Application of BAG2 serving as drug target and inhibitor of BAG2 in preparation of drug for treating keloid

Abstract

The invention discloses application of BAG2 as a drug target and an inhibitor thereof in preparation of a drug for treating keloid. The protein BAG2 specifically expressed by keloid fibroblasts is found through analysis of single cell data of keloid and normal skin. Through in-vitro experiments, BAG2 expression is inhibited, keloid fibroblast proliferative activity and collagen expression are significantly inhibited, and cell infiltration is also significantly weakened. A small molecule drug for targeted inhibition of BAG2 is further obtained through small molecule drug library screening and in-vitro experiments, so that the purpose of accurate and efficient treatment of keloids is achieved.

Description

Technical Field

[0001] The present invention relates to a new target BAG2 of keloid and an inhibitory drug thereof. Background Art

[0002] Scar formation is an effective repair system for wounds, but once a scar is formed, it cannot be completely removed. Scar tissue replaces the original normal tissue structure, affecting the appearance and even the normal function of the limbs. In particular, keloids that exceed the original scar boundary and are prone to recurrence cause pain, itching, dysfunction and significant aesthetic problems, which seriously affect the patient's daily life. Existing treatments for keloids usually include surgery, laser therapy, local drug therapy and other physical therapy methods. Among them, the first-line treatment options are drug injection therapy and scar excision combined with radiotherapy. These options can achieve certain therapeutic effects, but their side effects such as local pain, capillary dilation, and atrophy of the surrounding normal skin are unavoidable, and keloid lesions are prone to recurrence after drug withdrawal or irregular radiotherapy after surgery. At present, there is a lack of targeted treatment methods for the pathogenic genes and protein expressions of keloids. Since keloids have different gene and protein expression characteristics from the surrounding normal skin, the development of targeted drugs for keloids is expected to provide new precision treatment possibilities for them. Targeted therapy mainly intervenes in specific molecular targets or signaling pathways to achieve the purpose of alleviating lesions, reducing the volume of keloids and improving their appearance.

[0003] In the past, the development of targeted therapy for keloids was mainly centered around the TGF-β-Smad signaling pathway. TGF-β is expressed at higher levels in scar tissue than in normal skin and can promote collagen accumulation in scar fibroblasts. However, TGF-β is a multifunctional cytokine whose role in fibrotic diseases is complex and sometimes contradictory, showing inhibitory or promoting effects depending on the stage of the disease. However, due to its promotion of cell cycle arrest and apoptosis in healthy cells, TGF-β targeted therapy has a certain potential for systemic cytotoxicity, and its development has lagged behind. Further research is needed to explore targets specific to keloids.

[0004] Bcl-2-associated athanogene 2 (BAG2) is a member of the BAG gene family, which is characterized by containing a novel heat shock protein 70 nucleotide exchange factor domain, namely the "BNB" domain, which can mediate cell apoptosis, tumor growth, neuronal differentiation and stress response through various mechanisms. BAG2 plays an important role in the occurrence and development of different malignant tumors by interacting with related signaling molecules in the tumor microenvironment. Summary of the invention

[0005] The inventors found in their research that BAG2 is specifically and highly expressed in keloids, and inhibiting its gene expression can inhibit scar fibroblast proliferation and collagen secretion. Therefore, the present invention provides a new target for targeted treatment of keloids, namely, BAG2. That is, the use of BAG2 as a drug target in the preparation of a drug for the treatment of keloids.

[0006] In one aspect, the present invention provides use of a BAG2 inhibitor in the preparation of a medicament for treating keloid.

[0007] Furthermore, the BAG2 inhibitors include substances that downregulate or inhibit BAG2 gene expression, and BAG2 protein antagonists.

[0008] Preferably, the BAG2 protein antagonist is a small molecule compound.

[0009] Further preferably, the BAG2 inhibitor is selected from saikosaponin B1, DL-α-tocopherol, bazedoxifene acetate, siponimod, and bonecimod.

[0010] More preferably, the BAG2 inhibitor is selected from saikosaponin B1, bazedoxifene acetate, siponimod, and bonecimod.

[0011] In another aspect, the present invention provides a drug for treating keloid, comprising a BAG2 inhibitor.

[0012] In a preferred embodiment, the drug includes a substance that downregulates or inhibits the expression of the BAG2 gene.

[0013] In another preferred embodiment, the drug comprises a BAG2 protein antagonist.

[0014] Preferably, the BAG2 protein antagonist is a small molecule compound, including but not limited to one or more of saikosaponin B1, DL-α-tocopherol, bazedoxifene acetate, siponimod, and bonecimod.

[0015] More preferably, the BAG2 inhibitor is selected from one or more of saikosaponin B1, bazedoxifene acetate, siponimod, and bonecimod.

[0016] Furthermore, the drug also includes a pharmaceutically acceptable carrier or excipient.

[0017] Our research group analyzed single-cell data of keloids and normal skin and discovered BAG2, a protein specifically expressed by keloid fibroblasts. We inhibited BAG2 expression in vitro, and the proliferation activity and collagen expression of keloid fibroblasts were significantly inhibited, and their cell infiltration was also significantly weakened. We also further obtained small molecule drugs that target and inhibit BAG2 through small molecule drug library screening (SPR) and in vitro experiments, thereby achieving the goal of precise and efficient treatment of keloids.

[0018] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 are photos of BAG2 immunohistochemical staining of keloid (A) and normal skin (B), scale bar = 50 μm;

[0020] Figure 2 are the Western Blot results of BAG2 protein in keloid (KS) and normal skin (NS);

[0021] Figure 3 This is the proliferation curve of keloid fibroblasts detected by CCK8 after BAG2 interference;

[0022] Figure 4 This is the result of Western Blot detection of COL1, COL3 and α-SMA expression after BAG2 expression in scar fibroblasts was interfered;

[0023] Figure 5 This is the test result of the second round of SPR screening. The detection concentration is 100μM, and the red dot is Yangshen (BAG2rabbit mAb). DETAILED DESCRIPTION

[0024] Example 1 BAG2 is highly expressed in keloid cells

[0025] To explore the gene expression of BAG2 in different cell populations, we downloaded the single-cell dataset GSE181297 of keloid patients from the Gene Expression Omnibus (GEO). This dataset was generated by the Illumina NovaSeq 6000 platform and includes keloid and normal skin samples from humans. Data processing includes standardization, filtering low-quality cells, removing batch effects, selecting highly differential genes to reduce data dimensions, and analyzing the expression of BAG2 in different cell clusters. The results of single-cell data mining: It was found that the BAG2 gene was highly expressed in keloid fibroblasts, which is a gene expression feature unique to keloids. The gene is expressed at a low level in normal skin.

[0026] In clinical samples, BAG2 is also highly expressed in keloids in the form of protein, such as Figure 1 and 2 shown. Figure 1 A is a photo of BAG2 immunohistochemical staining of keloid, showing that most scar fibroblasts are positive, and B is a photo of BAG2 immunohistochemical staining of normal skin, showing that BAG2 expression is weak. Figure 2 Shown are Western Blot results, showing that BAG2 protein is highly expressed in keloid (KS) compared with normal skin (NS).

[0027] Example 2 In vitro experiments to inhibit BAG2 expression

[0028] BAG2 interference si-RNA was constructed to knock down BAG2 expression in keloid fibroblasts, and cell proliferation (CCK-8) was detected in comparison with the si-NC negative control and the untreated control, and the expression of keloid-related collagen deposition (COL1, COL3, α-SMA) was detected by Western Blot. Figure 3 As shown in Figure 2, after the expression of BAG2 was inhibited in keloid fibroblasts (green curve), the expression of keloid fibroblasts in the siBAG2 group was significantly inhibited from the third day. Figure 4 As shown, after inhibiting the expression of BAG2 in scar fibroblasts, the expressions of COL1, COL3 and α-SMA were reduced.

[0029] Example 3 Screening out compounds with strong affinity for BAG2 protein

[0030] In this example, a surface plasmon resonance (SPR) biosensor was used to detect compounds with high affinity to the BAG2 protein.

[0031] (1) Group SPR single concentration screening (first round of screening):

[0032] Protein immobilization was performed using the amino coupling method.

[0033] Protein coupling buffer solution: 1.0×PBS-P+ (pH 7.4).

[0034] Interaction buffer solution: 1.0×PBS-P+ (pH 7.4), 5% (v / v) DMSO.

[0035] The 50 compounds in each group were divided into 54 groups (the last group was 33 compounds), and the final concentration of this step was 10 μM.

[0036] (2) SPR single concentration screening (second round of screening):

[0037] Protein immobilization was performed using the amino coupling method.

[0038] Protein coupling buffer solution: 1.0×PBS-P+ (pH 7.4).

[0039] Interaction buffer solution: 1.0×PBS-P+ (pH 7.4), 5% (v / v) DMSO.

[0040] The signal values ​​of 54 groups of compounds at 10 μM were ranked, and the TOP2 (a total of 100 compounds) were selected for single-point screening one by one. The final detection concentration in this step was 100 μM.

[0041] The results of the second round of screening are as follows Figure 5 As shown, the detection concentration is 100 μM, and the compounds with signal value RU>20 are selected as high affinity compounds. Among them, the red dot is Yangshen (BAG2 rabbit mAb).

[0042] Through two rounds of SPR, six small molecules with high affinity to BAG2 were screened from the FDA and fibrosis small molecule libraries, namely Saikosaponin B1, DL-α-Tocopherol, Geraniin, Bazedoxifene acetate, Siponimod, and Ponesimod.

[0043] Example 4 Small molecule in vitro validation

[0044] The six small molecule compounds obtained in Example 3 were applied to keloid fibroblasts, and their inhibition rates on cells were detected using CCK-8. The results are shown in the following table.

[0045] Table 1 Cell inhibition rate of high affinity compounds

[0046]

[0047] n = 6, " / " indicates that the optical density (OD) of drug-treated cells exceeds the OD of the control, and the drug increases the cell survival rate. Cell inhibition rate = [1-(OD 药物 –OD 培养基 ) / (OD 对照 –OD 培养基 )]×100%.

[0048] The results of the experiment showed that the compounds saikosaponin B1, bazedoxifene acetate, siponimod and bonecimod had the highest inhibitory efficiency.

[0049] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. The use of BAG2 as a drug target in the preparation of drugs for the treatment of keloids.

2. Use of BAG2 inhibitors in the preparation of drugs for the treatment of keloids.

3. Use of the BAG2 inhibitor according to claim 2 in the preparation of a keloid treatment drug, characterized in that: The BAG2 inhibitors include substances that downregulate or inhibit BAG2 gene expression, and BAG2 protein antagonists.

4. Use of the BAG2 inhibitor according to claim 2 in the preparation of a keloid treatment drug, characterized in that: The BAG2 inhibitor is selected from saikosaponin B1, DL-α-tocopherol, bazedoxifene acetate, siponimod, and bonecimod.

5. Use of the BAG2 inhibitor according to claim 2 in preparing a drug for treating keloid, characterized in that: The BAG2 inhibitor is selected from saikosaponin B1, bazedoxifene acetate, siponimod, and bonecimod.

6. A drug for treating keloid, characterized in that: Including BAG2 inhibitors.

7. The drug for treating keloid according to claim 6, characterized in that: The drugs include substances that downregulate or inhibit the expression of BAG2 gene.

8. The drug for treating keloid according to claim 6, characterized in that: The drugs include BAG2 protein antagonists.

9. The drug for treating keloid according to claim 6, characterized in that: The drug comprises one or more of saikosaponin B1, DL-α-tocopherol, bazedoxifene acetate, siponimod, and bonecimod.

10. The drug for treating keloid according to claim 6, characterized in that: The medicament further includes a pharmaceutically acceptable carrier or excipient.

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