Molecular Docking of Phytochemicals against Streptococcus mutans Virulence Targets: A Proteomic Insight into Drug Planning

Streptococcus mutans ( S. mutans ) is the most prevalent and most associated with dental caries. Here we aim to identify, through an in silico study, potential bioactive molecules against S. mutans . Twenty-four bioactive molecules with proven action against S. mutans were selected: 1-methoxyficifolinol; 5,7,2 ′ ,4 ′ -tetrahydroxy-8-lavandulylflavanone (sophoraflavanone G); 6,8-diprenylgenistein; apigenin; artocarpesin; artocarpin; darbergioidin; dihydrobiochanin A; dihydrocajanin (5,2 ′ ,4 ′ -trihydroxy-7-methoxyisoflavanone); erycristagallin; Erystagallin; ferreirin; fisetin; kaempferol; licoricidin; licorisoflavan A; licorisoflavan C; licorisoflavan E; luteolin (3 ′ ,4 ′ ,5,7-tetrahydroxyflavone); malvidin-3,5-diglucoside; myricetin; orientanol B; quercetin; and quercitrin. Moreover, we selected nine important target proteins for the virulence of this microorganism to perform as drug targets: antigen I/II (region V) (PDB: 1JMM); Antigen I/II (carbox-terminal region) (PDB: 3QE5); of these assays will allow the incorporation of these phytoconstituents in products for oral use for the control of tooth decay.

Several drugs, currently available for use, have been discovered and improved with the aid of in silico tools, such as molecular dockings, zanamivir [12], imatinib [13], nelfinavir [14], and erdafitinib [15]. With the evolution of bioinformatics, biotechnology, and molecular biology, including the determination of protein structures by using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy, it has become increasingly easier to use in silico tools to predict functioning drugs. Thus, in the last 20 years, more than 60 different molecular docking software were developed by universities and companies [16].
Molecular docking programs have different approaches and their characterization is according to incremental construction approaches, including shape-based algorithms, genetic algorithms, the Monte Carlo method, and systematic search techniques [17][18][19][20].
Despite the evidence of the effectiveness and advantage of using molecular docking for drug discovery, studies in this area are still incipient for oral diseases [5], which justifies the performance of new studies. Streptococcus mutans (S. mutans), a gram-positive, aciduric, and acidogenic bacterium, is the most prevalent in the dental biofilm [21] and the most studied [22]. This microorganism has relevant virulence factors that enable colonization of the tooth surface, including its high capacity to form biofilms, causing the development of carious lesions [23]. The dominant defense systems of S. mutans for biofilm formation and caries development are its ability to adhere to the surface of teeth and produce acids, associated with its resistance to this environment without suffering damage [24]. Thus, preventing the formation of this microbial complex is one of the most targeted strategies for caries control [23].
Additionally, natural products have been a promising source of positive molecules for drug development over the years [25]. Therefore, plants are a promising source of new chemical compounds (phytochemicals) with high biological potential. Phytochemicals are a class of organic compounds synthesized in small amounts from secondary plant metabolism and are related to plant defense, growth, reproduction, and adaptation, among others. Its main classes of compounds are terpenes, alkaloids, and phenolic compounds [26,27].
In consequence, in this chapter, we performed, by molecular docking, a screening of molecules from plants that showed results of in vitro antimicrobial activity against S. mutans, to verify the possibility of interaction and inactivation of virulence factors of this bacteria.

Selection of the ligands
Ligands were selected from a literature search on phytoconstituents or plants with antimicrobial activity, in vitro, against S. mutans. The search was performed in the Pubmed database (http://www.ncbi.nlm.nih.gov/pubmed), using the following terms as keywords: S. mutans, natural products, and anti-cariogenic effects, without language specification or deadline. All articles that addressed the antimicrobial activity of phytoconstituents (isolated molecules) with action on S. mutans, or with activity related to the reduction of cariogenic dental biofilm, were considered relevant. After these filters, 24 articles remained that had defined chemical structures of molecules with an inhibitory effect against S. mutans. The molecules identified and selected for the study in these articles are shown in Figure 1.

Selection of protein targets in S. mutans
The first inclusion criterion was the selection of S. mutans target proteins with high relevance for the virulence of this microorganism [28]. The availability of the crystallographic structures resolved and available in the Protein Data Bank (PDB)  was the second inclusion criterion. The protein targets (receptors), their functions, PDB identifiers, and grid box coordinates are presented in Table 1.

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 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.

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 ligand-receptor 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.

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 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].

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].

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 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].
Quorum-sensing allows bacterial communication, providing benefits during host colonization, defense against competitors, and adaptation to the environment [43,49]. The chemical details of the signaling molecules of this system in S. mutans are known and only UA159sp has been identified as a signal peptide in wild-type S. mutans strain [44].
In a study, conducted by Syvitski et al. [50], peptides in which three or more residues were deleted from the C-terminal region of the signaling peptide UA159sp did not induce genetic competence and inhibited, by competition, the quorum sensing activated by UA159sp. Disruption of the amphipathic α-helix by replacing Phe-7, Phe-11, or Phe-15 residues with a hydrophilic residue resulted in a significant reduction or complete loss of peptide activity. In contrast to peptides truncated at the C-terminal region, these peptides with amino acid substitutions did not compete with UA159sp to activate quorum sensing, suggesting that disruption of the hydrophobic face of the α-helix structure results in a peptide that is not capable of binding to the receptor. Therefore, residues of the C-terminal region of the signaling peptide in the quorum-sensing system of streptococci are extremely important.
Quorum-sensing inhibitor drug design enables the development of more specific treatments for biofilm-dependent infectious diseases [51]. A benefit of using quorum sensing inhibitor drugs is that they are less susceptible to antimicrobial resistance than other antimicrobials, as they exert a lower selective pressure and do not directly kill bacterial cells [52].
Docking with the signaling peptide UA159sp PDB id: 2I2J identified as the best ligands the compounds: erystagallin (11) (Edock = −84.98 kJ/mol), erycristagallin (10) (Edock = −83.99 kJ/mol), and methoxyficifolinol (1) (Edock = −79.76 kJ/mol). In all ligands, the presence of hydrogen bonds with the Ser14 residue and steric interactions with the Ala18 residue is indicative of their importance for the stability of the interaction of these compounds with the active site ( Figure 5).

Signaling peptide TPC3 (PDB id: 2I2H)
TPC3 peptide is a signal peptide synthesized by the mutant strain of S. mutans JH1005 that also can activate the quorum-sensing system.

ATP binding protein ComA (PDB: 3VX4)
Quorum sensing is mediated by a signaling molecule autoinducer [53]. This system in some streptococcal species such as S. mutans is the ComABCDE pathway, in which inducing peptides are processed from the ComC precursor and exported to the extracellular space by ComA and ComB [43,54]. ComA is a bifunctional ATP-binding cassette transporter comprising three domains: an N-terminal peptidase domain (PEP), a transmembrane domain, and a C-terminal nucleotide linker domain [55][56][57]. PEP is a peptidase belonging to the cysteine protease family [55,[58][59][60].
Docking with the ATP binding protein ComA PDB id: 3VX4 identified as the best ligands the compounds: licorisoflavan A (16) (Edock = −-132.56 kJ/ mol), licoricidin (15) (Edock = −128.75 kJ/mol), and methoxyficifolinol (1) (Edock = −127.50 kJ/mol). When observing the interactions of the best ligands in the formed complexes, it was observed that hydrogen bonds with residues Thr568  and Ser563 and steric interactions with Lys567 are common, indicating that these interactions contributed to the reduction of the interaction energy and stabilization of the complexes (Figure 7).

Glucanosyucrase (PDB id: 3AIC)
Glucansucrases or glycosyltransferases (GTFs) are extracellular enzymes, produced by various bacteria, including S. mutans, that cleave sucrose into glucose and fructose and build sticky biofilm chains. The growth of the glucan chain was associated with adherence of one bacteria to another and the dental surface. Furthermore, modulate the diffusion of substances through the biofilm, which could occasionally serve as an extracellular energy reserve [61].
The glucanosucrase in S. mutans allows the metabolism of sucrose into lactic acid, which reduces the pH around the tooth, facilitating the dissolution of calcium phosphate from tooth enamel, which induces tooth decay [62]. These characteristics make the S. mutans glucanosucrase as one of the main and most studied targets for the development of new agents useful in the prevention of dental caries.
The docking study conducted out by Kim et al. [63] between rubusoside and S. mutans glucanosucrase (PDB id: 3AIC), identified residues Leu 433, Leu434, Ala478, Asp480, Glu515, Trp517, and Tyr916 as the main ones involved in the stabilization of the complex, and validated these residues as important anchoring sites for potential inhibitors of this enzyme.
Bhagavathy, Mahendiran, and Kanchana [64], performed molecular docking between seven phytochemical isolates of Psidium guajava and S. mutans glucanosucrase (PDB id: 3AIB) and demonstrated that the main residues involved in the formation of the complexes were Thr426, Ile427, Gln553, and Tyr978. These residues diverged from those identified in this study.
Opposing, Islam et al. [65] performed a molecular docking study between epigallocatechin gallate (EGCG) and the same S. mutans enzyme, glucanosucrase  (PDB id: 3AIB). The results showed that the main interactions that stabilize the complex of the ligand (EGCG) with the enzyme occurred between the amino acid residues Glu515 and Trp517, which were the same residues identified in our work, reinforcing the importance of these residues for the stabilization of the complex.

Dextranase (PDB id: 3VMO)
S. mutans dextranase is an enzyme that hydrolyzes the α-1,6 bonds of dextran and produces isomalto-oligosaccharides of different sizes for metabolic use [66,67]. This protein is composed of 850 aa residues with a molecular mass of 94.5 kDa, but it has multiple native and recombinant forms [68,69]. According to the sequencing of several enzymes in this family, dextranases are divided into five regions: a signal peptide sequence (N-terminal with 24 aa), a variable N-terminal region (Ser25-Asn99), a conserved region (Gln100-Ala615), a glucan binding site (Leu616-Ile732), and a C-terminal variable region (Asn733-Asp850) [70,71].
Some biochemical studies, based on the comparison of amino acid sequences with other glycosyltransferases, revealed that the Asp385 residue is essential for the catalytic reaction [72]. Besides, it was observed that Asp270 from cycloisomalto oligosaccharide glucanotransferases from Bacillus circulans T3040 [73] and Asp243 from endodextranase from Thermotoga lettingae TMO [74], corresponding to Asp385 from dextranase from Streptococcus mutans, were recognized as S. mutans residues catalytic. Molecular docking performed with the dextranase PDB id: 3VMO identified as the best ligands: licorisoflavan A (16) (Edock = −138.02 kJ/mol), malvidin-3,5-diglucoside (20) (Edock = −136.94 kJ μg/mol), and licoricidin (15) (Edock = −129.73 kJ/mol). Compounds 15 and 16 showed steric interactions in common with residues Tyr257 and Ala559 and showed steric interactions and hydrogen bonds with the key residue Asp385 which has already been identified as essential for catalytic reaction. Diglucoside 20, on the other hand, had a lower energy conformation distinct from compounds 15 and 16 and interacted with other amino acid residues in the active site of the enzyme (Figure 9).

Hemolysin (PDB id: 2RK5)
Hemolysins are exotoxins capable of promoting erythrocyte lysis. They are toxins produced by some species of streptococci [75] and contribute to the virulence process of S. mutans [76]. In S. mutans, alpha-and gamma-hemolytic strains are described [77], as well as beta-hemolytic [78].
Docking with hemolysin PDB id: 3VMO identified as the best ligands the compounds: erycristagallin (10) (Edock = −112.64 kJ/mol), erystagallin (11) (Edock = −104.10 kJ/mol), and methoxyficifolinol (1) (Edock = −100.63 kJ/mol). The steric interactions and hydrogen bonds with Asn21, Asn24, and Asp30 residues were common for compounds 10 and 11, and seem to be important for the stabilization of the complexes. Compound 1, despite belonging to the same chemical class as compounds 10 and 11, showed a more stable conformation in another position of the active site, consequently, is stabilized by interactions with different amino acid residues, but which contributed less to the stabilization of the complex (Figure 10).
Based on these findings, these selected compounds should have theirs in vitro and in vivo activities evaluated, to validate the efficiency of these compounds in inhibiting the virulence factors of planktonic S. mutans and in biofilms. The positive results in these tests will allow the incorporation of these phytoconstituents in toothpaste, mouthwashes, among others, and could be an effective alternative for the control of tooth decay.