In Silico Selection and In Vitro Evaluation of New Molecules That Inhibit the Adhesion of Streptococcus mutants through Antigen I/II

Abstract

Streptococcus mutans is the main early colonizing cariogenic bacteria because it recognizes salivary pellicle receptors. The Antigen I/II (Ag I/II) of S. mutans is among the most important adhesins in this process, and is involved in the adhesion to the tooth surface and the bacterial co-aggregation in the early stage of biofilm formation. However, this protein has not been used as a target in a virtual strategy search for inhibitors. Based on the predicted binding affinities, drug-like properties and toxicity, molecules were selected and evaluated for their ability to reduce S. mutans adhesion. A virtual screening of 883,551 molecules was conducted; cytotoxicity analysis on fibroblast cells, S. mutans adhesion studies, scanning electron microscopy analysis for bacterial integrity and molecular dynamics simulation were also performed. We found three molecules ZINC19835187 (ZI-187), ZINC19924939 (ZI-939) and ZINC19924906 (ZI-906) without cytotoxic activity, which inhibited about 90% the adhesion of S. mutans to polystyrene microplates. Molecular dynamic simulation by 300 nanoseconds showed stability of the interaction between ZI-187 and Ag I/II (PDB: 3IPK). This work provides new molecules that targets Ag I/II and have the capacity to inhibit in vitro the S. mutans adhesion on polystyrene microplates.

Keywords: Streptococcus mutans; adhesion proteins; antigen I/II; dental caries; molecular dynamics; structure-based virtual screening.

Figure 1

Crystals structures from the S. mutans Ag I/II protein (A). Model of S. mutans Ag I/II structure and predicted binding with human SAG (taken from [21]). (B) Crystal structure of A3VP1 protein from Ag I/II (3IPK) [21]. (C). Crystal structure C-domain protein from the Ag I/II (3QE5) [20]. (D). Schematic representation of the Ag I/II protein sequence (1566 aa) (Taken from [20]) and the description of the aa residues that constitute the crystals structures of the protein regions: 1—region V (PDB: 1JMM) [44]; 2—C-terminal domain [45]; 3—A3VP1 region (PDB: 3IPK) [21] and 4—the C-terminal region (PDB: 3QE5) [20].

Figure 2

Molecules structures selected by in silico analysis, which have affinity for Ag I/II and inhibitory potential of S. mutans adhesion. (A)-ZI-187; (B)-ZI-939; (C)-ZI-906; (D)-ZI-498.

Figure 3

Comparison of the interactions of three molecules with A3VP1 region (PDB: 3IPK) and C-terminal region (PDB: 3QE5) from Ag I/II of S. mutans. Hydrophobicity in surface form is shown for the A3VP1 region interacting with (A) ZI-187, (E) ZI-906 and (I) ZI-939 and C-terminal region interacting with (C) ZI-187, (G) ZI-906 and (K) ZI-939, where the color scale corresponds to that blue is very hydrophilic and red is very hydrophobic. Molecular interactions (to 3 Angstrom radius from the molecules) between residues from A3VP1 region with (B) ZI-187, (F) ZI-906 and (J) ZI-939 and from C-terminal region with (D) ZI-187, (H) ZI-906 and (L) ZI-939.

Figure 3

Comparison of the interactions of three molecules with A3VP1 region (PDB: 3IPK) and C-terminal region (PDB: 3QE5) from Ag I/II of S. mutans. Hydrophobicity in surface form is shown for the A3VP1 region interacting with (A) ZI-187, (E) ZI-906 and (I) ZI-939 and C-terminal region interacting with (C) ZI-187, (G) ZI-906 and (K) ZI-939, where the color scale corresponds to that blue is very hydrophilic and red is very hydrophobic. Molecular interactions (to 3 Angstrom radius from the molecules) between residues from A3VP1 region with (B) ZI-187, (F) ZI-906 and (J) ZI-939 and from C-terminal region with (D) ZI-187, (H) ZI-906 and (L) ZI-939.

Figure 4

Cytotoxicity assay on periodontal ligament fibroblast cells (PLF). (A). Evaluation of compounds solvent cytotoxicity (DMSO 1%, 0.1% and 0.01%). NT: Not-treated. (B). Cytotoxicity evaluation of compounds (100 µM) ZI-187 (p = 0.7372), ZI-939 (p = 0.8) and ZI-906 (p = 0.7964) on PLF, by laminar flow cytometry analysis with Propidium Iodide.

Figure 5

Surface adhesion of S. mutans-LT11 to a polystyrene microwell plate treated with molecules (A) ZI-187, (B) ZI-906 and (C) ZI-939. The asterisks represent the level of significance ** (p ≤ 0.01), *** (p ≤ 0.001) and **** (p ≤ 0.0001). All experiments were performed in triplicate and reproduced three separate times (the geometric figures above each column chart indicates each data).

Figure 6

Scanning electron microscopy of S. mutans-LT11 surface adhesion to a polystyrene microwell plate. Without treatment at 7000× (A) and 19,000× (B); and treated with 100 µM of molecule ZI-187, at of 4000× (C) and 11,000× (D).

Figure 7

Molecular dynamics simulations analysis. Root Mean Square Deviation (RMSD) (A), Root Mean Square Fluctuation (RMSF) (B), hydrogen bonds (C) and Radius of gyration (D) calculated for ZI-187/3IPK complex.