A synthetic sema4d / plexin b1 inhibitor and use thereof in the preparation of a medicament for treating and preventing ocular fundus vascular diseases

Abstract

The application belongs to the field of biological medicine, and discloses an artificially synthesized Sema4D / PlexinB1 inhibitor and application thereof in preparation of a medicine for treating and preventing fundus vascular diseases. The applicant screens Sema4D inhibitors capable of combining with sSema4D and blocking sSema4D-PlexinB1 by using an OBOC library, and finally obtains a protein small peptide KWIIVGA which has high affinity to sSema4D and has a good inhibiting effect on fundus vascular leakage and fundus vascularization of mice in a mouse experiment. The small peptide can effectively inhibit fundus vascularization and leakage, and provides a new idea for the treatment of fundus vascularization and leakage diseases such as diabetic retinopathy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an artificially synthesized Sema4D / PlexinB1 inhibitor and its application in preparing drugs for treating and preventing fundus vascular diseases. Background Art

[0002] Diabetic retinopathy (DR) is the most common and serious complication of diabetes. Within five years of developing diabetes, the incidence of DR reaches 25%, and after 15 years, approximately 80% of diabetic patients will develop DR.

[0003] The pathological process of DR is complex and includes immune inflammatory response, angiogenesis, vascular leakage, etc., which makes its treatment somewhat special. The purpose of inhibiting angiogenesis in the fundus is to inhibit abnormal blood vessels while restoring normal vascular function, providing blood supply to the area and reducing leakage. Therefore, not all inhibitors of angiogenesis are effective in reversing fundus vascular diseases, and the treatment results may be very different.

[0004] The semaphorin family of proteins, which plays a crucial role in the development of the nervous system, is currently believed to play a crucial role in angiogenesis and vascular development. Among them, the single-pass transmembrane protein Sema4D (Class 4 semaphorins) has been the subject of much debate regarding angiogenesis. Different inhibitors have specificity for different tissues and cells. For example, in tumors, Sema4D promotes cell migration and angiogenesis by binding to the PlexinB receptor, promoting the phosphorylation of Met and Ron (Valente 2009). However, in lung cancer, Sema4D binds to PlexinB1 and forms a complex with ERBB2, which inhibits cell migration by inhibiting Met (Swiercz 2008).

[0005] Currently, a variety of inhibitors targeting the Sema4D / PlexinB1 signaling pathway are available, including antibodies, interfering RNA, and lentiviruses. Anti-Sema4D antibodies include mouse antibodies (MAb67-2, BMA-12, etc.), human antibodies (pepinemab, etc.), and nanobodies (A9-VHH1, etc.), which play an important role in diseases such as tumors, neurological diseases, and osteoporosis. However, in a mouse model of diabetic retinopathy, MAb67-2 and A9-VHH1 were ineffective in reducing retinal angiogenesis and leakage, indicating that not all Sema4D inhibitors are effective in treating PDR.

[0006] In the present invention, the applicant has designed an artificial small peptide targeting the Sema4D / PlexinB1 signaling pathway that can effectively inhibit fundus angiogenesis and reduce fundus vascular leakage, providing a new strategy for the treatment of fundus angiogenesis and leakage. SUMMARY

[0007] The present application aims to provide a synthetic Sema4D / PlexinB1 inhibitor, which is a small peptide with an amino acid sequence of KWIIVGA.

[0008] Another object of the present application is to provide the use of the above-mentioned Sema4D / PlexinB1 inhibitor in the preparation of a medicament for treating and preventing ocular fundus vascular diseases.

[0009] In order to achieve the above-mentioned objects, the present application adopts the following technical measures:

[0010] The Sema4D inhibitor that can bind sSema4D and block sSema4D-PlexinB1 is screened by using an OBOC library, and finally a small peptide Sema4D inhibitor 2 with high affinity to sSema4D and good inhibitory effect on ocular fundus vascular leakage and ocular fundus neovascularization in mice is obtained, and the small peptide is KWIIVGA.

[0011] The small peptide of the present application can be directly synthesized by artificial synthesis, or can be synthesized by using the current conventional microbial means.

[0012] The scope of protection of the present application also includes:

[0013] A fusion protein obtained by fusing the Sema4D inhibitor 2 with a protein tag.

[0014] A gene encoding the Sema4D inhibitor 2 or the above-mentioned fusion protein.

[0015] An expression cassette, a recombinant vector, a recombinant microorganism or an isolated recombinant cell having the above-mentioned encoding gene.

[0016] A composition containing the Sema4D inhibitor 2.

[0017] The use of the Sema4D inhibitor 2, the fusion protein, the gene encoding the Sema4D inhibitor 2 or the above-mentioned fusion protein, the expression cassette having the above-mentioned encoding gene, the recombinant vector, the recombinant microorganism or the isolated recombinant cell, or the above-mentioned composition in the preparation of a medicament for treating and preventing ocular fundus vascular diseases.

[0018] Preferably, in the above-mentioned use, the ocular fundus vascular disease is caused by ocular fundus neovascularization.

[0019] Preferably, in the above-mentioned use, the ocular fundus vascular disease is caused by ocular fundus vascular leakage.

[0020] The application of the above-mentioned, preferably, the dosage form of the drug is powder, injection or nano preparation.

[0021] Compared with the prior art, the present application has the following advantages:

[0022] In the previous work, the applicant found that the Sema4D / PlexinB1 signal pathway inhibitor can effectively inhibit the ocular fundus neovascularization and leakage, and proved the effect of inhibiting Sema4D / PlexinB1 signal pathway on pathological neovascularization and leakage of ocular fundus in vivo, in vitro and gene knockout mice (CN109125731A), in the present application, the applicant artificially synthesized a small peptide inhibitor of Sema4D / PlexinB1 signal pathway aiming at this signal pathway, and the small peptide has a good inhibitory effect on ocular fundus vascular leakage and ocular fundus neovascularization of mice, and the present application provides a new treatment method for the treatment of ocular fundus vascular diseases. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Structure of five polypeptide Sema4D inhibitors screened from OBOC combinatorial peptide library.

[0024] Figure 2 Purpose of the inhibitory effect of Sema4D inhibitors on ocular fundus neovascularization of OIR model mice and ocular fundus vascular leakage of STZ model mice;

[0025] Wherein: A-B shows that Sema4D inhibitor 1, Sema4D inhibitor 2, Sema4D inhibitor 3, Sema4D inhibitor 4 and Sema4D inhibitor 5 are respectively administered to STZ model, and after 1 week of observation, only Sema4D inhibitor 2 has a significant effect.

[0026] C-F shows that Sema4D inhibitor 1, Sema4D inhibitor 2, Sema4D inhibitor 3, Sema4D inhibitor 4 and Sema4D inhibitor 5 are respectively administered to OIR model, and after 1 week of observation, only Sema4D inhibitor 2 has a significant effect. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to specific embodiments. The technical solutions described in the present invention are conventional solutions in the art unless otherwise specified. The reagents or materials described are all derived from commercial channels unless otherwise specified. In their previous work, the applicant discovered that Sema4D / PlexinB1 signaling pathway inhibitors can effectively inhibit fundus angiogenesis and leakage, and demonstrated the effect of inhibiting Sema4D / PlexinB1 signaling pathway on pathological fundus angiogenesis and leakage in vivo, in vitro, and in gene knockout mice (CN109125731A). Therefore, the applicant further artificially synthesized a small peptide that inhibits the Sema4D / PlexinB1 signaling pathway to provide a new therapeutic approach for the treatment of this disease.

[0028] Example 1:

[0029] Preparation and screening of Sema4D / PlexinB1 inhibitors:

[0030] In this example, the OBOC combinatorial peptide library was used to screen Sema4D inhibitors.

[0031] 1) Use the OBOC library to screen for Sema4D inhibitors that can bind to sSema4D and block sSema4D-PlexinB1.

[0032] The peptide sequences generated by the OBOC combinatorial peptide library consist of random 7 amino acids. TentaGel S resin beads are randomly coupled using 18 natural amino acids, theoretically allowing the construction of 18^7 different peptides. TentaGel S resin beads (approximately 10^6 beads) were placed in a peptide synthesis vessel, and the OBOC combinatorial library was screened using Sema4D as the target. Fluorescein isothiocyanate (FITC)-labeled sSema4D (FITC-sSema4D) was added to the peptide library and incubated with the TentaGel S resin beads for 6 hours. The beads were then washed thoroughly with DMF, water, and methanol. Finally, under fluorescence microscopy, beads with weak or no binding to FITC-sSema4D were excluded based on fluorescence intensity. Five beads with strong green fluorescence were selected as candidates for further investigation. After washing and treating the putative resin beads, the peptide compounds were cleaved from the beads by treatment with BrCN and analyzed by automated Edman sequencing. The protein sequences of Sema4D inhibitors 1-5 were determined and summarized as shown in Table 1. The structural formulas of inhibitors 1-5 are shown in Figure 1 shown.

[0033] Table 1 Sequences, molecular weights, and affinity Kd values ​​of five peptide Sema4D inhibitors screened from the OBOC combinatorial peptide library

[0034]

[0035] The structural formula of Sema4D inhibitor 2 is as follows:

[0036]

[0037] Sema4D inhibitors 1-5 in the above table were resynthesized by solid phase synthesis, and purified, analyzed, and their molecular weights were identified by high performance liquid chromatography (HPLC).

[0038] 2) Surface plasmon resonance imaging (SPRi) was used to measure the binding affinity of the screened peptide Sema4D inhibitors 1-5 to sSema4D.

[0039] Surface plasmon resonance imaging (SPRi) was used to measure the binding affinity of peptide Sema4D inhibitors 1-5 to sSema4D. The results showed that peptide Sema4D inhibitors 1-5 exhibited excellent binding affinity to sSema4D, with Kd values ​​of 6.32 × 10 −6 , 7.13× 10 −7 , 5.86× 10 −5 , 4.33× 10 −6 and 5.26× 10 −6 , with Sema4D inhibitor 2 having the strongest affinity.

[0040] Example 2:

[0041] Inhibitory effects of Sema4D inhibitors 1-5 on fundus vascular leakage in STZ model mice:

[0042] (1) Establishment of STZ model in mice

[0043] Eight-week-old C57BL / 6 mice were starved for 4 hours and then injected intraperitoneally with STZ (streptozotocin, 50 mg / kg) once a day for 5 consecutive days. Blood glucose was tested on the 7th day after the injection. A blood glucose level greater than 16.7 mmol / L was considered a successful model.

[0044] (2) Drug injection test

[0045] The STZ model mice were anesthetized at 5 months after modeling, and PBS (1 μl, i.e. Figure 2One week after injection, mice were injected with Evans blue dye (45 mg / kg) via the tail vein. Two hours later, mice were anesthetized and perfused with normal saline. Retinas were then removed and placed in formamide (70°C water bath) overnight for analysis using a microplate reader (wavelength 620-740 nm). Concentrations were calculated using a standard curve and converted to retinal weight and blood retinal levels. A portion of the eyeballs was fixed in 4% PFA at room temperature for 2 hours, and then retinal flat mounts were used for fluorescence imaging.

[0046] The results are as follows Figure 2 As shown in A and B: When the Sema4D inhibitors 1-5 provided by the present invention are used to treat fundus vascular leakage in STZ model mice, only Sema4D inhibitor 2 has significant therapeutic effect. Therefore, the Evans blue extraction experiment confirms that Sema4D inhibitor 2 can significantly inhibit fundus vascular leakage in the STZ model.

[0047] Example 3:

[0048] Inhibitory effects of Sema4D inhibitors 1-5 on fundus angiogenesis in OIR model mice:

[0049] (1) Establishment of mouse OIR model and drug injection experiment

[0050] After giving birth, 3-4 month old C57BL / 6 mother mice were placed in an oxygen chamber with 75% oxygen together with their 7-day old pups. After 12 days of birth, the pups and mother mice were removed and the pups were anesthetized and then injected with PBS (1 μl, i.e., Figure 2 P17-OIR group in middle C), Sema4D inhibitor 1 (2ug, 1ul in total), Sema4D inhibitor 2 (2ug, 1ul in total), Sema4D inhibitor 3 (2ug, 1ul in total), Sema4D inhibitor 4 (2ug, 1ul in total), and Sema4D inhibitor 5 (2ug, 1ul in total) were fed in normal air for 5 days; the pups were anesthetized on the 17th day after birth, and after cardiac perfusion with normal saline, the eyeballs were separated and fixed, and the retinas were peeled off for IB4 staining.

[0051] IB4 staining results Figure 2 As shown in C and D: When the Sema4D inhibitors 1-5 provided by the present invention are used to treat fundus angiogenesis in OIR model mice, only Sema4D inhibitor 2 has significant therapeutic effect. IB4 immunofluorescence staining shows that Sema4D inhibitor 2 can significantly inhibit fundus angiogenesis in the OIR model.

[0052] 2) Sema4D inhibitor 2 significantly inhibited the number of preretinal neovascularization cells in the OIR model (HE staining)

[0053] After giving birth, 3-4 month old C57BL / 6 mother mice were placed in an oxygen chamber with 75% oxygen together with their 7-day old pups. After 12 days of birth, the pups and mother mice were removed and the pups were anesthetized and then injected with PBS (1 μl, i.e., Figure 2 The P17-OIR group in Figure E), Sema4D inhibitor 1 (2ug, 1ul in total), Sema4D inhibitor 2 (2ug, 1ul in total), Sema4D inhibitor 3 (2ug, 1ul in total), Sema4D inhibitor 4 (2ug, 1ul in total), and Sema4D inhibitor 5 (2ug, 1ul in total) were fed in normal air for 5 days; the above mice were anesthetized on the 17th day after birth, and after cardiac perfusion with normal saline, their eyeballs were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned with HE staining. The number of vascular endothelial cells that broke through the inner limiting membrane of the retina was observed and counted under an optical microscope.

[0054] IB4 staining results Figure 2 As shown in Figures E and F: When Sema4D inhibitors 1-5 provided by the present invention were used to treat fundus angiogenesis in OIR model mice, only Sema4D inhibitor 2 had a significant therapeutic effect. HE staining showed that Sema4D inhibitor 2 could significantly inhibit the number of cells in the preretinal neovascularization.

Claims

1. A synthetic Sema4D / PlexinB1 inhibitor, which is a small peptide with an amino acid sequence of KWIIVGA.

2. A gene encoding the inhibitor according to claim 1.

3. An expression cassette, recombinant vector or recombinant microorganism comprising the gene according to claim 2.

4. A composition comprising the inhibitor according to claim 1.

5. Use of the inhibitor of claim 1, the gene of claim 2, the expression cassette, recombinant vector, recombinant microorganism comprising the gene of claim 2, or the composition of claim 4 in the preparation of a medicament for treating and preventing retinal vascular diseases, wherein the retinal vascular diseases are caused by retinal angiogenesis and / or retinal vascular leakage.

6. The use according to claim 5, characterized in that: The dosage form of the medicine is powder, injection or nano preparation.

Images