Application of brd7 as a target in the prevention and treatment of autoimmune disease drugs

Abstract

The application discloses application of BRD7 as a target in prevention and treatment of an autoimmune disease drug, and the drug is a BRD7 small molecule inhibitor, and the BRD7 small molecule inhibitor is BI-7273. The autoimmune disease is multiple sclerosis. It is verified through experiments that the BRD7 small molecule inhibitor has a good treatment effect on multiple sclerosis as the same as gRNA knockout BRD7. It is proved that the BRD7 protein activity and gene expression can be inhibited to inhibit T cell activation, reduce CD4 + T cell percentage, increase CD8 + T cell percentage, and inhibit CD8 + T cell activation, thereby inhibiting the autoimmune system, and playing a treatment effect on the autoimmune disease such as multiple sclerosis. The application discloses a new role of the BRD7 gene in immune cells and the immune system, and designs and prepares a drug or other treatment means for treating multiple sclerosis by taking the BRD7 gene or protein as a target.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an application of a drug targeting the bromodomain structural protein BRD7 in preventing and treating autoimmune diseases. Background Art

[0002] The brd7 gene encodes the BRD7 protein, which contains a highly conserved bromodomain that recognizes histone H3 and regulates its acetylation, participating in gene transcriptional regulation. BRD7 has been shown to be a core component of the SWI / SNF chromatin remodeling complex and participates in tumorigenesis and progression by influencing the transcriptional activity and expression of downstream genes. BRD7 can bind to YB1 and reduce its protein stability, thereby inhibiting breast cancer cell proliferation, tumor growth, and metastasis in vivo. The immunoregulatory functions of this protein remain incompletely elucidated. Studies have reported that BRD7 knockout mice are prone to inflammatory phenotypes, including genital inflammation, abdominal abscesses, and splenomegaly. BRD7 ablation has also been shown to activate the NF-κB signaling pathway, leading to altered secretion of inflammatory factors such as IL-6, TNFα, iNOS, and CXCL1, thereby contributing to the early onset of inflammation and the transition from inflammatory to cancer. Furthermore, BRD7 is ubiquitously expressed in immune cells, with particularly high expression in CD8+ T cells, suggesting that BRD7 may be involved in the development and progression of immune diseases. Autoimmune diseases are diseases caused by the immune system's response to the body's own components. Currently, there are more than 100 autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, lupus erythematosus, etc., which have become the third major disease after cardiovascular disease and tumors. These diseases are often chronic and difficult to cure. There are no effective preventive and therapeutic drugs in clinical practice, which eventually lead to disability or death. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), continuous clonal expansion of CD4 + 、CD8 + and γ + T cells. Myelin peptide MOG 35-55 Can specifically induce the expansion of CD4 + T cells, but in contrast, surrogate peptides extracted from yeast peptide-MHC libraries induced clonally expanded CD8 + T cells, these CD8 + T cells inhibit MOG-specific CD4 + T cell proliferation to suppress the progression of immune diseases. Therefore, spontaneous CD4 + T cells induce regulatory CD8 + Therefore, to regulate CD8 + Drugs targeting T cells can effectively prevent and treat multiple sclerosis.

[0003] It should be noted that the above introduction to the technical background is merely intended to provide a clear and complete description of the technical solutions of this application and facilitate understanding by those skilled in the art. Simply because these solutions are described in the background technology section of this application, it should not be assumed that the above technical solutions are well known to those skilled in the art. Summary of the Invention

[0004] In view of the above-mentioned shortcomings, the present invention provides an application of a drug targeting the bromodomain protein BRD7 in the prevention and treatment of autoimmune diseases. The present application experimentally verifies that BRD7 small molecule inhibitors have a good therapeutic effect on multiple sclerosis, just like knocking out BRD7 with gRNA. The present invention proves that inhibiting BRD7 protein activity and gene expression can inhibit T cell activation and reduce CD4 + T cell percentage, increased CD8 + T cell percentage, while suppressing CD8 + This discovery, published in the journal Nature Communications, reveals a new role for the BRD7 gene in immune cells and the immune system, and explores the potential for designing drugs or other treatments for multiple sclerosis using the BRD7 gene or protein as a target.

[0005] In order to achieve the above object, the present invention provides a method for treating autoimmune diseases by targeting the bromodomain protein BRD7. The drug is a BRD7 small molecule inhibitor, BI-7273, with a molecular weight of 353.51, CAS number 1883429-21-7, and a molecular formula of C 20 H 23 N3O3, its structural formula is shown in Ⅰ:

[0006]

[0007] According to one aspect of the present invention, the autoimmune disease is multiple sclerosis.

[0008] According to one aspect of the present invention, the targeting of the bromodomain structural protein BRD7 is to use chemical drugs to reduce the protein activity of BRD7 or to silence BRD7.

[0009] Beneficial effects of the present invention:

[0010] BRD7 gene knockout in this application can inhibit T cell activation and reduce CD4 + T cell percentage, increased CD8 + T cell percentage, while suppressing CD8 +T cells are activated, thereby suppressing the autoimmune system.

[0011] The present invention experiments have shown that conditional knockout of T cell BRD7 and intervention with BRD7 inhibitors can significantly improve the neurological function scores of EAE (MS animal model) mice. Conditional knockout of T cell BRD7 can improve nerve demyelination damage in EAE model mice. The present invention also proves that by inhibiting the expression of T cell BRD7 gene, the number of CD4 T cells in the spleen of EAE mice is reduced. + T cell percentage, increased CD8 + T cell percentage; at the same time, the spleen T cells of conditional T cell knockout BRD7 mice were stimulated with PHA, CD8 + The percentage of T cell activation marker CD86 decreased, but CD4 + There was no significant difference in the expression of MHC-II, a marker of T cell activation, indicating that BRD7 regulates CD8 + T cell activation plays a role in suppressing autoimmune responses, thereby playing a role in treating autoimmune diseases. This has led to the development of new autoimmune disease treatments: BRD7 gene-specific siRNA and BRD7 small molecule inhibitors. This application targets BRD7 and designs drugs for the treatment and / or prevention of multiple sclerosis. This reveals a new role for BRD7 in immune cells and the immune system, and provides new therapeutic targets and effective new drugs for the prevention and treatment of multiple sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a process flow chart for constructing BRD7-CKO mice according to an embodiment of the present invention;

[0013] Figure 2 The EAE model was constructed from WT mice (N=20) of the present invention using C57BL / 6 adult female mice, and the EAE symptom scores were performed every other day;

[0014] Figure 3 A is the EAE symptom score every other day for the EAE model constructed from WT mice (N=20) and C57BL / 6 adult female mice with conditional T cell BRD7 knockout (CKO, N=10) according to the embodiment of the present invention; Figure 3 B is the EAE model constructed from WT mice (N=20) and C57BL / 6 adult female mice with conditional T cell BRD7 knockout (CKO, N=10) in Example 2 of the present invention. After EAE symptom scoring every other day, spinal cord and brain samples of mice were collected, and LFB staining of paraffin sections was performed;

[0015] Figure 4A is an EAE model established by WT mice (N=10) and C57BL / 6 adult female mice with conditional T cell BRD7 knockout (CKO, N=10) in the present invention. After the EAE model scoring was completed, spleen cells were isolated and CD3 + Distribution of T cells; Figure 4 B is Figure 4 A local area of ​​CD4 + Distribution of T cells; Figure 4 C represents WT mice (N=10) and C57BL / 6 adult female mice with conditional T cell BRD7 knockout (CKO, N=10) to establish EAE models. After the EAE model scoring was completed, spleen cells were isolated and CD3 + T cell percentage; Figure 4 D is the EAE model established by WT mice (N=10) and C57BL / 6 adult female mice with conditional T cell BRD7 knockout (CKO, N=10). After the EAE model scoring was completed, spleen cells were isolated and CD4 + T cell percentage; Figure 4 E represents the EAE model established by WT mice (N=10) and C57BL / 6 adult female mice with conditional T cell BRD7 knockout (CKO, N=10). After the EAE model scoring was completed, spleen cells were isolated and CD8 + T cell percentage;

[0016] Figure 5 A represents the EAE model established in WT mice (N=10) and C57BL / 6 adult female mice with conditional T cell BRD7 knockout (CKO, N=10) as described in the present invention. After EAE model scoring, spleen cells were isolated and analyzed by flow cytometry for expression of the CD3+ cell activation marker CD86. Figure 5 B shows the EAE model established in WT mice (N=10) and C57BL / 6 adult female mice with conditional T cell BRD7 knockout (CKO, N=10) as described in the present invention. After EAE model scoring, spleen cells were isolated and the expression of CD8+ T cell activation marker CD86 was analyzed by flow cytometry. Figure 5 C represents the EAE model established in WT mice (N=10) and C57BL / 6 adult female mice with conditional T cell BRD7 knockout (CKO, N=10) as described in the present invention. After EAE model scoring, spleen cells were isolated and the expression of CD86, a CD4+ T cell activation marker, was measured by flow cytometry. Figure 5D represents an EAE model established using WT mice (N=10) and adult female C57BL / 6 mice with conditional T cell BRD7 knockout (CKO, N=10) as described in the present invention. After EAE model scoring, spleen cells were isolated and the expression of MHC-II, a CD4+ T cell activation marker, was analyzed by flow cytometry. DETAILED DESCRIPTION

[0017] To make the present invention easier to understand, the present invention is further described below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below are consistent with the meanings understood by professional and technical personnel in this field; unless otherwise specified, the raw materials and reagents involved in this article can be purchased from the market or prepared by known methods.

[0018] It should be noted that "Brd7-201" means that the Brd7 gene has 7 transcripts, and the transcript used in this application is transcript 201.

[0019] It should be noted that the BRD7 gene knockout mice of the present application are based on C57BL / 6 mice with their BRD7 gene knocked out, referred to as BRD7-CKO mice; the C57BL / 6 mice of the present application are referred to as WT mice.

[0020] It should be noted that the BRD7 gene-specific siRNA of the present application is a gRNA, specifically including gRNA1 and gRNA2. The sequence number of gRNA1 is: 5'-AGTGTGACCGTGAGACAGCG-3', as shown in SEQ ID NO.6; the sequence number of gRNA2 is: 5'-ATGTGCAAGGGCCAACCGAC-3', as shown in SEQ ID NO.7.

[0021] Example 1

[0022] CRISPR / Cas9 technology was used to construct T cell knockout BRD7 mice (BRD7-CKO mice). The flow chart is as follows Figure 1 shown.

[0023] The Brd7 gene has seven transcripts. The sense strand of the Brd7 gene DNA sequence is shown in SEQ ID NO. 1. Based on the structure of the Brd7 gene, exons 3 to 4 of the Brd7-201 (ENSMUST00000034085.7) transcript are the knockout region. The sequence numbers for exon 3 are shown in SEQ ID NO. 2, and the sequence numbers for exon 4 are shown in SEQ ID NO. 3. This region contains 188 base pairs of coding sequence, and knocking out this region will result in protein disruption. We used CRISPR / Cas9 technology to edit the Brd7 gene. The brief procedure is as follows: CRISPR / Cas9 system gRNA was microinjected into fertilized eggs of C57BL / 6JGpt mice. The resulting F0 mice were positive and confirmed by PCR and sequencing. Positive F0 mice were mated with C57BL / 6JGpt mice to generate a stable F1 mouse model. Stable F1 mice (flox-carrying mice) were mated with mice expressing Cre recombinase (a universal tool mouse) to knock out exons 3-4 of the BRD7 gene, resulting in loss of BRD7 function in T cells. The gRNA was designed as follows: the target sequence was edited with the BRD7Brd7-201 (ENSMUST00000034085.7) transcript, which is the positive strand of the DNA sequence of the Brd7 gene as shown in SEQ ID NO.1. The loxp insertion sites are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively. Among them, the gRNA of the CRISPR / Cas9 system includes gRNA1 and gRNA2, which are used to cut exon 3 and exon 4, respectively. The 5'-3' sequence number of gRNA1 is: 5'-AGTGTGACCGTGAGACAGCG-3', as shown in SEQ ID NO.6; the 5'-3' sequence number of gRNA2 is: 5'-ATGTGCAAGGGCCAACCGAC-3', as shown in SEQ ID NO.7.

[0024] It should be noted that Loxp tags are inserted on both sides of exons 3-4. Cre recognizes the Loxp tags inserted on both sides and knocks out the exon 3-exon 4 region within the tags, thereby achieving the purpose of gene editing.

[0025] It should be noted that due to the limited equipment of the applicant, we provided the above-mentioned plan for knocking out exons 3-4 of the BRD7 gene to Jiangsu Jicui Yaokang Biotechnology Co., Ltd. for processing, and prepared mice with knockout of exons 3-4 of the BRD7 gene (BRD7-CKO mice) for the experiments of this application, which are specifically shown in Evidence 1.

[0026] It should be noted that the above-mentioned mice in this application are only used for laboratory purposes and not for commercial use, and the gRNA of their CRISPR / Cas9 system has not been disclosed before this application.

[0027] It should be noted that the synthesis of gRNA in the CRISPR / Cas9 system of the present application is base synthesis, which is a common method for sequence synthesis and will not be described in detail here.

[0028] Example 2

[0029] EAE model was established using WT C57BL / 6 mice (N=20) or BRD7 knockout (BRD7-CKO) adult female mice of Example 1. The specific EAE modeling method was as follows: MOG 35-55 Dissolved in sterile PBS and mixed with complete Freund's adjuvant containing Mycobacterium tuberculosis to prepare an emulsion. The emulsion was injected subcutaneously in the neck and left and right hind limbs of the mouse, and pertussis toxin was injected into the tail vein at the same time. Pertussis toxin was injected into the tail vein again on the second day of modeling. The EAE simulated clinical multiple sclerosis symptom scoring criteria are: 0 points, no symptoms; 1 point, tail weakness; 2 points, tail weakness and partial limb weakness; 3 points, paralysis of one hind limb; 4 points; paralysis of both hind limbs; 5 points, dying or death. From the day of modeling (recorded as Day 0), they were randomly divided into two groups, 10 in each group, marked as control group and BI-727 group. Each mouse in the BI-727 group was given 180 mg / kg BI-7273 inhibitor orally every day, and each mouse in the control group was given the same volume of drinking water orally. The neurological function of EAE model mice was scored using a double-blind method every other day, and the scoring results are as follows. Figure 2 and Figure 3 As shown in A. Figure 2 and Figure 3 As shown in Figure A, the clinical neurological function scores of CKO mice were significantly lower than those of WT mice. The clinical neurological function scores of BI-727-treated mice were also significantly lower than those of WT mice that received the same volume of drinking water orally. This indicates that the clinical neurological function scores of BI-727-treated and CKO mice follow a similar pattern.

[0030] Example 3

[0031] EAE models were established using WT mice (N=10) and C57BL / 6 adult female mice with conditional T cell BRD7 knockout (CKO, N=10). The specific EAE modeling method and scoring criteria were the same as in Example 1. Starting from the day of modeling (recorded as Day 0), EAE symptoms were scored every other day. At the end of the disease, spinal cord and brain samples of the mice were collected and paraffin sections were stained with LFB. Figure 2-3It can be seen that compared with WT mice, the clinical neurological function scores of CKO-EAE mice were significantly reduced, and LFB staining showed that the demyelination lesions in the spinal cord and corpus callosum were significantly improved.

[0032] Example 4

[0033] After the EAE model was scored, spleen cells from the control group (N=3) and BRD7 knockout mice (N=4) were isolated and flow cytometry was used to detect the effect of T cell differentiation. T cells from the two groups were sorted by magnetic beads and stained with CFSE. PHA was added to stimulate the cells in vitro for 7 days, and T cell proliferation was detected by flow cytometry. Figure 4 As shown by Figure 4 It can be seen that compared with the spleen cells of WT-EAE model mice, the spleen CD3 + There was no difference in T cell percentage, CD4 + T cells decreased, CD8 + T cell upregulation.

[0034] Example 5

[0035] After the EAE model was scored, spleen cells from control mice (N=3) and BRD7 knockout mice (N=4) were isolated and T cells were sorted by magnetic beads. The cells were plated at 4M / mL, 500μL / well (24-well plate), and stimulated with PHA for 1 day in vitro. T cell activation (CD86 / MHC-II) was detected by flow cytometry. Figure 5 As shown by Figure 5 It can be seen that after 1 day of in vitro stimulation, more than 95% of T cells are viable cells; compared with WT mouse spleen T cells, the percentage of CD86 in CKO mouse spleen T cells decreased after PHA stimulation, indicating that the ability of T cell activation decreased, but there was no significant difference in MHC-II. These experimental results indicate that BRD7 activates T cells through CD8 + T cells play an immune regulatory role.

[0036] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. The use of bromodomain protein BRD7 as a target in the preparation of a drug for preventing and treating multiple sclerosis, characterized in that: The drug is a BRD7 small molecule inhibitor, and the BRD7 small molecule inhibitor BI-7273 has a structural formula as shown in I:

2. The use of the bromodomain protein BRD7 as a target in the preparation of a drug for the prevention and treatment of multiple sclerosis according to claim 1, characterized in that: The autoimmune disease is multiple sclerosis.

3. The use of the bromodomain protein BRD7 as a target in the preparation of a drug for the prevention and treatment of multiple sclerosis according to claim 1, characterized in that: The method of targeting the bromodomain structural protein BRD7 is to use chemical drugs to reduce the protein activity of BRD7 or silence BRD7.

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