Polypeptide in targeted binding with NGF (nerve growth factor) and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a targeted binding NGF polypeptide and application thereof. The polypeptide has an amino acid sequence as shown in a formula I. The polypeptide provided by the invention has high affinity with the NGF, and can inhibit the combination of the NGF and a receptor thereof, so as to prevent and / or treat diseases mediated by the NGF.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a polypeptide that targets and binds to NGF and applications thereof. Background Art

[0002] Nerve growth factor (NGF) is the first member of the neurotrophic factor family to be discovered. Nerve growth factor is a nerve growth regulator that has the dual biological functions of providing nutrition to neurons and promoting neurite growth. It plays an important regulatory role in the development, differentiation, growth, regeneration and functional expression of central and peripheral neurons. Nerve growth factor contains three subunits: α, β and γ. The β subunit is an active region composed of two single chains bound by non-covalent bonds.

[0003] For the nervous system, nerve growth factor has the functions of promoting neuron development, axon growth, transmitter synthesis and inhibiting nerve cell apoptosis; and it has been clinically developed for the treatment of certain nervous system diseases, such as glaucoma and Alzheimer's disease. In addition, for other systems such as cardiovascular, immune, and reproductive systems, nerve growth factor mainly regulates the function of the immune system, inhibits the mitosis of some tumor cells, and promotes wound healing.

[0004] NGF exerts its biological effects by binding to two different receptors: TrkA receptor and p75 receptor. p75 belongs to the tumor necrosis factor receptor superfamily and binds to all neurotrophic factors with the same affinity. It is a non-selective low-affinity receptor. TrkA is a 140kDa transmembrane glycoprotein encoded by the NTRK1 gene. It consists of an extracellular domain, a transmembrane region, and an intracellular region containing a tyrosine kinase domain. It belongs to the tyrosine kinase receptor family (including TrkA, TrkB, and TrkC). It is a specific high-affinity receptor for NGF, mediating most of the biological functions of NGF and serving as its functional receptor. NGF binding to TrkA receptor can lead to receptor dimerization and activate intrinsic kinase activity, thereby causing the activation of multiple signaling pathways, including Ras / MAPK, PI3K / Akt and PLCγ signaling pathways (Norman BH, McDermott J S. Targeting the nerve growth factor (NGF) pathway in drug discovery [J]. Journal of medicinal chemistry, 2017, 60 (1): 66-88), which are related to the pathophysiological process of pain and the occurrence, proliferation, angiogenesis and metastasis of tumors.

[0005] Studies have shown that peripheral nociceptors are activated to produce and release NGF under thermal, chemical or mechanical stimulation. After NGF binds to and activates TrkA receptors, it regulates the expression and activity of transient receptor potential cation channels (TRPV1) to promote Ca + Inward conduction of nociceptive pain. On the other hand, it promotes the expression of calcitonin gene-related peptide (CGRP) and substance P (SP). These neuropeptides are transported to the central terminal located in the dorsal horn of the spinal cord and released, and then bind to the NMDA receptors of central neurons to produce a long-term depolarization, leading to central sensitization. In addition, the formed NGF / TrkA complex will be internalized and retrogradely transported to the neuronal cell body, regulating the expression of bradykinin receptors (BK), acid-sensing ion channels (ASICs), voltage-gated sodium channels (VGSCs), and transient receptor potential cation channels (TRPV1) involved in the perception of pain. These cell surface receptors expression, thereby inducing peripheral sensitization and pain hypersensitivity. In addition, NGF stimulates mast cells to express histamine, 5-hydroxytryptamine, H + and NGF, forming a positive feedback loop, causing local amplification of pain signals (Shang X, Wang Z, Tao H. Mechanism and therapeutic effectiveness of nerve growth factor in osteoarthritis pain[J]. Therapeutics and clinical risk management, 2017, 13:951).

[0006] So far, a number of preclinical and clinical studies have shown that targeting the NGF-TrkA signaling pathway can effectively treat pain. US20130336964A1 discloses preclinical research data of an anti-TrkA monoclonal antibody CRB0089. In the experiment, complete Freund's adjuvant was injected into the right hind foot pad of male Wistar rats to induce mechanical hyperalgesia; CRB0089 (5 or 20 μg) was injected subcutaneously 72 hours later, and PWT was measured by using a Randall Selitto analgesia instrument 18 hours later to evaluate the in vivo analgesic activity of the drug. The results showed that CRB0089 had a significant analgesic effect in the complete Freund's adjuvant-induced inflammatory pain (CFA) model, and could dose-dependently reduce the mechanical hyperalgesia of rats after CFA injury. US20190263932A1 discloses in vivo and in vitro activity data of an anti-TrkA monoclonal antibody. The results also showed that the anti-TrkA antibody can effectively relieve pain. 1 mg / kg of anti-TrkA antibody can significantly improve the four pain indicators of rats after CFA induction.

[0007] In addition, studies have shown that TrkA participates in the occurrence and development of tumors in two ways. One is that the coding gene NTRK1 of TrkA fuses with other genes to produce chimeric oncogenes, resulting in TrkA kinase no longer being regulated and controlled by NGF and continuously activated. This structural rearrangement of TrkA is the direct cause of tumorigenesis. The other is that TrkA is overexpressed in a variety of tumors such as breast cancer, cervical squamous cell carcinoma, lung squamous cell carcinoma, epithelial ovarian cancer, head and neck squamous cell carcinoma, etc., and is overactivated in a way that depends on NGF, promoting tumor angiogenesis, perineural invasion (PNI) and epithelial-mesenchymal transition (EMT). Therefore, NGF or TrkA inhibitors can reverse NGF-induced tumor EMT by blocking the excessive activation of TrkA kinase by NGF, thereby inhibiting tumor invasion and metastasis.

[0008] Therefore, disease treatment targeting the NGF-TrkA signaling pathway is the research basis for developing analgesic or anti-tumor drugs.

[0009] Polypeptides are biologically active substances that constitute various cellular functions in the body. They have the characteristics of small relative molecular mass, high specificity, easy absorption, easy synthesis and modification, ability to enhance the body's immunity, and high safety. They have high application value in clinical treatment.

[0010] In view of this, it is of great significance to develop a drug peptide that can target and bind to NGF. Summary of the invention

[0011] The purpose of the present invention is to provide a drug polypeptide capable of targeting and binding to NGF, wherein the drug polypeptide has a high affinity to NGF.

[0012] In order to achieve the above object, the first aspect of the present invention provides a polypeptide targeting and binding to NGF or a pharmaceutically acceptable salt thereof, wherein the polypeptide has an amino acid sequence shown in Formula I:

[0013] X1-X2-C1-X3-R-X4-X5-Q-X6-X7-X8-W-X9-C2-X 10 -X 11 -X 12 Formula I,

[0014] Wherein, X1 is selected from W, M or F;

[0015] X2 is selected from Y, K, N, T or H;

[0016] X3 is selected from W, F, Y, H or M;

[0017] X4 is selected from M, E, Y, A, T, Q, V, K, L, or D;

[0018] X5 is selected from E, Q or H;

[0019] X6 is selected from F or W;

[0020] X7 is selected from H or Q;

[0021] X8 is selected from N, D or E;

[0022] X9 is selected from T, V, E, Q, S, I or A;

[0023] X 10 is selected from D, E, T, I, V, K or H;

[0024] X 11 Selected from Y, A, W, Q, G or H;

[0025] X 12 Selected from L, G, D, N, M, T, L, V, S, W, F, Y or H.

[0026] 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 targeting and binding to NGF as described in the first aspect above.

[0027] The third aspect of the present invention provides a vector, which contains the gene described in the second aspect.

[0028] The fourth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the third aspect.

[0029] The fifth aspect of the present invention provides a pharmaceutical composition, which contains a therapeutically effective amount of the polypeptide targeting and binding to NGF or a pharmaceutically acceptable salt thereof as described in the first aspect.

[0030] The sixth aspect of the present invention provides the use of at least one of the polypeptide targeting and binding to NGF described in the first aspect or its pharmaceutically acceptable salt, 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 drugs for preventing and / or treating diseases mediated by NGF.

[0031] The polypeptide provided by the present invention is obtained through phage screening, has a high affinity with NGF, and can effectively inhibit the binding of NGF to its receptor, thereby preventing and / or treating diseases mediated by NGF.

[0032] Other features and advantages of the present invention will be described in detail in the following detailed description. DETAILED DESCRIPTION

[0033] 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.

[0034] 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.

[0035] 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. Exemplary computer programs for comparing and aligning sequence pairs may 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.

[0036] As mentioned above, the first aspect of the present invention provides a polypeptide targeting and binding to NGF or a pharmaceutically acceptable salt thereof, wherein the polypeptide has an amino acid sequence shown in Formula I:

[0037] X1-X2-C1-X3-R-X4-X5-Q-X6-X7-X8-W-X9-C2-X 10 -X 11 -X 12 Formula I,

[0038] Wherein, X1 is selected from W, M or F;

[0039] X2 is selected from Y, K, N, T or H;

[0040] X3 is selected from W, F, Y, H or M;

[0041] X4 is selected from M, E, Y, A, T, Q, V, K, L, or D;

[0042] X5 is selected from E, Q or H;

[0043] X6 is selected from F or W;

[0044] X7 is selected from H or Q;

[0045] X8 is selected from N, D or E;

[0046] X9 is selected from T, V, E, Q, S, I or A;

[0047] X 10 is selected from D, E, T, I, V, K or H;

[0048] X 11 Selected from Y, A, W, Q, G or H;

[0049] X 12 Selected from L, G, D, N, M, T, L, V, S, W, F, Y or H.

[0050] The inventors of the present invention have found that when the position and / or order of one or more amino acid residues in C1, R, Q, W, C2 in the above formula I is changed, the affinity of the obtained polypeptide to NGF is significantly reduced. In view of this, the present invention provides the above polypeptide having the amino acid sequence shown in formula I.

[0051] Preferably, the polypeptide has a first amino acid sequence as shown in any one of SEQ ID NOs: 1-18; or, has an amino acid sequence having at least 90% sequence identity with the first amino acid sequence.

[0052] The first amino acid sequence shown in any one of SEQ ID NOs: 1-18 in the present invention is obtained by using phage display technology. Specifically, it is as follows:

[0053] A single-stranded DNA mutation random library was constructed, and the mutation random library was transferred into Escherichia coli strain SS320 cells infected with M13K07 helper phage to obtain a phage display library; NGF was used as the target protein, and the phage display library was used as the mobile phase for co-incubation, and the target polypeptide was obtained after washing, elution and amplification.

[0054] It should be noted that the present invention has no particular restrictions on the construction methods of vectors and phages, and those skilled in the art can make selections in combination with the known technical means in the art. A method is exemplarily provided in the present invention, and those skilled in the art should not be construed as limiting the present invention. Specifically, the method in Liu, B., et al. Improving the mutagenesis efficiency of the Kunkel method by codon optimization and annealing temperature adjustment. New Biotechnology. 56 (2019) can be referred to.

[0055] It should be noted that the present invention has no particular limitation on the method for constructing the random library of single-stranded DNA mutations, and those skilled in the art can select it in combination with the known technical means in the art. The present invention provides an exemplary construction method, which should not be construed as a limitation of the present invention by those skilled in the art. Specifically, it can be carried out with reference to the method in Tonikian, R., et al. Identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries. Nature Protocol. 2.6 (2007): 1368-1386.

[0056] It should be noted that the present invention has no particular limitation on the above-mentioned method for obtaining the target polypeptide, and those skilled in the art can select it in combination with the known technical means in the art. The present invention provides an exemplary screening method, which should not be construed as limiting the present invention by those skilled in the art. Specifically, the method can be performed with reference to Padmanaban, G., et al. Identification of peptides that selectively bind to myoglobin by biopanning of phage displayed-peptide library. Journal of Biotechnology. 2.6 (2007): 1368-1386.

[0057] Preferably, a disulfide bond is formed between C1 and C2.

[0058] Preferably, the N-terminus and / or C-terminus of the amino acid sequence of the polypeptide is modified with at least one connecting peptide in combination A; the combination A consists of the following connecting peptides:

[0059] SAAG, GSGS, GSGC, SAAGG, AGGGGSG, GAGGGGSG, GGGGS.

[0060] Further preferably, the combination A consists of the following connecting peptides:

[0061] SAAG, GSGS.

[0062] Particularly preferably, the C-terminus of the amino acid sequence of the polypeptide is modified with a connecting peptide SAAG; and the N-terminus of the amino acid sequence of the polypeptide is modified with a connecting peptide GSGS. The inventors of the present invention have found that the polypeptide in this preferred case has a higher affinity.

[0063] According to a preferred embodiment, the polypeptide has any one of the following structures:

[0064]

[0065] Preferably, the pharmaceutically acceptable salt is selected from at least one of trifluoroacetate, acetate, hydrochloride and phosphate.

[0066] In the present invention, the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 18 are shown in Table 1 below:

[0067] Table 1

[0068] Amino acid sequence SEQ ID NO: 1 WYCWRMEQFHNWTCDYM SEQ ID NO: 2 WKCWREQQFHNWTCEAV SEQ ID NO: 3 MYCWRYEQWHDWVCEW SEQ ID NO: 4 WNCFRAQQFHDWTCTQS SEQ ID NO: 5 WTCFRTQQFHEWTCIAV SEQ ID NO: 6 WNCWRQQQFHDWTCEYV SEQ ID NO: 7 WHCYRVHQFHDWECEYV SEQ ID NO: 8 FTCYRKEQFHDWTCIWG SEQ ID NO: 9 WTCWRLHQFQDWQCEWT SEQ ID NO: 10 WTCHRDQQFHEWQCVWL SEQ ID NO: 11 WKCWRKEQWHNWSCEWT SEQ ID NO: 12 MYCWRVQQFHDWQCDWM SEQ ID NO: 13 WHCYRVEQWHDWICKGG SEQ ID NO: 14 WYCWRDQQWHEWSCVHN SEQ ID NO: 15 WKCWRDEQFHDWSCHWG SEQ ID NO: 16 WTCWRLEQFHDWVCVGD SEQ ID NO: 17 WHCFREQQWHDWVCKGG SEQ ID NO: 18 WYCFRVEQFHEWACKQL .

[0069] 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 targeting and binding to NGF as described in the first aspect above.

[0070] As mentioned above, the third aspect of the present invention provides a vector, which contains the gene described in the second aspect.

[0071] As mentioned above, the fourth aspect of the present invention provides a host cell, which contains the vector described in the third aspect.

[0072] As mentioned above, the fifth aspect of the present invention provides a pharmaceutical composition, which contains a therapeutically effective amount of the polypeptide targeting and binding to NGF or a pharmaceutically acceptable salt thereof as described in the first aspect.

[0073] Preferably, the composition further contains a pharmaceutically acceptable carrier.

[0074] It should be noted that the present invention has no particular limitation on the type of the carrier, and those skilled in the art can select the carrier according to the technical means known in the art, as long as the polypeptide provided in the present invention can target NGF.

[0075] As described above, the sixth aspect of the present invention provides the use of at least one of the polypeptide targeting and binding to NGF described in the first aspect or its pharmaceutically acceptable salt, 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 NGF-mediated diseases.

[0076] Preferably, the NGF-mediated disease is selected from at least one of pain and cancer.

[0077] More preferably, the pain is chronic pain, and the cancer is selected from at least one of colorectal cancer, breast cancer, cervical squamous cell carcinoma, lung squamous cell carcinoma, epithelial ovarian cancer, and head and neck squamous cell carcinoma.

[0078] The present invention will be described in detail below by way of examples.

[0079] In the following examples, unless otherwise specified, all raw materials used were commercially available.

[0080] In the following examples, unless otherwise specified, the room temperature refers to 25±5°C.

[0081] Example 1: Screening of target polypeptides

[0082] After resuspending and washing the magnetic beads (streptavidin T1 magnetic beads, manufacturer: Invitrogen, catalog number 65602) according to the method in the manual, fix 60μg of NGF (target protein) to 105μL magnetic beads, and add 60μL of PBS, and the final total volume is 180μL. Place on a rotating mixer and mix at 4°C for 20±4h. After incubation, use a magnetic stand to precipitate the magnetic beads bound to the target protein, remove the supernatant, and wash the magnetic beads 4 times with 1mL of 0.1wt% Tween PBS (PBST). Subsequently, the magnetic beads were blocked with 0.5wt% BSA-PBS at room temperature for 30min. In the first round, 5μL of 1×10 11pfu of M13K07 helper phage library (dissolved in PBS), gently mix and incubate at 4°C for 1h. After incubation, unbound phages were repeatedly washed with PBST. In the next round of screening, the concentration of Tween was gradually increased from 0.1wt% to 0.4wt% at an increase of 0.1wt% / round. Phages bound to the target protein were obtained by incubation for 2min with 0.1M citrate (pH=3.1) for elution, and the phages were immediately neutralized with 1M Tris-HCl (pH=9.1). 20μL was taken for phage titer determination, and the remaining phages were amplified using Escherichia coli strain SS320. The phages were purified and used for the next round of screening. The amount of phage input in each round was 1×10 11 pfu.

[0083] Example 2: ELISA identification of target polypeptide

[0084] 1. Plate coating: Dilute SA to 5 μg / mL with 50 mM sodium bicarbonate (pH=8.5), take 50 μL and coat the ELISA plate at 37°C for 2 h;

[0085] 2. Blocking: Use TBST + 20 mg / mL BSA blocking solution to block the above ELISA plate at 4°C overnight;

[0086] 3. Washing: Wash the ELISA plate 3 times with 200 μL of washing solution (25 mM Tris-HCl (pH=7.2) + 150 mM NaCl + 0.1 wt% BSA + 0.05 wt% Tween 20), each washing for 3 min;

[0087] 4. Binding to target protein: 200 ng of NGF was incubated with the above ELISA plate at 37°C for 1 h;

[0088] 5. Washing: Wash three times with 200 μL of washing solution (25 mM Tris-HCl (pH=7.2) + 150 mM NaCl + 0.1 wt% BSA + 0.05 wt% Tween 20), each wash for 3 min;

[0089] 6. Combined with phage: 1×10 9 / 1×10 10 pfu of phage supernatant was incubated with NGF-coated ELISA plates at 37°C for 1 h;

[0090] 7. Washing: Wash 5 times with 200 μL of washing solution (25 mM Tris-HCl (pH=7.2) + 150 mM NaCl + 0.1 wt% BSA + 0.05 wt% Tween 20), each wash for 3 min;

[0091] 8. Binding antibody: Incubate 100 μL of 0.1 μg / mL M13K07 helper phage antibody with the phage in the above ELISA plate at 37°C for 1 h;

[0092] 9. Washing: Wash 5 times with 200 μL of washing solution (25 mM Tris-HCl (pH=7.2) + 150 mM NaCl + 0.1 wt% BSA + 0.05 wt% Tween 20), each wash for 3 min;

[0093] 10. Color development: Add 100 μL of TMB color development solution mixed with equal volumes of solution A and solution B, and develop the color for 5 minutes at room temperature;

[0094] 11. Stop: Add 100 μL of 2M H2SO4;

[0095] 12. Scanning: Use a microplate reader to read the OD450 to obtain the target polypeptide with high affinity to NGF.

[0096] Example 3: Synthesis of polypeptide compounds

[0097] The polypeptide compound and its derivatives provided by the present invention adopt a solid phase synthesis method to synthesize their linear precursors, and obtain a crude target polypeptide after cutting. The solid phase carrier is 2-Chlotrityl Resin resin. In the synthesis process, the 2-Chlotrityl Resin resin is first fully swollen in N,N-dimethylformamide (DMF), and then the swollen solid phase carrier and the activated amino acid derivative are repeatedly condensed → washed → Fmoc protection → washed → the next round of amino acid condensation to achieve the desired length of the synthesized polypeptide chain, and finally a mixed solution of trifluoroacetic acid: water: triisopropylsilane: anisyl thioether (v:v:v:v=90:2.5:2.5:5) is reacted with the solid phase resin to cleave the polypeptide from the solid phase carrier, and then the target polypeptide crude product is obtained after precipitation by frozen methyl tert-butyl ether. The target polypeptide crude product is purified and separated by a C18 reverse phase preparative chromatography column in a 0.1% trifluoroacetic acid acetonitrile / water system to obtain a pure product of the polypeptide and its derivatives. The following is a specific synthesis method of polypeptide compound 1, wherein Table 2 lists the reagents used in the synthesis.

[0098] Table 2

[0099]

[0100]

[0101] Synthesis of peptide compound 1:

[0102] Step 1: Coupling of the first amino acid Fmoc-Ser(tBu)-OH

[0103] Fully swell 0.1mmol of 2-Chlorotrityl chloride resin in DCM for 1h. Weigh 0.08mmol of Fmoc-Ser(tBu)-OH and 0.32mmol of DIEA and dissolve them in 5mL of DCM, add to the swollen resin, and react at room temperature for 2h. After the reaction is completed, add 10mL of blocking solution (DCM: methanol: DIEA (v:v:v = 85:10:5) and block at room temperature for 10min. Wash the blocked resin with DCM 5 times and then with DMF 5 times before use.

[0104] Step 2: Synthesis of linear precursor peptide chain

[0105] Linear precursor peptide chain of polypeptide compound 1:

[0106] SAAGWYCWRMEQFHNWTCDYM-GSGS.

[0107] The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the second position of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

[0108] (1) Fmoc-deprotect the swollen resin twice with 10 mL of a mixture of piperidine:DMF (v:v=20:80), each time for 8 min;

[0109] (2) Wash the resin with DMF 6-8 times until the pH of the liquid is neutral;

[0110] (3) Dissolve 0.5 mmol of Fmoc-AA, 0.5 mmol of HCTU and 1 mmol of NMM in DMF, then add the washed resin and react at room temperature for 1 h;

[0111] (4) Wash the resin 4-6 times with DMF before coupling the next amino acid;

[0112] After the synthesis of the linear precursor peptide chain, the resin was washed 5 times with DMF and then 5 times with DCM, and the washed resin was dried in a vacuum for later use.

[0113] Step 3: Cleavage of the linear precursor peptide chain

[0114] Add 10 mL of a freshly prepared mixture of trifluoroacetic acid: water: triisopropylsilane: thioanisole (v:v:v:v = 90:2.5:2.5:5) to the resin obtained in step 2 and shake for 2 h at room temperature. After the reaction is completed, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 times the volume of frozen MTBE to obtain a crude polypeptide. Wash the crude polypeptide with MTBE 3 times and then dry it in a vacuum for later use.

[0115] Step 4: Intramolecular disulfide bond formation

[0116] The crude product obtained in step 3 was fully dissolved in 20% (v:v) DMSO, and then 2mM GSH was added to 50mM ammonium bicarbonate buffer (pH=8.0, containing 30vol% acetonitrile), and the dissolved polypeptide solution was slowly added dropwise to the above buffer to a final concentration of 1mg / mL, and shaken at room temperature for 16h. The reaction results were monitored by LC-MS, and purification was directly carried out after the reaction was completed.

[0117] Step 5: Purification of crude peptide

[0118] The crude polypeptide was dissolved in 20 vol% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffer was phase A (0.1 wt% trifluoroacetic acid aqueous solution) and phase B (0.1 wt% trifluoroacetic acid acetonitrile solution). The chromatographic column was a BR-C18 (Saifen Technology) reversed-phase chromatographic column. During the purification process, the chromatograph detection wavelength was set to 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 product-related fractions were collected, and after HPLC identification of the purity, the fractions > 95% were combined and freeze-dried to obtain the pure polypeptide.

[0119] Step 6: Detection and characterization methods

[0120] The purified peptide from step 5 was subjected to analytical high performance liquid chromatography and liquid chromatography / mass spectrometry to determine the purity and intramolecular disulfide bond formation of the compound.

[0121] Other polypeptide compounds of the present invention can be synthesized by referring to the synthesis method of polypeptide compound 1.

[0122] Example 4: Determination of affinity of target polypeptide to NGF by ELISA

[0123] NGF protein (concentration of 0.2μg / mL) was fixed on a 384-well plate at 25μL / well; then the target polypeptide solutions were transferred to the aforementioned 384-well plate at 12.5μL / well using a workstation, and then the premix (TrkA protein (concentration of 1.2μg / mL) and peroxidase-conjugated affinity pure goat anti-human IgG-Fcγ fragment (concentration of 0.2μg / mL) were mixed at room temperature for 1h at a volume ratio of 1:1) in the aforementioned 384-well plate at 12.5μL / well, and the bubbles were removed by instant centrifugation and incubated in a 37℃ incubator for 2h. After discarding the liquid in the well, 80μL of 1×TBST washing solution with pH=7.4 was added to wash the plate 4 times, each time for 4min. The 384-well plate was drained, 25μL of TMB colorimetric solution was added to each well, and the plate was incubated in a 37℃ incubator for 30min. Finally, 25 μL of stop solution (1M HCl) was added to each well to terminate the reaction. The absorbance of each well at 450 nm was read using an ELISA reader. After calculating the inhibition rate of each target peptide concentration according to formula (1), the IC50 value of each peptide was calculated using graphpadprism software. The specific results are shown in Table 3. The inhibition rate was calculated as follows:

[0124] Inhibition% = (1-(absorbance of sample-absorbance of background) / (absorbance of positive-absorbance of background)) × 100%;

[0125] in,

[0126] Sample: refers to adding peptides into the reaction system;

[0127] Positive: refers to no peptide added to the reaction system;

[0128] Background: refers to the reaction system without adding polypeptide and NGF protein.

[0129] Table 3: IC values ​​of target peptides 50 value

[0130]

[0131]

[0132] It can be seen from the results in Table 3 that the polypeptide provided by the present invention has a high affinity with NGF and can effectively inhibit the binding of NGF-TrkA.

[0133] 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 targeting and binding to NGF or a pharmaceutically acceptable salt thereof, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO: 2 or 11.

2. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1, wherein: A disulfide bond is formed between the first cysteine ​​and the second cysteine ​​in the amino acid sequence of the polypeptide.

3. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein: The N-terminus and / or C-terminus of the amino acid sequence of the polypeptide is modified with at least one connecting peptide in combination A; The combination A consists of the following connecting peptides: SAAG, GSGS, GSGC, SAAGG, AGGGGSG, GAGGGGSG, GGGGS; Preferably, the combination A consists of the following connecting peptides: SAAG, GSGS.

4. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein: The C-terminus of the amino acid sequence of the polypeptide is modified by using the connecting peptide SAAG; and the N-terminus of the amino acid sequence of the polypeptide is modified by using the connecting peptide GSGS.

5. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein: The polypeptide has any one of the following structures:

6. 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 targeting and binding to NGF as described in any one of claims 1-5.

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

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

9. A pharmaceutical composition, characterized in that The pharmaceutical composition contains a therapeutically effective amount of the polypeptide targeting and binding to NGF as described in any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.

10. Use of at least one of the polypeptide targeting and binding to NGF according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, the gene according to claim 6, the vector according to claim 7, the host cell according to claim 8, and the pharmaceutical composition according to claim 9 in the preparation of a drug for preventing and / or treating NGF-mediated diseases; Preferably, the NGF-mediated disease is selected from at least one of pain and cancer; Preferably, the pain is chronic pain, and the cancer is selected from at least one of colorectal cancer, breast cancer, cervical squamous cell carcinoma, lung squamous cell carcinoma, epithelial ovarian cancer, and head and neck squamous cell carcinoma.

Images