Small peptides and their application in the preparation of drugs for treating retinal artery occlusion

Abstract

This invention discloses a small peptide and its application in the preparation of a drug for treating retinal artery occlusion. The amino acid sequence of the small peptide is shown in SEQ ID NO.1. The small peptide TAT-PQ of this invention effectively reduces apoptosis in mouse retinal artery cells by enhancing SUMOylation modification in retinal ganglion cells, inhibiting liquid-liquid phase separation of FXR1, and reducing the translation level of apoptosis-related proteins. Furthermore, TAT can directly introduce the fused small peptide into cells, solving the problem of drug entry into cells and greatly improving the convenience and effectiveness of administration. This small peptide is not immunogenic, reducing the risk of immune reactions and ensuring sustained effectiveness with secondary or subsequent administrations. Therefore, the small peptide of this invention has great application potential in the preparation of a drug for treating retinal artery occlusion.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a small peptide and its application in the preparation of a drug for treating retinal artery occlusion. Background Technology

[0002] Retinal artery occlusion (RAO) is an acute blinding eye disease, usually accompanied by massive apoptosis of retinal ganglion cells (RGCs), leading to vision loss. Although arterial thrombolysis can alleviate some RAO symptoms, its effectiveness remains limited. Especially during the reperfusion phase after thrombolysis, it can trigger retinal ischemia-reperfusion (RIR) injury, further exacerbating cell apoptosis and tissue damage.

[0003] There are currently no effective drugs available. Therefore, it is necessary to develop a drug for treating retinal artery occlusion. Summary of the Invention

[0004] The purpose of this invention is to provide a small peptide and its application in the preparation of drugs for treating retinal artery occlusion. This small peptide can significantly reduce apoptosis of retinal ganglion cells and alleviate visual function damage caused by RAO, and has great application prospects in the preparation of drugs for treating retinal artery occlusion.

[0005] To achieve the aforementioned objective, the present invention adopts the following technical solution:

[0006] In a first aspect of the invention, a small peptide is provided, the amino acid sequence of which is shown in SEQ ID NO.1 (YGRKKRRQRRR-DPAQAEAYTIYCQN).

[0007] Furthermore, the small peptide includes:

[0008] TAT penetration peptide: The amino acid sequence is shown in SEQ ID NO.2 (YGRKKRRQRRR), used to mediate intracellular delivery;

[0009] PQ domain: The amino acid sequence is shown in SEQ ID NO.3 (DPAQAEAYTIYCQN), which is used to competitively bind to Nedd4 and block its ubiquitination and degradation of Ubc9.

[0010] In a second aspect of the invention, a medicament for treating retinal artery occlusion is provided, the medicament comprising the small peptide described above.

[0011] Furthermore, the drug also includes pharmaceutically acceptable carriers or excipients.

[0012] Furthermore, the excipients include at least one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners, and colorants.

[0013] Furthermore, the dosage form of the drug includes at least one of granules, tablets, pills, capsules, injections, or dispersants.

[0014] Furthermore, the concentration of the small peptide in the drug is 0.1-10 μg / mL.

[0015] In a third aspect of the invention, the use of the small peptide is provided in the preparation of a medicament for treating retinal artery occlusion.

[0016] Furthermore, the retinal artery occlusion includes: visual impairment caused by retinal artery occlusion and apoptosis of retinal ganglion cells.

[0017] Furthermore, the drug enhances SUMOylation modification, inhibits the liquid-liquid phase separation of RNA-binding protein 1, and reduces the translation level of apoptosis-related proteins, thereby effectively alleviating the damage and apoptosis of retinal ganglion cells caused by retinal artery occlusion.

[0018] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0019] 1. This invention provides a small peptide and its application in the preparation of drugs for treating retinal artery occlusion. This small peptide can significantly reduce apoptosis of retinal ganglion cells and alleviate visual function damage caused by RAO, and has great application prospects in the preparation of drugs for treating retinal artery occlusion.

[0020] 2. By using the small peptide TAT-PQ of this invention to enhance SUMOylation modification in retinal ganglion cells, inhibiting FXR1 liquid-liquid phase separation, and reducing the translation level of apoptosis-related proteins, apoptosis in mouse retinal artery cells is effectively reduced. TAT can directly introduce the fused small peptide into cells, solving the problem of drug entry into cells and greatly improving the convenience and effectiveness of drug administration. This small peptide can directly penetrate the cell membrane, is not immunogenic, reduces the risk of immune reactions, and ensures sustained effectiveness for secondary and subsequent administrations. It is suitable for preparing drugs to treat recurrent acute ischemic attack (RAO) and ischemia-reperfusion injury. Attached Figure Description

[0021] Figure 1The retinal artery occlusion model of Example 1 resulted in a decrease in the level of Ubc9 protein and SUMOylation modification in mouse retinal ganglion cells; A: A comparison of bands in the retinal tissue of mice in the sham-operated group (Sham) and the UPOAO group, showing the level of SUMOylation modification (SUMO1 antibody) and Ubc9 protein expression (Ubc9 antibody) detected by Western blot.

[0022] B: Figure 1 A: Quantitative statistical bar chart (grayscale analysis); C: Immunofluorescence staining of retinal sections (anti-Ubc9 antibody, green fluorescence), showing the comparison of fluorescence intensity of retinal ganglion cell layer (GCL) between the Sham group and the UPOAO group, where Sham is the sham-operated group and UPOA is the retinal artery occlusion group.

[0023] Figure 2 Example 2 illustrates the reduction in Ubc9 protein and SUMOylation modification levels in R28 cells induced by glycogen deprivation; A: Flowchart of the procedure; B: Comparison of bands showing SUMOylation modification levels (SUMO1 antibody) and Ubc9 protein expression (Ubc9 antibody) in R28 cells from the control group and the OGD group using Western blot analysis; C: Immunofluorescence staining of R28 cells (anti-Ubc9 antibody, red fluorescence), showing the changes in Ubc9 fluorescence distribution in cells from the control group and the OGD group.

[0024] Figure 3 Example 2: Glucose deprivation induces a decrease in the SUMOylation level of the RNA-binding protein FXR1 in R28 cells; A: Procedure; B: Differential protein functional enrichment analysis of the two groups; C: Differential analysis results of proteins related to programmed cell death in retinal ganglion cells; D: Western blot detection of FXR1 SUMOylation level (IP: FXR1, WB: SUMO1); E: Figure 3 Quantitative statistical bar chart of D (grayscale analysis); F: Schematic diagram of FXR1 protein structure, labeled K88 and K663 sites.

[0025] Figure 4Example 3 shows that the loss of SUMOylation modification of FXR1 promotes its liquid-liquid phase separation and the expression of calcium signaling pathway-related genes; A: Live-cell imaging (GFP fluorescence), comparing the aggregate morphology of GFP-FXR1-WT and GFP-FXR1-DKR in HEK293T cells (arrows indicate aggregates); B: Sucrose gradient centrifugation Western blot, showing the distribution of FXR1-WT and FXR1-DKR in different density gradient fractions; C: RIP-Seq differential gene volcano plot, marking calcium signaling pathway enrichment; D: GO enrichment analysis bar chart of calcium signaling pathway genes; E: Bar chart of RIP-qPCR detection of the binding efficiency of FXR1-WT and FXR1-DKR to calcium signaling genes (P2rx2, Grm1, etc.); F: qPCR quantitative bar chart of calcium signaling genes in ribosomal multimer fractions.

[0026] Figure 5 In Example 4, P128 and Q139 are key amino acid sites for binding with Nedd4 in Ubc9; A: Immunoprecipitation map (IP: Flag, WB: HA), detecting the binding of Nedd4-HA and Ubc9-Flag; BD: Molecular docking model diagram (generated by Schrödinger software), showing the interaction interface between Ubc9 (surface structure) and Nedd4 (band structure), with P128 and Q139 sites labeled; E: Immunoprecipitation map (IP: Flag, WB: HA), comparing the binding efficiency of wild-type (WT) and mutants (P128A, Q139A, etc.) of Ubc9 with Nedd4.

[0027] Figure 6 Nedd4 in Example 5 promotes the ubiquitination and degradation of Ubc9.

[0028] Figure 7 Example 5 illustrates the TAT-PQ peptide's inhibition of Nedd4-Ubc9 binding. A: Amino acid sequence of the TAT-penetrating peptide and the amino acid sequence of the TAT-PQ peptide of this invention; B: Immunoprecipitation plot (IP: Flag, WB: HA), comparing the Nedd4-Ubc9 binding efficiency (band intensity change) in the TAT-PQ group and the control group; C: Western blot detection of Ubc9 protein levels in OGD-treated R28 cells (TAT-PQ group vs. control group).

[0029] Figure 8The TAT-PQ peptide from Example 6 was used to alleviate OGD-induced apoptosis in R28 cells. A: Western blot analysis of BAX and cleaved caspase 3 protein expression (TAT-PQ group vs. control group); B: Calcein-AM (green, live cells) / PI (red, dead cells) double-staining fluorescence image, showing changes in the proportion of apoptotic cells.

[0030] Figure 9 The TAT-PQ peptide in Example 6 was used to improve UPOAO-induced visual impairment in mice.

[0031] Figure 10 To illustrate how the TAT-PQ peptide in Example 6 alleviates UPOAO-induced apoptosis of mouse retinal ganglion cells, A: HE staining results of retinal cross sections in the Sham group (sham-operated group), UPOAO model group (retinal ischemia-injury group), UPOAO+TAT treatment group, and UPOAO+TAT-PQ treatment group; B: Double-labeled fluorescent staining results of retinal ganglion cell layer (GCL) in the Sham group, UPOAO group, UPOAO+TAT group, and UPOAO+TAT-PQ group. Detailed Implementation

[0032] The following detailed description of the embodiments and examples will illustrate the present invention in more detail, thereby making the advantages and various effects of the embodiments more clearly apparent. Those skilled in the art should understand that these detailed embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0033] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of this invention pertain. In the event of any conflict, this specification shall prevail.

[0034] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the embodiments of the present invention can be obtained by purchasing them on the market or by existing methods.

[0035] To solve the technical problem of this invention, the overall concept of this invention is as follows:

[0036] This invention has found that in retinal ischemia-reperfusion injury, Nedd4 increases the ubiquitination and degradation of Ubc9 protein and inhibits the SUMOylation of FXR1, a process that significantly exacerbates RGC apoptosis.

[0037] Next, this invention designs a small peptide, TAT-PQ, based on the amino acid sequence of the Ubc9-Nedd4 binding site, to competitively inhibit the binding of Ubc9 and Nedd4. TAT is derived from an 11-amino acid sequence (YGRKKRRQRRR) on a lentivirus, which can efficiently carry the fused peptide into the cell. PQ corresponds to the key amino acid sites (P128 and Q139) for Ubc9-Nedd4 binding. By competitively inhibiting the binding of Ubc9 and Nedd4, the TAT-PQ peptide reduces the ubiquitination and degradation of Ubc9, thereby increasing the protein level of Ubc9, promoting the SUMOylation modification of the FXR1 protein, and inhibiting its liquid-liquid phase separation.

[0038] Finally, experiments verified that this small peptide can significantly reduce retinal ganglion cell apoptosis and alleviate visual function impairment caused by RAO. The experimental results are as follows:

[0039] Cell model: Pretreatment with 10 μg / mL TAT-PQ significantly reduced the apoptosis rate of oxygen-glucose deprivation (OGD) R28 cells. Figure 8 B);

[0040] Animal model: Intravitreal injection of 1 μL TAT-PQ (1 mg / mL) into UPOAO mice resulted in visual function recovery ( Figure 9 ), retinal ganglion cell apoptosis decreased by more than 50% ( Figure 10 ).

[0041] The above results demonstrate that this small peptide has great application potential in the preparation of drugs for the treatment of retinal artery occlusion.

[0042] The present application will now be described in detail with reference to embodiments and experimental data.

[0043] Example 1: Effects of retinal artery occlusion on Ubc9 and SUMOylation

[0044] 1. Methods: (1) Establishment of UPOAO mouse model: The pterygopalatine artery of mice was blocked with suture occlusion for 2 hours, and the blood reperfusion time was 7 days. At the same time, a sham control group (Sham) was set up.

[0045] (2) Retinal tissue was taken for Western blot analysis to detect SUMOylation modification level (anti-SUMO1 antibody) and Ubc9.

[0046] Protein expression (anti-Ubc9 antibody). Parallel immunofluorescence staining of retinal sections (anti-Ubc9 antibody labeling of ganglion cell layer). 2. Results:

[0047] Western blot results are as follows Figure 1As shown in Figure AB, the intensity of the SUMO1 modified band in the UPOAO group was significantly weaker than that in the Sham group; the intensity of the Ubc9 protein band was significantly reduced. This indicates that SUMOylation and Ubc9 protein levels were significantly reduced in the retinal tissue of UPOAO model mice.

[0048] Immunofluorescence results as follows Figure 1 As shown in Figure C, Ubc9 expression was significantly reduced in retinal ganglion cells of UPOAO model mice.

[0049] Example 2: Regulation of Ubc9 and FXR1 in R28 cells by oxygen-glucose deprivation

[0050] 1. Method:

[0051] R28 cells were divided into a control group and an oxygen-glucose deprivation group (OGD treatment for 6 hours). Western blot was then used to detect SUMOylation modification and Ubc9 protein expression, where the SUMOylation modification level was calculated as (SUMOylation level in the experimental group) / (SUMOylation level in the control group) × 100%. Immunoprecipitation mass spectrometry analysis (using anti-SUMO1 antibody) was used to screen for differentially expressed proteins. 2. Results

[0052] R28 cells were subjected to oxygen-glucose deprivation treatment, and the changes in SUMOylation modification levels in the cells were detected by Western blot. Figure 2 As shown in Figure A, SUMOylation modification and the protein and levels of Ubc9 were significantly reduced in R28 cells subjected to oxygen-glucose deprivation. Immunofluorescence staining results are shown in Figure A. Figure 2 As shown in Figure B, Ubc9 is located in the cytoplasm and nucleus of R28 cells, and oxygen-glucose deprivation treatment significantly reduces Ubc9 expression.

[0053] Immunoprecipitation mass spectrometry (anti-SUMO1) was performed on OGD-treated R28 cells, and functional enrichment analysis was conducted on differentially expressed proteins between the two groups. Significant differences were found in hypoxia response, organic acid metabolism, cell surface receptor signaling pathways, and programmed cell death. Figure 3 (B). Considering that RGC death is the most important cause of visual impairment, we performed differential analysis on proteins related to programmed cell death of retinal ganglion cells (B). Figure 3 (C) Among them, the first 5 proteins Ndufs1, Fxr1, Ero1α, HK2, and Cul1 were selected for SUMOylation modification verification. The results showed that SUMOylation modification of FXR1 was the most significant. Figure 3 (DE). Further verification confirmed the presence of two SUMOylation modification sites, K88 and K663, in the FXR1 protein structure. Figure 3 (Middle F).

[0054] Example 3: SUMOylation deficiency of FXR1 promotes liquid-liquid phase separation

[0055] FXR1 is closely associated with messenger ribonucleoprotein particles (mRNPs) in polyribosomes during translation. FXR1 promotes mRNA translation by assembling target mRNAs into mRNPs through liquid-liquid phase separation (LLPS). To investigate the effect of SUMOylation modification on FXR1's LLPS, we constructed GFP-FXR1-WT or GFP-FXR1-DKR expression vectors, which were overexpressed in HEK293T cells along with Ubc9-HA and SUMO1-MyC, and the changes in GFP aggregates were observed in real time.

[0056] The results are as follows Figure 4 As shown in Figure A, SUMOylation modification inhibits GFP-FXR1 aggregate fusion. Further sucrose gradient centrifugation was used to separate RNPs, ribosomal subunits, and ribosomal multimers, and the protein content of FXR1 in layers of different densities was detected. Results are shown below. Figure 4 As shown in Figure B, FXR1-WT has a higher abundance in high-density aggregates, indicating that SUMOylation modification inhibits its formation in aggregates.

[0057] To further investigate the specific molecular mechanism by which SUMOylation of FXR1 regulates apoptosis in RGCs, RIP-Seq analysis was performed on R28 cells expressing FXR1-WT or FXR1-DKR. Figure 4 The calcium signaling pathway shown in Figure D was significantly enriched. Considering that aberrant calcium activation, especially mitochondrial calcium overload, plays a crucial role in hypoxia-induced apoptosis, we used RIP-qPCR to detect related calcium signaling pathways in R28KO cells expressing FXR1-WT or FXR1-DKR. The results... Figure 4 As shown in Figure E, FXR1-WT is enriched in binding to more calcium signaling pathway-related genes (P2rx2, Grm1, Sphk1, Chrm3, and Pde1b). The results are as follows... Figure 4 As shown in Figure F, this result was further confirmed in the isolated ribosomal polymers.

[0058] Example 4: Identification of binding sites between Nedd4 and Ubc9

[0059] In HEK294T cells, Nedd4-HA and Ubc9-Flag were co-expressed via plasmids, and an immunoprecipitation assay was performed to demonstrate the direct binding of Nedd4 to Ubc9. Results are as follows: Figure 5 As shown in the figure, the molecular docking model predicts that Nedd4 and Ubc9 have multiple binding sites.

[0060] Protein pretreatment: The crystal structures of the proteins were obtained from the RCSB PDB database (Ubc9: 2UYZ; Nedd4: P46934). The obtained protein crystals were pretreated using the Protein Preparation Wizard module of Schrödinger software, which performed protein pretreatment, regeneration of native ligands, hydrogen bond allocation optimization, protein energy minimization, and water removal. Protein-protein docking: The pretreated proteins were docked using the Protein-Protein docking module in Schrödinger. A lower score indicates a lower binding free energy between the ligand and the protein, and thus higher binding stability. Prime Energy analysis: Prime energy was calculated and analyzed for the active sites of the ligand and the protein. Prime energy can approximate the binding free energy between proteins; a lower binding free energy indicates higher binding stability. In this protein-protein docking test, pose4 had the lowest Prime Energy of -41183.4, suggesting a relatively stable binding conformation.

[0061] The predicted Nedd4 binding sites in Ubc9 were mutated to glycine A, and multiple expression vectors were constructed and transfected with HEK293T. Immunoprecipitation was then performed. Results are as follows: Figure 5 As shown in Figure E: Mutations in P128 and Q139 can inhibit the binding of Nedd4 to Ubc9.

[0062] Example 5: TAT-PQ inhibits Nedd4-mediated Ubc9 degradation

[0063] Nedd4-HA, Ubc9-Flag, and Ub-Myc were expressed in HEK293T cells. Under OGD treatment, and with simultaneous administration of an MG132 inhibitor, the ubiquitination level of Ubc9-Flag was measured. Results are as follows: Figure 6 As shown: Nedd4 promotes ubiquitination modification of Ubc9.

[0064] Based on the amino acid sites where Ubc9 binds to Nedd4 and P128 and Q138, the small peptide TAT-PQ was synthesized. Nedd4-HA and Ubc9-Flag were expressed in HEK293T cells. Immunoprecipitation assays were performed using TAT-PQ (10 ug / mL) or the control peptide TAT, and the results are as follows: Figure 7 As shown in Figure A: TAT-PQ inhibits the binding of Nedd4 to Ubc9. Retinal progenitor cell line R28 was treated with OGD, and simultaneously treated with TAT-PQ (10 ug / mL) and a control peptide. The results are as follows. Figure 7 As shown in Figure B: TAT-PQ inhibits the protein degradation of Ubc9.

[0065] Example 6: The therapeutic effects of TAT-PQ in reducing apoptosis and in animal models

[0066] 1. The retinal progenitor cell line R28 was treated with OGD and simultaneously treated with TAT-PQ (10 ug / mL) and control peptide. The apoptosis level, visual function and TUNEL staining of R28 cells in each group were detected.

[0067] like Figure 8 As shown in Figure A, TAT-PQ treatment reduced the expression of apoptosis markers BAX and cleaved caspase 3 in R28 cells. Figure 8 Figure B shows that C-AM / PI indicates that TAT-PQ treatment reduces apoptosis in R28 cells. C-AM labels surviving cells, while PI labels cells with impaired apoptosis. In summary, the TAT-PQ peptide alleviates OGD-induced apoptosis in R28 cells.

[0068] 2. Verify the restorative effect of TAT-PQ peptide on retinal nerve function in mice with retinal artery occlusion (UPOAO) model by quantifying light signal transmission capacity using b-wave amplitude.

[0069] The study was divided into four groups: sham surgery + TAT group, sham surgery + TAT-PQ group, UPOAO + TAT group, and UPOAO + TAT-PQ group. The drug concentration for all four groups was 1 mg / mL, and the volume was 1 uL. The modeling / drug administration time point was 24 hours before the UPOAO surgery.

[0070] Flash visual evoked potential (F-VEP) waveform as follows Figure 9 As shown, the amplitude of the TAT-PQ group recovered, indicating that the TAT-PQ small peptide improved the visual function impairment caused by UPOAO in mice.

[0071] TUNEL staining as Figure 10 As shown in Figure AB, the overall retinal thickness in the TAT-PQ group was greater than that in the TAT group, and the apoptosis of retinal ganglion cells was reduced (red fluorescence signal was weakened), indicating that the TAT-PQ small peptide alleviated the apoptosis of mouse retinal ganglion cells induced by UPOAO.

[0072] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0073] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present invention.

[0074] Obviously, those skilled in the art can make various modifications and variations to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. Therefore, if these modifications and variations to the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to include these modifications and variations.

Claims

1. A small peptide, characterized in that, The amino acid sequence of the small peptide is shown in SEQ ID NO.

1.

2. The small peptide according to claim 1, characterized in that, The small peptides include: TAT penetrating peptide: The amino acid sequence is shown in SEQ ID NO.2, and it is used to mediate intracellular delivery; PQ domain: The amino acid sequence is shown in SEQ ID NO.3, which is used to competitively bind to Nedd4 and block its ubiquitination and degradation of Ubc9.

3. A drug for treating retinal artery occlusion, characterized in that, The drug comprises the small peptides described in any one of claims 1-2.

4. The medicament for treating retinal artery occlusion according to claim 3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The medicament for treating retinal artery occlusion according to claim 4, characterized in that, The excipients include at least one of fillers, disintegrants, binders, lubricants, sweeteners, and colorants.

6. The medicament for treating retinal artery occlusion according to claim 3, characterized in that, The dosage form of the drug includes at least one of granules, tablets, pills, capsules, and injections.

7. The medicament for treating retinal artery occlusion according to claim 3, characterized in that, The concentration of the small peptide in the drug is 0.1-10 μg / mL.

8. Use of the small peptide according to any one of claims 1-2 in the preparation of a medicament for treating retinal artery occlusion.

9. The application according to claim 8, characterized in that, The retinal artery occlusion includes visual impairment and apoptosis of retinal ganglion cells caused by retinal artery occlusion.

10. The application according to claim 8, characterized in that, The drug enhances SUMOylation modification, inhibits the liquid-liquid phase separation of RNA-binding protein 1, and reduces the translation level of apoptosis-related proteins, thereby effectively alleviating the damage and apoptosis of retinal ganglion cells caused by retinal artery occlusion.

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