TW201600089A - Zhankuic acid A and analogs thereof and their use as an anti-inflammatory agent - Google Patents

Abstract

Zhankuic acid A (ZAA) is the major pharmacologically active compound of Taiwanofungus camphoratus. We analyzed the structure of human TLR4/MD-2 complex with ZAA by X-score and HotLig modeling approaches. Two antibodies against MD-2 were used to verify the MD-2/ZAA interaction. The inflammation and survival of the mice pretreated with ZAA and injected with LPS were monitored. The modeling structure shows that ZAA binds the MD-2 hydrophobic pocket exclusively via specific molecular recognition; the contact interface is dominated by hydrophobic interactions. Binding of ZAA to MD-2 reduced antibody recognition to native MD-2, similar to the effect of LPS binding. Furthermore, ZAA significantly ameliorated LPS-induced endotoxemia and Salmonella-induced diarrhea in mice. Our results indicate that ZAA, which can compete with LPS for binding to MD-2 as a TLR4/MD-2 antagonist, is a potential therapeutic agent for Gram-negative bacterial infections.

Description

Anthraquinone A and its analogues and use as anti-inflammatory agents

The present invention relates to a novel use of anthraquinone A and its analogs as an anti-inflammatory agent.

Lipopolysaccharide (LPS) is a glycolipid endotoxin composed of an amphiphilic component lipid A, a hydrophilic polysaccharide core, and an outermost segment of the O-antigen. LPS is usually present in the outer membrane of a variety of Gram-negative bacteria (1). The mammalian immune system recognizes LPS as a foreign molecule, which is the first step in alerting the host to the possibility of an invasive Gram-negative bacterial infection. In mammals, CD14 and TLR4/MD-2 complexes are involved in cellular recognition of LPS (2, 3). Binding of the TLR4/MD-2 complex to LPS triggers the TLR4/MAPK signaling pathway, which includes p38, ERK and JNK (4). LPS also causes an inflammatory response in quiescent cells, which promotes nuclear transfer of NF-κB and NF-κB-stimulated inflammatory genes through phosphorylation and subsequent degradation of IκBα, such as inducible nitric oxide synthesis. Enzymes (iNOS), TNF-α and IL-6 (5,6). Therefore, high LPS levels in the circulation can lead to severe septicemia, a life-threatening inflammatory complication. However, for LPS media Sexual inflammatory symptoms have not been effectively treated.

Taiwanofungus camphoratus (stout camphor fungus) is a parasitic fungus that grows only on the inner heartwood wall of the burdock tree (Cinnamomum kanehirai). This fungus has been widely used in traditional Chinese medicine to treat drug poisoning, diarrhea, abdominal pain, high blood pressure and cancer (7). The methanol extract of T. camphoratus has anti-inflammatory activity in microglia by inhibiting the expression of iNOS and cyclooxygenase-2 (COX2) (8). It has been shown that the main pharmacologically active compound ZAA in the fruiting bodies of Burdock burdock prevents inflammatory reactions due to human neutrophils and does not exert significant cytotoxicity (9). Furthermore, ZAA also exhibits potent anti-inflammatory activity by inhibiting the production of NO by LPS (10). However, the mechanism by which ZAA regulates and improves inflammation has not been fully elucidated.

In the present invention, we used a mouse model of diarrhea caused by S. choleraesuis to study the ability of ZAA to reduce inflammation caused by Gram-negative bacterial infection by blocking LPS. We used X-score and HotLig model methods (11, 12) to show that ZAA can act as a ligand for MD-2, thus inhibiting LPS/MD-2 interaction. Furthermore, ZAA inhibits the NF-κB signaling pathway and reduces the levels of TNF-α and IL-6 in vitro and in vivo. Intraperitoneal administration of ZAA protects mice against lung and kidney damage caused by LPS and diarrhea caused by Salmonella typhimurium. A series of ZAA analogs have also been studied and found to have anti-inflammatory activity in the expression of TNF-[alpha] caused by LPS.

The present invention suggests that ZAA and a series of ZAA analogs can have potential Acting as a therapeutic drug to prevent inflammatory diseases caused by Gram-negative bacterial infections.

According to an aspect of the present invention, the present invention provides a method for treating an inflammatory disease in a subject, which comprises administering a compound having the following formula (I) or a pharmaceutically acceptable salt thereof to a subject in need of such treatment : Wherein R ₁ is =O; R ₂ is =O, OCHO or OH; R ₃ is H or OH; R ₄ is -C(=CH ₂ )-C(CH ₃ )H-(C=O)OR _a , Wherein R _a is H or C _1-4 alkyl, or R ₄ is -(C=O)R _b , wherein R _b is C _1-4 alkyl; R ₅ is =O, OH or H; and R ₆ H or OH.

According to another aspect of the present invention, there is provided a use of a compound of the formula (I) or a pharmaceutically acceptable salt thereof for use as an active ingredient in the manufacture of a medicament for the treatment of a subject's inflammatory disease .

Preferably, the inflammatory disease is a mediating inflammatory condition of LPS.

Preferably, the inflammatory disease is caused by a bacterial infection and more preferably a Gram-negative bacterial infection.

Preferably, the inflammatory disease comprises lung damage caused by LPS or kidney damage caused by LPS.

Preferably, the inflammatory disease comprises diarrhea.

Preferably, the inflammatory disease comprises enteritis.

Preferably, the compound of formula (I) is selected from the group consisting of:

figure 1. ZAA inhibits the production of inflammatory related molecules in LPS and IFN-γ stimulated murine macrophages. (A) Murine peritoneal macrophage cell line was pretreated with or without ZAA for 1 hour, followed by incubation with LPS (0.5 μg/ml) for 24 hours. The total cell lysate is subjected to immunoblotting for detection of COX2 and iNOS. The relative expression levels of COX2 and iNOS proteins were quantified by density analysis and ImageJ software and normalized according to the β-actin reference band. (B) The murine peritoneal macrophage cell line was pretreated with ZAA for 1 hour, followed by incubation with LPS (0.5 μg/ml) or IFN-γ (50 Ng/ml) for 24 hours. The presence of nitrite in the culture medium was analyzed by Griess test and used as an indicator of NO level (n=4; *p<0.05 and ***p<0.001 for LPS or IFN-γ induced). cell). (C) The Raw264.7 cell line was co-transfected with pNFκB-Luc and pβ-actin-LacZ plastids. After 48 hours, the cell lines were treated with or without ZAA for 1 hour and then treated with LPS (0.5 μg/ml) for 24 hours. Total cell solubilized material was collected, and its luciferase activity was determined and normalized based on β-galactosidase activity. The values are mean ± SD (n=4; *p<0.05 and **p<0.01 vs. LPS-stimulated cells). (D and E) ZAA inhibits NF-κB, MAPK and Akt signaling pathways in LPS-stimulated Raw264.7 cells. These cell lines are at ZAA After 1 hour, it was stimulated with LPS (0.5 μg/ml) for 30 minutes. The protein indicated by the total cell lysate was examined by immunoblotting. The numbers shown in D under the ink dots and those shown in the table under the ink dots in E represent the relative performance of the density by the density analysis and ImageJ software and the normalization according to the β-actin reference band. quasi. Similar results were obtained in three separate experiments. N.D., unable to detect.

figure 2. ZAA interacts with the hydrophobic binding pocket of MD-2. (A) ZAA assumes a matching configuration to fit into the binding pocket that would otherwise be occupied by the LPS. The LPS binding pockets embedded in the MD-2 protein were modeled together with a MD-2 clipped surface model (PDB registration: 3FXI) and a ribbon model representation. The amino acid Gly110-Asn158, which is shown in magenta, is present in the immunogenic peptide fragment of MD-2 amino acid 110-160, which is in the ribbon-mode of MD-2 around the ZAA binding site. Gly110 and Asn158 are indicated by arrows. (B) shows the interaction of the binding of ZAA to the hydrophobic amino acid residue in the MD-2 binding pocket. (C) shows that within the complex structure of the 3FXI MD-2 model, the docked ZAA configuration (depicted in black) is superimposed on the terminal carbon chain of the LPS. (D) Recombinant human MD-2 protein (0.15 μg) was cultured with LPS (1 or 10 μg) or ZAA for 3 hours, subjected to non-denaturing PAGE (native PAGE), and to anti-MD-2 different epitopes The antibodies (amino acids 110-160 and 2-160) were immunostained. (E) Recombinant human TLR4/MD-2 complex (1 μg) was cultured with LPS (10 μg) or ZAA for 3 hours, subjected to non-denaturing PAGE, and against MD-2 (amino acid 110-160) The antibody is immunologically printed. Similar results were obtained in three separate experiments.

image 3. ZAA inhibits the production of TNF-α and IL-6 in Raw264.7 cells treated with LPS or Salmonella cholerae and in mice. (A and C) Rab264.7 cells (2 x 10 ⁴ cells/well) were cultured in Z-well plates for 1 hour in ZAS, followed by LPS (0.25 or 0.5 μg/ml) (A) or Salmonella cholerae (2 × 10 ³ CFU / well) (C) treatment. The TNF-α and IL-6 levels of the supernatant collected after 4 and 6 hours were evaluated by ELISA. (B) C3H/HeJ and C3H/HeN mice were pre-treated with 2 mg/kg ZAA intraperitoneally for 30 minutes followed by intraperitoneal injection of LPS (4 mg/kg). (D) C57BL/6 mice were pretreated with 10 mg/kg ZAA for 30 minutes followed by oral administration of Salmonella cholerae (2 x ¹⁰⁹ CFU/mouse). After 6 hours, the levels of TNF-α and IL-6 in plasma were measured by ELISA. The values are mean ± SD (n = 6-8; *p < 0.05, **p < 0.01, and *** p < 0.001). Similar results were obtained in at least three separate experiments. SC, Salmonella cholera.

Figure 4. ZAA reduces pathological changes caused by LPS in mice. (A-D) C3H/HeN and C3H/HeJ mice were pre-treated with ZAA (20 mg/kg) or vehicle for 30 minutes intraperitoneally, followed by intraperitoneal injection of LPS (4 mg/kg). After 10 hours, the mice were sacrificed and their lung and kidney tissues were removed. (A) A typical microscopic image showing a stained section of hematoxylin and eosin in lung and kidney tissues. (B) The number of PMNs infiltrated in each alveolar was observed by an optical microscope (original magnification × 200). The number of PMNs in each of the four randomly selected fields of view and the number of alveolar cells were normalized for each mouse. (C and D) ZAA reduces the level of BUN (C) and serum creatinine (D). The values shown in B-D are mean ± SD (n = 10; **p < 0.01 and *** p < 0.001). (E) C3H/HeN that has been pre-treated with ZAA (2 or 10 mg/kg) or vehicle for 30 minutes. C3H/HeJ mice were injected intraperitoneally with a lethal dose of LPS (20 mg/kg). Survival time was monitored and Kaplan-Meier survival curves in four groups were shown (n=10; ***p<0.001 vs. LPS-treated C3H/HeN mice). Similar results were obtained in at least three separate experiments.

Figure 5. ZAA improves diarrhea, weight loss and gastrointestinal infections caused by Salmonella typhimurium. (A and B) Oral anti-kanamycin-resistant Salmonella cholerae (2×10 ⁹ CFU) was administered orally in C57BL/6 mice pretreated with ZAA (2 or 10 mg/kg) or vehicle for 30 minutes. /mouse). After 2 days, diarrhea was scored on a scale of 0-3 (0 = normal pellets, 1 = slightly loose feces, 2 = loose feces, and 3 = watery diarrhea) (A). Body weight was recorded every 2 days for 2 weeks (n=9-12; **p<0.01 and ***p<0.001) (B). (C) After the Salmonella typhimurium infection, the stool samples were collected at 24-hour intervals until 96 hours, and the viable bacterial CFU counts were evaluated (n=10; **p<0.01 and ***p<0.001) ). (D and E) C57BL/6 mice that had been orally treated with ZAA (2 mg/kg) or vehicle for 30 minutes were orally administered with pCMV-Luc-transformed Salmonella cholerae (2 x 10 ⁸ CFU/mouse). After 48 hours, bioluminescence imaging of the mice was performed after injection of D-luciferin. The overall image is displayed in D. The photon flux scale is shown on the right. (E) Quantitative data for bioluminescence imaging. The amount of radiation is expressed as mean ± SD (*** p < 0.001). SC, Salmonella cholera.

Figure 6. ZAA is not a cytotoxin to rat peritoneal macrophages. (A) Cells were cultured for 72 hours at various ZAA concentrations. Cell viability was determined by tetrazolium colorimetric assay (MTS) and sulphorhodamine B (SRB) assay. The absorbances used in the MTS and SRB tests were measured at 490 and 590 nm, respectively. (B) Cells were cultured for 1 hour at various ZAA concentrations followed by LPS (0.5 micro G/ml) stimulated for 72 hours. Cell viability is determined by the SRB test. The values are the mean ± SD of at least three independent experiments (*p < 0.05 versus untreated cells).

Figure 7. ZAA is unable to inhibit the replication of Salmonella choleraesuis. Salmonella typhimurium is cultured in LB broth for 24 or 48 hours in the presence of various ZAA concentrations or in the presence of ampicillin as a positive control. Bacterial growth was assessed by measuring the absorbance at 600 nm and by the mean ± SD of at least three independent experiments. (***p<0.001 vs untreated cells).

Detailed description of the invention

The abbreviations used in the following examples are as follows: MD-2, myeloid differentiation factor-2; ZAA, anthuric acid A; CD14, differentiation cluster 14; iNOS, inducible nitric oxide synthase ; fMLP, N-methyl decyl-methionine decyl-alkamine-phenylalanine; PMA, phorbol-12-myristate-13-acetate; and ROS, active oxygenates.

Example:

Materials and methods

Cells, bacteria and mice

Raw264.7 murine macrophage cell line and attenuated Salmonella Intestinal subspecies intestinal serotype Salmonella enterica subsp.enterica serovar Choleraesuis (S. cholerae) (13) from the Bioresource Collection and Research Center (Hsinchu) , Taiwan (Taiwan) obtained. Male C3H/HeJ, C3H/HeN, and C57BL/6 mice (8 to 10 weeks old) were obtained from the National Laboratory Animal Center (Taiwan) (Taipei, Taiwan).

Platinum and reagents

NFκB reported that the plastid p-NFκB-Luc was purchased from Promega (Madison, WI). The pβ-actin-LacZ system is derived from the pFRL2 plastid (14) and is driven by the CMV promoter by a β-galactosidase expression assembly driven by the β-actin promoter. The firefly luminescent enzyme expression assembly. The pCMV-Luc reporter system was obtained from Addgene (Cambridge, MA). The pEGFP-N1 (ΔEGFP) quality system including the kanamycin-resistant gene was derived from pEGFP-N1 by deleting the EGFP coding region. Anti-COX2, iNOS and TLR4 anti-systems were purchased from Santa Cruz (Santa Cruz, CA). Anti-IkBα, ERK, JNK, Akt and p38, and phosphonium (p)-IkB kinase (IKK) α/β (pIKKα/β), pNF-κBp65, pERK, pJNK, pAkt and pp38 anti-systems from Cell Signaling (Danvers, MA) purchased.

Extraction and separation of fungal compounds

ZAA was isolated from Boletus edulis (10, 15) as previously described. Dissolve the compound in 40% cyclodextrin at a concentration of 2 mg/ml (Sigma-Aldrich, St. Louis, MO) was used as a raw material solution, stored at -20 ° C, and diluted with cell culture medium before each experiment. The final cyclodextrin concentration used in all experiments was less than 0.2%.

Anti-inflammatory molecule test

C57BL/6 mice were injected intraperitoneally with 3% hydrogen thioacetate and their peritoneal macrophages were collected 72 hours later. Macrophage cell lines were cultured in DMEM at 37 ° C in a humidified environment of 5% CO ₂ supplemented with 10% FBS and 50 μg/ml gentamicin. These cell lines were pretreated with or without ZAA for 1 hour and then incubated with LPS (Sigma-Aldrich; 0.5 μg/ml) or IFN-γ (PeproTech, Rocky Hill, NJ; 50 Ng/ml) for 24 hours. These cell lysates were subjected to SDS-PAGE to detect COX2 and iNOS expression. The nitrite (metabolite of NO) present in the culture medium was analyzed by the Griess test (Sigma-Aldrich) as previously described (16).

Immunoprinting analysis

The Raw264.7 cell line was treated with or without various ZAA concentrations for 1 hour, followed by stimulation with LPS (1 μg/ml) for 30 minutes and in RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, pH 8.0, 1 mM Na ₃ VO ₄ , 20 μg/ml leupeptin, 20 μg/ml anti-protease peptide, 1 mM PMSF, and 50 mM NaF) were homogenized. The cell lysate is obtained by a primary antibody against pIKKα/β, IkBα, pNF-κBp65, pERK, ERK, pJNK, JNK, pAkt, Akt, pp38, p38 and β-actin, followed by an appropriate secondary Antibodies were immunoblotted for analysis. Immunoreactive protein bands were detected using an Enhanced Chemiluminescence (ECL) kit (Pierce Biotechnology, Rockford, IL). The relative intensity of this protein band was quantified against β-actin normalization and using Image J software (available at http://rsb.info.nih.gov/ij/).

Reporter test

The secondary fused Raw264.7 cell line cultured in a 24-well plate was treated with the Neon Transfection System (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol, with p-NFκB-Luc and pβ-actin-LacZ plastids. Transfection. Forty-eight hours after transfection, cells were cultured in serum-free DMEM with or without ZAA (0.5 or 10 M) for 1 hour and then treated with LPS (1 μg/ml) for 24 hours. Cell lysates were collected and their luminescent enzyme activities were determined by the dual-lumen luminescent enzyme and β-galactosidase reporter gene assay system (Tropix, Bedford, MA). The relative luciferase activity was measured by dividing the luciferase activity by the β-galactosidase activity to normalize the transfection efficiency.

Molecular docking

Flexible Docking (17) using Dock 5.1 software. A Kollam partial charge is applied to the protein model used for force field calculations. Energy-optimized small molecule three-dimensional coordinates (18) were generated by Marvin 5.2.2 (available at http://www.chemaxon.com) and Ballon 0.6 software. Additionally, the Gasteiger partial charge (19) was calculated by applying OpenBabel 2.2.3 software. The parameters for the Dock program are set to iteratively generate 1000 orientations and 200 configurations in the MD-2 binding pocket. Docked configurations are re-rated and categorized by HotLig To predict protein-ligand interactions. HotLig is a molecular surface orientation scoring function that applies the Connolly surface of a protein to assess molecular interactions. First, the Connolly surface of the protein was calculated by the tool PscanMS in the HotLig package; then, the docked ligand configuration was input to analyze the molecular interaction and calculate the binding energy score. A graphic for providing a molecular model (20) was performed using Chimera software.

Nondenaturing PAGE

For in-tube binding assays, a predetermined amount of LPS or ZAA was sonicated for 3 minutes, and at 37 °C with recombinant human MD-2 (R & D, Minneapolis, MN; 0.15 μg) or recombinant human TLR4 /MD-2 complex (R &D; 1 μg) was incubated for 3 hours. The sample is subjected to non-denaturing PAGE, and the TLR4-linked or free-state MD-2 level is detected by immunoblotting with two anti-MD-2 antibodies, wherein the two anti-MD-2 amine groups are resistant. Rabbit 110-60 antibody (abcam, Cambridge, MA) and mouse monoclonal antibody (abcam) against MD-2 amino acid 2-160. The signal is detected by ECL.

ELISA for cytokine expression

Raw264.7 cell line was incubated with or without various ZAA concentrations for 1 hour, followed by LPS (0.25 or 0.5 μg/ml) or Salmonella cholerae [2×10 ³ colony forming units (CFU)/well] And 6 hours, the TNF-α and IL-6 levels in the supernatant were detected by ELISA kit (R & D).

Mouse model of inflammatory response and diarrhea caused by LPS or Salmonella cholerae

Mice were pretreated intraperitoneally with ZAA (20 mg/kg) or vehicle (0.2% cyclodextrin in physiological saline) for 30 minutes followed by intraperitoneal injection of LPS (4 mg/kg). After 6 hours, the expression level of cytokines in plasma was measured by ELISA. Ten hours after LPS treatment, the mice were sacrificed and the organs were excised and fixed in the formalin. The lungs and kidneys were embedded in paraffin, sectioned and stained with hematoxylin and eosin. Serum samples were collected to determine blood urea nitrogen (BUN) and serum creatinine levels. Salmonella cholerae (2 x ¹⁰⁹ CFU/mouse) transformed with pEGFP-N1 (ΔEGFP) was orally administered to mice orally pretreated with or without ZAA (2 or 10 mg/kg). After 6 hours, serum samples were collected for determination of TNF-[alpha] and IL-6 levels by ELISA. After infection with Salmonella cholerae, the stool samples were collected at 24-hour intervals until 96 hours. After overnight incubation at 37 ° C, Salmonella cholerae in the feces was quantified by plating a series of diluted stool samples on a kanamycin-containing agar plate and counting colonies. Daily monitoring of diarrhea symptoms and weight loss in mice. Diarrhea was scored according to the definition of diarrhea 0-3 (0 = normal pellets, 1 = slightly loose feces, 2 = loose feces and 3 = watery diarrhea) as previously described (21). The experimental method adheres to the rules of Animal Protection Act of Taiwan and is approved by the Laboratory Animal Care and Use Committee of National Cheng Kung University.

Septicemia model caused by LPS

C3H/HeN and C3H/HeJ mice were pretreated intraperitoneally with ZAA (2 or 10 mg/kg) or vehicle. After 30 minutes, LPS (20 mg/kg) was administered intraperitoneally. Mice were monitored every 2-4 hours until all C3H/HeN mice in the group not treated with ZAA and treated with LPS were deflated.

Non-invasive bioluminescence imaging of luminescent enzyme substrates

C57BL/6 mice were orally administered with ZAA (2 mg/kg) or vehicle, and 30 minutes later, pCMV-Luc-transformed Salmonella cholerae (2 x 10 ⁸ CFU/mouse) was orally administered. After 48 hours, the mice were injected intraperitoneally with D-luciferin potassium salt (Promega, 2.5 mg in 100 microliters). They were then anesthetized with 2% isoflurane. In vivo bioluminescence imaging and signal quantification were performed using the IVIS-200 system and its integrated acquisition and analysis software (Living Image V. 2.50) (Perkin Elmer, Fosty City, CA).

Statistical Analysis

Results are presented as mean ± standard deviation (SD). Student's unpaired T test and SigmaPlot ^TM software (Systat) analyze the statistical differences. A p value of less than 0.05 is considered statistically significant.

result

ZAA dose dependence inhibits the production of iNOS, COX2 and NO

First, we studied the anti-inflammatory properties of ZAA purified from the genus Burdock. During inflammation, a large amount of pro-inflammatory mediators, NO and prostaglandin E2 (PGE2) are produced by iNOS and COX2. ZAA is down-regulated by LPS in a dose-dependent manner COX2 and iNOS levels induced in murine peritoneal macrophages (Fig. 1A), and inhibition of NO production by LPS and IFN-γ (Fig. 1B). However, up to 30M of ZAA treatment for 72 hours did not exert any cytotoxic effects on intrinsic or LPS-activated macrophages, as determined by tetrazolium colorimetric (MTS) and sulfopyramine B (SRB) assays. (22), as shown in Figure 6.

ZAA blocks NF-κB, MAPK and Akt signaling pathways induced by LPS

To investigate the inhibitory role of ZAA in LPS-stimulated NF-κB signaling, we first examined its effect on the transcriptional activity of NF-κB. Figure 1C shows that ZAA inhibits NF-κB vector transcriptional activity in Raw264.7 cells, as determined by the luminescent enzyme reporter assay. Furthermore, LPS treatment stimulated NF-κBp65 phosphorylation, which was clearly prevented by ZAA (Fig. 1D). Similarly, phosphorylation by LPS of ERK, JNK, p38 and Akt was also inhibited by ZAA (Fig. 1E). These results together strongly suggest that ZAA inhibits LPS-stimulated NF-κB, MAPK and Akt signaling pathways.

ZAA interacts with the hydrophobic pocket of MD-2 to block LPS

It has been demonstrated that binding of MD-2 to the extracellular domain of TLR4 triggers LPS vector responses (23, 24). To investigate whether ZAA interrupted TLR4 signaling by competing with LPS binding to MD-2, we applied a molecular docking analysis in in silico to simulate the interaction between ZAA and MD-2. Previous studies have shown that a knowledge-based scoring function is a good method for predicting protein-ligand interactions, whereas empirically based scoring functions are more effective at predicting ligand-binding affinities (25, 26). So we use new knowledge The basic scoring program HotLig is used to predict the molecular interaction between ZAA and MD-2. Furthermore, we applied the empirically based scoring program X-score to predict its binding affinity. For the prediction of ligand binding position, HotLig showed a success rate of about 85% to 90% (12). On the other hand, X-score is reported as the best scoring function for protein-ligand affinity (25, 26) when compared to many other well-known scoring programs. Figure 2A together presents a trimmed surface model of MD-2 (PDB Login: 3FXI) and a ribbon model to depict LPS binding pockets embedded in MD-2 protein. It is worth noting that ZAA assumes a matching configuration for placement in previously identified LPS binding pockets. Figure 2B shows the interaction between ZAA and MD-2 forming hydrophobic amino acid residues (e.g., Ile, Val, Phe, Leu, and Tyr) of the LPS binding pocket. ZAA explicitly contacts the pocket by hydrophobic interaction (the semicircular radiant symbol in Figure 2B), with no hydrogen bonds. Additionally, as shown in Figure 2C, the molecular overlap of the ZAA-bound ZAA (depicted in black) and LPS (the co-crystallized ligand in the 3FXI MD-2 structure) means that the ZAA can be occupied otherwise. LPS's terminal carbon chain occupies space.

To assess the potential binding affinities of ZAA and LPS for MD-2, a common score analysis was performed using the X-score scoring function after binding position prediction was generated via HotLig (Table 1). The pKd (average HPScore, HMScore, and HSScore) predicted by ZAA was 7.83, while the LPS was 5.83 (Table 2). Therefore, we hypothesized that the matching molecular configuration of ZAA with the apparent hydrophobic interaction effect provides sufficient binding to stabilize the MD-2/ZAA complex. Therefore, ZAA can interfere with LPS/TLR4 signaling by competing with LPS for the combination of MD-2.

Mw, molecular weight; LogP, partition coefficient. HPScore=C0,1+CVDW,1*(VDW)+CHB,1*(H-bonding)+CHP*(hydrophobic pair)+CRT,1*(rotor) HMScore=C0,2+CVDW , 2*(VDW)+CHB, 2*(H-bond)+CHM*(hydrophobic matching)+CRT, 2*(rotor) HSScore=C0,3+CVDW,3*(VDW)+CHB,3 *(H-bond)+CHS*(hydrophobic surface)+CRT, 3*(rotor) X-Score=(HPScore+HMScore+HSScore)/3

Previous studies have demonstrated that LPS has similar affinities for MD-2 and TLR4/MD-2 complexes, suggesting that MD-2 is the major LPS binding component (27). Furthermore, the monomeric form of soluble recombinant MD-2 is stable in combination with LPS. The MD-2/LPS complex; this complex is sufficient to initiate TLR4-dependent activation (28, 29). Secondly, we performed immunoelectrophoresis with two anti-MD-2 antibodies with different epitopes, one of which was an antibody against MD-2 amino acid 110-160, and the other was anti-MD-2 amino acid 2 -160 monoclonal antibodies. ZAA binding to human MD-2 reduces the recognition of non-denatured MD-2 by both antibodies, similar to the effect of LPS binding (Fig. 2D). The recombinant human TLR4/MD-2 protein complex was then used to measure the binding efficiency of ZAA. Figure 2E shows that 10 micrograms of ZAA is approximately equal to 10 micrograms of LPS. Thus, ZAA can interfere with the recognition of MD-2 by anti-MD-2 antibodies by competitive binding to antibodies or by conformational changes in MD-2 proteins.

ZAA reduces the production of pro-inflammatory cytokines caused by LPS and Salmonella cholerae

Because LPS triggered the production of pro-inflammatory cytokines, secondly, we compared the effects of ZAA on the production of TNF-α and IL-6 caused by LPS and Salmonella typhimurium in vitro and in vivo. ZAA inhibited the production of TNF-[alpha] and IL-6 by LPS in Raw264.7 macrophages at two concentrations tested (Fig. 3A). To clarify whether this effect was also observed in vivo, ZAA was administered to mice 30 minutes before LPS injection. Although ZAA significantly reduced TNF-α and IL-6 production in C3H/HeN mice, a weaker response was observed in TLR4 signaling-deficient C3H/HeJ mice (Fig. 3B), suggesting that ZAA acts on LPS/ The TLR4/MD-2 pathway is selective.

Secondly, we explored the effects of both ZAA in vitro and in vivo on the production of vector-induced pro-inflammatory cytokines in Salmonella typhimurium. our The results showed that ZAA pretreatment for 1 hour effectively inhibited the production of TNF-α and IL-6 by Salmonella cholerae in Raw264.7 cells (Fig. 3C) and C57BL/6 mice (Fig. 3D).

ZAA attenuates lung and kidney damage and mortality caused by LPS

Because ZAA inhibits LPS-induced proinflammatory cytokine production and signaling pathways, we then explored that ZAA reduces organ pathology and mortality caused by LPS in vivo. We decided to express inflammatory responses in lung and kidney tissues due to their constructive TLR4 (30, 31). Infiltration of polymorphonuclear leukocytes (PMNs) in the lung was increased following administration of LPS to C3H/HeN mice; however, ZAA treatment significantly prevented lung accumulation of PMNs caused by LPS (Figs. 4A and 4B). Similarly, the extent of renal glomerulonephritis caused by LPS was significantly reduced in ZAA pretreated C3H/HeN mice (Fig. 4A). Consistent with renal failure, BUN and serum creatinine levels increased after LPS administration. ZAA significantly reduced the manufacture of both kidney damage markers (Figures 4C and 4D). Notably, LPS treatment did not elicit any pathological changes in TLR4 signaling-deficient C3H/HeJ mice (Figures 4A-4D).

To assess the protective efficacy of ZAA against lethality caused by LPS, C3H/HeN and C3H/HeJ mice were treated with ZAA or vehicle and challenged with LPS. ZAA clearly protects C3H/HeN mice against lethality and improves survival during endotoxemia (Fig. 4E), suggesting that ZAA generally has therapeutic potential against septicemia and Gram-negative bacterial infections caused by LPS.

ZAA improves clinical symptoms in mice infected with Salmonella cholerae

TLR4 plays a prominent role in host defense responses against Salmonella infection (32, 33). Previous studies have shown that mice lacking TLRs, particularly TLR4, are more resistant to Salmonella infection (32, 34), suggesting that LPS is blocked on the bacterial outer membrane, so TLR4/MD-2 interaction is a promising antibacterial response. To further confirm the anti-inflammatory properties of ZAA in vivo, C57BL/6 mice infected with Salmonella cholerae were treated with ZAA, and diarrhea and body weight were monitored separately for 2 and 2 weeks. Figure 5A shows that ZAA pretreatment reduces diarrhea scores in infected mice. ZAA treatment (10 mg/kg) dramatically reduced weight loss (Figure 5B), resulting in a return to normal body weight within 2 weeks. As shown in Figure 5C, the bacterial load in the feces of untreated mice gradually decreased over time. It is worth noting that the bacterial load of feces was dramatically reduced in dose- and time-dependent manner in ZAA-treated mice. At 96 hours after bacterial infection, fecal bacterial levels in mice treated with 10 mg/kg of ZAA were not detected. Furthermore, mice infected with Salmonella cholerae carrying the pCMV-Luc plastid had a weaker bioluminescence than the vehicle after oral treatment with ZAA (Figs. 5D and 5E). Together with these results, it was shown that ZAA is effective in inhibiting Salmonella cholerae infection and attenuating clinical symptoms in mice.

discuss

In the present invention, we used two different model methods and antibody recognition to show the interaction of ZAA with the hydrophobic pocket of MD-2 at the first time to block the LPS effect. We found that ZAA acts as a ligand for MD-2, thus inhibiting the interaction of LPS with MD-2. We also show the generality of ZAA The administration protects the mice from lung and kidney damage caused by LPS and enteritis and weight loss caused by Salmonella. Our results indicate that ZAA has anti-inflammatory activity and can be a potential therapeutic for septic shock.

The methanol extract of the fruiting body of Burdock burdock inhibits the production of COX2, iNOS and TNF-α in LPS/IFN-γ activated microglia (8), suggesting that its anti-inflammatory properties can be attributed to Inhibition of phosphorylation of ERK, JNK and NF-κB. The present invention investigates the anti-inflammatory properties of ZAA assuming that ZAA is the major pharmacologically active compound in the fruiting body. Previous studies have revealed that ZAA inhibits ROS production in human peripheral neutrophils activated by fMLP or PMA (9). Similarly, we demonstrate that ZAA regulates the production of NO in activated murine macrophages and attenuates the expression of pro-inflammatory mediators such as iNOS, COX2, TNF-[alpha], and IL-6.

Antibodies against the TLR4/MD-2 complex have been shown to be effective in treating acute inflammatory conditions evoked by LPS (35, 36). Molecules that block the formation of TLR4/MD-2 heterodimers and initiate inflammation have also recently been explored. For example, eritoran is a synthetic tetra-negative lipid A that competes with LPS for the same binding position in MD-2 and attenuates the formation of the LPS-activated receptor complex, which is successive Signal transmission through the serosa is suppressed (37). Unfortunately, eritoran's Phase III study showed no significant differences between the treatment and placebo groups (38). Recently, natural and synthetic chemical substances MD-2 oriented LPS antagonists (39) which are not related to the structure of bacterial lipid A have also been reported. The mechanism of binding to the MD-2 pocket can be divided into three general types: (1) competition for entry into the MD-2 pocket [eg, bis-ANS (1-anilinophthalene 8-sulfonate) and paclitaxel]; (2) covalent with the Cys133 residue in the MD-2 pocket Interactions (eg, N-芘马醯亚胺, auranofin, and JTT-705); and (3) linear alignment at the mouth of the inner portion of the bottom of the pocket (eg, JSH, curcumin) , xanthohumol (xanhohumol) and isoxanthohumol (isoxanthohumol). ZAA competes with LPS for entry into the MD-2 pocket, so the therapeutic efficacy of ZAA can be improved, probably by increasing its solubility in the blood, or by increasing its ability to target the MD-2 pocket via LPS binding protein recognition.

Salmonella is associated with human bacteremia, typhoid fever and enteritis. Most studies on the pathogenesis of Salmonella have used S. enterica subsp. enterica serovar Typhimurium (S. typhimurium) in mice. ) infection model. Although Salmonella typhimurium infection produces typhoid fever in mice, this microorganism specifically causes enteritis in humans (40). Because mice infected with Salmonella typhimurium did not develop diarrhea, the model of typhoid fever was not a good model for studying enteritis caused by Salmonella infection. On the other hand, Salmonella cholerae produces both enteritis and bacteremia in natural host pigs, but it also causes disease in humans (41). Furthermore, infection with Salmonella cholerae can cause bacteremia and death in mice (42, 43). Therefore, mice infected with Salmonella cholerae can provide a suitable model for bacteremia and enteritis caused by Salmonella. Because we have used the septicemia model caused by LPS to show that ZAA pretreatment can improve survival in mice before lethal LPS stimulation (Fig. 4E), we want to use the Salmonella porcine infection model to further study ZAA. Improve the effect of intestinal inflammation in mice. In the present invention, we use attenuated Salmonella cholera strains for use in swine plague Seedling candidates. Infection with this disease caused diarrhea and weight loss, making it suitable for studying the effects of ZAA on Salmonella infection. Our results revealed that ZAA pretreatment improved the clinical symptoms of mice infected with Salmonella choleraesuis in terms of diarrhea severity, weight changes, and bacterial load in feces.

In conclusion, we show that ZAA effectively improves the inflammatory response to experimental endotoxemia caused by free LPS or Salmonella cholerae, presumably due to its specific interaction with the MD-2 pocket in macrophages. Furthermore, the mitigation of inflammation can be attributed to a reduction in the level of Salmonella in the feces. However, ZAA has no direct inhibitory effect on the growth of Salmonella (Figure 7). Furthermore, our observations strongly support the assumption that ZAA can modulate diarrhea caused by Salmonella cholerae without the common side effects of many antibiotics (ie, the harmful immune response can be attributed to the killing of beneficial bacteria). In summary, in view of the above information and the supporting materials listed in Table 3, the present invention provides new insights into the mechanism by which ZAA and its analogs act as anti-inflammatory drugs for LPS vector infection.

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Claims (9)

A method of treating an inflammatory disease in a subject comprising administering a compound having the following formula (I) or a pharmaceutically acceptable salt thereof to a subject in need of such treatment: Wherein R ₁ is =O; R ₂ is =O, OCHO or OH; R ₃ is H or OH; R ₄ is -C(=CH ₂ )-C(CH ₃ )H-(C=O)OR _a , Wherein R _a is H or C _1-4 alkyl, or R ₄ is -(C=O)R _b , wherein R _b is C _1-4 alkyl; R ₅ is =O, OH or H; and R ₆ H or OH. The method of claim 1, wherein the inflammatory disease is a mediating inflammatory symptom of LPS. The method of claim 1, wherein the inflammatory disease is caused by a bacterial infection. The method of claim 3, wherein the bacterial infection is caused by Gram-negative bacteria. The method of claim 1, wherein the inflammatory disease comprises a lung caused by LPS Injury or kidney damage caused by LPS. The method of claim 1, wherein the inflammatory disease comprises diarrhea. The method of claim 1, wherein the inflammatory disease comprises enteritis. The method of claim 1, wherein the compound of formula (I) is anocetin A. The method of claim 1, wherein the compound of formula (I) is selected from the group consisting of: