Protein preparation

The 3-dimensional (3D) structures of HK2 (PDB ID: 5HEX) and GLS1 (PDB ID: 3VP1) were obtained from the Protein Databank [13]. To ensure the integrity and unobstructed nature of the protein structures used in molecular docking simulations, the HK2 and GLS1 protein structures underwent preprocessing using Discovery Studio (DS) 2021 software. This preprocessing involved the elimination of native ligands (Q27456136 for HK2 and BPTES for GLS1) and water molecules from the structures. The monomeric proteins were subjected to minimization and subsequently converted into the. pdbqt format in order to facilitate subsequent docking analysis.

Natural compound library retrieval and preparation

The Biopurify Phytochemicals subsets were obtained in the .sdf file format from the ZINC database. In the DS 2021 software, the 'Prepare Ligands module' was used to prepare a dataset of 2706 phytochemicals for subsequent docking analysis. This preparation involved performing energy minimization and applying a force field to ensure the suitability of the ligands for the docking analysis.

Molecular docking-based virtual screening

Virtual screening (VS) contributes to the expansion of compound databases by identifying active compounds and eliminating inactive ones prior to experimental validation. This method uses computational methods to analyze large datasets of three-dimensional molecule structures [14]. Autodock vina was used to screen the compound library against the enzyme active sites of HK2 and GLS1. DS 2021 accomplished further analyses of compound interactions and visual inspections, focusing on complexes with lower binding energy values to determine the most thermodynamically stable complexes.

Ligand interaction analysis

Following the docking-based VS, the resulting docked complex was subjected to additional analysis to examine the interaction between the ligand and the target proteins (HK2 and GLS1) using the 'analyze ligand poses' tool in DS software. The ligands' favorable, unfavorable, total charge, the total number of hydrophobic, and hydrogen bond interactions with the active site residues of the target proteins were computed.

Results

The main objective of this study was to specifically focus on two key enzymes, HK2 and GLS1, which are associated with RA. The 3D structure of both enzymes was available in the PDB database in complex with certain inhibitors, which helped to find the specific binding sites and screening of the potential ligands. In the search of potential natural inhibitors targeting both the targeted enzymes, ‘Biopurify Phytochemicals’ were utilized for the VS process.

During the screening procedure, the XYZ coordinates of the HK2 (5HEX) were established as 85.678242, 15.727273, and -102.007333, correspondingly. In addition, Q27456136 (co-crystal ligand) and a recognized inhibitor, Cypate-2DG, were employed as positive controls. Likewise, the XYZ coordinates of the GLS1 (3VP1) were determined to be 2.347000, -9.268778, and -0.893333. The co-crystal ligand BPTES and the GLS1 inhibitor Telaglenastat were used as positive controls.

During the VS process, it was observed that 34 phytochemicals exhibited higher binding energy compared to the control in the HK2 case, whereas 72 phytochemicals were identified in the GLS1 case. In the context of visual inspection and interaction analysis, it was observed that 13 compounds exhibited the best effectiveness and prevalence in targeting both HK2 and GLS1 (Table 1).

In this study, six of the 13 common ligands were described in detail with their binding interactions with the receptor molecules (Table 2). The receptor-ligand interactions of the six determined compounds with HK2 and GLS1 were found to facilitate various aspects of the interactions, such as Ligand SASA, total favorable and unfavorable interactions, charge, as well as the total number of hydrophobic and hydrogen bonds formed during the interaction.

Interaction with HK2

The common ligands that were found to have better efficacy in terms of binding energy were examined using 2D and 3D interaction analysis, as well as an examination of the ligand poses during receptor-ligand interactions. Figure 1 depicts the binding poses of the control compounds for HK2 enzyme, namely Cypate-2DG and Q27456136, which show that some of the residues such as ASN706, SER614, ASN683, ASP657, PRO513, PHE604, PHE602, THR680, ASN656, and others are the main binding residues in the active site of HK2.

The selected six ligands, namely, ZINC8234294, ZINC49888756, ZINC95627883, ZINC4096846, ZINC4098366, and ZINC49823084 and their superimposed interaction with active site residues and the positive controls Cypate-2DG and Q27456136 were displayed in figure 2.

Figure 2 illustrates that all selected compounds and positive controls are located within the identical binding pocket of the HK2 enzyme. Further, each of the selected common compounds and its 2D interaction with the active site residues of the HK2 is displayed in figure 3. It is glaringly obvious that the majority of active site residues interact with the six compounds via hydrogen bonds, hydrophobic interactions, van der Waals interactions, and other types of interactions.

Interaction with GLS1

Using 2D and 3D interaction analysis, as well as an examination of the ligand poses during receptor-ligand interactions, six common ligands with greater binding energy efficiency with both receptors (HK2 and GLS1) were investigated. Figure 4 illustrates the binding conformations of the control compounds, Telaglenastat and BPTES, with the GLS1 enzyme. The binding poses reveal that specific residues, such as LEU323, ASP327, GLU325, TYR94, PHE322, LEU321, and ASN324, among others, serve as the primary binding residues within the active site of GLS1.

All six of the specified compounds—ZINC000008234294, ZINC000049888756, ZINC000095627883, ZINC000004096846, ZINC000004098366, and ZINC000049823084 as well as their superimposed interactions with GLS1's active site residues and the positive controls Telaglenastat (green color) and BPTES were shown in Figure 5.

Similar to the case of HK2, Figure 6 depicts the 2D interaction between the active site residues of GLS1 and the selected common compounds. The six compounds (ZINC000008234294, ZINC000049888756, ZINC000095627883, ZINC000004096846, ZINC000004098366, and ZINC000049823084) establish interactions with most active site residues through various mechanisms, including hydrogen bonding, hydrophobic interactions, van der Waals interactions, and other means.

Figures & Tables

Discussion