SLC22A6-targeting polypeptide, antibody, composition and application of SLC22A6-targeting polypeptide, antibody and composition in treatment and diagnosis of atherosclerosis

Abstract

The invention relates to SLC22A6-targeted polypeptide, antibody, composition and application thereof in treatment and diagnosis of atherosclerosis, and researches find that expression of SLC22A6 in atherosclerotic endothelial cells is up-regulated, and glycolysis and lactic acid accumulation are promoted to drive H3K9 lactylation and activate SCD1 expression so as to mediate endothelial dysfunction and disease progression. Based on this, the invention provides a polypeptide and a monoclonal antibody which can specifically bind and inhibit the function of SLC22A6, and a pharmaceutical composition containing the polypeptide and the monoclonal antibody can be used for preventing or treating atherosclerosis. Meanwhile, the invention further provides a diagnostic kit containing the SLC22A6 detection reagent and a drug screening method taking the SLC22A6 as a target spot, and a new tool and strategy are provided for accurate intervention of atherosclerosis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to application of SLC22A6 as atherosclerosis treatment target, and polypeptides, antibodies, compositions targeting SLC22A6 and their application in treating and diagnosing atherosclerosis. BACKGROUND

[0002] Atherosclerosis, as the most common and deadly pathological basis of cardiovascular diseases, has a high incidence and mortality rate worldwide, which constitutes a major public health burden. In the complex process of the disease, endothelial dysfunction is recognized as an early event with core pathophysiological significance, often being a key link to start the progression of atherosclerosis. In recent years, the scientific community has paid increasing attention to the metabolic reprogramming of endothelial cells, and gradually recognized that it plays a key driving role in dysfunction. A significant feature is that endothelial cells exhibit high glycolytic activity. This enhanced glycolytic flux is not a simple energy metabolism adaptation, but an important mechanism to actively promote endothelial cell activation, inflammatory response and functional disorder. It is particularly worth noting that this excessive glycolysis can cause a significant increase in intracellular lactic acid levels, and lactic acid is no longer considered a simple metabolic waste. Emerging research shows that a new type of post-translational modification derived from lactic acid, histone lysine lactylation, plays a role in regulating gene expression and cell function, and the abnormality of this epigenetic mechanism is potentially associated with the development of atherosclerosis.

[0003] Among the solute carrier family, SLC22A6 protein, as an important organic anion transporter, mainly mediates the uptake of various acidic metabolites and organic anions in cells. Some observational studies have suggested that overexpression of SLC22A6 may be associated with endothelial dysfunction and related inflammatory damage. However, the specific role of SLC22A6 in the pathological environment of atherosclerosis, especially whether and how it participates in regulating lactic acid metabolism flux in endothelial cells, and then affects the epigenetic modification of histone lactylation, and ultimately drives the progression of atherosclerosis, the entire molecular pathway and causal chain have been unknown, and lack clear experimental explanation. This knowledge gap has largely limited our ability to develop new intervention strategies targeting this target. The starting point of the present application is based on the in-depth exploration of this unknown field, aiming to clarify the exact functional mechanism of SLC22A6 in atherosclerosis, and thus to open up new paths for diagnosis and treatment. SUMMARY

[0004] Technical problems solved: The present application aims to solve the problems in the prior art that the mechanism of SLC22A6 in atherosclerosis is unknown, there is a lack of specific treatment means targeting it, and there is a lack of related early diagnosis tools. By elucidating the new mechanism of SLC22A6 promoting disease progression by driving glycolysis-H3K9 lactylation-SCD1 signaling axis, a polypeptide targeting SLC22A6, an inhibitor and its application in treating and diagnosing atherosclerosis are provided.

[0005] Technical solutions: A polypeptide targeting SLC22A6 protein, which can specifically bind to SLC22A6 protein and inhibit its mediated organic anion transport function. The amino acid sequence of the above-mentioned polypeptide is shown in SEQ ID NO: 1. A monoclonal antibody or antigen binding fragment against SLC22A6 protein, which can specifically bind to SLC22A6 protein and inhibit its mediated organic anion transport function. A pharmaceutical composition comprising at least one of the above-mentioned polypeptide, the above-mentioned monoclonal antibody or antigen binding fragment, and a pharmaceutically acceptable carrier. The above-mentioned pharmaceutical composition is formulated as a preparation for preventing or treating atherosclerosis. A kit for diagnosing atherosclerosis, comprising reagents for detecting the expression level of SLC22A6 protein in a biological sample. The reagent is an antibody for detecting SLC22A6 protein, a primer or probe for detecting SLC22A6 mRNA, and the antibody is the monoclonal antibody or antigen binding fragment of claim 3. The use of a compound targeting SLC22A6 protein as an action target in the preparation of a drug for preventing or treating atherosclerosis. A method for screening candidate compounds for preventing or treating atherosclerosis, comprising the following steps: (a) contacting a test compound with a system expressing or containing SLC22A6 protein under test conditions; (b) detecting the effect of the test compound on the expression level or transport activity of SLC22A6 protein in the system; (c) screening compounds that can inhibit the expression or activity of SLC22A6 protein as the candidate compounds according to the detection result of step (b). In step (b), the effect of the test compound on the lactylation level of histone H3 lysine 9 in the system is also detected, and in step (c), further screening compounds that can reduce the lactylation level.

[0006] Beneficial Effects: This invention reveals the key mechanism of SLC22A6 in atherosclerosis. Based on the discovery of a novel mechanism by which SLC22A6 drives the glycolysis-lactation-SCD1 signaling axis, our designed targeting peptides and monoclonal antibodies can specifically intervene in the core links of this pathway, thus providing a new tool for precision treatment of the disease. These intervention tools themselves have the advantage of high specificity. Furthermore, they effectively synergize with diagnostic kits developed based on the same target. The diagnostic tools can identify potential beneficiaries of this targeted therapy at an early stage, while the treatment directly targets the abnormal pathway revealed by the diagnosis. This linkage between diagnosis and treatment provides a concrete path for achieving precision medicine in atherosclerosis. In addition, the established drug screening model based on SLC22A6 functional inhibition can efficiently screen lead compounds acting on this new pathway, which not only accelerates the new drug development process but also further validates and expands the therapeutic value of this target. Therefore, from the discovery of target mechanisms to the development of therapeutic products, and then to the establishment of diagnostic tools and screening platforms, the various components of this invention together constitute a complete technical system, providing a systematic solution with inherent synergistic effects for solving the diagnostic and treatment challenges of atherosclerosis. Attached Figure Description

[0007] Figure 1 To determine the expression and localization of SLC22A6 in atherosclerotic tissues.

[0008] Figure 2 To investigate the effects of SLC22A6 on the enrichment of H3K9la in the SCD1 promoter region and the influence of SLC22A6 expression level on H3K9la level in HUVECs.

[0009] Figure 3 The effects of SLC22A6 inhibitors on atherosclerotic plaque area and on plaque stability and endothelial function markers were investigated.

[0010] Figure 4 This is a schematic diagram illustrating the mechanism of action of SLC22A6 in atherosclerosis. Detailed Implementation

[0011] The following embodiments are provided to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way.

[0012] Example 1 Detection of SLC22A6 expression in atherosclerosis The expression of SLC22A6 in the aortic tissue of atherosclerotic mice and human atherosclerotic specimens was detected by RNA sequencing and immunohistochemistry. The results showed that SLC22A6 was significantly upregulated in atherosclerotic tissues compared with the control group, and was mainly expressed in CD31-positive endothelial cells.

[0013] Experimental Groups: Experimental group: ApoE - / - An atherosclerosis model was established in mice fed a high-fat diet for 16 weeks (n=10).

[0014] Control group: age-matched C57BL / 6J mice on a normal diet (n=10).

[0015] Methods and Results: 1. Protein level validation (Western Blot): Procedure: Total protein was extracted from mouse aorta and subjected to electrophoresis, membrane transfer, antibody incubation, and exposure development. Using GAPDH or β-Actin as internal controls, the grayscale values ​​of the target band and the internal control band were analyzed using ImageJ software to calculate the relative expression level of SLC22A6.

[0016] The results showed that the protein expression level of SLC22A6 in the aorta of the experimental group mice was 3.5 ± 0.4 times that of the control group (p < 0.01).

[0017] Immunohistochemistry: Procedure: Aortic sections were blocked. Then, primary antibodies against SLC22A6 and CD31 (an endothelial cell marker) were added, and the sections were incubated overnight at 4°C. The following day, the sections were developed using the corresponding secondary antibodies, and the cell nuclei were counterstained with DAIP.

[0018] Scoring and colocalization analysis: Laser confocal microscopy was used to observe and the colocalization coefficient (Pearson's coefficient) was calculated using its accompanying software to confirm the expression of SLC22A6 and CD31 in the same cells.

[0019] Mouse tissues: In the aortic sinus plaque region of the experimental group mice, SLC22A6 showed strong positive staining in CD31-positive endothelial cells (staining intensity score: 3.2±0.3), while the control group showed almost no staining (score: 0.4±0.1, p < 0.001). Double-labeled immunofluorescence co-localization analysis confirmed the co-expression of SLC22A6 and CD31.

[0020] Example 2: SLC22A6 regulates H3K9 lactation and downstream pathways Experimental methods: Western Blot: Human umbilical vein endothelial cells (HUVECs) were used. Groups were set up as follows: control group (normal culture medium), model group (treated with 50 μg / mL oxidized low-density lipoprotein (ox-LDL) for 24 hours), SLC22A6 knockdown group (transfected with siRNA followed by ox-LDL treatment), and SLC22A6 overexpression group (transfected with overexpression plasmid, ox-LDL not required). The protein levels of H3K9la, H3K9ac, and SCD1 in HUVECs and mouse aortic tissue were detected.

[0021] CUT&Tag: Use specific antibodies to capture genomic DNA fragments that bind to H3K9la modifications and perform high-throughput sequencing.

[0022] Results and data: In the aorta of atherosclerotic mice and in ox-LDL-treated HUVECs, H3K9la levels were significantly increased (2.5-fold and 3.1-fold, respectively), while conventional H3K9ac modification showed a slight decrease.

[0023] CUT&Tag sequencing analysis revealed that H3K9la was enriched 4.8-fold in the SCD1 gene promoter region in the lesion tissue. Correspondingly, the mRNA and protein levels of SCD1 were upregulated by 3.3-fold and 2.9-fold, respectively.

[0024] In HUVECs, knockdown of SLC22A6 reversed ox-LDL-induced H3K9la elevation. Conversely, overexpression of SLC22A6 mimicked the effects of ox-LDL.

[0025] Example 3 Functional validation of targeted peptides 1. Peptide design: Based on the substrate binding domain of SLC22A6, the peptide Pep-1 (sequence: SEQ ID NO: 1) was designed and synthesized.

[0026] 2. Peptide synthesis and processing: Synthesis: Pep-1 was synthesized by a professional company using solid-phase peptide synthesis and purified by high-performance liquid chromatography to a purity of >95%.

[0027] Cell treatment: In HUVECs, 100 μM Pep-1 was added for pretreatment for 2 hours, and then ox-LDL was added for co-culture for 24 hours.

[0028] 3. Functional assay: In the HUVEC model, Pep-1 (100 μM) treatment competitively inhibited lactate uptake with an inhibition rate of 45% (p < 0.05). Simultaneously, downstream H3K9la levels decreased by 40%, and SCD1 mRNA expression was downregulated by 55% (p < 0.05).

[0029] SEQ ID NO.1: MAFNDLLQQVGGVGRFQQIQVTLVVLPLLLMASHNTLQNFTAAIPTHHCRPPADANLSKNGGLEVWLPRDRQGQPESCLRFTSPQWGLPFLNGTEANGTGATEPCTDGWIYDNSTFPSTIVTEWDLVCSHRALRQLAQSL YMVGVLLGAMVFGYLADRLGRRKVLILNYLQTAVSGTCAAFAPNFPIYCAFRLLSGMALAGISLNCMTLNVEWMPIHTRACVGTLIGYVYSLGQFLLAGVAYAVPHWRHLQLLVSAPFFAFFIYSWFFIESARWHSSSGRL DLTLRALQRVARINGKREEGAKLSMEVLRASLQKELTMGKGQASAMELLRCPTLRHLFLCLSMLWFATSFAYYGLVMDLQGFGVSIYLIQVIFGAVDLPAKLVGFLVINSLGRRPAQMAALLLAGICILLNGVIPQDQSIV RTSLAVLGKGCLAASFNCIFLYTGELYPTMIRQTGMGMGSTMARVGSIVSPLVSMTAELYPSMPLFIYGAVPVAASAVTVLLPETLGQPLPDTVQDLESRWAPTQKEAGIYPRKGKQTRQQQEHQKYMVPLQASAQEKNGL

[0030] Example 4 In vivo efficacy verification Experimental methods: 1. Experimental design and dosing regimen: Animal grouping: Forty ApoE- / - mice (induced by a high-fat diet) were randomly divided into four groups (n=10): Model group: Daily intraperitoneal injection of an equal volume of solvent (such as PBS).

[0031] Low-dose treatment group: Daily intraperitoneal injection of SLC22A6 inhibitor (e.g., 5 mg / kg).

[0032] High-dose treatment group: Daily intraperitoneal injection of SLC22A6 inhibitor (e.g., 15 mg / kg).

[0033] Positive control group: For example, oral atorvastatin (10 mg / kg) daily.

[0034] Dosing period: Dosing begins in week 8 of a high-fat diet and continues until week 16, for a total of 8 weeks.

[0035] 2. Results and Data: Quantification of aortic plaque area: Oil Red O staining: After euthanizing the mice, the intact aorta was dissected, dissected along the longitudinal axis of the vessel, and fixed with 4% paraformaldehyde. Then, it was stained with 0.5% Oil Red O working solution for 30 minutes; this dye will stain the lipid plaques red.

[0036] Image analysis and calculation: Images were taken under a stereomicroscope, and ImagePro Plus software was used to calculate the percentage of the area of ​​the red-stained plaque on the entire aortic intima.

[0037] Patch stability analysis: Macrophage content: Frozen sections of the aortic root were stained with anti-CD68 antibody (macrophage marker) using immunofluorescence staining, and the content was quantified by calculating the percentage of positive areas.

[0038] Collagen content: Adjacent sections were stained with Sirius red, a dye that stains type I and type III collagen fibers bright yellow or red under polarized light. The proportion of collagen-positive area in the total plaque area was calculated through image analysis. Higher collagen content indicates more stable plaques.

[0039] Detection of markers of endothelial dysfunction: Blood was collected from the orbital venous plexus of mice, and the serum was separated by centrifugation after standing.

[0040] Using an enzyme-linked immunosorbent assay (ELISA) kit, and strictly following the instructions, the concentrations of soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1) in serum were measured. Compared with the model group, the aortic plaque area in the treatment group mice was reduced by 52% (p < 0.01).

[0041] Histological analysis showed that macrophage infiltration in the treatment group decreased by 48% and collagen content increased by 35%, indicating enhanced plaque stability.

[0042] The levels of sVCAM-1 and sICAM-1, markers of endothelial dysfunction, were also significantly reduced in the aorta of mice in the treatment group.

Claims

1. A polypeptide targeting the SLC22A6 protein, characterized in that, The polypeptide can specifically bind to the SLC22A6 protein and inhibit its mediated organic anion transport function.

2. The polypeptide according to claim 1, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

3. A monoclonal antibody or antigen-binding fragment against SLC22A6 protein, characterized in that, The monoclonal antibody or antigen-binding fragment can specifically bind to the SLC22A6 protein and inhibit its mediated organic anion transport function.

4. A pharmaceutical composition, characterized in that, It comprises at least one of the polypeptide of claim 1 or 2, the monoclonal antibody or antigen-binding fragment of claim 3, and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 4, characterized in that, It is formulated as a preparation for the prevention or treatment of atherosclerosis.

6. A reagent kit for diagnosing atherosclerosis, characterized in that, It contains reagents for detecting the expression level of SLC22A6 protein in biological samples.

7. The reagent kit according to claim 6, characterized in that, The reagents are antibodies for detecting SLC22A6 protein, primers or probes for detecting SLC22A6 mRNA, and the antibodies are the monoclonal antibodies or antigen-binding fragments as described in claim 3.

8. Application of compounds targeting SLC22A6 protein in the preparation of drugs for the prevention or treatment of atherosclerosis.

9. A method for screening candidate compounds for the prevention or treatment of atherosclerosis, characterized in that, Includes the following steps: (a) Under test conditions, the test compound is brought into contact with a system expressing or containing SLC22A6 protein; (b) The effect of the test compound on the expression level or transport activity of SLC22A6 protein in the system is detected. (c) Based on the detection results of step (b), compounds that can inhibit the expression or activity of SLC22A6 protein are screened as candidate compounds.

10. The method according to claim 9, characterized in that, In step (b), the effect of the test compound on the lactation level of histone H3 lysine at position 9 in the system is also detected, and in step (c), compounds that can reduce the lactation level are further screened.

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