Use of substances inhibiting the nitration of trx1 for the preparation of a medicament for the treatment of associated diseases

Abstract

This invention discloses the application of substances that inhibit Trx1 nitration in the preparation of drugs for treating Trx1 nitration-related diseases. This invention reveals for the first time that nitration modification of tyrosine residue 49 (Y49) of the Trx1 protein is a key pathological step in diseases such as ischemic stroke and myocardial ischemia-reperfusion injury. Inhibiting nitration at this site can effectively restore Trx1 reductase activity, enhance the binding of Trx1 to ASK1, and block the downstream ASK1-p38 / JNK apoptosis signaling pathway, thereby exerting a therapeutic effect. This invention provides a novel treatment strategy for Trx1 nitration-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to new uses of substances that inhibit Trx1 nitration, particularly to the application of drugs that treat Trx1 nitration-related diseases (such as ischemic stroke, myocardial ischemia-reperfusion injury, etc.) by inhibiting the nitration modification of Trx1 tyrosine at position 49, as well as related drug screening methods. Background Technology

[0002] Thioredoxin 1 (Trx1) is one of the core proteins in cells that maintain redox homeostasis. Through its active site Cys32-Gly-Pro-Cys35, it reversibly reduces oxidative substrates, protecting cells from oxidative stress damage. In addition to its antioxidant function, Trx1 also regulates cell survival signaling pathways through protein-protein interactions. Among these, the binding of Trx1 to apoptosis signal-regulated kinase 1 (ASK1) is a key node in regulating apoptosis: under physiological conditions, Trx1 binds to ASK1 and inhibits ASK1 kinase activity; when cells are subjected to oxidative or nitrifying stress, Trx1 undergoes post-translational modification, leading to its dissociation from ASK1. The released ASK1 is then autophosphorylated and activated, thereby activating the downstream p38 / JNK apoptosis signaling pathway.

[0003] Recent studies have found that excessive reactive nitrogen (RNS) production during ischemia-reperfusion injury can induce tyrosine nitration modification of Trx1. Nitration of tyrosine residue 49 (Y49) has been shown to lead to loss of Trx1 activity, but its pathological significance and potential for pharmacological intervention are not fully understood. The Y49 site is far from the catalytically active site C32 / C35 of Trx1, suggesting that this site may allosterically regulate Trx1 function by affecting its spatial conformation. However, no therapeutic strategies targeting Trx1 nitration modification have been reported, and there are no small molecule drugs that can specifically inhibit Trx1 nitration and restore Trx1 activity.

[0004] This invention reveals for the first time that Trx1-Y49 nitration is a key pathological step in diseases such as ischemic stroke and myocardial ischemia-reperfusion injury, and demonstrates that inhibiting nitration at this site can effectively restore Trx1 function and block downstream apoptosis signals. Based on this, this invention proposes a treatment strategy centered on inhibiting Trx1 nitration. Summary of the Invention

[0005] The purpose of this invention is to provide a new use for substances that inhibit Trx1 nitration, specifically: the use of substances that inhibit Trx1 nitration in the preparation of medicaments for treating related diseases.

[0006] Furthermore, the substance inhibiting Trx1 nitration is selected from any one or more of the following:

[0007] (a) A compound that binds to the 49th tyrosine residue (Y49) of Trx1;

[0008] (b) Compounds that inhibit Trx1 tyrosine nitration modification;

[0009] (c) Compounds that allosterically activate Trx1 reductase activity;

[0010] (d) Compounds that enhance the binding of Trx1 to ASK1;

[0011] (e) Compounds that inhibit the activation of the ASK1-p38 / JNK signaling pathway.

[0012] Furthermore, the substance that inhibits Trx1 nitration is a small molecule compound, polypeptide, antibody or its antigen-binding fragment, nucleic acid aptamer or protein.

[0013] Preferably, the small molecule compound includes tetramethylpyrazine and its derivatives, or Trx1-Y49 binding compounds obtained by virtual screening or high-throughput screening.

[0014] Furthermore, the related diseases are those associated with elevated Trx1 tyrosine nitration levels, including ischemic stroke, myocardial ischemia-reperfusion injury, neurodegenerative diseases (Parkinson's disease, Alzheimer's disease), diabetic complications, and atherosclerosis.

[0015] Furthermore, the substance that inhibits Trx1 nitration acts through one or more of the following mechanisms:

[0016] (a) It interacts with the Y49 residue of Trx1;

[0017] (b) Inhibit ONOO⁻-mediated Trx1 tyrosine nitration;

[0018] (c) The reduced active conformation of stable Trx1;

[0019] (d) Increase the spatial distance between the C32 and C35 catalytic active centers of Trx1.

[0020] Furthermore, the drug comprises a therapeutically effective amount of a substance that inhibits Trx1 nitration and a pharmaceutically acceptable carrier. The dosage form of the drug includes injections, tablets, capsules, granules, powders, topical preparations, or inhalation preparations.

[0021] Among them, candidate substances that can inhibit Trx1-Y49 nitration, restore Trx1 activity and enhance Trx1-ASK1 binding are candidate substances for treating related diseases.

[0022] Advantages and beneficial effects of the present invention:

[0023] (1) For the first time, it was revealed that Trx1-Y49 nitration modification is a pathological target for diseases such as ischemic stroke and myocardial ischemia-reperfusion injury, providing a new strategy for the treatment of related diseases.

[0024] (2) The first time, “inhibiting Trx1 nitration” was proposed as the core strategy of drug intervention, which broke through the non-specific limitations of traditional antioxidant and anti-apoptotic therapy.

[0025] (3) The scope of protection covers a variety of active substances and has broad industrial application value. Attached Figure Description

[0026] Figure 1 Pharmacodynamic validation of a representative substance that inhibits Trx1 nitration (tetramethylpyrazine, TMP)

[0027] A: TMP inhibits OGD / R-induced Trx1 nitrification in SH-SY5Y cells.

[0028] B: TMP restores OGD / R-induced Trx1 enzyme activity in SH-SY5Y cells.

[0029] C: TMP inhibits Trx1 nitration in the brain tissue of MCAO / R model rats.

[0030] D: TMP restores Trx1 enzyme activity in the brain tissue of MCAO / R model rats.

[0031] Figure 2 : Validation of the key role of the Trx1-Y49 site.

[0032] A: Molecular docking shows that TMP and Trx1-Y49 form π-π stacking interactions.

[0033] B: After Y49A site-directed mutation, the binding of TMP to Trx1 is significantly weakened.

[0034] After C:Y49A site-directed mutation, the effect of TMP in restoring Trx1 activity is weakened.

[0035] Figure 3 : Inhibits the regulatory effect of Trx1 nitration on the ASK1-p38 / JNK pathway.

[0036] A: TMP enhances Trx1-ASK1 binding in the brain tissue of MCAO / R model rats.

[0037] B: TMP inhibits the phosphorylation of ASK1, p38, and JNK in the brain tissue of MCAO / R model rats.

[0038] C: Neurological function scores showed that SIN-1 reversing TMP improved neurological deficits in MCAO / R model rats. Compared with the TMP group, the TMP + SIN-1 group had significantly higher neurological function scores.

[0039] D: TTC staining showed that SIN-1 (peroxynitrite donor, 1 mg / kg) reversed the effect of TMP on reducing the infarct volume in MCAO / R model rats. Compared with the TMP group, the infarct volume in the TMP + SIN-1 group was significantly increased. Detailed Implementation

[0040] To more clearly illustrate the technical solution of this invention, specific experimental examples are provided below for further explanation. These experimental examples are for illustrative purposes only and do not constitute a limitation on this invention. The following experiments use tetramethylpyrazine as a representative substance for inhibiting Trx1 nitration for verification, but the scope of protection of this invention is not limited thereto.

[0041] Experimental Example 1: Validation of the efficacy of a representative inhibitor of Trx1 nitrification (tetramethylpyrazine)

[0042] 1.1 Experimental Methods

[0043] Cellular experiments: SH-SY5Y cells were subjected to oxygen-glucose deprivation / reoxygenation / reoxygenation (OGD / R) treatment (glucose deprivation, hypoxia for 4 hours, reoxygenation / reoxygenation for 24 hours), while being administered different concentrations of tetramethylpyrazine (5, 10, 20 μM). Trx1 nitrification levels and enzyme activity were measured after treatment.

[0044] Animal experiments: MCAO / R model rats were intraperitoneally injected with ligustrazine (20, 40, 80 mg / kg) for 3 consecutive days. After sacrifice, the Trx1 nitration level and enzyme activity in the brain tissue were detected.

[0045] 1.2 Experimental Results

[0046] Cellular experiment results showed ( Figure 1 (A–B) OGD / R treatment significantly induced Trx1 nitration and reduced its enzyme activity in SH-SY5Y cells; tetramethylpyrazine treatment inhibited Trx1 nitration in a concentration-dependent manner. Figure 1 A), and restore Trx1 enzyme activity ( Figure 1 B).

[0047] Animal experiment results showed that ( Figure 1 In C–D rats, Trx1 nitration levels were elevated and enzyme activity was decreased in the brain tissue of MCAO / R model rats; treatment with tetramethylpyrazine significantly inhibited Trx1 nitration (C–D). Figure 1 C), and restore its enzyme activity (C) Figure 1 D).

[0048] The above results indicate that substances that inhibit Trx1 nitrification (represented by tetramethylpyrazine) can effectively antagonize ischemia-reperfusion-induced Trx1 nitrification inactivation.

[0049] Experimental Example 2: Validation of the key role of the Trx1-Y49 site

[0050] 2.1 Experimental Methods

[0051] Molecular docking: Molecular docking simulation of ligustrazine and Trx1 protein was performed using AutoDock software.

[0052] Site-directed mutagenesis: The Trx1-Y49A mutant (tyrosine at position 49 is mutated to alanine) was constructed and overexpressed in HEK293T cells either wild-type Trx1 or Y49A mutant.

[0053] Pull-down assay: Cell lysate was incubated with a tetramethylpyrazine photoaffinity probe, cross-linked with ultraviolet light, and then biotin was linked by a click chemical reaction. The protein was enriched with streptavidin magnetic beads and detected by Western blot.

[0054] Trx1 enzyme activity assay: Ligustrazine was added to the lysate of wild-type or Y49A mutant overexpressing cells, and the changes in Trx1 enzyme activity were detected.

[0055] 2.2 Experimental Results

[0056] Molecular docking results show ( Figure 2 A) Tetramethylpyrazine binds to the Y49 residue of Trx1 through π-π stacking interaction and forms a hydrogen bond with the neighboring K48.

[0057] The results of the pull-down experiment show that ( Figure 2 B), compared with wild-type Trx1, the Y49A mutation significantly weakens the binding of tetramethylpyrazine to Trx1.

[0058] Enzyme activity assay results showed that ( Figure 2 (C) In wild-type Trx1, tetramethylpyrazine significantly enhances Trx1 enzyme activity; however, in the Y49A mutant, this effect of tetramethylpyrazine is significantly weakened.

[0059] The above results confirm that Y49 is a key binding site for inhibiting Trx1 nitrates (represented by tetramethylpyrazine), and this site is crucial for the drug to restore Trx1 activity.

[0060] Experimental Example 3: The regulatory effect of inhibiting Trx1 nitration on the ASK1-p38 / JNK pathway

[0061] 3.1 Experimental Methods

[0062] Co-immunoprecipitation (Co-IP): SH-SY5Y cell lysate was immunoprecipitated using anti-Trx1 antibody, and the ASK1 content in the precipitate was detected by Western blot to assess Trx1-ASK1 binding.

[0063] Western blot: Detect the expression levels of p-ASK1, ASK1, p-p38, p38, p-JNK, and JNK proteins in cell lysates.

[0064] 3.2 Experimental Results

[0065] Co-IP results show that ( Figure 3 A) OGD / R treatment significantly reduced the binding of Trx1 and ASK1; treatment with tetramethylpyrazine significantly restored the binding between the two.

[0066] Western blot results showed that ( Figure 3 B), OGD / R treatment significantly induced phosphorylation of ASK1, p38, and JNK; tetramethylpyrazine treatment significantly inhibited the phosphorylation levels of the above proteins.

[0067] Furthermore, functional experiments further validated the pharmacodynamic correlation of the aforementioned molecular mechanisms. Figure 3 C–D). Neurological function scores showed ( Figure 3 C), tetramethylpyrazine significantly improved neurological deficits in MCAO / R model rats; SIN-1 significantly reversed this improvement. TTC staining results showed ( Figure 3 (D) Ligustrazine significantly reduced the infarct volume in MCAO / R model rats; however, when combined with SIN-1, the infarct-reducing effect of ligustrazine was significantly weakened, and the infarct volume rebounded.

[0068] The above results indicate that inhibiting Trx1 nitration can effectively block the activation of the downstream ASK1-p38 / JNK apoptosis signaling pathway by restoring Trx1-ASK1 binding. SIN-1-induced Trx1 nitration can reverse the above-mentioned molecular regulatory effects and overall efficacy of tetramethylpyrazine, further confirming that inhibiting Trx1 nitration is the core mechanism by which tetramethylpyrazine exerts its anti-ischemic stroke effect.

[0069] Industrial applicability

[0070] The "Application of Substances that Inhibit Trx1 Nitration in the Preparation of Drugs for Treating Related Diseases" provided by this invention has the following industrial applicability:

[0071] (1) This invention reveals for the first time that Trx1-Y49 nitration is a therapeutic target for diseases such as ischemic stroke and myocardial ischemia-reperfusion injury, providing a new direction for the development of related drugs.

[0072] (2) The drugs developed based on this invention can exert their effects by inhibiting Trx1 nitration, restoring Trx1 activity, and blocking the ASK1-p38 / JNK apoptosis pathway. They have clear targets and mechanisms, which are beneficial for precision treatment and clinical translation.

[0073] (3) The scope of protection of this invention covers a variety of active substances such as small molecule compounds, peptides, and antibodies, and has good patent layout value and market prospects.

Claims

1. Application of substances that inhibit Trx1 nitration in the preparation of drugs for treating related diseases.

2. The application according to claim 1, characterized in that, The substance that inhibits Trx1 nitration is selected from any one or more of the following: (a) A compound that binds to the 49th tyrosine residue (Y49) of Trx1; (b) Compounds that inhibit Trx1 tyrosine nitration modification; (c) Compounds that allosterically activate Trx1 reductase activity; (d) Compounds that enhance the binding of Trx1 to ASK1; (e) Compounds that inhibit the activation of the ASK1-p38 / JNK signaling pathway.

3. The application according to claim 1, characterized in that, The substance that inhibits Trx1 nitration is a small molecule compound, polypeptide, antibody or its antigen-binding fragment, nucleic acid aptamer or protein.

4. The application according to claim 3, characterized in that, The small molecule compounds include tetramethylpyrazine and its derivatives, or Trx1-Y49 binding compounds obtained through virtual screening or high-throughput screening.

5. The application according to claim 1, characterized in that, The related diseases are those associated with elevated Trx1 tyrosine nitration levels, including ischemic stroke, myocardial ischemia-reperfusion injury, neurodegenerative diseases, diabetic complications, and atherosclerosis.

6. The application according to claim 5, characterized in that, The neurodegenerative diseases mentioned include Parkinson's disease and Alzheimer's disease.

7. The application according to claim 1, characterized in that, The substance that inhibits Trx1 nitration works through one or more of the following mechanisms: (a) Interacts with the Y49 residue of Trx1; (b) Inhibit ONOO⁻-mediated Trx1 tyrosine nitration; (c) The reduced active conformation of stable Trx1; (d) Increase the spatial distance between the C32 and C35 catalytic active centers of Trx1.

8. The application according to claim 1, characterized in that, The drug contains a therapeutically effective amount of a substance that inhibits Trx1 nitration and a pharmaceutically acceptable carrier.

9. The application according to claim 8, characterized in that, The dosage forms of the drug include injections, tablets, capsules, granules, powders, topical preparations, or inhalation preparations.

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