Neuropeptide FF1 receptor binding molecules and their use in therapy

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly discloses a neuropeptide FF1 receptor binding molecule and application thereof in treatment. The 80 cyclopeptide SEQ ID No.1 with an activation effect on an NPFFR1 receptor is obtained through screening by a high-throughput screening technology, and the truncated peptide of the SEQ ID No.1 or a modification form of the truncated peptide is obtained through later optimization. In-vitro cellular level experiments show that the polypeptide can effectively activate the NPFFR1 receptor and has a good medical application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and specifically relates to a neuropeptide FF1 receptor binding molecule and its use in treatment. Background Art

[0002] Elshourbagy, NA and Bonini, JA and others almost simultaneously discovered two homologous NPFF receptors, namely NPFF1 and NPFF2 receptors, from rat and human brains. The two receptors have about 50% sequence homology. A large number of studies have shown that the two NPFF receptors belong to the GPCR family. In addition, studies have shown that NPFF receptors also have homology with orexin receptors and neuropeptide Y receptors. Existing studies have shown that although NPFF1 receptors and NPFF2 receptors are mainly distributed in central tissues such as the brain and spinal cord, and are less distributed in peripheral tissues, there are obvious differences between NPFF1 receptors and NPFF2 receptors, and there are also obvious differences between species.

[0003] Neuropeptide FF (NPFF) was isolated and purified from bovine thalamus in 1985. It is a C-terminal amidated octapeptide with an amino acid sequence of H-Phe-Leu-Phe-Glu-Pro-Glu-Arg-Phe-NH2 (FLFQPQRF-NH2). Pro-NPFFA and pro-NPFFB are considered to be two precursor proteins of the NPFF system. Studies have shown that peptides derived from pro-NPFFA, such as NPFF and NPAF, are more likely to bind to the NPFF2 receptor, while peptides derived from pro-NPFFB, such as NPVF, are more likely to bind to the NPFF1 receptor.

[0004] The NPFF system has been extensively studied for its involvement in the regulation of nociception and opioid analgesia. NPFF was described early as an anti-opioid peptide because it produces transient hyperalgesia, attenuates the analgesic effects of morphine, and enhances the overall morphine withdrawal syndrome. Moreover, pharmacological blockade of NPFF1 / 2 receptors has been shown to prevent the development of opioid-induced hyperalgesia (OIH) and analgesic tolerance, and reduce morphine withdrawal syndrome. In addition, NPFF has physiological activities that regulate pain perception, cardiovascular activity, neuroendocrine, gastrointestinal motility, inflammation, etc.

[0005] NPFF and its analogs have a bidirectional regulatory effect on pain. Injection of NPFF and its analogs above the spinal cord can inhibit the release of endogenous opioid peptides caused by stress response, thereby weakening analgesia and achieving an anti-opioid effect. However, intrathecal injection will produce analgesia or enhance the analgesic effect of opioids, and the analgesic effect can also be antagonized by the opioid receptor antagonist naloxone. Intraventricular injection of selective agonists of NPFF1 receptors can enhance the analgesic effect of morphine. The NPFF system plays a complex role in regulating pain, which is related to the injection site, the type of ligand and the dose. Anne Roussin et al. used NPFF1 receptor selective agonist NPVF and NPFF2 receptor selective agonist dNPA to explore the effect on body temperature. The results showed that intraventricular injection of NPVF can significantly reduce body temperature.

[0006] Pain is one of the most common symptoms in clinical patients, and drug therapy is currently the most effective treatment for pain. Traditional opioid analgesics such as morphine and fentanyl are mainly Mu-opioid receptor agonists, which can effectively relieve moderate / severe pain and are used as the first-line treatment for clinical analgesia. However, in addition to causing adverse reactions such as respiratory depression and constipation, long-term use of traditional opioids can also produce side effects such as analgesic tolerance and addiction, which seriously limits their scope of clinical application. Therefore, the development of new analgesics with high efficiency and low side effects is an important scientific issue that needs to be solved urgently.

[0007] Peptide drugs are a class of drugs with extremely fast market growth, and the peptide drug market has huge room for growth. Compared with small molecule drugs and biological agents, peptide drugs show unique advantages in drug formulation and lower side effects. Peptide drugs usually have higher target affinity. Therefore, it is very necessary to develop more peptide drugs targeting NPFF1 receptor to meet clinical needs. Summary of the invention

[0008] The purpose of the present invention is to provide a neuropeptide FF1 receptor binding molecule and its use in treatment.

[0009] In one aspect, the present invention provides a polypeptide, wherein the polypeptide is a polypeptide as shown in any one of the following (1) to (3):

[0010] (1) the amino acid sequence as described in SEQ ID No. 1 / or its modified form; or

[0011] (2) a truncated peptide of the polypeptide having the amino acid sequence as described in SEQ ID No. 1; or

[0012] (3) An amino acid sequence obtained by replacing, deleting or adding one or more amino acids to the amino acid sequence described in (1) or (2), and having the same or similar function as the amino acid sequence shown in SEQ ID No. 1.

[0013] The polypeptide of the present invention is a linear peptide or a head-to-tail cyclic peptide.

[0014] As a preferred embodiment of the present invention, the polypeptide is a polypeptide fragment consisting of 5 to 35 consecutive amino acids in the amino acid sequence as described in SEQ ID No.1.

[0015] As a preferred embodiment of the present invention, the polypeptide is a polypeptide fragment consisting of 26 to 35 consecutive amino acids in the amino acid sequence as described in SEQ ID No.1.

[0016] As a preferred embodiment of the present invention, the polypeptide can be selected from the amino acid sequences of SEQ ID No: 2 to SEQ ID No: 29.

[0017] As a preferred embodiment of the present invention, the polypeptide can be selected from the amino acid sequences shown in SEQ ID No: 3 to SEQ ID No: 6, SEQ ID No: 13, SEQ ID No: 16 to SEQ ID No: 20, SEQ ID No: 22, and SEQ ID No: 23.

[0018] As a preferred embodiment of the present invention, the polypeptide is a polypeptide fragment or a reverse polypeptide fragment consisting of 5 to 25 consecutive amino acids in the amino acid sequence as described in SEQ ID No.1.

[0019] As a preferred embodiment of the present invention, the polypeptide can be selected from the amino acid sequences of SEQ ID No: 30 to SEQ ID No: 50.

[0020] In another aspect, the present invention provides a polypeptide, wherein the polypeptide is a polypeptide as shown in (i) or (ii) below:

[0021] (i) truncated peptides of the amino acid sequences shown in SEQ ID No: 3, SEQ ID No: 6, SEQ ID No: 16 to SEQ ID No: 20, SEQ ID No: 22, and SEQ ID No: 23;

[0022] (ii) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (i), and having the same or similar function as the amino acid sequence shown in SEQ ID No. 1;

[0023] The polypeptide is a linear peptide or a head-to-tail cyclic peptide.

[0024] As a preferred embodiment of the present invention, the polypeptide is a polypeptide as shown in (i) or (ii) below:

[0025] (i) truncated peptides of the polypeptides having the amino acid sequences shown in SEQ ID No: 3, SEQ ID No: 6, SEQ ID No: 16 to SEQ ID No: 20, SEQ ID No: 22, and SEQ ID No: 23;

[0026] (ii) an amino acid sequence obtained by substituting, deleting or adding one amino acid to the amino acid sequence described in (i), and having the same or similar functions as the amino acid sequence described in (i);

[0027] The polypeptide is a linear peptide or a head-to-tail cyclic peptide.

[0028] As a preferred embodiment of the present invention, the polypeptide is a truncated peptide of the amino acid sequence shown in SEQ ID No: 3, SEQ ID No: 6, or SEQ ID No: 16.

[0029] As a preferred embodiment of the present invention, the polypeptide can be selected from the amino acid sequences of SEQ ID No: 30 to SEQ ID No: 50.

[0030] As a preferred embodiment of the present invention, the polypeptide can be selected from the amino acid sequences shown in SEQ ID No: 39 and SEQ ID No: 50.

[0031] Further preferably, the polypeptide can be selected from the amino acid sequence shown in SEQ ID No:39.

[0032] In another aspect, the present invention provides a polypeptide comprising an amino acid sequence as shown in SEQ ID No: 39, and an amino acid sequence having the same or similar function as the amino acid sequence as shown in SEQ ID No. 39.

[0033] Some of the polypeptides described in the present invention are shown in Table 1.

[0034] Table 1 Amino acid sequence of the polypeptide of the present invention

[0035]

[0036]

[0037] The polypeptides of the present invention may be modified, and the modified forms (usually without changing the primary structure) include: chemical derivatization forms of polypeptides in vivo or in vitro, including but not limited to acetylation, carboxylation, alkylation, acylation, and carbamylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the polypeptide or in further processing steps. This modification can be achieved by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylation enzyme). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, and phosphothreonine). Also included are polypeptides that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.

[0038] The polypeptides disclosed in the present invention, including their salts, may also exist in the form of their hydrates or in the form of containing their solvents (e.g., ethanol, DMSO, etc.), and may be used for crystallization. The compounds disclosed in the present invention may inherently or by design form solvates with pharmaceutically acceptable solvents (including water); therefore, the compounds of the present invention include solvated and unsolvated forms.

[0039] The neuropeptide FF1 receptor binding molecule is prepared by recombinant technology or solid phase synthesis.

[0040] As a preferred embodiment of the present invention, the preparation of the neuropeptide FF1 receptor binding molecule is to produce the polypeptide of the present invention by recombinant technology. The polynucleotide of the present invention can be used to express or produce the recombinant polypeptide of the present invention by conventional recombinant DNA technology. Since the polypeptide of the present invention is relatively short, it is possible to consider connecting multiple polypeptides in series, obtaining expression products after recombinant expression, and then forming the desired small peptide by enzyme cleavage and other methods.

[0041] As a preferred embodiment of the present invention, the preparation of the neuropeptide FF1 receptor binding molecule is to utilize a solid phase synthesis method to sequentially couple amino acids to a solid phase carrier one by one, and then cleave to obtain the target peptide.

[0042] Wherein, the solid phase carrier is preferably an amino resin.

[0043] In the solid phase synthesis, the amino deprotecting agent used is preferably a DMF solution of piperidine with a volume percentage of 20%, or a DMF solution of DBU with a volume percentage of 1%; the coupling agent used is a combination of HBTU, HOBt and DIEA, or a combination of DIC and HOBt, or a combination of PyBOP, HOBt and DIEA. The amount, ratio, timing and treatment of the amino deprotecting agent and coupling agent are well known to those skilled in the art.

[0044] In the cleavage process, the cleavage agent used is preferably a mixed solution of TFA and H2O at a volume ratio of 95:5, or a mixed solution of TFA, EDT, TIS, PhOH, and H2O at a volume ratio of 80:5:5:5:5, or a mixed solution of TFA, EDT, TIS, and H2O at a volume ratio of 92.5:2.5:2.5:2.5. The amount, ratio, timing, and treatment method of the above-mentioned cleavage agent are well known to those skilled in the art. Of course, the present invention is not limited to the above-mentioned specific process steps. Those skilled in the art can adapt and improve the above-mentioned process steps based on the idea of ​​solid phase synthesis to finally obtain the target polypeptide product of the present invention.

[0045] In another aspect, the present invention provides a polynucleotide molecule comprising a polynucleotide capable of encoding one or two of the above-mentioned polypeptides.

[0046] In another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned polypeptide or polynucleotide molecule and a pharmaceutically acceptable carrier.

[0047] The pharmaceutical composition may include pharmaceutically acceptable carriers, i.e., lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, etc., which are commonly used in preparing dosage forms, and, in addition to the above ingredients, may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0048] The pharmaceutical composition can be administered orally or parenterally (e.g., applied intramuscularly, intravenously, intraperitoneally, subcutaneously, intradermally, or topically) according to the desired method, and the dosage varies depending on the patient's condition and body weight, the extent of the disease, the form of the drug, the route of administration, and the time, but can be appropriately selected by those skilled in the art.

[0049] In another aspect, the present invention provides the use of the above-mentioned polypeptide, polynucleotide molecule or pharmaceutical composition in the preparation of a drug for treating diseases associated with the neuropeptide FF1 receptor.

[0050] As a preferred embodiment of the present invention, the disease associated with the neuropeptide FF1 receptor is acute pain or pathological pain and other types of pain diseases, including but not limited to acute pain or chronic pain, such as postoperative incision pain, inflammatory pain (including arthritis pain), neuralgia and cancer pain and other pathological pain. The polypeptide of the present invention has potential use in the treatment of various types of pain drugs with high efficiency and low side effects.

[0051] the term

[0052] Unless otherwise defined herein, scientific and technical terms used in this patent application shall have the meanings commonly understood by one of ordinary skill in the art.

[0053] The "peptide library" used in this article is a patented technology of Hunan Zhongsheng Quanpeptide Biochemical Co., Ltd. using PICT (Peptide Information Compression Technology). This technology uses biological means to compress peptide information, and can integrate the information of multiple peptides into one peptide, thereby achieving a relatively small library capacity containing a large amount of peptide information; using PICT technology to construct a cyclic peptide library containing nearly 73,000 80 amino acids. The specific construction method can be found in patents CN201580081102.3 and CN201780089941.9. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is the concentration response curve of the activation effect of SEQ ID NO.1 on NPFFR1 receptor in Example 1;

[0055] Figure 2 This is the concentration response curve of the activation effect of SEQ ID NO.30 to SEQ ID NO.38 on NPFFR1 receptor in Example 2;

[0056] Figure 3 This is the concentration response curve of the activation effect of SEQ ID NO.40 to SEQ ID NO.50 on NPFFR1 receptor in Example 2;

[0057] Figure 4 This is the concentration response curve of SEQ ID NO.39 in Example 2 on the activation of NPFFR1 receptor. DETAILED DESCRIPTION

[0058] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or manufacturer recommendations. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0059] Key reagents: peptide library (self-made), CHO-K1 cell line overexpressing NFPPR1, abbreviated as CHO-K1 / NPFF1 / Gα15 cells (GenScript), Calcium5 Assay Kit.

[0060] Synthesis Example

[0061] The polypeptide compound and its derivative provided by the present disclosure adopt a solid phase synthesis method to synthesize its linear precursor, and use the intramolecular native chemical ligation in the liquid phase for cyclization. The synthetic carrier is 2-Chlorotritylchloride resin. In the synthesis process, the 2-Chlorotrityl chloride resin is first fully swollen in dichloromethane (DCM), and then coupled with 2-(butylamino)ethanethiol linker. After coupling, capping is performed with methanol. The 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 (90: 2.5: 2.5: 5:, v: v: v: v) is reacted with the resin to cleave the polypeptide from the solid phase carrier, and then the solid crude product of the linear precursor is obtained after precipitation by frozen methyl tert-butyl ether. The crude product is further subjected to C-terminal thioesterification, and then cyclized in a neutral solution to obtain a crude polypeptide product. The crude polypeptide was purified and separated by C-18 reverse phase preparative chromatography in 0.1% trifluoroacetic acid acetonitrile / water system to obtain pure polypeptide and its derivatives.

[0062] Experimental reagents

[0063]

[0064]

[0065] Example 1. Preparation of the compound corresponding to SEQ ID No: 47

[0066]

[0067] Step 1: 2-(Butylamino)ethanethiol linker coupling

[0068] 220 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swelled in DCM for 1 hour. 0.12 mmol of 2-(butylamino)ethanethiol was added and reacted at room temperature for 2 hours. After the reaction was completed, DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 minutes for capping. After the reaction, the resin was washed with DCM 3 times and DMF 3 times.

[0069] Step 2: Synthesis of linear precursor peptide chain

[0070] SEQ ID No: 47 linear precursor sequence CRSRYYWASLAR

[0071] The linear precursor sequence of SEQ ID No: 47 was synthesized in the order from the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0072] · Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 10 min.

[0073] • Rinse the resin 6-8 times with DMF until neutral pH.

[0074] Dissolve 2 mmol Fmoc-AA, 2 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 3 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 h.

[0075] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0076] After the linear peptide synthesis, the resin was washed with DMF 3 times, IPA 2 times and MTBE 3 times. The resin was dried in vacuo.

[0077] Step 3: Cleavage of the linear precursor peptide chain

[0078] Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90: 2.5: 2.5: 5:, v: v: v: v) was added to the resin obtained in step 2 and shaken for 3 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 volumes of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

[0079] Step 4: Formation of the C-terminal sulfolipid functional group

[0080] The crude product obtained in step 3 was dissolved in 0.1% trifluoroacetic acid aqueous solution at a concentration of 3-4 mg / mL. The solution was placed in a 40°C water bath for 24 hours to perform C-terminal S-to-N acyl transfer to form a thioester group.

[0081] Step 5: Circularization of linear precursor chains

[0082] The reaction solution from step 4 was diluted dropwise 1:1 (v:v) into 2 mM reduced glutathione and 50 mM ammonium bicarbonate aqueous solution. Oscillating at room temperature for 24 h. After the reaction was completed, 2.5% (v:v) acetic acid was added.

[0083] Step 6: Peptide purification

[0084] After filtering through a 0.45um membrane, the product was separated using a reversed-phase high-performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column, and during the purification process, the chromatograph detection wavelength was set at 230nm, the flow rate was 15mL / min, and the gradient was 20-50% acetonitrile in 40min. 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.

[0085] Step 7: Detection and characterization methods

[0086] The purified peptide from step 6 was subjected to analytical HPLC and LC / MS to confirm the purity and compound backbone cyclization.

[0087] Example 2. Preparation of the compound corresponding to SEQ ID No: 39

[0088]

[0089] Step 1: Coupling of the first amino acid Fmoc-Arg(Pbf)-OH

[0090] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 hour. Fmoc-Arg(Pbf)-OH (0.08 mmol) and diisopropylethylamine (DIEA, 0.32 mmol) were weighed and dissolved in 5 ml of DCM and added to the resin to react at room temperature for 2 hours. After the reaction was completed, a blocking solution (10 ml) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 minutes for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

[0091] Step 2: Synthesis of linear precursor peptide chain

[0092] The linear precursor peptide chain of SEQ ID No: 39

[0093] ETQRYQAKYQQSDARYYWASL-AR

[0094] 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 A at the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0095] · Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0096] • Rinse the resin 6-8 times with DMF until neutral pH.

[0097] Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 h.

[0098] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0099] After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

[0100] Step 3: Cleavage of the linear precursor peptide chain

[0101] Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 2 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 volumes of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

[0102] Step 4: Peptide purification

[0103] The crude polypeptide was dissolved in 20% acetonitrile aqueous solution, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column, and during the purification process, the chromatograph detection wavelength was set at 230nm, the flow rate was 15mL / min, and the gradient was 20-50% acetonitrile in 40min. 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.

[0104] Step 5: Detection and characterization methods

[0105] The purified peptide from step 4 was subjected to analytical HPLC and LC / MS to determine the purity and molecular weight of the target product.

[0106] Biological test cases

[0107] Example 3 High Throughput Screening Process

[0108] 1.1. CHO-K1 / NPFF1 / Gα15 cell culture

[0109] 1.1.1. Cell recovery: Take out the cells from the liquid nitrogen tank and thaw them quickly in a 37℃ water bath. Transfer the cells to a 15mL centrifuge tube, slowly add 9mL of preheated thawing medium, centrifuge at 800 rpm for 5 minutes, and remove the supernatant medium. Resuspend the cells with 5mL of thawing medium, transfer to a T25 culture flask, and culture in an incubator at 37℃ and 5% CO2. Replace the culture medium with growth medium on the second day of cell recovery.

[0110] 1.1.2. Cell passaging: When the cells have grown all over the culture flask and reached 90% confluence, pass the cells. First, rinse the cells with DPBS, then add DPBS again and tap the cell flask to remove the cells from the flask wall; collect the cell suspension into a centrifuge tube, centrifuge at 800 rpm for 3-5 minutes, remove the supernatant medium; add 6-8 mL of fresh growth medium, resuspend the cells, pass the cells at a ratio of 1:3-1:8, and culture them in a 37°C, 5% CO2 incubator. Change the medium every 2-3 days after passaging.

[0111] 1.1.3. Cell freezing: When the cells have grown to 80-90% of the culture dish, rinse the cells with DPBS, then re-add DPBS and tap the cell bottle to remove the cells from the bottle wall; collect the cell suspension into a centrifuge tube, centrifuge at 800 rpm for 3-5 minutes, remove the supernatant medium; resuspend the cells with freezing medium, count the cells, and dilute the cells to 3-5×10 6 cells / mL. Each cryovial is filled with 1 mL of cell cryopreservation suspension. Place the cryovials filled with cells into a cryopreservation box, store the cryopreservation box in a -80℃ refrigerator overnight, and then transfer the cryovials to a liquid nitrogen tank.

[0112] 1.2. CHO-K1 / NPFF1 / Gα15 cell plating

[0113] 24 hours before the test, the CHO-K1 / NPFF1 / Gα15 cells in the culture flask were digested with 0.25% trypsin and suspended in cell culture medium. They were added to a black transparent bottom 384-well plate at a density of 6000 to 8000 cells per well using a dispenser, 25 μL per well, and cultured overnight at 37°C, 5% CO2.

[0114] 1.3.Calcium5 assay kit working solution preparation

[0115] On the day of the test, component A in the Calcium5 kit was dissolved in a calcium dye buffer (Loading buffer) and 250 mM probenecid was added to prepare a calcium dye solution containing 5 mM probenecid.

[0116] After the culture medium was removed, 50 μL of calcium dye solution was added to each well of the cell culture plate and incubated at room temperature for 2 hours.

[0117] 1.4. Dissolution and dilution of peptide library

[0118] 1.4.1. Dissolution of peptide library

[0119] 1.4.2. Place the 96-well deep-well plate of peptide library in a centrifuge at 4000rpm for 2-3 minutes. Use an automatic dispenser to add 200μL / well of ultrapure water to the 96-well deep-well plate. Seal with a silicone cover and place in a 95℃ water bath for 5 minutes. Note: At this time, the peptide concentration is approximately: 50μM.

[0120] 1.4.3. After dissolution, place the 96-well plate peptides in a centrifuge at 4000 rpm for 2-3 minutes.

[0121] 1.5. Peptide library dilution

[0122] After dissolution, the solution was transferred to a 384-well plate using a workstation and diluted to 10 μM using loading buffer.

[0123] 1.6. FLIPR detection

[0124] Two hours after the addition of Calcium 5 dye to the cells, the cell culture plate was removed and placed at room temperature in the dark for 10 minutes, and then placed in the FLIPR instrument together with the peptide solution plate for detection. NPFF was set as the agonist positive control.

[0125] 1.7. Confirmation of active 80-cyclic peptide

[0126] 1.7.1 Confirm the peptides screened in the initial screening according to the above experimental steps.

[0127] Through high-throughput screening, about 12 peptides that can activate the NPFFR1 receptor at the cellular level were screened out from nearly 73,000 80-cyclic peptides.

[0128] 1.7.2 Detect the EC of active peptides according to the above experimental steps 50 value

[0129] On the day of the test, the 12 active peptide stock solutions screened above were diluted to 15 μM (5× concentration) with 1× Loading buffer (containing 20 mM HEPES) (pH: 7.4), and then diluted 8-10 times in a gradient, with each concentration repeated to test the concentration response curve for NPFFR1 receptor activation and the EC of the active peptides. 50 value.

[0130] Among them, the 80-cyclic peptide SEQ ID No: 1 has the strongest activation effect. The experimental results are as follows Figure 1 , as shown in Table 2, SEQ ID No: 1EC50 The value is 29.83nM.

[0131] Table 2 EC values ​​of 80 cyclic peptides 50 result

[0132] SEQ ID No: <![CDATA[EC 50 (nM)]]> 1 29.83

[0133] Example 4 Screening of Derived Peptides

[0134] 1. Screening and confirmation of peptides derived from SEQ ID No: 1

[0135] The internal decompression technology was used to decompress the amino acids of 80 cyclic peptides (SEQ ID No: 1) and to design cyclic peptides or linear peptides with different amino acid sequences.

[0136] The screening was performed according to the screening steps in Example 1, and the decompression active polypeptides screened were subjected to EC 50 For verification, the test concentration was 1 μM or 100 nM as the starting concentration, and a gradient dilution was performed to 8 concentrations.

[0137] 2. Experimental results

[0138] The 80-ring peptide SEQ ID No: 1 with the strongest NPFFR1 receptor activation effect was decompressed, designed and screened, and multiple peptides with agonist activity were obtained. The activation rate of the active peptides relative to 0.3 μM NPFF is shown in Table 3, and the concentration response curve of the NPFFR1 receptor activation effect is shown in Figure 2 .

[0139] From the results, it can be seen that the amino acid sequences shown by SEQ ID No: 3, SEQ ID No: 6, SEQ ID No: 16 to SEQ ID No: 20, SEQ ID No: 22, and SEQ ID No: 23 have a strong effect on activating the NPFFR1 receptor.

[0140] The above-mentioned polypeptide with strong activity was further truncated and further screened according to the above-mentioned derivative peptide screening steps. The concentration response curve of the truncated peptide for NPFFR1 receptor activation was shown in Figure 3 , Figure 4 , E.C. 50 The experimental results are shown in Table 4. Among them, SEQ ID No: 39 has the strongest effect on activating NPFFR1. 50 was 0.11 nM, followed by SEQ ID No: 50, whose EC 50 It is 9.96nM.

[0141] Table 3 Activation rate of active peptides relative to 0.3 μM NPFF

[0142] SEQ ID No: Activation rate relative to 0.3 μM NPFF (%) SEQ ID No: Activation rate relative to 0.3 μM NPFF (%) 2 8.0 16 110.9 3 126.1 17 103.9 4 93.2 18 107.0 5 97.5 19 100.5 6 119.5 20 105.9 7 72.3 21 85.8 8 62.9 22 91.8 9 80.4 23 97.1 10 65.7 24 63.0 11 54.2 25 64.9 12 38.4 26 66.5 13 96.2 27 99.7 14 44.9 28 85.6 15 1.1 29 20.1

[0143] Table 4 EC of active peptides 50 result

[0144] SEQ ID No: <![CDATA[3 times of EC test 50 Average value (nM)]]> SEQ ID No: <![CDATA[3 - test EC 50 Average value (nM)]]> 30 709.43 41 49.43 31 158.62 42 67.27 32 160.33 43 79.30 33 144.50 44 137.27 34 209.63 45 166.60 35 479.37 46 98.24 36 227.13 47 91.49 37 91.96 48 255.67 38 95.62 49 38.78 39 0.11 50 9.96 40 124.12

[0145] It should be pointed out that the above is only a preferred embodiment of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide, characterized in that The polypeptide is a polypeptide as shown in any one of the following (1) to (3): (1) the amino acid sequence as described in SEQ ID No. 1 / or its modified form; or (2) a truncated peptide of the polypeptide having the amino acid sequence as described in SEQ ID No. 1; or (3) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (1) or (2), and having the same or similar function as the amino acid sequence shown in SEQ ID No. 1; The polypeptide is a linear peptide or a head-to-tail cyclic peptide.

2. The polypeptide according to claim 1, characterized in that The polypeptide is a polypeptide fragment consisting of 26 to 35 consecutive amino acids in the amino acid sequence as described in SEQ ID No.

1.

3. The polypeptide according to any one of claims 1 or 2, characterized in that The polypeptide can be selected from the amino acid sequences of SEQ ID No: 2 to SEQ ID No:

29.

4. The polypeptide according to claim 3, characterized in that The polypeptide can be selected from the amino acid sequences shown in SEQ ID No: 3 to SEQ ID No: 6, SEQ ID No: 13, SEQ ID No: 16 to SEQ ID No: 20, SEQ ID No: 22, and SEQ ID No:

23.

5. A polypeptide, characterized in that The polypeptide is a polypeptide as shown in (i) or (ii) below: (i) truncated peptides of the polypeptides having the amino acid sequences shown in SEQ ID No: 3, SEQ ID No: 6, SEQ ID No: 16 to SEQ ID No: 20, SEQ ID No: 22, and SEQ ID No: 23; (ii) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (i), and having the same or similar functions as the amino acid sequence described in (i); The polypeptide is a linear peptide or a head-to-tail cyclic peptide.

6. The polypeptide according to claim 5, characterized in that The polypeptide can be selected from the amino acid sequences of SEQ ID No: 30 to SEQ ID No:

50.

7. The polypeptide according to claim 6, characterized in that The polypeptide can be selected from the amino acid sequences shown in SEQ ID No: 39 and SEQ ID No:

50.

8. A polynucleotide molecule comprising a polynucleotide capable of encoding one or two of the polypeptides according to any one of claims 1 to 7.

9. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 7 or the polynucleotide molecule according to claim 8 and a pharmaceutically acceptable carrier.

10. Use of the polypeptide according to any one of claims 1 to 7, the polynucleotide molecule according to claim 8 or the pharmaceutical composition according to claim 9 in the preparation of a drug for treating diseases associated with the neuropeptide FF1 receptor.

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