Use of a sorting nexin 3 inhibitor for the preparation of a medicament for proliferative retinopathies

Abstract

The application belongs to the technical field of biomedicine, and particularly relates to an application of a sorting protein SNX3 inhibitor in preparation of a proliferative retinopathy drug. The application verifies that overexpression of a sorting protein (SNX3) induces pathological angiogenesis, and SNX3 is a new target point of a pathological neovascular disease. Based on a protein structure of SNX3, a small molecule inhibitor W1122 is identified. A chemical structural formula of the small molecule inhibitor W1122 is shown as formula 1, the small molecule inhibitor W1122 belongs to an imidazo[1,2-a]pyridine derivative, a chemical name is N-(1,1-dioxo-benzo[b]thiophene-6-yl)-2-(7-methoxy-2-phenylimidazo[1,2-a]pyridine-3-yl)acetamide, a chemical formula is C 24 H 19 N3O4S, a molecular weight is 445.49 g / mol, and the small molecule inhibitor W1122 can effectively inhibit retinal pathological angiogenesis. The results of the examples of the application show that the small molecule inhibitor W1122 can effectively prevent and treat proliferative retinopathy by inhibiting expression of SNX3, and it is indicated that the sorting protein SNX3 provides a new drug target point for treating proliferative retinopathy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a sorting protein SNX3 inhibitor W1122 in the preparation of a drug for proliferative retinopathy. Background Technology

[0002] Proliferative retinopathy is a leading cause of blindness in people of all ages, including newborns (retinopathy of prematurity), middle-aged individuals (proliferative diabetic retinopathy), and the elderly (age-related macular degeneration). Currently, the main clinical treatment for proliferative retinopathy is vascular endothelial growth factor (VEGF) antagonists. Although VEGF antagonists have shown some efficacy in treating these proliferative retinopathy conditions, many patients still experience primary resistance or a high relapse rate. Further research into the pathological mechanisms of proliferative retinopathy has revealed that myeloid cells (especially microglia and macrophages) are the main participants in pathological retinal angiogenesis. Since VEGF is almost not expressed in retinal microglia and macrophages, there is an urgent need to find new therapeutic targets that act on retinal microglia and macrophages.

[0003] Immune cells, particularly macrophages and microglia, act as immune surveillance guardians and are key effectors and regulators in the retinal microenvironment. They undergo a series of specific activation processes to respond to disruptions in retinal homeostasis caused by various stimuli or stresses, such as ischemia / hypoxia, neuroinflammation, traumatic stress, and degeneration. Activated immune cells frequently undergo apoptosis or death within the central nervous system to eliminate overactive inflammatory responses and maintain immune tolerance. New evidence suggests 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, remains largely unknown.

[0004] Therefore, efforts to discover key regulators of the necrosis-apoptosis pathway are crucial for identifying new drug screening targets. Summary of the Invention

[0005] The purpose of this invention is to provide an application of a sorting protein SNX3 inhibitor in the preparation of a drug for proliferative retinopathy. The small molecule inhibitor W1122 provided by this invention can bind to sorting protein SNX3, thereby inhibiting sorting protein SNX3 and its regulated downstream RIP1 / 3 signaling pathway, and promoting the transformation of sorting protein SNX3 as a new therapeutic target for proliferative retinopathy.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of a sorting protein SNX3 inhibitor in the preparation of drugs for proliferative retinopathy.

[0008] Preferably, the sorting protein SNX3 inhibitor is a small molecule inhibitor W1122; the small molecule inhibitor W1122 is an imidazo[1,2-a]pyridine derivative, chemically named N-(1,1-dioxobenzo[b]thiophene-6-yl)-2-(7-methoxy-2-phenylimidazo[1,2-a]pyridin-3-yl)acetamide, with the chemical formula 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:

[0009]

[0010] This invention also provides a small molecule inhibitor W1122, wherein the dissociation constant between the small molecule inhibitor W1122 and the target protein SNX3 is 2.6 μmol; the chemical structural formula of the small molecule inhibitor W1122 is shown in Formula 1:

[0011]

[0012] This invention also provides the application of the above-mentioned small molecule inhibitor W1122 in inhibiting or downregulating the expression of sorting protein SNX3.

[0013] Preferably, the small molecule inhibitor W1122 binds to the N-terminus of the sorting protein SNX3.

[0014] This invention also provides the application of the above-mentioned small molecule inhibitor W1122 in the preparation of drugs that inhibit RIP1 / 3 signaling.

[0015] Preferably, the small molecule inhibitor W1122 binds to the sorting protein SNX3 to inhibit RIP1 / 3 signaling.

[0016] This invention also provides the application of the above-mentioned small molecule inhibitor W1122 in the preparation of drugs that inhibit MMP12 gene expression.

[0017] The beneficial effects of this invention are:

[0018] This invention provides a potential therapeutic target for the treatment of proliferative retinopathy, namely the sorting protein SNX3.

[0019] This invention discovers a novel small molecule inhibitor, W1122, that inhibits or downregulates the expression of sorting protein SNX3. This small molecule inhibitor can bind to sorting protein SNX3, effectively inhibiting RIP1 / 3 signal transduction and alleviating retinal neovascularization in retinal diseases. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. The drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Figure 1 shows the results of experiments on the upregulation of sorting protein SNX3 expression in the retinas of patients with proliferative diabetic retinopathy and rodents with oxygen-induced retinopathy (OIR); (AB) shows the heatmap analysis results of SNX3 gene, myeloid cell markers, and necrosis-apoptosis-related genes expression in the neovascular membrane of the retina of patients with proliferative diabetic retinopathy. The data are from the reanalysis of previously published microarray datasets (GSE60436, A, n=3) or transcriptome sequencing datasets (GSE102485, B, n=2-4); (CF) shows the single-cell RNA sequencing analysis of CD11b in OIR mice. + F4 / 80 + (C) Snx3 gene expression results in retinal cells; (D) t-SNE distribution map of retinal cells in normoxic and OIR mice on day 17; (E) Cell type identification results based on marker abundance; (G) GO, KEGG and Reactome enrichment analysis of CD11b in OIR mice. + F4 / 80 + Pathway enrichment map of cells; (F) Violin diagram of Snx3 gene expression; (G) Representative images of CD11b, heterolectin B4 and DAPI immunofluorescence staining in OIR retina (n=4); (H) Laser capture microdissection technique for separating myeloid-enriched neovascular clusters: heterolectin and DAPI staining results in normoxic and OIR retinal frozen sections (scale bar: 50 μm, white dashed line is the laser-cut area, MΦ: myeloid cells); (I) RT-PCR analysis of Snx3 mRNA in laser-captured samples (n=4); (JM) Colocalization image of SNX3 (green) with macrophages / microglia (F4 / 80, J), myeloid cells (CD11b, L) and blood vessels (heterolectin) in whole retinal patches of OIR mice (NC: negative control, SNX3 fluorescence intensity quantified by ImageJ, K and M); (NQ) MACS sorting of CD11b +RT-PCR (O, n=6) and Western blot (PQ, n=3) analysis of cells. 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, and four asterisks represent P<0.0001).

[0022] Figure 2 Figure 1 shows the results of an experiment to reduce retinal neovascularization and vascular leakage in OIR mice by systemic or myeloid-specific knockout of SNX3; (AC) Retinal patch stained with isoglucan B4 showing areas of vascular occlusion (yellow) and neovascularization (white) (n=10, scale bar: 500 μm, original image; 200 μm, enlarged image); (D) Whole retinal staining showing hemorrhage points in control OIR mice (white arrow, n=6); (E) FITC-glucan perfusion assessment of blood-retinal barrier integrity (n=6, scale bar: 50 μm); (FG) Retinal albumin West ernblot analysis (n=6); (HJ) 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); (KL) Assessment of hemorrhage points and blood-retinal barrier (n=6); (MN) Albumin expression analysis (n=6). 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, and three asterisks in the figure represent P<0.001);

[0023] Figure 3 Figure 1 shows the results of an experiment on the inhibition of hypoxia-induced necrosis and apoptosis in myeloid cells and the release of pro-inflammatory / pro-angiogenic factors by SNX3 deficiency; (A) Single-cell sequencing results show that CD11b in OIR mice... + F4 / 80 + Enrichment of the cell necrosis-apoptosis pathway (threshold: gene expression in ≥10% of cells, corrected P<0.05, log2 fold change |>0.585); (BE) Immunofluorescence staining results, where C represents the pathway associated with Iba-1. + Colocalization and quantification of cells in retinal patch (scale bar: 20 / 5 micrometers, n = 5 × 3); (F) Flow cytometry sorting of CD11b + Western blot analysis of RIP1 / 3 and FGF2 in cells (n=4); (GH) RIP1 / 3 expression 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) Results of TUNEL co-staining with activated caspase-3 to detect necrosis and apoptosis (n=3×3); (K) CD11b in OIR mice+ Heatmap of pro-inflammatory / pro-angiogenic genes (n=4); (LP) Detection of Ripk1 / Ripk3 mRNA and cytokines in hypoxic microglia by enzyme-linked immunosorbent assay (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 represent P<0.01, and three asterisks represent P<0.001).

[0024] Figure 4 The experimental structure for downregulating SNX3 protein expression by enhancing RIP1 / 3 degradation is shown below; (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 μmol CoCl2) and after different time periods of cycloheximide treatment; relative protein levels were calculated by the ratio of band intensity at each time point to time 0 (n=3); (B) Immunofluorescence colocalization images of SNX3 and RIP1 / RIP3 / VPS35 in bone marrow-derived macrophages (red: SNX3, green: RIP1 / RIP3 / VPS35, scale bar: 5 μm); colocalization rate was analyzed using ImageJ software; (CD) Immunoprecipitation of FLAG antibody on Ad-FLAG-SNX3-infected bone marrow-derived macrophages (C) and microglia (D), and Western blot detection of SNX3 and RIP1 / VPS35. RIP3 / VPS35 interaction (non-immune IgG as control); (EF) Colocalization of RIP1 (E) or RIP3 (F) with lysosomal marker LAMP1 in bone marrow-derived macrophages of 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 bone marrow-derived macrophages induced by hypoxia after 24 hours of chloroquine (CQ, 10 ng / mL) treatment (n=4); (H) RIP1 / 3 expression in cell lysate and lysosomal fraction of chloroquine-treated bone marrow-derived macrophages induced by hypoxia (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 represent P<0.01, three asterisks represent P<0.001, and there was no significant difference in ns);

[0025] Figure 5Figure 1 shows the results of experiments on the inhibition of RIP1 / 3-mediated mitochondrial division and mtROS generation by SNX3 deficiency; (A) Oxygen consumption rate curves of Snx3WT and Snx3ΔMΦ bone marrow-derived macrophages after treatment with Nec-1 (10 μmol) / GSK872 (10 μmol) under normoxic (21% O2) or hypoxic (1% O2) conditions; (BC) Quantitative analysis of mitochondrial functional parameters (basal respiration, maximal respiration, etc.) (n = 5-8); (DE) Mitotracker RedFM staining shows the mitochondrial signal intensity in hypoxic bone marrow-derived macrophages (scale bar: 5 μm, n=10); (FG) transmission electron microscopy images show the mitochondrial morphology of bone marrow-derived macrophages under hypoxic conditions (scale bar: 500 nm) and the number of mitochondria per cell (n=8); (HI) Western blot analysis of phosphorylated DRP1 (p-DRP1) in bone marrow-derived macrophages under hypoxic conditions (n=4); (JK) Effects of Nec-1, GSK872, or Mdivi-1 treatment on p-DRP1 expression (n=4); (LM) Flow cytometry sorting of CD11b. + Western blot of p-DRP1 in cells (n=3); (N) MitoSOX staining and mtROS quantification in bone marrow-derived macrophages under hypoxic conditions treated with Mdivi-1 (scale bar: 50 μm, n=6); (O) MitoSOX staining of OIR mouse retina (scale bar: 100 μm, n=5, 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, and three asterisks in the figure represent P<0.001);

[0026] Figure 6Figure 1 shows the results of an experiment in which SNX3 deficiency downregulates MMP12 through inhibition of RIP1 / 3-mediated mitochondrial division; (A) Heatmap of matrix metalloproteinase (MMP) 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; an 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 normoxic; P<0.01 vs Snx3WT normoxic). Snx3WT hypoxia); MMP12 mRNA (C, RT-PCR, n=4) and protein (D, Western blot, n=3) expression in hypoxic bone marrow-derived macrophages treated with (CD) Nec-1, GSK872, Mdivi-1, or Mito-TEMPO (###P<0.001 vs) DMSO normoxic; one asterisk in the figure indicates P<0.01 / 0.001 vs DMSO hypoxia); (EH) Effect of bone marrow-derived macrophage conditioned medium (CM) on the budding of three-dimensional spheres of human retinal endothelial cells (HRECs) under hypoxic conditions after siRNA knockdown of Rip1 / Rip3 / Mmp12: (E) Representative image (scale bar: 50 μm); (F) Quantitative budding length (n=10); (G) Choroidal budding image (scale bar: 500 μm); (H) Quantitative budding area (n=8, data are expressed as mean ± standard error, ###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 Figure 1 shows the results of an experiment to inhibit reversible SNX3 overexpression-driven retinal neovascularization in OIR mice with RIP1 / 3; (A) Schematic diagram of experimental design: Snx3MΦ-TG (myeloid-specific SNX3 overexpression transgene) OIR mice were injected intravitreally with either the RIP1 inhibitor Nec-1 or the RIP3 inhibitor GSK872; (BD) Retinal patch stained with isoglucosin showing: (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) Area of ​​pathological neovascularization; (D) Quantitative analysis of vascular occlusion area (n=10, 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, and three asterisks in the figure represent P<0.001);

[0028] Figure 8 Figure 1 shows the experimental results of synergistic inhibition of retinal neovascularization by combination therapy of SNX3 inhibitor and anti-VEGF; (A) Structural diagram of W1122; (B) Molecular docking results of W1122 with SNX3-retromer (SNX3-VPS26-VPS35) (PDB: 5F0J); (C) Modeling results of W1122 with SNX3-retromer (PDB: 5F0J); Left figure: Binding mode of W1122 with SNX3-retromer, marking key residues around the W1122-SNX3-VPS26-VPS35 pocket, W112 Hydrogen bonds between the dual atoms and key VPS35 residues and the Pi cation are shown as dashed lines (pink and light green, respectively); Right panel: Detailed interaction between W1122 and pocket residues; (DE) Surface plasmon resonance (SPR) analysis results; (F) Cell thermal displacement analysis (CETSA) results; (G) Immunoprecipitation results; (HI) Representative staining image; (J) Western blot analysis results; (KL) TUNEL analysis and lysed caspase-3 staining results; (L) Culture image; (M) Received intravitreal (IVT) injection of W1122 and / or VEGF A schematic diagram of Nab OIR mice; (NP) a representative image of a heterolectin-stained retinal plain of OIR mice at P17; (N) quantification of neovascular (O) and avascular (P) regions, represented in white and yellow respectively (n = 10 retinas; scale bar: original image 500 μm, magnified image 200 μm, data are 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);

[0029] Figure 9 The figure shows the experimental results of the effect of W1122 on myeloid cell viability. (A) Cell viability of bone marrow-derived macrophages; (B) Cell viability of mouse retinal microglia (n=6, 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: no significant difference). Detailed Implementation

[0030] This invention provides a novel therapeutic target for proliferative retinopathy—the sorting protein SNX3, a member of the sorting connexin (SNX) family that participates in endosomal sorting and transport and is significantly upregulated in myeloid cell subsets of the retina in oxygen-induced retinopathy (OIR).

[0031] This invention demonstrates that the expression of the sorting protein SNX3 is significantly increased in retinal microglia / macrophages of mice with ocular irritation (OIR), primarily located within and around the neovascular plexus. Both global and myeloid-specific knockout of SNX3 inhibited retinal angiogenesis in OIR mice. Mechanistically, the SNX3 retromer interacts with RIP1 and RIP3, mediating their intracellular circulation and thus protecting them from lysosomal degradation.

[0032] Based on the protein structure of the sorting protein SNX3, this invention provides a small molecule inhibitor of SNX3, W1122. The small molecule inhibitor W1122 belongs to the imidazo[1,2-a]pyridine derivatives, with the chemical name N-(1,1-dioxobenzo[b]thiophene-6-yl)-2-(7-methoxy-2-phenylimidazo[1,2-a]pyridin-3-yl)acetamide, and the chemical formula C1122. 24 H 19 N3O4S has a molecular weight of 445.49 g / mol. The chemical structure of the small molecule inhibitor W1122 is shown in Formula 1.

[0033] The small molecule inhibitor W1122 has a dissociation constant (KD) of 2.6 μmol with its target protein SNX3. A smaller KD value indicates a tighter binding between the drug and the target protein, resulting in higher affinity. This small molecule inhibitor W1122 inhibits the protein expression of the sorting protein SNX3 by binding to its N-terminus, effectively suppressing retinal angiogenesis. Furthermore, it exhibits a synergistic anti-angiogenic effect when combined with anti-vascular endothelial growth factor therapy.

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

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

[0036] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention 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 distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0037] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

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

[0039] 1.1 Mendelian randomization (MR) was used to analyze the causal relationship between the expression of all members of the SNX gene family and retinal diseases, and to investigate the role of the SNX gene in retinal diseases.

[0040] The results of Mendelian randomization analysis showed that SNX3 levels were causally associated with a significantly higher incidence of diabetic retinopathy (odds ratio, OR = 3.8032, 95% confidence interval (CI) [1.0937, 13.2247], P = 0.036).

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

[0042] The results are as follows Figure 1 As shown in (AB), in both datasets, compared with the retinas of healthy controls, the retinas of patients with proliferative diabetic retinopathy showed significantly increased gene expression of SNX3, necrosis-related genes (RIPK1, RIPK3, and MLKL), and myeloid cell markers (C1QA, TREM2, ITGAM, and PTPRC), indicating that the expression of the sorting protein SNX3 is associated with myeloid cell necrosis and apoptosis and the development of proliferative diabetic retinopathy.

[0043] 1.3 A complete description of the transcriptional profile of SNX-related genes in myeloid cells of retinal diseases.

[0044] Single-cell transcriptome analysis was performed in OIR mice using the 10XGenomics platform. OIR mice are OIR-deficient mouse models that partially mimic certain aspects of retinopathy of prematurity and proliferative diabetic retinopathy in humans.

[0045] CD11b is an integrin molecule that typically binds to CD18 to form the CD11b / CD18 complex, participating in cell-cell adhesion and signal transduction. CD11b primarily marks monocytes and macrophages, which play crucial roles in the immune system, engulfing and digesting pathogens and participating in inflammatory responses and immune regulation. F4 / 80 is a cell membrane glycoprotein widely expressed in various mature macrophages, including Kupffer cells, Langerhans cells, microglia in the liver, and macrophages located in the peritoneum, lungs, intestines, thymus, spleen red pulp, and bone marrow stroma. F4 / 80 is a marker of mature mouse macrophages and is used in flow cytometry detection. + CD11b + It can effectively distinguish other types of monocytes.

[0046] CD11b-positive and F4 / 80-positive myeloid cells were enriched from the retinas (n=16) of normoxic or OIR mice by flow cytometry sorting. CD45+ cells were first screened from the total cell population. + Myeloid cells (n = 1545 cells) were then subjected to unsupervised clustering using the "Seurat" package for downstream analysis. The myeloid cells were clustered into four distinct clusters: resting microglia, activated microglia, macrophages, and monocytes. Results are as follows: Figure 1 As shown in (CD), genes highly expressed in cells of resting microglia (mainly from the retinas of control mice under normoxic conditions), such as P2ry12, Siglech, and Tmem119, are associated with microglia homeostasis, while markers highly expressed in cells of activated microglia (significantly enriched in the OIR retina), such as Cst7, Lpl, and Spp1, are also highly expressed.

[0047] Pathway enrichment analyses using GO, KEGG, and Reactome revealed that SNX-related pathways (e.g., early endosome, endosome membrane, and endocytic circulation) and necrotizing-apoptotic pathways were enriched in microglia / macrophages from OIR retina. Figure 1 As shown in (E). Among the SNX gene family members, SNX3 is the most significantly upregulated gene in the myeloid cells of the OIR mouse retina (P = 5.96E-50, Wilcoxon rank-sum test, OIR vs RA).

[0048] A violin diagram illustrating Snx3 gene expression is shown below. Figure 1(F) shows that the gene expression of key components of the SNX3 and reverse transcription complex (including Vps29 and Vps35) in macrophages and activated microglia of the OIR retina was significantly upregulated.

[0049] CD11b and heterolectin B4 in the OIR retina were observed using DAPI immunofluorescence staining. The immunofluorescence staining results are as follows: Figure 1 As shown in (G), CD11b was observed. + Myeloid cells aggregate in the neovascular plexus region of the OIR retina, as shown in a three-dimensional (3D) image of the planar 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 myeloid-rich neovascular plexuses from OIR retina. The results of heterolectin and DAPI staining in frozen sections of RA and OIR retina are shown in the figure below. Figure 1 As shown in (HI), SNX3 expression was increased in the neovascular plexus rich in myeloid cells in OIR mice compared with normal retinal vessels in control mice.

[0052] Immunoretinal staining of the entire retina revealed a significant increase in SNX3 expression levels in OIR retina, primarily observed in F4 / 80 and CD11b-positive macrophages / microglia. Figure 1 (JM) As shown. SNX3-positive macrophages / microglia aggregated within and around the neovascular plexus of the OIR retina. CD11b was sorted by MACS. + RT-PCR and Western blot analysis of cells revealed increased SNX3 expression in myeloid cells from OIR retina isolated using anti-CD11b microbeads. Figure 1 As shown in (NQ). In summary, the above results indicate that elevated SNX3 expression in retinal myeloid cells is associated with proliferative retinopathy in humans and rodents.

[0053] Example 2: Whole-body and bone marrow-specific SNX3 knockout reduces retinal angiogenesis and vascular leakage in OIR mice.

[0054] 2.1 Study on the role of SNX3 in retinal diseases

[0055] SNX3 flox / flox mice are homozygous genetically engineered mice in which loxP sites are inserted flanking both alleles of SNX3. 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 genetically engineered mice in which the Cre / ERT2 fusion gene is inserted at the Rosa26 safe site. Tamoxifen induction can activate Cre recombinase activity, achieving time-specific (drug-induced) and space-specific (promoter-dependent) gene editing, commonly used for conditional temporally controlled gene knockout or expression.

[0056] 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 newborn mice by tamoxifen administration. Rosa26 Cre / ERT2 littermates were used as wild-type (SNX3 WT) controls.

[0057] Experimental results are as follows Figure 2 As shown in (AC), retinal patch stained with hemagglutinin B4 reveals vascular occlusion areas (VO, yellow) and neovascularization (NV, white area). Tamoxifen treatment significantly reduced SNX3 expression in the retinas of SNX3iKO mice, confirming successful SNX3 knockout. Compared to mice with an OIR model induced by SNX3 WT (SNX3 WT OIR), mice with an OIR model induced by SNX3iKO at P17 (SNX3 iKO OIR) showed a significant reduction in the area of ​​neovascular plexuses and vascular occlusion areas in their retinas.

[0058] This study investigates whether SNX3 deficiency improves hypervascular permeability, a characteristic feature of pathological angiogenesis. Gross retinal photographs are shown below. Figure 2 As shown in (D), SNX3 iKO OIR mice exhibited less retinal hemorrhage compared to SNX3 WT OIR mice. Furthermore, compared to the control group, the retinas of SNX3 iKO OIR mice showed reduced retinal vascular permeability, confirmed by fluorescein angiography using FITC-glucan and AlexaFluor594-conjugated isoglucan B4 (IB4). The results of FITC-glucan perfusion assessment of blood-retinal barrier integrity are as follows: Figure 2 As shown in (E). Retinal protein imprint analysis indicated that the albumin level in the retina of the SNX3 iKO OIR (a marker of capillary leakage) was lower than that in the control group, such as... Figure 2 As shown in (FG).

[0059] 2.2 Determining the myeloid-cell-specific role of SNX3 in retinal NV

[0060] By mating SNX3 flox / flox mice with Lysm-Cre mice, myeloid-specific SNX3 knockout mice (SNX3ΔMΦ) and littermate control mice (SNX3 WT) were generated. Compared with SNX3 WT mice, SNX3 expression 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 vessels under normoxic conditions is not affected by the absence of SNX3 in myeloid cells. Similar to SNX3iKO, the retina of SNX3ΔMΦOIR mice at P17 showed decreased areas of neovascular clusters, avascular area, and vascular permeability compared to the control group. Figure 2 As shown in (HN), Figure 2 (HJ) is used for quantitative detection of retinal NV and avascular areas in Snx3ΔMΦOIR and Snx3WT OIR mice; Figure 2 (KL) is used for assessment of hemorrhage points and the blood-retinal barrier; Figure 2 (MN) represents the albumin expression analysis results.

[0062] In summary, these results indicate that myeloid cell-specific SNX3 knockout can significantly reduce retinal NV and vascular leakage in OIR.

[0063] Example 3: SNX3 deficiency can inhibit hypoxia-induced necrotizing apoptosis in myeloid cells and the subsequent expression and release of pro-inflammatory and pro-angiogenic cytokines.

[0064] 3.1 Analysis of the potential role of SNX3 in regulating myeloid cell necrosis and apoptosis during retinopathy

[0065] Pathway enrichment analysis of scRNA-seq data from myeloid cells of the retina of RA / OIR mice revealed that genes associated with the necrosis-apoptosis pathway and the SNX-retromeracile complex were significantly involved in microglia (especially active microglia) and macrophages in the OIR retina. Figure 3 As shown in (A).

[0066] Immunostaining revealed IBa1 within the neovascular plexus of the OIR retina. + The fluorescence intensity of RIP1 and RIP3, two key components of the necroptotic pathway in microglia / microphages, was significantly increased, as shown in Figure 1. Figure 3As shown in (BE). SNX3 deficiency significantly reduced OIR-induced RIP1 / 3 staining elevation.

[0067] Western blot analysis showed that CD11b isolated from the OIR retina + Protein levels of RIP1 / 3 in cells were upregulated, while SNX3 deficiency significantly reduced OIR-induced RIP1 / 3 expression levels, such as... Figure 3 As shown in (F). Mouse macrophages exposed to hypoxic conditions in vitro, as shown in... Figure 3 (G) and retinal microglia such as Figure 3 Similar results were obtained in (H). Conversely, the analysis of the effect of adenovirus-mediated SNX3 overexpression on RIP1 / 3 showed that SNX3 overexpression induced a significant increase in RIP1 / 3 expression in microglia, such as... Figure 3 As shown in (I).

[0068] In summary, both loss-of-function and gain-of-function studies indicate that SNX3 plays a crucial role in regulating RIP1 / 3 protein expression. Since the kinases RIP1 / 3 are key triggers for necroptosis, it is logical to determine whether SNX3 regulates necroptosis in myeloid cells. SNX3 knockout significantly counteracted necroptosis in hypoxic retinal microglia, and necroptosis (TUNEL) isolated from SNX3ΔMΦ mice was significantly reduced. + caspase-3 - As indicated by the decrease in cell number, the results of TUNEL co-staining with activated caspase-3 in the analysis of necrosis and apoptosis are as follows: Figure 3 As shown in (J).

[0069] 3.2 SNX3 affects the expression and release of pro-inflammatory and pro-angiogenic cytokines, mediating retinal angiogenesis.

[0070] First, CD11b isolated from SNX3ΔMΦOIR mice was analyzed. + Gene expression profile in retinal cells. CD11b in OIR mice. + The results of the heatmap of pro-inflammatory / pro-angiogenic genes are as follows: Figure 3 As shown in (K), SNX3 deficiency significantly reduced CD11b isolated from OIR mice. + mRNA expression of key pro-inflammatory and pro-angiogenic cytokines (including Il1b, Il6, Tnfα, and Fgf2) in myeloid cells.

[0071] ELISA results of Ripk1 / Ripk3 mRNA in microglia under hypoxic conditions are as follows: Figure 3As shown in (L), the protein levels of RIP1 / 3 were significantly reduced in SNX3 knockout myeloid cells, but their mRNA levels remained unchanged. Pro-inflammatory cytokines and FGF2 produced by cultured retinal microglia were assessed using ELISA. Figure 3 As shown in (MP), protein levels of IL-1β, TNF-α, IL-6, and FGF2 were elevated in the culture medium of hypoxic-exposed retinal microglia, but this effect was counteracted when SNX3 was knocked out.

[0072] Western blot analysis of FGF2 indicated that CD11b isolated from the SNX3ΔMΦOIR retina... + The expression of FGF2 protein in cells was reduced; similar results were obtained in hypoxic retinal microglia cultured from SNX3ΔMΦ mice, such as... Figure 4 (Q).

[0073] Overall, these data suggest that SNX3 promotes RIP1 / 3-mediated necrotizing apoptosis and the 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 Validation experiment on the effect of SNX3 knockout on the degradation of RIP1 / 3 proteins

[0076] SNX3 knockout downregulates RIP1 / 3 protein expression 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. Western blot analysis of RIP1 / 3 protein levels in Snx3WT and Snx3ΔMΦ bone marrow-derived macrophages after different durations of hypoxia (100 μmol CoCl2) and cycloheximide treatment is shown in the following results. Figure 4 As shown in (A), in SNX3 knockout bone marrow-derived macrophages, the protein level of RIP1 / 3 (but not the protein level of MLKL) decreased significantly faster than in control cells.

[0078] Immunofluorescence staining and co-immunoprecipitation (Co-IP) assays were used to assess protein colocalization and interactions. Immunofluorescence colocalization images of SNX3 and RIP1 / RIP3 / VPS35 in bone marrow-derived macrophages showed that RIP1 / 3 and reverse transcriptase proteins (SNX3 and VPS35) colocalized in macrophages such as... Figure 4 (B) and microglia are located in the same cell compartment.

[0079] Co-IP assays showed that macrophages transfected with FLAG-labeled SNX3, such as Figure 4 (C) and microglia such as Figure 4 In (D), SNX3 is physically related to RIP1 / 3 and VPS35.

[0080] 4.2 Verification of the interaction between SNX3 and RIP1 / 3 and its regulation of intracellular circulation and degradation

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

[0082] The colocalization results of RIP1(E) or RIP3(F) with the lysosomal marker LAMP1 in Snx3WT and Snx3ΔMΦ bone marrow-derived macrophages under hypoxic treatment are as follows: Figure 4 As shown in (EF), the loss of SNX3 enhanced the co-localization of RIP1 / 3 with LAMP1 and reduced the level of SNX3 protein, indicating that the loss of SNX3 facilitates the transport of RIP1 / 3 to lysosomes for degradation.

[0083] Chloroquine (CQ) was used to inhibit endosome and lysosomal degradation in myeloid cells. Western blot results of RIP1 / 3 protein in hypoxic-treated bone marrow-derived macrophages after 24 hours of chloroquine (CQ, 10 ng / mL) treatment are shown below. Figure 4 As shown in (G), CQ treatment increased the level of RIP1 / 3 protein in bone marrow-derived macrophages of control mice and restored the reduced level of RIP1 / 3 in SNX3-deficient macrophages.

[0084] To further determine whether SNX3 deficiency accelerates RIP1 / 3 degradation via the lysosomal pathway, the lysosomal fraction was isolated to detect RIP1 / 3 protein expression by Western blot analysis.

[0085] The study of RIP1 / 3 expression in CQ-treated hypoxic bone marrow-derived macrophage cell lysates and lysosomal fractions showed that in SNX3-deficient bone marrow-derived macrophages, RIP1 / 3 proteins were encapsulated and transported to lysosomes, such as... Figure 5 (H). Therefore, we identified RIP1 / 3 as a novel interacting protein of SNX3, and the absence of SNX3 promotes the transport of RIP1 / 3 to lysosomes for degradation.

[0086] Example 5. Knocking out 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 the Seahorse Cell Metabolism Analyzer

[0088] The oxygen consumption rate (OCR) curves and quantitative results of mitochondrial functional 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 normoxic (21% O2) or hypoxic (1% O2) conditions are shown below. Figure 5 As shown in (AC), hypoxia exposure leads to significant impairment in WT bone marrow-derived macrophages (OCR), including mitochondrial respiration and ATP production. These effects are reversed when SNX3 in macrophages is depleted. Similarly, administration of the RIP1 or RIP3 inhibitors Nec-1 or GSK872 alleviates the effects of hypoxia on WT macrophages.

[0089] In addition, Mitotracker Red FM staining showed the following results regarding mitochondrial signal intensity in hypoxic bone marrow-derived macrophages: Figure 5 As shown in (DE), hypoxic WT macrophages exhibited decreased fluorescence of mitochondrial-specific detection probes. Conversely, confocal imaging of SNX3ΔMΦ bone marrow-derived macrophages revealed an expanded mitochondrial network and enhanced mitochondrial signaling, indicating an increase in mitochondrial volume or number in the absence of SNX3.

[0090] Transmission electron microscopy (TEM) image analysis of mitochondrial morphology in hypoxic bone marrow-derived macrophages: Figure 5 As shown in (FG), transmission electron microscopy images of hypoxic-stimulated SNX3ΔMΦ bone marrow-derived macrophages reveal significantly enlarged mitochondrial structures, compared to smaller mitochondria observed in SNX3WT macrophages. The increase in mitochondrial number per cell was only observed in hypoxic SNX3WT macrophages; however, this was not observed in SNX3ΔMΦ cells. These data suggest that SNX3 influences mitochondrial fission. Consistent with this view, activation of the mitochondrial fission protein, dynein-associated protein 1 (DRP1), and its phosphorylation (p-DRP1) level are enhanced in hypoxic bone marrow-derived macrophages, whereas these levels are lower in the absence of SNX3 (Figure 5H-I) or in the presence of RIP1 or RIP3 inhibitors such as... Figure 5 In the case of (JK), this activation is weakened.

[0091] Based on in vitro study results, CD11b was sorted by MACS. + Western blot analysis results of p-DRP1 in cells are as follows: Figure 5 (LM) showed that CD11b isolated from the retina of OIR mice was significantly different from that in RA mice. + The level of p-DRP1 in cells was elevated, while the absence of SNX3 significantly downregulated OIR-induced DRP1 phosphorylation.

[0092] The results in summary 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] MitoSOX staining and mtROS quantification results in hypoxic bone marrow-derived macrophages treated with Mdivi-1 are as follows: Figure 5 As shown in (N), reactive oxygen species (ROS) were detected based on the fact that mitochondrial fission induces the production of mitochondrial ROS (mtROS). MitoSOX labeling showed a significant increase in mtROS 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 when SNX3 in macrophages was depleted. Consistent with in vitro results, OIR retina showed a significant increase in mtROS production compared to RA retina, while SNX3 deficiency in myeloid cells significantly attenuated OIR-induced mitochondrial superoxide production, such as... Figure 6 As shown in (O).

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

[0096] Snx3WT and Snx3ΔMΦ mouse CD11b + The results of the heatmap of matrix metalloproteinase (MMP) gene expression in cells are as follows: Figure 6 As shown in (A), Mmp12 is the most upregulated MMP gene in OIR mouse retinal myeloid cells, while its expression is suppressed in SNX3ΔMΦOIR retinal myeloid cells.

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

[0098] The effects of SNX3-mediated macrophage RIP1 / 3-MMP12 signaling activation on endothelial cell angiogenesis were evaluated. The effects of siRNA knockdown of Rip1 / Rip3 / Mmp12 on hypoxic-treated bone marrow-derived macrophage conditioned medium (CM) on three-dimensional spheroid budding of human retinal endothelial cells (HRECs) were assessed. Figure 7 As shown in (EH), the 3D spheroid budding assay demonstrated that conditioned medium (CM) from hypoxic-exposed SNX3 WT bone marrow-derived macrophages enhanced budding of human retinal endothelial cells (HRECs). The ability of CM from hypoxic-treated SNX3 WT macrophages to promote HREC budding was significantly attenuated when conditioned medium was used from SNX3 knockout bone marrow-derived macrophages or from bone marrow-derived macrophages treated with small interfering RNA (siRNA) of Rip1, Rip3, or Mmp12. The role of SNX3-induced upregulation of RIP1 / 3-MMP12 signaling in angiogenesis was further evaluated using an in vitro angiogenesis model. Conditioned medium in hypoxic-exposed WT bone marrow-derived macrophages increased choroidal explant budding, while the pro-angiogenic effect was reduced when SNX3 was knocked out or RIP1 / 3 and MMP12 were knocked down, supporting the view that SNX3-induced MMP12 expression in myeloid cells is a key regulator of ocular angiogenesis.

[0099] Example 6: RIP1 / 3 inhibition can alleviate angiogenesis induced by SNX3 overexpression in OIR mice.

[0100] This study used a function-gain approach to evaluate the effects of SNX3 overexpression in myeloid cells on retinal angiogenesis. Lysm-Cre mice are genetically engineered mice in which the expression of the Cre recombinase is driven by the lysozyme (LysM) promoter, primarily activated in myeloid cells (such as macrophages and neutrophils). This technology is widely used in immunological and inflammation-related research to achieve conditional gene knockout or expression in specific immune cell types. By crossing SNX3 transgenic mice with Lysm-Cre mice, myeloid-specific SNX3 transgenic mice (SNX3 MΦ-TG) were generated. This transgenic strain used the Cre / loxP system, where transgenic Cre expression was driven by the mouse-specific gene (Lyz2) promoter. The specific procedure is as follows: Figure 7 As shown in (A).

[0101] Results of retinal patch stained with heterolectin: Figure 8As shown in (BD), retinal myeloid cells from SNX3MΦ-TG mice exhibited robust SNX3 protein expression compared to littermate negative controls (SNX3WT). OIR SNX3 MΦ-TG mice showed significantly increased retinal neovascularization and avascularization areas compared to P17 control mice. However, intravitreal injection of a RIP1 inhibitor (Nec-1) or a RIP3 inhibitor (GSK872) significantly eliminated SNX3 overexpression-induced retinal NV, indicating that RIP1 / 3 signaling plays a downstream role in SNX3, regulating pathological angiogenesis in OIR.

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

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

[0104] VPS35 is the core subunit of the reverse transcription 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 forms a hydrogen bond with the Arg206 site of VPS35, while its benzothiophene structure forms a π-stacking with the Phe28 site. Furthermore, the Lys203 site of VPS35 can also establish a cation-π-stacking interaction with the benzene substituent of W1122, indicating a potential binding affinity and effective targeting between W1122 and the SNX3-reverse transcription complex.

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

[0106] To examine whether W1122 can mimic the effect of SNX3 deficiency on the inhibition of RIP1 / 3 signaling and retinal neovascularization. Figure 8 As shown in (JL), W1122 exhibited a strong inhibitory effect on hypoxia-induced expression of RIP1 / 3 and p-DRP1, mtROS production, and necrosis / apoptosis in bone marrow-derived macrophages. Consistent with SNX3 deficiency, W1122 significantly reduced both neovascularization and avascularization in the retina of OIR mice, as shown in [JL]. Figure 9 (MP).

[0107] The effect of different doses of W1122 on myeloid cell viability was detected by CCK-8 assay. Myeloid-derived macrophages and mouse retinal microglia were treated with W1122, and cell viability was observed 24 hours after treatment. Results are as follows: ​ As shown in (AB), W1122 at concentrations of 0.25–5 μmol did not exhibit significant toxicity to macrophages and microglia.

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

[0109] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of a sorting protein SNX3 inhibitor in the preparation of drugs for proliferative retinopathy, characterized in that, The sorting protein SNX3 inhibitor is a small molecule inhibitor, W1122. The small molecule inhibitor W1122 is an imidazo[1,2-a]pyridine derivative, chemically named N-(1,1-dioxobenzo[b]thiophene-6-yl)-2-(7-methoxy-2-phenylimidazo[1,2-a]pyridin-3-yl)acetamide, with the chemical formula 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: Formula 1.

2. The application of the small molecule inhibitor W1122 in the preparation of drugs for treating proliferative retinopathy, characterized in that, The drug inhibits or downregulates the expression of the sorting protein SNX3; The small molecule inhibitor W1122 is an imidazo[1,2-a]pyridine derivative, chemically named N-(1,1-dioxobenzo[b]thiophene-6-yl)-2-(7-methoxy-2-phenylimidazo[1,2-a]pyridin-3-yl)acetamide, with the chemical formula 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: Formula 1.

3. The application according to claim 2, characterized in that, The small molecule inhibitor W1122 binds to the N-terminus of the sorting protein SNX3.

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