Application of sorted protein SNX3 inhibitor in preparation of drugs for proliferative retinopathy

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of a sorted protein SNX3 inhibitor in preparation of drugs for proliferative retinopathy. The invention verifies that the overexpression of the sorted protein (SNX3) induces pathological angiogenesis and is a new target of pathological neovascular diseases, and a small-molecule inhibitor W1122 is identified based on the protein structure of the SNX3. The chemical structural formula of the small-molecule inhibitor W1122 is as shown in formula 1, the small-molecule inhibitor W1122 belongs to imidazo [1, 2-a] pyridine derivatives, the chemical name of the small-molecule inhibitor W1122 is N-(1, 1-dioxobenzo [b] thiophene-6-yl)-2-(7-methoxy-2-phenyl imidazo [1, 2-a] pyridine-3-yl) acetamide, the chemical formula of the small-molecule inhibitor W1122 is C24H19N3O4S, the molecular weight of the small-molecule inhibitor W1122 is 445.49 g / mol, and the small-molecule inhibitor W1122 can effectively inhibit pathological retinal angiogenesis. The result of the embodiment of the invention shows that the small-molecule inhibitor W1122 can effectively prevent and treat proliferative retinopathy by inhibiting the expression of SNX3, and shows that the sorted protein SNX3 provides a new drug target for treating the proliferative retinopathy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of a sorting nexin 3 (SNX3) inhibitor W1122 in the preparation of a medicament for proliferative retinopathy. Background Art

[0002] Proliferative retinopathy is one of the main causes of blindness in people of different ages, including neonates (retinopathy of prematurity), middle-aged people (proliferative diabetic retinopathy), and the elderly (age-related macular degeneration). Currently, the main therapeutic drug for treating proliferative retinopathy clinically is a vascular endothelial growth factor antagonist. Although the vascular endothelial growth factor antagonist shows certain efficacy in treating the above-mentioned proliferative retinopathies, there are still many patients with primary drug resistance or a high recurrence rate. With the further in-depth study of the pathological mechanism of proliferative retinopathy, it is found that myeloid cells (especially microglia and macrophages) are the main participants in pathological retinal angiogenesis. And vascular endothelial growth factor is hardly expressed in retinal microglia and macrophages. Therefore, there is an urgent need to find new therapeutic targets acting on microglia and macrophages in the retina.

[0003] Immune cells, especially macrophages and microglia, act as immune surveillance guards and are key effectors and regulators in the retinal microenvironment. They will undergo a series of specific activation processes to respond to the disruption of retinal homeostasis caused by various stimuli or stresses (such as ischemia / hypoxia, neuroinflammation, injury stress, and degeneration). Activated immune cells often undergo apoptosis or death in the central nervous system to eliminate overactive inflammatory responses and maintain immune tolerance. New evidence shows that receptor-interacting protein 1 / 3 (RIP1 / 3)-mediated necroptosis of retinal microglia is crucial for regulating pathological retinal angiogenesis. However, whether macrophages and microglia also undergo necroptosis during retinal angiogenesis, and how RIP1 / 3-mediated signal transduction in myeloid cells drives retinal angiogenesis are still largely unknown.

[0004] Therefore, working on discovering key regulators of the necroptosis pathway is crucial for finding new drug screening targets. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of a sorting nexin 3 inhibitor in the preparation of a medicament for proliferative retinopathy. The small molecule inhibitor W1122 provided by the present invention can bind to sorting nexin 3, thereby inhibiting sorting nexin 3 and the downstream RIP1 / 3 signaling pathway it regulates, and promoting the transformation of sorting nexin 3 as a new therapeutic target for proliferative retinopathy.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides an application of a sorting nexin 3 (SNX3) inhibitor in the preparation of a drug for proliferative retinopathy.

[0008] Preferably, the sorting nexin 3 (SNX3) inhibitor is a small molecule inhibitor W1122; the small molecule inhibitor W1122 is an imidazo[1,2-a]pyridine derivative, and its chemical name is N-(1,1-dioxobenzo[b]thiophen-6-yl)-2-(7-methoxy-2-phenylimidazo[1,2-a]pyridin-3-yl)acetamide, and its chemical formula is C 24 H 19 N3O4S, with a molecular weight of 445.49 g / mol; the chemical structural formula of the small molecule inhibitor W1122 is as shown in Formula 1:

[0009]

[0010] The present invention also provides a small molecule inhibitor W1122, and the dissociation constant of the small molecule inhibitor W1122 with the target protein SNX3 is 2.6 micromoles; the chemical structural formula of the small molecule inhibitor W1122 is as shown in Formula 1:

[0011]

[0012] The present invention also provides an application of the above small molecule inhibitor W1122 in inhibiting or down-regulating the expression of sorting nexin 3 (SNX3).

[0013] Preferably, the small molecule inhibitor W1122 binds to the N-terminus of sorting nexin 3 (SNX3).

[0014] The present invention also provides an application of the above small molecule inhibitor W1122 in the preparation of a drug for inhibiting RIP1 / 3 signal transduction.

[0015] Preferably, the small molecule inhibitor W1122 binds to sorting nexin 3 (SNX3) to inhibit RIP1 / 3 signal transduction.

[0016] The present invention also provides an application of the above small molecule inhibitor W1122 in the preparation of a drug for inhibiting the expression of MMP12 gene.

[0017] The beneficial effects of the present invention:

[0018] The present invention provides a potential therapeutic target, i.e., sorting nexin 3 (SNX3), for the treatment of proliferative retinopathy.

[0019] The present invention newly discovers a small molecule inhibitor W1122 that inhibits or down-regulates the expression level of sorting protein SNX3. This small molecule inhibitor can bind to sorting protein SNX3, effectively inhibit the signal transduction of RIP1 / 3, and alleviate retinal neovascularization in retinopathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. The drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0021] Figure 1 It is a graph showing the experimental results of the up-regulated expression of sorting protein SNX3 in the retinas of patients with proliferative diabetic retinopathy and oxygen-induced retinopathy (OIR) rodents; (A-B) are the results of heat map analysis of the expression of SNX3 gene, myeloid cell markers, and necroptosis-related genes in the retinal neovascular membranes of patients with proliferative diabetic retinopathy. The data are from the reanalysis of published microarray datasets (GSE60436, A, n = 3) or transcriptome sequencing technology datasets (GSE102485, B, n = 2-4); (C-F) single-cell RNA sequencing analysis of CD11b + F4 / 80 + The results of Snx3 gene expression in retinal cells; (C) t-SNE distribution map of retinal cells in normal oxygen and OIR day 17 mice; (D) cell type identification result map based on marker abundance; (E) GO, KEGG, and Reactome enrichment analysis of CD11b + F4 / 80 + Pathway enrichment map of cells; (F) violin plot of Snx3 gene expression; (G) representative images of CD11b, isolectin B4, and DAPI immunofluorescence staining in OIR retinas (n = 4); (H) laser capture microdissection technology to isolate myeloid cell-enriched neovascular clusters: isolectin and DAPI staining results in cryosections of normal oxygen and OIR retinas (scale bar: 50 microns, white dotted line is the laser cutting area, MΦ: myeloid cells); (I) Snx3 mRNA RT-PCR analysis map of laser capture samples (n = 4); (J-M) Co-localization images of SNX3 (green) with macrophages / microglia (F4 / 80, J), myeloid cells (CD11b, L), and blood vessels (isolectin) in whole retina spreads of OIR mice (NC: negative control, SNX3 fluorescence intensity quantified by ImageJ, K and M); (N-Q) MACS sorting of CD11b +RT-PCR of cells (O, n = 6) and Western blot (P-Q, n = 3) analysis diagrams. Data are expressed as mean ± standard error. (One asterisk in the figure represents P < 0.05, two asterisks represent P < 0.01, three asterisks represent P < 0.001; four asterisks represent P < 0.0001 in the figure).

[0022] Figure 2 Diagrams of experimental results showing that global or myeloid-specific knockout of SNX3 reduces retinal neovascularization and vascular leakage in OIR mice; (A-C) Retinal flat mounts stained with isolectin B4 showing occluded vessels (yellow) and neovascularization (white) areas (n = 10, scale bar: 500 μm, original; 200 μm, magnified); (D) Whole retina staining showing bleeding points in control OIR mice (white arrows, n = 6); (E) FITC-dextran perfusion to evaluate blood-retinal barrier integrity (n = 6, scale bar: 50 μm); (F-G) Western blot analysis of retinal albumin (n = 6); (H-J) Quantification of retinal neovascularization and avascular areas in Snx3 myeloid cell-specific knockout mice (Snx3ΔMΦ) and Snx3 myeloid cell non-knockout wild-type mice (Snx3WT) in the OIR model (n = 12); (K-L) Evaluation of bleeding points and blood-retinal barrier (n = 6); (M-N) Albumin expression analysis (n = 6). Data are expressed as mean ± standard error (One asterisk in the figure represents P < 0.05, two asterisks represent P < 0.01, three asterisks represent P < 0.001).

[0023] Figure 3 Diagrams of experimental results showing that SNX3 deficiency inhibits hypoxia-induced necroptosis of myeloid cells and release of pro-inflammatory / pro-angiogenic factors; (A) Single-cell sequencing results showing enrichment of the necroptosis pathway in CD11b + F4 / 80 + cells in OIR mice (threshold: genes expressed in ≥10% of cells, adjusted P < 0.05, log2 fold change |>0.585); (B-E) Immunofluorescence staining result diagrams, where C is the co-localization and quantification of cells with Iba-1 + in retinal flat mounts (scale bar: 20 / 5 μm, n = 5×3); (F) Flow cytometry sorting of CD11b + cells for Western blot of RIP1 / 3 and FGF2 (n = 4); (G-H) Expression of RIP1 / 3 in bone marrow-derived macrophages and retinal microglia under hypoxic conditions (n = 3); (I) Effect of adenovirus-mediated SNX3 overexpression on RIP1 / 3 (n = 3); (J) Analysis results of necroptosis detected by co-staining of TUNEL and activated caspase-3 (n = 3×3); (K) CD11b in OIR mice+ Cell pro - inflammatory / pro - angiogenic gene heatmap (n = 4); (L - P) Detection of Ripk1 / Ripk3 mRNA and cytokines by enzyme - linked immunosorbent assay in hypoxic microglia (n = 4); (Q) Analysis of FGF2 protein expression (n = 4), data are expressed as mean ± standard error (one asterisk in the figure represents P < 0.05, two asterisks in the figure represent P < 0.01, three asterisks in the figure represent P < 0.001);

[0024] Figure 4 Experimental structure diagram for SNX3 deletion down - regulating its protein expression by enhancing RIP1 / 3 degradation; (A) Western blot analysis of RIP1 / 3 protein levels in bone - marrow - derived macrophages from Snx3WT and Snx3ΔMΦ mice under hypoxic conditions (treated with 100 μM CoCl2) and after cycloheximide treatment for different times; relative protein amounts were calculated by the ratio of band intensity at each time point to that at time 0 (n = 3); (B) Immunofluorescence co - localization images of SNX3 with RIP1 / RIP3 / VPS35 in bone - marrow - derived macrophages (red: SNX3, green: RIP1 / RIP3 / VPS35, scale bar: 5 μm); co - localization rate was analyzed by ImageJ software; (C - D) Immunoprecipitation of Ad - FLAG - SNX3 - infected bone - marrow - derived macrophages (C) and microglia (D) with FLAG antibody, and Western blot was used to detect the interaction between SNX3 and RIP1 / RIP3 / VPS35 (non - immune IgG as a control); (E - F) Co - localization of RIP1 (E) or RIP3 (F) with lysosomal marker LAMP1 in bone - marrow - derived macrophages from Snx3WT and Snx3ΔMΦ mice under hypoxic conditions (green: RIP1 / 3, red: LAMP1, blue: DAPI, scale bar: 5 μm); (G) Expression of RIP1 / 3 protein in hypoxic - induced bone - marrow - derived macrophages after treatment with chloroquine (CQ, 10 ng / mL) for 24 hours (n = 4); (H) Expression of RIP1 / 3 in cell lysates and lysosomal fractions of chloroquine - treated hypoxic - induced bone - marrow - derived macrophages (β - Actin and LAMP1 were used as cytoplasmic and lysosomal markers respectively, data are expressed as mean ± standard error, one asterisk in the figure represents P < 0.05, two asterisks in the figure represent P < 0.01, three asterisks in the figure represent P < 0.001, ns indicates no significant difference);

[0025] Figure 5Experimental result graphs showing that SNX3 deficiency inhibits RIP1 / 3-mediated mitochondrial fission and mtROS production; (A) Oxygen consumption rate curves of Snx3WT and Snx3ΔMΦ bone marrow-derived macrophages treated with Nec-1 (10 μmol) / GSK872 (10 μmol) under normoxia (21% O2) or hypoxia (1% O2); (B-C) Quantification of mitochondrial function parameters (basal respiration, maximal respiration, etc.) (n = 5-8); (D-E) Mitotracker RedFM staining showing mitochondrial signal intensity in hypoxic bone marrow-derived macrophages (scale bar: 5 μm, n = 10); (F-G) Transmission electron microscopy images showing mitochondrial morphology (scale bar: 500 nm) and number of mitochondria per cell in hypoxic bone marrow-derived macrophages (n = 8); (H-I) Western blot analysis of phosphorylated DRP1 (p-DRP1) in hypoxic bone marrow-derived macrophages (n = 4); (J-K) Effects of Nec-1, GSK872 or Mdivi-1 treatment on p-DRP1 expression (n = 4); (L-M) Flow cytometry sorting of CD11b + Western blot of p-DRP1 in cells (n = 3); (N) MitoSOX staining and mtROS quantification in Mdivi-1-treated hypoxic bone marrow-derived macrophages (scale bar: 50 μm, n = 6); (O) MitoSOX staining of OIR mouse retinas (scale bar: 100 μm, n = 5, data are presented as mean ± SEM, one asterisk in the figure represents P < 0.05, two asterisks in the figure represent P < 0.01, and three asterisks in the figure represent P < 0.001);

[0026] Figure 6Experimental results figure for SNX3 deficiency downregulating MMP12 by inhibiting RIP1 / 3-mediated mitochondrial fission; (A) Heat map of matrix metalloproteinase (MMPs) gene expression in CD11b+ cells of Snx3 control and Snx3 myeloid cell-specific knockout mice (n = 4, #P < 0.001 indicates Snx3WT_OIR vs Snx3WT_RA; one asterisk in the figure indicates P < 0.01 indicates Snx3ΔMΦ_OIR vs Snx3WT_OIR); (B) RT-PCR analysis of MMPs in bone marrow-derived macrophages under hypoxic conditions (n = 4, ##P < 0.01, P < 0.001 vs Snx3WT normoxia; P < 0.01 vs Snx3WT hypoxia); (C-D) Expression of MMP12 mRNA (C, RT-PCR, n = 4) and protein (D, Western blot, n = 3) in hypoxic bone marrow-derived macrophages treated with Nec-1, GSK872, Mdivi-1 or Mito-TEMPO (P < 0.001 vs DMSO normoxia; one asterisk in the figure indicates P < 0.01 / 0.001 vs DMSO hypoxia); (E-H) Effect of conditioned medium (CM) of bone marrow-derived macrophages on three-dimensional spheroid sprouting of human retinal endothelial cells (HRECs) under hypoxic conditions after siRNA knockdown of Rip1 / Rip3 / Mmp12: (E) Representative images (scale bar: 50 μm); (F) Quantification of sprout length (n = 10); (G) Images of choroidal sprouting (scale bar: 500 μm); (H) Quantification of sprout area (n = 8, data are presented as mean ± SEM, P < 0.001 vs no Mφ-CM group, one asterisk in the figure represents P < 0.05, two asterisks in the figure represent P < 0.01, three asterisks in the figure represent P < 0.001 vs Snx3WT+siCtrl group);

[0027] Figure 7 Experimental results figure for inhibiting RIP1 / 3 can reverse SNX3 overexpression-driven retinal neovascularization in OIR mice; (A) Schematic diagram of the experimental design: Snx3MΦ-TG (myeloid cell-specific overexpression of SNX3 transgenic) OIR mice received intravitreal injection of the RIP1 inhibitor Nec-1 or the RIP3 inhibitor GSK872; (B-D) Isolectin-stained retinal flat mounts showed: (B) Representative images of retinal neovascularization (white) and avascular areas (yellow) in Snx3WT and Snx3MΦ-TG OIR mice (scale bar: 500 μm original image, 200 μm magnified image); (C) Pathological neovascularization area; (D) Quantification of the vascular occluded area (n = 10, data are presented as mean ± SEM, one asterisk in the figure represents P < 0.05, two asterisks in the figure represent P < 0.01, three asterisks in the figure represent P < 0.001);

[0028] Figure 8 Experimental result graphs for the synergistic inhibition of retinal neovascularization by the combination therapy of an SNX3 inhibitor and anti-VEGF; (A) Structure diagram of W1122; (B) Molecular docking results of W1122 and SNX3-retromer (SNX3-VPS26-VPS35) (PDB: 5F0J); (C) Modeling results of W1122 and SNX3-retromer (PDB: 5F0J); Left figure: Binding mode of W1122 and SNX3-retromer, marking the key residues around the W1122-SNX3-VPS26-VPS35 pocket. Hydrogen bonds and Pi cations between the heavy atoms of W1122 and the key residues of VPS35 are shown as dashed lines (pink and light green respectively); Right figure: Detailed interactions between W1122 and the pocket residues; (D-E) Surface plasmon resonance (SPR) analysis results; (F) Result graph of cellular thermal shift assay (CETSA); (G) Immunoprecipitation result graph; (H-I) Representative stained images; (J) Result graph of Western blot analysis; (K-L) Result graphs of TUNEL assay and cleaved caspase-3 staining; (L) Culture images; (M) Schematic diagram of OIR mice receiving intravitreal (IVT) injection of W1122 and / or VEGF Nab; (N-P) Representative images of isolectin-stained retinal flat mounts of OIR mice at P17; (N) Quantification of the neovascular (O) and avascular (P) regions, represented by white and yellow respectively, (n = 10 retinas; scale bar: 500 μm for the original image, 200 μm for the magnified image, data are mean ± SEM, one asterisk in the figure represents P < 0.05, two asterisks in the figure represent P < 0.01, three asterisks in the figure represent P < 0.001).

[0029] Figure 9 Experimental result graphs for the effect of W1122 on the viability of myeloid cells, (A) Cell viability of bone marrow-derived macrophages; (B) Cell viability of mouse retinal microglia (n = 6, data are presented as mean ± standard error, one asterisk in the figure represents P < 0.05, two asterisks in the figure represent P < 0.01, three asterisks in the figure represent P < 0.001, ns: no significant difference). Detailed implementation manners

[0030] The present invention provides a new therapeutic target - sorting protein SNX3 for proliferative retinopathy. This sorting protein SNX3 is a member of the sorting nexin (SNX) family, participates in endosomal sorting and trafficking, and is significantly upregulated in the myeloid cell subsets of the retina in oxygen-induced retinopathy (OIR).

[0031] The research of the present invention shows that the expression of sorting nexin SNX3 is significantly increased in retinal microglia / macrophages of mice with OIR, mainly located within and around the neovascular plexus. Both the global and myeloid cell-specific knockout of sorting nexin SNX3 inhibits retinal neovascularization in OIR mice. Mechanistically, SNX3-retromer interacts with RIP1 and RIP3, mediating their intracellular recycling and protecting them from lysosomal degradation.

[0032] Based on the protein structure of sorting nexin SNX3, the present invention provides a small molecule inhibitor W1122 of sorting nexin SNX3. The small molecule inhibitor W1122 belongs to imidazo[1,2-a]pyridine derivatives, and its chemical name is N-(1,1-dioxobenzo[b]thiophen-6-yl)-2-(7-methoxy-2-phenylimidazo[1,2-a]pyridin-3-yl)acetamide, and its chemical formula is C 24 H 19 N3O4S, with a molecular weight of 445.49 g / mol. The chemical structural formula of the small molecule inhibitor W1122 is shown in Formula 1.

[0033] The dissociation constant (KD) of the small molecule inhibitor W1122 and the target protein SNX3 is 2.6 micromoles. The smaller the KD value, the tighter the binding of the drug to the target protein and the higher the affinity. This small molecule inhibitor W1122 inhibits the protein expression of sorting nexin SNX3 by binding to the N-terminus of sorting nexin SNX3, effectively inhibits retinal angiogenesis, and can produce a synergistic anti-angiogenic effect in combination with anti-vascular endothelial growth factor therapy.

[0034] The small molecule inhibitor W1122 of the present invention can interact with the N-terminus of sorting nexin SNX3. The oxygen-sulfur double bond of the small molecule inhibitor W1122 can also form a hydrogen bond with the Arg206 site of vacuolar protein sorting-associated protein 35 (VPS35), and its benzothiophene structure forms a π-stacking with the Phe28 site. In addition, the Lys203 site of VPS35 can also establish a cation-π stacking interaction with the benzene substituent of the small molecule inhibitor W1122, indicating the potential binding affinity and effective targeting between the small molecule inhibitor W1122 and the SNX3-retromer complex.

[0035] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] In the embodiments of the present invention, the production processes, experimental methods or detection methods involved, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and definite in the relevant application fields. Those skilled in the art can understand the conventional process steps according to the names and apply the corresponding equipment, and implement them under conventional conditions or conditions recommended by the manufacturer.

[0037] There are no special restrictions on the sources of various instruments, equipment, raw materials or reagents used in the embodiments of the present invention. They are all conventional products that can be obtained through regular commercial channels, and can also be prepared according to the conventional methods well-known to those skilled in the art.

[0038] Example 1 Experiment on the correlation between SNX3 expression in retinal myeloid cells and proliferative retinopathy in humans and rodents

[0039] 1.1 Use Mendelian randomization (MR) to analyze the causal relationship between the expression of all members of the SNX gene family and retinopathy, and study the role of SNX genes in retinopathy

[0040] The results of the Mendelian randomization analysis showed that there was a causal relationship between the level of SNX3 and a significantly higher incidence of severe diabetic background retinopathy (odds ratio (OR) = 3.8032, 95% confidence interval (CI) [1.0937, 13.2247], P = 0.036).

[0041] 1.2 Analyze the expression of SNX3 in the microarray dataset (GSE60436) and RNA-seq sequencing dataset (GSE102485) of the retinal neovascularization membranes of patients with proliferative diabetic retinopathy

[0042] The results are as Figure 1 shown in (A - B). In these two datasets, compared with the retinas of healthy controls, the gene expressions of SNX3, necroptosis-related genes (RIPK1, RIPK3, and MLKL), and myeloid cell markers (C1QA, TREM2, ITGAM, and PTPRC) in the retinas of patients with proliferative diabetic retinopathy were significantly increased, indicating that the expression of the sorting protein SNX3 is related to myeloid cell necroptosis and the development of proliferative diabetic retinopathy.

[0043] 1.3 Describe the complete content of the transcriptional profiles of SNX-related genes in retinal myeloid cells

[0044] Single-cell transcriptome analysis was performed in OIR mice using the 10X Genomics platform. OIR mice are a murine model of oxygen-induced retinopathy that partially mimics certain aspects of preterm retinopathy of prematurity and human proliferative diabetic retinopathy.

[0045] CD11b is an integrin molecule that usually binds to CD18 to form the CD11b / CD18 complex, which is involved in cell adhesion and signal transduction. CD11b mainly labels monocytes and macrophages, which play important roles in the immune system, can phagocytose and digest pathogens, and participate in inflammatory responses and immune regulation. F4 / 80 is a glycoprotein on the cell membrane that is widely expressed in various mature macrophages, including Kupffer cells in the liver, Langerhans cells, microglial cells, and macrophages located in the peritoneal cavity, lung, intestine, thymus, red pulp of the spleen, and bone marrow stroma. F4 / 80 is a marker of mature murine macrophages. In flow cytometry assays, the use of F4 / 80 + CD11b + can effectively distinguish other types of monocytes.

[0046] CD11b-positive and F4 / 80-positive myeloid cells were enriched by flow sorting from the retinas (n = 16) of normoxic or OIR mice, respectively. First, CD45 + myeloid cells (n = 1545 cells) were selected from the total cell population, and then the "Seurat" package was used for unsupervised clustering for downstream analysis. Myeloid cells were clustered into four distinct clusters: resting microglia, activated microglia, macrophages, and monocytes. The results are shown in Figure 1 (C-D). Cells in the resting microglia cluster (mainly from the retinas of control mice under normoxic conditions) highly expressed genes such as P2ry12, Siglech, and Tmem119, etc., which are related to microglial homeostasis, while cells in the activated microglia cluster (significantly enriched in OIR retinas) highly expressed markers such as Cst7, Lpl, and Spp1, etc.

[0047] Pathway GO, KEGG, and Reactome enrichment analyses found that SNX-related pathways (such as early endosomes, endosomal membranes, and endocytic recycling) and the necroptosis pathway were enriched in microglia / macrophages from OIR retinas as shown in Figure 1 (E). Among the members of the SNX gene family, SNX3 was the most significantly upregulated gene in the myeloid cells of the retinas of OIR mice (P = 5.96E-50, Wilcoxon rank-sum test, OIR vs RA).

[0048] Violin plot of Snx3 gene expression is shown in Figure 1(F) shows that the gene expressions of SNX3 and key components of the retroviral complex (including Vps29 and Vps35) in macrophages and activated microglia of the OIR retina are significantly upregulated.

[0049] CD11b and isolectin B4 in the OIR retina were observed using DAPI immunofluorescence staining, and the immunofluorescence staining results are as Figure 1 (G) shows that CD11b + Myeloid cells were aggregated within the neovascular plexus region of the OIR retina, as shown in the three-dimensional (3D) image of the flat-mounted retina.

[0050] 1.4 Verification of SNX3 gene expression in activated myeloid cells in pathological retinal NV

[0051] Laser capture microdissection was used to isolate the neovascular plexus rich in myeloid cells from the OIR retina. The staining results of isolectin and DAPI in the frozen sections of the RA and OIR retina are as Figure 1 (H-I) shows that the expression of SNX3 in the neovascular plexus rich in myeloid cells of OIR mice is increased compared with that in the normal retinal vessels of control mice.

[0052] By immunostaining the whole retina, it was observed that the expression level of SNX3 in the OIR retina was significantly increased, mainly in F4 / 80 and CD11b-positive macrophages / microglia, as Figure 1 (J-M) shows. SNX3-positive macrophages / microglia were aggregated within and around the neovascular plexus of the OIR retina. By RT-PCR and Western blot analysis of CD11b + cells, it was observed that the expression of SNX3 in myeloid cells in the OIR retina isolated using anti-CD11b microbeads was increased, as Figure 1 (N-Q) shows. In summary, the above results indicate that the increased expression of SNX3 in retinal myeloid cells is related to proliferative retinopathy in humans and rodents.

[0053] Example 2 Global and bone marrow-specific SNX3 knockout reduces retinal neovascularization and vascular leakage in OIR mice

[0054] 2.1 Study on the role of SNX3 in retinopathy

[0055] SNX3 flox / flox mice refer to homozygous genetically engineered mice in which the two alleles of SNX3 are flanked by inserted loxP sites. When mated with mice expressing Cre recombinase, the Cre enzyme can specifically recognize and excise the gene sequence between the loxP sites, achieving conditional gene knockout. Rosa26 Cre / ERT2 mice are a type of genetically engineered mice in which the Cre / ERT2 fusion gene is inserted into the Rosa26 safe site. The activity of Cre recombinase can be activated by tamoxifen induction, enabling temporally specific (drug-induced) and spatially specific (promoter-dependent) gene editing, and is commonly used for conditional spatio-temporal control of gene knockout or expression.

[0056] The globally inducible SNX3 knockout mice (SNX3 f / f; Rosa26 Cre / ERT2) were generated by crossing SNX3 flox / flox mice with Rosa26 Cre / ERT2 mice. SNX3 gene deletion (SNX3 iKO) was induced in neonatal mice by tamoxifen administration. Rosa26 Cre / ERT2 littermate mice were used as wild-type (SNX3 WT) controls.

[0057] The experimental results are as Figure 2 (A-C) show that retinal flat mounts stained with isolectin B4 display vascular occluded regions (VO, yellow) and neovascularization (NV, white areas). After tamoxifen treatment, the expression of SNX3 in the retina of SNX3 iKO mice was significantly reduced, confirming successful SNX3 knockout. Compared with mice subjected to OIR modeling of SNX3 WT (SNX3 WT OIR), the areas of neovascular plexus and vascular occluded regions in the retina of mice subjected to OIR modeling of SNX3 iKO (SNX3 iKO OIR) at P17 were significantly reduced.

[0058] To investigate whether SNX3 deficiency could improve excessive vascular permeability, which is a characteristic of pathological angiogenesis. The gross retinal photographs are as Figure 2 (D) show that compared with SNX3 WT OIR mice, the retina of SNX3 iKO OIR mice had less retinal hemorrhage. In addition, compared with the control group, the retina of SNX3 iKO OIR mice showed reduced retinal vascular permeability, as confirmed by fluorescein angiography with fluorescein isothiocyanate (FITC)-dextran and AlexaFluor594-conjugated isolectin B4 (IB4). The results of FITC-dextran perfusion to evaluate blood-retinal barrier integrity are as Figure 2 (E) show. Retinal western blot analysis indicated that the albumin level (a marker of capillary leakage) in the retina of SNX3 iKO OIR was lower than that of the control group, as Figure 2 (F-G) show.

[0059] 2.2 Determine the specific role of SNX3 in myeloid cells in retinal NV

[0060] Myeloid-specific SNX3 knockout mice (SNX3ΔMΦ) and littermate control mice (SNX3 WT) were generated by mating SNX3 flox / flox mice with Lysm-Cre mice. Compared with SNX3 WT mice, the expression of SNX3 in bone marrow-derived macrophages (BMDMs) and retinal microglia cultured from SNX3ΔMΦ mice was significantly reduced, verifying the successful deletion of SNX3 in myeloid cells of SNX3ΔMΦ mice.

[0061] The physiological development of retinal blood vessels under normoxia was not affected by the deletion of SNX3 in myeloid cells. Similar to SNX3iKO, the retinal neovascular cluster area, avascular area, and vascular permeability of SNX3ΔMΦOIR mice were decreased compared with the control group at P17, as Figure 2 shown in (H-N), where Figure 2 (H-J) is the quantitative detection of retinal NV and avascular area in Snx3ΔMΦOIR and Snx3WT OIR mice; Figure 2 (K-L) is the evaluation of bleeding points and blood-retinal barrier; Figure 2 (M-N) is the analysis result of albumin expression.

[0062] The above results indicate that myeloid cell-specific SNX3 knockout can significantly reduce retinal NV and vascular leakage in OIR.

[0063] Example 3 Deletion of SNX3 can inhibit hypoxia-induced necroptosis of myeloid cells and subsequent expression and release of pro-inflammatory and pro-angiogenic cytokines

[0064] 3.1 Analysis of the potential role of SNX3 in regulating necroptosis of myeloid cells during retinal lesions

[0065] Pathway enrichment analysis of scRNA-seq data of retinal myeloid cells in RA / OIR mice found that genes related to the necroptosis pathway and SNX-retromer complex were significantly involved in microglia (especially active microglia) and macrophages in the OIR retina, as Figure 3 shown in (A).

[0066] Immunostaining found that the fluorescence intensity of two key components of the necroptosis pathway, RIP1 and RIP3, in IBa1 + microglia / macrophages located within the neovascular plexus of the OIR retina was significantly increased, as shown in Figure 3(as shown in (B-E)). SNX3 deficiency significantly attenuated the OIR-induced increase in RIP1 / 3 staining.

[0067] Western blot analysis showed that the protein levels of RIP1 / 3 were upregulated in CD11b + cells isolated from OIR retinas, while SNX3 deletion significantly reduced the OIR-induced RIP1 / 3 expression levels, as Figure 3 (F) shows. Similar results were obtained in mouse macrophages Figure 3 exposed to hypoxia in vitro as Figure 3 (G) and retinal microglia as Figure 3 (H). Conversely, the analysis of the effect of adenovirus-mediated SNX3 overexpression on RIP1 / 3 showed that SNX3 overexpression induced a substantial increase in RIP1 / 3 expression in microglia, as

[0068] (I) shows.

[0068] In summary, both loss-of-function and gain-of-function studies demonstrated that SNX3 plays a crucial role in regulating RIP1 / 3 protein expression. Since the kinase RIP1 / 3 is a key trigger for necroptosis, it is logical to determine whether SNX3 regulates necroptosis in myeloid cells. SNX3 knockout significantly counteracted necroptosis in retinal microglia exposed to hypoxia, as indicated by the reduced number of necroptotic (TUNEL + caspase-3 - ) cells isolated from SNX3ΔMΦ mice. The results of the analysis of necroptosis by co-staining TUNEL with activated caspase-3 are shown in Figure 3 (J).

[0069] 3.2 SNX3 affects the expression and release of pro-inflammatory and pro-angiogenic cytokines and mediates retinal neovascularization

[0070] First, the gene expression profiles in CD11b + retinal cells isolated from SNX3ΔMΦ OIR mice were analyzed. The heatmap results of pro-inflammatory / pro-angiogenic genes in CD11b + cells of OIR mice are shown in Figure 3 (K), indicating that SNX3 deficiency significantly reduced the mRNA expression of key pro-inflammatory and pro-angiogenic cytokines (including Il1b, Il6, Tnfα, and Fgf2) in myeloid cells of CD11b + isolated from OIR mice.

[0071] The ELISA results of Ripk1 / Ripk3 mRNA in microglia under hypoxic conditions are shown in Figure 3As shown in (L), the protein levels of RIP1 / 3 were significantly decreased in SNX3-knockout myeloid cells, but their mRNA levels remained unchanged. The pro-inflammatory cytokines and FGF2 produced by retinal microglia cultured in vitro were evaluated using ELISA. As Figure 3 shown in (M-P), the protein levels of IL-1β, TNF-α, IL-6, and FGF2 were increased in the culture medium of retinal microglia exposed to hypoxia, and this effect was abolished when SNX3 was knocked out.

[0072] Western blot analysis of FGF2 showed that the expression of FGF2 protein was decreased in CD11b + cells isolated from SNX3ΔMΦOIR retinas; similar results were obtained in hypoxic retinal microglia cultured from SNX3ΔMΦ mice, as Figure 3 (Q).

[0073] Overall, these data indicate that SNX3 promotes RIP1 / 3-mediated necroptosis and subsequent release of pro-inflammatory cytokines and the pro-angiogenic factor FGF2 from retinal myeloid cells.

[0074] Example 4 Regulation of RIP1 / 3 Protein Expression by SNX3

[0075] 4.1 Verification Test of the Promotion of RIP1 / 3 Protein Degradation by SNX3 Knockout

[0076] SNX3 knockout downregulates the expression of RIP1 / 3 protein but does not affect its transcriptional induction. Therefore, SNX3 knockout promotes the degradation of RIP1 / 3 protein.

[0077] Cycloheximide (CHX) was used to inhibit the synthesis of RIP1 / 3 protein. The results of Western blot analysis of RIP1 / 3 protein levels in Snx3WT and Snx3ΔMΦ bone marrow-derived macrophages after hypoxia (100 μM CoCl2) and cycloheximide treatment for different times are shown in Figure 4 (A). In SNX3-knockout bone marrow-derived macrophages, the protein level of RIP1 / 3 (but not that of MLKL) decreased significantly faster than that in control cells.

[0078] Immunofluorescence staining and immunoprecipitation (Co-IP) experiments were performed to evaluate protein co-localization and interaction. Immunofluorescence co-localization images of SNX3 with RIP1 / RIP3 / VPS35 in bone marrow-derived macrophages showed that RIP1 / 3 and the retromer proteins (SNX3 and VPS35) were in the same cellular compartment in macrophages as Figure 4 (B) and microglia.

[0079] Co-IP assays showed that SNX3 was physically associated with RIP1 / 3 and VPS35 in FLAG-tagged SNX3-transfected macrophages as Figure 4 (C) and microglia as Figure 4 (D).

[0080] 4.2 Validation of the interaction between SNX3 and RIP1 / 3 and regulation of their intracellular trafficking and degradation

[0081] Immunofluorescence analysis was performed using the lysosomal marker antibody LAMP1.

[0082] The co-localization results of RIP1 (E) or RIP3 (F) with the lysosomal marker LAMP1 in Snx3WT and Snx3ΔMΦ bone marrow-derived macrophages treated with hypoxia are shown as Figure 4 (E - F). Deletion of SNX3 enhanced the co-localization of RIP1 / 3 with LAMP1 and decreased the SNX3 protein level, indicating that deletion of SNX3 facilitated the transport of RIP1 / 3 to lysosomes for degradation.

[0083] Endosomal and lysosomal degradation in myeloid cells was inhibited using chloroquine (CQ). The Western blot results of RIP1 / 3 protein in bone marrow-derived macrophages treated with hypoxia after 24-hour treatment with chloroquine (CQ, 10 ng / mL) are shown as Figure 4 (G). CQ treatment increased the RIP1 / 3 protein level in bone marrow-derived macrophages from control mice and restored the decrease in RIP1 / 3 level in macrophages lacking SNX3.

[0084] To further determine whether deletion of SNX3 would accelerate RIP1 / 3 degradation through the lysosomal pathway, the lysosomal fraction was isolated to detect RIP1 / 3 protein expression by Western blot analysis.

[0085] The results of the study on the expression of RIP1 / 3 in cell lysates and lysosomal fractions of CQ-treated hypoxic bone marrow-derived macrophages showed that in bone marrow-derived macrophages lacking SNX3, RIP1 / 3 protein was encapsulated and transported to lysosomes, as Figure 4 (H). Therefore, we identified RIP1 / 3 as a new interacting protein of SNX3, and deletion of SNX3 promoted the transport of RIP1 / 3 to lysosomes for degradation.

[0086] Example 5. Knockout of SNX3 downregulates MMP12 by inhibiting the RIP1 / 3-DRP1-mitochondrial fission-ROS axis in hypoxic myeloid cells

[0087] 5.1 Detection of mitochondrial function using a Seahorse XF Cell Mito Stress Test

[0088] Oxygen consumption rate (OCR) curves and quantitative results of mitochondrial function parameters (basal respiration, maximal respiration, etc.) of Snx3WT and Snx3ΔMΦ bone marrow-derived macrophages after treatment with Nec-1 (concentration: 10 μmol) / GSK872 (concentration: 10 μmol) under normoxia (21% O2) or hypoxia (1% O2) are as follows Figure 5 (A-C) show that hypoxia exposure leads to significant impairment of (OCR) in WT bone marrow-derived macrophages, including mitochondrial respiration and ATP production. These conditions were reversed when SNX3 was depleted in macrophages. Similarly, administration of the RIP1 or RIP3 inhibitors Nec-1 or GSK872 alleviated the effects of hypoxia on WT macrophages.

[0089] In addition, Mitotracker Red FM staining shows the results of mitochondrial signal intensity in hypoxic bone marrow-derived macrophages as follows Figure 5 (D-E) show that WT macrophages exposed to hypoxia exhibit reduced fluorescence of the mitochondrial-specific detection probe. In contrast, confocal imaging of SNX3ΔMΦ bone marrow-derived macrophages shows that the cells exhibit an extended mitochondrial network and enhanced mitochondrial signal, indicating an increase in the volume or number of mitochondria in the absence of SNX3.

[0090] Transmission electron microscopy (TEM) image analysis of the mitochondrial morphology of hypoxic bone marrow-derived macrophages is as follows Figure 5 (F-G) show that transmission electron microscopy images of SNX3ΔMΦ bone marrow-derived macrophages stimulated by hypoxia show a significant increase in mitochondrial structure, with a smaller morphology observed in SNX3WT macrophages. Only in SNX3WT macrophages exposed to hypoxia does the number of mitochondria per cell increase; however, this is not observed in SNX3ΔMΦ cells. The data indicate that SNX3 affects mitochondrial fission. Consistent with this view, activation of the mitochondrial fission protein, dynamin-related protein 1 (DRP1), is enhanced in bone marrow-derived macrophages exposed to hypoxia, and its phosphorylation (p-DRP1) level is enhanced, while in the absence of SNX3 as shown in Fig. (5H-I) or in the presence of RIP1 or RIP3 inhibitors such as Figure 5 (J-K), this activation is attenuated.

[0091] Based on the in vitro study results, MACS sorting of CD11b + Western blot analysis results of p-DRP1 in cells are as follows Figure 5 (L-M) show that compared with RA mice, the level of p-DRP1 in CD11b + cells isolated from the retinas of OIR mice is elevated, while SNX3 deficiency significantly downregulates OIR-induced DRP1 phosphorylation.

[0092] The above results indicate that SNX3 mediates mitochondrial fission and dysfunction through the RIP1 / 3-DRP1 axis.

[0093] 5.2 Function of SNX3-mediated mitochondrial fission in myeloid cells during retinopathy

[0094] The results of MitoSOX staining and mtROS quantification in Mdivi-1-treated hypoxic bone marrow-derived macrophages are as Figure 5 (N) shown. According to the fact that mitochondrial fission induces the production of mitochondrial ROS (mtROS), reactive oxygen species (ROS) were detected. MitoSOX labeling showed that mtROS increased significantly in control mouse bone marrow-derived macrophages after hypoxia stimulation. However, this effect was attenuated when the mitochondrial fission inhibitor Mdivi-1 was present or SNX3 was depleted in macrophages. Consistent with the in vitro results, compared with the RA retina, mtROS production was significantly increased in the OIR retina, while SNX3 deletion in myeloid cells significantly attenuated OIR-induced mitochondrial superoxide production, as Figure 5 (O) shown.

[0095] MMP12 (matrix metalloproteinase 12, also known as macrophage elastase) is a member of the matrix metalloproteinase (MMPs) family and is mainly secreted by inflammatory cells such as macrophages. Its main function is to degrade extracellular matrix (ECM) components (such as elastin, fibronectin, etc.) and participate in tissue remodeling, inflammatory responses, and regulation of angiogenesis.

[0096] Heatmap results of the gene expression of matrix metalloproteinases (MMPs) in Snx3WT and Snx3ΔMΦ mouse CD11b + cells are as Figure 6 (A) shown. Mmp12 is the most upregulated MMP gene in the myeloid cells of the OIR mouse retina, and its expression is inhibited in the myeloid cells of the SNX3ΔMΦ OIR retina.

[0097] RT-PCR analysis results of MMPs in hypoxic-exposed bone marrow-derived macrophages in vitro are as Figure 6 (B) shown. In WT bone marrow-derived macrophages exposed to hypoxia, the mRNA level of Mmp12 increased significantly, but the mRNA levels of Mmp2 / 3 / 8 / 9 did not increase. However, when SNX3 was depleted in macrophages, the mRNA level decreased when macrophages were pretreated with Nec-1, GSK872, Mdivi-1, or the mitochondrial superoxide scavenger Mito-TEMPO, as Figure 6(C). Similar results were also obtained in retinal microglia exposed to hypoxic conditions. In addition, the change in MMP12 protein expression was consistent with the change in mRNA expression, as shown by Western blot Figure 6 (D).

[0098] Evaluate the angiogenic effect of SNX3-mediated activation of RIP1 / 3-MMP12 signaling in macrophages on endothelial cells. After knocking down Rip1 / Rip3 / Mmp12 with siRNA, the results of the effect of conditioned medium (CM) from hypoxic-treated bone marrow-derived macrophages on the sprouting of three-dimensional spheroids of human retinal endothelial cells (HRECs) are shown as Figure 6 (E-H). The 3D spheroid sprouting assay showed that conditioned medium (CM) from SNX3 WT bone marrow-derived macrophages exposed to hypoxia enhanced the sprouting of human retinal endothelial cells (HREC). When using conditioned medium from SNX3 knockout bone marrow-derived macrophages or bone marrow-derived macrophages treated with small interfering RNA (siRNA) of Rip1, Rip3, or Mmp12, the ability of CM from hypoxic-treated SNX3 WT macrophages to promote HREC sprouting was greatly attenuated. Further evaluate the angiogenic effect of SNX3-induced upregulation of RIP1 / 3-MMP12 signaling using an ex vivo angiogenesis model. Conditioned medium in hypoxic-exposed WT bone marrow-derived macrophages increased the sprouting of choroidal explants, while the pro-angiogenic effect was reduced when SNX3 was knocked out or RIP1 / 3 and MMP12 were knocked down, supporting the view that MMP12 expression in myeloid cells induced by SNX3 is a key regulator of ocular angiogenesis.

[0099] Example 6 Inhibition of RIP1 / 3 can reduce angiogenesis caused by overexpression of SNX3 in OIR mice

[0100] Use a gain-of-function method to evaluate the effect of overexpression of SNX3 in myeloid cells on retinal angiogenesis. Lysm-Cre mice are a type of genetically engineered mice in which the expression of Cre recombinase is driven by the lysozyme (LysM) promoter and is mainly specifically activated in myeloid cells (such as macrophages, neutrophils, etc.). It is used to achieve conditional gene knockout or expression in specific immune cell types and is widely used in immune and inflammation-related research. Cross SNX3 transgenic mice with Lysm-Cre mice to generate myeloid-specific SNX3 transgenic (SNX3 MΦ-TG) mice. This transgenic strain uses the Cre / loxP system, in which transgenic Cre expression is driven by the promoter of a mouse-specific gene (Lyz2). The specific process is shown as Figure 7 (A).

[0101] The results of isolectin-stained retinal whole mounts are shown as Figure 7As shown in (BD), retinal myeloid cells of SNX3MΦ-TG mice exhibited robust SNX3 protein expression compared with littermate negative controls (SNX3WT). Retinal neovascularization and avascular areas were significantly increased in OIR SNX3MΦ-TG mice compared with P17 control mice. However, intravitreal injection of RIP1 inhibitor (Nec-1) or RIP3 inhibitor (GSK872) significantly abolished SNX3 overexpression-induced retinal NV, indicating that RIP1 / 3 signaling functions downstream of SNX3 to regulate pathological angiogenesis in OIR.

[0102] Example 7 Structure and function of small molecule inhibitor W1122

[0103] Study the structure and function of the small molecule inhibitor W1122, such as Figure 8 (AC) shown.

[0104] VPS35 is a core subunit of the retromer complex, responsible for the recycling and sorting of membrane proteins from endosomes to the Golgi apparatus and plasma membrane, regulating protein transport, degradation and signal transduction. In addition to interacting with the N-terminus of SNX3, the oxygen-sulfur double bond of W1122 also forms a hydrogen bond with the Arg206 site of VPS35, while its benzothiophene structure forms a π stacking with the Phe28 site. In addition, the Lys203 site of VPS35 can also establish a cation-π stacking interaction with the benzene substituent of W1122, indicating that there is potential binding affinity and effective targeting between W1122 and the SNX3-retromer complex.

[0105] Surface plasmon resonance (SPR) analysis showed that W1122 exhibited moderate binding affinity to the full-length SNX3 protein, with a calculated dissociation constant (KD) of approximately 2.6 micromolar. Figure 8 (DE). Cellular thermal shift assay (CETSA) showed that in the presence of W1122, the stability of SNX3 protein increased significantly in the temperature range of 43°C to 51°C, as shown in Figure 8 (F) W1122 treatment downregulated hypoxia-induced SNX3 protein expression in bone marrow-derived macrophages and disrupted the interaction of SNX3 with RIP1 / 3 and VPS35, as shown in Figure 8 (G). In addition, immunofluorescence staining showed that W1122 reduced the co-localization of RIP1 / 3 and SNX3 under hypoxia stimulation, as shown in Figure 8 (HI). This indicates that W1122 can bind to SNX3 protein and function as a negative regulator and protein-protein interaction (PPI) inhibitor, hindering the scaffolding function of SNX3.

[0106] Check whether W1122 can mimic the effect of SNX3 deficiency on inhibiting RIP1 / 3 signaling and retinal neovascularization. As Figure 8 (J-L) shows, W1122 exhibits a strong inhibitory effect on the expression of RIP1 / 3 and p-DRP1, mtROS production, and necroptosis in hypoxia-induced bone marrow-derived macrophages. Consistent with SNX3 deficiency, the retinal neovascularization area and avascular area in OIR mice were significantly reduced by W1122, as Figure 8 (M-P).

[0107] The effect of different doses of W1122 on the viability of myeloid cells was detected by CCK-8 assay. Bone marrow-derived macrophages and mouse retinal microglia were treated with W1122, and their cell viability was observed after 24 hours of treatment. The results are as Figure 9 (A-B) shows that W1122 at a concentration of 0.25 - 5 μM has no significant toxicity to macrophages and microglia.

[0108] In summary, as an inhibitor of the sorting protein SNX3, W1122 can effectively inhibit RIP1 / 3 signaling and significantly inhibit retinal angiogenesis.

[0109] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a sorting nexin 3 (SNX3) inhibitor in the preparation of a drug for proliferative retinopathy.

2. The application according to claim 1, wherein The sorting nexin 3 (SNX3) inhibitor is a small molecule inhibitor W1122; the small molecule inhibitor W1122 is an imidazo[1,2-a]pyridine derivative with the chemical name of N-(1,1-dioxobenzo[b]thiophen-6-yl)-2-(7-methoxy-2-phenylimidazo[1,2-a]pyridin-3-yl)acetamide and the chemical formula of C 24 H 19 N3O4S, with a molecular weight of 445.49 g / mol; the chemical structural formula of the small molecule inhibitor W1122 is shown in Formula 1:

3. A small molecule inhibitor W1122, characterized in that, The dissociation constant of the small molecule inhibitor W1122 from the target protein SNX3 is 2.6 micromoles; the chemical structural formula of the small molecule inhibitor W1122 is shown in Formula 1:

4. Use of the small molecule inhibitor W1122 according to claim 3 in inhibiting or downregulating the expression of sorting nexin 3 (SNX3).

5. The application according to claim 4, wherein The small molecule inhibitor W1122 binds to the N-terminus of sorting nexin 3 (SNX3).

6. Use of the small molecule inhibitor W1122 according to claim 3 in the preparation of a drug for inhibiting RIP1 / 3 signal transduction.

7. The application according to claim 6, characterized in that The small molecule inhibitor W1122 binds to sorting nexin 3 (SNX3) and inhibits RIP1 / 3 signal transduction.

8. Use of the small molecule inhibitor W1122 according to claim 3 in the preparation of a drug for inhibiting the expression of MMP12 gene.

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