Use of jib-04 in the preparation of a drug for treating pulmonary alveolar proteinosis

Abstract

The application of JIB-04 in the preparation of a drug for treating pulmonary alveolar proteinosis relates to the technical field of biological medicine. Based on the epigenetic mechanism that menin in macrophages promotes the transcription and expression of GM-CSF through H3K36m3, and maintains the activation of alveolar macrophages and the homeostasis of alveolar surfactant, it is proved that the clinical application prospect of the small molecule inhibitor JIB-04 targets to improve the H3K36m3 modification in macrophages and the expression of GM-CSF for treating PAP. JIB-04 can effectively restore the chromatin H3K36m3 modification level and GM-CSF transcription caused by Men1 deletion, and restore the differentiation and function of damaged macrophages. Important theoretical and data support is provided for the application of JIB-04 in the targeted treatment of clinical PAP.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, and particularly relates to application of JIB-04 in preparation of a drug for treating pulmonary alveolar proteinosis. BACKGROUND

[0002] Pulmonary surfactant is a liquid film covering the alveolar wall, and its main functions are to reduce the surface tension of the alveolar during the lung inspiration process, prevent the alveolar from collapsing during the lung expiration process, and maintain the normal structure and function of the alveolar; the main chemical components are phospholipids (80%), cholesterol (10%) and surfactant proteins (10%) secreted from alveolar type 2 epithelial cells (AT2). The steady state of the pulmonary surfactant mainly depends on the balance between the synthesis, secretion, recycling, catabolism of the AT2 cell surfactant and the phagocytic clearance of the surfactant by the alveolar macrophages. GM-CSF (CSF2) is a glycoprotein cytokine, which binds to the receptors CSF2RA and CSF2RB on the surface of the macrophages, activates tyrosine protein kinase JAK2, and then activates STAT5, PU.1 and PPARγ signaling pathways, and is crucial for the normal biological functions of the macrophages such as maturation and differentiation and phagocytic function. The production of the GM-CSF antibody or the inactivating mutation of the GM-CSF receptor CSF2RA / CSF2RB will cause the phagocytic clearance dysfunction of the alveolar macrophages to the pulmonary surfactant, lead to the steady state disorder of the pulmonary surfactant, and cause the deposition of a large amount of metabolic products such as phospholipids, cholesterol and surfactant proteins in the alveolar cavity, thereby causing pulmonary alveolar proteinosis (PAP).

[0003] Pulmonary alveolar proteinosis (PAP) is a rare interstitial lung disease (ILDs) with the main clinical features of progressive dyspnea, hypoxemic respiratory failure, secondary infection, and so on, caused by the accumulation of surfactant in the alveolar space. At present, PAP is divided into three types according to the pathogenesis, clinical manifestations, diagnosis and treatment methods: 1. Primary; 2. Secondary; 3. Congenital, among which primary PAP is divided into autoimmune and hereditary. The two mechanisms of PAP are the increase of surfactant production and the dysfunction of alveolar macrophages leading to clearance disorder. Cytokine GM-CSF and its activated alveolar macrophages play an important role in the stability of alveolar surfactant, the stability of alveoli and the normal function of lungs (Pulmonary alveolar proteinosis. Nat Rev Dis Primers, 2019 Mar 7; 5(1): 16).

[0004] The clinical treatment goal of PAP is to slow down symptoms, improve oxygenation function and patient quality of life. Whole lung lavage is currently a common treatment for PAP, but there is no international standard for guiding operation, and repeated treatment is needed. During the lavage process, general anesthesia is required, causing discomfort and heavy economic burden for patients, and is a treatment that treats the symptoms but not the root cause (Long-term durable benefit after whole lung lavage in pulmonary alveolar proteinosis. The European respiratory journal, 2004, 23:526-531). The efficacy of GM-CSF replacement therapy is remarkable, with an overall response rate of 43%, and the main administration routes are inhalation and subcutaneous injection (Inhaled GM-CSF for Pulmonary Alveolar Proteinosis. N Engl J Med, 2019 Sep 5; 381(10): 923-932). The main treatment targeting GM-CSF antibodies is plasma exchange and rituximab (Rituximab), and the clinical trial of this strategy shows limited treatment effect and complex administration method (Rituximab therapy in pulmonary alveolar proteinosis improves alveolar macrophage lipid homeostasis. Respiratory research, 2012 Jun 14; 13(1): 46). PPARγ agonists targeting the CSF2-PU.1-PPARγ-ABCG1 pathway of alveolar macrophages are still in the preclinical research stage. At present, there is a lack of PAP treatment targeting small molecule intervention drugs based on the pathogenesis in clinical.

[0005] JIB-04 (CAS No. 199596-05-9, a small molecule inhibitor of Jumonji histone demethylase) can inhibit enzymatic activity in vitro biochemical level, tumor cells and tumor-bearing mice models, inhibit the proliferation of non-small cell lung cancer, breast cancer cells in vitro and in vivo, ultimately reduce the tumor burden in mice subcutaneously, prolong the survival of tumor-bearing mice. mRNA (messenger RNA) high-throughput sequencing (RNA-seq) found that JIB-04 regulated target gene expression more significantly in tumor tissue than in patient-derived normal tissue, that is, JIB-04 has strong tumor targeting, showing its application prospect in the field of human tumor treatment (A small molecule modulates Jumonji histone demethylase activity and selectively inhibits cancer growth. Nat Commun, 2013; 4: 2035). SUMMARY

[0006] The application aims to provide the application of JIB-04 in the preparation of a drug for treating pulmonary alveolar proteinosis.

[0007] JIB-04 treatment of wild type (Men1f / f) and Men1 knockout (Men1ΔM / ΔM) bone marrow-derived macrophages / BMDM and alveolar macrophages / AM, by RT-PCR and Western Blotting method proved that JIB-04 can target to restore the down-regulation of H3K36m3 modification level caused by Men1 deletion, and significantly up-regulate the expression of GM-CSF. Flow cytometry and cholesterol content detection proved that small molecule inhibitor JIB-04 can significantly restore the differentiation and cholesterol clearance disorder of macrophages caused by Men1 gene deletion.

[0008] Therefore, JIB-04 can be applied in the preparation of a drug for treating pulmonary alveolar proteinosis. JIB-04 can effectively restore the chromatin H3K36m3 modification level and GM-CSF transcription caused by Men1 deletion, and restore the differentiation and function of damaged macrophages. JIB-04 shows its good application prospect in macrophage differentiation and dysfunction related diseases such as PAP, and the method for treating PAP has the advantages of clear molecular mechanism, strong specificity and simple and efficient.

[0009] The application is based on the epigenetic mechanism that menin in macrophages promotes GM-CSF transcription and expression through H3K36m3, and maintains the activation of alveolar macrophages and the homeostasis of alveolar surfactant, and proves that small molecule inhibitor JIB-04 targets to improve H3K36m3 modification and GM-CSF expression in macrophages to treat PAP.

[0010] Compared with the prior art, the present application has the advantages of:

[0011] Cytokine GM-CSF expression defects will induce alveolar macrophage phagocytic clearance dysfunction of pulmonary surfactant, resulting in a large amount of pulmonary surfactant deposition in the alveolar cavity, and the occurrence of pulmonary alveolar proteinosis / PAP. The research basis of the present application is that we found that menin-regulated H3K36m3 histone modification is a key epigenetic mechanism for regulating GM-CSF transcription and maintaining the differentiation of lung macrophages, cholesterol clearance function and pulmonary surfactant homeostasis. On this basis, the present application proves that small molecule inhibitor JIB-04 can target to restore H3K36m3 modification of macrophages and GM-CSF expression caused by Men1 deletion, differentiation and cholesterol clearance dysfunction, and show good effect in treating PAP. The present application provides important theoretical and data support for the application of JIB-04 in the targeted treatment of clinical PAP. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 Men1 deletion induces pulmonary alveolar proteinosis. Among them, A. 10-month-old wild type (Men1 f / f ) and Men1 knockout (Men1 △M / △M ) mice were detected by lung Micro-CT, axial, coronal and sagittal sections; B. 10-month-old Men1 f / f and Men1 △M / △M mice were detected by lung tissue gross phenotype and pathology, hematoxylin-eosin (HE), periodic acid-schiff (PAS), alveolar surfactant protein A / B / C (SP-A / B / C); C. 2, 4, 12 months Men1 f / f and Men1 △M / △M mice were collected by bronchoalveolar lavage fluid (BALF), and the gross phenotype and protein concentration of bronchoalveolar lavage fluid were determined.

[0013] Figure 2 Men1 deletion leads to macrophage cholesterol clearance dysfunction. Among them, A. Men1 f / f and Men1 △M / △M bone marrow-derived macrophages / BMDM cholesterol (Cholesterol) content detection; B. Men1 f / f and Men1 △M / △M alveolar macrophages / AM cholesterol content detection.

[0014] Figure 3 Men1 deletion inhibits the maturation and differentiation of macrophages by down-regulating GM-CSF transcription. Among them, A. Men1 f / fand Men1 △M / △M Transcriptome high-throughput sequencing / RNA-Seq of peritoneal macrophages (PM) to screen Men1 downstream target genes; B. RT-PCR detection of Men1 f / f and Men1 △M / △M Expression of GM-CSF in alveolar macrophages / AM; C. RT-PCR detection of Men1 f / f and Men1 △M / △M Expression of GM-CSF in bone marrow-derived macrophages / BMDM; D. Men1 f / f and Men1 △M / △M BMDM were induced to differentiate into macrophages by M-GSF and GM-CSF respectively, and the number of mature differentiated macrophages (CD11b + ) was detected by flow cytometry.

[0015] Figure 4 Men1 promotes the transcriptional expression of GM-CSF through histone H3K36m3 modification. Among them, A. Chromatin immunoprecipitation ChIP primer position diagram of GM-CSF gene promoter region; B. ChIP detection of Men1 f / f and Men1 △M / △M Binding of menin in the promoter region of GM-CSF gene in BMDM; C. ChIP detection of Men1 f / f and Men1 △M / △M H3K36m3 modification level in the promoter region of GM-CSF gene in BMDM; D. ChIP detection of Men1 f / f and Men1 △M / △M Binding of SETD2 in the promoter region of GM-CSF gene in BMDM.

[0016] Figure 5 SETD2 promotes the transcriptional expression of GM-CSF through H3K36m3. Among them, A. RNA interference (RNAi) of Setd2 gene expression in BMDM, Western Blotting detection of SETD2 interference effect, H3K36m3, GM-CSF, menin and histone H3 expression; B. AM treated with different concentrations of SETD2 inhibitor / SNF, RT-PCR detection of GM-CSF expression; C. BMDM treated with different concentrations of SETD2 inhibitor / SNF, RT-PCR detection of GM-CSF expression.

[0017] Figure 6 H3K36m3 agonist JIB-04 restores the transcriptional expression of GM-CSF. Among them, A. Men1f / f and Men1 △M / △M A. RT-PCR detection of GM-CSF expression; B. Men1 cells treated with different concentrations of JIB-04 f / f and Men1 △M / △M A. RT-PCR detection of GM-CSF expression; B. Men1 cells treated with different concentrations of JIB-04

[0018] Figure 7 H3K36m3 agonist JIB-04 restores H3K36m3 modification and GM-CSF protein expression. Men1 cells treated with different concentrations of JIB-04 f / f and Men1 △M / △M BMDM, Western blot detection of H3K36m3, GM-CSF, menin and β-actin expression.

[0019] Figure 8 H3K36m3 agonist JIB-04 restores macrophage maturation and cholesterol clearance ability. Wherein, A. Men1 cells treated with JIB-04 f / f and Men1 △M / △M BMDM, flow cytometry detection of the number of mature macrophages (CD11b + ) ; B. Men1 cells treated with JIB-04 f / f and Men1 △M / △M BMDM, detection of cholesterol content. DETAILED DESCRIPTION

[0020] The following examples will further illustrate the present application with reference to the accompanying drawings.

[0021] Myeloid cell Men1 gene deletion leads to reduced protein stability of histone methyltransferase SETD2 (the only reported H3K36m3 modification enzyme), inhibits its chromatin-directed recruitment, resulting in reduced H3K36m3 modification of chromatin at specific sites, inhibits GM-CSF transcription, and ultimately causes macrophage development and dysfunction, leading to severe spontaneous PAP in mice. H3K36m3 agonist JIB-04 can effectively restore chromatin H3K36m3 modification levels and GM-CSF transcription caused by Men1 deletion, and restore the differentiation and function of damaged macrophages. JIB-04 shows good application prospects in macrophage differentiation and dysfunction related diseases such as PAP, and the method has the advantages of clear molecular mechanism, strong specificity and simple and efficient in treating PAP.

[0022] Preliminary work for this invention: (1) Using mouse imaging methods such as Micro-CT (Computed Tomography), histopathological staining, and bronchoalveolar lavage fluid monitoring, it was demonstrated that Men1 gene knockout in myeloid cells can induce spontaneous pulmonary alveolar proteinosis / PAP in mice. Figure 1 (2) Bone marrow-derived macrophages (BMDM) and alveolar macrophages (AM) were isolated. Analysis of cholesterol content revealed that Men1 gene deletion reduced the cholesterol clearance capacity of macrophages, leading to intracellular cholesterol deposition. Figure 2 (3) High-throughput mRNA (messenger RNA) sequencing (RNA-seq) screened the key downstream target gene GM-CSF of menin in macrophages, and by reverse transcription-polymerase chain reaction (RT-PCR), GM-CSF rescue experiments and flow cytometry, it was demonstrated that the Men1 gene regulates the maturation and differentiation of macrophages through the autocrine function of GM-CSF. Figure 3 The experiments, including chromatin immunoprecipitation (ChIP), SETD2 RNA interference (RNAi), Western blotting, and RT-PCR, confirmed that menin regulates the transcriptional expression of the GM-CSF gene through H3K36m3 modified by the histone methyltransferase SETD2. Figure 4 and 5 );

[0023] This invention utilizes JIB-04, a broad-spectrum small molecule inhibitor of Jumonji histone demethylase reported in the literature, to treat wild-type (Men1) f / f Men1 knockout (Men1) ΔM / ΔM Bone marrow-derived macrophages / BMDM and alveolar macrophages / AM were used to demonstrate, using RT-PCR and Western blotting, that JIB-04 can specifically restore the downregulation of H3K36m3 modification levels caused by Men1 deficiency and significantly upregulate GM-CSF expression. Figure 6 and 7 Flow cytometry and cholesterol level assays demonstrated that the small molecule inhibitor JIB-04 significantly restored macrophage differentiation and cholesterol clearance impairment caused by Men1 gene deletion, exhibiting a good therapeutic effect on pulmonary alveolar proteinosis. Figure 8).

[0024] 1. Men1 deletion induces pulmonary alveolar proteinosis

[0025] 10-month-old wild type (Men1 f / f ) and myeloid cell Men1 knockout (Men1 △M / △M ) mice were examined by Micro-CT. The Men1 knockout group showed abnormal imaging features, such as ground glass opacity, which is similar to human pulmonary alveolar proteinosis / PAP (Fig. A in Figure 1 ). The mice were sacrificed and the samples were collected. The lung of the control group was normal, and 18 / 31 (58%) of the Men1 knockout group showed obvious abnormalities, which were visible lung consolidation (Fig. B in Figure 1 ). Pathological examination of the lung showed that the abnormal lung tissue presented a large range of diffuse parenchymal lesions, and a large amount of Periodic acid-Schiff / PAS positive surfactant accumulated in the alveolar cavity; Immunohistochemistry (IHC) results showed that a large amount of surfactant protein A / B / C (SP-A / B / C) was deposited in the abnormal lung tissue area (Fig. B in Figure 1 ). The BALF of the mice was collected at 2, 4, and 12 months, respectively, for macroscopic observation and protein concentration determination. The results showed that the protein content of BALF increased significantly in Men1 knockout mice, and the BALF of 12-month-old (12M) mice showed the characteristic milk white color of clinical pulmonary alveolar proteinosis / PAP (Fig. C in Figure 1 ). Based on the above experimental evidence, two independent pathologists diagnosed the lung abnormalities of Men1 knockout mice as pulmonary alveolar proteinosis / PAP.

[0026] 2. Men1 deletion leads to macrophage cholesterol clearance disorder

[0027] Primary wild type Men1 f / f and Men1 △M / △M bone marrow-derived macrophages / BMDMs (matured and differentiated by M-CSF) and alveolar macrophages / AMs were used for cholesterol content detection. The results showed that Men1 knockout significantly up-regulated the total cholesterol, free cholesterol, and esterified cholesterol content in macrophages, suggesting that Men1 knockout led to the steady-state disorder of pulmonary surfactant, inducing pulmonary alveolar proteinosis / PAP in mice (Fig. A and B in Figure 2 ).

[0028] 3. Men1 deletion inhibits the maturation and differentiation of macrophages by down-regulating GM-CSF transcription and expression

[0029] Wild type Men1f / f and Men1 △M / △M Mouse peritoneal macrophages (PM) were subjected to transcriptome high-throughput sequencing / RNA-Seq to screen the downstream target genes of Men1 in macrophages. The screening results showed that Men1 knockout significantly reduced the transcription of the cytokine GM-CSF (GM-CSF gene) (Fig. A in the Figure 3 RT-PCR results of BMDM (M-CSF-induced mature differentiation) and AM further verified the positive transcriptional regulation of Men1 on GM-CSF (Fig. B and C in the Figure 3 BMDM was induced to mature differentiation by M-CSF or GM-CSF, and flow cytometry analysis found that Men1 knockout significantly inhibited the number of M-CSF-induced mature macrophages (CD11b + ); exogenous GM-CSF could restore the BMDM maturation differentiation disorder caused by Men1 knockout, suggesting that Men1 knockout inhibited the maturation differentiation of macrophages by reducing the autocrine GM-CSF of macrophages (Fig. D in the Figure 3

[0030] 4. Men1 promotes the transcriptional expression of GM-CSF through histone H3K36m3 modification

[0031] Chromatin immunoprecipitation / ChIP primers were designed in the promoter region of the GM-CSF gene (Fig. A in the Figure 4 f / f and Men1 △M / △M ChIP detection of BMDM cells found that menin was significantly bound in the promoter region of the GM-CSF gene, Men1 knockout significantly reduced the binding of menin and the modification level of H3K36m3 in the promoter region of the GM-CSF gene, and Men1 knockout also significantly weakened the binding of histone methyltransferase SETD2 (the only modification enzyme of H3K36m3) (Fig. B-D in the Figure 4

[0032] 5. SETD2 promotes the transcriptional expression of GM-CSF through H3K36m3

[0033] SETD2 SiRNA (si-Setd2) was transfected into BMDM, and the results of Western blotting detection showed that SETD2 interference significantly reduced the modification level of H3K36m3 and the expression level of GM-CSF protein (Fig. A in the Figure 5 ; and the inhibitor of SETD2 methyltransferase—SNF (CAS No. 58944-73-3) inhibited the transcriptional expression of GM-CSF in alveolar macrophages / AM and BMDM in a concentration gradient-dependent manner​​​Figure 5 (See Figures B and C). The above results suggest that SETD2 promotes the transcriptional expression of GM-CSF through histone H3K36m3 modification.

[0034] 6. The H3K36m3 agonist JIB-04 restores GM-CSF transcriptional expression.

[0035] JIB-04 (CAS No. 199596-05-9) Treatment for Men1 f / f and Men1 △M / △M AM and BMDM (M-CSF-induced maturation and differentiation), RT-PCR results showed that JIB-04 dose-dependently upregulated the transcriptional expression of GM-CSF in wild-type cells and significantly restored the transcriptional expression of GM-CSF in Men1 knockout cells. Figure 6 (Figures A and B in the text).

[0036] 7. The H3K36m3 agonist JIB-04 restores H3K36m3 modification and GM-CSF protein expression.

[0037] JIB-04 Processing Men1 f / f and Men1 △M / △M BMDM (M-CSF-induced maturation and differentiation), protein imprinting results showed that JIB-04 dose-dependently upregulated H3K36m3 modification levels and GM-CSF protein expression, completely restoring the decrease in H3K36m3 modification and GM-CSF expression caused by Men1 deficiency. Figure 7 ).

[0038] 8. The H3K36m3 agonist JIB-04 restores macrophage maturation, differentiation, and cholesterol clearance capabilities.

[0039] JIB-04 Processing Men1 f / f and Men1 △M / △M Flow cytometry analysis of BMDM (M-CSF-induced maturation and differentiation) revealed that JIB-04 can restore macrophage maturation and differentiation impairment (CD11b) caused by Men1 knockout. + (Number of positive cells) Figure 8 (Figure A in the figure). Cholesterol content detection results showed that JIB-04 could restore the defect in macrophage cholesterol clearance capacity caused by Men1 knockout, and the contents of total cholesterol, free cholesterol and esterified cholesterol were significantly reduced. Figure 8 (See Figure B in the figure). The above results suggest that JIB-04 has the ability to improve the normal differentiation of macrophages, enhance the clearance of alveolar surfactant by macrophages, and maintain alveolar surfactant homeostasis, making it a potential targeted drug for the treatment of pulmonary alveolar proteinosis / PAP.

[0040] The present application proves that the pathogenic gene MEN1 (Men1 in mice, encoding protein menin) of multiple endocrine neoplasia type I syndrome regulates the transcriptional expression of GM-CSF in macrophages, which has important biological functions in maintaining the maturation, development, activation of alveolar macrophages and the homeostasis of pulmonary surfactant. Importantly, the monogenic deletion of Men1 in myeloid cells can induce spontaneous pulmonary alveolar proteinosis / PAP in mice. Mechanism studies have shown that menin promotes GM-CSF transcription through H3K36m3 histone modification, and exhibits a key regulatory role in GM-CSF transcription. JIB-04, a broad-spectrum inhibitor of Jumonji histone demethylase, up-regulates GM-CSF by restoring H3K36m3 modification, and thus completely restores the maturation and differentiation of lung macrophages and the impairment of cholesterol clearance caused by the deletion of Men1 gene, and exhibits potential application prospects in macrophage activation. The present application provides potential application prospects of small molecule compound JIB-04 in treating macrophage function defect-related diseases such as PAP, and has the advantages of clear target, specific action, significant effect and the like.

[0041] In summary, based on the epigenetic mechanism that menin promotes the transcriptional expression of GM-CSF in macrophages through H3K36m3 and maintains the activation of alveolar macrophages and the homeostasis of pulmonary surfactant, the present application proves the clinical application prospects of small molecule inhibitor JIB-04 in targeting the increase of H3K36m3 modification and GM-CSF expression in macrophages for treating PAP.

Claims

1. Application of JIB-04 in the preparation of drugs for treating pulmonary alveolar proteinosis caused by dysfunction of the Men1 / H3K36m3 / GM-CSF axis.

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