Molecular dynamics simulation-driven focused virtual screening and experimental validation of Fisetin as an inhibitor of Helicobacter pylori HtrA protease

Molecular dynamics simulation-driven focused virtual screening and experimental validation of Fisetin as an inhibitor of Helicobacter pylori HtrA protease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular dynamics simulation-driven focused virtual screening and experimental validation of Fisetin as an inhibitor of Helicobacter pylori HtrA protease Li Gao, Xianqiong Jiang, Hongtao Duan, Yan Shen, Kui Gu, Kuilong Huang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5316707/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Feb, 2025 Read the published version in Molecular Diversity → Version 1 posted 15 You are reading this latest preprint version Abstract Helicobacter pylori (H. pylori, Hp) is a primary contributor to various stomach diseases, including gastritis and gastric cancer. This bacterium can colonize gastric epithelial cells, compromising their integrity and leading to the development of these conditions. While antibiotics are the mainstay of treatment for H. pylori infections, their widespread use has led to serious issues with drug resistance. High-temperature requirement A (HtrA), an important virulence factor of H. pylori, emerges as a promising target for the design of new inhibitors. In this study, we employed molecular docking, molecular dynamics simulation (MDs), and in vitro experimental validation to identify potential active components that specifically target HtrA, effectively combating H. pylori. Based on the results of molecular docking and literature reports, 8 potential natural compounds targeting HtrA were found: Camelliaside B, Rehmanioside A, Hesperidin, Quercetin, Fisetin, Colchicine, Apigenin and Geniposide. MDs further revealed that Fisetin, Geniposide, and Quercetin show promising stability and interactions with HtrA. Importantly, in vitro experiments, including casein hydrolysis assays and antibacterial sensitivity tests, demonstrated that Fisetin observably inhibited the hydrolysis of casein by HtrA and effectively suppressed the growth of H. pylori. Overall, our findings highlight Fisetin as a potential therapeutic drug that targets HtrA, offering new tack for the development of lead compounds and potential drugs against H. pylori infections. HtrA Fisetin Molecular dynamics simulation β-casein hydrolysis Helicobacter pylori Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Gastric cancer ranks as the fifth most common cancer globally and is the third leading cause of cancer-related mortality [1]. Helicobacter pylori (H. pylori), a spiral-shaped, microaerophilic, gram-negative bacterium, is a major risk factor for severe gastric disorders such as chronic gastritis, peptic ulcers, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer [2, 3]. While antibiotic therapy remains the cornerstone of H. pylori treatment, its effectiveness is diminishing due to the increasing prevalence of antibiotic resistance. This resistance arises from mutations in bacterial proteins, reduced membrane permeability, and the activation of efflux pumps, which together lower drug efficacy. The bacterium’s persistent colonization of the gastric mucosa underlies its role in various gastrointestinal diseases [4]. Therefore, novel therapeutic strategies targeting H. pylori's pathogenesis are urgently needed. H. pylori releases various virulence factors that enable it to invade, colonize, and infect the gastric mucosa, leading to conditions such as chronic gastritis and gastric cancer [5, 6]. One key virulence factor is High-temperature requirement A (HtrA), a multifunctional protein with an N-terminal protease domain and two C-terminal PDZ (postsynaptic density of 95 kD, Discs large and zonula occludens 1) domains [7, 8]. HtrA functions as both a chaperone and a protease, playing a vital role in protein quality control by managing the folding and degradation of misfolded proteins [9, 10]. This capability is essential for the survival and colonization of H. pylori under stress conditions like heat shock, extreme pH, and high salt concentrations, making HtrA a promising target for the development of anti-H. pylori therapies [11–14]. In addition to its protein quality control functions, HtrA cleaves key epithelial adhesion proteins, such as occludin, claudin-8, E-cadherin, and desmoglein-2, disrupting the epithelial barrier and facilitating H. pylori invasion [9, 12, 15–17]. Notably, the cleavage of E-cadherin not only weakens cell-cell junctions but also activates β-catenin, a transcription factor involved in cell proliferation, and enhances pro-inflammatory responses via NF-κB signaling [18]. Furthermore, HtrA has been shown to enhance the type IV secretion system (T4SS)-mediated translocation of the oncogenic protein CagA into host cells, exacerbating gastric disease development [19]. Given its pivotal role in pathogenesis, HtrA represents a critical target for novel anti-H. pylori drug development. Virtual screening has become a key tool in computer-aided drug design (CADD), allowing for the efficient identification of biologically active compounds against specific molecular targets [20]. Qualcomm Dose offers a rapid and effective approach to screen potential drug candidates, serving as a valuable supplement or alternative to traditional experimental methods [21]. In recent years, extensive research has led to the discovery of numerous natural compounds with important medicinal properties, showing promising results in anti-cancer, anti-inflammatory, and antiviral therapies [22, 23]. Previous studies have modeled the three-dimensional structure of HtrA, establishing its stable conformation and utilizing it as a receptor in virtual screening to identify potential lead compounds. For example, Gisbert Schneider's group introduced the virtual ligand concept to identify inhibitors of H. pylori HtrA, while R. Amutha, Tiago Rodrigues, and colleagues demonstrated inhibitory effects of selected lead compounds on HtrA secretion [17, 24–26]. Despite these advances, no clinically approved drugs or vaccines targeting HtrA have been developed, largely due to the protease’s complex structure. Recently, Professor Wei Liu’s team made a significant breakthrough by elucidating the monomer structure of the HtrA family and discovering a pH-dependent dynamic transition between trimeric and monomeric forms, accompanied by conformational changes [7]. These findings offer critical insights into the protease’s function in bacterial infections and lay the groundwork for drug discovery efforts targeting HtrA to treat H. pylori-related diseases. In this study, we conducted virtual screening of a natural product database using molecular docking and molecular dynamics simulations, identifying Fisetin, Geniposide, and Quercetin as promising HtrA inhibitors. Further validation through casein hydrolysis and drug sensitivity assays confirmed that Fisetin not only inhibits H. pylori growth but also exhibits anti-HtrA activity, supporting the reliability of our virtual screening results. 2. Materials and methods 2.1. H. pylori strains and culture conditions The H. pylori reference strain 26695 used in this study was kindly donated by Professor Zou Quanming from the China National Research Center for immunobiological Products, Army Medical University. Hp was cultured in Hp liquid medium supplemented with 10% fetal bovine serum (FBS; BasalMedia, Shanghai China) at 37°C under microaerobic conditions. The culture period lasted for 3 to 4 days. 2.2. Screening of natural compounds HtrA inhibitors 2.2.1. Receptor preparation The receptor protein structure was downloaded from the PDB website ( www.rcsb.org ), and the protein PDB ID was 7xs2. The protein structure is shown in Fig. 1 a, which mainly includes serine protease domain (amino acid residue sequence is 43–272), PDZ1 (amino acid residue sequence is 273–365) and PDZ2 domain (amino acid residue sequence is 366–475) [27, 28]. The macromolecule HtrA in the PDB is a protein that contains solvent molecules and non-standard residues. Therefore, it must be preprocessed and optimized by using AutoDock Vina tools, including removing water molecules, adding hydrogen atoms, repairing charges, adding force fields, applying energy minimization, and finally saving the structure in pdbqt format. 2.2.2. Receptor grid generation and AutoDock Vina virtual screening The latest research found that the catalytic center composed of His116, Asp147 and Ser221 residues located in the protease domain of helicobacter pylori HtrA plays an important role in substrate degradation activity [7]. As shown in Fig. 1 b. Therefore, we regard these three residues as the binding sites of HtrA protein and ligand. Then, we use the Grid module in AutoDock Vina software to fix the binding site residues at the centroid of the grid box, centering on the active site of the protein (center x = 68.231, center y = -9.75, center z = 45.696). Set the grid size to x = 22, y = 36, z = 38, which just covers the catalytic center residue, and save it as config.txt file after setting. About 7500 natural compounds were downloaded from the Topscience commercial natural products database. Firstly, the stable structure is constructed by using the MM2 force field to minimize energy in Chem 3D software, and it is saved in PDB format. Select it as ligand in AutoDock Vina software, keep all parameters default, and optimize all natural compounds in physical and chemical ways, including dehydration and hydrogenation, charge adjustment, ligand root determination and flexible torsion detection. The structures were then saved in pdbqt format for use in the Vina molecular docking program. After completing the preparation of receptor and ligand and the generation of grid box, docking was performed using the default parameters of AutoDock Vina. The results yielded nine default conformations for each ligand. Considering the maximum binding affinity of each docking ligand, the best binding conformation of the top 8 ligands was selected for 100 ns MDs to study the movement trajectory of Hp HtrA protein in the presence of natural compounds. 2.2.3. Molecular dynamics simulation (MDs) In this study, Amber 22 program was used to study molecular dynamics. Before MDs, the energy of these 8 natural compounds is minimized by HF/6-31G* optimization method in Gaussian 09, and the atomic partial charges were obtained from the electrostatic potential derived from Gaussian 09 by RESP fitting method in Amber 22. Antechamber and tleap modules are used to generate the parameters of generalized amber force field 2 (GAFF2) [29]. The complex system is placed in a periodic box composed of TIP3P water molecules, and the distance between the edge of the box and the solute surface is 10 Å. In order to restrain hydrogen bonds, SHAKE method is adopted. The whole system adopts Ff19SB position to parameterize the receptor, and neutralizes the residual charge in the complex by adding Na + or Cl − ions. The PMEMD.CUDA module in Amber 22 software package is used to perform MDs calculation [30, 31]. Sander module in Amber 22 is used for energy minimization and balance. Before MDs, the combination of steepest descent method and conjugate gradient method is used to minimize energy for each compound. The heating temperature is gradually raised from 0 to 303 K within 50 ps, and then enters the equilibrium stage and simulation stage at 303 K, with a time step of 2 fs. Use NVT (constant particle number, volume and temperature) ensemble to stabilize the density. Then start the unconstrained production stage, and run for 100 ns under the NPT ensemble of 1 atm and 303 K, saving 1000 conformations in total. Then, the equilibrium of receptor-ligand complex in MDs was evaluated by evaluating the root mean square deviation (RMSD), gyration radius (Rg), solvent accessible surface area (SASA), root mean square fluctuation of receptor (RMSF), distance of important residues and protein-ligand interaction parameters. The binding free energy of the simulation system is analyzed by using the script MMPBSA.py in Amber Tools software package. Based on the method of solvent accessibility and the force field of molecular mechanics, the interaction between each residue of protein and ligand was predicted by MDs sampling using MMPBSA (Poisson-Boltzmann surface area of molecular mechanics). Use Eq. (1) to calculate the total binding free energy. ∆G bind = G complex - G protein - G ligand (1) The free energy of protein-ligand complex and the free energy of separated protein and ligand are calculated by Eq. 2. ∆G bind = ∆E MM + ∆G PB + ∆G SA - T∆S = ΔE vdw + ΔE ele + ΔG PB + ΔG SA – TΔS (2) Among ∆G bind : free energy of binding; G complex : the free energy of the complex; G protein : the free energy of protein; G ligand : free energy of ligand; ΔE MM represents the gas phase (i.e. under vacuum) interaction energy between protein and ligand, which includes van der Waals ΔE vdw and electrostatic interaction ΔE ele ; ∆G PB : the difference between the polar solubility energy of protein-ligand complex and the sum of polar solvent energy of protein and ligand; ∆G SA : the difference between the nonpolar solubility free energy of the complex and the sum of the nonpolar solubility free energy of protein and ligand, T∆S: the entropy change of ligand binding conformation at T temperature. 2.3. Expression and purification of HtrA proteins The pET-28a(+)-HtrA expression plasmid used in this study was by Professor Wei Liu from the China National Research Center for Immunobiological Products, Army Medical University. The pET-28a (+)-HtrA plasmid was transformed into competent cells of E.coli BL21 (DE3) (Sangon Biotech, China) to express HtrA protein (His6). Bacteria were cultured in LuriaBertani broth supplemented with 50 µg/mL kanamycin at 37 ℃ until the OD 600 was in the range of 0.6 ~ 0.7, and then HtrA protein expression was induced by the addition of 0.3 mM isopropyl β-D-thiogalactopyranoside (IPTG) and incubated for 12 h at 16℃. The bacteria were collected by centrifugation at 8000 rpm for 10 min at 4℃. Bacteria were resuspended in buffer (25 mM Tris, 450 mM NaCl and 25 mM imidazole, pH 7.3) and lysed using a high-pressure cell crusher (ATS Engineering, Canada) until the bacterial fluid was clarified. The lysates were centrifuged and filtered, and the HtrA protein was purified by nickel affinity chromatography, and the samples were partitioned and stored at -20℃ for further use. Add 5× sodium dodecyl sulfate (SDS) buffer to the incubated samples and mix well. Boil at 95℃ for 5 min, subsequently separate the samples using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 15% polyacrylamide gel. Finally, the separated proteins were analyzed by staining with Coomassie Brilliant Blue Staining Solution G-250 (Beyotime Biotechnology, China). 2.4. Casein hydrolysis experiment A total of 20 µL of the hydrolysis reaction mixture was prepared by combining 5 µg of 5 µg β-casein (Solarbio, China), 1 µg HtrA and 50 mM HEPES (Sigma vetec, Germany), pH 7.5, followed by incubation in a 37°C water bath for 3 h. To halt substrate degradation, 5×SDS sample buffer was added, and the mixture was boiled at 95℃ for 5 min. The resulting products were analyzed using 15% SDS-PAGE and stained with Coomassie Brilliant Blue G-250. The data were processed and analyzed using Image J software and GraphPad Prism 9.1 (San Diego, USA). After confirming the hydrolytic activity of HtrA, Fisetin (Desite Biological Technology, China), Geniposide (Desite Biological Technology, China), and Quercetin (Desite Biological Technology, China) were prepared at a concentration of 200 µM and subsequently diluted to achieve final concentrations of 100 µM, 50 µM, 25 µM, and 12.5 µM. Another 20 µL hydrolysis reaction mixture was prepared by combining 5 µg of β-casein (Solarbio, China), 1 µg of HtrA, and the corresponding drug concentrations. This mixture was incubated in a 37℃ water bath for 3 h, then treated with 5×SDS and boiled at 95℃ for 5 min. The inhibitory effects of the drugs on the hydrolytic activity of HtrA protease were evaluated via SDS-PAGE and Coomassie Brilliant Blue G-250 staining. 2.5. Antimicrobial susceptibility tests in vitro The minimum inhibitory concentration (MIC) of Fisetin was determined using the broth microdilution method. In brief, 20 µL of Fisetin solution, with final concentrations ranging from 0.625 to 40 µM, and 80 µL of bacterial suspension (final concentration of 1 × 10⁶ CFU/mL) were added to and mixed in the wells of 96-well plate. The plates were then incubated in an anaerobic chamber at 37°C for 24 h, 48 h, and 72 h, respectively. Bacterial growth was monitored by measuring OD 600 using a microplate reader. The MIC was defined as the lowest concentration of Fisetin at which no visible microbial growth was detected. Amoxicillin (AMC, Huachu, China) at a final concentration of 0.125 mg/mL served as the positive control. 2.6. Statistical analysis The statistical analysis was conducted using a One-Way ANOVA (Bonferroni test) approach with Image J software and GraphPad Prism 9.1 (San Diego, USA). Statistical significance was defined as P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), and P ≤ 0.0001 (****). 3. Results and Discussion 3.1. The natural compounds with high affinity to HtrA Approximately 7,500 natural compounds were sourced from the Topscience commercial natural products database. Molecular docking was conducted using AutoDock Vina to evaluate the binding affinity between these compounds and the HtrA receptor, based on binding energy (ΔG, kcal/mol). The top eight compounds, selected for their docking scores below − 6 kcal/mol, were Camelliaside B, Rehmannioside A, Hesperidin, Quercetin, Fisetin, Colchicine, Apigenin, and Geniposide. Notably, all eight compounds have been previously reported in the literature for their anti-inflammatory, antibacterial, and gastric disease-related properties. Their binding energies with the HtrA protein ranged from − 10.9 to -6.7 kcal/mol (Table 1). The chemical structures of these compounds are shown in Fig. 2 . To further explore the interactions between these compounds and the HtrA protein, we performed molecular dynamics (MD) simulations and in vitro experimental validation. These analyses aim to provide deeper insights and expand the potential for drug discovery targeting HtrA. Table 1. The Affinity (kcal/mol) Information of 8 Natural Components with HtrA Protein No Name Cas Affinity (kcal/mol) Related diseases Reference 1 Camelliaside B 131573-90-5 -10.9 Antimicrobial, anti-oxidative, anti-inflammatory effects. [32] 2 Hesperidin 520-26-3 -9.1 Protective effects on oxidative stress, peptic ulcer and stomach injury in rats; Preventive effect of experimental model of acute lung inflammation; [33–39] 3 Rehmannioside A 81720-05-0 -8.0 Against Systemic Lupus Erythematosus;Psoriasis [40, 41] 4 Apigenin 8002-66-2 -7.5 Diabetes, amnesia and Alzheimer's disease, depression and insomnia; accelerate bone defects healing; Anti-proliferation of gastric cancer cells and inhibition of Helicobacter pylori colonization. [42–46] 5 Quercetin 117-39-5 -7.4 Inhibit liver and kidney damage; Therapeutic effect of gastrointestinal inflammation; Antitumor. [47–50] 6 Fisetin 345909-34-4 -7.0 Inducing apoptosis of head and neck cancer cells; Anti-cancer; Ulcerative colitis [51–53] 7 Colchicine 6962-03-4 -7.1 Gout, pericarditis and Behcet disease; High anti-proliferation and apoptosis-inducing effects on various types of cancer cell lines. [54–56] 8 Geniposide 24512-63-8 -6.7 Inhibition of helicobacter pylori; Alleviate acute kidney injury; Anti-inflammatory, anti-oxidative, anti-diabetic; Hepatoprotective; Cholagogic effects; Neuroprotective. [57–60] 3.2. Fisetin, Geniposide, and Quercetin exhibit high affinity for HtrA Molecular dynamics (MD) simulations, conducted for 100 ns using Amber 22, provided insights into the temporal fluctuations and conformational changes of the protein-ligand complexes, offering a deeper understanding of the interactions between the ligands and HtrA. As shown in Fig. 3 a, root mean square fluctuation (RMSF) analysis was used to assess the flexibility of the protein and the eight complexes. Most residues exhibited similar RMSF values, generally below 2 Å, indicating stable internal flexibility. However, in the Fisetin complex, conspicuous fluctuations were observed in the HtrA regions spanning GLU61–LYS92, GLY202–ILE210, and VAL350–LYS354, suggesting these regions may play functional roles in Fisetin binding. The dynamic stability of the complexes was evaluated through root mean square deviation (RMSD) of the protein backbone (Fig. 3 b, 3 c). After 20 ns, the RMSD values of most HtrA proteins stabilized, fluctuating between 3 and 6 Å, indicating that the natural compounds induced conformational stability in HtrA. Specifically, Fisetin, Geniposide, and Quercetin displayed RMSD values ranging from 0.5 to 3 Å, with average values of 0.91 Å, 1.51 Å, and 2.16 Å, respectively. The trajectory of Fisetin reached equilibrium after 10 ns, with the lowest and most stable RMSD values (0.43–1.26 Å), suggesting a more stable interaction with HtrA compared to other compounds. In contrast, compounds like Colchicine, Apigenin, and Rehmannioside A showed larger RMSD fluctuations, indicating less stable binding. Figure 3 d presents the radius of gyration (Rg), which reflects the compactness of the complexes. Fisetin, Geniposide, and Quercetin displayed lower Rg values (23.08 Å, 23.17 Å, and 23.36 Å), indicating more compact and stable structures. Similarly, solvent-accessible surface area (SASA) analysis revealed that the Fisetin and Colchicine complexes had lower SASA values (17991.78 ± 977.46 Ų and 18505.24 ± 1184.05 Ų), further supporting the higher stability of these complexes in the solvent environment (Fig. 3 e). Binding free energy was calculated using MM/PBSA to evaluate the affinity between the ligands and HtrA (Table 2 ). Fisetin, Geniposide, and Quercetin exhibited the lowest binding free energies (-82.61 kcal/mol, -73.70 kcal/mol, and − 68.33 kcal/mol, respectively), indicating the highest stability and strongest interactions with HtrA. These variations in binding energy are primarily attributed to electrostatic interactions (ΔE ele ) and polar solvation energy (ΔG PB ), with Fisetin showing weaker electrostatic interactions but a markedly higher polar solvent effect, suggesting a stronger affinity for the hydrophobic regions of the protein. Residue-level energy contribution analysis identified key amino acids involved in ligand binding (Fig. 3 f). For Fisetin, residues such as YAL140, ASP143, ILE271, and LEU139 played critical roles in stabilizing the complex through electrostatic and hydrophobic interactions. In Geniposide binding, residues GLU145, ARG334, and LEU333 were important contributors, while Quercetin formed key interactions with ASN416, GLN378, and GLY120. Figure 3 g and Fig. 4 show 2D and 3D interaction diagrams, respectively, confirming strong interactions between the ligands and HtrA. Collectively, the smaller RMSD, lower binding energy and higher docking scores all indicate that Fisetin, Geniposide and Quercetin exhibit the optimal affinity with the protein HtrA, all owing for stable binding. These three natural compounds may possess better HtrA inhibitory activity. Table 2 Analysis Table of Binding Free Energy of MM/PBSA of Different Natural Products (kcal/mol) Name ∆E vdw ∆E ele ∆G SA ∆G PB ∆G bind Fisetin -52.55 466.43 -82.61 -413.88 -82.61 Geniposide -55.02 386.03 -73.69 -331.02 -73.71 Quercetin -51.07 373.36 -75.33 -315.29 -68.33 Colchicine -67.31 480.52 -68.41 -403.21 -58.42 Hesperidin -56.38 585.80 -67.95 -516.32 -54.86 Camelliaside B -57.64 262.82 -54.92 -182.56 -32.30 Rehmannioside A -62.72 356.14 -85.40 -235.67 -27.66 Apigenin -54.30 548.97 -80.47 -434.67 -20.47 In the Table, ∆E vdw represents the van der Waals energy term; ∆E ele represents the electrostatic energy term; ∆G SA is the nonpolar energy term; ∆G PB represents the solvent energy term; ∆G bind represents the overall binding free energy. 3.3. Fisetin inhibits HtrA from hydrolyzing casein. The pET-28a(+)-HtrA plasmid was successfully transformed into BL21(DE3) cells, induced, expressed, purified, and concentrated. SDS-PAGE analysis confirmed that the molecular weight of the purified HtrA protein matched its theoretical size (Figure. S1a). Consistent with previous reports, casein was used as a substrate to assess the hydrolytic activity of HtrA in vitro. Incubation of 5 µg casein with 1 µg HtrA at 37°C for 3 hours demonstrated that HtrA effectively hydrolyzed casein, as verified by SDS-PAGE and coomassie brilliant blue staining (Figure. S1b). Molecular docking and dynamics simulations revealed stable interactions between HtrA and the natural compounds Fisetin, Geniposide, and Quercetin, contributing to the stability of the HtrA-ligand complex. We further tested the inhibitory effect of these three potential compounds against HtrA’s hydrolytic activity on casein. The results indicate that in the reaction system without HtrA or drugs, the molecular weight of β-casein is primarily concentrated in the range of 25 ~ 35 KD. However, after co-incubation with HtrA and β-casein, we observed the bands of 25 ~ 35 KD decreased evidently, while those of 15 ~ 25 KD increased clearly. Co-incubation with three natural compounds showed that Geniposide and Quercetin had no effect on the activity of HtrA, as there were no clear changes in the levels of β-casein or its hydrolysate (Figure. 5a–5f). In contrast, Fisetin exhibited a prominent concentration-dependent inhibition of HtrA’s casein hydrolytic activity. With the increase of the concentration of Fisetin, the inhibitory effect on HtrA activity became increasingly pronounced, which could markedly inhibit β-casein hydrolysis, mainly showing that the bands of 25 ~ 35 KD increased and the bands of 15 ~ 25 KD decreased (Figure. 5g–5i). 3.4. Fisetin inhibits the growth of Helicobacter pylori To further investigate Fisetin's impact on H. pylori growth, drug sensitivity assays were conducted at 24, 48, and 72 h. Fisetin effectively inhibited H. pylori growth, exhibiting a dose-dependent inhibitory effect. Amoxicillin at a final concentration of 0.125 mg/mL served as the positive control. The minimum inhibitory concentration required to achieve 80% inhibition (MIC₈₀) was determined to be 10 µM, with no difference observed over time (Fig. 6). These findings suggest that Fisetin, by inhibiting HtrA, holds potential as a therapeutic agent for H. pylori-related gastric diseases. 4. Discussion H. pylori infection is a major risk factor for the development of peptic ulcer disease, gastric adenocarcinoma, and a variety of other gastric and non-gastric conditions [61]. Currently, antibiotics are the primary treatment for H. pylori infections in clinical practice, and overall, the eradication of H. pylori has demonstrated satisfactory outcomes. However, the emergence of antibiotic resistance and the high recurrence rates associated with prolonged antibiotic therapy pose marked challenges[62–65]. Therefore, the identification of new therapeutic targets and the development of novel anti-H. pylori agents are urgently needed. HtrA is an important newly discovered active serine protease secreted by the gastric pathogen H. pylori [16]. HtrA has dual activity as both a molecular chaperone and serine protease within Helicobacter pylori. It plays a pivotal role in cellular protein quality control and is an essential gene product for the bacterium [66]. As a molecular chaperone, HtrA inhibits lysozyme aggregation in vitro, and the protein itself can be stabilized over a wide pH and temperature range [12]. In vivo, bacteria can refold or degrade misfolded proteins by overexpressing HtrA and other molecular chaperones to adapt to extreme environments, including prolonged heat stress, pH stress, high salt stress, oxidative stress, and reducing stress, and grow well even in the presence of antibiotics such as puromycin and metronidazole [7, 12, 67]. Based on the crucial role of HtrA in the pathogenesis of digestive tract-related diseases caused by H. pylori, along with its potential for therapeutic intervention and the current status of existing research, the development of drugs on targeting HtrA against H. pylori would be a valuable and meaningful drug discovery strategy. Currently, several inhibitors have already been identified that exhibit preliminary inhibitory activity against HtrA. Among them, Anna M Perna's research team developed the first HtrA peptide inhibitor by synthesizing cleavage site fragments and analogs that blocked the degradation of E-cadherin by H. pylori in gastric mucosal epithelial interstitial cells [68]. Jimin Hwang's research team found that the lead antichlamydial compound JO146 exhibited inhibitory activity against H.pylori with a minimum bactericidal concentration of 18.8 ~ 75.2 µg/mL. Furthermore, they found that the antimicrobial efficacy of JO146 was enhanced by encapsulating it in nanoparticles made from poly (lactic acid) - glycolic acid copolymers [69]. Sabine Bernegger et al. found that the combination of Zn and Cu ions effectively inhibits H. pylori HtrA activity [70]. Davoodbasha MubarakAli's research team docked H. pylori virulence factors by C-Docker algorithm and found that the derived peptide of the alga Tetradesmus sp had significant inhibitory activity against three virulence factors: CagA, VacA and HtrA [71]. Furthermore, regarding the HtrA target binding pocket, in addition to the active site cavity located around the catalytic Ser221, Anna Maria Perna et al. identified a second large pocket located at the interface between the PDZ1 structural domain and the serine protease domain of HtrA [72]. Notably, Krojer et al. found that the large cavity on the opposite side of the H6 helix, situated between the catalytic and PDZ1 structural domains, serves as a site for the metastable regulation of the hydrolytic chaperone activity of the DegP protein, which is the E. coli homologue of HtrA [73]. These results enhance our understanding of the functional role of this protease in bacterial infections and related mechanisms, while also provide new ideas for exploring HtrA-targeted therapies against H. pylori-related diseases. However, studies on natural compounds as HtrA inhibitors for H. pylori are quite limited. Despite the abundance of natural compounds that could offer more possibilities for H. pylori treatment, no natural compounds have been reported or screened as HtrA inhibitors. Therefore, the aim of this study was to identify effective and safe HtrA inhibitors derived from natural compounds in the hope of providing new ideas and approaches for the targeted treatment of H. pylori. Computational techniques for virtual screening based on docking have become an effective method for rapid discovery of new compounds from huge compound libraries [78]. In this study, a screening library was established using 7,500 commercially available natural compounds. The top 8 natural compounds with docking scores ≤ − 6 kcal /mol were selected based on molecular docking at the HtrA binding site using the AutoDock Vina docking method. To ensure the reliability of the experiments, 100 ns MDs were conducted after the virtual screening. Based on the RMSD, RMSF, Rg, SASA, and MMPBSA analyses of the composite system of the natural compounds with HtrA protein, the results indicated that the three natural compounds, Fisetin, Geniposide, and Quercetin exhibited the best affinity with HtrA and the composite system was more stable. By analyzing the energetic contributions of key amino acid residues at the HtrA active site and the interactions of the final conformation of the complex from 100 ns of MDs, we found that Fisetin exhibited the strongest interactions with HtrA's amino acid residues among the 8 selected natural compoundsnatural compoundsand the binding of Fisetin with HtrA was mainly mediated by electrostatic interactions, hydrophobic interactions, and van der Waals forces, with residues such as YAL140, ASP143, ILE271, LEU139, and THR142 playing crucial roles. This finding provides a basis for further screening of HtrA inhibitors. Subsequently, through experimental validation, we found that the binding of Fisetin to HtrA effectively inhibited HtrA-induced β-casein hydrolysis in a concentration-dependent manner. In addition, in vitro bacteriostatic assay showed that Fisetin inhibited the growth of H. pylori in a concentration-dependent manner, with a MIC 80 of 10 µM. In conclusion, our results indicate that Fisetin has a certain anti-H. pylori ability and inhibits the activity of HtrA to a certain extent. Notably, Huiqin Zhuo et al. found that Fisetin (10 µM) signally reduced TAGLN2-induced cytoplasmic ssDNA accumulation by inhibiting the TAGLN2-YBX1-AKT interaction, thereby enhancing the therapeutic sensitivity of gastric cancer [79]. Akash Sabarwal et al. demonstrate that Fisetin inhibits cell proliferation and induces mitochondria-dependent apoptosis in human gastric cancer cells [80]. Fisetin, a polyphenol with pleiotropic pharmacological properties, has shown promising anticancer activity in various types of cancers, including gastric, colon, breast, and liver cancers, and also possesses some antibacterial activity [81–83]. This provides some theoretical basis for the reliability of our experimental results, allowing us to continue screening HtrA inhibitors using the research methodology outlined in this paper to identify potential drugs for the eradication of HtrA. Meanwhile, there are some limitations in our study, on the one hand, we utilized a limited natural product library that does not comprehensively cover the diversity of natural compounds, on the other hand, the in vivo anti-H. pylori activity of Fisetin requires further investigation. 5. Conclusions Based on the findings from molecular docking, molecular dynamics simulations, and casein hydrolysis experiments, we discovered that Fisetin not only exhibits a high affinity for HtrA but also observably inhibits its hydrolytic activity, suggesting that Fisetin has potential as an inhibitor of HtrA. Additionally, we validated through MIC assays that Fisetin can suppress the growth of H. pylori. In summary, Fisetin may serve as a potential inhibitor of HtrA, potentially eliminating H. pylori infections by inhibiting the hydrolytic activity of HtrA and suppressing bacterial proliferation. This study integrates computational biology with experimental methods, opening new pathways for the exploration of targeted therapeutic agents against H. pylori infections. Declarations Author contributions Experimental Methods and Approaches: Li Gao, Xianqiong Jiang, Yan Shen, Kuilong Huang, Zhihua Lin; Literature Review and Investigation: Li Gao, Xianqiong Jiang, Yan Shen, Kuilong Huang; Experimental Procedures, Data Analysis, and Manuscript Preparation: Li Gao, Xianqiong Jiang, Hongtao Duan, and Rui Zhang; Experimental Supervision and Guidance: Yan Shen, Kuilong Huang, Kui Gu, Yuanqiang Wang, Mao Shu; Manuscript Revision: Yan Shen; Funding Support: Yan Shen and Zhihua Lin. All authors read and approved the final manuscript. Declaration of Interest Statemen The authors declare that there are no conflicts of interest related to this work. All authors have reviewed the manuscript and approved its submission. Funding This work was supported by natural Science Foundation of Chongqing China (CSTB2022NSCQ-MSX1493), Chongqing University of Technology Postgraduate Quality Development Action Plan Funding Results (gzlcx 20243219). 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Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5316707","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":373456778,"identity":"a3336b1d-f56e-47c3-b767-3cffa65e4577","order_by":0,"name":"Li Gao","email":"","orcid":"","institution":"Chongqing University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Gao","suffix":""},{"id":373456779,"identity":"7b0dcb32-05ad-48b9-936c-89ca8014fe44","order_by":1,"name":"Xianqiong Jiang","email":"","orcid":"","institution":"Chongqing University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Xianqiong","middleName":"","lastName":"Jiang","suffix":""},{"id":373456780,"identity":"c0df40b4-acc0-4ab7-8f74-bb0e90a48052","order_by":2,"name":"Hongtao Duan","email":"","orcid":"","institution":"Chongqing University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Hongtao","middleName":"","lastName":"Duan","suffix":""},{"id":373456781,"identity":"08005ccc-1f95-474a-96e2-624a6ad00b64","order_by":3,"name":"Yan Shen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYDACCQYGZiAlx8befIA0LcZ8PMcSSNOSOE8iR4E4HfKzm489Lsw5nN7GkMPA8KNiG2EtBneOpRvP3HY4t43h7AHGnjO3idAikWMmzQvSwtiXwMzYRoQW+Rn530Ba0tmYeQyI08JwI4cNpCWBjY1YLQY30sykZ25LN2zjYUs4SJRf5GckP5Mu3GYtLz//8cEHPyqIcRgENIPJA0SrB4I6UhSPglEwCkbBSAMA9ec5IWZu/okAAAAASUVORK5CYII=","orcid":"","institution":"Chongqing University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Yan","middleName":"","lastName":"Shen","suffix":""},{"id":373456782,"identity":"250c5c6b-f5a0-4207-a6bf-99437bd13d57","order_by":4,"name":"Kui Gu","email":"","orcid":"","institution":"Chongqing University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Kui","middleName":"","lastName":"Gu","suffix":""},{"id":373456783,"identity":"9403d2c2-78ce-4042-9d55-07e67472133a","order_by":5,"name":"Kuilong Huang","email":"","orcid":"","institution":"Chongqing University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Kuilong","middleName":"","lastName":"Huang","suffix":""},{"id":373456784,"identity":"b0b965ad-243f-4c92-8bcd-eedc43d75f05","order_by":6,"name":"Yuanqiang Wang","email":"","orcid":"","institution":"Chongqing University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yuanqiang","middleName":"","lastName":"Wang","suffix":""},{"id":373456785,"identity":"d2802cf6-0530-42d1-880d-caf7a51e62ac","order_by":7,"name":"Mao Shu","email":"","orcid":"","institution":"Chongqing University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Mao","middleName":"","lastName":"Shu","suffix":""},{"id":373456786,"identity":"50082032-afd8-4e53-bf73-33a211cf612f","order_by":8,"name":"Rui Zhang","email":"","orcid":"","institution":"Guizhou Provincial People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Zhang","suffix":""},{"id":373456787,"identity":"1e806e6b-c2c2-4d36-87a1-51863e527c16","order_by":9,"name":"Zhihua Lin","email":"","orcid":"","institution":"Chongqing University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Zhihua","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2024-10-23 07:38:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5316707/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5316707/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11030-025-11137-2","type":"published","date":"2025-02-23T15:57:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68253594,"identity":"eb8f342e-b7a5-46ff-81e3-20cf41434382","added_by":"auto","created_at":"2024-11-05 10:33:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":228032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCrystal structure diagram of HP HtrA. \u003c/strong\u003e(a) The whole structure of HP HtrA monomer includes protease domain and PDZ1 and PDZ2 domains. (b) HtrA protease domain, the active site is composed of three catalytic residues: Ser221, His116 and Asp147.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5316707/v1/330e61be182de19f7107ba2f.png"},{"id":68253588,"identity":"a1b94782-2a56-4687-8797-cf6698ab4de3","added_by":"auto","created_at":"2024-11-05 10:33:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":117068,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructures of 8 components selected by virtual screening\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5316707/v1/51728cf2d74beae616eb4f4a.png"},{"id":68253589,"identity":"eb9991bd-e062-4436-955b-2c8792a2be58","added_by":"auto","created_at":"2024-11-05 10:33:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":468802,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding stability analysis of 8 natural compounds in 100 ns MDs. \u003c/strong\u003e(a) RMSF of complexes with 8 natural compounds; (b) RMSD of HtrA; (c) RMSD of ligand in the pock; (d) Rg of complexes with 8 natural compounds; (e) SASA of complexes with 8 natural compounds; (f) MM/PBSA calculated free energy contribution of 10 critical residues; (g) 2D interaction diagram of the last conformation of the composite system from 100 ns MDs.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5316707/v1/7d99b97e1628ea2945953fc5.png"},{"id":68254319,"identity":"c320b8e4-ae4d-472c-87ca-401d44bd048a","added_by":"auto","created_at":"2024-11-05 10:41:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":737359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe 3D interaction diagram of the last conformation of the composite system from 100 ns MDs.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5316707/v1/970ad6257b7f80869e4e7c61.png"},{"id":68253590,"identity":"afd34e7f-4adc-4a8e-911f-dbc0b9d635d3","added_by":"auto","created_at":"2024-11-05 10:33:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":287443,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of Fisetin, Geniposide, and Quercetin on the hydrolysis of β-casein by HtrA. A hydrolysis system was prepared by mixing 5 μg β-casein, 1 μg of HtrA, and the respective drug concentrations, resulting in a total volume of 20 μL. The mixture was incubated in a water bath at 37°C for 3 h. 5× SDS sample buffer was added, and the mixture was boiled at 95°C for 5 minutes. The degradation products were analyzed using 15% SDS-PAGE and stained with Coomassie Brilliant Blue G-250. (a) ~ (c) effect of Geniposide on hydrolysis of casein by HtrA; (d) ~ (f) effect of Quercetin on hydrolysis of\u003cstrong\u003e \u003c/strong\u003ecasein by; (g) ~ (i) effect of Fisetin on hydrolysis of casein by HtrA\u003cstrong\u003e. \u003c/strong\u003eThe red arrows indicate the hydrolysis products of casein, while the yellow arrows represent the casein that has not undergone hydrolysis. All data are presented as Mean ± SD. *, **, ***, and **** indicate \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, and \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001, respectively.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5316707/v1/5a2e1c6cc4362b19c21a2bec.png"},{"id":68254318,"identity":"2627b862-858e-4aec-ab44-94ea7723e3ad","added_by":"auto","created_at":"2024-11-05 10:41:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":43147,"visible":true,"origin":"","legend":"\u003cp\u003eInhibitory effect of Fisetin on the growth of Helicobacter pylori 26695 strain inhibition rates of H. pylori by Fisetin at various concentrations (0.625 μM to 40 μM) following 24 h (a) 48 h (b) and 72 h (c) of treatment. Amoxicillin at a final concentration of 0.125 mg/mL served as the positive control. All data are presented as Mean ± SD.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5316707/v1/ded42a9593d5f60b9223c99a.png"},{"id":77052591,"identity":"05cd0a9f-959f-488a-a8f8-6d29e8a86190","added_by":"auto","created_at":"2025-02-24 16:16:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2715970,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5316707/v1/ca3047e5-a56c-4d43-8367-551a81cb475f.pdf"},{"id":68253595,"identity":"3d9636b4-d3d5-4562-a22c-413d3959c798","added_by":"auto","created_at":"2024-11-05 10:33:37","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":15944006,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5316707/v1/40942d34d1d04a27a2fc6968.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular dynamics simulation-driven focused virtual screening and experimental validation of Fisetin as an inhibitor of Helicobacter pylori HtrA protease","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGastric cancer ranks as the fifth most common cancer globally and is the third leading cause of cancer-related mortality [1]. Helicobacter pylori (H. pylori), a spiral-shaped, microaerophilic, gram-negative bacterium, is a major risk factor for severe gastric disorders such as chronic gastritis, peptic ulcers, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer [2, 3]. While antibiotic therapy remains the cornerstone of H. pylori treatment, its effectiveness is diminishing due to the increasing prevalence of antibiotic resistance. This resistance arises from mutations in bacterial proteins, reduced membrane permeability, and the activation of efflux pumps, which together lower drug efficacy. The bacterium\u0026rsquo;s persistent colonization of the gastric mucosa underlies its role in various gastrointestinal diseases [4]. Therefore, novel therapeutic strategies targeting H. pylori's pathogenesis are urgently needed.\u003c/p\u003e \u003cp\u003eH. pylori releases various virulence factors that enable it to invade, colonize, and infect the gastric mucosa, leading to conditions such as chronic gastritis and gastric cancer [5, 6]. One key virulence factor is High-temperature requirement A (HtrA), a multifunctional protein with an N-terminal protease domain and two C-terminal PDZ (postsynaptic density of 95 kD, Discs large and zonula occludens 1) domains [7, 8]. HtrA functions as both a chaperone and a protease, playing a vital role in protein quality control by managing the folding and degradation of misfolded proteins [9, 10]. This capability is essential for the survival and colonization of H. pylori under stress conditions like heat shock, extreme pH, and high salt concentrations, making HtrA a promising target for the development of anti-H. pylori therapies [11\u0026ndash;14]. In addition to its protein quality control functions, HtrA cleaves key epithelial adhesion proteins, such as occludin, claudin-8, E-cadherin, and desmoglein-2, disrupting the epithelial barrier and facilitating H. pylori invasion [9, 12, 15\u0026ndash;17]. Notably, the cleavage of E-cadherin not only weakens cell-cell junctions but also activates β-catenin, a transcription factor involved in cell proliferation, and enhances pro-inflammatory responses via NF-κB signaling [18]. Furthermore, HtrA has been shown to enhance the type IV secretion system (T4SS)-mediated translocation of the oncogenic protein CagA into host cells, exacerbating gastric disease development [19]. Given its pivotal role in pathogenesis, HtrA represents a critical target for novel anti-H. pylori drug development.\u003c/p\u003e \u003cp\u003eVirtual screening has become a key tool in computer-aided drug design (CADD), allowing for the efficient identification of biologically active compounds against specific molecular targets [20]. Qualcomm Dose offers a rapid and effective approach to screen potential drug candidates, serving as a valuable supplement or alternative to traditional experimental methods [21]. In recent years, extensive research has led to the discovery of numerous natural compounds with important medicinal properties, showing promising results in anti-cancer, anti-inflammatory, and antiviral therapies [22, 23].\u003c/p\u003e \u003cp\u003ePrevious studies have modeled the three-dimensional structure of HtrA, establishing its stable conformation and utilizing it as a receptor in virtual screening to identify potential lead compounds. For example, Gisbert Schneider's group introduced the virtual ligand concept to identify inhibitors of H. pylori HtrA, while R. Amutha, Tiago Rodrigues, and colleagues demonstrated inhibitory effects of selected lead compounds on HtrA secretion [17, 24\u0026ndash;26]. Despite these advances, no clinically approved drugs or vaccines targeting HtrA have been developed, largely due to the protease\u0026rsquo;s complex structure. Recently, Professor Wei Liu\u0026rsquo;s team made a significant breakthrough by elucidating the monomer structure of the HtrA family and discovering a pH-dependent dynamic transition between trimeric and monomeric forms, accompanied by conformational changes [7]. These findings offer critical insights into the protease\u0026rsquo;s function in bacterial infections and lay the groundwork for drug discovery efforts targeting HtrA to treat H. pylori-related diseases.\u003c/p\u003e \u003cp\u003eIn this study, we conducted virtual screening of a natural product database using molecular docking and molecular dynamics simulations, identifying Fisetin, Geniposide, and Quercetin as promising HtrA inhibitors. Further validation through casein hydrolysis and drug sensitivity assays confirmed that Fisetin not only inhibits H. pylori growth but also exhibits anti-HtrA activity, supporting the reliability of our virtual screening results.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. H. pylori strains and culture conditions\u003c/h2\u003e\n \u003cp\u003eThe H. pylori reference strain 26695 used in this study was kindly donated by Professor Zou Quanming from the China National Research Center for immunobiological Products, Army Medical University. Hp was cultured in Hp liquid medium supplemented with 10% fetal bovine serum (FBS; BasalMedia, Shanghai China) at 37\u0026deg;C under microaerobic conditions. The culture period lasted for 3 to 4 days.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Screening of natural compounds HtrA inhibitors\u003c/h2\u003e\n \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.1. Receptor preparation\u003c/h2\u003e\n \u003cp\u003eThe receptor protein structure was downloaded from the PDB website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.rcsb.org\u003c/span\u003e\u003c/span\u003e), and the protein PDB ID was 7xs2. The protein structure is shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea, which mainly includes serine protease domain (amino acid residue sequence is 43\u0026ndash;272), PDZ1 (amino acid residue sequence is 273\u0026ndash;365) and PDZ2 domain (amino acid residue sequence is 366\u0026ndash;475) [27, 28]. The macromolecule HtrA in the PDB is a protein that contains solvent molecules and non-standard residues. Therefore, it must be preprocessed and optimized by using AutoDock Vina tools, including removing water molecules, adding hydrogen atoms, repairing charges, adding force fields, applying energy minimization, and finally saving the structure in pdbqt format.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.2. Receptor grid generation and AutoDock Vina virtual screening\u003c/h2\u003e\n \u003cp\u003eThe latest research found that the catalytic center composed of His116, Asp147 and Ser221 residues located in the protease domain of helicobacter pylori HtrA plays an important role in substrate degradation activity [7]. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb. Therefore, we regard these three residues as the binding sites of HtrA protein and ligand. Then, we use the Grid module in AutoDock Vina software to fix the binding site residues at the centroid of the grid box, centering on the active site of the protein (center x\u0026thinsp;=\u0026thinsp;68.231, center y = -9.75, center z\u0026thinsp;=\u0026thinsp;45.696). Set the grid size to x\u0026thinsp;=\u0026thinsp;22, y\u0026thinsp;=\u0026thinsp;36, z\u0026thinsp;=\u0026thinsp;38, which just covers the catalytic center residue, and save it as config.txt file after setting. About 7500 natural compounds were downloaded from the Topscience commercial natural products database. Firstly, the stable structure is constructed by using the MM2 force field to minimize energy in Chem 3D software, and it is saved in PDB format. Select it as ligand in AutoDock Vina software, keep all parameters default, and optimize all natural compounds in physical and chemical ways, including dehydration and hydrogenation, charge adjustment, ligand root determination and flexible torsion detection. The structures were then saved in pdbqt format for use in the Vina molecular docking program. After completing the preparation of receptor and ligand and the generation of grid box, docking was performed using the default parameters of AutoDock Vina. The results yielded nine default conformations for each ligand. Considering the maximum binding affinity of each docking ligand, the best binding conformation of the top 8 ligands was selected for 100 ns MDs to study the movement trajectory of Hp HtrA protein in the presence of natural compounds.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.3. Molecular dynamics simulation (MDs)\u003c/h2\u003e\n \u003cp\u003eIn this study, Amber 22 program was used to study molecular dynamics. Before MDs, the energy of these 8 natural compounds is minimized by HF/6-31G* optimization method in Gaussian 09, and the atomic partial charges were obtained from the electrostatic potential derived from Gaussian 09 by RESP fitting method in Amber 22. Antechamber and tleap modules are used to generate the parameters of generalized amber force field 2 (GAFF2) [29]. The complex system is placed in a periodic box composed of TIP3P water molecules, and the distance between the edge of the box and the solute surface is 10 \u0026Aring;. In order to restrain hydrogen bonds, SHAKE method is adopted. The whole system adopts Ff19SB position to parameterize the receptor, and neutralizes the residual charge in the complex by adding Na\u003csup\u003e+\u003c/sup\u003e or Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e ions. The PMEMD.CUDA module in Amber 22 software package is used to perform MDs calculation [30, 31]. Sander module in Amber 22 is used for energy minimization and balance. Before MDs, the combination of steepest descent method and conjugate gradient method is used to minimize energy for each compound. The heating temperature is gradually raised from 0 to 303 K within 50 ps, and then enters the equilibrium stage and simulation stage at 303 K, with a time step of 2 fs. Use NVT (constant particle number, volume and temperature) ensemble to stabilize the density. Then start the unconstrained production stage, and run for 100 ns under the NPT ensemble of 1 atm and 303 K, saving 1000 conformations in total. Then, the equilibrium of receptor-ligand complex in MDs was evaluated by evaluating the root mean square deviation (RMSD), gyration radius (Rg), solvent accessible surface area (SASA), root mean square fluctuation of receptor (RMSF), distance of important residues and protein-ligand interaction parameters.\u003c/p\u003e\n \u003cp\u003eThe binding free energy of the simulation system is analyzed by using the script MMPBSA.py in Amber Tools software package. Based on the method of solvent accessibility and the force field of molecular mechanics, the interaction between each residue of protein and ligand was predicted by MDs sampling using MMPBSA (Poisson-Boltzmann surface area of molecular mechanics). Use Eq.\u0026nbsp;(1) to calculate the total binding free energy.\u003c/p\u003e\n \u003cp\u003e∆G\u003csub\u003ebind\u003c/sub\u003e = G\u003csub\u003ecomplex\u003c/sub\u003e - G\u003csub\u003eprotein\u003c/sub\u003e - G\u003csub\u003eligand\u003c/sub\u003e (1)\u003c/p\u003e\n \u003cp\u003eThe free energy of protein-ligand complex and the free energy of separated protein and ligand are calculated by Eq.\u0026nbsp;2.\u003c/p\u003e\n \u003cp\u003e∆G\u003csub\u003ebind\u003c/sub\u003e = ∆E\u003csub\u003eMM\u003c/sub\u003e + ∆G\u003csub\u003ePB\u003c/sub\u003e + ∆G\u003csub\u003eSA\u003c/sub\u003e - T∆S\u003c/p\u003e\n \u003cp\u003e= \u0026Delta;E\u003csub\u003evdw\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;\u0026Delta;E\u003csub\u003eele\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;\u0026Delta;G\u003csub\u003ePB\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;\u0026Delta;G\u003csub\u003eSA\u003c/sub\u003e \u0026ndash; T\u0026Delta;S (2)\u003c/p\u003e\n \u003cp\u003eAmong ∆G\u003csub\u003ebind\u003c/sub\u003e: free energy of binding; G\u003csub\u003ecomplex\u003c/sub\u003e: the free energy of the complex; G\u003csub\u003eprotein\u003c/sub\u003e: the free energy of protein; G\u003csub\u003eligand\u003c/sub\u003e: free energy of ligand; \u0026Delta;E\u003csub\u003eMM\u003c/sub\u003e represents the gas phase (i.e. under vacuum) interaction energy between protein and ligand, which includes van der Waals \u0026Delta;E\u003csub\u003evdw\u003c/sub\u003e and electrostatic interaction \u0026Delta;E\u003csub\u003eele\u003c/sub\u003e; ∆G\u003csub\u003ePB\u003c/sub\u003e: the difference between the polar solubility energy of protein-ligand complex and the sum of polar solvent energy of protein and ligand; ∆G\u003csub\u003eSA\u003c/sub\u003e: the difference between the nonpolar solubility free energy of the complex and the sum of the nonpolar solubility free energy of protein and ligand, T∆S: the entropy change of ligand binding conformation at T temperature.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Expression and purification of HtrA proteins\u003c/h2\u003e\n \u003cp\u003eThe pET-28a(+)-HtrA expression plasmid used in this study was by Professor Wei Liu from the China National Research Center for Immunobiological Products, Army Medical University. The pET-28a (+)-HtrA plasmid was transformed into competent cells of E.coli BL21 (DE3) (Sangon Biotech, China) to express HtrA protein (His6). Bacteria were cultured in LuriaBertani broth supplemented with 50 \u0026micro;g/mL kanamycin at 37 ℃ until the OD\u003csub\u003e600\u003c/sub\u003e was in the range of 0.6\u0026thinsp;~\u0026thinsp;0.7, and then HtrA protein expression was induced by the addition of 0.3 mM isopropyl \u0026beta;-D-thiogalactopyranoside (IPTG) and incubated for 12 h at 16℃. The bacteria were collected by centrifugation at 8000 rpm for 10 min at 4℃. Bacteria were resuspended in buffer (25 mM Tris, 450 mM NaCl and 25 mM imidazole, pH 7.3) and lysed using a high-pressure cell crusher (ATS Engineering, Canada) until the bacterial fluid was clarified. The lysates were centrifuged and filtered, and the HtrA protein was purified by nickel affinity chromatography, and the samples were partitioned and stored at -20℃ for further use. Add 5\u0026times; sodium dodecyl sulfate (SDS) buffer to the incubated samples and mix well. Boil at 95℃ for 5 min, subsequently separate the samples using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 15% polyacrylamide gel. Finally, the separated proteins were analyzed by staining with Coomassie Brilliant Blue Staining Solution G-250 (Beyotime Biotechnology, China).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Casein hydrolysis experiment\u003c/h2\u003e\n \u003cp\u003eA total of 20 \u0026micro;L of the hydrolysis reaction mixture was prepared by combining 5 \u0026micro;g of 5 \u0026micro;g \u0026beta;-casein (Solarbio, China), 1 \u0026micro;g HtrA and 50 mM HEPES (Sigma vetec, Germany), pH 7.5, followed by incubation in a 37\u0026deg;C water bath for 3 h. To halt substrate degradation, 5\u0026times;SDS sample buffer was added, and the mixture was boiled at 95℃ for 5 min. The resulting products were analyzed using 15% SDS-PAGE and stained with Coomassie Brilliant Blue G-250. The data were processed and analyzed using Image J software and GraphPad Prism 9.1 (San Diego, USA). After confirming the hydrolytic activity of HtrA, Fisetin (Desite Biological Technology, China), Geniposide (Desite Biological Technology, China), and Quercetin (Desite Biological Technology, China) were prepared at a concentration of 200 \u0026micro;M and subsequently diluted to achieve final concentrations of 100 \u0026micro;M, 50 \u0026micro;M, 25 \u0026micro;M, and 12.5 \u0026micro;M. Another 20 \u0026micro;L hydrolysis reaction mixture was prepared by combining 5 \u0026micro;g of \u0026beta;-casein (Solarbio, China), 1 \u0026micro;g of HtrA, and the corresponding drug concentrations. This mixture was incubated in a 37℃ water bath for 3 h, then treated with 5\u0026times;SDS and boiled at 95℃ for 5 min. The inhibitory effects of the drugs on the hydrolytic activity of HtrA protease were evaluated via SDS-PAGE and Coomassie Brilliant Blue G-250 staining.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Antimicrobial susceptibility tests in vitro\u003c/h2\u003e\n \u003cp\u003eThe minimum inhibitory concentration (MIC) of Fisetin was determined using the broth microdilution method. In brief, 20 \u0026micro;L of Fisetin solution, with final concentrations ranging from 0.625 to 40 \u0026micro;M, and 80 \u0026micro;L of bacterial suspension (final concentration of 1 \u0026times; 10⁶ CFU/mL) were added to and mixed in the wells of 96-well plate. The plates were then incubated in an anaerobic chamber at 37\u0026deg;C for 24 h, 48 h, and 72 h, respectively. Bacterial growth was monitored by measuring OD\u003csub\u003e600\u003c/sub\u003e using a microplate reader. The MIC was defined as the lowest concentration of Fisetin at which no visible microbial growth was detected. Amoxicillin (AMC, Huachu, China) at a final concentration of 0.125 mg/mL served as the positive control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6. Statistical analysis\u003c/h2\u003e\n \u003cp\u003eThe statistical analysis was conducted using a One-Way ANOVA (Bonferroni test) approach with Image J software and GraphPad Prism 9.1 (San Diego, USA). Statistical significance was defined as \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 (*), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01 (**), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001 (***), and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.0001 (****).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. The natural compounds with high affinity to HtrA\u003c/h2\u003e \u003cp\u003eApproximately 7,500 natural compounds were sourced from the Topscience commercial natural products database. Molecular docking was conducted using AutoDock Vina to evaluate the binding affinity between these compounds and the HtrA receptor, based on binding energy (ΔG, kcal/mol). The top eight compounds, selected for their docking scores below \u0026minus;\u0026thinsp;6 kcal/mol, were Camelliaside B, Rehmannioside A, Hesperidin, Quercetin, Fisetin, Colchicine, Apigenin, and Geniposide. Notably, all eight compounds have been previously reported in the literature for their anti-inflammatory, antibacterial, and gastric disease-related properties. Their binding energies with the HtrA protein ranged from \u0026minus;\u0026thinsp;10.9 to -6.7 kcal/mol (Table\u0026nbsp;1). The chemical structures of these compounds are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. To further explore the interactions between these compounds and the HtrA protein, we performed molecular dynamics (MD) simulations and in vitro experimental validation. These analyses aim to provide deeper insights and expand the potential for drug discovery targeting HtrA.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eTable\u0026nbsp;1. The Affinity (kcal/mol) Information of 8 Natural Components with HtrA Protein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCas\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAffinity (kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRelated diseases\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCamelliaside B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e131573-90-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-10.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAntimicrobial, anti-oxidative, anti-inflammatory effects.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[32]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHesperidin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e520-26-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-9.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProtective effects on oxidative stress, peptic ulcer and stomach injury in rats; Preventive effect of experimental model of acute lung inflammation;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[33\u0026ndash;39]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRehmannioside A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e81720-05-0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAgainst Systemic Lupus Erythematosus;Psoriasis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[40, 41]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApigenin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e8002-66-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDiabetes, amnesia and Alzheimer's disease, depression and insomnia; accelerate bone defects healing; Anti-proliferation of gastric cancer cells and inhibition of Helicobacter pylori colonization.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[42\u0026ndash;46]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQuercetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e117-39-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-7.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInhibit liver and kidney damage; Therapeutic effect of gastrointestinal inflammation; Antitumor.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[47\u0026ndash;50]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFisetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e345909-34-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInducing apoptosis of head and neck cancer cells; Anti-cancer; Ulcerative colitis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[51\u0026ndash;53]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColchicine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e6962-03-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGout, pericarditis and Behcet disease; High anti-proliferation and apoptosis-inducing effects on various types of cancer cell lines.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[54\u0026ndash;56]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeniposide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e24512-63-8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-6.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInhibition of helicobacter pylori; Alleviate acute kidney injury; Anti-inflammatory, anti-oxidative, anti-diabetic; Hepatoprotective; Cholagogic effects; Neuroprotective.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[57\u0026ndash;60]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Fisetin, Geniposide, and Quercetin exhibit high affinity for HtrA\u003c/h2\u003e \u003cp\u003eMolecular dynamics (MD) simulations, conducted for 100 ns using Amber 22, provided insights into the temporal fluctuations and conformational changes of the protein-ligand complexes, offering a deeper understanding of the interactions between the ligands and HtrA. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, root mean square fluctuation (RMSF) analysis was used to assess the flexibility of the protein and the eight complexes. Most residues exhibited similar RMSF values, generally below 2 \u0026Aring;, indicating stable internal flexibility. However, in the Fisetin complex, conspicuous fluctuations were observed in the HtrA regions spanning GLU61\u0026ndash;LYS92, GLY202\u0026ndash;ILE210, and VAL350\u0026ndash;LYS354, suggesting these regions may play functional roles in Fisetin binding.\u003c/p\u003e \u003cp\u003eThe dynamic stability of the complexes was evaluated through root mean square deviation (RMSD) of the protein backbone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb,\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). After 20 ns, the RMSD values of most HtrA proteins stabilized, fluctuating between 3 and 6 \u0026Aring;, indicating that the natural compounds induced conformational stability in HtrA. Specifically, Fisetin, Geniposide, and Quercetin displayed RMSD values ranging from 0.5 to 3 \u0026Aring;, with average values of 0.91 \u0026Aring;, 1.51 \u0026Aring;, and 2.16 \u0026Aring;, respectively. The trajectory of Fisetin reached equilibrium after 10 ns, with the lowest and most stable RMSD values (0.43\u0026ndash;1.26 \u0026Aring;), suggesting a more stable interaction with HtrA compared to other compounds. In contrast, compounds like Colchicine, Apigenin, and Rehmannioside A showed larger RMSD fluctuations, indicating less stable binding.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed presents the radius of gyration (Rg), which reflects the compactness of the complexes. Fisetin, Geniposide, and Quercetin displayed lower Rg values (23.08 \u0026Aring;, 23.17 \u0026Aring;, and 23.36 \u0026Aring;), indicating more compact and stable structures. Similarly, solvent-accessible surface area (SASA) analysis revealed that the Fisetin and Colchicine complexes had lower SASA values (17991.78\u0026thinsp;\u0026plusmn;\u0026thinsp;977.46 \u0026Aring;\u0026sup2; and 18505.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1184.05 \u0026Aring;\u0026sup2;), further supporting the higher stability of these complexes in the solvent environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eBinding free energy was calculated using MM/PBSA to evaluate the affinity between the ligands and HtrA (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Fisetin, Geniposide, and Quercetin exhibited the lowest binding free energies (-82.61 kcal/mol, -73.70 kcal/mol, and \u0026minus;\u0026thinsp;68.33 kcal/mol, respectively), indicating the highest stability and strongest interactions with HtrA. These variations in binding energy are primarily attributed to electrostatic interactions (ΔE\u003csub\u003eele\u003c/sub\u003e) and polar solvation energy (ΔG\u003csub\u003ePB\u003c/sub\u003e), with Fisetin showing weaker electrostatic interactions but a markedly higher polar solvent effect, suggesting a stronger affinity for the hydrophobic regions of the protein.\u003c/p\u003e \u003cp\u003eResidue-level energy contribution analysis identified key amino acids involved in ligand binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). For Fisetin, residues such as YAL140, ASP143, ILE271, and LEU139 played critical roles in stabilizing the complex through electrostatic and hydrophobic interactions. In Geniposide binding, residues GLU145, ARG334, and LEU333 were important contributors, while Quercetin formed key interactions with ASN416, GLN378, and GLY120. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e show 2D and 3D interaction diagrams, respectively, confirming strong interactions between the ligands and HtrA.\u003c/p\u003e \u003cp\u003eCollectively, the smaller RMSD, lower binding energy and higher docking scores all indicate that Fisetin, Geniposide and Quercetin exhibit the optimal affinity with the protein HtrA, all owing for stable binding. These three natural compounds may possess better HtrA inhibitory activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalysis Table of Binding Free Energy of MM/PBSA of Different Natural Products (kcal/mol)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e∆E\u003csub\u003evdw\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e∆E\u003csub\u003eele\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e∆G\u003csub\u003eSA\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e∆G\u003csub\u003ePB\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e∆G\u003csub\u003ebind\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFisetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-52.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e466.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-82.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-413.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-82.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGeniposide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-55.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e386.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-73.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-331.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-73.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuercetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-51.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e373.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-75.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-315.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-68.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColchicine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-67.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e480.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-68.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-403.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-58.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHesperidin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-56.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e585.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-67.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-516.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-54.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCamelliaside B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-57.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e262.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-54.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-182.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-32.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRehmannioside A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-62.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e356.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-85.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-235.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-27.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eApigenin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-54.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e548.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-80.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-434.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-20.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn the Table, ∆E\u003csub\u003evdw\u003c/sub\u003e represents the van der Waals energy term; ∆E\u003csub\u003eele\u003c/sub\u003e represents the electrostatic energy term; ∆G\u003csub\u003eSA\u003c/sub\u003e is the nonpolar energy term; ∆G\u003csub\u003ePB\u003c/sub\u003e represents the solvent energy term; ∆G\u003csub\u003ebind\u003c/sub\u003e represents the overall binding free energy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Fisetin inhibits HtrA from hydrolyzing casein.\u003c/h2\u003e \u003cp\u003eThe pET-28a(+)-HtrA plasmid was successfully transformed into BL21(DE3) cells, induced, expressed, purified, and concentrated. SDS-PAGE analysis confirmed that the molecular weight of the purified HtrA protein matched its theoretical size (Figure. S1a). Consistent with previous reports, casein was used as a substrate to assess the hydrolytic activity of HtrA in vitro. Incubation of 5 \u0026micro;g casein with 1 \u0026micro;g HtrA at 37\u0026deg;C for 3 hours demonstrated that HtrA effectively hydrolyzed casein, as verified by SDS-PAGE and coomassie brilliant blue staining (Figure. S1b).\u003c/p\u003e \u003cp\u003eMolecular docking and dynamics simulations revealed stable interactions between HtrA and the natural compounds Fisetin, Geniposide, and Quercetin, contributing to the stability of the HtrA-ligand complex. We further tested the inhibitory effect of these three potential compounds against HtrA\u0026rsquo;s hydrolytic activity on casein. The results indicate that in the reaction system without HtrA or drugs, the molecular weight of β-casein is primarily concentrated in the range of 25\u0026thinsp;~\u0026thinsp;35 KD. However, after co-incubation with HtrA and β-casein, we observed the bands of 25\u0026thinsp;~\u0026thinsp;35 KD decreased evidently, while those of 15\u0026thinsp;~\u0026thinsp;25 KD increased clearly. Co-incubation with three natural compounds showed that Geniposide and Quercetin had no effect on the activity of HtrA, as there were no clear changes in the levels of β-casein or its hydrolysate (Figure. 5a\u0026ndash;5f). In contrast, Fisetin exhibited a prominent concentration-dependent inhibition of HtrA\u0026rsquo;s casein hydrolytic activity. With the increase of the concentration of Fisetin, the inhibitory effect on HtrA activity became increasingly pronounced, which could markedly inhibit β-casein hydrolysis, mainly showing that the bands of 25\u0026thinsp;~\u0026thinsp;35 KD increased and the bands of 15\u0026thinsp;~\u0026thinsp;25 KD decreased (Figure. 5g\u0026ndash;5i).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Fisetin inhibits the growth of Helicobacter pylori\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate Fisetin's impact on H. pylori growth, drug sensitivity assays were conducted at 24, 48, and 72 h. Fisetin effectively inhibited H. pylori growth, exhibiting a dose-dependent inhibitory effect. Amoxicillin at a final concentration of 0.125 mg/mL served as the positive control. The minimum inhibitory concentration required to achieve 80% inhibition (MIC₈₀) was determined to be 10 \u0026micro;M, with no difference observed over time (Fig.\u0026nbsp;6). These findings suggest that Fisetin, by inhibiting HtrA, holds potential as a therapeutic agent for H. pylori-related gastric diseases.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eH. pylori infection is a major risk factor for the development of peptic ulcer disease, gastric adenocarcinoma, and a variety of other gastric and non-gastric conditions [61]. Currently, antibiotics are the primary treatment for H. pylori infections in clinical practice, and overall, the eradication of H. pylori has demonstrated satisfactory outcomes. However, the emergence of antibiotic resistance and the high recurrence rates associated with prolonged antibiotic therapy pose marked challenges[62\u0026ndash;65]. Therefore, the identification of new therapeutic targets and the development of novel anti-H. pylori agents are urgently needed.\u003c/p\u003e \u003cp\u003eHtrA is an important newly discovered active serine protease secreted by the gastric pathogen H. pylori [16]. HtrA has dual activity as both a molecular chaperone and serine protease within Helicobacter pylori. It plays a pivotal role in cellular protein quality control and is an essential gene product for the bacterium [66]. As a molecular chaperone, HtrA inhibits lysozyme aggregation in vitro, and the protein itself can be stabilized over a wide pH and temperature range [12]. In vivo, bacteria can refold or degrade misfolded proteins by overexpressing HtrA and other molecular chaperones to adapt to extreme environments, including prolonged heat stress, pH stress, high salt stress, oxidative stress, and reducing stress, and grow well even in the presence of antibiotics such as puromycin and metronidazole [7, 12, 67].\u003c/p\u003e \u003cp\u003eBased on the crucial role of HtrA in the pathogenesis of digestive tract-related diseases caused by H. pylori, along with its potential for therapeutic intervention and the current status of existing research, the development of drugs on targeting HtrA against H. pylori would be a valuable and meaningful drug discovery strategy. Currently, several inhibitors have already been identified that exhibit preliminary inhibitory activity against HtrA. Among them, Anna M Perna's research team developed the first HtrA peptide inhibitor by synthesizing cleavage site fragments and analogs that blocked the degradation of E-cadherin by H. pylori in gastric mucosal epithelial interstitial cells [68]. Jimin Hwang's research team found that the lead antichlamydial compound JO146 exhibited inhibitory activity against H.pylori with a minimum bactericidal concentration of 18.8\u0026thinsp;~\u0026thinsp;75.2 \u0026micro;g/mL. Furthermore, they found that the antimicrobial efficacy of JO146 was enhanced by encapsulating it in nanoparticles made from poly (lactic acid) - glycolic acid copolymers [69]. Sabine Bernegger et al. found that the combination of Zn and Cu ions effectively inhibits H. pylori HtrA activity [70]. Davoodbasha MubarakAli's research team docked H. pylori virulence factors by C-Docker algorithm and found that the derived peptide of the alga Tetradesmus sp had significant inhibitory activity against three virulence factors: CagA, VacA and HtrA [71]. Furthermore, regarding the HtrA target binding pocket, in addition to the active site cavity located around the catalytic Ser221, Anna Maria Perna et al. identified a second large pocket located at the interface between the PDZ1 structural domain and the serine protease domain of HtrA [72]. Notably, Krojer et al. found that the large cavity on the opposite side of the H6 helix, situated between the catalytic and PDZ1 structural domains, serves as a site for the metastable regulation of the hydrolytic chaperone activity of the DegP protein, which is the E. coli homologue of HtrA [73]. These results enhance our understanding of the functional role of this protease in bacterial infections and related mechanisms, while also provide new ideas for exploring HtrA-targeted therapies against H. pylori-related diseases. However, studies on natural compounds as HtrA inhibitors for H. pylori are quite limited. Despite the abundance of natural compounds that could offer more possibilities for H. pylori treatment, no natural compounds have been reported or screened as HtrA inhibitors. Therefore, the aim of this study was to identify effective and safe HtrA inhibitors derived from natural compounds in the hope of providing new ideas and approaches for the targeted treatment of H. pylori.\u003c/p\u003e \u003cp\u003eComputational techniques for virtual screening based on docking have become an effective method for rapid discovery of new compounds from huge compound libraries [78]. In this study, a screening library was established using 7,500 commercially available natural compounds. The top 8 natural compounds with docking scores\u0026thinsp;\u0026le;\u0026thinsp;\u0026minus;\u0026thinsp;6 kcal /mol were selected based on molecular docking at the HtrA binding site using the AutoDock Vina docking method. To ensure the reliability of the experiments, 100 ns MDs were conducted after the virtual screening. Based on the RMSD, RMSF, Rg, SASA, and MMPBSA analyses of the composite system of the natural compounds with HtrA protein, the results indicated that the three natural compounds, Fisetin, Geniposide, and Quercetin exhibited the best affinity with HtrA and the composite system was more stable. By analyzing the energetic contributions of key amino acid residues at the HtrA active site and the interactions of the final conformation of the complex from 100 ns of MDs, we found that Fisetin exhibited the strongest interactions with HtrA's amino acid residues among the 8 selected natural compoundsnatural compoundsand the binding of Fisetin with HtrA was mainly mediated by electrostatic interactions, hydrophobic interactions, and van der Waals forces, with residues such as YAL140, ASP143, ILE271, LEU139, and THR142 playing crucial roles. This finding provides a basis for further screening of HtrA inhibitors. Subsequently, through experimental validation, we found that the binding of Fisetin to HtrA effectively inhibited HtrA-induced β-casein hydrolysis in a concentration-dependent manner. In addition, in vitro bacteriostatic assay showed that Fisetin inhibited the growth of H. pylori in a concentration-dependent manner, with a MIC\u003csub\u003e80\u003c/sub\u003e of 10 \u0026micro;M. In conclusion, our results indicate that Fisetin has a certain anti-H. pylori ability and inhibits the activity of HtrA to a certain extent. Notably, Huiqin Zhuo et al. found that Fisetin (10 \u0026micro;M) signally reduced TAGLN2-induced cytoplasmic ssDNA accumulation by inhibiting the TAGLN2-YBX1-AKT interaction, thereby enhancing the therapeutic sensitivity of gastric cancer [79]. Akash Sabarwal et al. demonstrate that Fisetin inhibits cell proliferation and induces mitochondria-dependent apoptosis in human gastric cancer cells [80]. Fisetin, a polyphenol with pleiotropic pharmacological properties, has shown promising anticancer activity in various types of cancers, including gastric, colon, breast, and liver cancers, and also possesses some antibacterial activity [81\u0026ndash;83]. This provides some theoretical basis for the reliability of our experimental results, allowing us to continue screening HtrA inhibitors using the research methodology outlined in this paper to identify potential drugs for the eradication of HtrA. Meanwhile, there are some limitations in our study, on the one hand, we utilized a limited natural product library that does not comprehensively cover the diversity of natural compounds, on the other hand, the in vivo anti-H. pylori activity of Fisetin requires further investigation.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eBased on the findings from molecular docking, molecular dynamics simulations, and casein hydrolysis experiments, we discovered that Fisetin not only exhibits a high affinity for HtrA but also observably inhibits its hydrolytic activity, suggesting that Fisetin has potential as an inhibitor of HtrA. Additionally, we validated through MIC assays that Fisetin can suppress the growth of H. pylori. In summary, Fisetin may serve as a potential inhibitor of HtrA, potentially eliminating H. pylori infections by inhibiting the hydrolytic activity of HtrA and suppressing bacterial proliferation. This study integrates computational biology with experimental methods, opening new pathways for the exploration of targeted therapeutic agents against H. pylori infections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental Methods and Approaches: Li Gao, Xianqiong Jiang, Yan Shen, Kuilong Huang, Zhihua Lin; Literature Review and Investigation: Li Gao, Xianqiong Jiang, Yan Shen, Kuilong Huang; Experimental Procedures, Data Analysis, and Manuscript Preparation: Li Gao, Xianqiong Jiang, Hongtao Duan, and Rui Zhang; Experimental Supervision and Guidance: Yan Shen, Kuilong Huang, Kui Gu, Yuanqiang Wang, Mao Shu; Manuscript Revision: Yan Shen; Funding Support: Yan Shen and Zhihua Lin. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interest Statemen\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest related to this work. All authors have reviewed the manuscript and approved its submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by natural Science Foundation of Chongqing China (CSTB2022NSCQ-MSX1493), Chongqing University of Technology Postgraduate Quality Development Action Plan Funding Results (gzlcx 20243219).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank the China National Research Center for Immunobiological Products and Army Medical University for providing us with the H. pylori 26695 strain and the pET-28a(+)-HtrA expression plasmid, as well as for the technical support during the expression and purification of the HtrA protein.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eE.C. Smyth, M. Nilsson, H.I. Grabsch, N.C. van Grieken, F. 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Clausen, Structural basis for the regulated protease and chaperone function of DegP, Nature (2008), https://doi.org/10.1038/nature07004.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-diversity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"modi","sideBox":"Learn more about [Molecular Diversity](http://link.springer.com/journal/11030)","snPcode":"11030","submissionUrl":"https://submission.nature.com/new-submission/11030/3","title":"Molecular Diversity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"HtrA, Fisetin, Molecular dynamics simulation, β-casein hydrolysis, Helicobacter pylori","lastPublishedDoi":"10.21203/rs.3.rs-5316707/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5316707/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHelicobacter pylori (H. pylori, Hp) is a primary contributor to various stomach diseases, including gastritis and gastric cancer. This bacterium can colonize gastric epithelial cells, compromising their integrity and leading to the development of these conditions. While antibiotics are the mainstay of treatment for H. pylori infections, their widespread use has led to serious issues with drug resistance. High-temperature requirement A (HtrA), an important virulence factor of H. pylori, emerges as a promising target for the design of new inhibitors. In this study, we employed molecular docking, molecular dynamics simulation (MDs), and in vitro experimental validation to identify potential active components that specifically target HtrA, effectively combating H. pylori. Based on the results of molecular docking and literature reports, 8 potential natural compounds targeting HtrA were found: Camelliaside B, Rehmanioside A, Hesperidin, Quercetin, Fisetin, Colchicine, Apigenin and Geniposide. MDs further revealed that Fisetin, Geniposide, and Quercetin show promising stability and interactions with HtrA. Importantly, in vitro experiments, including casein hydrolysis assays and antibacterial sensitivity tests, demonstrated that Fisetin observably inhibited the hydrolysis of casein by HtrA and effectively suppressed the growth of H. pylori. Overall, our findings highlight Fisetin as a potential therapeutic drug that targets HtrA, offering new tack for the development of lead compounds and potential drugs against H. pylori infections.\u003c/p\u003e","manuscriptTitle":"Molecular dynamics simulation-driven focused virtual screening and experimental validation of Fisetin as an inhibitor of Helicobacter pylori HtrA protease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-05 10:33:32","doi":"10.21203/rs.3.rs-5316707/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-11T20:04:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-09T18:01:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-09T00:31:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-08T17:01:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-03T13:54:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"340245866664901394181333012298426104684","date":"2024-10-31T20:13:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"236135947743216300576621764687856332968","date":"2024-10-30T04:52:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"256311029538336113453843230398177220479","date":"2024-10-29T20:20:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"252902270951743347650335936788919324389","date":"2024-10-29T20:17:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"128983818930891531827895835471373314570","date":"2024-10-29T20:05:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"56444970350448764302367118943452015736","date":"2024-10-29T17:58:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-29T17:30:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-25T16:30:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-25T06:38:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Diversity","date":"2024-10-23T07:31:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-diversity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"modi","sideBox":"Learn more about [Molecular Diversity](http://link.springer.com/journal/11030)","snPcode":"11030","submissionUrl":"https://submission.nature.com/new-submission/11030/3","title":"Molecular Diversity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f95ff261-a8aa-4ad7-b668-6b720c6cdb5a","owner":[],"postedDate":"November 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-24T16:02:07+00:00","versionOfRecord":{"articleIdentity":"rs-5316707","link":"https://doi.org/10.1007/s11030-025-11137-2","journal":{"identity":"molecular-diversity","isVorOnly":false,"title":"Molecular Diversity"},"publishedOn":"2025-02-23 15:57:31","publishedOnDateReadable":"February 23rd, 2025"},"versionCreatedAt":"2024-11-05 10:33:32","video":"","vorDoi":"10.1007/s11030-025-11137-2","vorDoiUrl":"https://doi.org/10.1007/s11030-025-11137-2","workflowStages":[]},"version":"v1","identity":"rs-5316707","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5316707","identity":"rs-5316707","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}