Polypeptide for inhibiting combination of RANK and RANKL and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a polypeptide for inhibiting combination of RANK and RANKL and application of the polypeptide. The amino acid sequence of the polypeptide is shown as SEQ ID NO: 1-3. The polypeptide provided by the invention can effectively inhibit the combination of RANK and RANKL 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 in particular to a polypeptide for inhibiting the binding between RANK and RANKL and an application thereof. Background Art

[0002] RANK (receptor activator of NF-κB), also known as nuclear factor-κB receptor activator, is a member of the tumor necrosis factor TNF (tumor necrosis factor) superfamily. RANK contains 616 amino acids and consists of a C-terminal cytoplasmic region, an N-terminal extracellular region, a signal peptide and a transmembrane region. It is mainly expressed on the surface of osteoclast precursor cells, mature osteoclasts and dendritic cells. With the deepening of research, the presence of RANK has also been detected on the surface of some cancer cells (such as breast cancer and prostate cancer).

[0003] RANKL (receptor activator of NF-κB ligand) is a ligand for the receptor activator of nuclear factor-κB. It is a type II transmembrane protein that exists in a soluble form released by type II membrane protein or extracellular protease hydrolysis.

[0004] RANK specifically binds to RANKL, and the signaling pathways mediated by them play a core role in many physiological and pathological processes. Under normal physiological conditions, the combination of RANK and RANKL is essential for the fine regulation of osteoclast differentiation, maturation, and bone remodeling. It precisely regulates and maintains the dynamic balance of bone metabolism. For example, during the bone growth and development stage of children, this pathway coordinates the construction and remodeling of bone tissue to ensure normal bone growth and morphological shaping.

[0005] However, when this pathway is abnormally activated, it can trigger a series of serious bone-related diseases. In osteoporosis, RANKL expression is significantly upregulated, overstimulating osteoclast activity, causing bone absorption to far exceed bone formation, resulting in a rapid loss of bone mass, destruction of bone tissue microstructure, and greatly increasing the risk of fractures. According to statistics, about 200 million women worldwide are affected by osteoporosis, and the incidence rate rises significantly with age. In addition, in the process of bone metastasis of various malignant tumors, tumor cells secrete a large amount of RANKL, inducing excessive activation of osteoclasts and destroying bone tissue. At the same time, the released bone matrix growth factor further promotes the proliferation and migration of tumor cells, forming a vicious circle. For solid tumors such as breast cancer and prostate cancer, about 70% of advanced patients will have bone metastasis, which seriously affects the quality of life and prognosis of patients.

[0006] At present, the main treatment for the RANK / RANKL pathway is monoclonal antibody drugs, such as Denosumab, which is already on the market. Although this type of drug has achieved certain efficacy in clinical applications and can effectively inhibit RANKL activity and reduce bone resorption, it also has many limitations. First, monoclonal antibody drugs have a large molecular weight and are difficult to penetrate tissue barriers. The drug concentration reaching the lesion site is limited, which affects the therapeutic effect. Its high production cost makes it difficult for many patients to bear the burden for a long time, limiting its widespread clinical application. Secondly, monoclonal antibodies are usually administered subcutaneously or intravenously, which is inconvenient to use and requires professional medical staff to operate, increasing the patient's medical burden. In addition, monoclonal antibodies, as exogenous proteins, have potential immunogenicity and may trigger an immune response in the body, leading to serious adverse reactions, such as allergic reactions, infusion reactions, etc. Some patients have to interrupt treatment because they cannot tolerate it.

[0007] In view of the shortcomings of existing treatment methods, there is an urgent clinical need to develop a class of peptide drugs that can effectively inhibit the binding of RANK and RANKL. Peptide drugs have the advantages of relatively small molecular weight, good tissue penetration, strong specificity, and low immunogenicity. They are expected to overcome the shortcomings of monoclonal antibody drugs and provide a safer, more effective and economical treatment for bone-related diseases.

[0008] Therefore, the development of a new drug that can specifically inhibit the binding of RANK and RANKL has important clinical significance and market value. Summary of the invention

[0009] The purpose of the present invention is to provide a pharmaceutical polypeptide capable of inhibiting the binding of RANK and RANKL, wherein the pharmaceutical polypeptide has a high affinity with RANK and / or RANKL.

[0010] To achieve the above object, the first aspect of the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof for inhibiting the binding of RANK to RANKL, wherein the polypeptide is a polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1-3 or a variant thereof.

[0011] 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 RANK and RANKL as described in the first aspect.

[0012] The third aspect of the present invention provides a vector, which contains the gene described in the second aspect.

[0013] The fourth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the third aspect.

[0014] The fifth aspect of the present invention provides a pharmaceutical composition, which contains a therapeutically effective amount of the polypeptide for inhibiting the binding between RANK and RANKL as described in the first aspect or a pharmaceutically acceptable salt thereof.

[0015] The sixth aspect of the present invention provides the use of at least one of the polypeptide inhibiting the binding of RANK and RANKL described in the first aspect or a pharmaceutically acceptable salt thereof, the gene described in the second aspect, the vector described in the third aspect, the host cell described in the fourth aspect, and the pharmaceutical composition described in the fifth aspect in the preparation of a drug for preventing and / or treating diseases related to the signal pathway binding to RANK and RANKL.

[0016] The polypeptide provided by the present invention is obtained through screening, and can effectively inhibit the binding of RANK and RANKL, thereby preventing and / or treating diseases related to the signal pathways thereof.

[0017] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a graph showing the ELISA results of the polypeptides with amino acid sequences shown in SEQ ID NOs: 1-3 provided by the present invention inhibiting the binding between RANK and RANKL; Figure 2 It is a graph showing the ELISA results of the polypeptides provided by the present invention containing the amino acid sequences shown in SEQ ID NOs: 4-11 inhibiting the binding between RANK and RANKL. DETAILED DESCRIPTION

[0019] The endpoints and any values ​​of the ranges disclosed in this article 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 endpoint values ​​of each range, the endpoint values ​​of each range and the 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 as specifically disclosed in this article.

[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 RANK to RANKL, wherein the polypeptide is a polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1-3 or a variant thereof.

[0022] In the present invention, the variant includes a polypeptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1-3; it also includes a polypeptide obtained by connecting a tag protein or modification to the amino terminus and / or carboxyl terminus of the amino acid sequence shown in any one of SEQ ID NOs: 1-3; and it also includes a polypeptide with the same function obtained by substituting, deleting and / or adding the amino acid sequence shown in any one of SEQ ID NOs: 1-3.

[0023] In the present invention, the "sequence identity" refers to the degree to which two sequences (e.g., amino acids) have the same residues at the same position after alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" means that the amino acid sequence has X% identity with SEQ ID NO: Y, and is described as X% of the residues in the amino acid sequence being identical to the residues of the sequence disclosed in SEQ ID NO: Y. Typically, such calculations are performed using computer programs. Computer programs for comparing and aligning sequence pairs can exemplarily be ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN or GCG (Devereux et al., 1984). In addition, when determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure, which has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Such "conservative" amino acids may be amino acids known in the art.

[0024] It should be noted that the present invention has no particular limitation on the type of the tag protein, and those skilled in the art can select it according to the technical means known in the art. The tag protein will not affect the activity of the polypeptide provided by the present invention. In practical application, those skilled in the art can choose whether to connect the tag protein to the amino terminus and / or carboxyl terminus of the amino acid sequence according to the needs.

[0025] In the present invention, the modifications include amino modification, hydroxylation modification, carboxylation modification, carbonylation modification, amidation modification, alkylation modification, phosphorylation modification, glycosylation modification, cyclization modification, biotinylation modification, acetylation modification, esterification modification, fluorescent group modification, polyethylene glycol modification and immobilization modification.

[0026] The amino acid sequence shown in any one of SEQ ID NOs: 1-3 in the present invention is a polypeptide having the function of inhibiting the binding between RANK and RANKL 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 RANKL and its marker, a reactant II containing RANK, and a reactant III capable of undergoing a colorimetric reaction with the marker to obtain a detection solution; finally, using an ELISA instrument to detect the absorbance of the detection solution at a specific wavelength, and obtaining the target polypeptide based on the inhibition rate calculated based on the absorbance.

[0027] 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 may make their selections based on technical means known in the art. A preferred specific implementation is exemplarily provided in the following text of the present invention, which should not be construed as a limitation on the present invention by those skilled in the art.

[0028] In the present invention, the polypeptide library is a polypeptide library containing nearly 73,000 polypeptides of 80 amino acids constructed by Hunan Zhongsheng Quanpeptide Biotechnology Co., Ltd. using PICT (Peptide Information Compression Technology) technology. The specific construction method thereof can be found in CN107849737A and CN111727194A.

[0029] It should be noted that the present invention has no particular limitation on the method for synthesizing the polypeptide, and those skilled in the art can select the method according to the technical means known in the art. For example, a preferred method for synthesizing polypeptides is exemplified in the following text of the present invention, which should not be construed as a limitation on the present invention by those skilled in the art.

[0030] Preferably, the polypeptide further comprises a polypeptide having an amino acid sequence of 20-40 amino acids obtained by disassembling the amino acid sequence shown in any one of SEQ ID NOs: 1-3.

[0031] More preferably, the polypeptide obtained by disassembly is a polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 4-11.

[0032] According to a particularly preferred embodiment, the first amino acid and the last amino acid in the amino acid sequence of the polypeptide form a ring via a peptide bond.

[0033] Preferably, the pharmaceutically acceptable salt is selected from at least one of trifluoroacetate, acetate, hydrochloride and phosphate.

[0034] In the present invention, the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 11 are shown in Table 1.

[0035] Table 1

[0036] 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 RANK and RANKL as described in the first aspect above.

[0037] As mentioned above, the third aspect of the present invention provides a vector, which contains the gene described in the second aspect.

[0038] As mentioned above, the fourth aspect of the present invention provides a host cell, which contains the vector described in the third aspect.

[0039] As mentioned above, the fifth aspect of the present invention provides a pharmaceutical composition, which contains a therapeutically effective amount of the polypeptide for inhibiting the binding of RANK and RANKL as described in the first aspect above, or a pharmaceutically acceptable salt thereof.

[0040] Preferably, the composition further comprises an excipient.

[0041] 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 the excipient according to technical means known in the art, as long as the polypeptide provided in the present invention can target RANK and / or RANKL.

[0042] As described above, the sixth aspect of the present invention provides the use of at least one of the polypeptide inhibiting the binding of RANK and RANKL described in the first aspect or a pharmaceutically acceptable salt thereof, the gene described in the second aspect, the vector described in the third aspect, the host cell described in the fourth aspect, and the pharmaceutical composition described in the fifth aspect for the preparation of a drug for preventing and / or treating diseases related to the signal pathway binding to RANK and RANKL.

[0043] Preferably, the disease associated with the signaling pathway in which RANK and RANKL are combined is selected from at least one of anaphylactic shock, skin allergic reaction, respiratory allergic reaction, digestive tract allergic reaction, pathological cardiac remodeling and dysfunction.

[0044] More preferably, the disease associated with the signaling pathway in which RANK and RANKL are combined is at least one selected from osteoporosis, bone cancer, osteolytic tumors, and rheumatoid arthritis.

[0045] Further preferably, the osteoporosis includes familial osteoporosis, senile or postmenopausal osteoporosis, osteoporosis caused by glucocorticoids, drug-induced osteoporosis and immobilization osteoporosis; the osteolytic tumor includes bone metastasis, giant cell tumor of bone, and multiple myeloma.

[0046] Particularly preferably, the bone metastasis cancer includes breast cancer, lung cancer, and prostate cancer.

[0047] The present invention will be described in detail below by way of examples.

[0048] In the following examples, unless otherwise specified, all raw materials used were commercially available.

[0049] In the following examples, unless otherwise specified, the room temperature refers to 25±5°C.

[0050] Experimental reagents: Reagent 1: Biotinylated Human TNFSF11 / RANKL Protein, His, AvitagTM, activetrimer (purchased from Beijing Biopsies Biotechnology Co., Ltd.); Reagent 2: Human RANK / TNFSF11A Protein, Mouse IgG2a Fc Tag (purchased from Beijing Biopsies Biotechnology Co., Ltd.); Reagent 3: Streptavidin-Eu (purchased from AAT Bioquest); Reagent 4: Goat Anti-mouse IgG Fc-Alexa 647 (purchased from Jackson ImmunoResearchLaboratories); Reagent 5: Streptavidin, HRP Conjugated (purchased from Thermo).

[0051] Example 1: TR-FRET screening of peptides Dissolution of peptide library: Peptides in the peptide library were placed in a 96-deep-well plate, centrifuged at 4000 rpm for 3 min, and then 200 μL / well of ultrapure water (peptide concentration was 50 μM) was added; then the plate was sealed with a silicone cap and placed in a 95°C water bath for 5 min, and then centrifuged at 4000 rpm for 3 min; Dilution of peptide library: The peptides after centrifugation were transferred to a 384-well plate using a workstation, and the peptide concentration was diluted to the experimental concentration (10 μM) using loading buffer (Tris-HCl buffer, pH=7.4) to obtain each mixed solution; Peptide screening: 2μM of the above mixed solutions, 1nM of reagent 1, and 1nM of reagent 2 were added to a 384-well plate at 4μL / well, and then centrifuged to remove bubbles. Then, the premixed solution of reagent 3 and reagent 4 was added at 8μL / well (mixed at room temperature for 1h at a volume ratio of 1:1), and then centrifuged to remove bubbles; then incubated at room temperature in the dark for 2h. The detection was performed using an ELISA reader (model cytation5), with an excitation light of 320nm, and the fluorescence signal values ​​of each well at wavelengths of 665nm and 620nm were detected. After calculating the inhibition rate of each peptide concentration according to the formula, the IC50 value of each peptide was calculated using GraphPad prism software, and 3 target peptides were screened, namely, the peptides with amino acid sequences as shown in SEQ ID NO: 1-3; wherein, the calculation formula for the inhibition rate is: Inhibition%=(1-(sample signal value-background signal value) / (positive signal value-background signal value))×100%; where, Sample: refers to adding peptides into the reaction system; Positive: refers to no peptide added to the reaction system; Background: refers to the reaction system without adding peptide, reagent I and reagent II; Signal value: refers to the ratio of the fluorescence signal value of each well at a wavelength of 665nm to the fluorescence signal value of the corresponding well at a wavelength of 620nm.

[0052] Example 2: Synthesis of polypeptides According to the three target polypeptides obtained in Example 1, polypeptides containing an amino acid sequence of 5-80 amino acids were designed and synthesized according to the following method: Step 1: Gene construction The DNA sequence of the target polypeptide is designed as overlapping oligonucleotide primers and synthesized. The full-length target DNA sequence is obtained by PCR reaction, and the sequence is connected to the expression vector pET15b-sumo for fusion expression with the intein. The aforementioned vector is then transferred into Escherichia coli for culture, the plasmid is extracted and sequenced, and the sequencing results are analyzed to ensure that they are consistent with the designed target sequence.

[0053] Step 2: Peptide expression The above-constructed bacterial solution was inoculated into the auto-induction medium, and after adding 1 / 1000 ampicillin (mother solution concentration: 100 mg / mL), shaking culture was carried out at 37°C and 200 rpm overnight to induce expression.

[0054] Step 3: Peptide purification The overnight induced bacterial solution was poured into urea and dissolved in a 60°C water bath for 5 minutes, then 6M NaOH was added for ultrasonic disruption (3s, 8s interval, 3min, 60% power), then 6M HCL was added for neutralization and then incubated with 25mL nickel magnetic beads for 1h. After strong magnetic adsorption to remove the supernatant, it was resuspended, washed 3 times and eluted with 500mM imidazole, and the obtained eluate was self-sheared at 35°C overnight. The sheared solution was loaded onto a well-balanced C18 reverse phase column (600mg), washed with 5wt% acetonitrile + 1wt% formic acid and then eluted with 50wt% acetonitrile, and the obtained eluate was subjected to SDS-PAGE electrophoresis. The molecular weight of the polypeptide was detected by mass spectrometry, and the purity of the polypeptide was detected by HPLC.

[0055] Example 3: Further ELISA screening of polypeptides Reagent 2 was diluted to 0.125 μg / mL with coating solution (0.05 M carbonate buffer (pH = 9.6)) and added to a 384-well plate at a volume of 25 μL / well for overnight coating at 4°C. Then, after washing 4 times with washing solution (a mixture of Tween-20 and TBS buffer (pH=7.4) prepared at a mass to volume ratio of 0.05%), blocking solution (a mixture of BSA and TBS buffer (pH=7.4) prepared at a mass to volume ratio of 2%) was added for blocking at 37°C; after washing 4 times with the above washing solution, a mixture of the peptide to be tested in gradient dilution, 0.06μg / mL of reagent 1 and reagent 5 mixed at a volume ratio of 1:1 was added in sequence, and incubated at 37°C for 1h, the unbound part was washed with the above washing solution, and 25μL of TMB (manufacturer: Solarbio, catalog number: PR1210) was added to each well, and the color was continued to be developed at 37°C for 30min, and finally 25μL of stop solution (1M HCl) was added to each well to terminate the reaction. The absorbance of each well at 450nm was read using an enzyme reader (model: cytation5). After calculating the inhibition rate of each polypeptide concentration according to the formula, the IC50 value of each polypeptide was calculated using GraphPad prism software, and 8 target polypeptides were screened, namely, polypeptides with amino acid sequences as shown in SEQ ID NO: 4-11; wherein, the calculation formula of the inhibition rate is: Inhibition%=(1-(absorbance of sample-absorbance of background) / (absorbance of positive-absorbance of background))×100%; where, Sample: refers to adding peptides into the reaction system; Positive: refers to no peptide added to the reaction system; Background: refers to the reaction system without adding peptide and reagent II.

[0056] Table 2: IC50 values ​​of some peptides

[0057] Figure 1 and Figure 2 The ELISA results of the polypeptides of the amino acid sequences shown in SEQ ID NO: 1-11 provided by the present invention inhibiting the binding of RANK to RANKL are shown in the figure. In the figure, Concentration represents the concentration of the polypeptide, and Inhibition represents the inhibition rate. The IC50 value of each polypeptide can be calculated from the figure, and some results are shown in Table 2.

[0058] It can be seen from the results in Table 2 that the polypeptide provided by the present invention can effectively inhibit the binding of RANK and RANKL. The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof for inhibiting the binding of RANK to RANKL, characterized in that: The polypeptide is a polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1-3 or a variant thereof.

2. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1, wherein: The polypeptide also includes a polypeptide having an amino acid sequence of 20-40 amino acids obtained by disassembling the amino acid sequence shown in any one of SEQ ID NOs: 1-3.

3. The polypeptide or pharmaceutically acceptable salt thereof according to claim 2, wherein: The polypeptide obtained by the disassembly is a polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 4-11.

4. The polypeptide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein: The first amino acid and the last amino acid in the amino acid sequence of the polypeptide form a ring through a peptide bond.

5. A gene, characterized in that The nucleotide sequence of the gene is a nucleotide sequence capable of encoding the amino acid sequence of the polypeptide that inhibits the binding of RANK and RANKL as described in any one of claims 1 to 4.

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

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

8. A pharmaceutical composition, characterized in that The pharmaceutical composition contains a therapeutically effective amount of the polypeptide for inhibiting the binding between RANK and RANKL according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

9. Use of at least one of the polypeptide inhibiting the binding of RANK and RANKL according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, the gene according to claim 5, the vector according to claim 6, the host cell according to claim 7, and the pharmaceutical composition according to claim 8 in the preparation of a drug for preventing and / or treating diseases related to the signal pathway of the binding of RANK and RANKL.

10. The use according to claim 9, wherein: The disease associated with the signaling pathway in which RANK and RANKL are combined is selected from at least one of osteoporosis, bone cancer, osteolytic tumors, and rheumatoid arthritis.

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