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 that inhibits the binding of RANK to RANKL and applications thereof. Background Art

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

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

[0004] RANK specifically binds to RANKL, and the signaling pathways it mediates play a central role in numerous physiological and pathological processes. Under normal physiological conditions, the binding of RANK and RANKL is crucial for the fine-tuning of osteoclast differentiation, maturation, and bone remodeling, precisely regulating and maintaining the dynamic balance of bone metabolism. For example, during the skeletal growth and development stage of children, this pathway coordinates the construction and remodeling of bone tissue, ensuring 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 resorption to far exceed bone formation, resulting in a rapid loss of bone mass, destruction of bone tissue microstructure, and a significant increase in the risk of fractures. According to statistics, approximately 200 million women worldwide are affected by osteoporosis, and the incidence rate rises significantly with age. In addition, during the bone metastasis process 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 factors further promote the proliferation and migration of tumor cells, forming a vicious cycle. In solid tumors such as breast cancer and prostate cancer, about 70% of patients with advanced disease will develop bone metastasis, which seriously affects the patient's quality of life and prognosis.

[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 therapeutic effects 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, affecting the therapeutic effect. Its high production cost makes it difficult for many patients to afford it in the long term, 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 burden on patients to seek medical treatment. 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] Given the shortcomings of existing treatments, there is an urgent clinical need to develop peptide drugs that can effectively inhibit the binding of RANK and RANKL. Peptide drugs offer advantages such as 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 more 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 objectives, 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 comprising the gene described in the second aspect.

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

[0014] The fifth aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the polypeptide for inhibiting the binding of 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 or a pharmaceutically acceptable salt thereof as described in the first aspect, the gene as described in the second aspect, the vector as described in the third aspect, the host cell as described in the fourth aspect, and the pharmaceutical composition as described in the fifth aspect in the preparation of a drug for preventing and / or treating diseases related to the signaling pathway involving the binding of 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 their signaling pathways.

[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 This is a graph showing the ELISA results of the polypeptides with amino acid sequences as shown in SEQ ID NOs: 1-3 provided by the present invention inhibiting the binding between RANK and RANKL;

[0019] Figure 2 This is a graph showing the ELISA results of the polypeptides provided by the present invention comprising the amino acid sequences shown in SEQ ID NOs: 4-11 inhibiting the binding between RANK and RANKL. DETAILED DESCRIPTION

[0020] 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.

[0021] 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.

[0022] As mentioned above, the first aspect of the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof that inhibits the binding of RANK to RANKL. 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.

[0023] 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.

[0024] As used herein, "sequence identity" refers to the degree to which two sequences (e.g., amino acids) have identical 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 is X% identical to SEQ ID NO: Y, and is described as X% of the residues in the amino acid sequence being identical to the residues in the sequence disclosed in SEQ ID NO: Y. Such calculations are typically performed using computer programs. Exemplary computer programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), 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, one 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 a 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.

[0025] It should be noted that the present invention does not particularly limit the type of the tag protein, and those skilled in the art can select it based on technical means known in the art. The tag protein will not affect the activity of the polypeptide provided by the present invention. In actual application, those skilled in the art can choose whether to attach the tag protein to the amino terminus and / or carboxyl terminus of the amino acid sequence according to their needs.

[0026] In the present invention, the modifications include amino modification, hydroxylation modification, carboxyl modification, carbonyl 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.

[0027] The amino acid sequence shown in any one of SEQ ID NOs: 1-3 in the present invention is a polypeptide having the effect 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 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 enzyme-labeled 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.

[0028] 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.

[0029] In the present invention, the peptide library is a library containing nearly 73,000 peptides of 80 amino acids constructed using PICT (Peptide Information Compression Technology) technology by Hunan Zhongsheng Quanpeptide Biotechnology Co., Ltd. The specific construction method can be found in CN107849737A and CN111727194A.

[0030] 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.

[0031] 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.

[0032] More preferably, 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.

[0033] 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.

[0034] Preferably, the pharmaceutically acceptable salt is selected from at least one of trifluoroacetate, acetate, hydrochloride and phosphate.

[0035] In the present invention, the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 11 are shown in Table 1.

[0036] Table 1

[0037]

[0038]

[0039] 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.

[0040] As mentioned above, the third aspect of the present invention provides a vector containing the gene described in the second aspect.

[0041] As mentioned above, the fourth aspect of the present invention provides a host cell, which contains the vector described in the third aspect.

[0042] 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 RANK to RANKL as described in the first aspect or a pharmaceutically acceptable salt thereof.

[0043] Preferably, the composition further contains an excipient.

[0044] 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 the polypeptide provided in the present invention can target RANK and / or RANKL.

[0045] As described above, the sixth aspect of the present invention provides the use of at least one of the polypeptide or pharmaceutically acceptable salt thereof for inhibiting the binding of RANK and RANKL described in the first aspect, 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 preparing a drug for preventing and / or treating diseases related to the signaling pathway binding to RANK and RANKL.

[0046] Preferably, the disease associated with the signaling pathway in which RANK and RANKL are combined is at least one selected from anaphylactic shock, skin allergic reaction, respiratory allergic reaction, digestive tract allergic reaction, pathological cardiac remodeling and functional disorder.

[0047] 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.

[0048] Further preferably, the osteoporosis includes familial osteoporosis, senile or postmenopausal osteoporosis, glucocorticoid-induced osteoporosis, drug-induced osteoporosis and immobilization osteoporosis; the osteolytic tumor includes bone metastasis, giant cell tumor of bone and multiple myeloma.

[0049] Particularly preferably, the bone metastasis cancer includes breast cancer, lung cancer, and prostate cancer.

[0050] The present invention will be described in detail below through examples.

[0051] In the following examples, unless otherwise specified, all raw materials used were commercially available.

[0052] In the following examples, unless otherwise specified, the room temperature refers to 25±5°C.

[0053] Experimental reagents:

[0054] Reagent 1: Biotinylated Human TNFSF11 / RANKL Protein, His, Avitag™, active trimer (purchased from Beijing Biopsies Biotechnology Co., Ltd.);

[0055] Reagent 2: Human RANK / TNFSF11A Protein, Mouse IgG2a Fc Tag (purchased from Beijing Biopsies Biotechnology Co., Ltd.);

[0056] Reagent 3: Streptavidin-Eu (purchased from AAT Bioquest);

[0057] Reagent 4: Goat Anti-mouse IgG Fc-Alexa 647 (purchased from Jackson ImmunoResearch Laboratories);

[0058] Reagent 5: Streptavidin, HRP Conjugated (purchased from Thermo).

[0059] Example 1: TR-FRET screening of peptides

[0060] 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.

[0061] 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.

[0062] Peptide screening: 2 μM of each of the above mixed solutions, 1 nM of reagent 1, and 1 nM of reagent 2 were added to a 384-well plate at 4 μL / well, followed by instant centrifugation to remove bubbles. Then, a premix of reagent 3 and reagent 4 was added at 8 μL / well (mixed at room temperature for 1 hour in a volume ratio of 1:1) and then centrifuged to remove bubbles; the plate was then incubated at room temperature in the dark for 2 hours. Detection was performed using an enzyme reader (model cytation5), with an excitation light of 320 nm, and the fluorescence signal values of each well at wavelengths of 665 nm and 620 nm were detected. After calculating the inhibition rate of each polypeptide concentration according to the formula, the IC50 value of each polypeptide was calculated using GraphPadprism software, and 3 target polypeptides were screened, namely polypeptides with amino acid sequences as shown in SEQ ID NO: 1-3; wherein, the calculation formula for the inhibition rate is:

[0063] Inhibition% = (1-(sample signal value - background signal value) / (positive signal value - background signal value)) × 100%; where,

[0064] Sample: refers to the addition of peptides into the reaction system;

[0065] Positive: refers to the absence of peptide in the reaction system;

[0066] Background: refers to the reaction system without adding peptide, reagent I and reagent II;

[0067] Signal value: refers to the ratio of the fluorescence signal value of each well at a wavelength of 665 nm to the fluorescence signal value of the corresponding well at a wavelength of 620 nm.

[0068] Example 2: Synthesis of polypeptide

[0069] Based on 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:

[0070] Step 1: Gene Construction

[0071] 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.

[0072] Step 2: Peptide expression

[0073] The correctly constructed bacterial solution was inoculated into the auto-induction medium, and after adding 1 / 1000 ampicillin (mother solution concentration: 100 mg / mL), the culture was shaken at 37°C and 200 rpm overnight to induce expression.

[0074] Step 3: Peptide purification

[0075] The overnight induced bacterial solution was poured into urea and dissolved in a 60°C water bath for 5 minutes, followed by the addition of 6M NaOH for ultrasonic disruption (3s of disruption, 8s of interval, 3 minutes of duration, 60% power), and then neutralized with 6M HCL and incubated with 25mL nickel magnetic beads for 1 hour. After strong magnetic adsorption to remove the supernatant, the solution was resuspended, washed 3 times, and eluted with 500mM imidazole. The resulting eluate was subjected to self-shearing at 35°C overnight. The sheared solution was loaded onto a well-equilibrated C18 reverse phase column (600mg), washed with 5wt% acetonitrile + 1wt% formic acid, and then eluted with 50wt% acetonitrile. The resulting 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.

[0076] Example 3: Further ELISA screening of polypeptides

[0077] Reagent 2 was diluted to 0.125 μg / mL with coating solution (0.05 M carbonate buffer (pH = 9.6)), and then added to a 384-well plate at a volume of 25 μL / well for coating at 4°C overnight. The cells were then washed four times with a wash buffer (a mixture of 0.05% Tween-20 and TBS buffer (pH 7.4) at a mass-to-volume ratio) and blocked with a blocking buffer (a mixture of 2% BSA and TBS buffer (pH 7.4) at a mass-to-volume ratio) at 37°C. After another four washes with the wash buffer, a serial dilution of the test peptide and a 1:1 volume ratio mixture of 0.06 μg / mL Reagent 1 and Reagent 5 were added sequentially. The cells were incubated at 37°C for 1 h. Unbound matter was then washed again with the wash buffer. 25 μL of TMB (Solarbio, Cat. No. PR1210) was added to each well and the cells were incubated at 37°C for 30 min 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 (cytation5). After calculating the inhibition rate of each peptide concentration according to the formula, the IC50 value of each peptide was calculated using GraphPadprism software. Eight target peptides were screened, namely, peptides with amino acid sequences as shown in SEQ ID NOs: 4-11. The inhibition rate was calculated using the following formula: Inhibition% = (1-(absorbance of sample - absorbance of background) / (absorbance of positive sample - absorbance of background)) × 100%; wherein,

[0078] Sample: refers to the addition of peptides into the reaction system;

[0079] Positive: refers to the absence of peptide in the reaction system;

[0080] Background: refers to the reaction system without the addition of peptide and reagent II.

[0081] Table 2: IC50 values of some peptides

[0082] IC50 (μM) SEQ ID NO: 1 0.4 SEQ ID NO: 2 1.6 SEQ ID NO: 3 0.4

[0083] Figure 1 and Figure 2 This figure shows the ELISA results of the polypeptides of the amino acid sequences set forth in SEQ ID NOs: 1-11 provided herein inhibiting the binding of RANK to RANKL. 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. Some of the results are shown in Table 2.

[0084] From the results in Table 2, it can be seen that the polypeptide provided by the present invention can effectively inhibit the binding of RANK and RANKL.

[0085] 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 RANK to RANKL, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO: 3 or SEQ ID NO:

11.

2. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1, 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.

3. 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 RANK and RANKL according to claim 1 or 2.

4. A carrier, characterized in that The vector contains the gene according to claim 3.

5. A host cell, characterized in that The host cell contains the vector according to claim 4.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition contains a therapeutically effective amount of the polypeptide for inhibiting the binding of RANK and RANKL according to claim 1 or 2 or a pharmaceutically acceptable salt thereof.

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