Drug likeness and bioactivity score
The concept “drug-likeness” is emphasized in pharmaceutical research in recent years as it’s believed that successful prediction of drug-like properties in early stage will pay off in the drug development at the later stages. The Lipinski rule of five, “Ro5” is the most favorable technique to identify the compounds that are similar to the conventional drug. If the violation is 0 or 1, the compounds are assumed to bind effectively to the receptor and was rejected when the violation number exceeds 2. Seventeen compounds that are present in C. oblonga seeds are selected (Table 1) for this study and are filtered through Lipinski rule, including the reference compound Doxorubicin.
Table 1
In silico-based determination of pharmacokinetics of the selected compounds and Doxorubicin (Standard compound) using online server Molinspiration.
Sr. No.
|
Bioactive compounds
|
Molecular weight
(g/mol)
|
Hydrogen bond acceptor count (HBA)
|
Hydrogen bond donor count (HBD)
|
XlopP
|
N violations
|
Reference Compound
|
1.
|
Doxorubicin
|
543.52
|
12
|
7
|
0.57
|
3
|
Bioactive Compounds from C. oblonga
|
1.
|
3-O- caffeoylquinic acid
|
354.31
|
9
|
6
|
-0.45
|
1
|
2.
|
4-O- caffeoylquinic acid
|
354.31
|
9
|
6
|
-0.67
|
1
|
3.
|
5-O- caffeoylquinic acid
|
354.31
|
9
|
6
|
-0.45
|
1
|
4.
|
3,5-O-di caffeoylquinic acid
|
516.46
|
12
|
7
|
1.42
|
3
|
5.
|
Vicenin-2
|
594.52
|
15
|
11
|
-2.10
|
3
|
6.
|
Lucenin-2
|
610.52
|
16
|
12
|
-2.59
|
3
|
7.
|
Stellarin-2
|
624.55
|
16
|
11
|
-2.28
|
3
|
8.
|
Isoschaftoside
|
564.50
|
14
|
10
|
-1.68
|
3
|
9.
|
Schaftoside
|
564.50
|
14
|
10
|
-1.68
|
3
|
10.
|
Chrysoeriol
|
300.27
|
6
|
3
|
2.28
|
0
|
11.
|
Kaempferol-3-O-glucoside
|
448.38
|
11
|
7
|
0.12
|
2
|
12.
|
Kaempferol-3-O-rutinoside
|
578.52
|
14
|
8
|
-0.10
|
3
|
13.
|
Quercetin-3-O-galactoside
|
464.38
|
12
|
8
|
-0.36
|
2
|
14.
|
Quercetin-3-O-glucoside
|
464.38
|
12
|
8
|
-0.36
|
2
|
15.
|
Quercetin-3-O-rutinoside
|
610.52
|
16
|
10
|
-1.06
|
3
|
16.
|
Catechin
|
290.27
|
6
|
5
|
1.37
|
0
|
17.
|
Mangiferin
|
422.34
|
11
|
8
|
-0.16
|
2
|
Only 5 of these compounds including 3CQA (3-O- caffeoylquinic acid), Catechin, 4CQA (4-O- caffeoylquinic acid), Chrysoeriol and 5CQA (5-O- caffeoylquinic acid) are screened which follows the Lipinski drug likeness criteria and hence they are continued for further evaluations. Compounds that debased the Lipinski’s rules more than once could have a bioavailability complication.
Two of our filtered compounds Chrysoeriol and Catechin show better bioavability in comparison to the standard drug Doxorubicin. The drug is expected to bind the biological targets; thus, bioactivity of the compounds contributes to the overall potential to qualify the compounds as a drug that are determined by the combination of kinase inhibitor, G-protein-coupled receptor, protease and enzyme inhibitor, ion channel modulators and nuclear receptor ligands. According to the general concept, greater the bioactivity scores the probability of being biologically active of the individual compound is higher (Table 2). If the bioactivity score for the metal complexes is higher than 0.0, then the complex is believed to be biologically active and if its value lies between -5.0 and 0.0, then the complex is active moderately while the compounds trusted to be inactive when bioactivity score lies below -5.0 [31]. 3CQA, 4CQA and 5CQA acids shows highly bioactive towards the nuclear receptor ligand. All our compounds show moderate activity toward other biological receptors (>-0.50). The results demonstrate that 3CQA and 5CQA shows better bioactivity score than doxorubicin.
Table 2
Bioactivity score of the bioactive compounds and its complex evaluated using online server Molinspiration.
Sr. No.
|
Compounds
|
GPCR
|
Ion channel modulator
|
Kinase inhibitor
|
Nuclear receptor ligand
|
Protease inhibitor
|
Enzyme inhibitor
|
1.
|
Doxorubicin
|
0.20
|
-0.20
|
-0.07
|
0.32
|
0.67
|
0.66
|
2.
|
3-O- caffeoylquinic acid
|
0.29
|
0.14
|
-0.00
|
0.74
|
0.27
|
0.62
|
3.
|
4-O- caffeoylquinic acid
|
0.18
|
0.02
|
-0.10
|
0.66
|
0.14
|
0.49
|
4.
|
5-O- caffeoylquinic acid
|
0.29
|
0.14
|
-0.00
|
0.74
|
0.27
|
0.62
|
5.
|
Chrysoeriol
|
-0.05
|
-0.14
|
0.25
|
0.32
|
-0.26
|
0.21
|
6.
|
Catechin
|
0.41
|
0.14
|
0.09
|
0.60
|
0.26
|
0.47
|
ADMET prediction of the compounds in comparison with the conventional drug Doxorubicin
Unwanted ADMET properties of the new drug candidate are one of the reasons for many developmental failures hence to survive the phase I clinical trials these problems required to be identify in earlier stages of processes. The hurdle encounter in traditional ADME and toxicity testing is (multistep and time-consuming) is addressed by employing combinatorial chemistry and computation studies. Hence for the determination of pharmacokinetics or pharmacology properties of the compound, several chemical parameters were evaluated to determine their compliance with the standard range by using in silico tools. Those properties comprise of blood-brain barrier penetration, aqueous solubility, MDCK cell permeability, Human Intestinal Absorption (% HIA), Toxicity, Caco-2 and skin permeability and Cytochrome P450 2D6 binding. These properties were projected to be essential for the invention of novel bioactive compounds, thus on investigate the failure of lead competitors that will cause toxicity in its inactive form or one can possibly penetrate the intestinal layer. Thus, on the basis of the evaluation our compounds show mutagenicity and moderate absorption/permeation properties, though doxorubicin is non-mutagenic but is carcinogenic in both mouse and rat. CQA shows non-carcinogenicity in mouse and carcinogenicity in rat (except 4-O- caffeoylquinic acid is non carcinogenic in both rat and mice), whereas catechin is carcinogenic in both mouse and rats (Table 3). Our compounds have values below one for BBB which shows its inactivity for central nervous system and PPB below 90 that represents weak interaction (except catechin that shows strong bonding) with PPB. Thus, from the interpretation of our results 3CQA, 4CQA, 5CQA and Chrysoeriol shows the promising results and are further evaluated for docking and its stability.
Table 3
The ADMET profiling of the bioactive compounds and the reference compound (doxorubicin) using online server tool PreADMET.
|
Toxicity
|
Absorption
|
Distribution
|
Metabolism
|
Compounds
|
Mutagenicity
|
Carcinogenicity
|
HIA
|
Caco2
|
MDCK
|
Skin
|
PPB
|
BBB
|
CYP2D6
|
Mouse
|
Rat
|
Reference Compound(s)
|
Doxorubicin (31703)
|
NM
|
C
|
C
|
31.95
|
17.73
|
1.02
|
-4.69
|
32.79
|
0.03
|
NON
|
Bioactive compounds in Cydonia oblonga seeds
|
3-O- caffeoylquinic acid (1794427)
|
M
|
NC
|
C
|
20.427
|
18.71
|
4.51
|
-3.89
|
41.96
|
0.033661
|
NON
|
4-O- caffeoylquinic acid (5315599)
|
M
|
NC
|
NC
|
20.427
|
19.6166
|
0.746
|
-3.895
|
43.977
|
0.0331
|
NON
|
5-O- caffeoylquinic acid (5280633)
|
M
|
NC
|
C
|
20.427
|
18.71
|
4.51
|
-3.89
|
41.96
|
0.0336
|
NON
|
Chrysoriol (5280666)
|
M
|
C
|
NC
|
88.18
|
5.18
|
37.45
|
-4.14
|
90.87
|
0.088
|
NON
|
Catechin (73160)
|
M
|
C
|
C
|
66.707
|
0.656
|
44.38
|
-4.29
|
100.00
|
0.39
|
NON
|
Molecular docking of the ligands and standard drug with pTEN and HBx protein
For the better understanding protein-ligand complex structural basis, molecular docking was carried out using AutoDock Tool 4.2 which is a very convenient and cheap tool to study protein-ligand interactions. The results output files provide the free binding energy of all ten conformation for each protein-ligand interaction which is utilized to generate the best docked complex. 3CQA, 4CQA, 5CQA and Chrysoeriol are the compounds concealed after the pharmacokinetic determination. These compounds are docked against pTEN and HBx along with the standard drug doxorubicin. The docked result of the standard drugs shows the free energy of binding, -3.46 and -5.69 kcal/mol with inhibition constant of 2.92 mM and 67.50 uM with pTEN and HBx, respectively. The caffeoylquinic acid derivatives shows the best binding affinity against pTEN (-7.53, -7.42, -7.49 of 3CQA, 4CQA and 5CQA, respectively) while HBx shows somewhat similar affinity with all the ligands (3CQA, 4CQA, 5CQA and Chrysoeriol, respectively shows -5.94, -5.87, -6.01 and -5.83, respectively) (Table 4).
Table 4
AutoDock score of the ligands and doxorubicin against both pTEN and HBx.
Bioactive compounds
|
pTEN (1D5R)
|
HBx (3MS6)
|
Free Energy of Binding (kcal/mol)
|
Inhibition Constant, Ki
|
Free Energy of Binding
|
Inhibition Constant, Ki
|
Doxorubicin (31703)
|
-3.46
|
2.92 mM
|
-5.69
|
67.50 uM
|
3-O- caffeoylquinic acid (1794427)
|
-7.53
|
3.03 µM
|
-5.94
|
44.33 uM
|
4-O- caffeoylquinic acid (5315599)
|
-7.42
|
2.58 µM
|
-5.87
|
49.51 uM
|
5-O-caffeoylquinic acid (5280633)
|
-7.49
|
3.24 µM
|
-6.01 kcal/mol
|
39.54 uM
|
Chrysoeriol (5280666)
|
-6.90
|
8.74 µM
|
-5.83
|
39.91 uM
|
The residues that are reported to be present at the active site in pTEN are HCXXGXXR motifs, while the residue Cys-124 and Arg-130 plays a critical role in pTEN catalytic activity and residue His-123 and Gly-127 are essential for the conformation P-lop of pTEN [32,33]. Additionally, Asp-92 residue present in “WPD” loop of pTEN behaves as basic acid to promote the phenolic oxygen protonation of the tyrosyl leaving group [34]. On the other hand, the binding pocket of HBx in the novel structure 3MS6 contain residues Leu-5, His-8, Thr-12, Thr-12, Thr-36, His-41, Glu-68, Ser-69, Asp-70, Asn-71, Ile-74, His-79, Asp-80 and His-87. The detailed evaluation of the binding pockets of caffeoylquinic acid derivatives with both pTEN and HBx is given in Table 5. As we compare the binding pocket of our docked results (Fig. 1 and Fig. 2) the residues involved in Doxorubicin are ALA126, ASP92, ARG130, CYS124, GLY127, GLY129, HIS93, ILE168, LYS125, LYS128, LYS330, THR167 and similar residues are seen in other ligands binding pockets (Fig. 1) against docking with protein pTEN. Hence this shows that are ligands are binding in the vary pocket where the novel pTEN (1D5R) structure reported to have active sites. Our standard complex with pTEN shows 5 hydrogen bond interactions while caffeoylquinic acid derivatives shows hydrogen bonding more than Doxorubicin-pTEN complex, which also attributes to the stability of the complex.
Table 5
Active site pocket residues and Hydrogen bonds interaction between protein pTEN (1D5R) and ligand revealed through molecular docking
Compounds
|
Hydrogen bonds between protein and ligand
|
Residues
|
pTEN
|
HBx
|
pTEN
|
HBx
|
Doxorubicin
|
A:ALA126:HN -:UNK0:O7
A:LYS128:HN -:UNK0:O7
A:ARG130:HH21 -:UNK0:O8
:UNK0:H63 -A:ASP92:OD2
|
:UNK0:H55 -:THR12:OG1
:UNK0:H61 - A: VAL84: O
A:ILE76:HN3-:UNK0:O10
A:HIS79:HE2:B - :UNK0:O6
|
ASP92, HIS93 CYS124, LYS125, ALA126, GLY127, LYS128, GLY129, ARG130, THR167, ILE168, LYS330
|
HIS8, THR12, ASN15, ILE18, VAL65, CYS66, ASN73, ILE74, ILE76, GLN77, HIS79, VAL84, ALA85, VAL86, LYS88
|
3-O- caffeoylquinic acid
|
A:HIS93:HE2 - :UNK0:O3
A:CYS124:SG - :UNK0:O8
A:ALA126:HN - :UNK0:O9
A:LYS128:HZ3 - :UNK0:O7
A:ARG130:HN - :UNK0:O8
A:ARG130:HE - :UNK0:O8
A:ARG130:HH21 - :UNK0:O9
A:LYS330:HZ3 - :UNK0:O2
A:LYS330:HZ3 - :UNK0:O5
:UNK0:H36 - A:ASP326:O
:UNK0:H42 - A:CYS124:SG
|
:UNK0:H34-A: VAL84: O
:UNK0:H42 - A:THR12:OG1
:UNK0:H43 - A:THR12:OG1
A:ILE76:HN3 - :UNK0:O3
A:ILE76:HN3 - :UNK0:O4
A:HIS79:HE2:B - :UNK0:O1
A:HIS79:HE2:B - :UNK0:O7
A:LYS88:HZ3 - :UNK0:O5
|
ASP92, HIS93, CYS124, LYS125, ALA126, GLY127, LYS128, GLY129, ARG130, THR131, THR167, ILE168, GLN171, ASP326, LYS327, ASN329, LYS330
|
HIS8, THR12, ILE76, GLN77, HIS79, VAL84, ALA85, VAL86, LYS88
|
4-O- caffeoylquinic acid
|
A:HIS93:HE2 - :UNK0:O4
A:CYS124:SG - :UNK0:O6
A:ALA126:HN - :UNK0:O6
A:LYS128:HN - :UNK0:O6
A:LYS128:HZ3 - :UNK0:O7
A:ARG130:HH21 - :UNK0:O5
A:LYS330:HZ3 - :UNK0:O8
:UNK0:H33 - A:ASP92:OD2
:UNK0:H42 - A:ASP326:O
|
:UNK0:H35 - A: VAL84: O
A:ILE76:HN3 - :UNK0:O2
A:ILE76:HN3 - :UNK0:O5
A:HIS79:HE2:B - :UNK0:O1
A:HIS79:HE2:B - :UNK0:O4
A:VAL86:HN - :UNK0:O6
|
ASP92, HIS93, CYS124, LYS125, ALA126, GLY127, LYS128, GLY129, ARG130, THR167, ILE168, GLN171, ASP326, LYS327, ASN329, LYS330
|
LEU5, HIS8, LEU9, THR12, VAL64, VAL65, ILE74, ILE76, GLN77, HIS79, VAL84, ALA85, VAL86
|
5-O- caffeoylquinic acid
|
:UNK0:H33 - A:THR167:OG1
:UNK0:H36 - A:THR167:OG1
:UNK0:H42 - A:CYS124:SG
A:CYS124:SG - :UNK0:O8
A:ALA126:HN - :UNK0:O9
A:LYS128:HZ3 - :UNK0:O7
A:ARG130:HN - :UNK0:O8
A:ARG130:HE - :UNK0:O8
A:ARG130:HH21 - :UNK0:O9
A:THR167:HG1 - :UNK0:O2
A:LYS330:HZ1 - :UNK0:O2
A:LYS330:HZ1 - :UNK0:O5
|
A:THR12:HG1 - :UNK0:O8
A:ILE76:HN1 - :UNK0:O4
A:ILE76:HN3 - :UNK0:O3
A:HIS79:HE2:B - :UNK0:O7
A:VAL86:HN - :UNK0:O4
A:LYS88:HZ3 - :UNK0:O5
:UNK0:H35 - A: VAL86: O
|
ASP92, HIS93, CYS124, LYS125, ALA126, GLY127, LYS128, GLY129, ARG130, THR131, THR167, ILE168, GLN171, LYS330
|
HIS8, LEU9, THR12, VAL21, ILE74, ILE76, GLN77, HIS79, VAL84, ALA85, VAL86, LYS88
|
The pocket residues involve in HBx-doxorubicin complex (Table 5) are HIS8, ASP11, THR12, ASN15, ILE18, VAL65, CYS66, ASN73, ILE74, ILE76, GLN77, HIS79, VAL84, ALA85, VAL86 and LYS88; and 4 hydrogen bonds. Similarly, HIS8, THR12, ILE76, GLN77, HIS79, VAL84, ALA85, VAL86 and LYS88 are present in HBx-3-O- caffeoylquinic acid; LEU5, HIS8, LEU9, THR12, VAL64, VAL65, ILE74, ILE76, GLN77, HIS79, VAL84, ALA85 and VAL86 in HBx-4CQA and HBx-5CQA contain HIS8, LEU9, THR12, VAL21, ILE74, ILE76, GLN77, HIS79, VAL84, ALA85, VAL86 and LYS88 in the binding pocket with 8, 6 and 7 hydrogen bonds respectively (Fig. 2). Hence it can be concluded that the binding of the ligands is in the similar pocket region as our standard drugs doxorubicin is binding with the HBx.
Molecular dynamics simulation to analyze the complex stability
Initially, our docking results revealed that the screened compounds including 3CQA, 4CQA, 5CQA and Chrysoeriol could be potential inhibitors. 3CQA and 5CQA were, however, seen to be promising. Therefore, we employed MD simulation for better comprehending the molecular mechanism which may be involved in the analysis. An unbiased simulation was performed for each of the two protein-ligand complexes. A valuable insight was provided by the 100 ns long MD simulation related to the pTEN and HBx structural dynamics with different ligand compounds. For example, the energy of interaction between proteins and ligands illustrates the profound binding between them and well-built interactions between them are characterized by the higher number of hydrogen bonds.
MD trajectories were analyzed for all the related atoms in the backbone and ligands for RMSD determination and assessment of average fluctuations i.e., the RMSF of each residue of the protein in a time-dependent manner for all molecules in the backbone. One of the key criteria for testing the equilibrium of MD directions is the RMSD. To begin with, we determined the RMSD from the initial docked position of the individual compound. The smaller the variance, the individual compounds have a better docked conformation. In other words, during computation, the ligand remains in its initial conformation. The average RMSD value of pTEN with 3CQA and 5CQA for 100 ns trajectory calculated to be 0.245 nm and 0.215 nm, respectively. At first, there is a small fluctuation in the trajectory but later the degree of fluctuation reaches equilibrium (Fig. 3 (a)). The pTEN complexes with 3CQA shows high level of fluctuation in a residue at a position 1-29, 504-518, 1041-1098, 1673, 1681, 3212-3217 and 4093-4115, while 5CQA shows high level of fluctuation in residue position at 1-29, 1086-1098, 3203, 3210 and 4062-4134, with average fluctuation to be 0.107 nm and 0.114 nm, respectively. The standard drug complex pTEN-Doxorubicin (pTEN-Dox) average RMSD value determine to be 0.213 nm with large number of residues showing high level fluctuation at position 1-53, 3201-3214 and 4081-4117 with average RMSF value about 0.100 nm. The residue at position between 4093 to 4115shows the highest fluctuation in all the pTEN complexes. In comparison with standard, pTEN-5CQA shows more stability than pTEN-3CQA and pTEN-Dox as they have less variation throughout the 100 ns simulation and the system reaches equilibrium after 10 ns as compared to pTEN-Dox approximately and pTEN-3CQA. However, the system pTEN-5CQA shows higher flexibility followed by pTEN-3CQA complex and lastly pTEN-Dox complex system (Fig. 3 (c)).
The standard-HBx complex system have average RMSD value about 0.376 nm, which reaches equilibrium after 30 ns. However, HBx-Dox complex shows higher deviation and lowest flexibility compare to HBx-3CQA and HBx-5CQA complex system Fig. 3(b) & 3(d). The residue with highest fluctuation values includes 1-20, 823-847, 916-946 and 968-998, while the complex has average RMSF value to be 0.139. The RMSD value for HBx complexes with 3CQA and 5CQA has average fluctuations about 0.336 nm and 0.352 nm, respectively and the complex system reaches to equilibrium after 30-40 ns. The residue at position 1-30, 345-347, 733-851, 914-925 and 968-1005 shows high level of fluctuation in HBx-3CQA and at residue 1-20, 172, 190 and 797-821in HBx-5CQA complex system with the highest deviation. The RMSF graph of HBx with ligands shows an increase in fluctuation compare to Doxorubicin, which might be due to the ligand adjustment in the binding pocket. Whereas, HBx-3CQA shows higher fluctuation in residues than to HBx-5CQA complex at the active region compare to the standard RMSF.
Protein compactness and Hydrogen bond analysis
The compactness and the folding behavior of the protein during the simulation is analyzed by utilizing the structural parameters of the 3D protein for computing the Radius of gyration (Rg), which provides insight to the protein packaging during the simulation time period [35]. The average Rg for pTEN with ligand 3CQA and 5CQA complexes calculated to be 2.242 and 2.237 nm, respectively. Similarly, the HBx ligand complexes has 1.268 nm and 1.260 nm average Rg value, respectively. The Rg plot of pTEN and HBx of ligands shows the decreased Rg value compare to the standard pTEN-Dox complex (2.234 nm) and Hbx-Dox complex (1.285 nm). However, there is no structural shift observed in pTEN after ligand binding compare to HBx protein and thus suggesting the higher stability of the ligand complexes compare to standard complexes throughout the trajectory (Fig. 4(a) & (b)).
To evaluate the dynamic of protein folding-unfolding under the solvent environment, SASA plot (solvent-accessible surface area) was calculated and investigated [36]. The average SASA value of pTEN with Dox, 3CQA and 5CQA were calculated as 17.309, 17.334 and 17.407 nm, respectively. Similarly, the HBx protein SASA against standard and ligand (3CQA and 5CQA) computed as 8.645, 8.597 and 8.634 nm, respectively. The SASA pots shows a slight raise in protein-ligand complex compare to standard, which is possibly due to the exposure of some surface residues. However, it immediately reached equilibrium after 1ns without any shift in structure during the whole run, depicting the protein stability before and after the ligand binding (Fig. 4 (c) & (d)).
To validate the complex stability the intramolecular bonds of hydrogens also play a key role [37]. The intramolecular hydrogen bonds within 0.35 nm dynamics were determined in protein pTEN and HBx. The number of hydrogen bonds in pTEN with Dox, 3CQA and 5CQA had maximum 236, 238 and 238, respectively and that of HBx complexes were computed to be maximum around 61, 66 and 67, respectively. The pTEN-ligand and Hbx-ligand complex shows a slight increase in intramolecular bonds compare to the Doxorubicin complex of both proteins, which is suspected due to the protein’s higher compactness (Fig. 5 (a) & (b)). Similarly, the intermolecular hydrogen bonds show the similar number of bonds supporting the molecular docking analysis. The number of bonds involve in protein and compounds binding pocket to be around 4 to 6 for all complexes. Hence, supporting higher stability of the ligand complexes in comparison to the standard drug Doxorubicin throughout the trajectory (Fig. 5 (c) & (d)). Hence, the docking and dynamics analysis of the hydrogen bonds depict that binding of 3-CQA and 5CQA with pTEN and HBx is more significantly stable than that with the protein-Dox as there are least variation in system throughout the 100 ns simulation.