i. Design of Ligand Library
Based upon the available literature ligands like Vernonioside-A, B, A1, A2, A3, B2, B3, A4, luteolin, stigmasterol, β-sitosterol, rhein, aloe-emodin etc. were shortlisted for generating a ligand library. The two-dimensional structure of these ligands was generated by obtaining isomeric SMILES from PubChem and converting them into two-dimensional structure using ChemDraw 8.0[49]. These two-dimensional structures of all the shortlisted ligands were utilized for generation of their three-dimensional structure followed by energy minimization process.
ii. Target Identification
Reverse transcriptase, HIV-protease, and α-glucosidase are the viral enzymes that plays an important role in the viral lifecycle of HIV virus within human body and by inhibiting these enzymes we can interrupt the further maturation of HIV virus within the human body. The three-dimensional structure model of reverse transcriptase, HIV protease, and α-glucosidase for executing the docking studies were procured from protein databank. The crystallized structure model of viral reverse transcriptase (pdb id: 1ep4) was revealed by X-ray diffraction technique at a resolution of 2.50 Å by using E. coli as an expression system. Structural model of viral reverse transcriptase consists of two macromolecular chains of 560 amino acids complex with a carbamate-based inhibitor molecule S-1153. The crystallized structure model of viral protease enzyme (pdb id: 7doz) was revealed by X-ray diffraction technique at a resolution of 1.91 Å by using E. coli as an expression system. Structural model of viral protease consists of a single macromolecular chain of 203 amino acids complex with an inhibitor molecule nelfinavir. The crystallized structure model of viral α-glucosidase (pdb id: 7kb6) was revealed by X-ray diffraction technique at a resolution of 2.20 Å by using H. sapiens as an expression system. Structural model of viral α-glucosidase consists of four macromolecular chains out of which chain A and C consists of 977 amino acids, chain B and D consists of 554 amino acids and complex with valiolamine derivatives as a potent inhibitor.
iii. Molecular Docking Studies
Three-dimensional structural model of all the separated macromolecular targets used in the current study were redocked against their reference ligand which were complexed in their crystallized structure for the validation of the utilized docking protocol. After successful validation the prepared molecular ligand library was computationally screened against each of the macromolecular targets used in the current study. After completion of the virtual screening of the ligand library the best lead molecule is selected based upon the minimum binding energy within the predefined range of -5 to -15 kcal/mol[41, 36, 50, 44–46, 51]. The binding score of each of the ligand of the ligand library against each macromolecular target is tabulated in Table I.
Table I. Binding score obtained for each of the ligand of the designed ligand library against each of the shortlisted macromolecular target involved in the treatment of HIV.
Analyzing the docking score obtained after the computational screening of designed library clearly showing that the vernonioside-A2 and Stigmasterol is showing best binding affinity against all the macromolecular targets used in the current study.
iv. Pharmacokinetic and Toxicological Evaluation
Physicochemical and pharmacokinetic factors control drug transport within the human body. These regulatory characteristics are critical for a drug molecule's pharmacological expression via pharmacodynamics and toxicological qualities. As per to the Lipinski's rule of five, compound Vernonioside-A2 is having the considered parameters within the desired range, i.e., MW < 500, <10 HBA, < 5 HBD, TPSA 20–130 Å2, < 5.0 LogP. The reported physicochemical features imply that Vernonioside-A2 has a drug-like candidature with optimized pharmacokinetics. P-glycoprotein acts as a physiological barrier for xenobiotics, drugs, and toxins, has not shown substrate-like expression for the ligand Vernonioside-A2.
Vernonioside-A2 has not demonstrated substrate-like expression for the majority of cytochrome P450 isoenzymes. AMES, hERG-I inhibition, skin sensitization, Tetrahymena Pyriformis toxicity, hepatotoxicity, and minnow toxicity are only a few of the toxicity pathways that the ligand constantly demonstrates a high inclination for elimination from the body with a very few expressions. Overall, the expected pharmacokinetics, and toxicological profile of Vernonioside-A2 based upon physicochemical parameters fits well in the prescribed range, indicating its promising future as a candidate for therapeutic development. Table II lists the pharmacokinetics, and toxicological features of the shortlisted compounds.
Table II. Pharmacokinetic and toxicological analysis of screened leads against viral reverse transcriptase enzyme.
Property
|
Descriptor
|
Vernonioside A2
|
Stigmasterol
|
Vernonioside A3
|
Oleanolic acid
|
β-sitosterol
|
Vernonioside B1
|
Vernonioside A1
|
Chrysosplenol D
|
Cassiabrevone
|
Picetannol
|
MW
|
(g/mol)
|
632.791
|
412.702
|
630.775
|
456.711
|
414.718
|
632.791
|
632.791
|
360.318
|
530.529
|
244.246
|
LogP
|
-
|
2.3866
|
7.8008
|
2.5948
|
7.2336
|
8.0248
|
2.3866
|
2.3866
|
2.6026
|
3.8782
|
2.6794
|
Rotatable bond
|
-
|
6
|
5
|
6
|
1
|
6
|
6
|
6
|
4
|
3
|
2
|
HBA
|
-
|
10
|
1
|
10
|
2
|
1
|
10
|
10
|
8
|
9
|
4
|
HBD
|
-
|
5
|
1
|
4
|
2
|
1
|
5
|
5
|
3
|
7
|
4
|
TPSA
|
(Å)2
|
265.314
|
186.349
|
264.681
|
201.354
|
187.039
|
265.314
|
265.314
|
146.955
|
222.567
|
103.706
|
Absorption
|
Water solubility (mol/L)
|
-4.208
|
-6.781
|
-4.033
|
-3.493
|
-6.871
|
-3.831
|
-4.208
|
-3.505
|
-2.913
|
-2.801
|
Absorption
|
Caco2 permeability
|
-0.359
|
1.218
|
-0.428
|
1.305
|
1.205
|
-0.48
|
-0.359
|
1.053
|
0.204
|
0.961
|
Absorption
|
Intestinal absorption (%) (human)
|
53.934
|
95.372
|
61.921
|
100
|
94.866
|
46.152
|
59.934
|
74.86
|
84.306
|
88.692
|
Absorption
|
Skin Permeability (Log Kp)
|
-2.741
|
-2.794
|
-2.74
|
-2.723
|
-2.794
|
-2.737
|
-2.741
|
-2.735
|
-2.735
|
-2.778
|
Absorption
|
P-glycoprotein substrate
|
Yes
|
No
|
Yes
|
No
|
No
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Absorption
|
P-glycoprotein I inhibitor
|
Yes
|
Yes
|
Yes
|
No
|
No
|
Yes
|
Yes
|
No
|
Yes
|
No
|
Absorption
|
P-glycoprotein II inhibitor
|
No
|
Yes
|
No
|
Yes
|
Yes
|
No
|
No
|
No
|
Yes
|
No
|
Distribution
|
VDss (human)
|
-0.368
|
0.224
|
-0.391
|
-1.062
|
0.24
|
-0.626
|
-0.368
|
0.365
|
-1.123
|
0.018
|
Distribution
|
Fraction unbound (human)
|
0.324
|
0
|
0.32
|
0
|
0
|
0.336
|
0.324
|
0.145
|
0.338
|
0.154
|
Distribution
|
BBB permeability
|
-1.405
|
0.787
|
-1.416
|
-0.351
|
0.797
|
-1.259
|
-1.405
|
-1.489
|
-1.589
|
-0.838
|
Distribution
|
CNS permeability
|
-3.797
|
-1.701
|
-3.827
|
-1.047
|
-1.754
|
-3.884
|
-3.797
|
-3.453
|
-3.466
|
-2.306
|
Metabolism
|
CYP2D6 substrate
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Metabolism
|
CYP3A4 substrate
|
No
|
Yes
|
Yes
|
Yes
|
Yes
|
No
|
No
|
No
|
No
|
No
|
Metabolism
|
CYP1A2 inhibitor
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Yes
|
No
|
Yes
|
Metabolism
|
CYP2C19 inhibitor
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Metabolism
|
CYP2C9 inhibitor
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Yes
|
Metabolism
|
CYP2D6 inhibitor
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Metabolism
|
CYP3A4 inhibitor
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Excretion
|
Total Clearance (log ml/min/kg)
|
0.385
|
0.618
|
0.316
|
-0.081
|
0.628
|
0.383
|
0.385
|
0.72
|
0.272
|
0.123
|
Excretion
|
Renal OCT2 substrate
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Toxicity
|
AMES toxicity
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Yes
|
No
|
No
|
Toxicity
|
Max. tolerated dose (human) (log mg/kg/day)
|
-2.352
|
-0.6
|
-1.719
|
0.612
|
-0.555
|
-1.861
|
-2.352
|
1.085
|
0.297
|
0.519
|
Toxicity
|
hERG I inhibitor
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Toxicity
|
hERG II inhibitor
|
Yes
|
Yes
|
Yes
|
No
|
Yes
|
Yes
|
Yes
|
No
|
Yes
|
No
|
Toxicity
|
Oral Rat Acute Toxicity (LD50) (mol/kg)
|
4.146
|
2.313
|
3.858
|
2.764
|
2.326
|
3.724
|
4.146
|
2.267
|
2.339
|
1.638
|
Toxicity
|
Oral Rat Chronic Toxicity (LOAEL) (mg/kg/day)
|
2.671
|
0.846
|
2.817
|
2.177
|
0.829
|
2.836
|
2.671
|
2.349
|
3.454
|
1.789
|
Toxicity
|
Hepatotoxicity
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Toxicity
|
Skin Sensitisation
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Toxicity
|
T. Pyriformis toxicity (mg/L)
|
0.285
|
0.458
|
0.285
|
0.285
|
0.454
|
0.285
|
0.285
|
0.289
|
0.285
|
0.622
|
Toxicity
|
Minnow toxicity
|
1.819
|
-1.993
|
0.85
|
-1.872
|
-2.12
|
1.051
|
1.819
|
0.168
|
3.446
|
1.115
|
v. Molecular Dynamic Simulation
Vernonioside-A2 was revealed as one of the most stabilized ligands after running MD simulation for a brief period of time. Thus, in order to determine the stability of the complex ligand Vernonioside-A2 within the macromolecular target, the same technique has been amplified for a longer length of 100 ns. The protein backbone's stability and structural shifts are measured using the RMSD during the simulation period. Trajectories obtained after performing the MD simulation of the macromolecular complex Vernonioside-A2 has revealed their stability throughout the 100 ns simulation with an average RMSD value ranging from 3–7 Å for macromolecular backbone and 6–8 Å for the complex ligand. RMSD of the complex ligand Vernonioside-A2 as well as the macromolecule was demonstrated stable behavior with a very little fluctuation as shown in Figure I.
RMSF value of the macromolecule was calculated by considering the fluctuation of the macromolecular Cα atoms to identify the movement of the amino acids with respect to their initial position. The movement of amino acids in the active site is inversely correlated with variations, and vice versa. The amino acid residues are displayed on the x-axis of the RMSF plot, and their RMSF value is displayed on the y-axis. RMSF value observed for the macromolecular backbone was found to be in the range of 1.5–7.5 Å. After evaluation, it was shown that the RMSF value for the complex system was lower for the active amino acid residues with a mean alteration for the ligand Vernonioside A2 was found to be 1.5–3.5 Å, confirming the acceptable fluctuations within the macromolecular active site. RMSF of the viral enzyme complexed with Vernonioside-A2 detected during MD simulation of 100 ns was represented in Figure II.
SSE analysis during whole simulation process revealed that the macromolecular structure had a total of 41.21% of SSE, out of which 26.05% of alpha-helices as well as 15.16% of beta-sheets, which may remain conserved during the simulation. The stability of the protein ligand complexes is due to the formation of hydrogen bonds, hydrophobic contacts, and ionic interactions throughout the MD simulation. To measure the stability of ligand Vernonioside-A2, the intensity of these interactions were observed during the whole timeframe of the simulation. The interaction of the ligand Vernonioside-A2 against the viral reverse transcriptase enzyme were analyzed throughout the simulation process and found that ligand molecules interact with Leu100, Lys102, Val106, Val179, Tyr181, Tyr188, Glu224, Pro225, Phe227, Trp229, Leu234, and Tyr318 amino acid by hydrophobic interaction, and amino acid Lys103, His235, and Pro236, forms a hydrogen bond with the ligand Vernonioside-A2. The ligand interaction observed between the ligand Vernonioside-A2 and macromolecular target reverse transcriptase was demonstrated in Figure III.
RMSD value of the bound ligand Vernonioside-A2 was found to be within the adequate range of 2–3 Å, concluding a few or no oscillation of the vernonioside-A2 withing the macromolecular pocket throughout the simulation. Radius of gyration (rGyr) of ernonioside-A2 was found to be 6.5 Å. Molar surface area (MolSA) of the complex Vernonioside-A2 was observed in the range of 540–550 Å2 having average value of 545 Å2. Ligand’s solvent accessible surface area (SASA) was observed within 160–320 Å2 and polar surface area (PSA) for the complexed Vernonioside-A2 was found to be in a range of 270–290 Å2.