Protein preparation

The protein structure used as a prerequisite for molecular docking was obtained from the Protein Data Bank, with the PDB ID 2QV4 file depicting the three-dimensional structure of human pancreatic α-amylase complexed with Acarbose derivative (named as QV4) at a resolution of 1.97 angstroms [11]. This structural model was created by removing heteroatom coordinates, water molecules, and co-crystallized ligand molecules, and the resulting protein structure was saved in.pdb format.

Compound library preparation

The investigation incorporated a widely recognized botanical species, Zingiber officinale. A comprehensive investigation was carried out within the LOTUS database to identify bioactive compounds originating from this particular species. The compounds that have been found were downloaded in the .sdf format and subsequently subjected to processing in order to facilitate their utilization in further virtual screening.

Virtual screening

PyRx version 0.8 [12], which uses Autodock Vina as the underlying docking engine, was used for molecular docking for virtual screening. The docking grid center was defined as X = 12.384745, Y = 48.136073, and Z = 26.209218. Subsequently, the binding affinities for the most favorable poses were tabulated, and the interaction details were visualized utilizing the Discovery Studio (DS) Visualizer. QV4 (co-crystal ligand) and montbretin A (MbA), a human pancreatic α-amylase inhibitor [11] was used as a positive control for this study.

Molecular properties and molecular descriptors estimation

The molecular properties and molecular descriptors for the selected five compounds were accessed from the LOTUS database (https://lotus.naturalproducts.net/).

Results

A virtual screening methodology was employed in order to identify a putative natural inhibitor of α-amylase. A total of 383 compounds were obtained from a LOTUS database derived from Zingiber officinale. The secondary structure of α-amylase, specifically the ProMotif, is characterized by the presence of 8 β-sheets, 5 βαβ units, 6 β-hairpins, 3 β-bulges, 24 β-strands, 23 α-helices, 13 helix-helix interactions, 48 β-turns, 7 γ-turns, and 5 disulfide bonds. The catalytic residues responsible for enzymatic activity are located at positions D197-D300-E233, D197-E233, and D197-E233 (Figure 1).

A structure-based virtual screening approach was employed to screen the active pockets of α-amylase against the 383 compounds that were retrieved. After conducting virtual screening and conducting a thorough analysis of the screened compounds, which involved evaluating binding energies and visually inspecting their 2D and 3D interactions, it was determined that 14 compounds exhibited greater efficacy in terms of binding energies (Table 2).

Further investigation was undertaken on the top five prominent compounds that were identified, specifically LTS0006138, LTS0117761, LTS0176515, LTS0102243, and LTS0018665. Figure 2 illustrates the binding conformations of the five compounds under investigation, as well as two positive controls (QV4 and MbA), within the active pocket of α-amylase (Figure 2). The comprehensive analysis of docked complexes demonstrated that these selected compounds exhibited interactions within the identical catalytic pocket of the target protein, comparable to the positive controls.

The positive control MbA was found to interact with Leu162, Tyr151, Pro54, Thr163, Asn53, Ile51, Trp58, Gln63, His299, Asp300, Asn298, Glu233, Arg195, Asp197, Tyr62, Gly104, Leu165, Trp59, Asp356, and His305 residues of α-amylase; while the co-crystal inhibitor QV4 interacted with Trp58, Leu162, His305, His299, Asp300, Arg195, Asp197, Glu233, Ala198, Tyr62, Gln63, Trp59, Leu165, Glu60, Ala106, Pro54, Ile51, Asn53, Val107, Gly104, and Thr163 residues of α-amylase (Figure 3).   

LTS0176515 was found to interact with Arg421, Val401, Pro332, Arg398, Ser289, Gly334, Asp290, Arg252, Ser3, Pro4, Thr11, Phe335, Asp402, and Gly403 residues of α-amylase (Figure 4). LTS0018665 interacted with Trp58, Tyr62, Trp59, His299, Asn298, Arg195, Asp300, Glu233, Ala198, and Asp197 residues of α-amylase (Figure 4). LTS0006138 was found to bind with Gly9, Gly403, Arg421, Ser289, Arg398, Pro332, Gly334, Tyr333, Arg252, Asp290, Asp402, Phe335, Thr11, Pro4, Thr6, and Arg10 residues of α-amylase
(Figure 4). LTS0117761 interacted with Phe335, Pro4, Arg252, Asp290, Gly334, Ser289, Pro332, Gly403, Arg398, Arg421, Thr11, Asp402, Gly9, Arg10, and Thr6 residues of α-amylase (Figure 4). Furthermore, LTS0102243 was found to interact with Ile235, Asp300, Glu233, Arg195, His299, Asp197, Trp59, Tyr62, His305, Trp58, Asn298, Phe256, Asn301, Ala307, and Gly306 residues of α-amylase (Figure 4). 

The additional comprehensive data regarding the selected 5 compounds, including their molecular properties (Table 3) and molecular descriptors (Table 4), was obtained from the LOTUS database. Molecular properties including the number of total atoms, heavy atoms, bond count, carbon atoms, minimal number of rings, as well as maximal number of rings were estimated for these compounds.

Drug-like characteristics are evaluated using molecular descriptors during compound design. Size, shape, polarity, and chemical reactivity are all numerical representations of molecule attributes. Various descriptors were calculated for the selected substances, typically comprising the NP (natural product)-likeness score, AlogP, and evaluation for Lipinski's rule violation. These descriptors collectively offered a comprehensive assessment of the compound's drug-like characteristics.

Figures & Tables

Discussion