AHR ligand 5-methoxy-2-methyl-3-indoleacetic acid and application thereof

Abstract

The invention belongs to the technical field of aromatic hydrocarbon receptor ligands, and particularly relates to an AHR ligand 5-methoxy-2-methyl-3-indoleacetic acid and application thereof. According to the invention, a compound 5-methoxy-2-methyl-3-indoleacetic acid (MMIA) is used as an aromatic hydrocarbon receptor (AHR) ligand, and the application of the MMIA in the aspects of enhancing the stability of the AHR, promoting the AHR to enter a cell nucleus, relieving colitis and the like is disclosed. The result of the embodiment shows that the MMIA can be specifically combined and activate the AHR; mMIA gavage treatment can play an anti-inflammatory role and effectively relieve colitis symptoms. AHR is a key molecule for regulating and controlling the steady state of the intestinal tract, can inhibit proinflammatory pathways such as NF-kappa B and the like, reduce secretion of proinflammatory factors such as IL-6 and TNF-alpha, and also can repair damaged mucosa, restore the integrity of the intestinal barrier and improve related symptoms of the colitis from the source.

Description

Technical Field

[0001] This invention belongs to the field of aromatic hydrocarbon acceptor ligand technology, specifically relating to an AHR ligand 5-methoxy-2-methyl-3-indoleacetic acid and its applications. Background Technology

[0002] Aromatic hydrocarbon receptors (AHRs) are ligand-dependent transcription factors widely expressed in various human tissues and cells. They play important regulatory roles in microbial-host interactions and maintaining physiological homeostasis, and are also a research hotspot and potential target in fields such as tumor immunotherapy and metabolic regulation.

[0003] In the absence of ligand binding, AHR forms a cytoplasmic complex with molecules such as heat shock protein 90 (HSP90) and AHR interacting protein (XAP2). Upon binding to a specific ligand, AHR undergoes a conformational change, enters the nucleus, and forms a heterodimer with aryl hydrocarbon receptor nuclear transloin (ARNT), thereby regulating the transcription of downstream target genes and completing the signal transduction process.

[0004] Currently known AHR ligands exhibit structural diversity, including environmental pollutants, plant natural products, and metabolites from humans or symbiotic microorganisms. Among these, indole compounds produced by microbial metabolism of tryptophan have been reported as endogenous AHR ligands. Studies show that these ligands primarily drive receptor activation by binding to the PAS-B domain of AHR and interacting with its conserved amino acid residues.

[0005] Although some indole derivatives have been shown to activate AHR and participate in physiological regulation, existing known AHR ligands have shortcomings in terms of binding specificity and stability. Therefore, developing novel AHR ligands with novel structures and well-defined activities is of great significance for further elucidating the regulatory mechanisms of the AHR signaling pathway and expanding its targeted applications. Summary of the Invention

[0006] The purpose of this invention is to provide an aromatic hydrocarbon acceptor (AHR) ligand, 5-methoxy-2-methyl-3-indoleacetic acid (MMIA), and its application as an AHR ligand.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The present invention provides an AHR ligand comprising 5-methoxy-2-methyl-3-indoleacetic acid (MMIA).

[0009] The present invention also provides the use of 5-methoxy-2-methyl-3-indoleacetic acid as an AHR ligand.

[0010] Preferably, its use as an AHR agonist.

[0011] This invention also provides the following applications:

[0012] Application of 5-methoxy-2-methyl-3-indoleacetic acid in enhancing the stability of AHR.

[0013] Application of 5-methoxy-2-methyl-3-indoleacetic acid in promoting AHR entry into the cell nucleus.

[0014] Application of 5-methoxy-2-methyl-3-indoleacetic acid in promoting AHR binding to XRE reaction elements.

[0015] Application of 5-methoxy-2-methyl-3-indoleacetic acid in promoting the expression of downstream target genes of AHR.

[0016] Application of 5-methoxy-2-methyl-3-indoleacetic acid in inhibiting the NF-κB pro-inflammatory pathway, reducing the secretion of IL-6 and TNF-α pro-inflammatory factors, repairing damaged mucosa, or restoring the integrity of the intestinal barrier.

[0017] The use of 5-methoxy-2-methyl-3-indoleacetic acid in the preparation of anti-inflammatory drugs. Preferably, the anti-inflammatory effect is for the treatment or relief of colitis.

[0018] The present invention also provides a pharmaceutical composition for treating or relieving colitis, the pharmaceutical composition comprising 5-methoxy-2-methyl-3-indoleacetic acid.

[0019] Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0020] Beneficial effects:

[0021] This invention is the first to discover that compound 5-methoxy-2-methyl-3-indoleacetic acid (MMIA) is an AHR ligand, and this has been confirmed by biochemical and molecular biological experiments. MMIA is an indoleacetic acid compound with a robust AHR-binding core structure, making it a stable tool molecule for studying the AHR mechanism.

[0022] This invention discloses the application of MMIA in alleviating colitis, demonstrating significant application potential in this field. Examples confirm that its gavage administration exerts an anti-inflammatory effect, effectively relieving colitis symptoms. AHR is a key molecule in intestinal homeostasis regulation; it can inhibit pro-inflammatory pathways such as NF-κB, reduce the secretion of pro-inflammatory factors such as IL-6 and TNF-α, and repair damaged mucosa, restoring intestinal barrier integrity, thus fundamentally improving colitis-related symptoms. Compared to existing drugs and potential ligands, MMIA, with its unique structure, enhances the binding specificity and stability of AHR, precisely regulates the inflammatory microenvironment, reduces side effects, and possesses the safety advantages of its naturally derived structure, laying the foundation for clinical translation.

[0023] The results of this invention clearly demonstrate that MMIA can specifically bind to and activate AHR. Its 5-position methoxy and 2-position methyl substitutions enhance hydrophobicity, and its unique structure forms a differentiated conformation, improving binding specificity and stability. This structural advantage supports its ability to maintain activity and exert efficient anti-inflammatory effects in the intestine, ensuring continuous targeting of AHR to alleviate colitis in a complex physiological environment.

[0024] The discovery of MMIA in this invention fills a research gap in indoleacetic acid compounds as AHR ligands, overcomes the limitations of natural product discovery, enriches the diversity of ligand libraries, and provides a new model for exploring the association between ligand structure and signal specificity. Its potential to alleviate colitis expands the research scope of AHR targeting, provides new ideas and candidate molecules for the development of targeted drugs for inflammatory bowel disease, and has significant academic and clinical value.

[0025] The MMIA of this invention, as a novel AHR ligand, has advantages such as well-defined structural characteristics, strong binding specificity, stable signal activation, and the ability to exert ligand function without additional structural optimization. It can not only provide a reliable tool molecule for the precise analysis of the AHR signaling pathway regulation mechanism, but also enrich the structural diversity of the AHR ligand library. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 Figure showing the results of enhancing AHR stability for MMIA.

[0028] Figure 2 The figure shows the results of MMIA enhancing AHR stability in a dose-dependent manner.

[0029] Figure 3 The image shows the results of Western blotting analysis to show how MMIA promotes AHR entry into the cell nucleus.

[0030] Figure 4 Immunofluorescence results of MMIA promoting AHR entry into the cell nucleus.

[0031] Figure 5 Resulting diagram of MMIA promoting AHR binding to XRE reaction elements.

[0032] Figure 6 Figure showing the results of MMIA promoting the expression of downstream target genes of AHR.

[0033] Figure 7 The graph shows the results of the Disease Activity Index (DAI score) and colon length in mice in Example 6.

[0034] Figure 8 The image shows the results of the mouse colon tissue structure and inflammatory factor levels in Example 6.

[0035] Figure 9 This shows the distribution of dead cells in the mouse colon tissue in Example 6. Detailed Implementation

[0036] This invention provides an aromatic hydrocarbon acceptor (AHR) ligand, 5-methoxy-2-methyl-3-indoleacetic acid (MMIA), and its application as an AHR ligand.

[0037] The 5-methoxy-2-methyl-3-indoleacetic acid (MMIA) of this invention is a structurally modified derivative of indoleacetic acid. With indole-3-acetic acid as the parent nucleus, methoxy is substituted at the 5-position and methyl is substituted at the 2-position. It retains the core skeleton that binds to AHR and forms a differentiated spatial conformation through unique substituent groups.

[0038] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0039] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0040] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0041] Example 1

[0042] HCT116 cells in logarithmic growth phase were harvested, digested with trypsin, and then injected at a concentration of 1.3 × 10⁻⁶. 6Cells were seeded into 6cm dishes (4mL per dish) and cultured for 16h. The culture medium was discarded, and fresh medium was added. 0.2mM MMIA was added to one dish, and an equal volume of DMSO was added to another. After 3 hours of treatment, the cells were digested with trypsin, collected, and the culture medium was discarded. 650µL of PBS containing 1mM PMSF was added to each tube. The 650µL resuspension was aliquoted into 200µL PCR tubes (10 tubes, 50µL each). Ten tubes were treated with PCR at temperature gradients of 51, 51.2, 51.5, 52, 52.4, 52.8, 53.2, 53.6, 54, and 54.5℃ for 3 min each. The resuspension was repeatedly freeze-thawed three times with liquid nitrogen, centrifuged at 12000rpm at 4℃ for 15 min, and the supernatant was collected. Protein loading buffer was added, and the mixture was incubated at 70℃ for 10 min. AHR stability was detected by Western blotting. The results showed that ( Figure 1 MMIA can enhance the stability of AHR.

[0043] Example 2

[0044] HCT116 cells in logarithmic growth phase were digested with trypsin and then divided into 2.5 × 10⁶ cells per well. 5 (1 ml per well) cells were seeded into a 12-well plate and cultured for 16 hours. The culture medium was then discarded and replaced with the following drug-containing culture medium:

[0045] MMIA was diluted to 3.2 mM, 1.6 mM, 0.8 mM, 0.4 mM, 0.2 mM, 0.1 mM, 0.05 mM, 0.025 mM, 0.0125 mM, and 0 mM in cell culture medium and treated for 3 hours. Cells were then digested with trypsin, collected, and the culture medium discarded. 50 µL of PBS containing 1 mM PMSF was added to each tube, and 10 tubes were treated with a PCR instrument at 52 °C for 3 min. The cells were resuspended three times by repeated freeze-thaw cycles in liquid nitrogen, centrifuged at 12000 rpm at 4 °C for 15 min, and the supernatant was collected. Protein loading buffer was added, and the cells were incubated at 70 °C for 10 min. AHR stability was detected by Western blotting. The results showed that ( Figure 2 MMIA enhances the stability of AHR in a dose-dependent manner.

[0046] Example 3

[0047] HCT116 cells in logarithmic growth phase were harvested, digested with trypsin, and then divided into 6.25 × 10⁶ cells per well. 5Two milliliters (2 ml per well) were seeded into 6-well plates and cultured for 16 hours. The culture medium was then discarded, and the cells were treated with 0.2 mM MMIA for 0, 6, and 9 hours. After the treatment time, nuclear and cytoplasmic proteins were extracted, and the expression of AHR in the nucleus and cytoplasm was detected by Western blotting. The results showed that ( Figure 3 MMIA can promote AHR entry into the cell nucleus.

[0048] Place a sliding plate into a 24-well plate, with each well containing 1.25 × 10⁻⁶ plates. 5 500 µL of cells per well were seeded into a 24-well plate and cultured for 16 h. The culture medium was then discarded, and the cells were treated with 0.2 mM MMIA for 0, 6, and 9 hours. After the treatment time was met, the cells were collected, fixed, and immunofluorescence was performed. The scale bar was 200 µm. The results showed that ( Figure 4 MMIA can promote AHR entry into the cell nucleus.

[0049] Example 4

[0050] HCT116 cells in logarithmic growth phase were harvested, digested with trypsin, and then injected at a concentration of 1.3 × 10⁻⁶. 6 (4 ml per dish) Cells were seeded into 6 cm dishes and cultured for 16 h. The culture medium was then discarded. A luciferase reporter gene plasmid (pGL4.42-luc2P-XRE) carrying a heterologous biological response element (XRE) and a Renilla luciferase internal control plasmid (pRL-TK) carrying a TK promoter were co-transfected into the cells. Eight hours after transfection, the cells were digested and the cells were divided into two wells at a density of 2.5 × 10⁶ cells / well. 5 1 ml of cells per well was seeded into a 12-well plate and cultured for 16 h. The culture medium was then discarded, and the cells were treated with 0.2 mM MMIA for 0, 1, 3, and 6 hours. After the treatment time was met, a dual-luciferase reporter assay was performed. The results showed that ( Figure 5 MMIA can promote the binding of AHR to XRE reactive elements.

[0051] Example 5

[0052] HCT116 cells in logarithmic growth phase were harvested, digested with trypsin, and then divided into 6.25 × 10⁶ cells per well. 5 Two cells per well were seeded into 6-well plates and cultured for 16 hours. The culture medium was then discarded, and the cells were treated with 0.2 mM MMIA for 9 hours. After the treatment time, cellular RNA was extracted, and the expression of downstream target genes CYP1A1, CYP1A2, AHRR, and TIPARP of AHR was detected by RT-qPCR. The results showed that ( Figure 6 MMIA can promote the expression of downstream target genes of AHR.

[0053] Example 6

[0054] (1) Forty C57BL / 6 mice (16-18g) were randomly divided into four groups, named as follows: blank control group (CON), model group (dextran sulfate sodium DSS), drug control group (MMIA) and intervention group (DSS + MMIA); each group consisted of 10 mice.

[0055] (2) The blank control group (CON), the model group (DSS), the drug control group (MMIA) and the DES intervention group (DSS + MMIA) were treated. The intervention group (DSS + MMIA) was treated as follows: from day 1 to day 17 of the experiment, the DES intervention group (DSS + MMIA) was given 200 μL of MMIA solution by gavage every day and had free access to distilled water. From day 8 to day 14 of the experiment, the group was required to drink 3% DSS solution.

[0056] The treatment method for the model group (DSS) was as follows: from day 1 to day 17 of the experiment, 200 μL of physiological saline was administered by gavage daily; from day 8 to day 14 of the experiment, 3% DSS water was allowed to be drunk freely.

[0057] The treatment method for the drug control group (MMIA) was as follows: From day 1 to day 17 of the experiment, the drug control group (MMIA) was given 200 μL of MMIA solution by gavage daily and had free access to distilled water.

[0058] The blank control group (CON) was treated as follows: During the experiment, the control group was given 200 μL of physiological saline by gavage every day and had free access to distilled water.

[0059] During the modeling period, the changes in diarrhea index of mice in each group were detected.

[0060] After modeling, 500 μL of blood was collected from the eyes of the blank control group (CON), the model group (DSS), the drug control group (MMIA), and the MMIA intervention group (DSS + MMIA). Serum was separated and ELISA was used to detect cytokines. A whole colon (from the terminal cecum to the anus) was harvested, its length measured, and its appearance observed. One cm of mid-colon tissue was fixed, dehydrated, embedded, and stained with hematoxylin and eosin (HE). HE staining of colon tissue from different groups of mice was observed and scored.

[0061] At the end of the modeling process, the DAI score of mice in the DSS group was significantly increased, while the Disease Activity Index (DAI score) of mice in the MMIA intervention group (DSS + MMIA) was decreased. Figure 7 A) By Figure 7 As shown in B and C, the colon length of mice in the DSS model group was significantly shortened, while the colon length of mice in the MMIA intervention group (DSS + MMIA) was significantly increased.

[0062] Depend on Figure 8 As shown in Figure A, the colonic tissue structure of the MMIA intervention group (DSS + MMIA) was similar to that of the normal group, with clear structures in each layer of the intestinal tissue; the mucosal epithelium was intact, the epithelial cells had normal morphology, and no obvious inflammatory changes were observed. In contrast, DSS-induced colonic tissue in mice showed significant inflammatory damage, and the colonic tissue damage score of the MMIA intervention group (DSS + MMIA) was significantly lower than that of the DSS group (see Figure A). Figure 8 B).

[0063] Depend on Figure 8 C, D, and E show that the levels of inflammatory factors in the MMIA intervention group (DSS+MMIA) were significantly lower than those in the model group (DSS).

[0064] Depend on Figure 9 TUNEL staining showed that the level of intestinal epithelial cell death in the MMIA intervention group (DSS+MMIA) was significantly lower than that in the model group (DSS). TUNEL staining is used to detect cell apoptosis; more green signals indicate more cell death and more severe intestinal damage.

[0065] Therefore, MMIA can alleviate the symptoms of acute colitis and colonic tissue damage, reduce the level of inflammatory factors, and significantly relieve colitis.

[0066] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An AHR ligand, characterized in that, The AHR ligand includes 5-methoxy-2-methyl-3-indoleacetic acid. Application of 2,5-methoxy-2-methyl-3-indoleacetic acid as an AHR ligand.

3. The application according to claim 2, characterized in that, The AHR ligand is an AHR agonist. Application of 4,5-methoxy-2-methyl-3-indoleacetic acid in enhancing the stability of AHR. Application of 5,5-methoxy-2-methyl-3-indoleacetic acid in promoting AHR entry into the cell nucleus. Application of 6,5-methoxy-2-methyl-3-indoleacetic acid in promoting AHR binding to XRE reaction elements. Application of 7,5-methoxy-2-methyl-3-indoleacetic acid in promoting the expression of downstream target genes of AHR. Application of 8,5-methoxy-2-methyl-3-indoleacetic acid in inhibiting the NF-κB pro-inflammatory pathway, reducing the secretion of IL-6 and TNF-α pro-inflammatory factors, repairing damaged mucosa, or restoring the integrity of the intestinal barrier. Application of 9,5-methoxy-2-methyl-3-indoleacetic acid in the preparation of anti-inflammatory drugs.

10. A pharmaceutical composition for treating or relieving colitis, said pharmaceutical composition comprising 5-methoxy-2-methyl-3-indoleacetic acid.

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