A polypeptide for inhibiting the binding of SOST to LRP6 and its application

Abstract

The present invention relates to the field of biomedicine, and more particularly to a polypeptide for inhibiting the binding of SOST to LRP6 and its use. The amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1. The polypeptide provided by the present invention can effectively inhibit the binding of SOST to LRP6, thereby preventing and / or treating diseases related to the signaling pathways thereof.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a polypeptide for inhibiting the binding of SOST to LRP6 and applications thereof. Background Art

[0002] Sclerostin (SOST), a glycoprotein secreted by osteocytes, is an important regulator of bone metabolism. It inhibits the Wnt / β-catenin signaling pathway, thereby suppressing the differentiation and function of osteoblasts and reducing bone formation.

[0003] Low-density lipoprotein receptor-related protein 6 (LRP6) is a key co-receptor in the Wnt signaling pathway. In classical Wnt signaling, Wnt protein binds to Frizzled receptor and LRP6, triggering a series of signal cascade reactions, promoting the proliferation and differentiation of osteoblasts and maintaining normal bone metabolism balance.

[0004] SOST is a natural inhibitor of the Wnt signaling pathway. It binds to the first and third β-propeller regions of the LRP5 receptor, blocking the interaction between the Wnt ligand and the receptor, thereby inhibiting the Wnt signaling pathway, leading to reduced bone formation, decreased bone mass, and osteoporosis, which seriously affects human health.

[0005] In addition to osteoporosis, abnormal binding of SOST to LRP6 is also closely associated with the development and progression of diseases such as osteoarthritis and tumor bone metastasis. In osteoarthritis, overexpression of SOST inhibits subchondral bone repair and exacerbates joint damage. During tumor bone metastasis, tumor cells may regulate SOST expression, affecting the bone microenvironment and promoting tumor cell colonization and growth. Therefore, the development of a safe and effective drug that inhibits the binding of SOST to LRP6 is of great clinical significance for the treatment of these bone metabolism-related diseases.

[0006] Currently, the main inhibitors of SOST in clinical practice are monoclonal antibodies (such as Romosozumab). Although they have shown good effects in promoting bone formation, they also have the following limitations:

[0007] First, antibody drugs require complex mammalian cell expression systems and cumbersome purification processes, resulting in high treatment costs. Second, antibodies, as exogenous proteins, may trigger immune responses, affecting the safety and effectiveness of long-term use. Third, the half-life of antibodies is too long. Although its long half-life (approximately 2-3 weeks) can reduce the frequency of dosing, it also increases the risk of potential side effects (such as cardiovascular events) and makes it difficult to quickly adjust the dose in emergency situations (such as fracture healing). Fourth, antibodies have a large molecular weight (approximately 150kDa) and limited ability to penetrate bone tissue, which may affect the efficacy of the drug.

[0008] Compared to antibodies, peptide drugs offer advantages such as smaller molecular weight, better tissue penetration, lower production costs, and reduced immunogenicity, making them an ideal alternative to antibodies for inhibiting the interaction between SOST and LRP6. Peptide-based strategies targeting LRP6 are already available. For example, Novartis has developed a fusion protein comprising two LRP6 single-chain antibodies (scFvs) and human serum albumin, which specifically binds to propeller regions 1 and 3 of LRP6 and inhibits Wnt signaling. However, these constructs primarily target the binding of Wnt ligands to LRP6, rather than specifically blocking the interaction between SOST and LRP6.

[0009] While existing technologies have demonstrated the feasibility of peptides targeting LRP6, small molecule peptides specifically designed to block the binding of SOST to LRP6 remain unresolved. In particular, peptides with high affinity and specificity have yet to be reported. Furthermore, existing LRP6-targeting peptides primarily focus on inhibiting Wnt signaling, whereas the present invention aims to activate the Wnt pathway by selectively blocking the inhibitory effects of SOST. This strategy may offer improved safety and specificity.

[0010] Therefore, developing a class of peptide drugs that can efficiently and specifically inhibit the binding of SOST to LRP6 can not only overcome the limitations of existing antibody therapies, but also provide a safer and more economical treatment option for bone diseases such as osteoporosis, and has important clinical application value and market prospects. Summary of the Invention

[0011] The purpose of the present invention is to provide a pharmaceutical polypeptide capable of inhibiting the binding of SOST to LRP6, wherein the pharmaceutical polypeptide has a high affinity with SOST and / or LRP6.

[0012] To achieve the above objectives, the first aspect of the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof for inhibiting the binding of SOST to LRP6. The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1.

[0013] The second aspect of the present invention provides a gene, the nucleotide sequence of which is a nucleotide sequence capable of encoding the amino acid sequence of the polypeptide that inhibits the binding of SOST to LRP6 as described in the first aspect.

[0014] The third aspect of the present invention provides a vector comprising the gene described in the second aspect.

[0015] The fourth aspect of the present invention provides a host cell, which contains the vector described in the third aspect.

[0016] The fifth aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the polypeptide for inhibiting the binding of SOST to LRP6 as described in the first aspect or a pharmaceutically acceptable salt thereof.

[0017] The polypeptide provided by the present invention is obtained through screening and can effectively inhibit the binding of SOST to LRP6, thereby preventing and / or treating diseases related to the signaling pathway thereof.

[0018] Other features and advantages of the present invention will be described in detail in the following detailed description. DETAILED DESCRIPTION

[0019] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0020] It should be noted that, unless otherwise defined herein, the scientific and technical terms used in the present invention should have the meanings commonly understood by those skilled in the art.

[0021] As mentioned above, the first aspect of the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof for inhibiting the binding of SOST to LRP6. The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1.

[0022] The amino acid sequence shown in SEQ ID NO: 1 in the present invention is a polypeptide having the ability to inhibit the binding between SOST and LRP6, which is detected from a polypeptide library by high-throughput screening technology. The steps of discovering the polypeptide include: dissolving and diluting the polypeptide library to obtain a mixed solution; then sequentially mixing the mixed solution with a reactant I containing SOST and its marker, a reactant II containing LRP6, and a reactant III capable of generating a colorimetric reaction with the marker to obtain a detection solution; finally, using a microplate reader to detect the absorbance of the detection solution at a specific wavelength, and obtaining the target polypeptide based on the inhibition rate calculated according to the absorbance.

[0023] It should be noted that the present invention has no particular limitation on the methods of dissolution and dilution, and those skilled in the art can make their selections based on technical means known in the art. A preferred specific embodiment is exemplified below in the present invention, which should not be construed as a limitation on the present invention by those skilled in the art.

[0024] The peptide library described in the present invention is a library containing nearly 73,000 peptides of 80 amino acids, constructed by Hunan Zhongsheng Quanpeptide Biotechnology Co., Ltd. using PICT (Peptide Information Compression Technology) technology. The specific construction method can be found in CN107849737A and CN111727194A.

[0025] It should be noted that the present invention does not particularly limit the method for synthesizing the polypeptide, and those skilled in the art may select a method based on techniques known in the art. For example, a preferred method for synthesizing the polypeptide is exemplified below, which should not be construed as limiting the present invention.

[0026] Preferably, the polypeptide further comprises a polypeptide obtained by disassembling the amino acid sequence shown in SEQ ID NO: 1; the amino acid sequence of the disassembled polypeptide is shown in any one of SEQ ID NOs: 2-91.

[0027] According to a particularly preferred embodiment, the first amino acid and the last amino acid in the amino acid sequence of the polypeptide having the amino acid sequence shown in any one of SEQ ID NOs: 1-91 form a ring via a peptide bond.

[0028] Preferably, the pharmaceutically acceptable salt is selected from at least one of trifluoroacetate, acetate, hydrochloride and phosphate.

[0029] In the present invention, the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 91 are shown in Table 1.

[0030]

[0031]

[0032]

[0033] As mentioned above, the second aspect of the present invention provides a gene, the nucleotide sequence of which is a nucleotide sequence capable of encoding the amino acid sequence of the polypeptide that inhibits the binding of SOST to LRP6 as described in the first aspect.

[0034] As mentioned above, the third aspect of the present invention provides a vector containing the gene described in the second aspect.

[0035] As mentioned above, the fourth aspect of the present invention provides a host cell, which contains the vector described in the third aspect.

[0036] As mentioned above, the fifth aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the polypeptide for inhibiting the binding of SOST to LRP6 or a pharmaceutically acceptable salt thereof as described in the first aspect.

[0037] Preferably, the composition further contains an excipient.

[0038] It should be noted that the present invention has no particular limitation on the type of the excipient, and those skilled in the art can select it according to technical means known in the art, as long as it can enable the polypeptide provided in the present invention to target SOST and / or LRP6.

[0039] The present invention will be described in detail below through examples.

[0040] In the following examples, unless otherwise specified, all raw materials used were commercially available.

[0041] In the following examples, unless otherwise specified, the room temperature refers to 25±5°C.

[0042] Experimental reagents:

[0043] Reagent 1: Human SOST Protein, biotinylated (homemade);

[0044] Reagent 2: Human LRP6 Protein, Mouse IgG2a Fc Tag (purchased from Beijing Biopsies Biotechnology Co., Ltd.);

[0045] Reagent 3: Peroxidase-conjugated AffiniPure™ Goat Anti-Mouse IgG, Fcγ Fragment (purchased from Jackson Immuno Research Laboratories).

[0046] Example 1: ELISA screening of polypeptides

[0047] Dissolution of the peptide library: Peptides in the peptide library were placed in a 96-deep-well plate, centrifuged at 4000 rpm for 3 minutes, and then 200 μL / well of ultrapure water (peptide concentration 50 μM) was added. The plate was then sealed with a silicone cap and incubated in a 95°C water bath for 5 minutes, followed by centrifugation at 4000 rpm for 3 minutes.

[0048] Dilution of peptide library: The peptides after centrifugation were transferred to a 384-well plate using a workstation and diluted to the experimental concentration (10 μM) using loading buffer (Tris-HCl buffer, pH = 7.4) to obtain various mixed solutions.

[0049] Peptide screening: SA protein (2 μg / mL) was coated in a 384-well plate at a volume of 25 μL / well, and blocking solution (a mixture of BSA and TBS buffer (pH = 7.4) prepared at a mass to volume ratio of 2%) was added to block at 37°C, and capture reagent 1 (0.5 μg / mL) was added; then 12.5 μL / well was transferred to a new 384-well plate and incubated at 37°C for 1 hour; then the peptide to be tested and 0.25 μg / mL of the peptide to be tested were added in a gradient dilution at a volume of 12.5 μL / well, respectively. mL of reagent 2 was added and incubated at 37°C for 1 hour. Then, 0.1 μg / mL of reagent 3 was added at 25 μL / well and incubated at 37°C for 1 hour. After discarding the solution in the wells, the plate was washed four times with 80 μL of TBST (pH 7.4) per well, with a 4-minute interval between each wash. After draining, 25 μL of TMB (Solarbio, Cat. No. PR1210) was added to each well and incubated at 37°C for 30 minutes for color development. Finally, 25 μL of stop solution (1 M HCl) was added to each well to terminate the reaction. The absorbance of each well was read at 450 nm using a microplate reader (Cytation 5). The inhibition rate of each peptide concentration was calculated according to the formula, and the IC50 value of each peptide was calculated using GraphPad Prism software. A target peptide, the peptide with the amino acid sequence of SEQ ID NO: 1, was screened. The inhibition rate was calculated as follows:

[0050] Inhibition%=(1-(absorbance of sample-absorbance of background) / (absorbance of positive-absorbance of background))×100%; where,

[0051] Sample: refers to the addition of peptides into the reaction system;

[0052] Positive: refers to the absence of peptide in the reaction system;

[0053] Background: refers to the reaction system without the addition of peptide and reagent 1.

[0054] Example 2: Synthesis of polypeptide

[0055] Based on the target polypeptide obtained in Example 1, a polypeptide containing an amino acid sequence of 5-80 amino acids was designed and synthesized according to the following method:

[0056] Step 1: Gene Construction

[0057] Overlapping oligonucleotide primers are designed for the target peptide's DNA sequence and synthesized. PCR is then performed to obtain the full-length target DNA sequence. This sequence is then ligated into the expression vector pET15b-sumo for fusion expression with the intein. The vector is then transformed into E. coli and cultured. The plasmid is then extracted and sequenced, and the sequencing results are analyzed to ensure consistency with the designed target sequence.

[0058] Step 2: Peptide expression

[0059] The correctly constructed bacterial solution was inoculated into the autoinduction medium, and after adding 1 / 1000 ampicillin (stock solution concentration: 100 mg / mL), the culture was shaken at 37°C and 200 rpm overnight to induce expression.

[0060] Step 3: Peptide purification

[0061] The overnight induced bacterial suspension was poured into urea and dissolved in a 60°C water bath for 5 minutes. 6M NaOH was then added for ultrasonic disruption (3 seconds, 8 seconds interval, 3 minutes, 60% power). The suspension was then neutralized with 6M HCl and incubated with 25 mL of nickel magnetic beads for 1 hour. The supernatant was removed by strong magnetic adsorption and resuspended. The suspension was washed three times and eluted with 500 mM imidazole. The resulting eluate was autogenously sheared at 35°C overnight. The sheared solution was loaded onto a 600 mg equilibrated C18 reverse-phase column, washed with 5 wt% acetonitrile + 1 wt% formic acid, and then eluted with 50 wt% acetonitrile. The eluate was analyzed by SDS-PAGE. Peptide molecular weight was determined by mass spectrometry, and peptide purity was determined by HPLC.

[0062] Step 4: Peptide purification

[0063] The peptide obtained in step 3 was dissolved in 20 wt% acetonitrile-water solution, filtered through a 0.45 μm membrane, and separated using a reversed-phase high-performance liquid chromatography system with a buffer consisting of phase A (0.1 wt% trifluoroacetic acid in water) and phase B (0.1 wt% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen Technology) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was from 20% to 50% phase B over 0-40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions with >95% purity were combined and lyophilized to obtain the pure peptide.

[0064] Example 3: Further ELISA screening of polypeptides

[0065] The designed polypeptides were screened according to the method in Example 1 to obtain 90 target polypeptides, namely polypeptides whose amino acid sequences are shown in SEQ ID NOs: 2-91.

[0066]

[0067]

[0068] From the results in Table 2, it can be seen that the polypeptide provided by the present invention can effectively inhibit the binding of SOST to LRP6.

[0069] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof for inhibiting the binding of SOST to LRP6, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The polypeptide also includes a polypeptide obtained by disassembling the amino acid sequence shown in SEQ ID NO: 1; the amino acid sequence of the disassembled polypeptide is shown in any one of SEQ ID NO: 2, 4, 9-15, 21, 27, 40, 42-53, 59, 61-64, 66-77, and 82.

3. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: The first amino acid and the last amino acid in the amino acid sequence of the polypeptide form a ring through a peptide bond.

4. A gene, characterized in that The nucleotide sequence of the gene is a nucleotide sequence that can encode the amino acid sequence of the polypeptide that inhibits the binding of SOST to LRP6 as described in any one of claims 1 to 3.

5. A carrier, characterized in that The vector contains the gene according to claim 4.

6. A host cell, characterized in that The host cell contains the vector according to claim 5.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition contains a therapeutically effective amount of the polypeptide for inhibiting the binding of SOST to LRP6 according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

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