**2.3 Molecular docking analysis**

Molecular modeling was performed as described by Rodrigues et al. [29]. Using Hyperchem v. 8.0.3, the chemical structures of all compounds of interest (ligands) were drawn and their geometric structures were optimized using the MM+ force field. Subsequently, a new geometry optimization was performed based on the AM1 semi-empirical method (Austin Model 1). The optimized structures were subjected to conformational analysis using Spartan software for Windows 10.0. The Monte Carlo computational method with 1000 interactions, 100 optimization cycles, and 10 conformations with the lowest energy level was selected. The dihedral angles were evaluated by rotation according to the standard conditions (default) of the program, in which the number of simultaneous variations was 1–8, acyclic chains were subjected to rotations from 60 to 180°, and the torsion rings, to rotations from 30 to 120°. The conformations with the lowest minimum energies were selected and saved in .sdf format. Receivers (protein target) were obtained from the PDB. Receiver, PDB id, and selected three-dimensional coordinates for docking are described in **Table 1**. Docking simulations were performed in AutoDock 4.2 software. The preparation of receptors and ligands was performed using VEGA ZZ 3.0.1 and MOLEGRO Molecular Viewer 2.5 software. Initially, ligand and receptor structures were saved in .pqbqt format to be used in docking calculations. Then, PyRx 0.9 software was used to assist in the docking steps and the analysis of the results. The "grid maps", which represent the boxes with three-dimensional coordinates determined for each receiver, were calculated with AutoGrid. Each ligand was docked inside its "grid" with the Lamarckian algorithm implemented in the AutoDock software. The genetics-based algorithm ran 12 simulations per ligand with 2,500,000 energy ratings and a maximum number of 54,000 generations. The crossover rate was increased to 0.8, the gene mutation rate was 0.02, and the number of individuals in each population was 200. All other parameters were left with the default AutoDock settings. The results for each calculation were analyzed to obtain the affinity energy of docking score (Edock) in kcal/mol values for each ligand conformation in its respective complex; structure inaccuracies were ignored in the calculations. To verify the number and positions of hydrogen

*Molecular Docking of Phytochemicals against* Streptococcus mutans *Virulence Targets… DOI: http://dx.doi.org/10.5772/intechopen.101506*

bonds and non-covalent interactions between each ligand conformation and the catalytic residues of the receptors, the software PyMOL 1.4 and Molegro Molecular Viewer 2.5 were used.

**Figure 1.** *Selected compounds for molecular docking in* S. mutans *target proteins.*


#### **Table 1.**

*Names of macromolecules (receivers), identifier in the Protein Data Bank (PDB id), and selected threedimensional coordinates for docking.*

### **3. Molecular docking screening results**

Molecular docking is an *in silico* methodology that makes it possible to simulate the orientation and conformations (poses) of a ligand near the active site of a target macromolecule, evaluating intermolecular forces, such as hydrophobicity, Van der Waals forces, hydrogen bonds, interactions electrostatic, and ionic bonds. Moreover, this methodology provides the energy of interaction (docking scores), the types of interaction, and the amino acid residues involved in the formation of the ligandreceptor complex [30].

The scores are used as a reference to rank the most stable poses of the ligand. Therefore, the lower the score value, the stronger and more stable the interaction with the selected target. The role and functioning of each of the nine selected S. mutans target proteins are briefly presented below, along with the presentation of the three best ligands for each of the proteins.

#### **3.1 Adhesins**

#### *3.1.1 Region V of antigen I/II (PDB id: 1JMM)*

The protein-antigen AgI/II is an adhesin present in the cell wall of S. mutans, which recognizes and binds to salivary glycoproteins on the tooth surface, enabling the formation of dental biofilm [31, 32]. Anti-AgI/II antibodies block the adhesion and colonization of S. mutans in the oral cavity [33, 34], justifying the interest in this adhesin in studies aimed at the development of an anticaries therapy [35].

AgI/II adhesin exhibits a functional supramolecular architecture on the cell surface [36], as well as an unusual tertiary structure, where a central variable domain (V-domain) appears like the tip of a formed stem by intertwined and flanked

*Molecular Docking of Phytochemicals against* Streptococcus mutans *Virulence Targets… DOI: http://dx.doi.org/10.5772/intechopen.101506*

regions rich in alanine and proline [37]. The carboxy-terminal domain (C-domain), connected to a small N-terminal domain that attaches to the cell wall through an anchoring region [38]. AgI/II binding sites for DMBT1 agglutinin are located in the V-domain and C-domain [39].

Docking identified as the best ligands for antigen I/II (V-region) PDB id: 1JMM were the compounds: maldivin-3,5-diglucoside (20) (Edock = −160.78 kJ/mol), licorisoflavan C (17) (Edock = −151.50 kJ/mol), and erystagallin (11) (Edock = −139.85 kJ/ mol). Common steric interactions in the complexes formed between Ser818 and Ser697 residues and compounds 17 and 20 were observed. As well as between residue Trp818 and compounds 11 and 17 (**Figure 2**).

#### *3.1.2 Antigen I/II (carboxy-terminal) (PDB id: 3QE5)*

The carboxy-terminal domain of antigen I/II, as well as other proteins in this family, can bind salivary glycoproteins, extracellular matrix molecules, and ligands from other bacteria. This category of proteins is not exclusive to **S. mutans**. Homologous proteins subsist in other *Streptococci* [40].

The I/II antigen is highly conserved and may be associated with M protein in other streptococcal species. The carboxy-terminal region (with 800–1540 amino acid residues) includes proline-rich (P) repeats, conferring hydrophobicity, a transmembrane domain (with 1537–1556 amino acid residues), and an LPXTG motif required for anchorage to the cell wall catalyzed by sortase [32, 41].

The phytochemicals with the most promising linkages with the antigen I/II (carboxy-terminus) PDB id: 3QE5 were: erycristagallin (10) (Edock = −128.98 kJ/ mol), sophoraflavanone G (2) (Edock = −105.77 kJ/mol), and erystagallin (11) (Edock = −105.16 kJ/mol). All compounds had in common hydrogen bonds with the Lys1120 residue and steric interactions with the Thr1118 residue, thus indicating that these amino acids are important for minimizing the binding energies and stabilizing the complexes (**Figure 3**).

#### *3.1.3 Spap (PDB id: 3OPU)*

The Spap protein, also called P1, is a multifunctional adhesin that mediates the sucrose-independent adhesion of bacteria to salivary film glycoproteins on the tooth

#### **Figure 2.**

*Representations of the interactions between the three best ligands (compounds 20, 17, and 11) and the amino acid residues of the antigen I/II (V-region) PDB id: 1JMM. Blue dashed lines represent hydrogen bonds and red dashed lines represent steric interactions.*

#### **Figure 3.**

*Representations of the interactions between the three best ligands (compounds 10, 2, and 11) and the amino acid residues of antigen I/II (carboxy-terminal) PDB id: 3QE5. Blue dashed lines represent hydrogen bonds and red dashed lines represent steric interactions.*

surface. Like other extracellular proteins, this adhesin can produce amyloid, which, in turn, is present in dental biofilms. Thus, this protein directly interferes with the facilitation and adhesion of cariogenic bacteria [21, 42].

The best interactions with Spap PDB id: 3OPU occurred with the compounds: sophoraflavanone G (2) (Edock = −136.98 kJ/mol), erystagallin (11) (Edock = −134.89 kJ/mol), and licorisoflavan (18) (Edock = −129.64 kJ/mol). The common interactions between these ligands and the active site of the protein, which contributed to the low values of the scores of these molecules, are the steric interactions with residues Lys1261 and Pro1210, and hydrogen bonds with residue Asp1208 (**Figure 4**).

#### **3.2 Quorum sensing-associated signaling proteins**
