1.8	-5.1	4.9
15	Gallic acid	370	-5.5	0.6	-4	4.4	-4.2	2.4
16	Methyl gallate	7428	-5.7	1.6	-5.4	1.7	-4.8	2.4
17	Trans,Trans- Farnesol	445070	-5.7	1.3	-5.3	1.3	-5.4	1.6
18	Digallic acid	341	-5.6	1.1	-5.5	1.2	-5.3	2.5
19	Myricetin	5281672	-5.6	2.7	-5.2	1.7	-5.2	1.6
20	Ellagic acid	5281855	-5.5	0.6	-4.8	0.8	-4.9	1.8
21	Ethyl gallate	13250	-5.3	1.3	-4.2	1.0	-4.7	1.4
22	Thymoquinone	10281	-5.0	1.3	-4.4	1.0	-4.4	0.6
23	Acacetin	5280442	-5.4	2.8	-5 .0	1.3	-5.2	1.9
24	Apigenin	5280443	-5.4	1	-5.0	2.5	-4.9	1.4
25	Chrysin	5281607	-5.1	1.6	-4.8	1.6	-4.9	1.2
26	Kaempferol	5280863	-5.4	2.5	-5.2	1.4	-5.2	0.9
27	Luteolin	5280445	-5.3	2.4	-5.2	1.1	-5.0	1.4
28	Naringetol	439246	-5.7	1.5	-5.1	0.9	-5.1	1.1
29	Quercetin	5280343	-5.6	1.2	-5.0	2.4	-5.4	1.2
30	Tricetin	5281701	-5.3	2.0	-5.2	0.6	-5.2	1.1
31	Guggulsterone E	6439929	-5.5	2.0	-4.9	1.2	-5.2	3.1
32	Benzylisothiocyanat e	2346	-5.2	3.9	-4.2	1.2	-4.4	1.6
33	harmine	5280953	-5.5	2.4	-4.5	2.1	-4.7	1.0
34	lycorine	72378	-5.4	4.9	-4.8	2.3	-5.2	1.5
35	Eugenol	3314	-5.4	1.0	-4.2	1.1	-4.5	0.9
36	Butenolides	386024422	-5.6	1.4	-4.5	0.9	-4.5	1.3
37	Sulforaphane	5350	-5.3	2.3	-4.4	1.6	-4.7	1.2
38	Thymol	6989	-4.5	1.0	-4.1	3.9	-4.5	1.5
39	Thymohydroquinone	95779	-4.8	2.9	-4.4	1.2	-4.4	1.9
Antiviral agents
40	Velpatasvir	67683363	-9.3	1.7	-	-	-5.5	2.0
41	IDX-184	135565589	-8.8	2.5	-	-	-6.6	4.0
42	Oseltamivir	65028	-6.5	2.1	-	-	-5.1	2.2

Abbreviations: S= binding score in kcal/mol, RdRp= RNA-dependent-RNA polymerase, RBD= Receptor binding Domain of virus spike protein

3.3 Interactions of ligands and RNA-dependent RNA polymerase (RdRp)

As shown in Table 3, the docking results of glycyrrhizin show the lowest binding affinity (–7.9) among all of the screened compounds that forming many hydrogen bonds, alkyl, and carbon hydrogen bond (Figure 1). Docking of glycyrrhizin is considered successful with RMSD 2 (dockings with RMSDs 2Å are considered a successful, docking with RMSD 2–3Å are considered a partial success) ⁵¹. The activity of glycyrrhizin on SARS- CoV–2 polymerase, aligned with previous studies, confirmed the activity of glycyrrhizin on the replication of SARS-CoV and polymerase of influenza virus ⁵²^,⁵³. Rutin with RdRp active site showed the second-lowest binding energy, at –7.8; this was a result of the formation of two conventional hydrogen bonds, two pi-alkyl, one pi-donor hydrogen bond, three pi-cation bonds, and five carbon-hydrogen bonds with residues located at the conserved pocket of RdRp ⁵⁴. The docking of rutin is considered successful with RMSD 2 Å (Table 3) (Figure 2).

Baicalin formed a strong interaction with protein active site, which scored low binding energy (–7.6) and RMSD 1.3. Baicalin strong interaction and well configuration in protein pocket occurred due to the presence of six hydrogen bonds with distances less than 3.33 Å. In addition to the presence of H bonds, there were many other types of bonds, such as pi-alkyl, pi-sigma, Pi-cation, pi-anion, pi-pi t-shaped, pi-lone pair, carbon hydrogen bond (Table 3) (Figure 3).

Table 3. Interaction of active compounds with SARS-CoV-2 RdRp.

Compounds (PubChem ID)	S	RM SD	Protein ligands interaction
Compounds (PubChem ID)		RM SD	Type of interaction	Amino acid residues (No. of bonds)	Distance (Å)
Glycyrrhizin (14982)	-7.9	2.1	Conventional hydrogen bonds	TYR619 (1), LYS621 (1), ASP684 (1), ALA688 (1), ARG624, ARG553(1)	3.03, 2.61, 2.95, 2.42, 2.97, 2.41
				ASP760 (2)	2.75, 2.10
			Alkyl	ARG555, ALA688	3.49, 4.95
			Carbon Hydrogen bonds	THR687(1), ASP623 (3), ARG533(1)	2.47, 2.65, 2.22, 2.25, 2.53,
Rutin (5280805)	-7.8	2.0	Conventional hydrogen bonds	TYR619(1), ARG553(1)	2.27, 2.16
			Pi-Alkyl	LYS621 (1), ARG553	4.47, 4.2
			Pi-Donor Hydrogen bond	PRO620 (1)	2.82
			Pi-Cation	LYS621(1), ARG553 (2)	4.52, 3.89
			Carbon Hydrogen bond	ASP760 (2), ASP623(1), LYS621 (2)	2.51, 2.66, 2.60, 2.91, 2.80
Baicalin (64982)	-7.6	1.3	Conventional hydrogen bonds	TYR455 (1), ALA554 (1)	3.33, 1.99
				ARG553 (2)	2.44, 2.85
				ARG624 (2)	2.33, 2.26, 3.72
			Pi- Alkyl	MET542(1)	5.22
			Pi-Sigma	ALA558(1)	2.37
			Pi-Cation	ARG624 (2)	3.56, 4.25
			Pi-Anion	ASP623 (2)	3.17, 3.38
			Pi-Pi T-shaped	TYR456 (1)	5.64
			Pi-lone Pair	TYR456(1)	2.83
			Carbon Hydrogen bonds	ASP452 (1), LYS621(1)	2.56, 2.44
1,6-di-O-galloyl- beta-D-glucose (440221 )	-7.5	2.9	Conventional hydrogen bonds	ASP452 (1), ASP623 (1), TYR456(1), LYS545(1), ARG555(1)	2.38, 2.08, 2.69, 4,42, 2.23
				THR680 (2)	1.71, 2.87
				THR556 (2)	2.83, 2.10
			Pi-Cation	ARG553 (1)	3.60
			Pi-Anion	ASP623 (1), ARG624 (2)	4.44, 3.79, 4.09
			Pi-Alkyl	ALA558	4.67
Pyropheophorbide A (161456)	-7.3	1.4	Conventional hydrogen bonds	ARG553 (2)	2.50, 3.35
Pyropheophorbide A (161456)			Conventional hydrogen bonds	THR556 (1), LYS621 (1), ASP760 (1)	2.62, 3.11, 1.97
			Pi- Alkyl	ARG553 (1), LYS621 (1), TYR455 (2)	4.76, 4.93, 4.24, 5.25
			Alkyl	CYS622 (1), ARG554 (1), ARG553 (1)	4.61, 4.46, 3.89

			Pi-Sigma	ARG553 (1)	2.64
			Pi-Cation	ARG553 (1), LYS621 (1)	3.37, 2.55
			Pi-Anion	ASP623 (1)	3.28
Pheophorbide A (253193)	-7.3	1	Conventional hydrogen bonds	ARG553 (1), ASN691 (1)	2.90, 2.08
				THR556 (3)	3.19, 2.56, 2.28
			Alkyl	PRO620 (1)	5.08
			Pi- Alkyl	TYR455 (1)	3.83
			Pi-Cation	ARG553 (3)	3.66, 3.67, 3.90
			Pi-Anion	ASP623 (2)	3.22, 3.51
Beta-Sitosterol (222284)	-7.0	1.5	Conventional hydrogen bonds	ALA688 (1)	2.87
			Alkyl	LYS621 (1), ARG624 (1), ALA688 (1)	5.04, 4.88, 5.31
Vitexin (5280441 )	-6.5	2.4	Conventional hydrogen bonds	ARG553 (1), THR556 (1), ASP618 (1), LYS621 (1), ASP623 (1)	3.37, 2.94, 2.22, 3.11, 2.30
				TYR455 (2)	3.03, 2.80
			Pi-Cation	ARG553 (2)	4.03, 4.54
			Pi-Anion	ARG624 (1)	4.842
			Carbon Hydrogen bonds	ALA554, LYS621 (2)	2.46, 2.44, 3.30

1,6-di-O-galloyl-beta-D-glucose, a natural product found in an abundant amount in Acacia nilotica, has a previously reported activity on HIV–1 reverse transcriptase (RT) ²⁹. Our study demonstrated that this molecule shows a promising anti-SARS-CoV–2 polymerase activity, forming nine-strong H bonding, pi-cationic, and pi-anionic interaction with four amino acids, and pi-alkyl bond (Table 3 and Figure 4).

Pyropheophorbide A and pheophorbide A are compounds found in many plants, and they have possess a significant inhibitory activity against H1N1, H3N2, H5, and B influenza viruses ⁵⁵. Both compounds score low docking energy (–7.3) and form many types of interactions with the SARS-CoV–2 RdRp enzyme (Table 3) (Figures 5 and 6).

0000With low binding energy (–7), three alkyl bonds, and 2.87Å distanced hydrogen bond, beta- sitosterol, tightly blocked the active site of the RdRp enzyme (Table 3 and Figure 7). This good interaction aligned with a previously reported capacity of beta-sitosterol to inhibit the enzymatic activity of SARS 3CLpro ⁵⁶.

Vitexin interacts with RdRp through seven hydrogen bonds, two pi-cationic bonds, one pi- anionic bond, and two carbon-hydrogen bonds (Table 3 and Figure 8).

Three antiviral agents (velpatasvir, IDX–184, oseltamivir, and ribavirine), showed a high activity against SARS-CoV–2 RdRp. Velpatasvir is a hepatitis C RNA polymerase inhibitor ⁵⁷, that shows here, a high affinity against the SARS-CoV–2 RdRp enzyme, with –9.3 binding energy and 1.7 RMSD. Similarly, IDX–184 previously reported with activity against RdRps of HCV, MERS and SARS HCoVs, and Zika virus ⁵⁸, and showed –8.8 docking energy. This finding agrees with Elfiky, (2020), who reported –9 docking energy of IDX–184 with SARS-CoV–2 RdRp ⁵⁸ (Table 3).

3.3.1. Interactions of ligands and RBD

Natural ligands showed numerous interactions with the following amino acids; THR345, ARG403, ASP405, GLN409, GLY416, LYS417, ILE418, TYR421, GLY496, TYR453,

TYR495, GLY504, and TYR505 (Table 4), these residues located at the binding site of RBD ¹. Glycyrrhizin was the most active compound on RBD that scored the lowest energy –7.3 kcal/mol and an RMSD within the acceptable range (2.3Å). Glycyrrhizin forms a strong H bonding with exposed TYR505, which has known contact with the ACE2 receptor ⁵⁹. Glycyrrhizin which creates alkyl interaction with LYS417, is one of the most important mutant residues in SARS-CoV–2 RBD ¹, which may contribute to its increased binding affinity to ACE2 receptors. In addition, glycyrrhizin interacts with another important residue (TYR453) that interacts with HIS34 of ACE2. Blocking of these important residues may interfere with the virus binding to human cells (Figure 9). Rutin created two conventional hydrogen bonds with TYR453 (ACE2 binding residue) and ILE418, located at the hydrophobic pocket of RBD ⁵⁹.

The presence of pi-alkyl and carbon-hydrogen bonds in the RBD active site make rutin the second active compound with binding energy –6.9 (Figure 10). Pheophorbide A makes a hydrogen bond with GLN409, in addition to alkyl and pi-alkyl bonds with different aliphatic amino acids in the active site of RBD (Table 4) (Figure 11).

Table 4. Interaction of active compounds with SARS-CoV-2 RBD (6VSB) and Human ACE2 receptor (6VW1).

Compounds (PubChem ID)		RBD		ACE2
Compounds (PubChem ID)	Type of interaction	Amino acid residues (No. of bonds)	Distance (Å)	Amino acid residues (No. of bonds)	Distance (Å)
Glycyrrhizin (14982)	Conventional hydrogen bonds	TYR505 (1)	3.31	ASP350 (1)	2.71
	Alkyl	LYS417 (1)	4.85	LEU391(1), ALA99(1), LEU100(1)	4.96, 4.35, 4.68
	Carbon hydrogen bond	GLY496 (2)	2.76, 3.08	ALA99(1)	2,54
	Pi- Alkyl	TYR453, TYR495	4.18, 5.13	LEU391(1), ARG393 (2)	4.97
	Pi-Pi Stacked	-	-	PHE40(1)	5.49

Rutin (5280805)	Conventional hydrogen bonds	TYR453 (1), ILE418(1),	2.70, 2.73 HIS34, ASP30, and LYS353	ASP30 (2), ARG393 (2), ASN33(1)	2.31, 2.94, 2.20, 2.43, 2.96
	Pi-Alkyl	TYR421 (1), ARG403 (2)	5.25, 4.37, 4.97	PRO389	4.94
	Carbon Hydrogen bond	GLY416(1)	3.58	GLN388, ALA387	2.50, 2.53
	Pi-Sigma	-	-	PRO389	2.48
	Alkyl	-	-	ALA386, PPRO389	4.22
Pheophorbide A (253193)	Conventional hydrogen bonds	GLN409(1)	3.19	ARG393 (2), ASP30	2.03, 3.07, 2.0
	Alkyl	ILE418 (1)	5.18	VAL93,AL A387, LEU29	5.11, 4.48, 4.94
	Pi- Alkyl	TYR505 (1) (1), ARG403 (2), TYR453(1), TYR495(1), ILE418(1)	5.41, 4.71, 5.08, 4.30, 5.06, 5.18	PRO389 (2)	5.36, 5.27
	Amide-Pi Stacked	ASP405(1), GLY504 (2)	5.05, 4.80, 5.41

3.3.2. Interactions of ligands and ACE2 receptor

Several studies have reported that SARS-CoV–2 enters its target cells through angiotensin- converting enzyme 2 (ACE2) ⁶⁰^,⁶¹. ACE2 is highly expressed in the mouth and tongue, which facilitates the entry of the virus into the body ⁶⁰. The blocking of the ACE2 receptor could reduce its binding affinity to viral spike protein attachment. We identified three compounds (glycyrrhizin, rutin, and pheophorbide A), with a high binding affinity (–7.8, –6.9, -and –6.5 kcal/mol, respectively) to the ACE2 receptor. These compounds created an interaction with the following residues: PHE4, LEU29, ASP30, ASN33, VAL93, ALA99, LEU100, ALA387, ASP350, ALA387, GLN388,

PRO389, LEU391, and ARG393. Some of them (such as ASP30) have an essential role in RBD binding ⁵⁰^,⁶² (Table 4) (Figures 12, 13, and 14).

3.4. Predicted ADMET profiles of compounds

Beta-sitosterol, pheophorbide A, and pyropheophorbide A are of high (more than 95%) human intestinal absorption (HIA) values, which indicates their high absorbance rate in the human intestinal tract. They also showed high permeability to Caco–2 (cells derived from a colon cancer cell) and MDCK (cells derived from canine kidney cells) cell models when compared with antiviral drugs (velpatasvir, IDX–184, and oseltamivir). Rutin and 6-Bis-O- galloyl-beta-D-glucose show the lowest intestinal absorption rate (less than 7%), indicating their reduced intestinal absorption rate. This finding agrees with previous clinical studies that show the low absorption rate of rutin ⁶³^,⁶⁴ (Table 5).

Drug distribution depends on a drug’s ability to penetrate the blood-brain barrier (BBB), as well as its degree of plasma protein binding (PPB) ⁶⁵. Most of our compounds, as predicted by PreADMET, have a high binding affinity to plasma protein (up to 100%), and good penetration of the BB, compared with reference antiviral agents (Table 5).

Table 5. ADMET predicted profile of top eight natural compounds as compared to three reference antiviral molecules
	Natural compounds								References antiviral compounds
Aadme	1,6-Bis-O- galloyl- beta-D- glucose	beta- Sitosterol	Rutin	Pheophorbi de A	Pyropheoph orbide A	Glycyrrhizi n	Baicalin	Vitexin	Velpata svir	IDX-184	Oseltamivi r
Absorption
HIA (%)	2,65	100	2,86	96,15	95,66	9,46	32,42	31.37	92,72	26,39	87,16
Caco2 (mm/sec)	6,9455	52,3734	7,9127	21,1468	22,0961	20,7977	11,5594	5.49	23,86	0,6315	14,12
MDCK (mm/sec)	0,4922	8,8572*	0,3269	0,0434*	0,0439	0,0434*	0,1478	0.54	0,043	0,044	5,028
Pgp inhibition	Non	Inhibitor	Non	Non	Inhibitor	Non	Non	Non	Inhibitor	Non	Non
Distribution
BBB (%)	0,0313	19,8883	0,0286*	0,0337	0,3105	0,0365*	0,0252	0.039	0,2710	0,069	0,123
Plasma Protein Binding (%)	86,2074	100	43,8979	87,2730	88,0027	82,4843	75,6919	61.32	88,7939	36,8052	37,83
Metabolism
CYP_2C19_inhibition	Inhibitor	Non	Inhibitor	Non	Non	Non	Non	Inhibitor	Non	Non	Non
CYP_2C9_inhibition	Inhibitor	Inhibitor	Inhibitor	Non	Non	Inhibitor	Inhibitor	Inhibitor	Inhibitor	Non	Non
CYP_2D6_inhibition	Non	Non	Non	Non	Non	Non	Non	Non	Non	Non	Inhibitor
CYP_2D6_substrate	Non	Non	Non	Non	Non	Non	Non	Non	Non	Non	Substrate
CYP_3A4_inhibition	Inhibitor	Inhibitor	Inhibitor	Inhibitor	Inhibitor	Inhibitor	Inhibitor	Inhibitor	Inhibitor	Inhibitor	Non
CYP_3A4_substrate	Weakly	Substrate	Weakly	Substrate	Substrate	Substrate	Weakly	Weakly	Substrat e	Substrate	Weakly
Bioavailability
Buffer solubility(mg/L)	129005	1,8010	28,8864	0,0951	0,0296	173820	77684	196.168	0,1263	3,9685**	1204,74
Pure water solubility(mg/L)	8527,4	0,0015	217,2070	0,3530	0,2631	0,0555	361,9440	344.563	0,0000	6,6251	2517,58
Skin Permeability	-4,3906	-0,5934	-4,6667	-3,3780	-3,4464	-1,8968*	-4,3741	-4.611	- 3,0823*	-2,9751*	-3,3491

Toxicity assays
Algae at	0,0009	0,0072	0,0070	0,0020	0,0035	0,0003	0,0192	0.029	0,0001	0,0044	0,0817
Ames test	non-mutagen	mutagen	non- mutagen	mutagen	mutagen	non- mutagen	mutagen	non- mutagen	mutagen	non- mutagen	mutagen
Carcino Mouse	positive	negative	positive	positive	positive	positive	negative	positive	negative	negative	negative
Carcino Rat	negative	negative	negative	positive	positive	positive	positive	negative	negative	negative	negative
Daphnia at	0,0027	0,4737	2,5526	0,0060	0,0045	0,1218	0,3948	0,78	0,0009	0,4436	0,4499
hERG inhibition	Low risk	ambiguous	ambiguous	Low risk	ambiguous	ambiguous	ambiguou s	High risk	High risk	ambiguou s	Low risk

The cytochrome P450 (CYP) enzymes play a major role in the first phase of drug metabolism and detoxification of harmful substances in cells, such as drugs and toxins ⁶⁶. Inhibition of CYP enzymes could lead to toxic side effects or a decrease in drug effectiveness ⁶⁷. Pheophorbide A, and pyropheophorbide A showed low toxicity to CYP enzymes, followed by glycyrrhizin, beta-Sitosterol, baicalin, and vitexin, which were inhibitors for two of six CYP enzymes predicted by PreADMET. 1, 6-Bis-O-galloyl-beta-D-glucose and rutin show the highest inhibitory effect for CYP enzymes. The nhibitory effect of rutin to CYP3A4 is in agreement with a previous study that found that rutin is one of the most potent CYP34A4 inhibitors ⁶⁶ (Table 5).

Four compounds (1, 6-Bis-O-galloyl-beta-D-glucose, glycyrrhizin, rutin, and vitexin) were predicted by the Ames test to have no carcinogenicity and mutagenicity.

The discovery and development of novel anti-SARS-CoV–2 drugs to fight the global pandemic is of worldwide urgency. In this study, we discovered eight potential SARS-CoV–2 inhibitors (glycyrrhizin, rutin, baicalin, 1, 6-di-O-galloyl-beta-D-glucose, pyropheophorbide A, pheophorbide A, beta-Sitosterol, and vitexin). These outcomes indicate that these compounds are potential candidates to be utilized in lead optimization for the development of anti-SARS- CoV–2 drugs.

Funding

This project was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia under grant No (D-658-130-1441)

Conflicts of interest

None of the authors has financial interests or conflicts of interest related to this research

Acknowledgement

The authors thanks LetPub and Hassuna Altayeb from Al Ittihad Newspaper for their free language editing service.

Authors’ contributions

HNA, LB, KC and MAZ designed the study, NHA and KC performed the docking study, HNA, LB, KC, OSB and MAZ wrote the manuscript, all the authors approved the final version of the manuscript.

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Potential activity of a selected natural compounds on SARS-CoV-2 RNA-dependent-RNA polymerase, and binding affinity of the receptor-binding domain (RBD)

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

Table1: Selected natural products displayed antiviral activities

3. Results And Discussion

Conclusion

Declarations

References

Status:

Version 1