Use of AP1B1 allosteric activators of ethyl gallate

Abstract

The invention belongs to the technical field of medicine and pharmacy, and particularly relates to application of an AP1B1 allosteric activator of ethyl gallate. In the invention, AP1B1 is a beta 1 subunit (AP1B1) of a linking factor associated protein complex 1 (AP-1), and is an essential component for assembling a functional AP-1B complex. The ethyl gallate is used as an allosteric activator of AP1B1 and is specifically combined with an allosteric pocket of the AP1B1 to induce the AP1B1 to generate conformational change, so that the vesicle transportation function of an AP1B complex is activated, and the substrate-side membrane protein sorting capacity is recovered. A target dependence experiment verifies that the effect of ethyl gallate on promoting tight junction protein ZO-1 expression and membrane localization in AP1B1 knocked-down Caco-2 cells is remarkably weakened, and it is prompted that the intestinal barrier protection function of ethyl gallate depends on AP1B1. The invention provides a new strategy for developing medicines for treating intestinal barrier injury related diseases.

Description

Technical Field

[0001] This invention belongs to the fields of medical and pharmaceutical technology, specifically relating to the use of ethyl gallate AP1B1 allosteric activator. Background Technology

[0002] The integrity of the intestinal epithelial barrier is crucial for maintaining homeostasis, preventing the invasion of exogenous substances, and preventing systemic inflammation. The core of this barrier function depends on the establishment of intestinal epithelial cell polarity and the stability of tight junctions between cells. Adaptor-associated protein complex 1B (AP-1B) maintains barrier homeostasis by mediating basolateral protein sorting; its dysfunction leads to "leaky gut" and chronic inflammation, which is a core pathological mechanism of inflammatory bowel disease (IBD). The AP-1B complex is composed of β1, γ, σ1, and μ1B subunits. Among them, μ1B (encoded by AP1M2) imparts epithelial specificity and directly recognizes sorting signals, acting as a functional determinant. The β1 subunit of adaptor-associated protein complex 1 (AP1B1), serving as a shared structural scaffold for AP-1A / B, is an essential component for the assembly of a functional AP-1B complex. Studies have shown that AP1M2 deficiency leads to barrier dysfunction and spontaneous colitis in mouse models of colitis; clinical sample studies have also confirmed that patients with Crohn's disease have reduced AP1M2 expression in the colonic epithelium and are accompanied by barrier defects.

[0003] However, current clinical treatments for intestinal barrier dysfunction (such as probiotics and anti-inflammatory agents) are mostly indirect or symptomatic supportive, with limited efficacy. Crucially, although the scaffolding role of AP1B1 in the assembly of the AP-1B complex has been elucidated, there are currently no lead compounds or drugs that can directly target AP1B1 and repair the sorting mechanism upstream through allosteric activation. Therefore, developing molecules that can allosterically activate AP1B1 and promote the formation of functional AP-1B complexes holds promise for restoring intestinal epithelial barrier function and providing a primary intervention strategy for the treatment of Crohn's disease and other IBDs. Summary of the Invention

[0004] The purpose of this invention is to provide the use of ethyl gallate as an AP1B1 allosteric activator. This invention has experimentally verified that ethyl gallate is a lead compound for allosteric activator, opening up a new therapeutic pathway for the development of original drugs to treat diseases related to intestinal barrier damage.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the use of ethyl gallate or a pharmaceutically acceptable salt thereof in the preparation of AP1B1 allosteric activator.

[0006] Preferably, the dosage form of the AP1B1 allosteric activator includes tablets, capsules, granules, oral liquids, or injections.

[0007] Preferably, the AP1B1 allosteric activator further includes pharmaceutically acceptable excipients.

[0008] This invention provides the use of an ethyl gallate probe or a pharmaceutically acceptable salt thereof in the preparation of AP1B1 allosteric activator, the structure of which is shown in Formula I: Formula I.

[0009] Preferably, the dosage form of the AP1B1 allosteric activator includes tablets, capsules, granules, oral liquids, or injections.

[0010] Preferably, the AP1B1 allosteric activator further includes pharmaceutically acceptable excipients.

[0011] This invention provides the use of an ethyl gallate probe or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating intestinal barrier dysfunction, the structure of which is shown in Formula I: Formula I.

[0012] Preferably, the drug for treating intestinal barrier dysfunction includes drugs for treating inflammatory bowel disease.

[0013] Preferably, the dosage form of the drug for treating intestinal barrier dysfunction includes tablets, capsules, granules, oral liquids, or injections.

[0014] Preferably, the medicament for treating intestinal barrier dysfunction further includes pharmaceutically acceptable excipients.

[0015] This invention provides the use of ethyl gallate (EG, chemical name ethyl 3,4,5-trihydroxybenzoate) and ethyl gallate probes in the preparation of AP1B1 allosteric activators. AP1B1 is the β1 subunit of adaptor-associated protein complex 1 (AP-1), a shared structural scaffold of AP-1A / B, and an essential component for the assembly of the functional AP-1B complex. Experiments in this invention show that EG, as an allosteric activator of AP1B1, induces conformational changes in AP1B1 by specifically binding to its allosteric pockets (key sites including Pro192, Leu197, and Asn228). This allosteric effect activates the vesicle transport function of the AP-1B complex, enhancing its ability to sort proteins on the basolateral membrane. Furthermore, target-dependent experiments show that in AP1B1 knockdown Caco-2 cells, the effect of EG in promoting the expression and correct membrane localization of the tight junction protein ZO-1 is significantly weakened, indicating that its function in protecting the intestinal barrier depends on AP1B1.

[0016] In summary, the mechanism of action of EG can be described as follows: As an allosteric activator of AP1B1, EG induces a conformational change in AP1B1 by specifically binding to its allosteric pocket, thereby activating the vesicle transport function of the AP-1B complex, restoring the protein sorting ability of the basolateral membrane, and subsequently repairing tight junctions between cells, thus improving the intestinal epithelial barrier. This tandem mechanism, from molecular interaction to cellular function, provides a mechanistic explanation for EG's efficacy in improving intestinal barrier defects and its anti-colitis effects, and offers a new strategy for developing drugs to treat diseases related to intestinal barrier damage. Attached Figure Description

[0017] Figure 1 Example 1 shows the synthetic route of the ethyl gallate probe (AD-EG) with the structure shown in Formula I in this invention; Figure 2 This is a graph showing the efficacy verification results of EG and AD-EG in protecting the intestinal barrier in Example 2; Figure 3 This is a diagram of the fluorescent labeling experiment of AD-EG in Example 3; Figure 4 This is a volcano diagram of the potential target proteins of EG in Example 4; Figure 5 The results are from the competitive pull-down experiment in Example 5; Figure 6 The results of the drug affinity target stability experiment in Example 6; Figure 7 This is a graph showing the results of the cell thermal displacement analysis experiment in Example 7; Figure 8 The results of the AP1B1(1-584) recombinant protein experiment in Example 8; Figure 9 The SPR analysis in Example 9 showed the binding of EG to wild-type AP1B1 (1–584); Figure 10 The mass spectrum obtained from the LC-MS / MS analysis in Example 10; Figure 11 This is a graph showing the molecular docking results in Example 11; Figure 12 The results of SPR analysis of EG and P192A mutant proteins in Example 13; Figure 13 The results of SPR analysis of EG and L197A mutant proteins in Example 13; Figure 14 The results of SPR analysis of EG and N228A mutant proteins in Example 13; Figure 15The results of SPR analysis of the binding of EG to the P192A / L197A / N228A triple mutant protein in Example 13; Figure 16 This is a scan result of tryptophan in Example 14; Figure 17 This is a graph showing the RMSD results in Example 15; Figure 18 The results of the RMSF analysis in Example 15; Figure 19 This is a visualization of the molecular dynamics results from Example 15; Figure 20 This is a diagram showing the efficacy verification results after target knockdown in Example 16. Detailed Implementation

[0018] This invention provides the use of ethyl gallate and its pharmaceutically acceptable salts in the preparation of AP1B1 allosteric activators.

[0019] This invention provides the use of an ethyl gallate probe in the preparation of AP1B1 allosteric activator, the structure of which is shown in Formula I: Formula I.

[0020] In this invention, unless otherwise specified, all raw materials / components are commercially available products well-known to those skilled in the art. This invention does not have specific requirements for the preparation method of the ethyl gallate probe with the structure shown in Formula I; it employs... Figure 1 The preparation can be carried out using the conventional preparation process shown.

[0021] In this invention, the dosage form of the AP1B1 allosteric activator preferably includes tablets, capsules, granules, oral liquids, or injections. In this invention, the AP1B1 allosteric activator also includes pharmaceutically acceptable excipients.

[0022] This invention provides the use of an ethyl gallate probe in the preparation of a drug for treating intestinal barrier dysfunction, the structure of which is shown in Formula I: Formula I.

[0023] In this invention, the drug for treating intestinal barrier dysfunction preferably includes a drug for treating inflammatory bowel disease.

[0024] In this invention, the dosage form of the medicament for treating intestinal barrier dysfunction preferably includes tablets, capsules, granules, oral liquids, or injections. In this invention, the medicament for treating intestinal barrier dysfunction also includes pharmaceutically acceptable excipients.

[0025] Unless otherwise stated, the terms used in this application have the following definitions, and the definitions of terms not referred to below are as commonly understood by those skilled in the art to which this invention pertains.

[0026] This invention relates to the compound ethyl gallate (EG), chemically named ethyl 3,4,5-trihydroxybenzoate, which has the following chemical structure: .

[0027] Ethyl gallate probe (AD-EG) has the following chemical structure: .

[0028] Because EG and AD-EG structures contain multiple phenolic hydroxyl groups (especially the relatively acidic 3,4,5-trisubstituted phenolic hydroxyl system), the compounds of this invention can react with pharmaceutically acceptable bases to form stable base addition salts. Therefore, in this invention, the term "pharmaceutically acceptable salt" specifically refers to the salts formed by ethyl gallate and ethyl gallate probes with relatively non-toxic, pharmaceutically acceptable bases, including but not limited to sodium salts, potassium salts, calcium salts, magnesium salts, methylamine salts, ethylamine salts, diethylamine salts, ethanolamine salts, N-methylglucosamine salts, and dibenzylethylenediamine salts.

[0029] In this invention, the term "treatment" should be interpreted broadly, including but not limited to: preventing, delaying, controlling, alleviating, improving, or curing intestinal barrier damage and related diseases through the AP1B1 activation mechanism. In this invention, the term "AP1B1 allosteric activator" refers to a compound that can specifically bind to the allosteric pocket of the β1 subunit (AP1B1) of the adaptor-associated protein complex 1 and induce a conformational change therein, thereby activating the vesicle transport function of the AP-1B complex and restoring the protein sorting ability of the basolateral membrane.

[0030] In this invention, the term "intestinal barrier damage-related diseases" refers to diseases whose pathological processes are closely related to impaired intestinal mucosal barrier function, including but not limited to: inflammatory bowel disease (such as ulcerative colitis and Crohn's disease), irritable bowel syndrome, necrotizing enterocolitis, chemotherapy or radiotherapy-induced intestinal mucositis, infectious enteritis, and liver disease-related intestinal barrier dysfunction.

[0031] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1: Synthesis of an ethyl gallate bifunctional probe To identify the direct target of ethyl gallate (EG) in protecting the intestinal barrier, this invention follows... Figure 1The synthetic route shown (Yu W, Liao M, Chen Y, et al. Photoaffinity labelling-based chemoproteomic strategy identifies PEBP1 as the target of ethyl gallate against macrophage activation) Chem Commun (Camb). (January 24, 2023;59(8):1022-1025.) To synthesize the ethyl gallate probe (AD-EG), firstly, 1-benzoyl-4-piperidinylmethylamine hydrochloride and 3-(3-(but-3-yn-1-yl)-3H-bisacrylidine-3-yl)propionic acid were dissolved in dichloromethane and stirred for 24 hours. Subsequently, EG and sodium hydride were dissolved in tetrahydrofuran, and the resulting solution was added to the previous mixture. Then, N,N'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) were added. The reaction mixture was stirred at 25°C in the dark for 24 hours. Finally, the product AD-EG was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:10, v / v) to obtain a white solid product.

[0033] 1H NMR (600 MHz, CDCl3) δ 7.53 (d, J = 1.9 Hz, 1H), 7.38 (d, J = 1.9Hz, 1H), 4.35 (q, J = 7.1 Hz, 2H), 2.47 (t, J = 7.3 Hz, 2H), 2.20 (t, J = 7.7Hz, 2H), 2.07 – 2.03 (m, 5H), 1.84 (t, J = 7.7 Hz, 2H), 1.74 (d, J = 7.3 Hz, 1H), 1.68 (t, J = 7.4 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H). 13C NMR (151 MHz, CDCl3) δ 177.16, 170.73, 165.85, 145.27, 140.11, 137.81, 122.51, 116.31, 114.48, 82.48, 69.56, 69.32, 61.23, 32.16, 28.50, 27.70, 14.28, 13.25, 1.03, 0.00. Example 2: Validation of the efficacy of EG and AD-EG in protecting the intestinal barrier Caco-2 cells were cultured in 6-well plates at 37°C. When the Caco-2 cells reached 80%–90% confluence, the medium was replaced with serum-free DMEM containing 4% DSS to establish a cell damage model. The drug-treated groups were treated with 10, 25, and 50 μM EG or AD-EG, respectively, in serum-free DMEM containing 4% DSS for 24 hours. After 24 hours, cells were collected and lysed, and protein solutions were collected. Protein quantification was performed using BCA, and the concentrations of all protein solutions were adjusted to be consistent. 6× loading buffer was added, and immunoblotting analysis was performed to determine the expression of tight junction proteins Occludin and ZO-1.

[0034] like Figure 2 The results showed that the expression of Occludin and ZO-1 decreased after DSS modeling. After treatment with EG and AD-EG, the expression of Occludin and ZO-1 was significantly upregulated, demonstrating that EG and AD-EG have the ability to protect the intestinal barrier.

[0035] Example 3: Fluorescent labeling experiment of AD-EG To verify the function of AD-EG, a fluorescent labeling experiment was performed. The specific procedure was as follows: Caco-2 cells were divided into three groups: a blank control group (untreated), a binding group (treated with 50 μM AD-EG for 2 hours), and a competition treatment group (pretreated with 100 μM EG for 2 hours, then co-incubated with 50 μM AD-EG). After treatment, the cells were washed twice with PBS and irradiated under 365 nm UV light for 10 minutes to cross-link the probe. Cells were then lysed using NP-40 lysis buffer, with 1% protease inhibitor mixture added to the lysis buffer. Protein concentration was measured and normalized. For the copper-catalyzed click chemistry reaction, cell lysate containing 1 mg of protein was taken, and 3 μL of 5-TAMRA-azide (20 mM, dissolved in DMSO), 24 μL of TCEP (14.4 mg / mL), and 3.5 μL of TBTA (34 mM, dissolved in DMSO) were added sequentially. 11.6 μL of CuSO4 (50 mM) was added to initiate the reaction, and the reaction was carried out at room temperature for 1 hour. The labeled proteins were precipitated by centrifugation at 6000 × g, washed with cold methanol, and then denatured by heating at 98 °C for 10 min in 0.2% SDS buffer. Finally, the samples were separated by SDS-PAGE and analyzed by intragel fluorescence imaging using a chemiluminescent gel imaging system (ChemiDoc XRSSystem, Bio-Rad, USA).

[0036] like Figure 3The results show that the strong fluorescent labeling signal observed using the 5-TAMRA-azide reporter system can be significantly suppressed under free EG competition conditions, confirming that AD-EG can specifically label potential targets in living cells.

[0037] Example 4: Discovery of direct target of ethyl gallate (EG) based on chemical proteomics To elucidate the direct target of ethyl gallate (EG), this invention employs known chemical proteomics methods. Specifically, it uses methods such as... Figure 1 The EG probe (AD-EG) prepared by the shown synthetic route was used for target identification in Caco-2 cells. Caco-2 cells were divided into a binding group and a competition group. The binding group was treated with 50 μM AD-EG for 2 hours, while the competition group was pretreated with 100 μM EG for 2 hours, followed by the addition of 50 μM AD-EG. After treatment, all cells were washed twice with PBS and irradiated with UV (365 nm, 10 min) to achieve photocrosslinking. After cell lysis and protein concentration normalization, a click chemistry reaction with biotin-PEG3-azide was performed. The biotinylated protein was precipitated, resuspended in 0.2% sodium dodecyl sulfate (SDS) in PBS solution, and incubated with streptavidin-coated magnetic beads at room temperature for 3 hours. Subsequently, the captured protein was digested with trypsin on the magnetic beads, and the resulting peptides were analyzed by LC-MS / MS. Proteins with an enrichment factor ≥1.5 and a p-value ≤0.05 were defined as significantly enriched proteins.

[0038] like Figure 4 The results showed that the adaptor protein β subunit AP1B1 was significantly and specifically enriched under these conditions.

[0039] Example 5: Competitive pull-down and Western Bllot verification of binding specificity.

[0040] To confirm the specificity of EG binding to AP1B1, this invention conducted competitive chemical proteomics experiments, which were ultimately validated by Western blotting. The specific steps are as follows: Caco-2 cells were divided into a blank group, a binding group, and a competition group. The blank group received no treatment, the binding group was treated with a 50 μM AD-EG probe for 2 hours, and the competition group was pretreated with 100 μM free EG for 2 hours, followed by co-incubation with 50 μM AD-EG. All three groups of cells were washed with PBS and irradiated under 365 nm UV light for 10 minutes to allow in-situ photocrosslinking of the probe with adjacent proteins. After cell lysis, the probe-bound protein complex was biotinylated using a click chemistry reaction with biotin-PEG3-azide. Subsequently, the biotinylated proteins were affinity-enriched (pull-down) using streptavidin magnetic beads. After elution, the enriched protein complexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. Western blot analysis was performed using an anti-AP1B1 specific antibody.

[0041] like Figure 5 The results showed that no specific bands appeared in the blank control group. In the binding group, the AD-EG probe effectively enriched a clear AP1B1 protein band; however, in the competition group, the AP1B1 enrichment signal was significantly weakened or completely disappeared because pretreatment with excess free EG competitively blocked the binding of AD-EG to AP1B1. These results directly and visually confirm the specific interaction between EG and AP1B1 at the protein level.

[0042] Example 6: Drug Affinity Target Stability (DARTS) experiment to verify binding specificity.

[0043] To verify the direct binding of EG to AP1B1 protein, this invention conducted a drug affinity target stability experiment based on an established method. The process is briefly described below: Caco-2 cells were lysed using NP-40 lysis buffer to obtain total protein lysates. The lysates were then co-incubated with a series of concentration gradients of EG (50, 100, 200, 400 μM) at room temperature for 1 hour. Subsequently, the lysates were co-incubated with different concentrations of EG (0, 50, 100, 200, 400 μM) at room temperature for 1 hour. Following this, each group (except for a separately set DMSO solvent control group) underwent limited enzymatic digestion with a final concentration of 5 μg / mL of protease, and incubation was continued at room temperature for 20 minutes. The enzymatic digestion was terminated by adding SDS-PAGE loading buffer and heating. Finally, the stability of AP1B1 protein was assessed using Western blotting with an anti-AP1B1 specific antibody.

[0044] like Figure 6 The experimental results showed that, compared with the control group not treated with EG, the AP1B1 protein band signal was significantly enhanced in the EG-incubated samples, and this protective effect was EG concentration-dependent. This result directly demonstrates that EG can specifically bind to endogenous AP1B1 protein in cell lysates, thereby stabilizing its conformation and enhancing its resistance to protease degradation.

[0045] Example 7: Cellular thermal displacement analysis (CETSA) experiment to verify binding specificity.

[0046] Cellular thermal displacement analysis was used to verify the interaction between EG and AP1B1 protein. The specific experiments were as follows: Caco-2 cells were collected, and cell lysates were prepared by repeated freeze-thaw cycles in liquid nitrogen. The lysates were then divided into two treatment groups: a solvent control group (DMSO) and an experimental group, both incubated with 50 μM EG at 4°C for 1 hour. After incubation, each sample was aliquoted and heated to a series of gradient temperatures (ranging from 40°C to 67°C) using a T100 thermal cycler (Bio-Rad, USA) for 3 minutes. After heat stress, the soluble protein fraction was collected by centrifugation, and Western blotting analysis was performed using a specific anti-AP1B1 antibody to assess the thermal stability of the AP1B1 protein.

[0047] like Figure 7 The results showed that the thermal stability of AP1B1 protein was significantly increased after EG treatment, proving that the drug has specific binding to AP1B1 protein.

[0048] Example 8: Expression and purification of wild-type recombinant protein with AP1B1 core domain (1-584).

[0049] To precisely locate the binding interface between ethyl gallate (EG) and the target AP1B1, this invention first prepared a protein targeting its core functional domain, AP1B1(1–584). A plasmid encoding a wild-type truncated form of AP1B1(1–584) fused with an N-terminal His tag was transformed into *E. coli* BL21(DE3) competent cells for expression. After induction at 20°C for 12 hours using 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG), the bacterial cells were collected by centrifugation. The cells were sonicated and centrifuged, and the supernatant was purified using a Ni-NTA affinity chromatography column. The target protein was eluted with a buffer containing a linear gradient of imidazole (10–250 mM). The collected protein was concentrated and the buffer was replaced with PBS. Identification was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining, confirming the acquisition of high-purity recombinant AP1B1(1–584) protein (e.g., ...). Figure 8This provides a material basis for subsequent research on molecular interactions.

[0050] Example 9: Surface plasmon resonance (SPR) technique to verify the bonding strength between EG and AP1B1 (1-584).

[0051] To accurately quantify the interaction kinetics between ethyl gallate (EG) and the target AP1B1, this embodiment employs SPR (Sequencing-Reactive Protein) assays. Specifically, using a Biacore T200 instrument, the carboxymethyl dextran matrix of the CM5 sensor chip was first activated with an EDC / NHS mixed solution. Then, purified recombinant AP1B1 protein (120 μg / mL, dissolved in 10 mM pH 4.5 sodium acetate buffer) was immobilized on the chip surface as a ligand, and the remaining active sites were blocked with ethanolamine-HCl. All analyses were performed at 25°C and a constant flow rate of 30 μL / min. EG solutions of different concentrations, dissolved in PBS buffer containing 5% DMSO, were sequentially passed through the sample and reference cells containing the immobilized protein, and the binding and dissociation processes were monitored in real time. The equilibrium dissociation constant KD was obtained.

[0052] like Figure 9 The experiment showed that the KD value of EG binding to AP1B1 was 1.48 μM, confirming that there is a high-affinity specific interaction between the two.

[0053] Example 10: Mass spectrometry-based probe-target crosslinking site analysis To directly identify the specific binding region of the ethyl gallate (EG) derivative probe AD-EG to the truncated target protein AP1B1 (1-584) at the molecular level, this example performed cross-linking and mass spectrometry analysis. The specific method is as follows: Purified recombinant AP1B1 (1-584) protein was co-incubated with DMSO (control group) or 50 µM AD-EG probe at 4°C for 2 hours, followed by irradiation with 365 nm UV light in an ice bath for 10 minutes to covalently cross-link the probe with adjacent amino acid residues. The cross-linked sample was denatured at 98°C, separated by SDS-PAGE, and the gel band corresponding to AP1B1 (1-584) was excised and digested with in-gel trypsin. The obtained peptides were analyzed using a Q-Exactive HF mass spectrometer coupled with a UltiMate 3000 RSLCnano system: the peptides were first enriched on a C18 capture column (100 µm × 2 cm), and then separated on an analytical column (75 µm × 15 cm) at a flow rate of 300 nL / min using a linear gradient of 2%–40% mobile phase B (acetonitrile containing 0.1% formic acid) for 70 minutes. Mass spectrometry was performed in positive ion mode, with a full scan mass range of 350–1500 m / z and a resolution of 60,000. Raw data were retrieved from the UniProt database using Proteome Discoverer software (version 2.2) with the following parameters: maximum number of missed cleavage sites of 2, precursor mass error of 10 ppm, fragment mass error of 0.02 Da, cysteine ​​carbamoyl methylation as a fixed modification, methionine oxidation as a variable modification, and a custom modification of +318.1098 Da for relevant amino acids. Finally, the Percolator algorithm was used to filter the peptide identification results to ensure a false discovery rate of less than 1%. This analysis aimed to accurately identify the covalent cross-linking sites of the AD-EG probe on the AP1B1 protein.

[0054] like Figure 10 The results showed that LC-MS / MS analysis identified a covalently modified peptide (residues 248-272). The peptide sequence was confirmed by alignment with theoretical fragment ions, and a mass shift consistent with AD-EG crosslinking was observed, with serine 258 (S258) and serine 271 (S271) identified as the major modification sites. This suggests that this region is directly involved in binding or is adjacent to the binding pocket of EG.

[0055] Example 11: Molecular docking simulation predicts the binding site of EG and AP1B1 To predict the binding mode of ethyl gallate (EG) to the domains near the target AP1B1 peptide modification at the atomic level, this embodiment uses Schrödinger 2018 software for molecular docking simulation. First, the AP1B1 crystal structure was obtained from the PDB database and optimized using the "Protein Preparation Wizard" module; simultaneously, the three-dimensional structure of EG was optimized using the "LigPrep" module. The binding pocket was predicted using the "SiteMap" tool, and a docking grid with a side length of 20 Å was generated centered on this pocket (coordinates x=-17.332, y=32.139, z=44.032). The "Induced Fit Docking" (IFD) protocol was used to account for protein flexibility, and accurate calculations were performed in "Glide XP" mode. Finally, the optimal docking conformation was selected and analyzed using PyMOL software.

[0056] like Figure 11 The results show that EG can form stable hydrogen bonds and other interactions with key amino acid residues (such as Pro192, Leu197, and Asn228) in the active pocket of AP1B1. This result theoretically clarifies the binding mode and provides a direct basis for subsequent site-directed mutagenesis experiments.

[0057] Example 12: Expression and purification of AP1B1 core domain (1-584) mutant recombinant protein Based on molecular docking predictions, EG binds to a region on the surface of the AP1B1 core domain (1-584) and forms key interactions with proline residues at position 192 (Pro192), leucine residues at position 197 (Leu197), and asparagine residues at position 228 (Asn228). To experimentally verify the necessity of these amino acid residues in binding, this invention constructed corresponding single-site alanine mutants (P192A, L197A, N228A) and triple alanine mutants (P192A / L197A / N228A). The expression and purification of these mutant recombinant proteins were carried out in the same manner as described in Example 8, providing materials for subsequent binding experiments to directly verify whether the above sites are functional binding sites for EG.

[0058] Example 13: SPR verification of the binding strength of EG to the mutant AP1B1 (1-584).

[0059] To accurately verify at the molecular level whether Pro192, Leu197, and Asn228 are key amino acid residues for the binding of EG to AP1B1, this invention determined the binding affinity of EG to each mutant of the AP1B1 core domain (1-584) using surface plasmon resonance (SPR) technology. The specific experimental method is the same as in Example 9.

[0060] like Figure 12 , 13 , Figure 14 , Figure 15 The results showed that, compared to the wild-type protein, all mutants resulted in a significant decrease in binding affinity, exhibiting a stepwise loss: the equilibrium dissociation constant (KD) for EG binding to single-point mutants (P192A, L197A, N228A) increased to approximately 10 μM; while the KD value of the triple mutant (P192A / L197A / N228A) increased dramatically to 45.5 μM. This data directly demonstrates that Pro192, Leu197, and Asn228 are key sites for maintaining high-affinity binding between EG and AP1B1, and their contributions to binding have an additive effect, thus experimentally confirming the binding mode predicted by molecular docking.

[0061] Example 14: Protein allosteric analysis based on tryptophan endogenous fluorescence spectroscopy To obtain direct evidence of protein conformational changes caused by EG binding to AP1B1 in solution, this example uses tryptophan endogenous fluorescence spectroscopy for analysis. Purified AP1B1(1-584) protein was diluted to 5 μM with PBS and mixed with a gradient of EG solutions at final concentrations of 12.5–200 μM in pretreated, cleaned quartz microwells. Using a multi-mode microplate reader, fluorescence spectra at emission wavelengths of 310–500 nm were scanned and recorded at an excitation wavelength of 280 nm, with background correction performed using EG solutions of corresponding concentrations.

[0062] like Figure 16 The results showed that EG significantly quenched the endogenous tryptophan fluorescence of AP1B1 protein in a concentration-dependent manner. This phenomenon directly demonstrates that EG binding alters the microenvironment of tryptophan residues, inducing local or global three-dimensional conformational changes in AP1B1, thus providing crucial biophysical and chemical evidence for EG as an allosteric regulator of AP1B1.

[0063] Example 15: Analysis of Allosteric Mechanism Based on Molecular Dynamics Simulations To elucidate the dynamic structural basis of the ethyl gallate (EG)-induced allosteric reaction of AP1B1 at the atomic scale, this embodiment performed all-atom molecular dynamics simulations. The simulations began with the structure of the EG-AP1B1(1–584) complex obtained from the aforementioned docking, using the AMBER 14 software package, based on quantum chemistry (B3LYP / 6-31G). Based on the calculated precise force field parameters of EG, a periodic boundary simulation system containing TIP3P aqueous solution was constructed. After stepwise energy minimization, heating, and equilibrium, the system underwent a 200 ns isothermal and isobaric production simulation at 300 K and 1 atm.

[0064] To quantitatively assess the dynamic changes in protein conformation, this invention performed root mean square deviation (RMSD) and root mean square fluctuation (RMSF) analysis on the simulated trajectory. Figure 17 As shown, the overall conformational RMSD of the complex system after equilibrium is comparable to that of the individual protein system, indicating that EG binding did not significantly affect the global structural stability of AP1B1. Figure 18 As shown, the binding of EG can be detected as altering the local conformational flexibility of the N-terminal functional domain of AP1B1 (which is involved in the binding of the downstream effector protein Arf1), thus changing its RMSF distribution pattern. Figure 19 The molecular dynamics visualization shows the local conformational changes after EG binds to AP1B1. These results indicate that EG, as an allosteric regulator, exerts its function by modulating the conformational dynamics of key functional domains of AP1B1. This provides a favorable conformational basis for the subsequent interaction between AP1B1 and Arf1, and thus offers a molecular-level understanding of the functional regulation of the AP-1B complex.

[0065] Example 16: Target knockdown to verify the targeting effect of EG on AP1B1 To confirm that the adaptor protein AP1B1 is a necessary target for the function of ethyl gallate (EG), this embodiment validated the function by constructing an AP1B1 knockdown cell model. Specifically, AP1B1-specific siRNA or control siRNA was transfected into Caco-2 cells using Lipofectamine RNAiMAX reagent. After culturing for 48 hours, the knockdown efficiency was verified by Western blotting, resulting in a cell line with stable low expression of AP1B1. An inflammation model was established in this knockdown cell line and a normal control cell line, and EG was administered. The efficacy was evaluated by detecting the expression of the tight junction protein ZO-1.

[0066] like Figure 20 The results showed that EG significantly promoted ZO-1 expression in control cells, while this effect was significantly weakened in AP1B1 knockdown cells, thus directly demonstrating at the functional level that AP1B1 is a key target for EG to exert its anti-colitis efficacy.

[0067] In summary, AP1B1 is the β1 subunit of adaptor-associated protein complex 1 (AP-1), a shared structural scaffold of AP-1A / B, and an essential component for the assembly of the functional AP-1B complex. This invention demonstrates that EG, as an allosteric activator of AP1B1, induces conformational changes in AP1B1 by specifically binding to its allosteric pockets (key sites including Pro192, Leu197, and Asn228). This allosteric effect activates the vesicle transport function of the AP-1B complex, restoring the basolateral membrane protein sorting ability. Based on this, target-dependent experiments showed that in AP1B1 knockdown Caco-2 cells, the effect of EG in promoting the expression and correct membrane localization of the tight junction protein ZO-1 was significantly weakened, indicating that its function in protecting the intestinal barrier depends on AP1B1. This tandem mechanism, from molecular interaction to cellular function, provides a mechanistic explanation for EG's efficacy in improving intestinal barrier defects and combating colitis.

[0068] 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. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of ethyl gallate or pharmaceutically acceptable salt thereof in the preparation of an AP1B1 allosteric activator.

2. Use according to claim 1, characterized in that, The dosage form of the AP1B1 allosteric activator includes tablets, capsules, granules, oral liquids or injections.

3. Use according to claim 2, characterized in that, The AP1B1 allosteric activator further comprises pharmaceutically acceptable adjuvants.

4. Use of an ethyl gallate probe in the preparation of an AP1B1 allosteric activator, characterized in that, The structure of the ethyl gallate probe is shown as formula I: Formula I.

5. Use according to claim 4, characterized in that, The dosage form of the AP1B1 allosteric activator includes tablets, capsules, granules, oral liquids or injections.

6. Use according to claim 5, characterized in that, The AP1B1 allosteric activator further comprises pharmaceutically acceptable adjuvants.

7. Use of an ethyl gallate probe or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of intestinal barrier dysfunction, characterized in that, The structure of the ethyl gallate probe is shown as formula I: Formula I.

8. Use according to claim 7, characterized in that, The drug for treating intestinal barrier dysfunction includes drugs for treating inflammatory bowel disease.

9. Use according to claim 7, characterized in that, The dosage form of the drug for treating intestinal barrier dysfunction includes tablets, capsules, granules, oral liquids or injections.

10. Use according to claim 9, characterized in that, The drug for treating intestinal barrier dysfunction further comprises pharmaceutically acceptable adjuvants.

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