The molecular alignment is done according to the electrostatic and structural properties of the active site of AChE. The steric, electrostatic, and hydrophobic fields were mapped onto the active binding pocket of AChE to better understand AChE and ligand interactions. Early inhibition research was mainly focused on ligands binding in the active site. The recent efforts have focused on finding novel ligands that bind to both sites in order to search for more potent reversible inhibitors. Prediction of interactions between small molecules and proteins is a crucial step to decipher many biological processes and plays a critical role in drug discovery.

Among 500 herbal lead compounds top 10 ligands were subsequently analyzed for binding pattern with AChE using AutoDock methods on the basis of binding energy score and further analyzed for possible molecular interactions with AChE using LigPlot program and visualized by Python Molecular Viewer (PyMol) (Table 1) and as per our previous studies [33]. Cannabis plant's secondary metabolite C₂₈H₃₄N₂O₆ (CID: 1990283) was found interacting with active site residue Phe288 of AChE through one hydrogen bond with 2.98 Å and nine hydrophobic interactions, obeying Lipinski's rule of five, having lowest minimum binding energy of −12.61 Kcal/Mol, log⁡⁡P = 2.91, inhibition constant (K _i) = 570.38 nM, and total intermolecular energy of −13.49 Kcal/Mol while second rank derivative (CID: 1991460) was interacting through two hydrogen bonds and eight hydrophobic interactions with active site residues Phe288 and Asp121, obeying Lipinski's Rule as well, having second lowest minimum energy of −11.18 Kcal/Mol, log⁡⁡P value = 1.24, inhibition constant (K _i) = 6.40 nM, and total intermolecular energy of −11.75 Kcal/Mol (Figure 1 and Table 1). On the basis of complex scoring and interactions with the active site residue and binding ability it was deciphered that THC derivative specifically CID: 1990283 could be promising inhibitor of AChE.

Since molecular docking provides only a static view of protein-ligand interaction, hence we also performed molecular dynamics simulation on ChE/C28H34N2O6 complexes in order to study the interactions in motion, fluctuation in residues, and movement in any specific domain or separation of protein ligand complex. The overall goal of this simulation step was to account for protein flexibility and movement that cannot be achieved in the docking simulation also to check the stability of the complex interactions [34]. Figure 2 shows the interactions of the docked C₂₈H₃₄N₂O₆ with AChE in a 1000 ps simulated snapshot. Two hydrogen bonds with Ser343 and Lys346 with 5 hydrophobic bond interactions have been formed during course of simulation in water (Table 2 and Figure 2). On careful observation by aligning the two docked structures, the presimulated one with the 1000 ps simulated snapshot, it was found that the ligand substantially moves towards these residues to form additional H-bonds. Moreover, it was observed that the ligand was slide down into the deep gorge cavity to stabilize contacts with active site residues and to avoid further fluctuations in structural confirmations.

To examine in detail the ligand-receptor interactions of AChE/C₂₈H₃₄N₂O₆ complex and to estimate the dynamic stabilities of the hydrogen bonds facilitating the inhibitor in the active site of AChE, we calculated the time evolutions of the associated interatomic distances and contacts. For this complex, two hydrogen bonds with Ser343 and Lys346 and 5 hydrophobic were observed, with percentage occupancy over 50 for the entire simulation. The minimum binding energy was decreased from −12.61 to −8.64 Kcal/Mol but more importantly two hydrogen bonds were found instead of one (Figures 1 and 2). We also calculated RMSDs between Cα of AChE trajectories recorded every 1000 ps and Cα of their X-ray crystal structure. The RMSDs for the trajectory of AChE in complex with C₂₈H₃₄N₂O₆ were also calculated using its initial docked structure as a reference. The simulation length used in the entire study was long enough to allow rearrangement of side chains of the native as well as the drug complex protein thus facilitating the most stable binding mode. The folding of biological macromolecules is among the main problems of molecular biology and biophysics although having a huge set of possible conformations; a protein assumes its unique stable spatial structure within a time. The molecular spatial packing of amino acid residues is an important aspect of protein stability, which was evaluated by calculating radius of gyration. The higher the value of gyration is, the less is the compactness [34]. At aqueous environment condition gyration value reaches 0.40 nm for AChE/C₂₈H₃₄N₂O₆ complex (Figure 4). Hence, AChE structure was found to be less compact and more accessibility of binding pocket residues to our ligand C₂₈H₃₄N₂O₆. The greater degree of movement has been observed in the AChE, when simulated under aqueous condition.

Based on the results from AChE/C28H34N2O6 complex it appears that molecular interactions of C28H34N2O6 with the residues Ser343, Ser340, and Lys346 in AChE are important for inhibitory activity. A comparison between the conformations obtained from docking and molecular dynamics simulations showed substantial changes in binding conformations. These results indicate initial receptor-ligand interaction observed in docking can be limited due to the receptor rigid docking algorithm but the conformational changes and interactions observed after simulation runs are more energetically favoured and better representations of derivative poses in receptor.