Application of ZBP1 small-molecule inhibitor in preparation of medicine for treating myocardial ischemic diseases

Abstract

The invention discloses an application of a ZBP1 small-molecule inhibitor with a structure shown as a formula I or a formula II and a pharmaceutically acceptable salt thereof in preparation of a medicine for treating myocardial ischemic diseases. The ZBP1 small molecule inhibitor as shown in the formula I or the formula II has remarkable ZBP1 inhibition activity, can effectively improve the activity of myocardial cells, and has a relieving effect on myocardial cell hypoxia / reoxygenation (HR) damage of mice. I / II

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of ZBP1 small molecule inhibitors in the preparation of drugs for treating myocardial ischemia. Background Technology

[0002] Myocardial infarction poses a serious threat to human health, and restoring myocardial blood supply is the most effective strategy for improving myocardial infarction. Although reperfusion therapy can effectively restore myocardial blood supply and reduce mortality in myocardial infarction patients, post-reperfusion induced myocardial oxidative stress, release of inflammatory factors, and mitochondrial dysfunction lead to cardiomyocyte death, causing myocardial ischemia / reperfusion injury (MI / R), ultimately resulting in worsening of the patient's condition or even death. Due to the very limited clinical treatment strategies, MI / R has become a major unresolved problem in clinical practice. Clinical research results show that anti-inflammatory and antioxidant drugs (such as allopurinol, h-SOD, abciximab, and lopiroximide) can alleviate myocardial damage and heart failure after reperfusion in myocardial infarction patients; however, they have no significant effect on improving post-operative clinical risk events and survival. Therefore, implementing cardiac protection after reperfusion will help promote post-operative recovery in myocardial infarction patients, and seeking more effective treatment strategies to prolong post-operative survival is urgent. Summary of the Invention

[0003] Purpose of the invention: To address the problems existing in the prior art, this invention provides the application of ZBP1 small molecule inhibitors and their pharmaceutically acceptable salts in the preparation of drugs for treating myocardial ischemia. By inhibiting the activity of ZBP1, the ZBP1 small molecule inhibitors inhibit cardiomyocyte death and can effectively improve cardiomyocyte vitality, thus showing promising application in the treatment of myocardial ischemia.

[0004] Technical Solution: This invention provides the application of a ZBP1 small molecule inhibitor and its pharmaceutically acceptable salt in the preparation of a medicament for treating myocardial ischemia, wherein the small molecule inhibitor is... or .

[0005] The aforementioned ischemic myocardial disease is myocardial ischemia-reperfusion injury.

[0006] The pharmaceutically acceptable salt is a salt formed by the small molecule inhibitor and a pharmaceutically acceptable acid or base.

[0007] Preferably, the pharmaceutically acceptable acid is hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, citric acid, or ferulic acid.

[0008] Preferably, the pharmaceutically acceptable base is sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, or ethylenediamine.

[0009] "Pharmaceutically acceptable salts" refer to salts of compounds prepared by reacting a compound with a relatively non-toxic acid or base, containing specific substituents. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts, or similar salts. When a compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free form of the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogen phosphates, dihydrogen phosphates, sulfuric acid (forming sulfates or bisulfates), hydroiodic acid, phosphorous acid, etc.); and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid. Acids such as citric acid, tartaric acid, and methanesulfonic acid; organic acid salts also include salts of organic acids such as amino acids (e.g., arginine) and glucuronic acid. Certain compounds contain both basic and acidic functional groups, thus allowing them to be converted into either a base or acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner, and then the parent compound is separated, thereby regenerating the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as different solubilities in polar solvents.

[0010] Pharmaceutically acceptable salts can be synthesized from parent compounds containing an acid radical or a base using conventional chemical methods. Generally, such salts are prepared by reacting these compounds, in their free acid or base form, with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of both. Non-aqueous media such as ethers, ethyl acetate, ethanol, isopropanol, or acetonitrile are generally preferred.

[0011] The pharmaceutical composition of the present invention for treating myocardial ischemia contains the aforementioned small molecule inhibitor as an active ingredient and a pharmaceutically acceptable carrier.

[0012] Preferably, the dosage form of the pharmaceutical composition is tablets, capsules, powders, pills, granules, injections, oral liquids, syrups, inhalers, ointments, patches, or suppositories.

[0013] "Pharmaceutically acceptable carriers" are excipients widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods to facilitate the dissolution of the active ingredient at a desired rate after administration to a subject, or to promote the effective absorption of the active ingredient after administration to a subject. The pharmaceutical excipients may be inert fillers or provide a function, such as stabilizing the overall pH of the composition or preventing the degradation of the active ingredient. The pharmaceutical excipients may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0014] The pharmaceutical compositions described in this invention can be prepared using any method known to those skilled in the art, based on the disclosure. For example, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0015] The pharmaceutical compositions of the present invention can be administered in any form, including by injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, local or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration.

[0016] The pharmaceutical compositions of the present invention may also be controlled-release or sustained-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral dosage forms include, but are not limited to, powders, capsules, tablets, soft capsules, and tablets. Examples of liquid dosage forms for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical dosage forms include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of parenteral dosage forms include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection.

[0017] Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and tablets.

[0018] The use of the pharmaceutical composition of the present invention in the preparation of a medicament for treating myocardial ischemia.

[0019] The aforementioned ischemic myocardial disease is myocardial ischemia-reperfusion injury.

[0020] Mechanism of Invention: ZBP1 (Z-DNA Binding Protein 1) is a Z-DNA and Z-RNA binding protein. Initially reported as an interferon-gamma (IFNG)-induced tumor-associated protein, it is highly expressed in IFNG- or lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages. Recent studies have demonstrated that ZBP1 can promote cell death by activating panapoptotic bodies. Therefore, this invention...

[0021] Inhibiting ZBP1 can effectively reduce cell death and play a role in treating myocardial ischemia-reperfusion injury.

[0022] Mechanism of Invention: ZBP1 (Z-DNA Binding Protein 1) is a Z-DNA and Z-RNA binding protein. Initially reported as an interferon-gamma (IFNG)-induced tumor-associated protein, it is highly expressed in mouse peritoneal macrophages stimulated by IFNG or lipopolysaccharide (LPS). Recent studies have demonstrated that ZBP1 promotes cell death by activating panapoptotic bodies. Inhibition of ZBP1 effectively reduces cell death; therefore, the inventors hypothesized that ZBP1 inhibitors could treat myocardial ischemia-reperfusion injury. In this patent, the full-length ZBP1 protein structure was predicted using AlphaFold, and the binding sites for molecular docking were predicted using the sitemap function of Schrödinger software to screen the Specs database. Four compounds were ultimately selected as potential ZBP1 inhibitors. In vitro cellular activity evaluation of the compounds was used to verify their alleviating effect on hypoxia / reoxygenation (HR) injury induced in mouse cardiomyocytes; only two of the compounds showed an alleviating effect.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The ZBP1 small molecule inhibitor of the present invention has significant ZBP1 inhibitory activity and can effectively improve cardiomyocyte viability and alleviate hypoxia / reoxygenation (HR) injury of mouse cardiomyocytes. (2) The ZBP1 small molecule inhibitor of the present invention has the application prospect of treating myocardial ischemic diseases, especially myocardial ischemia-reperfusion injury. Attached Figure Description

[0024] Figure 1 This diagram shows the binding of compounds I-1 to I-4 to the ZBP1 protein.

[0025] Figure 2 The graph shows the heart rate (HR) damage of cardiomyocytes caused by compounds I-1, I-2, I-3, and I-4. Detailed Implementation

[0026] The technical solution of the present invention will be further described below with reference to the embodiments.

[0027] Example 1

[0028] Virtual screening of ZBP1 inhibitors:

[0029] The full-length structure of the human ZBP1 protein was predicted using AlphaFold, and then the binding sites for molecular docking were predicted using the sitemap function of Schrödinger software to screen the Specs database. First, Glide's high-throughput screening (HTVS) mode was used for semi-flexible docking, and the top 10% of compounds by score were retained. Next, Glide SP mode was used for molecular docking, and the top 25% of compounds were retained for Glide XP mode docking. Finally, the top 40% of compounds were retained for induced fit docking. Cluster analysis was performed on the compounds based on the induced fit docking results to ensure structural diversity. Finally, the four compounds with the highest scores (I-1 ~ I-4, see Table 1 and ...) were selected. Figure 1 Experiments were conducted to verify this.

[0030] Table 1. Results of compound molecular docking

[0031]

[0032] Example 2

[0033] In vitro cell activity evaluation of the compounds of this invention:

[0034] Hypoxia / reoxygenation (HR) injury was induced in mouse HL-1 cardiomyocytes, followed by CCK8 cell viability assays to preliminarily evaluate the inhibitory activity of the compound on ZBP1.

[0035] Test Method: First, HL-1 cardiomyocytes were cultured in glucose-free medium with 1% O2, and simultaneously incubated with compounds I-1, I-2, I-3, and I-4 (10 μM) to induce hypoxia. After 4 hours of culture, the medium was replaced with high-glucose medium, and the cells were again incubated with compounds I-1, I-2, I-3, and I-4 (10 μM) for 6 hours, followed by reoxygenation at 21% O2. Then, the medium in the 96-well plates was discarded, and the cells were washed with PBS. CCK8 assay reagent was added to each well, and the cells were incubated for 1 hour. The absorbance at 450 nm was then measured using a microplate reader to calculate cell viability. The test results are as follows: Figure 2 As shown.

[0036] Depend on Figure 2 The results of the CCK8 experiment show that compounds I-1 and I-4 have a mitigating effect on heart rate (HR) damage in cardiomyocytes.

[0037] Example 3

[0038] Evaluation of the protein binding activity of the compounds of this invention:

[0039] The inhibitory activity of the compound against ZBP1 was preliminarily evaluated using surface plasmon resonance (SPR) technology.

[0040] Test method: Surface plasmon resonance (SPR)

[0041] (1) Preparation of solutions and buffer solutions

[0042] Preparation of 1 × PBS-P: Dilute 10 × PBS-P (Cytiva) 10 times with deionized water, that is, add 1 part of 10 × PBS to 9 parts of deionized water for dilution;

[0043] Preparation of 0.95 × PBS-P containing 5% DMSO: Take 475 mL of 1 × PBS-P buffer, add 25 mL of DMSO, shake well and set aside.

[0044] (2) Preparation of ZBP1 protein working stock solution

[0045] The ZBP1 protein was diluted to 1000 μg / mL with 1 × PBS-P.

[0046] (3) Preparation of compound working stock solution

[0047] 1) Dilute the compound to 20 times its highest concentration using 100% DMSO to obtain a stock solution of the compound;

[0048] 2) Dilute the compound stock solution 20-fold with 1×PBS-P buffer, i.e., add 1 part of the compound stock solution to 19 parts of 1×PBS-P buffer for dilution;

[0049] 3) After the sample with the highest compound concentration was filtered through a microporous membrane, it was diluted down to the lowest concentration using a 3-fold concentration gradient with 0.95×PBS-P buffer containing 5% DMSO.

[0050] (4) Protein coupling

[0051] 1) Using 1 × PBS-P as the running buffer, take 90 μL of EDC and NHS, mix them thoroughly, and inject them into channels 1 and 2 of the CM5 chip. The contact time is set to 600 s.

[0052] 2) Take 10 μL of ZBP1 protein working stock solution, add 190 μL of pH 5.5 sodium acetate buffer to dilute to 50 μg / mL, and then inject it into channel 2 of the CM5 chip until a value of 5000 RU is reached.

[0053] 3) Take 120 μL of ethanolamine and inject it into channels 1 and 2 of the CM5 chip. The contact time is set to 420 s.

[0054] (5) Affinity test

[0055] 1) Replace the running buffer with 0.95 × PBS-P containing 5% DMSO, set the binding time to 150 s, and the dissociation time to 300 s;

[0056] 2) Based on the preliminary experimental results, the compound concentration gradient was set to cover 10 × K d up to 0.1 × K d At least 5 consecutive concentrations;

[0057] 3) Take 200 μL of working stock solution of each concentration of compound and select LMW single-cycle kinetics mode for detection.

[0058] (6) Fitting of affinity Kd value

[0059] Import the data into Biacore T200 Evaluation Software, click solvent correction, use the Surface bound of the Kinetics / Affinity module, select Curve: 2-1 corr, and then use the Kinetics method to fit the data.

[0060] (7) Fitting of affinity Kd value.

[0061] The test results are shown in Table 2.

[0062] Table 2. Binding of compounds to ZBP1 protein

[0063]

[0064] As shown in Table 2, the SPR experimental results indicate that compounds I-1 and I-4 have good molecular binding activity to ZBP1.

Claims

1. The use of ZBP1 small molecule inhibitors and their pharmaceutically acceptable salts in the preparation of drugs for treating myocardial ischemia, characterized in that, The small molecule inhibitor is or .

2. The application according to claim 1, characterized in that, The aforementioned ischemic myocardial disease is myocardial ischemia-reperfusion injury.

3. The application according to claim 1, characterized in that, The pharmaceutically acceptable salt is a salt formed by the small molecule inhibitor and a pharmaceutically acceptable acid or base.

4. The application according to claim 1, characterized in that, The pharmaceutically acceptable acids are hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, citric acid, or ferulic acid.

5. The application according to claim 1, characterized in that, The pharmaceutically acceptable base is sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, or ethylenediamine.

6. A pharmaceutical composition for treating ischemic myocardial disease, characterized in that, The pharmaceutical composition contains the small molecule inhibitor of claim 1 as the active ingredient and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, The dosage form of the pharmaceutical composition is tablets, capsules, powders, pills, granules, injections, oral liquids, syrups, inhalers, ointments, patches, or suppositories.

8. The pharmaceutical composition according to claim 6, characterized in that, The carrier is an adhesive, suspending agent, emulsifier, diluent, filler, granulating agent, glue, disintegrant, lubricant, anti-adhesion agent, flow aid, wetting agent, gelling agent, absorption delay agent, dissolution inhibitor, reinforcing agent, adsorbent, buffer, chelating agent, preservative, colorant, flavoring agent, and sweetener.

9. Use of the pharmaceutical composition of claim 6 in the preparation of a medicament for treating myocardial ischemia.

10. The application according to claim 9, characterized in that, The aforementioned ischemic myocardial disease is myocardial ischemia-reperfusion injury.

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