Application of 3-bromopyruvic acid in preparation of medicine for treating atherosclerosis or complications thereof

Abstract

The invention belongs to the technical field of biological medicines, and relates to application of 3-bromopyruvic acid in preparation of medicines for treating atherosclerosis or complications thereof, and the medicines are medicines for treating atherosclerosis and / or medicines for preventing atherosclerosis complications. The 3-bromopyruvic acid reduces the volume of aorta root plaques, reduces aorta lipid deposition and relieves inflammation in the treatment of atherosclerosis diseases by inhibiting the interaction of sTREM2-HSP90 beta proteins, and is expected to solve the problem that the existing atherosclerosis clinical treatment medicines cannot effectively solve the risk of residual inflammation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of 3-bromopyruvic acid (3-BrP) or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the treatment or prevention of atherosclerosis or its complications. Background Technology

[0002] Atherosclerosis is a common vascular disease characterized by lipid deposition in the arterial wall, chronic inflammation, and plaque formation. It is the main pathological basis for cardiovascular and cerebrovascular events such as myocardial infarction and ischemic stroke. The pathogenesis of atherosclerosis involves multiple steps, including endothelial dysfunction, lipid infiltration, immune cell infiltration, and chronic inflammation. Among these, the sustained activation of inflammatory signaling pathways plays a central role in plaque formation, progression, and instability. Currently, clinical treatment mainly relies on lipid-lowering drugs such as statins. Although these drugs can effectively reduce blood lipid levels, a considerable number of patients still have residual inflammation risks, making it difficult to completely control disease progression. Therefore, developing novel therapeutic drugs that directly target inflammatory pathways is of significant clinical importance. Summary of the Invention

[0003] This invention discovers that 3-bromopyruvate reduces aortic root plaque volume, decreases aortic lipid deposition, and alleviates inflammation in the treatment of atherosclerotic diseases by inhibiting the interaction between sTREM2 and HSP90β proteins. Based on this discovery, this invention provides the use of 3-bromopyruvate in the preparation of medicaments for treating atherosclerosis or its complications, aiming to address the problem that existing clinical treatments for atherosclerosis cannot effectively address the risk of residual inflammation.

[0004] The technical solution provided by this invention is as follows: In a first aspect, the present invention provides the use of an sTREM2-HSP90β interaction inhibitor in the preparation of a medicament, wherein the medicament is: (i) Medications used to treat atherosclerosis; and / or, (ii) Drugs used to prevent complications of atherosclerosis.

[0005] In conjunction with the first aspect of the invention, in some embodiments, the sTREM2-HSP90β interaction inhibitor is 3-bromopyruvic acid or a pharmaceutically acceptable salt thereof.

[0006] In conjunction with the first aspect of the invention, in some embodiments, the pharmaceutically acceptable salt is one or more of sodium 3-bromopyruvate, potassium 3-bromopyruvate, calcium 3-bromopyruvate, magnesium 3-bromopyruvate, ammonium 3-bromopyruvate, arginine 3-bromopyruvate, meglumine 3-bromopyruvate, and lysine 3-bromopyruvate.

[0007] In conjunction with the first aspect of the invention, in some embodiments, the atherosclerosis is atherosclerosis caused by a high-fat diet.

[0008] In conjunction with the first aspect of the present invention, in some embodiments, the atherosclerosis manifests as at least one of (1) to (2): (1) Plaque formation at the root of the aorta; (2) Aortic lipid deposition.

[0009] In conjunction with the first aspect of the present invention, in some embodiments, the atherosclerosis further manifests as at least one of (3) to (5): (3) Levels of IL-1β, IL-6, and TNF-α were higher than healthy levels; (4) CCL2 levels are higher than healthy levels; (5) The levels of total cholesterol, triglycerides and low-density lipoprotein cholesterol in the blood rise sharply.

[0010] In conjunction with the first aspect of the present invention, in some embodiments, the treatment of atherosclerosis includes at least one of (1) to (4): (1) Reduce the volume of plaque at the aortic root; (2) Reduce lipid deposition in the aorta; (3) Reduces the levels of IL-1β, IL-6, and TNF-α in the blood; (4) Reduce CCL2 levels in the blood.

[0011] In conjunction with the first aspect of the present invention, in some embodiments, the atherosclerotic complication is one or more of the following: acute myocardial infarction, ischemic stroke, unstable angina, stable angina, transient ischemic attack, peripheral artery disease and related diseases.

[0012] In conjunction with the first aspect of the invention, in some embodiments, the drug is an injection and / or an oral preparation.

[0013] In a second aspect, the present invention provides a pharmaceutical composition comprising: (i) a therapeutically effective amount of 3-bromopyruvate or a pharmaceutically acceptable salt thereof; and (ii) Lipid-lowering active ingredients.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects: This invention reveals a novel mechanism by which 3-BrP regulates vascular inflammation by inhibiting the interaction between sTREM2 and HSP90β. In the treatment of atherosclerosis, it does not rely on lipid-lowering mechanisms but directly targets vascular inflammation pathways, reducing plaque volume in the aortic root, decreasing lipid deposition in the aorta, and alleviating inflammation. It is expected to solve the problem that existing clinical drugs for the treatment of atherosclerosis cannot effectively address the risk of residual inflammation. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 High-throughput screening and functional validation of inhibitors targeting the sTREM2-HSP90β interaction; among which: (A) Schematic diagram of the principle and process of SPRi high-throughput screening based on 3D-carbene chip. The left side of the figure shows the physical model of screening using surface plasmon resonance imaging (SPRi) technology: incident light passes through a prism to generate surface plasmon waves on the metal surface, and the binding of sTREM2 protein to candidate small molecule compounds (compounds) covalently fixed on the chip surface in the sample cavity (Inlet to Outlet) is monitored in real time by detecting changes in the intensity of reflected light; (B) Initial screening Western Blot results of sTREM2-induced expression of inflammatory factors in BMDM. The figure shows the effects of different candidate small molecule compounds on the expression of pro-inflammatory factors pro-IL-1β, mature IL-1β, IL-6, and TNF-α under sTREM2 (2 nM) stimulation. α-Tubulin was used as an internal control. (CF) Quantitative bar chart analysis of the inhibition of inflammatory factor expression by candidate compounds; (C) is the relative quantitative analysis of the expression levels of pro-IL-1β protein by each group of compounds; (D) is a relative quantitative analysis of the expression levels of mature IL-1β protein by each group of compounds; (E) is the relative quantitative analysis of the effect of each compound on the expression level of IL-6 protein; (F) shows the relative quantitative analysis of the effects of each compound on the expression level of TNF-α protein; the data in the figure are expressed as Mean ± SEM, and the dashed line represents the level in the sTREM2 stimulation group; (G)3-BrP competitively inhibits the binding of sTREM2 to HSP90β via GST-pulldown validation; the figure shows the changes in the Myc-HSP90β band in the precipitate after adding different candidate compounds to the in vitro interaction system. The input group shows that the initial protein amount is consistent, and the experimental group results show that the Myc-HSP90β band is the lightest after treatment with 3-BrP (T5882), proving that it has the highest blocking efficiency.

[0017] (H) Statistical graph of protein immunoassay in GST-pulldown experiment to verify the compounds among the 7 core candidate compounds that have inhibitory effects on sTREM2-HSP90β interaction. The results showed that only 3-BrP (T5882) showed a significant inhibitory effect, and the difference was statistically significant.

[0018] Figure 2 Analysis of the binding characteristics and molecular mechanism of 3-BrP and sTREM2; where: (A) Surface plasmon resonance (SPR) sensor image, showing the real-time response curve of 3-BrP binding to immobilized sTREM2 protein and the fitted dissociation constant (KD value). (B) Microscale thermophoresis (MST) analysis curves, independently verifying the binding affinity of 3-BrP to sTREM2 and the measured KD value; (C) A three-dimensional model of molecular docking between 3-BrP and sTREM2 protein, showing the binding pocket of 3-BrP (green rod) embedded in the intermediate domain of sTREM2 (purple cartoon), and demonstrating in detail the molecular forces of hydrogen bonds formed between 3-BrP and key amino acid residues of sTREM2 (such as ASP87, THR94, ARG33).

[0019] Figure 3 : The inhibitory effect of 3-BrP on the sTREM2-mediated IκBα / NF-κB signaling pathway and the expression of inflammatory factors; among which: (A) Statistical graph of the effect of different concentrations of 3-BrP on the viability of bone marrow-derived macrophages (BMDM) as detected by CCK-8 assay; (B) Immunoblotting of the effects of different concentrations of 3-BrP treatment on sTREM2-induced phosphorylation levels of IκBα and NF-κB p65, as well as on the pro-IL-1β, IL-1β, IL-6 and TNF-α protein cytokines. (C) Quantitative statistical histogram of NF-κB P65 phosphorylation levels; (D) Quantitative statistical bar chart of IκBα phosphorylation level; showing the concentration-dependent inhibitory effect of 3-BrP; (E) Concentration-dependent inhibition statistics of 3-BrP on sTREM2-induced pro-IL-1β protein expression; (F) Statistical graph of concentration-dependent inhibition of sTREM2-induced inflammatory cytokine IL-1β protein expression by 3-BrP; (G)3-BrP concentration-dependent inhibition of sTREM2-induced inflammatory cytokine IL-6 protein expression statistical graph; Statistical graph of concentration-dependent inhibition of sTREM2-induced inflammatory factor TNF-α protein expression by (H)3-BrP.

[0020] Figure 4 The effect of 3-BrP on the stability of the HSP90β / IKKs complex; where: (A) Immunoprecipitation assay showing the immunoblot of representative proteins of HSP90β binding to IKKα, IKKβ and IKKγ after 3-BrP treatment; (B) Quantitative statistical bar chart of the binding efficiency of the HSP90β / IKKs complex shows that 3-BrP significantly reduces the stability of the complex.

[0021] Figure 5 Effects of in vivo 3-BrP intervention on the progression of atherosclerotic plaques in ApoE- / - mice; among which: (A) Representative frontal images of the mouse aorta stained with Oil Red O (ORO) after treatment with different doses of 3-BrP (0, 0.25, 0.5, 1 mg / kg), visually demonstrating the plaque deposition situation; (B) Quantitative statistical analysis of the overall plaque burden of the aorta (the percentage of ORO-positive area to the total aortic area) in a bar chart; (C) Representative images of H&E staining and Oil Red O staining of aortic root sections from each group of mice; (D) Quantitative statistical analysis of plaque area at the aortic root; (E) Quantitative statistical analysis of lipid deposition in aortic root plaques (percentage of ORO-positive area to total plaque area).

[0022] Figure 6 The effect of in vivo intervention of 3-BrP on the level of systemic inflammation; among which: (A) Quantitative statistical graph of plasma levels of inflammatory factors IL-1β, IL-6 and TNF-α in mice after treatment with different doses of 3-BrP by ELISA; (B) Quantitative statistical graph of the levels of chemotactic mediators CCL2, CCL5, CXCL1 and CXCL3 in mouse plasma after treatment with different doses of 3-BrP.

[0023] Figure 7 Safety assessment of in vivo treatment with 3-BrP; (A) Statistical chart of the trend of body weight change in mice in each group during treatment with different doses of 3-BrP; (B) Statistical graph of serum lipid profiles (total cholesterol TC, triglycerides TG, low-density lipoprotein cholesterol LDL-c, and high-density lipoprotein cholesterol HDL-c) in mice after treatment; (C) Representative images of H&E-stained pathological sections of major organs (heart, liver, spleen, lung, kidney, and small intestine) of mice after treatment, used to assess histological changes; (D) Quantitative statistical graph of alanine aminotransferase (ALT) levels, a biomarker of liver and kidney function, in mouse serum after treatment; (E) Quantitative statistical graph of the level of aspartate aminotransferase (AST), a biomarker of liver and kidney function in mouse serum, after treatment; (F) Quantitative statistical graph of the level of alkaline phosphatase (ALP), a biomarker of liver and kidney function, in mouse serum after treatment; (G) Quantitative statistical graph of serum creatinine (CREA), a biomarker of liver and kidney function, in mice after treatment; (H) Quantitative statistical graph of serum blood urea nitrogen (BUN) levels, a biomarker of liver and kidney function, in mice after treatment. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0025] Triggering receptor 2 (TREM2) is expressed in myeloid cells such as macrophages and participates in the regulation of inflammatory responses and cell survival signaling through its soluble form (sTREM2). This invention demonstrates that the interaction between sTREM2 and heat shock protein 90β (HSP90β) may play an important role in atherosclerosis-related inflammation and apoptosis. HSP90β, as a molecular chaperone, participates in the folding and stabilization of various signaling proteins, and its aberrant expression is closely related to the regulation of inflammatory responses, cellular stress, and apoptosis.

[0026] Previous research in this invention, using co-immunoprecipitation-affinity purification mass spectrometry (Co-IP / AP-MS), identified for the first time a specific binding between sTREM2 and HSP90β in an inflammatory microenvironment. Further GST-pulldown experiments confirmed a direct interaction between the two. Mechanistic studies revealed that sTREM2 enhances the binding stability of the HSP90β-IKK kinase complex, promoting activation of the IκBα / NF-κB signaling pathway, thereby driving macrophage polarization towards a pro-inflammatory phenotype. In a macrophage-specific Hsp90ab1 gene knockout atherosclerotic mouse model, the pro-atherosclerotic effect of sTREM2 was significantly inhibited, further validating the key regulatory function of this interaction in disease progression.

[0027] Currently, treatment strategies for atherosclerosis, in addition to lipid regulation, increasingly focus on intervening in inflammatory pathways and immune regulatory mechanisms. However, therapeutic molecules capable of precisely targeting key inflammatory signaling nodes and possessing a clear mechanism of action remain limited. To identify small-molecule inhibitors that can specifically intervene in the sTREM2–HSP90β protein interaction, this invention constructs a high-throughput screening platform based on this interaction system. A library containing 3180 compounds was systematically screened using surface plasmon resonance (SPR) technology, and 3-bromopyruvic acid (3-BrP) was identified as a small-molecule candidate capable of effectively and specifically disrupting the binding of sTREM2–HSP90β. Further molecular docking and binding kinetic analysis showed that 3-BrP has a high affinity for sTREM2, and its binding may involve specific amino acid residues in the intermediate domain of sTREM2. 3-BrP, as a known glycolysis inhibitor and alkylating agent, has shown anticancer activity in tumor research by interfering with energy metabolism and inducing apoptosis. However, its function as an inhibitor of the sTREM2–HSP90β interaction in the regulation of atherosclerotic inflammation has not been previously reported, and its therapeutic potential and mechanism of action in this disease model remain unclear.

[0028] Therefore, based on the above high-throughput screening results, further exploration of the protective effect and mechanism of 3-BrP as an inhibitor of sTREM2–HSP90β interaction in atherosclerosis models will not only help reveal new mechanisms of atherosclerosis pathogenesis, but also provide high-value lead compounds and experimental evidence for developing novel therapeutic strategies targeting key protein interactions.

[0029] This invention systematically evaluated the inhibitory effect of 3-BrP on the interaction between sTREM2 and HSP90β and its therapeutic potential through in vitro protein interaction experiments, cell models, and animal models of atherosclerosis. The results showed that 3-BrP can directly bind to the sTREM2 protein, effectively blocking the interaction between sTREM2 and HSP90β, thereby disrupting the stability of the HSP90β / IKKs complex, inhibiting the activation of the IκBα / NF-κB signaling pathway, and ultimately significantly reducing the release of inflammatory factors from macrophages. In an ApoE- / - atherosclerotic mouse model, 3-BrP intervention dose-dependently reduced aortic plaque area and decreased plasma levels of inflammatory factors such as IL-1β, IL-6, and TNF-α without causing significant changes in lipid metabolism or hepatotoxicity or nephrotoxicity, indicating that it exerts its therapeutic effect through an anti-inflammatory mechanism and has good biocompatibility.

[0030] Therefore, 3-BrP can serve as a specific inhibitor of the sTREM2-HSP90β interaction, enabling the construction of drug screening models based on this target to screen candidate compounds for the prevention, alleviation, and / or treatment of atherosclerosis. 3-BrP itself can also be used as an active ingredient in the preparation of drugs for the prevention, alleviation, and / or treatment of atherosclerosis. For example, using 3-BrP as the parent core structure, its pharmacokinetic properties can be optimized through chemical modification to develop small molecule inhibitors with higher affinity and selectivity; or it can be combined with suitable pharmaceutical excipients to prepare injectable formulations for the clinical treatment of atherosclerosis and related inflammatory vascular diseases.

[0031] This invention provides the use of 3-bromopyruvic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament, characterized in that the medicament is (i) a medicament for treating atherosclerosis; and / or, (ii) a medicament for preventing complications of atherosclerosis. The medicament treats atherosclerosis or prevents complications of atherosclerosis by inhibiting the interaction between sTREM2 and HSP90β proteins.

[0032] In some embodiments of the present invention, the pharmaceutically acceptable salt is one or more of sodium 3-bromopyruvate, potassium 3-bromopyruvate, calcium 3-bromopyruvate, magnesium 3-bromopyruvate, ammonium 3-bromopyruvate, arginine 3-bromopyruvate, meglumine 3-bromopyruvate, and lysine 3-bromopyruvate.

[0033] In some embodiments of the present invention, the atherosclerosis is atherosclerosis caused by a high-fat diet.

[0034] In some embodiments of the present invention, the atherosclerosis is manifested as at least one of (1) to (2): (1) Plaque formation at the root of the aorta; (2) Aortic lipid deposition.

[0035] In some embodiments of the present invention, the atherosclerosis further manifests as at least one of (3) to (5): (3) Levels of IL-1β, IL-6, and TNF-α were higher than healthy levels; (4) CCL2 levels are higher than healthy levels; (5) The levels of total cholesterol, triglycerides and low-density lipoprotein cholesterol in the blood rise sharply.

[0036] In some embodiments of the present invention, the treatment of atherosclerosis includes at least one of (1) to (4): (1) Reduce the volume of plaque at the aortic root; (2) Reduce lipid deposition in the aorta; (3) Reduces the levels of IL-1β, IL-6, and TNF-α in the blood; (4) Reduce CCL2 levels in the blood.

[0037] In conjunction with the first aspect of the present invention, in some embodiments, the atherosclerotic complication is one or more of the following: acute myocardial infarction, ischemic stroke, unstable angina, stable angina, transient ischemic attack, peripheral artery disease and related diseases.

[0038] In some embodiments of the present invention, the drug is an injection and / or an oral preparation.

[0039] The present invention also provides a pharmaceutical composition comprising: (i) a therapeutically effective amount of 3-bromopyruvate or a pharmaceutically acceptable salt thereof; and (ii) Lipid-lowering active ingredients.

[0040] The technical solution provided by the present invention will be described in detail below with reference to the embodiments.

[0041] Example 1: High-throughput screening and molecular identification of sTREM2-HSP90β interaction inhibitors 1.1 High-throughput small molecule screening based on the SPRi platform was performed using the Plexera PlexArray HT surface plasmon resonance imaging (SPRi) system.

[0042] Chip fabrication: A compound library containing 3180 small molecules (Selleck, Catalog No. L1300) was covalently immobilized on the surface of a functionalized biochip.

[0043] Protein sample: sTREM2 protein was dissolved in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 0.05% Surfactant P20, pH 7.4).

[0044] Screening process: Protein samples are flowed over the chip surface at a flow rate of 5 μL / s for 150 seconds to bind to the phase, followed by 300 seconds of dissociation phase in the run buffer, and finally regenerated for 200 seconds using regeneration buffer (alternating between 10 mM Glycine-HCl pH 2.0 and 50 mM Glycine-NaOH pH 9.5).

[0045] Data Analysis: Binding kinetics were analyzed using Plexera SPR Data Analysis Model software, initially screening 69 candidate compounds with significant physical binding signals to sTREM2. For the 69 bound molecules screened by SPRi, their biosafety and pharmacological background were assessed by reviewing literature databases. Cellular-level anti-inflammatory experiments were conducted to exclude molecules with high cytotoxicity or no significant anti-inflammatory activity, ultimately focusing on 20 core candidate compounds.

[0046] 1.2 Verification of Cellular Anti-inflammatory Activity BMDM extraction and induction: Femur and tibia of C57BL / 6 mice were taken, bone marrow was aseptically rinsed, and after erythrocyte lysis, differentiation was induced for 6-7 days in DMEM medium containing 10% FBS and 50 ng / mL M-CSF, with the medium changed every 3 days.

[0047] Activity screening: Inflammation was induced in BMDM by prestimulation with LPS (100 ng / mL) + IFNγ (20 ng / mL), and then BMDM was treated with a mixture of candidate compounds and sTREM2 protein pre-incubated in EP tubes. Compounds with anti-inflammatory activity were screened by detecting the expression of inflammatory factors by Western blotting.

[0048] Further analysis of the inhibitory effects of the four inflammatory factors revealed seven core candidate compounds (Eganelisib, Farnesol, Tenuigenin, Semulgin, 3-BrP, Salmeterol, and Xanthurenic Acid) for subsequent validation.

[0049] 1.3 GST-pulldown competitive inhibition experiment and molecular identification To identify the compound among the seven that directly interferes with the sTREM2-HSP90β interaction, a GST-pulldown experiment was performed. Lysates of 293T cells overexpressing GST-sTREM2 were constructed. Equal volumes of these lysates were pre-incubated with each of the seven candidate compounds, and then incubated overnight at 4°C with lysates of 293T cells overexpressing Myc-HSP90β using a vertically rotating shaker. The complex was pulled down using GST beads, washed, and then analyzed by Western blotting (anti-GST and anti-Myc antibodies). Results are as follows: Figure 1 As shown in G, among the seven compounds, only 3-BrP (T5882) was able to significantly inhibit the binding of GST-sTREM2 to Myc-HSP90β, thus being confirmed as a specific inhibitor of the sTREM2-HSP90β interaction.

[0050] Example 2: Verification of the binding characteristics and molecular mechanism of 3-BrP and sTREM2 To accurately quantify the binding affinity between 3-BrP and sTREM2, this invention employs surface plasmon resonance (SPR), microscale thermophoresis (MST), and molecular docking techniques for analysis. The specific steps are as follows: 2.1 Biacore SPR kinetic measurement Precise quantification using the Biacore system: Immobilization: Recombinant sTREM2 protein (50 μg / mL, dissolved in pH 4.5 sodium acetate buffer) was immobilized on a CM5 chip by amine coupling, with the response value controlled between 500 and 1000 RU.

[0051] Assay: The running buffer was PBS-P+ (containing 5% DMSO, pH 7.4). 3-BrP was prepared into a series of concentration gradients and injected at a flow rate of 30 μL / min, binding for 150 seconds, followed by dissociation. The chip was regenerated using 10 mM Glycine-HCl (pH 2.0).

[0052] Analysis: The KD value was calculated using Biacore Insight Evaluation Software through steady-state affinity and kinetic analysis (1:1 binding model).

[0053] 2.2 MST (Microscale Thermophoresis) Validation Using the NanoTemper Monolith NT.115 instrument: System: The buffer solution consisted of 50 mM HEPES (pH 7.0), 50 mM NaCl, 0.01% Tween-20, and 2 mM MgCl2.

[0054] Detection: His-tagged sTREM2 protein was mixed with serially diluted 3-BrP, incubated at room temperature for 5 minutes, and then inserted into a hydrophilic capillary for detection. The infrared laser power was set to a level that could induce heating without damaging biomolecules (maximum 24 mW), and thermophoretic behavior was recorded over 30 seconds.

[0055] 2.3 Molecular docking AutoDock Vina (version 1.1.2) software was used. The extracellular domain of sTREM2 (residues 1-168) was set as the receptor, and 3-BrP as the ligand. The structure was processed using AutoDock Tools (hydrogen addition, charge calculation), and the results were visualized and analyzed using PyMOL (v2.6.0) and Discovery Studio.

[0056] SPR results show ( Figure 2 A) 3-BrP can bind to immobilized sTREM2 protein in a dose-dependent manner, and the dissociation constant was calculated by fitting. K D The value was 38.2 μM. MST experimental results ( Figure 2 B) This corroborates the findings of SPR, which were measured... K D The value was 19.0 μM, which together confirmed the existence of a moderate-strength direct binding between 3-BrP and sTREM2.

[0057] Molecular docking simulation results show that ( Figure 2 (C) 3-BrP primarily binds to a hydrophobic pocket within the intermediate domain (MD) of the sTREM2 protein, forming a stable hydrogen bond network with key amino acid residues such as ASP87, THR94, and ARG33. This binding site may be the interface through which sTREM2 interacts with HSP90β, thus structurally explaining why 3-BrP can competitively inhibit the sTREM2-HSP90β interaction.

[0058] Example 3: 3-BrP inhibits the sTREM2-HSP90β / IKK / NF-κB inflammatory signaling pathway at the cellular level. 3.1 Preparation of experimental materials and cell culture BMDM cell origin and differentiation induction: Wild-type C57BL / 6 mice aged 6-8 weeks (purchased from Vital River Pharmaceuticals, Beijing) were euthanized by cervical dislocation, and the femur and tibia were aseptically isolated. The epiphyses at both ends of the bones were removed, and the bone marrow cavity was flushed with sterile PBS using a syringe to collect the bone marrow cell suspension. After treatment with erythrocyte lysis buffer, the cells were resuspended in DMEM complete medium containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 50 ng / mL recombinant mouse M-CSF (macrophage colony-stimulating factor). The cells were seeded in culture dishes and incubated at 37°C in a 5% CO2 incubator for 6-7 days, with the medium replaced with fresh M-CSF every 3 days until mature bone marrow-derived macrophages (BMDM) were obtained.

[0059] 3-BrP preparation: 3-BrP was purchased from TargetMol. Before the experiment, 3-BrP powder was dissolved in sterile PBS (phosphate buffered saline, pH 7.4) to prepare a stock solution, which was then filtered through a 0.22 μm filter for sterilization. Note: 3-BrP solutions should be prepared fresh and protected from light to prevent degradation and inactivation in the solution.

[0060] 3.2 Cell viability assay (CCK-8 assay) Induced mature BMDM cells were digested and seeded in 96-well plates (density 5 × 10⁶ cells / well). 4 (Number of cells / well) and incubate overnight. Discard the old medium and add fresh DMEM medium (containing 10% FBS) with different concentrations (5 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM) of 3-BrP, and treat for 12 hours. After treatment, aspirate the medium from the wells and add 100 μL of serum-free medium containing 10% CCK-8 reagent to each well, and incubate at 37°C in the dark for 1-2 hours. Measure the absorbance (OD value) at 450 nm using a microplate reader. Results are as follows: Figure 3 As shown in Figure A, 3-BrP did not exhibit significant cytotoxicity against BMDM within the concentration range of 5–100 μM.

[0061] 3.3 Signaling pathways and inflammatory factor detection (1) Cell grouping and treatment: After starving BMDM cells (serum-free DMEM) for 4 hours, they were divided into the following groups: Blank control group (Control, PBS); sTREM2 stimulation group (200 nM sTREM2 protein); sTREM2 + different concentrations of 3-BrP treatment groups (5 μM, 10 μM, 20 μM, and 40 μM of 3-BrP were co-incubated with sTREM2 before being added to cells) (Note: sTREM2 protein was dissolved in PBS buffer).

[0062] (2) Western Blot detection: Cellular proteins were collected 2 hours after stimulation, and key proteins of the NF-κB signaling pathway (p-IκBα, p-p65, and their total protein) were detected. Results showed ( Figure 3 BD), sTREM2 stimulation alone significantly induced phosphorylation of IκBα and p65 (Ser536), while 3-BrP inhibited this activation process in a concentration-dependent manner. Cells and supernatant were collected 6 hours after stimulation, and precursors and mature forms of inflammatory factors were detected. Results showed ( Figure 3 EH), the expression of sTREM2-induced pro-inflammatory factors pro-IL-1β, IL-1β, IL-6 and TNF-α was significantly inhibited by 3-BrP.

[0063] 3.4 Immunoprecipitation (Co-IP) assay for complex stability To elucidate the upstream mechanism of 3-BrP, this invention investigated its effect on the stability of the HSP90β / IKKs complex using co-immunoprecipitation (Co-IP) assay. The specific method is as follows: Total protein was extracted from BMDM cells using IP lysis buffer containing a protease inhibitor. 500 μg of protein lysis buffer was used as input, and the remaining lysis buffer was incubated with 2 μg of anti-HSP90β specific antibody at 4°C overnight by rotation. Subsequently, Protein A / G magnetic beads were added and incubated at room temperature for 2 hours. The magnetic beads were washed five times with lysis buffer, and then eluted by boiling with 1×SDS loading buffer. Western blotting was used to detect the levels of IKKα, IKKβ, and IKKγ in the immunoprecipitate. Figure 4 As shown, sTREM2 stimulation enhanced the binding of HSP90β to IKKα, IKKβ, and IKKγ, while 3-BrP treatment significantly disrupted the formation of this complex. This indicates that 3-BrP inhibits the interaction between sTREM2 and HSP90β, thereby destabilizing the HSP90β / IKKs complex and ultimately blocking the activation of the NF-κB pathway.

[0064] Example 4: 3-BrP slows the progression of atherosclerosis and demonstrates good safety in animal models. 4.1 Animal Model Construction and Drug Administration Modeling: 7-week-old males ApoE - / -Mice were acclimatized and fed a high-cholesterol diet (40% fat calories, 1.25% cholesterol, Diet #XT108C) for 8 weeks to induce atherosclerosis.

[0065] Grouping and administration: Mice were randomly divided into a model group (Vehicle) and low, medium, and high dose 3-BrP groups. During the modeling period, drug intervention was performed (intraperitoneal injection at doses of 0.25 mg / kg, 0.5 mg / kg, and 1 mg / kg, twice a week, with sterile PBS as the solvent and control) for 4 weeks.

[0066] 4.2 Gross Oil Red O staining of the aorta After the intervention, the mice were euthanized, and their hearts were perfused with PBS. The entire aorta (from the aortic root to the iliac bifurcation) was harvested, peripheral fat was removed, and the aorta was longitudinally dissected to expose the intima. En face staining was performed using Oil Red O (Servicebio), and after photographing, ImageJ was used to quantitatively analyze the plaque area (percentage of total lumen area). Results are as follows: Figure 5 As shown, compared with the control group, all treatment groups with different doses of 3-BrP showed a dose-dependent reduction in the proportion of plaque deposition area. Figure 5 A, B).

[0067] 4.3 Pathological analysis of aortic root sections Heart samples were dehydrated with 20% sucrose, embedded in OCT, and flash-frozen in liquid nitrogen. Serial frozen sections with a thickness of 5 μm were prepared using the aortic root as the section surface. H&E and ORO staining were performed on the aortic root plaques. Results analysis showed a significant reduction in plaque area and lipid deposition. Figure 5 CE).

[0068] 4.4 Plasma biochemistry and safety assessment ELISA test results show ( Figure 6 The levels of inflammatory factors (IL-1β, IL-6, TNF-α) and chemokine (CCL2) in the plasma of mice treated with AB and 3-BrP were significantly reduced.

[0069] Simultaneously, this invention systematically evaluated the systemic effects of 3-BrP. Plasma samples were collected, and the levels of total cholesterol, triglycerides, LDL-c, and HDL-c were detected using an enzymatic reagent kit (Nanjing Jiancheng). Serum ALT, AST, ALP (liver function), CREA, and BUN (kidney function) levels were detected using a fully automated biochemical analyzer (Shenzhen Rayto) to assess drug safety.

[0070] The results showed that during the entire intervention period, the body weight of mice in each group ( Figure 7A) and serum lipid profile (total cholesterol, triglycerides, LDL-c, HDL-c, ... Figure 7 B) There were no significant differences, indicating that the anti-atherosclerotic effect of 3-BrP is independent of lipid-lowering or affects the overall metabolism of important organs (heart, liver, spleen, lung, kidney, small intestine) H&E stained sections ( Figure 7 C) No obvious pathological damage was found, and serum liver and kidney function markers (ALT, AST, ALP, CREA, BUN) were normal. Figure 7 The levels of DH were all within the normal range, demonstrating that 3-BrP has good biocompatibility at the stated dose.

[0071] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. Use of 3-bromopyruvic acid or a pharmaceutically acceptable salt thereof for the manufacture of a medicament, characterized in that, The drug is: (i) a drug for treating atherosclerosis; and / or, (ii) a drug for preventing atherosclerosis complications.

2. Use according to claim 1, characterized in that: The drug treats atherosclerosis or prevents atherosclerosis complications by inhibiting the sTREM2 and HSP90β protein interaction.

3. Use according to claim 1, characterized in that: The pharmaceutically acceptable salt is one or more of sodium 3-bromopyruvate, potassium 3-bromopyruvate, calcium 3-bromopyruvate, magnesium 3-bromopyruvate, ammonium 3-bromopyruvate, arginine 3-bromopyruvate, meglumine 3-bromopyruvate, and lysine 3-bromopyruvate.

4. Use according to claim 1, characterized in that: The atherosclerosis is atherosclerosis caused by a high-fat diet.

5. Use according to claim 1, characterized in that: The atherosclerosis is manifested by at least one of (1) and (2): (1) plaque formation at the aortic root; (2) aortic lipid deposition.

6. Use according to claim 5, characterized in that: The atherosclerosis is also manifested by at least one of (3) to (5): (3) IL-1β, IL-6, and TNF-α levels are higher than healthy levels; (4) CCL2 levels are higher than healthy levels; (5) total cholesterol, triglycerides, and low-density lipoprotein cholesterol levels in the blood are sharply increased.

7. Use according to claim 1, characterized in that: The treatment of atherosclerosis includes at least one of (1) to (4): (1) reducing the volume of plaque at the aortic root; (2) reducing aortic lipid deposition; (3) reducing IL-1β, IL-6, and TNF-α levels in the blood; (4) reducing CCL2 levels in the blood.

8. Use according to claim 1, characterized in that: The atherosclerosis complications are one or more of acute myocardial infarction, ischemic stroke, unstable angina, stable angina, transient ischemic attack, peripheral arterial disease, and related diseases.

9. Use according to claim 1, characterized in that: The drug is an injection and / or an oral agent.

10. A pharmaceutical composition, characterized by: The pharmaceutical composition comprises: (i) a therapeutically effective amount of 3-bromopyruvic acid or a pharmaceutically acceptable salt thereof; and (ii) a lipid-lowering active ingredient.

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