Introduction

Staphylococcus aureus (S. aureus) is a common and opportunistic pathogenic bacterium [1] that is widely present in nature. The bacterium can cause infectious diseases [2], including pneumonia, endocarditis, and bacteremia [3], which can lead to death [4]. S. aureus frequently causes community and hospital infections [5]. The main drugs used to treat S. aureus infections are antibiotics [6]. A methicillin-resistant S. aureus strain (MRSA) was first discovered in 1961 [7]. Genomic plasticity and antibiotic abuse have led to increased antibiotic resistance [8]. MRSA isolates are resistant to all available penicillins and other β-lactam antimicrobials [9]. MRSA not only expresses virulence factors [10] but also utilizes a complex mechanism to resist drugs [11]. In 2019, MRSA caused more than 100,000 deaths worldwide in more than 200 countries and regions. Therefore, the development of novel anti-MRSA treatment models is essential.

Currently, proteins related to bacterial growth, drug resistance, and virulence factors have become important targets for combating S. aureus [12,13,14,15]. Dalal et al. [16] screened three inhibitor molecules of FmtA (ofloxacin, roflumilast, and furazolidone) via virtual screening. Further research revealed that drugs and FmtA can be stably combined, thereby enhancing the thermostability of this protein. Kumari et al. screened five inhibitors that stably bind to ribosome biogenesis GTP-binding (YsxC) via molecular docking [17]. The results demonstrated that inhibitors can be effectively screened using theoretical calculations. In recent years, methods that specifically block bacterial pathogenicity have become a frontier hotspot in research against S. aureus infection. This strategy treats bacterial infections by reducing pathogenicity in hosts [18]. Previous research found that serine/threonine kinase/phosphatase (Stk1/Stp1) is involved in regulating the phosphorylation and dephosphorylation of Cys in MgrA and SarA [19] and plays an important role in the regulation of virulence [20]. Stp1 participates in many important pathways, such as bacterial cell wall biosynthesis, virulence factor regulation, and pathways linked with antibiotic sensitivity [21]. Previous studies also showed that after bacteria delete the Stp1 gene, their ability to produce hemolysin is reduced [22]. A murine infection model showed that a Stp1 deletion mutant reduced hepatic abscess formation and tissue necrosis [21], indicating that Stp1 is closely related to the pathogenicity of S. aureus and could offer a new target for drug action.

Stp1 belongs to the metal-dependent phosphatase/protein phosphatase 2C (PP2C) family. The structure of Stp1 is similar to those of other PP2C phosphatases [23]. Its core is a β-sandwich consisting of two antiparallel β sheets, which are flanked by a pair of antiparallel helices on either side [22]. The active site is located on top of the β-sandwich and contains four metal ions close to the catalytic site; these ions are responsible for linking water molecules and assist in the catalytic function [23]. Knowledge on the protein structure facilitates further research on drug screening and binding mechanisms.

Echinacoside (ECH) is derived from the herb Echinacea species. Echinacea is a prominent dietary supplement used worldwide. Caffeic acid derivatives are bioactive compounds of Echinacea. Cichoric acid and ECH are important components of caffeic acid derivatives and show major pharmacological properties [24]. Previous studies have shown that ECH exhibits a variety of biological activities, including antioxidative [25], antihyperglycemic [26], anti-inflammatory [27], anticancer [28], and antidepressant [29] activities. However, few studies have investigated the antimicrobial effects of ECH. Via virtual screening, we found that ECH has the potential to inhibit Stp1 activity. Furthermore, phosphatase activity experiments revealed that ECH exhibits the highest inhibitory effect. Subsequently, molecular dynamics simulations and binding free-energy calculations revealed the binding mode of Stp1 and ECH, and fluorescence quenching experiments confirmed these results. This study provides new methods and ideas for the discovery of novel Stp1 inhibitors.

Materials and methods

Bacterial strains and materials

S. aureus strain USA300 was deposited in the laboratory. Escherichia coli strains DH5α and BL21 (DE3) were purchased from TIANGEN BIOTECH (Beijing, China) Co., Ltd. Echinacoside was purchased from Chengdu Herbpurify (Chengdu, China) Co., Ltd. Dimethyl sulfoxide (DMSO) was purchased from Sigma–Aldrich (St. Louis, USA).

Plasmid construction, protein expression, and purification

The S. aureus USA300 genome was used as the template for PCR [30]. The amplified gene and pET28a vector were digested at 37 °C for 2 h with restriction endonucleases BamHI and XhoI. These two digested fragments were connected with T4 ligase and named PET-28a-Stp1. Construction of the mutant plasmid was performed using a QuikChange™ site-directed mutagenesis kit [31]. After point mutation was performed, the plasmid was digested with DpnI restriction endonuclease and sequenced [32]. The primers used in the experiment are listed in Table 1.

Table 1 Oligonucleotide primers used in this study
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The sequenced plasmid was transfected into BL21 (DE3) cells for expression analysis. The bacteria were cultured in a medium at 37 °C until the OD600 nm reached 0.5. After adding isopropyl β-D-thiogalactoside to promote the expression of the target protein, the bacteria were cultured at 16 °C overnight. The bacteria were collected by centrifugation and subsequently sonicated. The cell lysate was centrifuged at 12,000 × g for 40 min, and the supernatant was loaded onto a Ni-NTA agarose column [33] that was previously equilibrated with binding buffer (20 mM Tris, pH = 8.0). The column was washed with washing buffer (20 mM Tris, 10 mM imidazole, pH 8.0). After washing, the His-tagged protein was eluted with elution buffer (20 mM Tris, 100 mM imidazole, pH 8.0). The elution solution was concentrated by a Millipore Amicon filter to obtain Stp1 protein.

Virtual screening

AutoDock Vina 4.0 software was used for virtual screening by docking compound to Stp1 [34]. A total of 15,362 natural compounds were obtained from the Zinc database. The 2D structure files of small molecules were converted into 3D structure files that can be used in AutoDock Vina. The 3D structure of Stp1 was downloaded from the RCSB Protein Data Bank (protein ID:5F1M). AutoDock Tools was used to add polar hydrogen atoms to Stp1 and distribute part of the charge generated by Kollman’s combined atoms. Subsequently, a grid box surrounding the hydrolyzed active region was constructed as a ligand docking site. To improve the computational efficiency, the AutoDock Vina search space was minimized to cover the binding site with the following dimensions: center_x = 47.248 Å, center_y = 101.299 Å, and center_z = 195.568 Å; size_x = 14 Å, size_y = 40 Å, and size_z = 16 Å. The ligands were docked individually to the active region of Stp1. The Lamarckian genetic algorithm was used for the entire calculation process during virtual screening [35]. The target protein [36] was always rigid, and all the twisted bonds of the inhibitor could rotate freely [37]. A grid box was created, including metal ions at the active site of the Stp1 receptor, which was positioned on the ligand’s center of mass. After each ligand in the ligand library was docked to the active site of Stp1, the binding affinity (kcal/mol) of the ligand-Stp1 system was obtained. Binding affinity is an indicator used to assess the ability of ligands to bind receptor proteins. The values obtained for the binding affinity were sorted from low to high, and the top 50 ligands were selected for structural analysis. Small molecules that are closely bound to the protein activity center and can establish a strong interaction are commonly selected for phosphatase assays.

Phosphatase assay

As described in a previous study [19], the drug was detected using a phosphatase activity assay. The purified Stp1 was preincubated in a 96-well plate with different concentrations of drug in buffer at 25 °C for 10 min. p-Nitrophenyl phosphate (PNPP) was then added to the mixture at a final concentration of 0.8 mM. The samples were incubated for 20 min at 25 °C. The total volume of the mixture was 200 μL. After the reaction was complete, the absorbance was measured at 405 nm using a spectrophotometer. The group containing PNPP and the drug served as the negative control. A group without drug addition was used as a positive control [30]. The absorbance was measured three times at 492 nm after NaOH was added to determine the point at which the reaction was neutralized and completed. Enzyme activity = (A−A0)/(A100−A0) × 100%, where A represents the absorbance of inhibitor groups and A0 is the negative control measurement. A100 was used as a positive control.

Molecular dynamics simulation

The Gromacs 4.5.5 software package with the Amberff99sb force field and TIP3P water model was used for the molecular dynamics simulation [38]. The Stp1 structure file contained Mn2+ coordinates. Given that the Amberff99sb force field did not contain the Mn2+ force field, the parameters of Mn2+ were added to some files (ions.itp, ffnonbonded.itp, and atomtypes.atp) to establish the force field [39]. The initial phase of the Stp1-inhibitor system involved energy relaxation. In this phase, 2000 steps were maximum descending energy minimizations, followed by 2000 steps of conjugate gradient energy minimizations. Subsequently, the temperature and pressure of the system were controlled by 200 ps and 500 ps molecular dynamic runs, while position restriction was applied to the proteins and ligands to allow relaxation of the solvent molecules. Next, a 200 ns MD was run with no position restriction on the solute. The trajectory equilibration was reflected by the balance of various quantities, such as root mean square deviation (RMSD) relative to the initial structure, internal protein energy, and fluctuations calculated at different time intervals. After a 200 ns simulation, the binding energy of the protein and ligand was analyzed using the molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) method [40]. We evenly selected a total of 100 snapshots from 10 ns on the equilibration MD trajectory at intervals of 100 ps. A detailed description of this method is provided in the Supplementary Materials.

Fluorescence quenching

The binding constants (KA) of the ligand to the binding site of the wild-type and mutant Stp1 were calculated by fluorescence quenching. Stp1 protein solution (3 mL) and inhibitors were added to a 1 cm quartz cuvette, mixed, and incubated for 10 min. The fluorescence spectra of the mixed solutions were obtained using a fluorescence spectrophotometer. The excitation and emission slits were 5.0 nm, and the excitation wavelength was 280 nm. The calculation expression is r/Df = n KA − r KA [41], where r is the amount of ligand per mol of protein-bound material (r ≈ ΔF/F0), Df is the free concentration of quercetin, and n refers to the quantity of binding sites.

Bioinformatics prediction

The SwissADME program (http://www.swissadme.ch) from the Swiss Institute of Bioinformatics can predict the absorption, distribution, metabolism, excretion (ADME), and pharmacokinetic properties of drugs [42]. Accessing http://www.swissadme.ch in a web browser directly displays the submission page of SwissADME. The structure of the ligand was entered, and the analysis result was obtained. The software reveals molecular properties from physicochemical properties, pharmacokinetics, and medicinal chemistry friendliness.

Results and discussion

ECH inhibits Stp1 activity

Using virtual screening, we found that ECH (Fig. 1a) has the potential to inhibit Stp1. Therefore, phosphatase activity was measured using Stp1 at different drug concentrations. The results showed that ECH strongly inhibited Stp1 activity (Fig. 1b). As the drug concentration increased, the activity of Stp1 gradually decreased. At an ECH concentration of 2 μg/mL, the inhibition rate was 16.00%. When the ECH concentration was 16 μg/mL, the degree of inhibition exceeded 50%.

Fig. 1
figure 1

Inhibition of Stp1 by ECH. a Structure of ECH. b Inhibitory effect of ECH on Stp1

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To further study the mechanism by which ECH inhibits Stp1, enzyme kinetic assays were carried out. As shown in Fig. 2, the KM values in the reaction changed. Therefore, we conclude that ECH is a competitive inhibitor of Stp1.

Fig. 2
figure 2

Lineweaver−Burk plot of Stp1 activity against different concentrations of ECH indicates that ECH is a competitive inhibitor

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Virtual screening analysis

To screen the potential inhibitors of Stp1, Stp1 and the ligands were docked to obtain the binding affinity [43]. According to previous reports, verbascoside (VBS) exerts a significant inhibitory effect on Stp1 by binding to the active site of Stp1 [30]. Molecular docking experiments found that the binding affinity of Stp1-VBS was 7.4 kcal/mol, which was used as the cutoff value to select the active compound. According to the binding affinity, the top 50 ligands were selected. As previously mentioned, small molecules that were closely bound to the protein activity center and could establish a strong interaction were selected by the PyMOL and LigPlus software [33]. Therefore, these candidate compounds showed the potential to become an inhibitor of Stp1 [13]. Subsequently, a total of nine molecules were selected as candidate compounds, and enzyme activity experiments were conducted to verify their inhibitory activity (Table 2). The docking results are shown in Fig. 3. Enzyme activity experiments showed that ECH exhibits a significant inhibitory effect on Stp1.

Table 2 Binding affinity and inhibition ratio of 9 candidate compounds
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Fig. 3
figure 3

Ligand binding poses at the active site of Stp1

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Determination of the binding mode of Stp1 with ECH

In this study, the potential binding mode of the Stp1-ECH complex was determined using molecular docking, molecular dynamics simulations, and binding free energy calculations. According to the results of the virtual screening and activity experiment, ECH can bind to the active site of Stp1 and effectively inhibit the hydrolytic activity of Stp1. A three-dimensional structure of the Stp1-ECH complex obtained by molecular docking was used as the initial coordinates for the molecular dynamics simulations. Based on the molecular docking results, a 200 ns molecular dynamics simulation of the Stp1-ECH complex was conducted to explore the underlying mechanism. The RMSD is an important indicator for analyzing the structural stability of complexes [16]. To investigate the stability of the complex system during the simulation [12], the RMSD values of the complex and free proteins during a 200 ns simulation were calculated (Fig. 4a). The RMSD values of the Stp1-ECH complex (~ 0.28 nm) and ECH (~ 0.12 nm) were smaller than that of free Stp1. Therefore, the combination of Stp1 and ECH was more stable. After 25 ns, the RMSD value of the system stabilized. Therefore, the complex system reached equilibrium during the 180–200 ns simulation, and the trajectory of the molecular dynamics simulation could be used for subsequent analysis. After a 200 ns molecular dynamics simulation, the stable binding mode of the ECH and Stp1 complex system was obtained. ECH bonded to the active region of Stp1 via hydrogen bonding and hydrophobic interactions (Fig. 4b). ECH, especially the benzene ring, was tightly bound by Met39, Gly41, Asp120, Asn162, and Ile163 of Stp1, as shown in Fig. 4b. The side chains of Met39 and the carbonyl group of Asn162 tightly bonded the benzene ring of ECH at the binding site. The carboxyl group of Asp 120 strongly interacted with the phenolic hydroxyl group of ECH. As revealed by PyMOL software, compared to other inactive site amino acids, Gly41 and Ile163 were closer to ECH; thus, these two amino acids and the drug may interact.

Fig. 4
figure 4

This paper demonstrates the potently competitive binding mode of Stp1 and ECH through molecular modeling. a RMSD plot of free protein, Stp1-ECH, and ECH in 200 ns. b Stable three-dimensional (3D) structure of Stp1 binding with ECH based on MD simulation. The hydrogen-bonding interactions are shown as yellow dashed lines

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Table 3 shows that the Stp1-VBS complex exhibited negative van der Waals (~ −26.32 kcal/mol), electrostatic (~ −23.75 kcal/mol), and SASA (~ −3.75 kcal/mol) energies, resulting in a total binding energy of −16.63 kcal/mol. Compared with Stp1-VBS, the Stp1-ECH complex exhibits a lower binding energy. Therefore, the combination of Stp1 and ECH is more stable.

Table 3 Binding free energies (kcal/mol) between Stp1 and compounds by the MM/PBSA method
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The root mean square fluctuation (RMSF) reflects the fluctuation in each residue and reveals the flexibility of the system [44]. The average RMSF values of Stp1-ECH and free Stp1 were 0.10 and 0.13 nm, respectively. Compared with the free protein, the binding site residues in the complex system exhibited a smaller RMSF value, indicating that the residues of the binding site in the complex system were less flexible (Fig. S1). These residues became more rigid due to binding with ECH. The radius of gyration (Rg) was used to characterize the compactness of the complex. As seen in Fig. S2, the Rg of ECH-Stp1 is lower than that of free Stp1, which is consistent with previous results.

Confirmation of the binding mode of ECH to Stp1

To further analyze the interaction between ECH and Stp1, we applied the MM/PBSA method to calculate the electrostatic (ΔEele), van der Waals (ΔEvdw), solvation (ΔEsol), and total contribution (ΔEtotal) of the residues to the binding free energy. As shown in Fig. 5, most of the decomposed energy interactions originated from van der Waals forces, which resulted from the strong hydrophobic interactions between the protein and ligand.

Fig. 5
figure 5

Decomposed binding free energies of the residues in the binding site. The histogram chart shows the van der Waals (black), electrostatic (red), solvation (blue), and total (green) contributions for the complexes

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As shown in Fig. 5, Ile163 exhibited significant van der Waals contributions (~ −2.43 kcal/mol), resulting in binding energy contributions of −2.76 kcal/mol. This indicates that Ile163 and ECH exhibit a strong hydrophobic interaction. In addition, Met39 and Gly41 exhibited electrostatic (~ −0.63 and −0.07 kcal/mol), van der Waals (~ −1.78 and −0.83 kcal/mol), and solvation (~ −0.12 and −0.01 kcal/mol) force contributions, resulting in strong binding free energy contributions (−2.53 and −0.91 kcal/mol). This was primarily attributed to the strong hydrophobic interaction between Met39 and Gly41 with the benzene ring of ECH. Although the van der Waals and solvation forces were unfavorable, Asp120 provided a strong electrostatic contribution, resulting in a ΔEtotal value of −1.19 kcal/mol. This indicates that hydrogen bonds could form between Asp120 and ECH. In addition, Asn162 provided significant van der Waals and electrostatic contributions, resulting in binding energy contributions of −1.38 kcal/mol. Therefore, hydrophobic interactions occurred between Asn162 with ECH due to strong hydrogen bonding between Asn162 and ECH. Decomposition of the binding free energy further indicated that the amino acid residues in the binding site of Stp1 to ECH were Met39, Gly41, Asp120, Asn162, and Ile163.

Hydrogen bonding is a strong interaction that plays an important role in the stability of protein–ligand complexes [17]. To further study hydrogen bonding between Stp1 and its ligands, the number of hydrogen bonds was determined using a trajectory file. As shown in Fig. 6a, the number of hydrogen bonds fluctuated between four and five during the simulation, suggesting that the ligand formed four or five hydrogen bonds with the amino acid residues of Stp1. As shown in Fig. 6b, according to the LigPlus software, ECH can form four strong hydrogen bond interactions with the amino acid residues of proteins, which confirms the above conclusion. Stp1 and VBS can directly form a stable hydrogen bond [30], and there are more hydrogen bonds in the Stp1-ECH complex than the Stp1-VBS complex.

Fig. 6
figure 6

Interaction between ECH and Stp1 demonstrated in the competitive binding mode. a Number of hydrogen bonds between ECH and Stp1. b Interaction between ECH and the residues of binding sites of Stp1 identified using LigPlus software

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The distance between the ligand ECH and the amino acid of Stp1 was also calculated and analyzed. As shown in Fig. 7, the distances between the inhibitor ECH and the binding site amino acids were less than 0.2 nm, further indicating that ECH interacts strongly with amino acids Met39, Gly41, Asp120, Asn162, and Ile163. According to previous reports [45], these amino acids are located in the active region of Stp1. Therefore, ECH inhibits the hydrolytic activity of Stp1 via competitive inhibition.

Fig. 7
figure 7

a Distance between all residues of Stp1 and ECH. b Distances between the residues in the binding sites of Stp1 and ECH

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Identification of the binding mode of ECH and Stp1

To verify the theoretical calculation results, the Met39 and Gly41 residues of Stp1 were mutated to alanine (Ala) to obtain mutants M39A-Stp1 and G41A-Stp1, respectively. After protein expression and purification, the mutant protein was obtained and used for subsequent phosphatase hydrolysis and fluorescence quenching experiments. The binding constants (KA) of ECH and Stp1 were calculated by the fluorescence quenching method. The binding constants (KA) of the WT-Stp1-ECH, M39A-ECH, and G41A-ECH systems were 7.76, 2.81, and 5.73 × 105 L/mol, respectively. The binding affinity of the mutant decreased compared to the wild-type, resulting in the following binding order: WT-Stp1 > G41A-Stp1> M39A-Stp1. This indicates that the wild-type protein was the most suitable to bind to ECH.

As shown in Fig. 8, the phosphatase assays revealed that the inhibitory activity of ECH on mutant Stp1 was lower than that on WT-Stp1. Therefore, the mutation of the two amino acid residues, namely, Met39 and Gly41, directly reduced the inhibitory ability of ECH. Mutating the binding site residues reduced the binding affinity of the inhibitor to Stp1, thus resulting in loss of inhibitory activity. Therefore, the experimental results further verified that a reliable complex structure was produced by molecular dynamics simulations.

Fig. 8
figure 8

Enzyme inhibition assays of ECH against wild-type-Stp1, M39A-Stp1, and G41A-Stp1

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Lipinski and ADMET properties of ECH

The SwissADME software can predict Lipinski and ADME based on a molecular structure. Given that the values of fraction Csp3 (Fraction Csp3 = 0.57) and log S (ESOL) (log S = −2.63) were within the standard value range, the solubility and saturation of ECH were adequate. Therefore, ECH can be easily dissolved and formulated, which is conducive to absorption. Conversely, the Log Po/w values were not within the standard range, and the saturation was poor. Based on pharmacokinetic studies, the gastrointestinal absorption of ECH was low. However, ECH can pass through the blood–brain barrier in permanent middle cerebral artery occlusion in rats [46].

According to Lipinski’s rule, ECH is not suitable for oral administration due to the relative molecular mass and the number of hydrogen bond receptors, which exceeds the standard. The prediction results are consistent with previous experimental studies. Utilizing absorption promoters and new formulation technologies can improve bioavailability. Li et al. formulated ECH into a phospholipid complex to enhance its bioavailability [47]. Shen et al. used verapamil and clove oil to enhance the intestinal absorption and oral bioavailability of ECH [48].

Moreover, ECH is a natural compound. The medicinal history of plants rich in ECH shows that its adverse reactions and toxic effects are relatively minor. Animal and cell experiments have shown that ECH exhibits good activity in the treatment of osteoporosis [49] and liver injury [50]. ECH can be employed to develop new anti-infective drugs using nano-based drug delivery systems.

Conclusions

ECH is a natural compound with numerous biological activities. In this study, using virtual screening and enzyme activity assays, we found that ECH is a novel Stp1 inhibitor. Further studies showed that ECH binds to the active center of Stp1, preventing binding to Stp1 by substrate competition and thus reducing enzyme activity. This was mainly due to residues Met39, Gly41, Asp120, Asn162, and Ile163 of Stp1. The benzene ring of ECH also plays an important role in complex binding. These results provide information useful for the design and development of antimicrobial drugs targeting S. aureus and will thus be useful for the discovery and design of new Stp1 inhibitors.