Structural and Active site Analysis of COVID-19 Mpro complexed with N3 Inhibitor (PDB ID: 6LU7)
X-ray crystallographic structure of SARS-CoV-2 Mpro (PDB ID: 6LU7) contains 306 amino acid residues complexed with an inhibitor (N3-(N-[(5-Methylisoxazol-3-Yl)Carbonyl]Alanyl-L-Valyl-N~1~-((1r,2z)-4-(Benzyloxy)-4-Oxo-1-{[(3r)-2-Oxopyrrolidin-3-Yl]Methyl}But-2-Enyl)-L-Leucinamide). It consists of 23%, 31%, 45% and 28% α-helix, β-sheets, Coil and Turns respectively. The resolution of the protease as revealed by X-ray diffraction was 2.16 Å, crystal dimension is a = 97.93 Å, b = 79.48 Å and c = 51.08 Å with angles α (900), β (114.550), and γ (900) respectively. R-values (free, work, and observed) are 0.235, 0.202, and 0.204 respectively while the Total Accessible Surface Area (TASA) on the protease is 14043.1 (Å). There are three (3) domains which are: Domain I (residues 8-110), Domain II (residues 102-184), and Domain III (residues 185-200.SARS-CoV-2 Mpro active site is located in the cleft between Domain I and II and contains a Cys-His catalytic dyad. Amino acid residue at the active site are as follows Thr24, Thr25, Thr26, His41, Met49, Tyr54, Phe140, Leu141, Asn142, Gly143, Ser144, Cys145, His163, His164, Met165, Glu166, Leu176, Pro168, His172, Asp187, Arg188, Gln189, Thr190, Ala191, and Gln192[14].
Molecular Docking Analysis
Recent developments in drug discovery have led to a renewed interest in the computational study which involves the use of algorithms and programs for predictions of therapeutic interventions in biological processes[15]. Molecular Docking is a structure-based drug design approach that predicts binding interactions between ligand molecules and target receptor at the binding site [16]. It is an important virtual screening tool which could screen several thousands of ligands against the target as well as identify potential inhibitors of the target receptor with speed and accuracy [17].
To investigate potential inhibitor of SARS-CoV-2 Mpro, AutoDock Vina (MGL tools- 1.5.6), PyMOL Console Edu, and Biovia Discovery studio 4.5 were used. The results obtained from the docking of selected saponins and tannins against SARS-CoV-2 Mpro were as shown in table 1. Binding affinity is a reflection of the inhibitory activity of the plant extract against SARS-CoV-2 Mpro. It is apparent from this table that most of the selected saponins and tannins had a higher binding affinity and inhibitory activity against SARS-CoV-2 Mpro compared to Remdesivir and Dexamethasone which are standard drugs used in the management of this pandemic disease. Binding affinity (BA) for the selected saponins range between -8.3 kcal/mol and -7.1 kcal/mol while those of tannins range between -9.0 kcal/mol and -4.7 kcal/mol, it can be seen that selected tannins had far greater binding affinity compared to saponins, with Punicalagin (-9.0 kcal/mol) having the outstanding inhibitory activity.
Table 1: Binding Affinities and inhibition constant of selected saponins and tannins
SAPONINS
|
TANNINS
|
S/N
|
Ligands
|
Binding Affinity(ΔG) Kcal/Mol
|
Inhibition Constant
(Ki), µm
|
Ligands
|
Binding Affinity(ΔG), Kcal/Mol
|
Inhibition Constant
(Ki), µm
|
1
|
Priverogenin A
|
-8.3
|
0.83
|
Punicalagin
|
-9.0
|
0.25
|
2
|
Arjunic acid
|
-8.1
|
1.16
|
Punicalin
|
-8.6
|
0.50
|
3
|
Theasapogenol B
|
-8.1
|
1.16
|
Ellagic acid
|
-8.4
|
0.70
|
4
|
Euscaphic Acid
|
-8.0
|
1.37
|
Corilagin
|
-8.2
|
0.98
|
5
|
Camelliagenin C
|
-7.8
|
1.93
|
Gallagic acid
|
-8.1
|
1.16
|
6
|
Medicagenic Acid
|
-7.8
|
1.93
|
Remdesivir
|
-7.6
|
2.70
|
7
|
Protoescigenin
|
-7.8
|
1.93
|
Terflavin B
|
-7.6
|
2.70
|
8
|
Arjunolic acid
|
-7.7
|
2.28
|
Catechin
|
-7.5
|
3.20
|
9
|
Asiatic acid
|
-7.7
|
2.28
|
Chebulinic acid
|
-7.5
|
3.20
|
10
|
Protobassic acid
|
-7.7
|
2.28
|
Hexahydroxydiphenic acid
|
-6.4
|
20.45
|
11
|
Dexamethasone
|
-7.7
|
2.28
|
Gallic acid
|
-5.5
|
93.34
|
12
|
Arjugenin
|
-7.6
|
2.70
|
N3Inhibitor
|
-5.6
|
78.85
|
13
|
Polygalacic acid
|
-7.6
|
2.70
|
Catechol
|
-4.7
|
359.95
|
14
|
Primulagenin A
|
-7.6
|
2.70
|
|
|
|
15
|
Remdesivir
|
-7.6
|
2.70
|
|
|
|
16
|
Soyasapogenol B
|
-7.6
|
2.70
|
|
|
|
17
|
Tomentosic acid
|
-7.6
|
2.70
|
|
|
|
18
|
Presenegenin
|
-7.1
|
6.28
|
|
|
|
19
|
N3Inhibitor
|
-5.6
|
78.85
|
|
|
|
Drug-likeness Analysis of the selected compounds
Evaluation of physicochemical properties and drug-likeness of potential active compounds is an important step in drug discovery, as proposed by Lipinski, an effective oral therapeutic drug must obey the ‘rule of five’ with not more than one (1) violation, this is because an orally bioavailable drug must possess molecular weight (MW) ≤ 500Da, hydrogen bond donor (HBDs) ≤ 5, hydrogen bond acceptor (HBAs) ≤ 10 and logP (octanol-water partition coefficient) ≤ 5[18]. These descriptors of oral bioavailability are important as they predict the permeability and absorption of such drug across a biological membrane such as an epithelium cell, partition coefficient value (LogP) is especially important in predicting intestinal absorption of such a drug[19]. Considering the first ten (10) of the tannins and saponins (Table 1) in order of binding affinity, efficacy, and safety profile using the Molinspiration online (http://www.molinspiration.com/) and ADMET SAR-2 web-server[20], Ellagic acid, Arjunic Acid, Theasapogenol B, and Euscaphic Acid were selected as hit compounds for further analysis and are coded C1, C2, C3, and C4 respectively. Although Punicalagin, Punicalin, and Priverogenin A had better inhibitory activity and binding affinity than ellagic acid, they were shunned based on toxicity and the possibility of poor absorption and permeability across a biological membrane.
Drug-likeness of the selected hits were evaluated with Molinspiration online (http://www.molinspiration.com/) as shown in Table 2, it is apparent from the table that none of the selected hits had more than one violation of the ‘rule of five’ which is an indication of good oral bioavailability and permeability. It is also interesting to note that the selected hit had better drug-like properties compared to Remdesivir (http://www.molinspiration.com/).
Table 2: Drug-Likeness of Selected Hit Compounds
Compounds
|
Heavy atoms (HA)
|
Molecular Weight (MW)
|
RO5 violations
|
Hydrogen bond donor (HBD)
|
Hydrogen bond acceptor (HBA)
|
miLog P
|
C-1
|
22
|
302.19
|
0
|
4
|
8
|
0.94
|
C-2
|
35
|
488.71
|
0
|
4
|
5
|
4.89
|
C-3
|
35
|
490.73
|
0
|
5
|
5
|
4.10
|
C-4
|
35
|
488.71
|
0
|
4
|
5
|
4.93
|
C-1= Ellagic Acid, C-2= Arjunic Acid, C-3= Theasapogenol B, C-4= Euscaphic Acid
ADMET properties of the selected hit
Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) profile of a molecule is an important assay in the early stage of drug discovery. ADMET data enhance the selection and identification of molecules with optimum safety profile at a therapeutic dose along the discovery process rather than at the final stage, as this help in avoiding waste of time and precious resource on drug molecules that may eventually be discarded[21]. ADMET profile of selected saponins and tannins as computed by ADMET SAR-2 web-server [20] were as shown in Table 3, as part of the drug ADMET profile, a drug molecule should have good human intestinal absorption (HIA), solubility (log S) range between -1 and -5, should be a non-inhibitor of cytochrome P450 enzyme, and should be non-Ames toxic. Others include non-carcinogenicity, non-inhibition of hERG, and no or low level of toxicity [21]. All the selected hit, C1, C2, C3, and C4 are well absorbed in the human intestine, only Theasapogenol B (C3) was found to cross the blood-brain barrier, although an oral drug does not necessarily need to cross the blood-brain barrier, only central nervous system target drug need to[22]. It was also found from the prediction that all the four (4) selected hit compounds were non-inhibitor of the microsomal enzyme (cytochrome P450), which is an indication of good metabolism of the drug in the hepatocytes [23]. The potential of a drug molecule to cause mutation in DNA is revealed by Ames toxicity value and could be a major reason for excluding a drug molecule along the discovery process [24], and non-carcinogenic. Similarly, the hits compound possesses type III acute oral toxicity values (slightly toxic) which could easily be converted to type IV (nontoxic) during hit-lead optimization. The human ether-a-go-go related gene (hERG) potassium ion channel plays important role in cardiac repolarization, blockage of which may be caused by inherited mutation or some drug molecules, leading to long QT syndrome and an eventual death[25]. Interestingly all the hit compounds are not blockers of the hERG potassium channel.
Table 3: ADMET Prediction of Selected Compounds
Absorption& Distribution
|
C-1
|
C-2
|
C-3
|
C-4
|
BBB (+/-)
|
0.6372
(BBB-)
|
0.3145
(BBB-)
|
0.8187
(BBB+)
|
0.5278
(BBB-)
|
HIA+
|
98.15%
|
96.43 %
|
97.21%
|
97.46%
|
Aqueous Solubility(LogS)
|
-3.144
|
-4.446
|
-3.753
|
-4.129
|
Metabolism
|
|
|
|
|
CYP450 2C19
Inhibitor
|
0.8017
Non-Inhibitor
|
0.8826
Non-Inhibitor
|
0.8633
Non-Inhibitor
|
0.8799
Non-Inhibitor
|
CYP450 1A2
Inhibitor
|
0.5914
Non-Inhibitor
|
0.8863
Non-Inhibitor
|
0.8936
Non-Inhibitor
|
0.7582
Non-Inhibitor
|
CYP450 3A4
Inhibitor
|
0.9078
Non-Inhibitor
|
0.8734
Non-Inhibitor
|
0.8723
Non-Inhibitor
|
0.7415
Non-Inhibitor
|
CYP450 2C9
Inhibitor
|
0.5591
Non-Inhibitor
|
0.8938
Non-Inhibitor
|
0.8595
Non-Inhibitor
|
0.8493
Non-Inhibitor
|
CYP450 2D6
Inhibitor
|
0.9575
Non-Inhibitor
|
0.9476
Non-Inhibitor
|
0.9368
Non-Inhibitor
|
0.9607
Non-Inhibitor
|
Excretion
|
|
|
|
|
Biodegradation
|
0.8250
Not biodegradable
|
0.8500
Not biodegradable
|
0.9250
Not biodegradable
|
0.8250
Not biodegradable
|
Toxicity
|
|
|
|
|
AMES Mutagenesis
|
0.8200
Non-Ames Toxic
|
0.9000
Non-Ames Toxic
|
0.8600
Non-Ames Toxic
|
0.8600
Non-Ames Toxic
|
Acute Oral Toxicity
|
0.6020
III
|
0.6470
III
|
0.7710
III
|
0.7326
III
|
Eye Irritation (YES/NO)
|
YES
|
NO
|
NO
|
NO
|
Eye Corrosion (YES/NO)
|
NO
|
NO
|
NO
|
NO
|
hERG Inhibition
|
0.8048
NO
|
0.5631
NO
|
0.4360
NO
|
0.5439
NO
|
Carcinogenicity
|
1.0000
Non-Carcinogenic
|
1.0000
Non-Carcinogenic
|
0.9857
Non-Carcinogenic
|
0.9286
Non-Carcinogenic
|
C-1= Ellagic Acid; C-2= Arjunic Acid; C-3= Theasapogenol B; C-4= Euscaphic Acid
Bioactivity of the selected compounds
Shown in Table 4 are the bioactivity properties of the four (4) selected hit. The inverse relationship existing between binding Energy and inhibition constant is as shown in equation 1, indicating that the higher the binding energy the lower the inhibition constant. A potential hit compound is expected to have inhibition constant values ranging between 0.1-1.0µM and not more than 10nM for a drug[23]. Inhibition constant values of the hit compounds range from 0.70 to 1.37 µM. This observation revealed that all the four (4) selected compounds are qualified as hit with Ellagic acid (0.70 µM) being the most potent of all. Ligand Efficiency (LE), Fit Quality (FQ), and Ligand-efficiency-dependent lipophilicity (LELP) were also calculated according to equation 2-5, interestingly all the hit compounds had values within the recommend fit quality (≥0.8)[26].
Table 4: Bioactivity analysis of the selected compounds
BIOACTIVITY
|
C-1
|
C- 2
|
C- 3
|
C- 4
|
AutoDock Vina docking score (kcal/mol)
|
-8.40
|
-8.10
|
-8.10
|
-8.00
|
Ki (µM)
|
0.70
|
1.16
|
1.16
|
1.37
|
miLog P
|
0.94
|
4.89
|
4.10
|
4.80
|
Ligand Efficiency (LE) /kcal/mol/heavy atom)
|
0.38
|
0.23
|
0.23
|
0.23
|
LE- Scale
|
0.43
|
0.29
|
0.29
|
0.29
|
Fit Quality (FQ)
|
0.89
|
0.81
|
0.81
|
0.80
|
Ligand-efficiency-dependent lipophilicity (LELP)
|
2.47
|
21.13
|
17.72
|
21.00
|
Amino acid interaction of selected compounds
Shown in Table 5 and Figure 1are the docking scores including binding affinity, inhibition constant, and amino acid interaction of the selected hit compared with standard drugs, it is obvious from the table that all the selected hit compounds had a higher binding affinity to the SARS-CoV-2 Mpro and a much better inhibition constant, showing that they could be a better alternative to remdesivir and dexamethasone even with their drug-likeness and ADMET profile. It is also observed that Ellagic acid (Figure 2) was involved in hydrogen bond interaction with the SARS-CoV-2 Mpro in the same binding pocket as the native ligand N3 inhibitor with Thr190, Gln189, Glu166, Phe140, Asn142, Cys145, Gly143, and Ser144 as amino acid residue. Amino acid involved in electrostatic/hydrophobic interaction includes Glu166, Met165, Cys145, His163, Leu141, and His172. It is important to note that the Cys-His catalytic dyad interaction was also present as was in the native ligand interaction with the target receptor.
Table 5: Docking Score of Selected Hits
Ligands
|
Binding Affinity (ΔG), kcal/mol
|
COX-2 Receptor
amino acids forming H-bond with ligands (H-Bond Distance, Å)
|
Electrostatic/
Hydrophobic Interactions involved
|
Inhibition
Constant
(Ki), µM
|
Remdesivir
|
-7.6
|
Lys137 (2.7Å), Asp289 (3.2Å), Thr199 (3.3Å), Leu287 (2.1Å, 3.1Å)
|
Tyr237, Asn238
|
2.28
|
Dexamethasone
|
-7.7
|
Lys137 (2.7Å), Asp289 (3.3Å), Asp289 (3.1Å)
Thr199 (3.3Å), Leu287 (2.7Å), Leu271 (3.5)
|
Leu286
|
2.70
|
Ellagic acid
|
-8.4
|
Thr190 (3.5Å), Gln189 (3.3Å, 2.7Å), Glu166 (3.0Å), Phe140 (2.8Å, 2.1Å), Asn142 (2.8Å, 3.1Å), Cys145 (2.2Å), Ser144 (2.2Å), Gly143 (2.4Å, 2.3Å)
|
Glu166, Met165, Cys145, His163, Leu141, His172
|
0.25
|
Arjunic acid
|
-8.1
|
Asp289 (3.2Å),
Leu287 (3.2Å)
|
Leu272, Leu286
|
1.16
|
Theasapogenol B
|
-8.1
|
Lys137 (2.0Å), Lys137 (2.5Å), Asp197 (3.4Å), Asp197 (2.9Å), Asp289 (3.5Å), Asp289 (3.5Å), Arg131 (2.8Å), Thr198 (3.0Å), Thr199 (2.6Å)
|
Tyr239, Leu272, Met276, Leu286, Leu287
|
1.16
|
Euscaphic Acid
|
-8.0
|
Thr199 (2.2Å), Asp289 (3.0Å), Asp289 (3.1Å), Arg131 (2.5Å)
|
Leu272, Leu286, Leu287
|
1.37
|
Figure 1: The bar chart showing the binding energies and inhibition constants of selected hits as significant potential inhibitor of SARS-CoV-2 main protease
Figure 2: Amino acid interactions of Ellagic acid with the active site (binding pocket) of SARS-CoV-2 main protease