Identification of Potential Glucosyltransferase Inhibitors from Cinnamic Acid Derivatives Using Molecular Docking Analysis: A Bioinformatics Study

Background: Dental caries is one of the most common oral chronic diseases. Streptococcus mutans is the main pathogenic bacteria playing a role in degrading the mineral texture of the teeth. Glucosyltransferase (GTFase) of S. mutans is responsible for producing glucan, which is the main exopolysaccharide found in the cariogenic biofilms. Further, previous studies have reported that cinnamic acid diminished biofilm formation of S. mutans. Therefore, we hypothesized that cinnamic acid and its derivatives might act as GTFase inhibitors. Methods: The binding affinity of a total of 12 plant-based compounds including cinnamic acid and its 11 derivatives to the GTFase active site were examined by utilizing the AutoDock tool. The possible interactions between top-ranked cinnamic acid derivatives and the residues within the GTFase catalytic site were also taken into consideration. Results: Five of the cinnamic acid derivatives including rosmarinic acid (RA), cynarine, chlorogenic acid (CGA), caffeic acid 3-glucoside, and N-p-coumaroyltyramine demonstrated inhibitory effects on GTFase at nanomolar concentration. Stabilizing interactions such as π–π stack pairing and pi-charge interactions were detected between top-ranked GTFase inhibitors and residues within the enzyme active site. Conclusions: The present study suggests that RA, cynarine, CGA, caffeic acid 3-glucoside, and N-pcoumaroyltyramine might have protective effects on dental caries, and therefore, may be considered as antitooth caries compounds.

compounds in this study are presented in Table 1.

Structural Preparation
The three-dimensional structure of GTFase was collected from the Protein Data Bank (PDB ID: 3AIE) with a criteria of X-ray resolution = 2.10 Å, which is available at https:// www.rcsb.org. A total of eight subunits were included within the 3AIE file. Chain A, with a total of 844 residues, was chosen for computational dockings. It should be noted that the molecular energy of GTFase was optimized prior to molecular docking analysis by using the Swiss-PdbViewer version 4.1.0, which is available at http://www. expasy.org/spdbv (20).

Molecular Docking and Post-docking Analyses
A windows-based computer (with the criteria of installed memory: 32 GB, processor: Intel Core i7, and system type: 64-bit) was used for in silico simulations. The AutoDock tool imposes limited flexibility on the protein. It uses an accurate free energy force field based on a Lamarckian genetic algorithm, leading to a rapid ligand conformation prediction within the binding site and estimating the Gibbs free binding energy from the following algorithm (28)(29)(30): ∆G binding = Intermolecular Energy + Total Internal Energy + Torsional Free Energy -Unbound System's Energy The active site of GTFase was considered as a receptor for the ligands. The grid box settings in the AutoDock tool (included spacing, 0.375 Å; X-dimension, 58; Y-dimension, 74; Z-dimension, 52; X-center, 190.161; Y-center, 46.104; and Z-center, 191.584. A total of 14 amino acids were identified to be located within the GTFase catalytic site from the Ito and colleagues' study (22), including Tyr430, Leu433, Leu434, Arg475, Asp477, Asn481, Glu515, Trp517, Arg540, His587, Asp588, Asp909, Tyr916, and Gln960. It is worth mentioning that a total of 50 runs were set for each ligand.
For each ligand, the lowest ∆G binding within the largest cluster of results was considered for post-docking analyses including protein-ligand complex imaging and interaction mode study. The BIOVIA Discovery Studio Visualizer version 19.1.0.18287 (https://discover.3ds.com/discoverystudio-visualizer-download) was used for visualizing the two-dimensional images of interactions between topranked inhibitors and residues within the GTFase active site as well as demonstrating the three-dimensional docked pose of the top-ranked CA derivatives.

Binding Affinity and Interaction Modes Between GTFase and Small Molecules
The Gibbs free energy changes of interactions between GTFase and the studied compounds were estimated using the AutoDock tool to identify potential GTFase inhibitors for combating dental caries. According to the results, a total of five cinnamic acid derivatives including RA, cynarine, CGA, caffeic acid 3-glucoside, and N-pcoumaroyltyramine were predicted to bind to the GTFase catalytic site at the nanomolar scale (nM); therefore, these cinnamic acid derivatives were considered as topranked GTFase inhibitors in the present study. It was also estimated that CAPE, o-Coumaric acid, and CA could inhibit the GTFase activity at the micromolar scale (uM). Moreover, ferulic acid, sinapinic acid, p-coumaric acid, and cinnamic acid revealed a dismal affinity to the GTFase active site, based on the inhibition constant values (Ki) calculated for these molecules that were predicted to be at the millimolar (mM) scale. In addition, acarbose demonstrated the highest binding affinity to the GTFase active site among control inhibitors followed by maltose and WP1066. Moreover, the ΔG binding of GTFase with RA, cynarine, and CGA was predicted to be more negative than that of WP1066, suggesting that these three compounds can attach to the GTFase catalytic site more tightly than the WP1066 (Figure 2).
The estimated ∆G binding and Ki values for all tested compounds in this study are presented in Table 2. The details of energies among top-ranked cinnamic acid derivatives and GTFase catalytic site are illustrated in Table 3. The interaction modes between top-ranked cinnamic derivatives   and the residues within the GTFase active sites were also taken into consideration (Table 4). Accordingly, cynarine and N-p-coumaroyltyramine demonstrated the greatest number of hydrogen and hydrophobic interactions, respectively. It should be noticed that the H-bonds with the criteria of distance > 5 Å were not considered significant, and consequently, were removed from Table 4. Figure 3 illustrates the two-dimensional images of these interactions as well as the three-dimensional docked pose of topranked ligands. Figure 4 demonstrates all interactions between top-ranked cinnamic acid derivatives and their corresponding amino acids in a unique network achieved by Cytoscape version 3.8.0 software (https://cytoscape.org/ download.html) (31).

Discussion
Tooth decay is one of the most common chronic diseases worldwide (32). It is a multifactorial disorder in which matrix metalloproteinases and S. mutans are most responsible for degrading the organic and mineral texture of the teeth, respectively (33,34). GTFase of S. mutans plays an essential role in biofilm formation, leading to more bacterial cohesion, acid production, and dental caries (11)(12)(13)35). To discover potential GTFase inhibitors, the binding affinity of several plant-based compounds including cinnamic acid and its 11 derivatives with GTFase catalytic sites were estimated using a molecular docking approach.
The obtained results predicted that RA, cynarine, CGA, caffeic acid 3-glucoside, and N-p-Coumaroyltyramine could potentially inhibit the GTFase active site at the nanomolar scale. In addition, it was found that three of these compounds (i.e., RA, cynarine, and CGA) were more tightly bonded to the enzyme compared with WP1066 as one of the standard inhibitors of the enzyme. CA is a water-soluble metabolite that can be synthesized in herbs with several beneficial properties such as antioxidant, antiviral, antibacterial, antitumorigenic, as well as liver and cardiovascular protective effects (36). Sorgi et al (37) conducted a study to examine CA's antibacterial and anti-inflammatory properties in macrophage response against S. mutans. The authors demonstrated that S. mutans displayed an antibacterial effect at the half-maximal inhibitory concentration (IC 50 ) = 2.938 mM without illustrating cytotoxicity. Moreover, CA led to downregulation of nitrite, tumor necrosis factor alpha, and prostaglandin E 2 through the nuclear factor kappa B dependent pathway, demonstrating its anti-inflammatory effects within the macrophages. Furthermore, Nakamura et al (38) reported that CA solution significantly increased the antibacterial effect of 385 nM LED irradiation against cariogenic S. mutans biofilms. According to the present  Caffeic acid 3-glucoside demonstrated three hydrogens one hydrophobic and one electrostatic interaction with the Asp477, Glu515, and Asp588 inside the GTFase active site. A pi-charge was detected between caffeic acid 3-glucoside and Asp588 (5.86 Å). It is worth mentioning that the π-π stack pairing, pi-charge, and salt bridges are the most stabilizing connections among ligands and proteins (33). RA is a well-known antioxidant compound that exhibits antipathogenic activity in plants. It is an ester of CA and (R)-(+)-3-(3, 4-dihydroxy phenyl) lactic acid originating from L-phenylalanine and L-tyrosine, respectively. Many other beneficial properties have also been reported for RA including antinociceptive and neuroprotective effects. Zdarilová et al (45) carried out a study to examine the effects of Prunella vulgaris L. extract (PVE) and RA, the main compound of PVE, on lipopolysaccharide -induced inflammation and oxidative impairment in human gingival fibroblasts. They reported that PVE and RA led to reduced reactive oxygen species production, resulting in down-regulation of interleukin 1b, interleukin 6, tumor necrosis factor-a, and inducible nitric oxide synthase. The authors demonstrated that PVE and RA could significantly reduce lipopolysaccharide-induced damages in gingival fibroblasts due to their anti-inflammatory properties. Therefore, they may be used for therapeutic purposes in periodontal diseases. Further, previous studies have reported a link between inflammatory periodontal diseases and dental plaque (46).
According to the present results, RA demonstrated the highest binding affinity to GTFase active site among 12 cinnamic acid derivatives. It was predicted that RA could attach to the GTFase catalytic site at the nanomolar concentration (Ki = 212.34 nM) with a salient ∆G binding of −9.10 kcal/mol. It revealed three hydrogen, two electrostatic, and one miscellaneous interactions with the  Possible GTFase Inhibition by Cinnamic Acid Derivatives Asp477, Glu515, Tyr916, and Asp909 within the GTFase active site. Further, the interactions among Glu515, Asp909, and SA were of pi-charge type. CGA is an ester of CA and quinic acid with antioxidant activity (47,48) which is mainly found in coffee, apples, berries, pears, and aubergines (49). Previous studies have shown that coffee has exhibited anti GTFase activity in S. mutans, leading to dental caries prevention (11,50). Moreover, Lin et al (51) triggered a study to examine the effect of CGA on tooth decay in rats. The authors investigated the antibacterial properties of CGA on S. mutans ATCC 10449 and S. sobrinus OMZ65.The obtained results revealed that the MIC and MBC of S. mutans were 2.5 and 7.5 mg/mL, respectively. Therefore, the authors suggested that CGA may be considered as a potential anti tooth caries compound by inhibiting the growth of S. mutans. Hu et al (52) found that CGA elevated the osteogenic differentiation of human dental pulp stem cells through the Wnt signaling pathway. The authors suggested that CGA may be useful for alveolar bone damage repairmen in patients with periodontal disease. According to the results of the present study, CGA formed two hydrogen, one hydrophobic, and one pi-charged interactions with Asn481, Trp517, and Asp909 within the GTFase active site. It was also estimated that CGA can attach to the GTFase catalytic domain at the nanomolar concentration (Ki = 419.70 nM) with a ∆G binding of -8.70 kcal/mol. N-p-coumaroyltyramine is a phenolic compound primarily found in Tribulus terrestris that has demonstrated several pharmaceutical properties such as anti-cariogenic effect against S. mutans. According to previous studies, T. terrestris have significantly reduced the growth, adhesion, acid production, as well as synthesis of glucan within the S. mutans (53,54). According to the results of the present study, N-p-Coumaroyltyramine could block the GTFase activity at the nanomolar scale (Ki = 864.04 nM) with a ∆G binding = -8.27 kcal/mol, suggesting the potential anti-tooth decay property of the compound. N-p-Coumaroyltyramine demonstrated four hydrogen and three hydrophobic interactions with Ala478, Asn481, His587, Gln592, Phe907, and Tyr916 inside the GTFase catalytic site.
Cynarine is a polar component mainly found in the roots of Echinacea angustifolia (55). It revealed a considerable binding affinity to the GTFase catalytic site (∆G binding = −8.97 kcal/mol) and was found to inhibit the enzyme activity at the nanomolar scale (Ki = 265.18 nM). Cynarine displayed five hydrogen and two hydrophobic interactions with the Asn481, Glu509, Trp517, Ser589, Asp593, Asp909, and Tyr916 within the GTFase active site. It should be noted that the electrostatic between Trp517 and cynarine is of pi-pi stack pairing type.
Previous studies have demonstrated that acarbose and maltose are potent GTFase inhibitors (22). Moreover, Tsurumaki et al (23) reported that WP1066 (PubChem ID: 11210478), a well-known JAK/STAT3 signaling pathway inhibitor, revealed inhibitory effects on ceramide GTFase. Therefore, these three compounds were considered as control inhibitors of GTFase in this study. Acarbose showed a salient binding affinity to the GTFase active site with ∆G binding and Ki values of -13.45 kcal/mol and 138.56 picomolar (pM), respectively. It demonstrated eight hydrogen bonds with the Gly428, Gly429, Asp477, Glu515, Trp517, Ser518, Asn481, and Asp909 residues within the GTFase catalytic site. In addition, maltose revealed a high binding affinity to the GTFase active site. The ∆G binding and Ki values for this compound were calculated to be −10.94 kcal/mol and 9.53 nM, respectively. It illustrated four hydrogen interactions with the Asn481 and Gln592 residues within the GTFase active site. Further, WP1066 formed one hydrogen, four hydrophobic, and two pi-charge interactions with the Leu433, Ala478, Glu515, Trp517, His587, Asp588, and Asp509 within the GTFase active site. The ∆G binding and Ki values of WP1066 regarding the enzyme were calculated to be −8.58 kcal/mol and 511.17 nM, respectively.

Conclusions
The present study suggests that RA, cynarine, CGA, caffeic acid 3-glucoside, and N-p-coumaroyltyramine potentially have inhibitory effects on GTFase of S. mutans at nanomolar concentration. Therefore, these compounds may be helpful for preventing dental caries; however, these findings should be confirmed by wet-lab techniques.