2.3.2. Molecular Docking Study with α-Glucosidase

The crystal structure of isomaltase from *Saccharomyces cerevisiae* (PDB ID: 3A4A) was used for the docking study. The 3D crystalline structures of α-glucosidase from *Saccharomyces cerevisiae* (maltase, EC 3.2.1.20) are unavailable in the PDB. However, there are crystalline X-ray structures of isomaltase or α-methylglucosidase have been deposited in the PDB. Previous studies have used isomaltase with a PDB ID 3AJ7 or 3A4A for molecular docking. This structure has a high-resolution X-ray structure, high sequence identity (72.51%) and sequence similarity score (0.54) with the *Saccharomyces cerevisiae* α-glucosidase MAL32 (UniProt entry P38158) [42,43]. Additionally, the authors identified several differences between residues lining the binding pockets of α-glucosidase and isomaltase, such as Phe157/Tyr158, Asp307/Glu204, Asp408/Glu411, Thr215/Val216, Ala278/Gln279, Val303/Thr306 or Ala178/Cys179, respectively. Most of these differences involve very similar residues. Hence we decided to use the crystal structure of isomaltase from *Saccharomyces cerevisiae* (PDB ID: 3A4A) for the molecular docking study with α-glucosidase.

The sequence alignment between α-glucosidase from bakers yeast (GI number 411229) and isomaltase (PDB ID: 3A4A) from *S. cerevisiae* have the structure identity and similarity of 73% and 85%, respectively. The key interactions of acarbose with *S. cerevisiae* α-glucosidase and *S. cerevisiae* isomaltase are quite similar.

The binding energy, interacting residues including H-bond interacting residues and Van der Waals interacting residues, along with the number of H-bonds with the crystal structure of isomaltase from *S. cerevisiae* for compounds **2–7,** are presented in Table S1. Karaviloside VI (**2**) had a binding energy of −10.54 kcal/mol and occupied the active region of isomaltase by interacting with sixteen amino acid residues (Table S1). In the conformation of isomaltase–karaviloside VI complex, the compound was able to establish four hydrogen bonds, including Glu332, Ala281, and Leu313. These hydrogen bonds overtly strengthened the interaction between karaviloside VI and isomaltase. The above interactions resulted in an inhibition constant of 12.48 nM. The 3D schematic interaction of karaviloside VI (**2**) is shown in Figure 6A. Indeed, the docking analysis of some cucurbitane triterpenes from Chinese bitter melon was carried out in our previous study [18,19] showed that compounds were surrounded by residues Glu277, His351, and Asp352, which are part of the catalytic residues of isomaltase.

**Figure 6.** The 3D protein-ligand interactions for (**A**) karaviloside VI (**2**), (**B**) karaviloside VIII (**3**), (**C**) momordicoside L (**4**), (**D**) momordicoside A (**5**), (**E**) charantoside XV (**6**), and (**F**) kuguaglycoside C (**7**) in the binding sites of isomaltase. Black dotted lines indicate hydrogen bonds between compounds and amino acid residues. Ligands in the active sites are denoted in green color. Active site residues are shown in white color.

The 3D schematic interaction of karaviloside VIII (**3**) is shown in Figure 6B. Karaviloside VIII was oriented toward the core of the binding pocket and interacted closely with the important seven amino acid residues in the active site (Table S1). According to the Autodock 4.2 simulation results, the isomaltase–karaviloside VIII inhibitor complex

showed −10.56 kcal/mol binding energy. Karaviloside VIII made six hydrogen bonds with Glu277 (two bonds), Gln279 (two bonds), Lys156, and Leu313. Moreover, two hydrogen bonds with Glu277 with a bond length of 1.4 and 1.5 Å, which is a key interaction to inhibit enzyme to a greater extent.

Momordicoside L (**4**) was surrounded by fourteen amino acid residues at the catalytic site of isomaltase. The 2D schematic and 3D interactions of momordicoside L and isomaltase are shown in Figure 6C. Momordicoside L generated the best docking pose with a minimum binding energy of −8.28 kcal/mol, and the inhibition constant of 852.19 nM. The ligand was surrounded by catalytic residue Asp307, and the interaction was likely crucial for inhibition of isomaltase. Eight conventional hydrogen bonds were observed between compound **4** and the isomaltase catalytic site residues, including Pro320, Pro312, His280, Ser304, Asn302, Ala281, and Glu232.

Similarly, momordicoside A (**5**) was bound to isomaltase's active site with minimum binding energy −12.48 kcal/mol. Figure 6D clearly shows how momordicoside A interacted with twenty-one crucial amino acid residues (Table S1). Compound **5** was able to establish six hydrogen bonds with Glu411, Ser314, and Glu332. The binding energy and inhibition constant for compound **5** are 706.7 pM and 12.48 kcal/mol, respectively.

The binding mode of charantoside XV (**6**) in the active site of isomaltase was shown in Figure 6E. The ligand was stabilized in an enzyme's active site by interacting with nine amino acid residues (Table S1). The compound was able to form seven hydrogen bonds with Pro312, Ser240 (two hydrogen bonds), Asp242 (three hydrogen bonds), and Asp307. Finally, the binding energy was −10.37 kcal/mol, and the inhibition constant was 25.02 nM.

The refined docking of kuguaglycoside C (**7**) with isomaltase generated the best pose with a minimum binding energy of −8.23 kcal/mol. The 3D schematic in Figure 6F shows that kuguaglycoside C established several hydrogen bonds within the isomaltase enzymatic pocket and interacted with sixteen amino acid residues (Table S1). Three conventional hydrogen bonds were established between kuguaglycoside C and the active pocket of isomaltase, Ser282, Glu332, and Asp242. The 2D docking images of all compounds are shown in the Supporting Information Figures S22 and S23.
