Studies have shown that E. faecalis can lead to persistent infections and reinfections in root canals, often resulting in treatment failures (Zoletti et al. 2006; Ali et al. 2020; Voit et al. 2022; Carlesi et al. 2023). Biofilm formation is a significant virulence factor for E. faecalis, enabling the bacterium to colonize various surfaces and resist antimicrobial treatments (Alghamdi and Shakir 2020). The esp and ace genes play important roles in the initial attachment and aggregation of E. faecalis cells, which are critical steps in biofilm formation (Francisco et al. 2001; Francisco et al. 2021).

Recently, targeted aPDT has emerged as a promising adjuvant treatment for combating microbial biofilms (Ribeiro et al. 2022). For effective targeted aPDT, selecting an appropriate photosensitizer is crucial. A clinically effective photosensitizer should possess a certain degree of hydrophilicity to prevent accumulation and should specifically bind to target regions on microbial cells. This specificity enhances internalization at the target site and minimizes unwanted off-target effects (Ghorbani et al. 2018).

In this in silico study, Kojic acid and Parietin are proposed as natural photosensitizers that interact with Ace and Esp proteins, which are essential for the formation and development of E. faecalis biofilms. In silico analysis, utilizing computational methods and algorithms to investigate biological systems, can be instrumental in identifying target sites on microbial proteins.

Predicting the secondary and three-dimensional structures of the target proteins is crucial for future analysis. In this study, random coils were identified as the optimum scaffolded structure in the designed secondary structures of Ace (50.15%) and Esp (48.48%) proteins. The modeled three-dimensional tertiary structure of the target proteins showed high quality, as assessed by various methods including Ramachandran Plot analysis, Verify3D, ERRAT, and ProSA-web servers. Furthermore, molecular dynamics simulations were conducted to verify the stability of the Ace and Esp structures, and the results indicated that the modeled structures of both proteins were stable, making them reliable targets for Kojic acid and Parietin.

The signal peptide pathway in bacteria is an essential process for transport of proteins across the cell membrane, allowing for their proper function and survival (Schneewind and Missiakas 2014). In Gram-positive bacteria like E. faecalis, there are two primary pathways for protein transport across the cell membrane: the secretion (Sec) pathway and twin-arginine translocation (Tat) pathway (van Wely et al. 2001; Tseng et al. 2009). Both pathways are responsible for the transport of proteins that contain a signal peptide, a short amino acid sequence that directs the protein to the appropriate pathway. The Sec pathway is the most commonly used pathway for protein transport in bacteria, responsible for the translocation of unfolded proteins across the membrane. This pathway involves the SecA ATPase, which binds to the protein’s signal peptide and facilitates its transfer across the cytoplasmic membrane. The translocation process requires the presence of a protein channel known as the SecYEG complex, which forms a pore in the membrane. The SecYEG complex is composed of three subunits: SecY, SecE, and SecG, which work in concert to translocate the protein through the membrane (Beckwith 2013; Tsirigotaki et al. 2017).

In contrast, the Tat pathway is responsible for transporting folded proteins that are too large to be moved by the Sec pathway. This pathway involves the proteins TatA, TatB, and TatC, which form a protein channel that allows the transport of folded proteins across the membrane (Goosens et al. 2014; Frain et al. 2019). The Tat pathway is energized by the proton motive force across the membrane (Goosens et al. 2014). In E. faecalis, the signal peptide pathways for both Sec and Tat are similar to those in other bacteria. The signal peptide is recognized by the appropriate pathway, directing the protein to either the Sec or Tat system. Once the protein is translocated across the membrane, it can either be folded and functional or undergo further modified by other cellular machinery. In this study, SignalP was used to analyze the protein sequences and predict the pathway used for their transport across the cell membrane. The results indicated that a greater proportion of Ace signal peptides are associated with the Sec/SPI pathway compared to the Tat/SPI and Sec/SPII pathways. In contrast, Esp signal peptides were found across all three pathways, but at a lower frequency. These findings provide insight into the sub-cellular localization and transport mechanisms of Ace and Esp within the bacterial cell, revealing that Ace is located in the cell wall while Esp is found in the cytoplasm.

In this study, the structure-based method using AutoDock4.2 software package was used to predict the active sites of Ace and Esp proteins. The active site of a protein is a critical region where chemical reactions occur. It is typically a small, specific region of the protein that interacts with other molecules, such as substrates, cofactors, or inhibitors (Brooks et al. 1985; Bray et al. 2009; Barnsley and Ondrechen 2022; Weinstein et al. 2023). Predicting the location and properties of the active site is important for understanding the function of a protein, designing inhibitors or drugs, and predicting the effect of mutations or variations on protein function (Sankararaman et al. 2010). Active site prediction involves identifying and characterizing the key residues that contribute to the active site, as well as the shape and electrostatic properties of the site (Barnsley and Ondrechen 2022).

In the current study, the PPI network of Ace and Esp proteins was assessed. PPIs provide a way to visualize and study the complex interactions between proteins, allowing researchers to gain insights into the functional properties of individual proteins as well as the overall function of the network (Rao et al. 2014; Sevimoglu and Arga 2014). Furthermore, PPIs can be used to identify potential drug targets for various diseases. By targeting proteins that are critical nodes in disease-associated PPIs, researchers can potentially disrupt the function of the network and treat the disease (Feng et al. 2017). In this study, we identified critical nodes that play important roles in maintaining the overall stability and function of the network. There was a sum of 22 nodes in the PPI network of Ace and Esp. These nodes are often proteins with a high degree of connectivity, meaning they interact with many other proteins in the network. Disrupting these node proteins due to the aPDT can have a significant impact on the overall function of the network.

Moreover, we evaluated the molecular dynamics simulation of the Ace and Esp proteins. The importance of the prediction of molecular dynamics simulation in proteins is to provide insights into the structural and functional properties of proteins (Sinha et al. 2022). In silico studies that determine a protein’s deformability enable researchers to explore how conformational changes in the protein can affect its function (Haspel et al. 2010). Many drugs, including photosensitizers, operate by binding to specific protein targets and changing their conformation, either by stabilizing or destabilizing their structures. As the results of the current study showed, the Esp protein exhibited the highest deformability, characterized by numerous peaks. Furthermore, Esp displayed the highest number of eigenvalues, which can be used to predict the protein’s normal modes of motion (4.887245e-04). In contrast, the flexibility of various regions of the Ace protein, as determined by its B-factor (also known as the temperature factor), was more than the Esp protein. Other factors evaluated were the variance plot, the elastic network model, and the covariance matrix. The variance plot describes the distribution of fluctuations in the protein’s structure and serves as a measure of the protein’s flexibility (López-Blanco et al. 2011; López-Blanco et al. 2014). The elastic network model is a computational method that allows for the investigation of the collective motions and dynamics of a protein, which are critical for its function (Fuglebakk et al. 2013). The covariance matrix of a protein also provides valuable insights into the protein’s functional dynamics (López-Blanco et al. 2011). Proteins are dynamic molecules that undergo conformational changes in order to perform their biological functions, and the covariance matrix can help identify regions of the protein that are involved in these dynamic processes (Fuglebakk et al. 2013). According to the results of this study, the most cumulative variances and a good correlation between residues with the flexible springs were observed in Ace protein. Overall, the findings showed that both proposed proteins have stable structures and functional features.

There is currently a lack of information regarding the implications of molecular dynamics simulations on the stability and functional dynamics of proteins during aPDT. However, further research using molecular dynamics simulations has the potential to enhance our understanding of protein stability during aPDT by providing detailed insights into the dynamic behavior of proteins under conditions that mimic physiological and therapeutic environments. Based on literature (Pikkemaat et al. 2002; Salsbury 2010; Sinha et al. 2022), it is expected that molecular dynamics simulations can allow researchers to observe the time-dependent movements of proteins, revealing how structural fluctuations can affect stability. This is crucial during aPDT, as proteins may encounter ROS that can induce conformational changes. By analyzing the trajectories from molecular dynamics simulations, researchers can identify flexible regions within proteins that may be more susceptible to destabilization or denaturation during aPDT. On the other hand, molecular dynamics simulations can assess how proteins respond to thermal stress and photosensitizers used in aPDT. By simulating conditions that mimic these stresses, researchers can evaluate the resilience of proteins like Ace and Esp, determining their likelihood of maintaining functional integrity during treatment. Molecular dynamics simulations can provide insights into how proteins interact with photosensitizers and other therapeutic agents. Understanding these interactions at an atomic level helps in predicting how aPDT will affect protein function and stability, as the binding of ligands can alter the conformational landscape of proteins. The role of water molecules in stabilizing protein structures is highlighted in molecular dynamics studies. Water can mediate interactions between amino acids and influence the overall stability of protein complexes. This is particularly relevant in aPDT, where the generation of reactive species can alter the hydration shell around proteins, potentially impacting their stability and function.

Furthermore, we conducted molecular docking and ADME/Tox analysis to assess the mechanism of action of Kojic acid and Parietin as potential inhibitors of E. faecalis biofilm formation during the aPDT process. Molecular docking is used to study and predict the intermolecular interactions between a small molecule (ligand) and a macromolecule (receptor) (Meng et al. 2011). According to the findings, Parietin has significantly lower binding energies (-6.88 and − 7.83 Kcal/mol) compared to Kojic acid (-4.76 and − 5.97 Kcal/mol) for both Ace and Esp proteins. Lower binding energies indicate stronger binding affinity between the ligand and the protein. Parietin interacts with more active site residues (7 for Ace and 9 for Esp) compared to Kojic acid (2 for Ace and 6 for Esp). A greater number of interactions with active site residues can contribute to the stronger binding affinity of Parietin. The inhibitory constants (Ki) for Parietin are much lower than those for Kojic acid, indicating that Parietin is a more potent inhibitor of both Ace and Esp proteins. Lower Ki values suggest stronger inhibition at lower concentrations. The differences in binding energies and inhibitory constants between the two compounds can be attributed to their structural differences. Parietin, being a larger and more complex molecule, may form more favorable interactions with the proteins, leading to stronger binding and inhibition compared to the smaller and simpler Kojic acid molecule. The lower binding energies, higher number of active site interactions, and lower inhibitory constants suggest that Parietin has a stronger binding affinity and inhibitory potency towards Ace and Esp proteins compared to Kojic acid, based on the provided docking simulation data.

The ADME/Tox profiling of photosensitizers is determined to evaluate their pharmacokinetic properties. ADME/Tox analysis revealed that both natural compounds were soluble in water. The percentages of Kojic acid and Parietin that would be absorbed through the human intestine (HIA%) were predicted to be 91.39 and 99.21%, respectively. Both compounds lack blood-brain barrier (BBB) permeability and exhibit high gastrointestinal absorption without binding to blood plasma proteins. These compounds, with human oral bioavailability, were also predicted to be non-inhibitor and non-substrate of P-gp. Except for CYP1A2, which was inhibited by Parietin, all CYP3A4, CYP2C9, CYP2D6, CYP2C19, and CYP1A2 were predicted to not be inhibited and metabolized by Kojic acid and Parietin. The toxicity data showed a toxicity class of 3 and 5 with LD50 values of 550 and 5000 mg/kg for Kojic acid and Parietin, respectively. Kojic acid was found to be non-hepatotoxic, non-immunotoxic, and non-cytotoxic, while Parietin is non-hepatotoxic, non-carcinogenic, non-toxic to the respiratory system, and non-cytotoxic.

The ADME/Tox analysis revealed that both Kojic acid and Parietin have a molecular mass of less than 500 Da (142.11 and 284.26 g/mol, respectively), adhering to the Lipinski rule. The molecular weight of a drug is important as it affects the absorption rate and amount in the body. Furthermore, both photosensitizers have a suitable hydrogen bond donor count of 2 and a hydrogen bond receptor count of less than 10, indicating strong binding strength. They also exhibit high lipophilicity, defined as the partition coefficient (logP), which is less than 3, indicating good absorption. In drug development, lipophilicity and molecular weight are often increased to improve the affinity and selectivity of the therapeutic candidate. The molar refractivity of both photosensitizers falls within the acceptable range of 40 to 130 (33.13 for Kojic acid and 75.25 for Parietin).

While in silico studies can provide valuable insights into the behavior of biological systems, the results may not always be accurate or reliable since they are based on theoretical models and assumptions. Therefore, it is essential to verify the predictions of in silico studies via in vitro study followed by in vivo examination to confirm their validity and relevance to the actual biological systems. When the results of in silico studies are examined in vitro, they can be compared to actual experimental data, and any discrepancies or limitations can be identified and addressed. This helps to refine the computational models and algorithms used in silico studies, improving their accuracy and reliability. Therefore, in this study, we obtained the MICs of two natural photosensitizers against E. faecalis and evaluated their effects along with blue laser irradiation during targeted aPDT on the expression of genes involved in E. faecalis biofilm formation in vitro. As the results showed, the MICs of Parietin and Kojic acid against E. faecalis were 16 µg/mL and 64 µg/mL, respectively. Traditional antibiotics commonly used to treat E. faecalis infections include penicillin (MICs 8–16 µg/mL), ampicillin (MICs 8–16 µg/mL), amoxicillin (MICs 8–16 µg/mL), Ampicillin/sulbactam (MICs ≤ 32/16 µg/mL), vancomycin (MICs 4–32 µg/mL), and tigecycline (MIC ≤ 0.5 µg/mL). The emergence of resistant strains, such as vancomycin-resistant enterococci (VRE), has led to the need for alternative therapies like combination regimens or newer antibiotics including linezolid (MICs 2–8 µg/mL) (CLSI guideline 2019). It is evident that the MICs of Parietin and Kojic acid against E. faecalis are higher than those of most traditional antibiotics used for this pathogen. However, it is important to highlight that in aPDT, the activation of photosensitizers by light of a specific wavelength generates ROS. This process significantly enhances the efficacy of the photosensitizers, resulting in a reduction of their MICs against microorganisms during aPDT.

In addition, the expression of ace and esp genes in E. faecalis was significantly downregulated following treatment with aPDT using sub-MIC concentrations of Kojic acid and Parietin, combined with one min of blue laser irradiation, compared to the control group. The qRT-PCR results provide compelling evidence that both Kojic acid and Parietin, particularly when combined with laser light, significantly reduce the expression of the ace and esp genes in E. faecalis. This downregulation can have implications for understanding the potential therapeutic applications of these compounds, especially in contexts where inhibition of these genes is desirable.

The use of natural photosensitizers, such as Kojic acid and Parietin, during targeted aPDT offers several benefits. They are biocompatible, have improved targeting capabilities, are less toxic, reduce resistance, and are often readily available and cost-effective compared to synthetic photosensitizers, making them an attractive option for clinical use. Nevertheless, further research is necessary to explore their full potential as natural photosensitizers in aPDT.

This study found that Kojic acid and Parietin, with drug-likeness properties, could effectively interact with Ace and Esp proteins with strong binding affinities. Targeted aPDT using these compounds could inhibit the biofilm growth of E. faecalis by significant downregulation of the expression of ace and esp genes. As a result, natural photosensitizers-mediated aPDT can be considered a promising adjunctive treatment against endodontic infections. It is necessary to validate the potential antimicrobial activities of these natural compounds through further in vitro and in vivo studies to confirm their effectiveness and ensure their safety profiles for their use in the clinical setting. It is also suggested that the molecular dynamics simulations on the stability and functional dynamics of Ace and Esp proteins during aPDT assessed.