Application of E3 ubiquitin ligase inhibitor in preparation of allergic rhinitis medicine

Abstract

The invention relates to the technical field of biological medicines, in particular to application of an E3 ubiquitin ligase inhibitor in preparation of an allergic rhinitis medicine. It is found for the first time that the E3 ubiquitin ligase inhibitor inhibits ubiquitination degradation of CLDN1 to enhance the barrier function and relieve nasal symptoms, meanwhile, the E3 ubiquitin ligase inhibitor can be used for preventive medication in the high-risk exposure period, the first defense line is reinforced in advance, the probability of invasion of exogenous pathogens is effectively reduced, the attack frequency and severity of diseases are reduced, and the treatment effect of the E3 ubiquitin ligase inhibitor is improved. And a new target and a new way are provided for prevention and treatment of allergic rhinitis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of E3 ubiquitin ligase inhibitors in the preparation of drugs for allergic rhinitis. Background Technology

[0002] As the primary interface between the human respiratory tract and the external environment, the nasal mucosa plays a crucial role in defending against the invasion of the submucosa by the physical and chemical barriers formed by its epithelial cell layer, protecting against allergens (such as pollen and dust mites), pathogenic microorganisms (including influenza viruses, coronaviruses, and bacteria), and environmental pollutants. The integrity of this barrier is highly dependent on the tight junction structures between epithelial cells. Claudin-1 protein, the most widely distributed and functionally critical transmembrane component of the tight junction family, is responsible for sealing intercellular spaces, maintaining barrier density, and limiting peripheral permeability. Clinical and pathological studies have fully demonstrated that in inflammatory diseases such as allergic rhinitis, the expression level of Claudin-1 in nasal mucosal epithelial cells is significantly reduced or abnormally internalized (e.g., translocated from the cell membrane to the cytoplasm), leading to the disintegration of tight junction function and the formation of a leaky epithelial state. This barrier defect allows exogenous antigens to easily penetrate the mucosa, continuously stimulating the immune system, triggering and exacerbating the inflammatory cascade, and causing recurrent symptoms.

[0003] Although there are currently a variety of drugs available for the treatment of allergic rhinitis, chronic sinusitis and nasal inflammation in clinical practice, the existing technology still has the following significant limitations and unmet clinical needs.

[0004] Firstly, current mainstream nasal medications mainly consist of corticosteroids (such as budesonide and mometasone furoate) and antihistamines. These drugs primarily work by inhibiting downstream immune inflammatory responses or blocking inflammatory mediator receptors, thereby relieving symptoms such as nasal congestion, runny nose, and sneezing. However, these drugs cannot directly repair the damaged physical barrier of the nasal mucosa. Because the epithelial barrier (tight junctions) is not substantially restored, once medication is discontinued, external allergens or pathogens can easily re-penetrate the mucosa, leading to recurrent inflammation that is difficult to cure.

[0005] Secondly, while nasal corticosteroids are the gold standard for treatment, long-term, high-frequency use can lead to problems such as nasal dryness, crusting, and nosebleeds, and may even cause mucosal atrophy or decreased sense of smell. Some patients (especially children and those with a fear of hormones) have poor adherence to long-term use of steroid medications. Furthermore, while antihistamines are fast-acting, they are often accompanied by discomfort such as nasal dryness and a bitter taste in the mouth, and they do not improve mucosal defense function.

[0006] Finally, existing non-pharmacological nasal products (such as saline nasal rinses and barrier gels) can only provide mechanical cleaning or temporary physical coverage, failing to penetrate deep into cells to regulate the stability of tight junction proteins. Crucially, the current technological system completely lacks interventions targeting the Claudin-1 ubiquitination degradation pathway—in the pathology of allergic rhinitis, excessive ubiquitination of Claudin-1 mediated by a specific E3 ubiquitin ligase is the direct cause of its protein level decline, but currently no drugs can specifically target this enzyme to block the degradation process, thereby rescuing Claudin-1 expression and rebuilding the nasal mucosal biological barrier. This fundamental technological gap results in a lack of clinical treatment strategies to repair the epithelial barrier at its source and block the inflammatory cycle, severely limiting the effectiveness of curing allergic rhinitis. Therefore, seeking new and more effective diagnostic and treatment technologies has significant social implications. Summary of the Invention

[0007] To overcome the problems existing in related technologies, this invention provides the application of E3 ubiquitin ligase inhibitors in the preparation of drugs for allergic rhinitis. By inhibiting the ubiquitination and degradation of CLDN1 through E3 ubiquitin ligase inhibitors, barrier function can be enhanced and nasal symptoms can be relieved. At the same time, it can also be used for prophylactic medication during high-risk exposure periods. By strengthening the first line of defense in advance, the probability of invasion by exogenous pathogens can be effectively reduced, and the frequency and severity of disease attacks can be reduced.

[0008] This invention provides the application of E3 ubiquitin ligase inhibitors in the preparation of drugs for allergic rhinitis.

[0009] As a preferred embodiment, the E3 ubiquitin ligase inhibitor includes compound 1 represented by structural formula I and / or compound 2 represented by structural formula II; The structural formula I is shown below: Formula I; The structural formula II is shown below: Formula II.

[0010] As a preferred embodiment, the dosage form of the allergic rhinitis medication is nasal drops, nasal spray, aerosol, or powder.

[0011] As a preferred option, the allergic rhinitis medication is administered orally, intranasally, by injection, or topically.

[0012] As a preferred embodiment, the raw materials for preparing the allergic rhinitis drug include solvents.

[0013] As a preferred embodiment, the solvent comprises, by volume fraction, 10% solvent and 90% physiological saline.

[0014] As a preferred embodiment, the solvent includes at least one of dimethyl sulfoxide, PEG300, PEG400 and Tween-80.

[0015] As a preferred embodiment, the raw materials for preparing the allergic rhinitis drug also include pharmaceutically acceptable excipients.

[0016] As a preferred embodiment, the concentration of the E3 ubiquitin ligase inhibitor in the allergic rhinitis drug is 0.25 μM to 56 μM.

[0017] As a preferred embodiment, the concentration of compound 1 in the allergic rhinitis medication is 0.25 μM to 10 μM, and the concentration of compound 2 in the allergic rhinitis medication is 3.5 μM to 56 μM.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention upstreams the therapeutic target to the intracellular ubiquitin-proteasome system (UPS), directly blocking the ubiquitination and degradation pathway of the key tight junction protein CLDN1 by inhibiting the activity of specific E3 ubiquitin ligases, thereby rescuing and maintaining the normal expression of CLDN1 on the cell membrane at the molecular level. Experimental verification shows that compound 1 dose-dependently restores CLDN1 protein expression in IL-4-treated human nasal mucosal epithelial cells (HNEpCs) within the concentration range of 0.25-10 μM, with the maximum rescue effect achieved at concentrations of 1-5 μM; compound 2 also dose-dependently restores CLDN1 expression within the concentration range of 3.5-56 μM, with significant effects at concentrations ≥7 μM; simultaneously, both compounds 1 and 2 dose-dependently reduce CLDN1 ubiquitination levels in an IL-4-induced human nasal mucosal epithelial cell inflammation model; most importantly, WWP1 / WWP2, as specific E3 ubiquitin ligases for CLDN1, is an original scientific discovery of this invention. This invention utilizes Pull... Down-target protein sequencing screened out E3 enzymes that potentially interact with CLDN1. Further verification in an IL-4-induced nasal mucosal epithelial inflammation model showed that their interaction with CLDN1 was significantly enhanced. After confirming the core function of WWP1 / WWP2 in mediating CLDN1 ubiquitination and degradation through cell function experiments, small molecule compounds 1 and 2 that could inhibit their activity were selectively screened. Neither of them affected the protein expression level of the E3 ubiquitin ligases WWP1 / WWP2, confirming that their mechanism of action is to inhibit enzyme activity rather than change ligase expression, thus achieving precise targeted regulation.

[0019] 2. The active ingredients of the two compounds used in this invention are both non-steroidal small molecule compounds that do not exert their effects through the glucocorticoid receptor pathway. This completely avoids common adverse reactions of hormonal drugs, such as nasal dryness, crusting, nosebleeds, mucosal atrophy, or decreased sense of smell, as well as potential systemic side effects. The formulation is mild and non-irritating, and will not induce drug-induced rhinitis. It is particularly suitable for long-term use in sensitive populations such as children and the elderly, significantly improving patient compliance.

[0020] 3. This invention regulates the homeostasis of protein metabolism within epithelial cells through pharmacological means, promoting the reconstruction and reinforcement of tight junction structures, thus forming an active biological barrier enhancement effect. This effect does not depend on the physical retention of the formulation in the nasal cavity. The strengthened tight junctions can effectively resist the penetration of allergens such as pollen and dust mites, as well as pathogenic microorganisms such as influenza viruses and coronaviruses into the submucosa, providing more durable and deeper endogenous defense protection than physical barrier agents such as saline rinsing solutions and barrier gels.

[0021] 4. This invention is the first to apply E3 ubiquitin ligase inhibitors to the field of nasal local administration, filling the gap in existing technologies for specific drugs targeting the key pathological link of CLDN1 over-degradation. This application provides a novel target and drug class for the prevention and treatment of allergic rhinitis, chronic sinusitis, and viral upper respiratory tract infections, greatly expanding the pharmaceutical application scope of E3 ubiquitin ligase inhibitors and possessing significant clinical translational value.

[0022] 5. The formulation of the present invention can maintain high-level expression of CLDN1 and ensure the density of the nasal mucosal barrier: during the onset of diseases such as allergic rhinitis, it can be used as an adjunct treatment to accelerate the healing of damaged mucosa; during high-risk exposure periods such as pollen season and flu season, it can be used preventively to strengthen the first line of defense of the nasal mucosa in advance, reduce the probability of invasion of exogenous pathogens, and reduce the frequency and severity of disease attacks, thus having clinical value in achieving integrated prevention and treatment. Attached Figure Description

[0023] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the invention.

[0024] Figure 1 This is the Western blot result of NCS2805 dose-dependently restoring IL-4-induced decreased CLDN1 protein expression, as shown in Example 1 of this invention; Figure 2 This is the Western blot result of Heclin dose-dependent restoration of IL-4-induced decreased CLDN1 protein expression, as shown in Example 2 of this invention. Figure 3 The results of immunoprecipitation-immunoblotting are shown in Example 3 of this invention, which demonstrate the effect of NCS2805 and Heclin on inhibiting IL-4-induced CLDN1 ubiquitination. Detailed Implementation

[0025] Preferred embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While preferred embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0026] Statistical analysis in this invention: Quantitative data are expressed as mean ± standard deviation (mean ± SD). Multiple comparisons of quantitative data were performed using one-way ANOVA. Statistical analysis was conducted using GraphPad Prism 9.5 software. P < 0.05 was considered statistically significant, #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001. ns indicated no statistically significant difference.

[0027] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.

[0028] Source: NCS2805 used in this invention has the molecular formula C 14 H 14 O4, CAS number 4371-34-0, was purchased from MedChemExpress (MCE); the Heclin used in this invention has the molecular formula C 17 H 17 NO3, CAS number 890605-54-6, was purchased from MedChemExpress (MCE).

[0029] Example 1 1. Drug composition Active ingredient: NCS2805, at concentrations of 0.25 μM, 0.5 μM, 1 μM, 5 μM, and 10 μM; Pharmaceutically acceptable excipients: 90% physiological saline, 10% DMSO; Dosage form: liquid.

[0030] 2. Testing 2.1 Experiment Human nasal mucosal epithelial cells (HNEpCs) were treated with 10 ng / mL IL-4 (an inflammatory factor) for 48 hours to induce CLDN1 ubiquitination. Then, the cells were treated with the above concentration of the drug for 1 hour. A DMSO control group and a blank control group were set up.

[0031] 2.2 Test Method Western blotting was used to detect the relative expression levels of CLDN1, WWP1, and WWP2 proteins in HNEpCs after each treatment group. The results are as follows: Figure 1 As shown.

[0032] 2.3 Test Results like Figure 1 As shown, NCS2805 dose-dependently restored IL-4-induced decreased CLDN1 protein expression, achieving the maximum rescue effect at concentrations of 1–5 μM, while the expression levels of WWP1 and WWP2 proteins showed no significant change, confirming that its mechanism of action is the inhibition of E3 ubiquitin ligase activity.

[0033] Compared with the negative control group, ####P<0.0001; compared with the IL-4+DMSO group, ##P<0.01, ###P<0.001, ####P<0.0001 (n=3 independent experiments, data are expressed as mean ± SEM). Compared with the solvent control group under IL-4 induction, ##P<0.01, ###P<0.001, ####P<0.0001.

[0034] Example 2 1. Drug composition Active ingredient: Heclin, at concentrations of 3.5 μM, 7 μM, 14 μM, 28 μM, and 56 μM; Pharmaceutically acceptable excipients: 90% physiological saline, 10% DMSO; Dosage form: liquid.

[0035] 2. Testing 2.1 Experiment Same as in Example 1, the drug was treated with concentrations of 3.5 μM, 7 μM, 14 μM, 28 μM, and 56 μM for 1 hour, and a DMSO control group and a blank group were set up.

[0036] 2.2 Test Method Same as Example 1.

[0037] 2.3 Test Results like Figure 2As shown, Heclin dose-dependently rescued IL-4-induced decreased CLDN1 protein expression, and a significant recovery was observed at concentrations ≥7 μM. Furthermore, there were no significant changes in the expression levels of WWP1 and WWP2 proteins, confirming that its mechanism of action is the inhibition of E3 ubiquitin ligase activity.

[0038] Compared with the control group, ####P<0.0001; compared with the IL-4+DMSO group, #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001 (n=3 independent experiments, data are expressed as mean ± SEM). Compared with the solvent control group under IL-4 induction, ##P<0.01, ###P<0.001, ####P<0.0001.

[0039] Example 3 1. Drug composition Active ingredients: NCS2805, at concentrations of 0.5 μM and 1 μM, respectively; Heclin at concentrations of 14 μM and 28 μM, respectively; Pharmaceutically acceptable excipients: 10% dimethyl sulfoxide (DMSO) and 90% physiological saline (volume fraction).

[0040] 2.1 Experiment Human nasal mucosal epithelial cells (HNEpCs) were treated with 10 ng / mL IL-4 (an inflammatory factor) for 48 hours to induce CLDN1 ubiquitination. Subsequently, MG132 (final concentration 10 μM) was added to inhibit the degradation of ubiquitinated proteins and accumulate ubiquitinated products for subsequent Western blotting detection. The cells were treated with the above-mentioned NCS2805 (0.5 μM, 1 μM) and Heclin (14 μM, 28 μM), and a DMSO control group and a blank control group were set up.

[0041] 2.2 Testing Immunoprecipitation (IP) and immunoblotting (IB) assays: Cell lysis: Total protein was extracted using RIPA lysis buffer containing protease inhibitors; IP reaction: Take 500 μg of total protein, add anti-CLDN1 antibody (1:100 dilution), and incubate overnight at 4°C; add Protein A / G agarose beads and incubate at 4°C for 2 hours; Washing and elution: Wash the agarose beads three times with PBS, add SDS loading buffer and boil for 5 minutes; IB assay: The eluted products were subjected to SDS-PAGE electrophoresis, and after transfer to a membrane, they were detected with anti-ubiquitin antibody and anti-CLDN1 antibody; 20 μg of total protein was directly taken from the Input group for IB assay (GAPDH was used as an internal control).

[0042] 2.3 Test Results like Figure 3 As shown, this experiment investigated the effects of NCS2805 and Heclin on CLDN1 ubiquitination levels in the presence of the proteasome inhibitor MG-132. Compared with the blank control group, IL-4 stimulation significantly increased CLDN1 ubiquitination levels (manifested as enhanced high-molecular-weight diffuse bands in the Ubiquitin blot). The addition of NCS2805 (0.5 μM, 1 μM) or Heclin (14 μM, 28 μM) resulted in a dose-dependent decrease in CLDN1 ubiquitination levels, while the DMSO control group showed no significant change. Simultaneously, the IB results of the Input group showed uniform GAPDH bands, indicating consistent protein loading amounts across groups; the CLDN1 band intensity in the IP products was consistent, demonstrating uniform immunoprecipitation efficiency. Therefore, NCS2805 and Heclin effectively block IL-4-induced CLDN1 ubiquitination, which is the core mechanism by which they inhibit CLDN1 degradation and maintain protein stability.

[0043] In summary, this invention represents a key breakthrough in the treatment strategy for allergic rhinitis, moving from downstream anti-inflammatory symptomatic relief to upstream barrier repair addressing the underlying cause. By utilizing NCS2805 and Heclin to specifically inhibit the activity of E3 ubiquitin ligase, the ubiquitination pathway of Claudin-1 (CLDN1) protein is directly blocked. This precise regulation significantly improves the stability and expression level of CLDN1 protein in nasal mucosal epithelial cells, promotes the reconstruction and reinforcement of tight junction structures, blocks the invasion of allergens and pathogens at the source, reduces the risk of inflammation recurrence, and achieves a fundamental cure.

[0044] This invention provides a new, safe, non-steroidal treatment option, addressing the risks of nasal mucosal atrophy, dryness, bleeding, and systemic side effects associated with long-term use of nasal corticosteroids. It also avoids the intracellular protein accumulation toxicity caused by non-specific inhibition of broad-spectrum proteasome inhibitors such as MG132. The active ingredient, NCS2805 / Heclin, is a non-steroidal small molecule that does not act through corticosteroid receptors. While enhancing mucosal barrier function, it avoids the common adverse reactions of steroid drugs, making it particularly suitable for children, the elderly, and patients requiring long-term nasal care, significantly improving medication adherence.

[0045] This invention fills a gap in drug development targeting the ubiquitination regulation mechanism of tight junction proteins. Compared to existing technologies that lack specific drugs targeting key pathological steps in CLDN1 degradation, this invention is the first to apply E3 ubiquitin ligase inhibitors to nasal topical administration. By validating the effectiveness of NCS2805 and Heclin, the feasibility and efficiency of regulating epithelial barrier permeability via the upstream ubiquitination pathway are demonstrated, providing a novel target and drug class for the prevention and treatment of allergic rhinitis and viral respiratory infections.

[0046] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. Application of E3 ubiquitin ligase inhibitors in the preparation of drugs for allergic rhinitis.

2. The application of the E3 ubiquitin ligase inhibitor according to claim 1 in the preparation of allergic rhinitis drugs, characterized in that, The E3 ubiquitin ligase inhibitor includes compound 1 shown in structural formula I and / or compound 2 shown in structural formula II; The structural formula I is shown below: Equation I; The structural formula II is shown below: Formula II.

3. The application of the E3 ubiquitin ligase inhibitor according to claim 1 in the preparation of allergic rhinitis drugs, characterized in that, The dosage form of the allergic rhinitis medication is nasal drops, nasal spray, aerosol, or powder.

4. The application of the E3 ubiquitin ligase inhibitor according to claim 1 in the preparation of allergic rhinitis drugs, characterized in that, The allergic rhinitis medication can be administered orally, intranasally, by injection, or topically.

5. The application of the E3 ubiquitin ligase inhibitor according to claim 1 in the preparation of allergic rhinitis drugs, characterized in that, The raw materials for preparing the allergic rhinitis drug include solvents.

6. The application of the E3 ubiquitin ligase inhibitor according to claim 5 in the preparation of allergic rhinitis drugs, characterized in that, The solvent, by volume fraction, comprises 10% solvent and 90% physiological saline.

7. The application of the E3 ubiquitin ligase inhibitor according to claim 6 in the preparation of allergic rhinitis drugs, characterized in that, The solvent includes at least one of dimethyl sulfoxide, PEG300, PEG400 and Tween-80.

8. The application of the E3 ubiquitin ligase inhibitor according to claim 5 in the preparation of allergic rhinitis drugs, characterized in that, The raw materials for preparing the allergic rhinitis medication also include pharmaceutically acceptable excipients.

9. The application of the E3 ubiquitin ligase inhibitor according to claim 1 in the preparation of allergic rhinitis drugs, characterized in that, The concentration of the E3 ubiquitin ligase inhibitor in the allergic rhinitis medication is 0.25 μM to 56 μM.

10. The use of the E3 ubiquitin ligase inhibitor according to claim 9 in the preparation of allergic rhinitis drugs, characterized in that, The concentration of compound 1 in the allergic rhinitis medication is 0.25 μM to 10 μM, and the concentration of compound 2 in the allergic rhinitis medication is 3.5 μM to 56 μM.

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