Identification of Potential Anti-tooth-decay Compounds From Organic Cinnamic Acid Derivatives by Inhibiting Matrix Metalloproteinase-8: An In Silico Study

Background: Matrix metalloproteinase-8 (MMP-8) is the most abundant member of the MMP family in human dentin. It takes a part in the normal physiology of tissue remodeling and wound healing, while the overexpression/hyperactivity of this protein leads to several oral disorders, including dental caries and peri-implant inflammation/diseases, and therefore, MMP-8 inhibition may have therapeutic effects. Accordingly, the current study aimed to identify potential MMP-8 inhibitors from cinnamic acid derivatives. Methods: The binding affinity of cinnamic acid and its several derivatives to the MMP-8 active site were estimated using the AutoDock 4.0 software. The pharmacokinetics, toxicity, and bioavailability of top-ranked MMP-8 inhibitors were also predicted by utilizing bioinformatics web tools. Results: Five of the studied components, including chlorogenic acid (CGA), caffeic acid 3-glucoside, rosmarinic acid, N-p-Coumaroyltyramine, and caffeic acid phenethyl ester (CAPE) demonstrated a salient affinity of binding to the MMP-8 catalytic site (∆G binding < -10 kcal/mol). It was estimated that these compounds can inhibit the MMP-8 at the nanomolar concentration, and therefore, were considered as top-ranked MMP-8 inhibitors. Finally, none of the top-ranked components revealed a considerable side effect and thus were found to be suitable for oral use. Conclusions: The results of the present study suggested that CGA, caffeic acid 3-glucoside, rosmarinic acid, N-p-coumaroyltyramine, and CAPE might have protective effects on tooth decay and peri-implant inflammation/diseases.

inflammatory, anticancer, and antibacterial properties (12)(13)(14). Additionally, cinnamic acid can result in several derivatives with many beneficial effects, including antiinflammatory, antimicrobial (15), antidiabetic (16), and anticancer (17) activities. Several previous studies (18)(19)(20) have experimentally confirmed the anti-bacterial effects of cinnamic acid and its several derivatives on Streptococcus mutans and Porphyromonas gingivalis. S. mutans and P. gingivalis are well known as the main pathogens that are responsible for the initiation/progression of dental caries and periodontitis, respectively (20)(21)(22). Therefore, the biological efficacy of cinnamic acid and its derivatives have been considered for scientists regarding designing/ discovering drug candidates for therapeutic aims in various disorders (23). In the present study, it was suggested that cinnamic acid and its derivatives may be effective compounds in the inhibition of MMP-8. Thus, this study was designed based on molecular docking simulations to examine the binding affinity of cinnamic acid and its several derivatives to the catalytic site of the MMP-8.

Structural Preparation and Molecular Docking
The structure of the MMP-8 and the ligands tested in the current study, including cinnamic acid and a total of 11 cinnamic acid derivatives, were downloaded from the Structural Bioinformatics database (https://www.rcsb. org) and the PubChem database (https://pubchem.ncbi. nlm.nih.gov), respectively (24,25). The Protein Data Bank file with the ID of 4QKZ contained the three-dimensional structure of MMP-8, as well as the inhibitor of the MMP-8 (named QZK) in the Pochetti et al study with the criteria of X-ray resolution of 1.2 Å (https://www.rcsb.org/ structure/4QKZ). Energy optimization was applied before molecular docking simulations for MMP-8 and all ligands. All docking operations were performed by utilizing the AutoDock software, version 4.0 (http://autodock.scripps. edu) (26). The AutoDock estimates the binding energy (∆G binding ) between the ligand and the receptor using the Lamarckian genetic algorithm. The catalytic site of the MMP-8 was considered a docking pocket. The details of energy optimization, grid box options, and the residues identified within the catalytic domain of the MMP-8 are reported in our previous study (27).
As shown in Figure 1, cinnamic acid is an organic aromatic carboxylic acid (11) with several pharmaceutical characteristics, including antioxidant, antimicrobial (13), anti-inflammatory, antidiabetic (28), and anticancer effects (14). Although this acid could be synthesized by the enzymatic deamination of phenylalanine (29), it is naturally produced in herbs (30). Several derivatives of cinnamic acid are achieved by the modification of the benzene ring and the acrylic acid group (12,23,31). In this study, several features were considered for ligand selection from cinnamic acid derivatives. To this end, being a herb was the main character because of its low side effect and high availability (32). In addition, demonstrating antibacterial effects against tooth caries-related bacteria in previous studies was considered as another important feature of the components.

Drug-likeness Study
The Rule of Five (RO5), which has been presented by Lipinski et al (33), was considered to predict the druglikeness of the tested compounds in the present study using the PubChem database. According to the RO5, the orally administered drugs must confirm at least three of the incoming physical/chemical properties (Mass ≤ 500 g/ mol, Log of the partition coefficient between octanol and water (LogP) ≤ 5, number of accepting H-bonds ≤ 10, and number of the H-bond donor ≤ 5).

Absorption, Distribution, Metabolism, Excretion, and Toxicity
The absorption, distribution, metabolism, excretion (ADME), in addition to the toxicity (ADMET) of the top-ranked inhibitors, were taken into consideration by applying SwissADME (http://www.swissadme.ch/) and the PreADMET (https://preadmet.bmdrc.kr/) webservers. The carcinogenicity of the compounds in rats and mice and the possible inhibitory effect of the components on the human ether-a-go-go-related gene channel of the heart were predicted to evaluate the toxicity of the ligands. Several pharmacokinetic characteristics of the components were referred to the ADME, including the gastrointestinal absorption, blood-brain barrier permeability, possible inhibition of the cytochrome P-450, and possible substrate for the P-glycoprotein. SwissADME applies several vigorous algorithms such as support vector machine, the Ward method, and a reciprocal nearest neighbor algorithm to achieve more reliable results (34).

Affinity of Binding Between the MMP-8 and Small Molecules
Among 12 ligands tested in the present study, a total of five compounds revealed a salient binding affinity to the MMP-8 catalytic site with the criteria of ∆G binding ≤ -10 kcal/mol, including chlorogenic acid (CGA), caffeic acid 3-glucoside, rosmarinic acid, N-p-coumaroyltyramine, and caffeic acid phenethyl ester (CAPE). Therefore, these cinnamic acid derivatives were considered as top-ranked

MMP-8 Inhibition by Cinnamic Acid Derivatives
MMP-8 inhibitors. The inhibition constant (Ki) value for these components was predicted to be at the nanomolar (nM) concentration. According to our previous research (27), the binding affinity and the Ki value for the control component (QZK) were estimated to be -9.45 kcal/mol and 118.80 nM. Hence, the results of the molecular docking analysis represented that the affinity of binding between the top-ranked cinnamic acid derivatives, as well as the cynarin, and the MMP-8 catalytic domain is more than that of QZK. Table 1 presents the ∆G binding and the Ki value of all components evaluated in this study. Figure 2 illustrates the binding affinity between cinnamic acid and its derivatives, the control inhibitor, and the MMP-8 catalytic site. For post-docking analysis, the interaction modes between top-ranked MM-P8 inhibitors and the residues within the catalytic site of the MMP-8 were screened by utilizing the BIOVIA Discovery Studio Visualizer 19.1.0.18287 (https:// discover.3ds.com/discovery-studio-visualizer-download). Table 2 and Figure 3 demonstrate these interactions as a table and figure, respectively. Figure 4 depicts all the interactions between top-rank cinnamic acid derivatives and their corresponding residues in a unique graph by the Cytoscape software (https://cytoscape.org/download. html) (35). Figure 5 illustrates the number of interactions calculated for each top-ranked MMP-8 inhibitor called a degree.

Bioavailability of Top-ranked Compounds
All chemical and physical characteristics of the topranked MMP-8 inhibitors were analyzed based on the RO5. Interestingly, all of them were found to agree with Lipinski's law, and therefore, CGA, caffeic acid 3-glucoside, rosmarinic acid, N-p-coumaroyltyramine, and CAPE were confirmed to be suitable for oral use (Table 3).

Pharmacokinetics and Toxicity of Top-ranked Compounds
The ADMET prediction study revealed no considerable toxicity for the top-ranked cinnamic acid derivatives. However, CGA and caffeic acid 3-glucoside were found to be safer than the other top-ranked compounds. Furthermore, N-p-Coumaroyltyramine and CAPE showed higher gastrointestinal absorbance compared to other compounds (Table 4).

Discussion
The enhanced expression and/or activity of MMP-8 is

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Archive of SID associated with several human disorders, including oral cavity and peri-implant inflammation/diseases. In the present study, molecular docking analysis was conducted to estimate the binding affinity of several natural compounds to the MMP-8 catalytic domain from cinnamic acid and its derivatives to discover drug candidates for MMP-8 inhibition.   CGA is a secondary metabolite in herbs with several pharmaceutical properties (e.g., antioxidant, antibacterial, cardioprotective, neuroprotective, and anti-inflammatory characteristics). Moreover, CGA can go through the bacteria cell and release the components of the cytoplasm, leading to bacteria death (36)(37)(38). Therefore, this compound has been widely used for tooth decay prevention. Palaniraj et al (19) examined the possible anti-biofilm effects of CGA when loaded to calcium phosphate-chitosan nanoparticles in restorative dentistry and reported that CGA significantly enhanced biofilm degradation up to 68%. Likewise, CGA revealed no toxicity effect on HaCaT cells up to 40 μg/mL. In the present study, CGA showed a considerable binding affinity to the MMP-8 catalytic domain with the ∆G binding of -11.81 kcal/mol. It was also estimated that this compound can inhibit the MMP-8 at the nanomolar scale Ki = 2.22 nM. CGA demonstrated three hydrophobic and two hydrogen bonds with Leu160, Val194, His197, Asn218, and Tyr219 within the MMP-8 catalytic domain. According to the potential inhibitory effect of CGA on MMP-8, in addition to the anti-bacterial activity of this component, CGA might be considered as a useful compound in restorative dentistry with protective effects against dental caries.

Ribeiro et al (18) studied the anti-bacterial effects of
Similarly, Yamamoto and Ogawa (39) investigated the antimicrobial activity of perilla seed extracts against several bacteria involved in the pathogenesis of tooth caries and periodontitis, including oral streptococci and different strains of P. gingivalis (20). They concluded that rosmarinic acid revealed stronger antibacterial activity against various strains of P. gingivalis compared with oral streptococci. According to our results, rosmarinic acid can potentially connect to the MMP-8 catalytic site with a noticeable ∆G binding and Ki of -11.03 kcal/mol and 8 nM, respectively. Rosmarinic acid demonstrated two hydrogen and two hydrophobic interactions with Ile159, His197, Tyr219, and Ala220 within the MMP-8 active site. It may be concluded that rosmarinic acid has several protective effects on dental caries and periodontitis. However, confirmation is needed in this regard.
In another study, Kuramoto et al (40) found that CAPE significantly enhanced the expression and/or activity of the vascular endothelial growth factor (VEGF), nuclear factor-kappa B (NF-κB) transcription factor, and VEGF receptor-(VEGFR-) 2 in rat odontoblast cells (KN-3 cells), leading to elevated mineralization activity in KN-3 cells. Based on our findings, CAPE demonstrated a salient binding affinity to the MMP-8 catalytic domain with a ∆G binding of -10.23 kcal/mol. CAPE formed three hydrogen interactions and one hydrophobic interaction with His197, Pro217, Asn218, and Tyr219 inside the MMP-8 catalytic site. According to the findings of previous research, in addition to our results, it may be declared that CAPE could be considered as a new organic compound with conservative and regenerative properties in dental pulpal tissue as well as anti-tooth caries effects by inhibiting the MMP-8, and therefore, CAPE might be a useful compound in restorative dentistry (40). It is noteworthy that propolis is a rich source of CAPE (41,42). Further, the ∆G binding and Ki for caffeic acid 3-glucoside were evaluated to be -11.26 kcal/mol and 5.57 nM, suggesting a considerable affinity of binding between caffeic acid 3-glucoside and the MMP-8 active site. Caffeic acid 3-glucoside formed seven hydrogen and two hydrophobic interactions with Gly158, Ala161, Val194, His197, Glu198, Ala213, Leu214, Pro217, and Ala220 inside the MMP-8 catalytic domain.

Conclusions
In general, it was estimated that five of the cinnamic acid derivatives, including CGA, caffeic acid 3-glucoside, rosmarinic acid, N-p-Coumaroyltyramine, and CAPE, can connect to the MMP-8 catalytic site at the nanomolar concentration with the criteria of ∆G binding < -10 kcal/ mol, and therefore, were introduced as potential MMP-8 effective inhibitors. Additionally, these components all agreed with Lipinski's RO5 and represented no significant toxicity, and thus may be beneficial for preventive/ therapeutic aims in dentistry. Eventually, His197 was found to be the most active residue within the MMP-8 catalytic site. However, validation is inevitable in the future.