Polypeptide for inhibiting combination of SOST and LRP6 and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to polypeptide for inhibiting combination of SOST and LRP6 and application of the polypeptide. The amino acid sequence of the polypeptide is shown as SEQ ID NO: 1. The polypeptide provided by the invention can effectively inhibit the combination of SOST and LRP6, so as to prevent and / or treat diseases related to a signal channel of the polypeptide.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a polypeptide that inhibits the binding of SOST to LRP6 and its applications. Background Art

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

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

[0004] As a natural inhibitor of the Wnt signaling pathway, SOST binds to the first and third β-propeller regions of the LRP5 receptor, blocking the interaction between Wnt ligands and receptors, thereby inhibiting the Wnt signaling pathway, resulting in reduced bone formation, decreased bone mass, and osteoporosis, seriously affecting human health.

[0005] In addition to osteoporosis, abnormal binding of SOST to LRP6 is also closely related to the occurrence and development of diseases such as osteoarthritis and tumor bone metastasis. In osteoarthritis, overexpression of SOST inhibits the repair of subchondral bone and exacerbates joint damage; during tumor bone metastasis, tumor cells may affect the bone microenvironment by regulating the expression of SOST, promoting the colonization and growth of tumor cells. Therefore, developing a safe and effective drug that inhibits the binding of SOST to LRP6 has important 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 show good bone formation-promoting effects, they also have the following limitations: First, antibody drugs require complex mammalian cell expression systems and cumbersome purification processes, resulting in high treatment costs. Second, as foreign proteins, antibodies 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 (about 2 - 3 weeks) can reduce the dosing frequency, it also increases the risk of potential side effects (such as cardiovascular events), and it is difficult to quickly adjust the dose in emergency situations (such as fracture healing). Fourth, the molecular weight of antibodies is large (about 150 kDa), and their penetration ability into bone tissue is limited, which may affect the efficacy.

[0007] Compared with antibodies, polypeptide drugs have the advantages of small molecular weight, good tissue penetration, low production cost, low immunogenicity, etc., and are ideal alternatives to antibodies for inhibiting the interaction between SOST and LRP6. In the prior art, there are already polypeptide strategies targeting LRP6. For example, Novartis developed a fusion protein containing two LRP6 single-chain antibodies (scFv) and human serum albumin, which can specifically bind to the propeller 1 and 3 regions of LRP6 and inhibit Wnt signal transduction. However, such constructs mainly target the binding of Wnt ligands to LRP6, rather than specifically blocking the interaction between SOST and LRP6.

[0008] Although the feasibility of polypeptides targeting LRP6 has been confirmed in the prior art, small molecule polypeptides specifically designed to block the binding between SOST and LRP6 are still blank. In particular, polypeptides with high affinity and specificity have not been reported. In addition, existing LRP6-targeting polypeptides mostly focus on inhibiting Wnt signals, while the present invention aims to activate the Wnt pathway by selectively blocking the inhibitory effect of SOST, and this strategy may have better safety and specificity.

[0009] Therefore, developing a class of polypeptide drugs that can efficiently and specifically inhibit the binding between SOST and LRP6 can not only overcome the limitations of existing antibody therapies, but also provide safer and more economical treatment options for bone diseases such as osteoporosis, and has important clinical application value and market prospects. Summary of the Invention

[0010] The object of the present invention is to provide a pharmaceutical polypeptide that can inhibit the binding between SOST and LRP6, and this pharmaceutical polypeptide has a high affinity for SOST and / or LRP6.

[0011] To achieve the above object, the first aspect of the present invention provides a polypeptide that inhibits the binding between SOST and LRP6 or a pharmaceutically acceptable salt thereof, and the amino acid sequence of this polypeptide is as shown in SEQ ID NO: 1.

[0012] The second aspect of the present invention provides a gene, and the nucleotide sequence of this gene is the nucleotide sequence capable of encoding the amino acid sequence of the polypeptide that inhibits the binding between SOST and LRP6 described in the first aspect above.

[0013] The third aspect of the present invention provides a vector, and this vector contains the gene described in the second aspect above.

[0014] The fourth aspect of the present invention provides a host cell, and this host cell contains the vector described in the third aspect above.

[0015] The fifth aspect of the present invention provides a pharmaceutical composition, which contains a therapeutically effective amount of the polypeptide that inhibits the binding of SOST to LRP6 described in the aforementioned first aspect or a pharmaceutically acceptable salt thereof.

[0016] The polypeptide provided by the present invention is obtained by screening, and it can effectively inhibit the binding of SOST to LRP6, thereby being able to prevent and / or treat diseases related to its signaling pathway.

[0017] Other features and advantages of the present invention will be described in detail in the subsequent specific implementation section. Specific Implementation Modes

[0018] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0019] 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.

[0020] As mentioned above, the first aspect of the present invention provides a polypeptide that inhibits the binding of SOST to LRP6 or a pharmaceutically acceptable salt thereof, and the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1.

[0021] The amino acid sequence shown as SEQ ID NO: 1 in the present invention is a polypeptide detected by high-throughput screening technology from a polypeptide library and having the effect of inhibiting the binding of SOST to LRP6. The discovery steps of the polypeptide include: dissolving and diluting the polypeptide library to obtain a mixed solution; then successively mixing the mixed solution with reactant I containing SOST and its label, reactant II containing LRP6, and reactant III capable of undergoing a color reaction with the label to obtain a detection solution; finally, using an enzyme-labeled instrument to detect the absorbance of the detection solution at a specific wavelength, and obtaining the target polypeptide according to the inhibition rate calculated from the absorbance.

[0022] It should be noted that the present invention has no particular limitation on the methods of the dissolution and dilution, and those skilled in the art can select according to the known technical means in the art. An exemplary specific implementation mode is provided in the following text of the present invention, and those skilled in the art should not understand it as a limitation to the present invention.

[0023] In the present invention, the polypeptide library is a polypeptide library containing nearly 73,000 polypeptides of 80 amino acids constructed by Hunan Zhongcheng Peptide Biotechnology Co., Ltd. using the PICT (Peptide Information Compression Technology) technology. The specific construction method can be specifically referred to CN107849737A and CN111727194A.

[0024] It should be noted that the present invention does not particularly limit the synthesis method of the polypeptide, and those skilled in the art can select according to the known technical means in the art. Exemplarily, a preferred polypeptide synthesis method is provided exemplarily in the following text of the present invention, and those skilled in the art should not understand it as a limitation to the present invention.

[0025] Preferably, the polypeptide further includes a polypeptide disassembled from 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.

[0026] According to a particularly preferred embodiment, the first amino acid and the last amino acid in the amino acid sequence of the polypeptide with the amino acid sequence shown in any one of SEQ ID NOs: 1-91 are cyclized through a peptide bond.

[0027] Preferably, the pharmaceutically acceptable salt is selected from at least one of trifluoroacetate, acetate, hydrochloride, and phosphate.

[0028] In the present invention, the amino acid sequences shown in SEQ ID NO: 1-SEQ ID NO: 91 are shown in Table 1.

[0029]

[0030]

[0031]

[0032] As described above, the second aspect of the present invention provides a gene, and the nucleotide sequence of the gene is the nucleotide sequence capable of encoding the amino acid sequence of the polypeptide that inhibits the binding of SOST to LRP6 described in the first aspect above.

[0033] As described above, the third aspect of the present invention provides a vector, and the vector contains the gene described in the second aspect above.

[0034] As described above, the fourth aspect of the present invention provides a host cell, and the host cell contains the vector described in the third aspect above.

[0035] As described above, the fifth aspect of the present invention provides a pharmaceutical composition, which contains a therapeutically effective amount of the polypeptide that inhibits the binding of SOST to LRP6 or a pharmaceutically acceptable salt thereof described in the aforementioned first aspect.

[0036] Preferably, the composition further contains excipients.

[0037] 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 according to the known technical means in the art, as long as the polypeptide provided in the present invention can target SOST and / or LRP6.

[0038] The present invention will be described in detail below by way of examples.

[0039] In the following examples, unless otherwise specified, the raw materials used are commercially available.

[0040] In the following examples, unless otherwise specified, the room temperature means 25 ± 5 °C.

[0041] Experimental reagents: Reagent 1: Human SOST Protein, biotinylated (self-made); Reagent 2: Human LRP6 Protein, Mouse IgG2a Fc Tag (purchased from Beijing Protein Innovation Co., Ltd.); Reagent 3: Peroxidase-conjugated AffiniPure™ Goat Anti-Mouse IgG, Fcγ Fragment (purchased from Jackson Immuno Research Laboratories).

[0042] Example 1: ELISA screening of polypeptides Dissolution of the polypeptide library: Place the polypeptides in the polypeptide library in a 96-well deep-well plate, centrifuge at 4000 rpm for 3 min, and then add 200 μL / well of ultrapure water (polypeptide concentration is 50 μM); then seal with a silica gel lid, incubate in a 95 °C water bath for 5 min, and then centrifuge at 4000 rpm for 3 min; Dilution of the polypeptide library: Transfer the centrifuged polypeptides to a 384-well plate using a workstation, and dilute the polypeptide concentration to the experimental concentration (10 μM) with loading buffer (loading buffer: Tris-HCl buffer, pH = 7.4) to obtain each mixture; Screening of polypeptides: Coat a 384-well plate with SA protein (2 μg / mL) at a volume of 25 μL per well. After adding the blocking solution (a mixture of BSA and TBS buffer (pH = 7.4) prepared at a mass-to-volume ratio of 2%) and incubating at 37°C for blocking, add capture reagent 1 (0.5 μg / mL). Then transfer it to a new 384-well plate at a volume of 12.5 μL per well and incubate at 37°C for 1 h. Next, add the polypeptides to be tested diluted in gradients and reagent 2 (0.25 μg / mL) at a volume of 12.5 μL per well in sequence and incubate at 37°C for 1 h. Then add reagent 3 (0.1 μg / mL) at a volume of 25 μL per well and incubate at 37°C for 1 h. After discarding the solutions in the wells, add 80 μL of TBST washing solution with pH = 7.4 to each well and wash the plate 4 times, with an interval of 4 min each time. After drying, add 25 μL of TMB (manufactured by Solarbio, catalog number PR1210) to each well and continue to incubate at 37°C for 30 min for color development. Finally, add 25 μL of termination solution (1 M HCl) to each well to terminate the reaction. Use a microplate reader (model cytation5) to read the absorbance of each well at 450 nm. After calculating the inhibition rate of each polypeptide concentration according to the formula, use GraphPad prism software to plot a graph and calculate the IC50 value of each polypeptide. One target polypeptide is screened, that is, the polypeptide with the amino acid sequence as shown in SEQ ID NO: 1. Among them, the formula for calculating the inhibition rate is: Inhibition% = (1 - (Absorbance of sample - Absorbance of background) / (Absorbance of positive - Absorbance of background)) × 100%; where, Sample: refers to adding polypeptides to the reaction system; Positive: refers to not adding polypeptides to the reaction system; Background: refers to not adding polypeptides and reagent 1 to the reaction system.

[0043] Example 2: Synthesis of polypeptides Design polypeptides with amino acid sequences containing 5 - 80 amino acids based on the one target polypeptide obtained in Example 1 and synthesize them according to the following method: Step 1: Gene construction Design the DNA sequence of the target polypeptide as overlapping oligonucleotide primers and synthesize them. Obtain the full-length target DNA sequence through PCR reaction, and ligate the sequence into the expression vector pET15b-sumo for fusion expression with an intein. Then transfer the aforementioned vector into Escherichia coli for culture, extract the plasmid and sequence it, and analyze the sequencing results to ensure consistency with the designed target sequence.

[0044] Step 2: Polypeptide expression The correctly constructed bacterial solution above was inoculated into the self-inducing medium. After adding 1 / 1000 of ampicillin (stock solution concentration: 100 mg / mL), it was cultured overnight with shaking at 37 °C and 200 rpm for induced expression.

[0045] Step 3: Polypeptide purification The overnight-induced bacterial solution was poured into urea and dissolved in a water bath at 60 °C for 5 min. Then, 6 M NaOH was added for ultrasonic disruption (disrupting for 3 s, with an interval of 8 s, for a total of 3 min and a power of 60%). Then, 6 M HCl was added for neutralization, and then it was incubated with 25 mL of nickel magnetic beads for 1 h. After strongly magnetically adsorbing and removing the supernatant, it was resuspended, washed 3 times, and then eluted with 500 mM imidazole. The obtained eluate was self-cleaved overnight at 35 °C. The cleaved solution was loaded onto a pre-equilibrated C18 reversed-phase column (600 mg), washed with 5 wt% acetonitrile + 1 wt% formic acid, and then eluted with 50 wt% acetonitrile. The obtained eluate was detected by SDS-PAGE electrophoresis. The molecular weight of the polypeptide was detected by mass spectrometry, and the purity of the polypeptide was detected by HPLC.

[0046] Step 4: Polypeptide refinement The polypeptide obtained in Step 3 was dissolved in an aqueous solution of 20 wt% acetonitrile, filtered through a 0.45 μm membrane, and then separated using a reversed-phase high-performance liquid chromatography system. The buffer solutions were Phase A (0.1 wt% trifluoroacetic acid aqueous solution) and Phase B (0.1 wt% trifluoroacetic acid acetonitrile solution). Among them, the chromatographic column was a BR-C18 (Sepax Technologies) reversed-phase chromatographic column. During the purification process, the detection wavelength of the chromatograph was set at 230 nm, the flow rate was 15 mL / min, and the gradient was that Phase B changed from 20% to 50% within 0 - 40 min. The relevant fractions of the product were collected, and after identifying the purity by HPLC, the fractions with a purity > 95% were combined and freeze-dried to obtain the pure polypeptide product.

[0047] Example 3: Further ELISA screening of polypeptides The designed polypeptides were screened according to the method in Example 1, and 90 target polypeptides were obtained, namely polypeptides with amino acid sequences as shown in SEQ ID NO: 2 - 91.

[0048]

[0049]

[0050] As can be seen from the results in Table 2, the polypeptides provided by the present invention can effectively inhibit the binding of SOST to LRP6.

[0051] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope 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 polypeptide obtained by disassembling is shown in any one of SEQ ID NO: 2-91.

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 having the amino acid sequence shown in any one of SEQ ID NOs: 1-91 form a ring via 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 as described in any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

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