1. ADMET Analysis of compounds isolated from Allium sativum
The nine compounds isolated from Allium sativum underwent ADMET analysis. The abbreviation ADME stands for the processes of Absorption, Distribution, Drug Metabolism, and Excretion. It elucidates the process of how a substance or medication is absorbed, distributed, metabolised, and excreted in the body. The oral absorption rate is significantly influenced by the crucial features of solubility and permeability. Once the drug has dissolved, it quickly spreads throughout the entire body and is distributed into different compartments. Drug metabolism refers to the enzymatic breakdown or alteration of a drug molecule, which is then eliminated from the body. The Lipinski's five rule, often known as the Pfizer rule of five, mandates that oral medications must meet certain criteria to be considered for approval. These criteria include a molecular weight below 500, less than five hydrogen bond donors and acceptors, and a distribution ratio below five. All nine compounds adhere to Lipinski's rule of five which is shown in Table 1.
Table 1
ADMET Analysis of nine compounds isolated from Allium sativum
Name of Compound
|
Molecular Weight
|
GI Absorption
|
BBB permeant
|
Log Kp (skin permeation)
|
Lipinski
|
Alliin
|
177.22 g/mol
|
High
|
No
|
-9.89 cm/s
|
Yes; 0 violation
|
Allicin
|
162.27 g/mol
|
High
|
Yes
|
-6.36 cm/s
|
Yes; 0 violation
|
E-Ajoene
|
234.40 g/mol
|
High
|
No
|
-6.52 cm/s
|
Yes; 0 violation
|
Z-Ajoene
|
234.40 g/mol
|
High
|
No
|
-6.52 cm/s
|
Yes; 0 violation
|
2-Vinyl-4H-1,3-dithiin
|
144.26 g/mol
|
High
|
Yes
|
-5.55 cm/s
|
Yes; 0 violation
|
Diallyl sulfide
|
114.21 g/mol
|
High
|
Yes
|
-5.46 cm/s
|
Yes; 0 violation
|
Diallyldisulfide
|
146.27 g/mol
|
High
|
Yes
|
-5.63 cm/s
|
Yes; 0 violation
|
Diallyltrisulfide
|
178.34 g/mol
|
High
|
Yes
|
-5.51 cm/s
|
Yes; 0 violation
|
Allyl methyl sulfide
|
88.17 g/mol
|
High
|
Yes
|
-5.76 cm/s
|
Yes; 0 violation
|
2. Molecular Docking
The selected nine molecules from the Allium sativum were docked at the active site of the alpha synuclein. The binding affinity of the nine compounds lies between − 5.1 and − 10.8 kcal/mol, as mentioned in Table 2, and 3D visualized results of top 3 binding affinities is shown in Fig. 1 but in our study allicin and E-ajone show binding affinity of -10.4 kcal/mol. So, for further in vitro study we took alliin, allicin, ajone and diallyl disulfide.
Table 2
Binding Affinities of all nine compounds
Name of isolated compound from Allium sativum
|
PubChem CID
|
Binding Affinities
(kcal/mol)
|
Alliin
|
9576089
|
-10.8
|
Allicin
|
65036
|
-10.4
|
E-Ajoene
|
5386591
|
-10.4
|
Z-Ajoene
|
9881148
|
-5.1
|
2-Vinyl-4H-1,3-dithiin
|
133337
|
-6.0
|
Diallyl sulfide (DAS)
|
11617
|
-6.8
|
Diallyldisulfide (DADS)
|
16590
|
-10.8
|
Diallyltrisulfide (DATS)
|
16315
|
-7.5
|
Allyl methyl sulfide (AMS)
|
66282
|
-6.2
|
One hundred nanoseconds of molecular dynamics simulation of the protein target and the best compound-complex were carried out and Desmond was used to examine simulation trajectories. MD trajectory analysis calculated RMSD, RMSF, and protein-ligand interactions. Figure 1 shows how the RMSD values of ligand-bound protein’s carbon alpha atoms have changed over time. According to the RMSD plot, the proteins in the all five complexes reached stability at 20 ns. Following that, the RMSD values fluctuate around about 1.0 Angstrom during the simulation length, which is totally acceptable. The structure appeared to be stable for the most part throughout the experiment. Complex 1xq8-65036 showed the lowest average RMSD among the individual complexes under examination, suggesting that it was the most stable. Stability was likewise shown by Complex 1xq8-121922, but with somewhat higher RMSD values.
The Ligands fit to protein remained steady throughout the simulation after getting equilibrium up to 20 ns. In some instances, from 50 ns to 90 ns, the RMSD values increased and decreased abruptly for 1xq8-5386591 complexed ligand (pink representation in Fig. 2). This could be attributed to a ligand mode flip; after equilibrium was reached, the ligand RMSD stayed constant for the length of the simulation.
RMSF value of protein coupled to the ligand is shown in Fig. 3. As determined by MD trajectories, higher peaks indicate residues that belong to loop regions or N and C-terminal zones due to flexibility of loops as compared to other secondary structures. Ligand binding site residues of target protein represent lower RMSF values, indicate stable ligand-protein binding. Alpha-helices and beta-strands observed throughout the simulation as basic secondary structure elements (SSE).
Hydrogen bonds make up the majority of significant ligand-protein interactions determined by MD simulations. Figure 3 shows the total hydrogen bonds between the target and selected lead compound concerning time. The figure shows significant number of hydrogen bonds formed between ligand and the protein. Important residues for making interaction throughout simulation were GLU_28, LYS_32, LYS_34, and GLU_35 for the complex 1xq8-6074. No significant hydrogen bonds were detected but hydrophobic interaction was most important for 1xq8-16590 complex. For 1xq8-65036 these are MET_1, LYS_21, LYS_58, THR_59, and GLN_62 residues crucial. In complex 1xq8-121922, LYS_10, GLU_28, GLY_84, GLY_86, SER_87 and LYS_96 are vital hydrogen bond making residues. LYS_21, ALA_89, and THR_92 residues are significant in terms of hydrogen bonds for 1xq8-5386591 complex. We calculated MM-GBSA for each complex. Total deltaG of binding affinity is represented in Table 3. Compound ID 65036 and 5386591 showed Stronger energy.
The radius of gyration of a protein is quantitative measure of protein overall shape and compactness. It is defined the distance between point of rotation and the point where energy transfer has greatest impact. Following is the Table 3 representing average radius of gyration for each ligand complexes protein with its standard deviation. The fluctuation in RoG over time is shown in Fig. 5. The protein shoed compact three-dimensional structure throughout time of simulation.
Table 3
Ligand bound proteins’ average radius of gyration with standard deviation
Ligand Bound Protein
|
Average RoG with Standard deviation
|
1xq8-6074
|
15.52 ± 2.05
|
1xq8-16590
|
13.93 ± 1.87
|
1xq8-65036
|
17.08 ± 2.03
|
1xq8-121922
|
16.54 ± 2.46
|
1xq8-5386591
|
15.13 ± 2.93
|
Principal Component Analysis (PCA) describes the dynamics of proteins.11 Calculations were made on the trajectory motions observed during MD simulations. The stability was demonstrated in the first 20 modes of motion by plotting the eigenvalues (protein) against the eigenmode (eigenvector index). Eigenvalues span the hyperspace apparent eigenvector fluctuations. The target protein's overall mobility is regulated by eigenvectors with higher eigenvalues in simulated analyses. PC3 has a more miniature structure than PC1 and PC2, and because of its low variability, it thought more stable protein ligand-binding complexes. Simple PC subspace clustering showed that all of the groups' conformations varied. The blue color shows the greatest mobility, followed by white for moderate mobility and red for less flexibility.13
Table 4
Total MM-GBSA calculated for each complex
Complexes
|
MMGBSA_dG_Bind (Kcal/mol)
|
1xq8-6047
|
-20.30926372
|
1xq8-16590
|
-20.38183743
|
1xq8-65036
|
-28.98871704
|
1xq8-121922
|
-21.19605566
|
1xq8-5386591
|
-24.63823394
|
MTT Assay of selected compound from molecular docking
Alliin, allicin, ajone and diallyl disulfide of concentration (5 & 10 µM), effective protection against the rotenone cytotoxicity measured as MTT reduction or cell viability % which is shown in Fig. 6. The below mentioned graph describes the neurotoxic role of rotenone, taken in doubling concentrations starting from 0.1–20 micromolar concentration, and decreased viability is observed on the increase of concentration of rotenone and all the values are calculated in triplicates and one-way analysis has a significant value of 0.001. The data is further subjected for checking its IC50 value for further experiments, and the IC 50 value is 37.43 at 5mM and 0.25mM concentrations so, further experiments are conducted by considering 5mM of rotenone. IC50 is calculated by XY analysis of non-linear regression model, adding bell-shaped curve which is shown in Fig. 3. The four graphs in Fig. 4 represent MTT assay on four compounds that have a neuroprotective role against rotenone acid as we need to prove that these four compounds namely alliin, allicin, ajone and diallyl disulfide are non toxic to SHSY5Y neuroblastoma cell lines, graphs are made by experimenting with concentration lower 0.1 to higher 10 mM. All the graph values are plotted in duplicates comparing to control and, one-way ANOVA showed significant cytotoxicity with p > 0.05 and p > 0.01 and p.0.001 .and 5mM and 10mM have not shown any toxicity on neuroblastoma cell lines and near to similarity as like control. Figure 6 is also MTT assay done on SHSY5Y by comparing control, rotenone and rotenone added with alliin, allicin, ajone and diallyl disulfide. All the values are taken in duplicates, one-way is conducted with the significant p-value.>0.001, and all experiments are independently done.