Molecular Docking
In this work, docking results were ranked based on these factors, 1) maximum hydrogen bond interaction with active site residues; 2) minimum binding energy value; 3) the total number of interactions; 4) bond distance. A total of 234 compounds previously reported from Goniothalamus species were screened (Table S1) and the top three ranked compounds were selected. Firstly, the validation of the docking procedure was done before carrying out molecular docking for the selected ligands. A co-crystalline ligand extracted from a selected crystallographic protease structure was re-docked into the same binding pocket. Based on the validation of the docking protocol with co-crystalline ligand, the RMSD value for 1OKE (1.16 Å), 1R6A (0.72 Å), and 3VWS (1.73 Å) were below the 2.0 Å threshold. NS3/NS2B developed an interesting active site that has a highly conserved catalytic triad (His51, Asp75, and Ser135) that is of prime functional importance. Any interaction that takes place in this active site will result in the inhibition of viral replication [2].
The top-ranked compounds were ranked based on their minimum docking energy value and a maximum number of interactions with active site residues. The molecules that can disrupt virus-host binding and inhibiting the entry of virus particles into target cells can be considered as potential inhibitors. Envelope protein is a vital protease because it is crucial for the initial attachment of viral particles to host cell receptors. Thus, any compound that can disrupt E protein activity can already be considered as a lead compound towards anti-DENV drug discovery [19]. The chemical structure of top ranked compounds was drawn and listed in figure 1. The results of docking studies showed that compounds 96, 97, and 149 can bind well to the active site of DENV E protein. Among these three, xylomaticinone was selected for having a minimum number of docking energy values and a maximum number of interactions with the crucial amino acid residues. However, due to the large molecular weight (MW≤500), acetogenin compounds like 96 and 97 have poor pharmacokinetic properties and therefore it was not further pursued as potential inhibitors for envelope protein.
Compound 149 passes all the Lipinski rule’s criteria and has the docking energy value of -45.9 kcal/mol (Table 1). This compound binds to the enzyme through hydrogen bonding with the active residue such as Lys128 and Thr48 while being stabilized by other residues through hydrophobic interactions. The ligand-enzyme complex was stabilized by a carbon-hydrogen bond interaction with Thr48 at 2.54 Å and 2.83 Å (Fig. 2). The compound is further stabilized through more contacts in the form of π−π interactions with the binding site amino acids, Ala50, Pro53, and Ile270. These π−π interactions are necessary to obtain stronger and more selective binding interactions. This ligand-enzyme complex also displayed the presence of electrostatic attraction in the form of a salt bridge in protein (Lys128-1.69 Å). The binding of compound 149 to envelope protein prevents protein-protein interaction between DENV-2 and host cells during infection. Hence, this compound has potential in preventing the attachment and entry process.
NS3/NS2B was considered to have a central role in enhancing viral replication in the host cells. Allosteric pockets of NS3/NS2B protease were located close to its catalytic triad (His51, Asp75, Ser135), so any close interaction with residues in an allosteric pocket will disrupt the activities of the protease [61]. Residues His51, Asp75, Gly133, Ser135, Gly151, Asn152, and Gly153 are located in the active site regions, and it is crucial for ligand interactions with NS3/NS2B protease inhibitors while residues Lys74, Leu76, Asn152, Trp83, Leu149, Gly148, and Glu88 are important residue near to allosteric pockets for NS3/NS2B [19,20]. Compounds 100 and 155 both have interactions with the catalytic triad (His51) and exhibited the minimum docking energy value, but these two compounds have several violations towards drug-likeness test due to the large molecular weight and large value of octanol-water partition coefficient so, these two acetogenins were not further investigated. The results showed that 149 produced a docking energy value of -25.9 kcal/mol. Even though 149 does not interact with the catalytic triad, this compound has several interactions with the active residues (Lys74, Leu76, and Asn152) in the allosteric pocket that is crucial for an inhibitor. The ligand-enzyme complex was stabilized by a conventional hydrogen bond (N---HO) of hydrogen from the hydroxyl group of a compound with the nitrogen of amine from residue Asn152 and Lys74. Compound 149 also forms carbon-hydrogen bonding with other residues, Gly87 (2.50 Å), Gly87 (2.56 Å), and Val147 (2.50 Å).
This complex was further stabilized by π−π interactions with the binding sites involving Ala166, and Leu76 (Fig. 3). Furthermore, salt bridge interaction between lysine (Lys74-1.69 Å) residues and electrostatic charge at the oxygen of hydroxyl group from the ligand side chain also play an important role to stabilize the complex. Thus, any interaction with these residues in the allosteric pocket of the active site will enhance the inhibition activities in the processing of polyprotein during the replication of virus dengue.
Besides, both NS5 methyltransferase and NS5 RdRp are crucial for the inhibition of viral replication [7]. Compound 149 showed the best docking interaction for both proteases due to the maximum number of interactions with active residues and without having any violation for the drug-likeness test. Compound 149 showed the best docking interaction with IR6A with a docking energy value of -53.6 kcal/mol. The stability of the ligand-enzyme complex is based on the carbon-hydrogen bond with the following active residues (Ser150, Lys29, Pro152, and Lys22). The results displayed π−π interactions with the binding sites involving Phe25 and Pro152 (Fig. 4). This complex was further stabilized by two electrostatic attractions between oppositely charged residues that are sufficiently close to each other (Lys22-1.63 Å and Lys29-1.69 Å). Compound 149 also showed the best docking interaction with the 3VWS protease enzyme with a docking energy value of -56.8 kcal/mol. This compound binds to the enzyme through conventional hydrogen bonding with the active residue Thr413 and Asn405 and is further stabilized by other residues through hydrophobic interactions. This compound is able to form π−π interactions with Trp795 (5.00 Å) and attractive charge interaction with Arg792 at 2.88 Å (Fig. 5). As mentioned above, compound 149 is bound to a common active site residue that is structurally and functionally important for NS5 dengue proteases.
Eighteen flavones showed favorable binding energy of less than -56.8 kcal/mol. Overall, from the docking results, compound 149 was observed to have the highest affinity with E, NS3/NS2B, NS5 methyltransferase, and NS5 RdRp. This compound successfully fits in the binding site of the protein and forms the most number of interactions with the active site residues at the lowest docking energy value. Compound 149 is a quercetin derivative, which is known as 3-O-methylquercetin because of the presence of four hydroxy and one methoxy group. The presence of hydroxyl groups at C5, C7, C3' and C4' on the phenyl rings produces a resonance phenomenon. The electrons from the hydroxyl group are delocalized into the aromatic ring. Hence, this facilitates the formation of hydrogen bonding with the residues in the protein binding sites. In addition, the methoxy group at the C3 position could enhance the overall binding interactions (Fig. S1). This compound was reported to display antioxidant and free radical scavenging activities [21]. Besides, 3-O-methylquercetin also displayed several in vitro and in vivo biological activities such as anti-inflammatory, neuroprotective, bronchodilatory, vasodilatory, antinociceptive, immunomodulatory, antitumor, and antiviral [22].
The acetogenins were observed to have a minimal docking energy value lower than -59.0 kcal/mol. This is due to the long alkyl chain in the skeleton that could enhance the interactions with the protein target. However, acetogenins normally have molecular weights of more than 500, which causes difficulty in terms of fitting in the protein binding site and not being absorbed through the gastrointestinal tract. Styryl-lactones are the dominant class of compounds in this genus, but they were found to exhibit poor binding energy of less than -33.1 kcal/mol while alkaloids were observed to have even lower binding energy than -25.8 kcal/mol.
Table 1 Result of top-three ranked phytochemicals screened against the targeted protein (HB-IR: Hydrogen bond Interaction; HDP-IR: Hydrophobic Interaction; PI-IR: Pi-Interaction)
Protein
(PDB ID)
|
Ligand
|
Docking energy (kcal/mol)
|
HB-IR-Å
|
HDP-IR-Å
|
PI-IR-Å
|
Residue/
Distance (Å)
|
Type of interaction
|
Residue/
Distance (Å)
|
Type of interaction
|
Residue/
Distance (Å)
|
Type of interaction
|
E
(1OKE)
|
96
|
-46.1
|
Thr189 (2.29)
Leu191 (2.42)
Thr189 (2.97)
|
Conventional H-bond
Conventional H-bond
Carbon H-bond
|
Gln271 (2.31)
Ala50 (2.53)
Ala205 (4.57)
Leu198 (5.37)
Leu198 (5.37)
|
Unfavorable donor-donor
Unfavorable donor-donor
Alkyl
Alkyl
Alkyl
|
Phe193 (5.16)
|
Pi-alkyl
|
97
|
-48.4
|
Ala50 (1.97)
Gly190 (2.60)
|
Conventional H-bond
Carbon H-bond
|
Leu191 (4.11)
Leu277 (4.38)
Pro53 (4.62)
Ala50 (5.16)
Leu207 (5.25)
|
Alkyl
Alkyl
Alkyl
Alkyl
Alkyl
|
Phe279 (5.18)
|
Pi-alkyl
|
149
|
-45.9
|
Pro53 (2.44)
Thr48 (2.54)
Thr48 (2.83)
|
Carbon H-bond
Carbon H-bond
Carbon H-bond
|
Lys128 (1.69)
|
Salt bridge
|
Ala50 (3.10)
Ala50 (4.13)
Ala50 (4.33)
Pro53 (4.94)
Ala50 (5.01)
Ile270 (5.48)
|
Pi-donor
Pi-alkyl
Pi-alkyl
Pi-alkyl
Pi-alkyl
Pi-alkyl
|
NS3/NS2B (2FOM)
|
100
|
-36.7
|
Glu88 (1.98)
Glu86 (2.74)
Asn152 (2.77)
|
Conventional H-bond
Carbon H-bond
Carbon H-bond
|
Ala164 (3.50)
Val154 (4.93)
Ile123 (4.94)
|
Alkyl
Alkyl
Alkyl
|
His51 (4.19)
|
Pi-alkyl
|
149
|
-25.9
|
Asn152 (2.32)
Lys74 (2.56)
Val147 (2.50)
Gly87 (2.50)
Gly87 (2.56)
|
Conventional H-bond
Conventional H-bond
Carbon H-bond
Carbon H-bond
Carbon H-bond
|
Lys74 (1.69)
|
Salt bridge
|
Ala166 (2.78)
Gol202 (2.99)
Leu76 (5.02)
Lys74 (5.43)
|
Pi-sigma
Pi-lone pair
Pi-alkyl
Pi-alkyl
|
155
|
-29.3
|
Asn152 (1.79)
Gol202 (1.93)
Glu88 (1.99)
Gol202 (2.07)
Ile165 (2.23)
Ile165 (2.31)
|
Conventional H-bond
Conventional H-bond
Conventional H-bond
Conventional H-bond
Conventional H-bond
Carbon H-bond
|
-
|
-
|
His51 (5.35)
|
Pi-alkyl
|
NS5 methyltransferase (1R6A)
|
96
|
-41.0
|
Lys22 (1.76)
Ser150 (2.36)
Glu149 (2.85)
|
Conventional H-bond
Conventional H-bond
Carbon H-bond
|
-
|
-
|
-
|
-
|
149
|
-53.6
|
Ser150 (2.54)
Pro152 (2.56)
Ser150 (2.64)
|
Carbon H-bond
Carbon H-bond
Carbon H-bond
|
Lys22 (1.63)
Lys29 (4.93)
|
Salt bridge
Salt bridge
|
Pro152 (5.23)
Phe25 (5.60)
|
Pi-alkyl
Pi-Pi-T-shaped
|
187
|
-42.6
|
Lys29 (5.64)
Lys181 (5.46)
Lys181 (6.24)
Ser150 (3.71)
Arg212 (3.75)
Lys22 (3.01)
Lys22 (3.76)
SO4904 (3.37)
Thr213 (5.18)
|
Conventional H-bond
Conventional H-bond
Conventional H-bond
Conventional H-bond
Conventional H-bond
Conventional H-bond
Conventional H-bond
Conventional H-bond
Carbon H-bond
|
Pro152 (4.29)
|
Alkyl
|
-
|
-
|
NS5 RdRp (3VWS)
|
95
|
-54.8
|
Ser796 (2.80)
His798 (3.27)
|
Conventional H-bond
Carbon H-bond
|
Lys401 (4.77)
Ile797 (5.19)
|
Alkyl
Alkyl
|
-
|
-
|
96
|
-59.0
|
Arg792 (2.88)
|
Conventional H-bond
|
Arg352 (4.92)
|
Alkyl
|
His800 (5.08)
|
Pi-alkyl
|
149
|
-56.8
|
Thr413 (3.08)
Asn405 (3.32)
|
Conventional H-bond
Conventional H-bond
|
Arg792 (2.88)
|
Attractive charge
|
Trp795 (5.00)
|
Pi-Pi-T-shaped
|
Table 2 Result of top-ranked reported protein inhibitor screened against E, NS3/NS2B, NS5 methyltransferase, NS5 RdRp DENV proteins with their respective docking energy value and interacting residue in the binding site.
Protein
|
PDB ID
|
Inhibitor
|
State
|
Mode of action
|
Docking energy
(kcal/mol)
|
Reference
|
Envelope (E)
|
1OKE
|
DO2
|
D02 inhibits the virus life cycle. It is also reduced viral replication activity
|
E protein βOG pocket
|
-37.3
|
51
|
PO2
|
P02 inhibits viral replication at micromolar concentrations.
|
E protein βOG pocket
|
-26.3
|
51
|
A5
|
A5 has low micromolar activity against DENV
|
E protein βOG pocket
|
-25.9
|
52
|
NS3/NS2B
|
2FOM
|
Compound 23i
|
Compound 23i showed DENV NS2B-NS3 protease inhibition activity. This compound showed antiviral activity against DENV in vitro and maybe a good lead for discovering new therapeutic agents for DENV.
|
Competitive inhibition protease
|
-32.6
|
53
|
BP13944
|
BP13944 inhibited viral replication and RNA synthesis in all four DENV serotypes.
|
Competitive inhibition protease
|
-28.4
|
54
|
Compound 32
|
This compound has an inhibitory effect on DENV replication and is determined in a dose-dependent manner. Cytotoxicity is cell culture is unknown.
|
Competitive inhibition protease
|
-27.5
|
55
|
NS5 methyltransferase
|
1R6A
|
NSC14778
|
NSC14778 is a low-micromolar inhibitor
of DENV-2 MTase.
|
MTase active site. The ligand binds to the SAM site
|
-35.2
|
56
|
Sinefungin
|
The affinity for Sinefungin is approximately six times higher than for SAM. Sinefungin is non-cell permeable.
|
MTase active site. The ligand binds to the SAM site
|
-25.5
|
57
|
Guanosine monophosphate (GMP)
|
GMP is non-cell permeable.
|
MTase GTP pocket
|
-24.1
|
57
|
NS5 RdRp
|
3VWS
|
ddGTP
|
ddGTP showed low micromolar IC50 values in in vitro DENV-2 RdRp activity tests using a poly(rC) template
|
RdRp non-nucleoside
|
-53.5
|
58
|
Balapiravir
|
Balapiravir failed to be effective for patients with DENV. No efficacy was found in the phase II Clinical trial
|
Cytidine nucleoside
|
-48.5
|
57
|
NITD 203
|
NITD 203 showed in vivo toxicity after 2 weeks of administration in rats and dogs.
|
RdRp non-nucleoside
|
-42.4
|
59
|
For comparison purposes, twelve reported protease inhibitors obtained from previous studies (Table 2) were docked into the specific target protein. Their result showed that the docking energy value ranged from -24.0 to -53.0 kcal/mol. Most of the reported inhibitors showed different interactions with the target protein of the binding pockets. Among these reported inhibitors, compound 23i was observed to have no interaction at all with the catalytic triad but produces hydrogen bond interactions with amino acid residues Asn152, Asn167, and Ala166. Other than that, for envelope DENV protein, DO2 was found to interact with crucial amino acid residues (Lys128 and Ala50) and another active amino acid (Pro53 and Ile270). For NS5 methyltransferase DENV protein, NSC14778 exhibited interaction with three crucial amino acid residues and several interactions with other active amino acids. These inhibitors can form hydrogen bonds with Lys14, Ser214, and Lys22, π-interaction with Phe25, and salt-bridge interactions with Lys14 and Lys22.
In addition, ddGTP inhibits the NS5 RdRp DENV protein by forming a hydrogen bond interaction with crucial amino acid (Lys401) and several interactions with other active site residues (Arg792, Gly601, Trp795, Asn492, Asn405, Thr413, Val603, and Phe412). Moreover, it also has the ability to interact with Cys331 (Fig. 8). From the findings, most of the top-ranked phytochemicals from Goniothalamus sp. exhibited good binding interaction as compared to reported inhibitors based on the docking energy value and the maximum number with crucial amino acids. Several compounds showed a maximum interaction with crucial amino acid residues that makes them potential lead compounds for anti-dengue drug discovery.
Drug-likeness and ADMET
Drug scan tools at Molinspiration and SwissADME server predict the drug-likeness of the proposed DENV inhibitors. All ligands were checked for their physicochemical properties through the filter of Lipinski rule of 5. The nine descriptors include octanol-water partition coefficient (Log-P), polar surface area (TPSA), number of non-hydrogen atoms, molecular weight (MW), number of hydrogen bond acceptors [O and N atoms], number of hydrogen bond donors [OH and NH groups], number of rules of 5 violations, number of rotatable bonds and molecular volume are taken into consideration. Among these phytochemicals, only 149 passed all the drug-likeness tests without any violation (Table 3). The remaining acetogenins had some violations because of the molecular weight of more than 500, Log-P value bigger than 5 and exceeding the number of hydrogen bond donors (≤5). The phytochemical that successfully passed all these parameters is predicted and considered as potential lead compounds with the good pharmacokinetic property.
Based on the results, compound 149 (C16H12O7) is the only phytochemical that passed the drug-likeness analyses without any violations. ADMET analyses were further conducted to predict the inhibitor's capability of drug-likeness. This analysis was conducted based on several parameters, physicochemical property, absorption, distribution, metabolism, and toxicity. These parameters have then been evaluated based on the number of thresholds. The results showed that all the acetogenins compounds are not qualified for certain parameters. However, the ADME prediction result for compound 149 inhibitors were predicted to pass the ADMETsar threshold of drug ability (Table 4).
Table 3 Molecular physicochemical descriptors analysis of nine ligands using Molinspiration online software tools.
Ligands
|
Log Pa
|
TPSAb
|
Nato msc
|
MWd
|
noNe
|
nOH NHf
|
Nviolationsg
|
Nrotbh
|
Volumei
|
95
|
9.48
|
72.84
|
43
|
606.97
|
5
|
1
|
2
|
28
|
659.42
|
96
|
9.45
|
93.07
|
46
|
651.03
|
6
|
2
|
2
|
30
|
701.07
|
97
|
9.32
|
93.07
|
45
|
637.00
|
6
|
2
|
2
|
29
|
684.26
|
100
|
8.48
|
113.29
|
45
|
638.97
|
7
|
3
|
2
|
28
|
675.51
|
149
|
1.96
|
120.36
|
23
|
316.26
|
7
|
4
|
0
|
2
|
257.61
|
155
|
8.51
|
153.75
|
46
|
656.94
|
9
|
5
|
2
|
31
|
678.80
|
187
|
7.52
|
173.98
|
47
|
672.94
|
10
|
6
|
3
|
31
|
686.85
|
aOctanol-Water partition coefficient (expressed as LogP, acceptable range: ˂5), bPolar surface area, cNumber of non-hydrogen atoms, dMolecular weight (acceptable range: <500), eNumber of hydrogen bond acceptors [O and N atoms] (acceptable range: ≤10), fNumber of hydrogen bond donors [OH and NH groups] (acceptable range: ≤5),gNumber of Rule of 5 violations, hNumber of rotatable bonds, iMolecular volume.
The ADME prediction result for compound 149 showed some solubility (Log S) with value -3.88. The distribution coefficient P (Log P) for this compound was 1.96 (should be between -0.7 and + 5.0). The compounds were unable to permeate BBB and act as non-substrate of P-glycoprotein. However, this compound was considered as an inhibitor for enzyme CYP1A2, CYP23A4, CYP2C9 and CYP2D6 subtypes. From this in-silico ADME analysis, compound 149 also was predicted to have no hepatotoxicity and did not have the plasma protein binding (PPB) ability. The results of Lipinski’s calculation and ADME analysis indicated that compound 149 is the most selected among others due to the zero violations in Lipinski’s rule of five and it was also predicted as a potential inhibitor by ADME analysis.
Table 4 ADMET Profiling Enlisting Absorption, Distribution, Metabolism and Toxicity related drug-likeness parameters.
Models
|
95
|
96
|
97
|
100
|
149
|
155
|
187
|
- Physicochemical Property
|
|
|
|
|
|
|
|
Solubility (Log S)
|
-9.68
(Poorly soluble)
|
-9.77
(Poorly soluble)
|
-9.41
(Poorly soluble)
|
-7.95
(Poorly soluble)
|
-3.88
(Soluble)
|
-7.04
(Poorly soluble)
|
-6.52
(Poorly soluble)
|
Distribution coefficient P (Log P)
|
9.48
|
9.45
|
9.32
|
8.48
|
1.96
|
8.51
|
7.52
|
- Absorption
|
|
|
|
|
|
|
|
Caco2 permeability (Lop Papp)
|
Caco2+
|
Caco2-
|
Caco2-
|
Caco2+
|
Caco2+
|
Caco2-
|
Caco2-
|
P-glycoprotein inhibitor
|
Non-inhibitor
|
Non-inhibitor
|
Non-inhibitor
|
Non-inhibitor
|
Non-inhibitor
|
Non-inhibitor
|
Non-inhibitor
|
P-glycoprotein substrate
|
Substrate
|
Substrate
|
Substrate
|
Substrate
|
Non-substrate
|
Non-substrate
|
Non-substrate
|
Human Intestinal absorption (HIA)
|
HIA-
|
HIA-
|
HIA-
|
HIA-
|
HIA+
|
HIA-
|
HIA-
|
- Distribution
|
|
|
|
|
|
|
|
Plasma Protein Binding (PPB)
|
PPB-
|
PPB-
|
PPB-
|
PPB-
|
PPB-
|
PPB-
|
PPB-
|
Blood Brain Barrier Permeability (BBB)
|
BBB-
|
BBB+
|
BBB-
|
BBB-
|
BBB-
|
BBB-
|
BBB-
|
- Metabolism
|
|
|
|
|
|
|
|
CYP1A2 substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
CYP3A4 substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
CYP2C9 substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
CYP2C19 substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
CYP2D6 substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
Non-Substrate
|
CYP1A2 inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
Inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
CYP23A4 inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
Inhibitor
|
Inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
CYP2C9 inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
Inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
CYP2C19 inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
CYP2D6 inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
Inhibitor
|
Non-Inhibitor
|
Non-Inhibitor
|
- Toxicity
|
|
|
|
|
|
|
|
heRG Blockers
|
Non-blockers
|
Non-blockers
|
Non-blockers
|
Non-blockers
|
Non-blockers
|
Non-blockers
|
Non-blockers
|
Human hepatotoxicity (H-HT)
|
HHT-
|
HHT-
|
HHT-
|
HHT-
|
HHT-
|
HHT-
|
HHT-
|
AMES mutagenicity
|
Ames-
|
Ames-
|
Ames-
|
Ames-
|
Ames-
|
Ames-
|
Ames-
|
Skin Sensitization, (r)LLNA
|
Non-sensitizer
|
Non-sensitizer
|
Non-sensitizer
|
Non-sensitizer
|
Non-sensitizer
|
Non-sensitizer
|
Non-sensitizer
|
Drug Induced Liver Injury (DILI)
|
DILI-
|
DILI-
|
DILI-
|
DILI+
|
DILI+
|
DILI-
|
DILI-
|
Molecular Dynamic
To evaluate the dynamics behavior of macromolecules at the molecular and atomic levels, all protein-ligand complexes were examined using MD simulations. From the docking results, compound 149 was observed to have the strongest affinity with E, NS3/NS2B, NS5 methyltransferase, and NS5 RdRp. To investigate the dynamic behavior of the docking result, the docked complexes of 149 against E, NS3/NS2B, NS5 methyltransferase, and NS5 protein were subjected through MD simulation. The system can be monitored using root-mean-square deviation (RMSD) that gives information equilibration on structural conformation throughout the simulation. If fluctuations happen towards the end of the simulation and the changes are larger than 3 Å, it indicates that the protein undergoes a large conformational change, and the ligand is not stable in the binding pocket of the protein during the simulations.
The relative fluctuation in the RMSD of Cα carbon atoms (Cα-RMSD) for 149 bound to 1OKE protein (Fig. 9A) was observed for the first 42 ns. Between 42-90 ns, the RMSD of the 1OKE protein slightly decreases while 149 shows some marginal increase with a small fluctuation at the end with an RMSD value of 0.75 Å. In the case of 2FOM (Fig. 9B), the RMSD showed a marginal decrease for 5 ns followed by a slight fluctuation for 38 ns. Then, the RMSD of 149 increases to 0.8 Å and converges at the end of the simulation. Similarly, the RMSD of 1R6A (Fig. 9C) slightly fluctuate for the first 35 ns followed by fluctuation between 35 to 45 ns and continue to fluctuate marginally between 45 to 85 ns. The RMSD starts to converge at 85 ns to the end of the simulation (RMSD: 0.4 Å). The RMSD for 3VWS (Fig. 9D) showed the most stable ligand-protein complex due to the shorter time taken to reach convergence. At first, the RMSD showed a marginal increase for 25 ns before it started to converge throughout the simulation (RMSD: 0.8 Å).
Root-mean-square fluctuation (RMSF) is useful to indicate the local changes along with the mobility of the protein during the simulation. In the case of 1OKE (Fig. 10A), a higher fluctuation was observed at the C-terminus residues up to 3.6 Å. Residues Thr48, Lys128, and Ala50 were recognized as a binding residue for 1OKE that showed lesser fluctuation. Unlike 149 complexed with 2FOM (Fig. 10B), N-terminus residues showed a higher fluctuation of up to 3.0 Å. Residues 10-15 and 40-45 tend to show more fluctuation as compared to the binding site residues (Lys74, Leu76, Gly87, Val147, Asn152, and Ala166). In the case of NS5 methyltransferase (Fig. 10C), higher fluctuation was observed at the C-terminus residues up to 2.8 Å. The RMSF for residues 40-50, 100-105 and 165-170 tend to show high fluctuation as compared to active site residues of Leu20, Lys14, Asn18, Lys29, Phe25, Leu17. Compound 149 complexed with 3VWS (Fig. 10D) was observed to have a marginally higher fluctuation at the N-terminus and the fluctuation was minimal for binding site residue, so it showed higher stability during the simulation.
To understand the conformational evolution of the ligand, the torsional conformations of each rotatable bond in the ligand throughout the simulation run were calculated (Fig. 11). The angle of each bond was illustrated by the dartboard plots while the probability of the torsions as a function of angle were illustrated by the histograms. The dartboard plot was placed in the centre of the radial plot at the beginning of the simulation trajectories while radially outwards at the time evolution. The angular coordinate was the torsional angle while the radial coordinate was the torsion rotatable bond [63]. The relationship between the torsional angle changes and interactions are directional proportional. This relationship was discovered while combining with the results on interactions observed through the docking model (Fig. 6 and Fig. 11). The results for compound 149 in the binding pocket of envelope dengue protein (Fig. 6A and Fig. 11A) displayed torsional angle changes in bond e and d, promoted by the hydrogen bond interaction between the hydroxyl group and residue Thr48. In a similar way, the torsional angle changes in bond c correspond to the hydrogen bond interaction between the hydroxyl group and residue Pro53. The torsional angle also changes in bond b corresponding to the π-π interaction between the aromatic ring and residue Ala50. The dartboard plots and the bar charts showed that bond c is rigid while bonds b, d, and e were more flexible.
In Fig. 6B and Fig. 11B, ligand 149 in the binding cavity of NS3/NS2B dengue protein showed the torsional angle changes in bond a and e promoted by the hydrogen bond interaction between the hydroxyl group and residue Asn152, Lys74, Gly87 and Val147. The torsional angle changes in bond c were promoted by the hydrophobic (salt-bridge) interaction between the hydroxyl group and residue Lys74. The torsional angles also change in bond b corresponding to the π-π interaction between the aromatic ring and residues Ala166, Leu76, Lys74 and Gol202. The bar charts and dial plots showed that the bonds b and c were rigid while bond a and e are more flexible.
The torsional angle for compound 149 in the binding site of NS5 methyltransferase was shown in combination of Fig. 6C and Fig. 11C. The Torsional angle changes in bond e promotes the hydrogen bond interaction between the hydroxyl group and residue Ser150. The torsional angle changes in bond c and d were promoted by the hydrophobic interaction between the hydroxyl group and residues Lys22 and Lys29. The torsional angles also change in bond b correspond to the π-π interaction between the aromatic ring and residue Phe25. From the bar charts and dial plots, bond b, d and e were flexible compared to bond c which was more rigid.
Ligand 149 in the binding pocket of NS5 RdRp (Fig. 6D and Fig. 11D) showed torsional angle changes in bond a and c promoted by the hydrogen bond interaction between the hydroxyl group and residues Asn405 and Thr413. The torsional angles in the bond a corresponded to the hydrophobic interaction between hydroxyl group and residues Arg792. In addition, the torsional angle also changes in bond b promoted by the π-π interaction between aromatic ring and residue Trp795. The dial plots and bar charts showed that the bonds a, b, and c are more flexible. It was shown that ligand 149 in the binding pocket of NS5 RdRp had more rotatable bonds which indicates that the molecule is flexible, and the complex is stable throughout the simulation.
To illustrate the stability of the selected ligand in the binding pocket of the proteins during the simulation, these six properties were analyzed: 1) root mean square deviation (RMSD) of a ligand concerning the reference confirmation; 2) radius of gyration (rGyr) as an indicator of extendedness of a ligand; 3) intramolecular hydrogen bond (intra-HB) as the number of internal hydrogen bonds (HB) within a ligand molecule; 4) molecular surface area (MolSA) calculated with 1.4 Å probe radius; 5) solvent accessible surface area (SASA) is the surface area of a biomolecule that is accessible to a solvent; 6) polar surface area (PSA) is a solvent accessible surface area in a molecule contributed only by oxygen and nitrogen atoms [62]. As shown in Fig. 12A, the RMSD value of compound 149 in the active site of envelope protein was below 1.5 Å. Striking fluctuation occurs at 20-30 ns and 45-100 ns. This could lead to instability of the ligand in the active site of the protein. The RMSD value for compound 149 in the active site of NS3/NS2B protein and NS5 methyltransferase (Fig. 12B and Fig. 12C) slightly fluctuates at the first 5 ns at 0.8-1.2 Å, before it starts to stabilize at 0.8 Å from 5-38 ns and the value was lower than 0.5 Å after 38 ns. For compound 149 in the binding site of NS5 methyltransferase, the RMSD value showed a marginal fluctuation for the first 35 ns. The RMSD value showed presence of minor fluctuation throughout the remaining simulation run. The RMSD value of ligand 149 in the active site of NS5 RdRp (Fig. 12D) showed some fluctuations for the first 20 ns and then it starts to reach convergence after 20 ns at 1.2 Å.
The results of changes in the rGyr of ligand bound to the active site of the proteins are shown in Fig. 13. The rGyr value for the ligand compound 149 in the binding pocket of the envelope protein (Fig. 13A) was stable at 3.65-3.80 Å from 0 to 100 ns. The rGyr value for compound 149 in the binding pocket of NS3/NS2B protein (Fig. 13B), NS5 methyltransferase protein (Fig. 13C), and NS5 RdRp protein (Fig. 13D) showed a constant value at 3.70-3.80 Å from 0 to 100 ns. These constant values predicted a steady behaviour and compactness of the structure that exerts a great effect on the quality of the substrate binding to the active site.
The consistency of the ligand during the simulation run was also indicated by intraHB, MolSA, PSA, and SASA. The properties of MolSA and PSA for compound 149 with all targeted proteins produced a consistent value throughout the simulation run. This constant value discovered the consistency of the ligand in the binding pocket throughout the simulation run. Additionally, the solvent-accessible surface area (SASA) was also calculated. This analysis measured the surface area of a molecule accessible by a water molecule. An increase in SASA values from the initial pose indicates that the ligand was exiting the binding pocket or sticking out into water while decreasing value of SASA indicates that more of the molecule was buried in the protein. To pursue the stability of the ligand in the binding pocket of protein, lower scores of SASA are priority. According to Fig. 14A, the SASA analysis for ligand 149 in envelope protein showed a constant value at 180 Å2 for the first 40 ns. However, the instability of the SASA value was observed from 40 ns to the end of the simulation run. The SASA analysis of compound 149 in NS3/NS2B (Fig. 14B) showed some fluctuation (100-200 Å2) for the first 40 ns but then it showed a decreasing score at 50 Å2 until the end of the simulation run. The SASA value for ligand 149 in NS5 methyltransferase (Fig. 14C) showed a marginal decrease for the first 5 ns. Then, it showed a constant value at 5 to 37 ns. After that, the SASA value was observed to slightly fluctuate throughout the simulation at 180-240 Å2. For the ligand 149 in NS5 RdRp protein (Fig. 14D), the SASA analysis showed some fluctuations for the first 20 ns. After 20 ns, SASA was observed to form a steady behaviour at 30 Å2 throughout the simulation process.
The comparison of the protein-ligand contacts from docking results and molecular dynamic simulation are important to predict the stability and the interactions in the binding site. Based on the docking result of compound 149 with envelope protein, the complex was stable due to H-bond interaction with residues Thr48 and Pro53, hydrophobic interaction with residue Lys128 and π-π interaction with residues Ala50, Ile270 and Pro53. In comparison, the molecular dynamic simulation result (Fig. 15A and Fig.16A) showed that this complex was able to maintain H-bond interaction with Asn18, Phe25 and Glu149 at 99%, 70% and 90% of the simulation time respectively. The presence of interactions with new residues in the simulation run showed the instability of the compound 149 in the binding site of envelope protein. For the compound 149 in NS3/NS2B, the complex was stable due to H-bond with residues Asn152, Lys74, Val147 and Gly87, hydrophobic interaction with Lys74 and π-π interaction with residues Ala166, Leu76, Lys74 and Ala166, while for the molecular dynamic analysis (Fig. 15B and Fig.16B), interaction with Lys74 was observed to maintain at 32% of the simulation run. In addition, the interaction with residues Asp75 (60%), Leu149 (100%) and Asn152 (20%) was also present during the simulation run.
The docking result of complex 149 in NS5 methyltransferase showed the H-bond interaction with Ser150 and Pro152, hydrophobic interaction with Lys22 and Lys29 and π-π interaction with Phe25 and Pro152. However, the interaction with residues Lys22 and Lys29 was only maintained for less than 10% of the simulation run (Fig. 15C and Fig. 16C). Interestingly, the presence of new interaction with active residues Lys14 (62%), Asn18 (99%), Glu149 (30%) and Ser150 (10%) was also observed. For the NS5 RdRp, docking results showed that compound 149 forms a H-bond interaction with residues Thr413 and Asn405, hydrophobic interaction with residue Arg792 and π-π interaction with residue Trp795. In comparison with molecular dynamic simulation (Fig. 15D and Fig. 16D), the interaction with residues Asn405, Thr413, Trp795 and Arg792 was maintained at <10%, 20%, 48%, 100% of the simulation run respectively. In addition, new interaction with active residues Val411, Arg352, Ser796 and His801 was also observed.
Based on the RMSD and RMSF value, the complex of 149 with protein 3VWS was more stable due to the shorter time to reach convergence and minor fluctuation of the active residues during the simulation run. Throughout the simulation, protein-ligand interaction can be monitored. The stability of the specific interaction of the protein-ligand complex was recorded during the simulation. Based on the result, the complex 149 with 3VWS was stable as compared to other protein targets due to the ability to maintain the most number of interactions with active residues Asn405, Thr413, Trp795 and Arg792 during the simulation run. In addition, the six properties analysis to predict the stabilities of the selected ligand in the binding pocket also showed that it takes a shorter time to reach convergence. It shows that ligand 149 can interact well and it is stable in the binding site of the NS5 RdRp proteins.