Targeted TRPV1 polypeptide and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses polypeptide targeting TRPV1 and application. The polypeptide has an amino acid sequence as shown in a formula I: Asp-Gln-X1-X2-X3 (formula I), wherein X1 is selected from any one of natural or non-natural substances in Phe, Trp, His, Lys and Arg; x2 is selected from any natural or non-natural one of Phe, Trp, His, Lys and Arg; and X3 is selected from any one of natural or non-natural substances in Asn, Ser and Met. As a TRPV1 antagonist, the polypeptide is novel in structure and clear in action mechanism, has good selectivity and inhibitory activity, and can be used for developing drugs for treating diseases such as pain, xerophthalmia, pruritus and skin allergy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to polypeptides targeting TRPV1 and applications thereof. Background Art

[0002] TRPV1 (transient receptor potential vanilloid 1) is a non-selective cation channel widely involved in physiological processes such as pain perception, inflammatory responses, and temperature regulation. TRPV1 consists of four subunits arranged symmetrically around a central ion channel. Each subunit contains six transmembrane helices (S1-S6), with S5-S6 forming the pore region. Both the N-terminus and C-terminus are located intracellularly. TRPV1 functions as a molecular receptor, activated by a range of physical and chemical stimuli to transmit pain and itch. It is primarily distributed in nociceptive sensory neurons (such as the dorsal root ganglion and trigeminal ganglion), but is also present in non-neuronal tissues (such as keratinocytes, mast cells, and intestinal epithelial cells), mediating sensations such as tingling, itching, burning, and pain, as well as afferent responses to chemical stimuli. TRPV1 is implicated in the pathogenesis of numerous diseases, including pain, inflammation, cardiovascular disease, cough, psychiatric disorders, and diabetes. According to incomplete statistics, there are currently 106 TRPV1-targeting drugs in the world. Of these, 31 are in the preclinical stage, 49 have been discontinued or are progressing at no rate, 3 have been approved for marketing, and 15 are in the clinical stage. These drugs encompass a variety of drug types, including small molecules, synthetic peptides, and siRNAs, with small molecules dominating the market, comprising nearly 89. Furthermore, all three currently marketed drugs are small molecules. TRPV1 has been most extensively studied in the context of pain, with both its agonists and antagonists producing analgesic effects. High concentrations of capsaicin activate TRPV1, leading to neuronal desensitization and exhibiting analgesic effects in various animal models. Qutenza (capsaicin patch) has been approved by the FDA for neuropathic pain. However, adverse effects on blood pressure, respiratory function, and other reflex pathways have been observed at high doses, making it difficult to distinguish between efficacy and toxicity. Therefore, TRPV1 agonists are currently primarily used as topical analgesics. ACD-440 is a TRPV1 antagonist developed by AlzeCure Pharma for the topical treatment of patients with peripheral neuropathic pain. ACD440 completed a positive Phase Ib study in 2021. It showed very good tolerability and safety, and this substance is applied to the skin as a gel and has an effective and long-lasting analgesic effect. In addition to pain management, the TRPV1 antagonist developed by South Korea's Amorepacific for the topical treatment of atopic dermatitis pruritus has completed Phase III clinical trials. A siRNA drug targeting TRPV1, developed by Sylentis, is used to treat dry eye disease and is administered in the form of an aqueous eye drop solution. Tivanisiran is currently in Phase III trials to evaluate the safety of treating dry eye disease and dry eye disease caused by Sjögren's syndrome.

[0003] However, drug development targeting TRPV1 faces numerous challenges. While traditional small-molecule inhibitors have made some progress, they suffer from poor selectivity and significant side effects. In contrast, peptide inhibitors are emerging with their high affinity and specificity. Currently, there are relatively few reports of peptide inhibitors targeting TRPV1. Parentide (DD04107), a TRPV1 antagonist developed by Bcn Peptides SA and DiverDrugs SL, is currently in Phase II clinical trials for the treatment of neuropathic pain. Therefore, there is an urgent need to develop new methods and approaches for rapid screening and efficient identification of active peptide antagonists with potential to target TRPV1. Summary of the Invention

[0004] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and provide a polypeptide targeting TRPV1 and its application. The polypeptide is a pentapeptide with good selectivity and inhibitory activity, and can be used to develop drugs for treating diseases such as pain, dry eye, itching and skin allergies.

[0005] To achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention is to provide a polypeptide targeting TRPV1 or a pharmaceutically acceptable salt thereof, having an amino acid sequence shown in Formula I: Asp-Gln-X 1 -X 2 -X 3 (Formula I), X 1 Any natural or non-natural one selected from Phe, Trp, His, Lys, and Arg; X 2 Any natural or non-natural one selected from Phe, Trp, His, Lys, and Arg; X 3 Any one selected from Asn, Ser, and Met, whether natural or unnatural.

[0006] Furthermore, the amino acid sequence of the polypeptide is selected from any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

[0007] The second aspect of the present invention is to provide a pharmaceutical composition comprising at least the above-mentioned polypeptide or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0008] Furthermore, the pharmaceutical composition is formulated for topical, oral, ocular, intravenous, intravitreal, subconjunctival or subcutaneous administration.

[0009] Furthermore, the pharmaceutical composition is in the form of a gel, lotion, cream, tablet, capsule, pill, solution, eye drops, spray, bandage, contact lens, depot preparation, injection, implant or sustained release preparation.

[0010] The third aspect of the present invention is to provide use of the above-mentioned polypeptide or an acceptable salt thereof in the preparation of a medicament for treating pain.

[0011] The fourth aspect of the present invention is to provide use of the above-mentioned polypeptide or an acceptable salt thereof in the preparation of a medicament for treating dry eye.

[0012] The fifth aspect of the present invention is to provide use of the above-mentioned polypeptide or an acceptable salt thereof in the preparation of a medicament for treating skin itching or allergy.

[0013] Furthermore, the amount of the polypeptide or its pharmaceutically acceptable derivative is in the range of 0.1 μM to 100 μM.

[0014] The sixth aspect of the present invention is to provide use of the above-mentioned polypeptide or an acceptable salt thereof in the preparation of cosmetics.

[0015] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects: (1) The present invention uses structure-based virtual screening molecular docking technology to screen out novel structural polypeptide molecules with TRPV1 inhibitory activity from a self-built pentapeptide virtual library, shortening the R&D cycle and saving costs for large-scale screening, and providing multiple lead molecules targeting TRPV1 for subsequent peptide drug development.

[0016] (2) The TRPV1 antagonists screened by the present invention have novel structures, clear mechanisms of action, good selectivity and inhibitory activity, and can be used to develop drugs for the treatment of diseases such as pain, dry eye, itching and skin allergies.

[0017] (3) The lead molecule reported in this invention further expands the polypeptide template for targeting TRPV1 antagonists, and the production method is simple, which is conducive to various modifications and transformations. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Figure 1 shows the results of establishing a virtual pentapeptide library and the screening process. Figure A: Results of establishing a virtual pentapeptide library: 20 natural amino acids were randomly generated into pentapeptide sequences using Python code and saved in Fasta format to form a pentapeptide sequence database. These sequences were then converted into 2D structures using the software RDKit and saved in SMILES (smi) format. Finally, the three-dimensional structures of the pentapeptides were generated using the LigPrep module of the Schrödinger software, which simultaneously preprocessed the ligand library. Figure B: HTVS refers to a docking method using the Glide module, which allows for rapid ligand screening. Figure 2 This is a diagram of the docking combination of P1-P5; Figure 3 This is the solid phase synthesis, separation and purification chromatogram and mass spectrometry identification diagram of P5; Figure 4 Schematic diagram of the inhibitory effect of P5 on TRPV1 channel current; Figure 5 This is the trace graph of the selectivity of 100 μM P5 for TRPV3, av1.5, Nav1.7, Kv4.2, and hERG; Figure 6 This is a diagram showing the therapeutic effect of P5 on skin allergy inflammation caused by capsaicin. DETAILED DESCRIPTION

[0019] To make the objects, technical solutions, and advantages of the present invention more apparent, the following describes the specific embodiments of the present invention in further detail with reference to the specific examples and accompanying drawings. Where specific test methods, instruments, or conditions are not specified in the examples, the methods or conditions described in the literature in the art or the product specifications were used. Where the manufacturer of the reagents or instruments is not specified, all are commercially available conventional products.

[0020] The present invention provides a polypeptide targeting TRPV1 or a pharmaceutically acceptable derivative thereof, having an amino acid sequence shown in Formula I: Asp-Gln-X 1 -X 2 -X 3 (Formula I), X 1 Any natural or non-natural one selected from Phe, Trp, His, Lys, and Arg; X 2 Any natural or non-natural one selected from Phe, Trp, His, Lys, and Arg; X 3 Any one selected from Asn, Ser, and Met, whether natural or unnatural.

[0021] The polypeptides of the present invention include derivatives of the above polypeptides. Such derivatives can be used directly as active substances or as prodrugs.

[0022] Furthermore, the derivatives of the present invention can be obtained by adding (modifying) or replacing specific amino acid groups in the polypeptide, such as hydrogen atoms, hydroxyl groups, carboxyl groups, amino groups, and imino groups, with known substituents. Examples of amino acid modifications within the polypeptide include, but are not limited to, chemical modifications such as glycosylation, acetylation, phosphorylation, and lipid attachment.

[0023] The polypeptides of the present invention also include substances obtained by mutating the above-mentioned polypeptides. That is, the mutants of the present invention can be obtained by deleting amino acids in the polypeptide or replacing or adding other amino acids. The number of amino acids deleted or replaced or added with other amino acids can be a peptide with 80% homology, preferably 90% homology, relative to the polypeptide of the present invention, and at least has TRPV1 inhibitory activity. It should be noted that in order to improve the activity of the polypeptide of the present invention, to make the effect of the present invention last, and / or to increase the storage stability of the polypeptide of the present invention, the polypeptide of the present invention or the amino acids constituting it can be appropriately changed. For example, chemical modifications of the amino acids of the polypeptide of the present invention can be performed, part of the amino acids constituting the polypeptide can be deleted, replaced with other amino acids, and / or new amino acids can be added.

[0024] The abbreviations of the 20 amino acids used in the present invention are shown in Table 1 below: Table 1. Corresponding abbreviations of 20 amino acids.

[0025]

[0026] Pharmaceutically acceptable salts are not particularly limited and may include salts with inorganic bases, organic bases, inorganic acids, organic acids, and basic or acidic amino acids. Examples of inorganic base salts include alkali metal salts such as sodium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; and aluminum and ammonium salts. Examples of organic base salts include salts with alkylamines such as trimethylamine and triethylamine; salts with heterocyclic amines such as pyridine and picoline; salts with alkanolamines such as ethanolamine, diethanolamine, and triethanolamine; salts with cycloalkylamines such as cyclohexylamine and dicyclohexylamine; and salts with alkylenediamine derivatives such as N,N'-dibenzylethylenediamine. Examples of inorganic acid salts include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid. Examples of salts of organic acids include salts with monocarboxylic acids such as formic acid, acetic acid, trifluoroacetic acid, and propionic acid; salts with polycarboxylic acids such as fumaric acid, oxalic acid, and maleic acid; salts with hydroxycarboxylic acids such as tartaric acid, citric acid, succinic acid, and malic acid; salts with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid; and salts with benzoic acid. Examples of salts of neutral amino acids include salts with glycine, valine, and leucine; salts of basic amino acids include salts with arginine, lysine, and ornithine; and salts of acidic amino acids include salts with aspartic acid and glutamic acid.

[0027] Among the above, preferred are polypeptides formed by the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, or pharmaceutically acceptable salts thereof.

[0028] The method for producing the polypeptide, derivative, and pharmaceutically acceptable salt thereof of the present invention is not particularly limited, and for example, known chemical synthesis methods and genetic engineering synthesis methods can be used.

[0029] When the above-mentioned amino acid sequence is synthesized by chemical synthesis, any chemical synthesis method can be used, and known peptide synthesis methods such as solid phase synthesis and liquid phase synthesis can be used. In addition, commercially available peptide synthesis equipment can be used for synthesis.

[0030] The present invention also provides a pharmaceutical composition comprising at least one of the above polypeptides or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0031] The term "pharmaceutically acceptable carrier" refers to a carrier used to administer a therapeutic agent. The term refers to pharmaceutical carriers that do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not unduly toxic upon administration. Such carriers are well known to those of ordinary skill in the art. Such carriers include, but are not limited to, saline, buffered solutions, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.

[0032] Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol, and ethanol. Additionally, these carriers may contain auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like. Typically, therapeutic compositions are formulated as injectables, such as liquid solutions or suspensions; they may also be formulated as solid forms suitable for incorporation into solutions or suspensions or liquid carriers prior to injection. Once formulated, the compositions of the present invention may be administered via conventional routes, including, but not limited to, intramuscular, intravenous, subcutaneous, intradermal, or topical administration. The subject to be prevented or treated may be an animal, particularly a human.

[0033] In preferred embodiments, the pharmaceutical composition is formulated for topical, oral, ocular, intravenous, intravitreal, subconjunctival, or subcutaneous administration.

[0034] In a preferred embodiment, the pharmaceutical composition takes the form of a gel, lotion, cream, tablet, capsule, pill, solution, eye drops, spray, bandage, contact lens, depot formulation, injection, implant or sustained release formulation.

[0035] These pharmaceutical compositions can be formulated according to conventional methods by mixing, diluting or dissolving, and occasionally suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers are added, and the formulation process can be carried out in a conventional manner according to the dosage form.

[0036] The pharmaceutical composition of the present invention can also be administered in the form of a sustained-release formulation. For example, the polypeptide of the present invention or a pharmaceutically acceptable salt thereof can be incorporated into a pill or microcapsule with a sustained-release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated. Examples of sustained-release polymers include ethylene-vinyl acetate copolymers, polyhydroxymethylacrylate (polyhydrometaacrylate), polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, lactic acid-glycolic acid copolymers, and the like. Preferred examples include biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.

[0037] The pharmaceutical preparation should be compatible with the mode of administration. The medicament of the present invention can also be used with other synergistic therapeutic agents (including before, during, or after use). When using a pharmaceutical composition, a safe and effective amount of the drug is administered to the desired subject (such as a human or non-human mammal). The safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 8 milligrams per kilogram of body weight. Preferably, the dosage is about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0038] The present invention also provides the use of the above-mentioned polypeptide or an acceptable salt thereof in the preparation of a medicament for treating pain. Pain includes chronic pain conditions, acute and chronic neuralgia, acute and chronic inflammatory pain, diabetic peripheral neuropathic pain, hypersensitivity postoperative pain, eye pain, musculoskeletal disorders, overactive bladder, erythromelalgia, cancer pain, and osteoarthritis pain.

[0039] The present invention also provides the use of the above polypeptide or an acceptable salt thereof in preparing a drug for treating dry eye. Dry eye includes dry eye syndrome.

[0040] The present invention also provides the use of the aforementioned polypeptide or an acceptable salt thereof in the preparation of a medicament for treating skin itching or allergies. Skin itching or allergies include chronic pruritus, such as atopic dermatitis (AD)-related pruritus, prurigo nodularis, and rosacea, and allergic skin diseases such as urticaria and contact dermatitis.

[0041] The present invention also provides the use of the above-mentioned polypeptide or an acceptable salt thereof in the preparation of cosmetics for allergy relief, such as any one of essence, cream, toner, ointment, lotion, and facial mask.

[0042] Example 1 Establishment of a virtual pentapeptide library Step 1: Obtain a 5-peptide database in FASTA format: Use Python code to randomly combine 20 amino acids, including alanine (A), cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y), and save them in FASTA format with the combination number. A total of 20 5 Pentapeptide.

[0043] Step 2: Batch construction of pentapeptide molecular structures: Based on RDKIT software ( https: / / www.rdkit.org / docs / index / html ), run the python code on the server, generate the corresponding pentapeptide molecular structure from the above pentapeptide sequence, and establish a SMILES structure library of the pentapeptide molecular structure.

[0044] Example 2 Virtual screening of pentapeptides targeting TRPV1 1. Ligand data preparation Using the Schrödinger software's LigPrep module, SMILES-formatted pentapeptide structures were batch-converted to pentapeptides with spatial structures on the server, generating different conformations. Output files were in SDF format. LigPrep parameters were set as follows: specifying the input and output file locations; selecting force field "16 (OPLS3e)"; setting "EPIK" to "Yes" for ionization; setting "DETERMINE_CHIRALITIES" to "No" for determining chirality; setting "GNORE_CHIRALITIES" to "No" for ignoring chirality; and setting "NUM_STEREOISOMERS" to "1" for the number of generated spatial conformations.

[0045] 2. Receptor data preparation (1) Obtaining the TRPV1 channel structure: The rTRPV1 channel structure with PDB ID 5IS0 was selected from the PDB protein structure database (https: / / www.rcsb.org / ). The structure had a resolution of 3.43 Å and was resolved by cryo-electron microscopy.

[0046] (2) rTRPV1 structure processing: The protein preparation wizard module of Schrödinger software was used to optimize the structure of rTRPV1 protein, such as deleting water molecules, adding hydrogen atoms, adding missing side chain residues, and charge correction. Energy minimization was performed using the OPLS3 force field at pH 7.3.

[0047] (3) Setting up the receptor docking box: Based on the Receptor Grid Generation in the Glide module of the Schrödinger software, the docking sites Gly643, Met644, Gly645, Asp646, Leu647, Glu648, Phe649, and Thr650 were selected for customization, and a docking box with an outer box side length of 20 Å and an inner box side length of 10 Å was generated.

[0048] 3. Molecular docking The prepared pentapeptide structure was molecularly docked with the pre-processed receptor protein docking box through the Ligand Docking of the Glide module of the Schrödinger software. The locations of the receptor protein docking box file "GRIDFILE" and the ligand pentapeptide file "LIGANDFILE" were specified, and the docking score was used using HTVS. The system was run in multi-threaded mode on the server using the command line.

[0049] Select the top 30% peptides in the HTVS score and use the SP scoring function for docking. Set the location of the receptor protein docking box file "GRIDFILE" and the ligand pentapeptide file "LIGANDFILE". Set the maximum number of poses written from the docking run "NREPORT" to 5000, set whether to generate each residue interaction term "WRITE_RES_INTERACTION" to "True", and set the data readout format "WRITE_CSV" to "True".

[0050] 4. Filter results After the initial screening of 200,000 pentapeptides by HTVS docking, the top 30% of the pentapeptides were selected for further standard SP docking. Among the SP scores, 5 peptides scored below -10, and the top 15 pentapeptides were selected (see Table 2). The binding regions of the candidate peptides P1-P5 are all located outside the pore region composed of S5-S6. The binding of the candidate pentapeptides to TRPV1 is shown in Figure 2. Figure 2 As shown, peptides P1-P3 all bind to one or more amino acid residues in the TRPV1 pore selector (G643-M644-G645) by inserting lysine side chains into the pore region, forming hydrogen bonds to block current. Peptides P4 and P5, on the other hand, insert into the pore region using tryptophan and lysine side chains, respectively. Peptides P1-P5 were selected for subsequent bioactivity validation.

[0051] Table 2: Pentapeptide docking scoring results

[0052] Example 3 Peptide activity verification.

[0053] (1) Peptide solid phase synthesis Linear peptides were synthesized using the Fmoc solid-phase peptide synthesis method, using Rink Resin as a carrier, sequentially synthesizing from the C-terminus to the N-terminus, with Fmoc (9-fluorenylmethoxycarbonyl) as the amino protecting group. After the last amino acid was coupled, the polypeptide chain was cleaved with a lysis buffer, then precipitated with glacial ether and identified by mass spectrometry, followed by separation and purification by RP-HPLC. Figure 3 Shown are the solid phase synthesis, separation and purification chromatogram and mass spectrometry identification diagrams of P5.

[0054] (2) Verification of inhibitory activity targeting rTRPV1.

[0055] After 3-4 passages, revived HEK293T cells are ready for transfection experiments. When the cell density reaches 80-90%, discard the culture medium, rinse once with PBS, and add 2 mL of serum-free culture medium (Opti-MEM). Prepare Solution A: 240 μL of serum-free culture medium + 10 μL of lipofectamine 2000 per well (total volume 250 μL) and incubate at room temperature for 5 minutes. Prepare Solution B: Add 4 ng of rTRPV1 channel plasmid to 250 μL of serum-free culture medium and incubate at room temperature for 5 minutes. Mix Solution A and Solution B thoroughly and incubate at room temperature for 20 minutes. Gently add the rTRPV1-liposome mixture to the cells and incubate in a 37°C, 5% CO2 incubator for 4-6 hours. After incubation, replace the cells with DMEM supplemented with 10% FBS for 12 hours before use for subsequent electrophysiological experiments.

[0056] Whole-cell patch clamp recordings were performed at room temperature (20–25°C). The recording and data acquisition amplifier used was an EPC10 USB Amplifier (HEKA, Elektronik, Lambrecht, Germany), and the data recording and control software was PatchMaster (HEKA, Elektronik, Lambrecht, Germany). Glass electrodes were pulled from microelectrode glass capillaries (1.5 mm outer diameter, 1.05 mm inner diameter, 10 mm length, Wuhan Micro-Tan Scientific Instrument Co., Ltd.) using a two-step process on a PC-10 electrode puller (NARISHIGE, Tokyo, Japan). The pulling temperature was adjusted to maintain the electrode's water resistance between 2 and 3 MΩ. The cell was sealed with pressure, and the resistance after complete sealing was generally > GΩ. Fast capacitance was compensated, and then recording was switched to WHOLE-CELL mode. The membrane was ruptured by opening the three-way valve, and slow capacitance was compensated. The series resistance, R-series, was maintained at less than 10 MΩ. The cells are clamped at a specific voltage, the series resistance and leakage capacitance are compensated, and after the resistance value stabilizes, stimulation is given according to the set stimulation method and the channel current is recorded.

[0057] When measuring the inhibitory activity of candidate pentapeptide drugs on the transient potential vanilloid channel TRPV1, the cells were maintained at 0 mV and -60 mV for 40 s, with an interval of 2 s between each stimulation. After 0.1 μM capsaicin (Cap) was used to stimulate a stable current, the candidate peptide drug prepared with the same concentration of capsaicin was added.

[0058] The synthesized candidate pentapeptide drugs (P1, P2, P3, P4, and P5) were prepared into 1 mM stock solutions in ddH2O. The TRPV1 channel agonist capsaicin (Cap) was diluted to 0.1 μM in the corresponding extracellular solution as a stimulation solution. A 100 μM working solution of the candidate pentapeptide drugs was prepared with the same concentration of capsaicin for activity testing. TRPV1 whole-cell patch clamp electrophysiological recordings were performed at -60 mV for 40 s, with a 2-s interval between each stimulation. To test the TRPV1 channel activity of the candidate pentapeptide drugs, a 0.1 μM concentration of capsaicin (Cap) was added through the perfusion system to stimulate the current. Once the stimulated current stabilized, the candidate peptide drug was added. At a test concentration of 100 μM, the inhibition rates of P1 were 48.88±7.02%, P2 were 52.61±10.56%, P3 were 80.01±11.99%, P4 were 47.5±11.8%, and P5 were 86.57±6.05%. All five candidate pentapeptides inhibited TRPV1 channel currents, with P3 and P5 showing the best inhibitory effects. These two pentapeptides will be used as lead drugs for subsequent studies.

[0059] The inhibitory effects of the lead peptide drugs P3 and P5 were tested to determine their IC50 values, and the current trace graphs and concentration-dose effect curves of different drug concentrations were drawn, such as Figure 4 As shown: IC50 of P3 = 11.44 μM, IC50 of P5 = 0.71 μM.

[0060] Example 4 The P5 peptide targets other channels selectively.

[0061] For P3 and P5 selective analysis of Kv4.2 and hERG, cells were held at -80 mV, and currents were induced at voltages of 0 mV, +30 mV, +50 mV, and +60 mV for 300 ms, with a 5-second interval between each stimulus. For P3 and P5 selective analysis of Nav1.5 and Nav1.7, cells were held at -90 mV, and currents were induced at 0 mV for 50 ms, with a 5-second interval between each stimulus.

[0062] The results are as follows Figure 5As shown, at concentrations of 10 μM and 100 μM, P3 and P5 had no significant inhibitory effects on Nav1.5 and Nav1.7. They exhibited some inhibitory effects on TRPV3 and TRPV4. At 100 μM, P3 exhibited approximately 37.7% inhibition of TRPV3 and 38.67% inhibition of TRPV4. At 100 μM, P5 exhibited 30.12% inhibition of TRPV3 and 32.94% inhibition of TRPV4. However, P3 exhibited a strong inhibitory effect on potassium channels: the IC50 for Kv4.2 was 6.50 μM, while its inhibitory effect on Kv4.3 was weak, reaching an inhibition rate of 42.47 ± 11.47 μM at 100 μM. The IC50 of P5 against Kv4.3 is 2.43 μM, while its inhibitory effect on Kv4.2 is not strong. At 100 μM, the inhibition rate is 38.63±7.13 μM.

[0063] Example 5 The allergic effect of P5 on skin inflammation.

[0064] Human skin patch test Six male or female volunteers aged 18 to 40 years old, who were in good health and had no history of skin diseases were recruited (all volunteers were informed of the experimental informed consent form) and a simultaneous drug administration model was performed on the skin of the inner left and right arms of the volunteers.

[0065] Simultaneous drug administration model: configuration: ① control group (Control): 0.8 mM capsaicin (Cap) + normal saline ② experimental group: 0.8 mM capsaicin (Cap) + 5 mMP5 ③ positive drug group: 0.8 mM capsaicin (Cap) + 0.1 mM capsaicin (CPZ). After mixing, add 20 μL of each of the three groups of reagents to the filter paper of the spot tester, stick it on the skin of the inner left arm of the subject, and take pictures and record it at 1 h and 3 h.

[0066] The results of the simultaneous drug administration model are as follows Figure 6 As shown in the results, compared with the control group, P5 can significantly reduce the pain, burning or itching caused by capsaicin stimulation, while reducing the degree and range of skin redness and swelling, and delaying the onset of these discomforts, indicating that in this model, P5 has a significant effect on capsaicin-induced skin allergy inflammation.

[0067] Example 6 Effects on the rat dry eye model.

[0068] SCOP, as a lacrimal gland cholinergic receptor inhibitor, can reduce tear production and establish an animal model of aqueous humor-deficient dry eye. Therefore, a rat dry eye model was established by subcutaneous injection of SCOP solution. Female SD rats that had been adaptively fed for 7 days were divided into a blank group, a model group, a low-dose P5 peptide group, a high-dose P5 peptide group, and a positive control group (0.1% sodium hyaluronate eye drops), with 10 rats in each group. Except for the blank group, the other groups were subcutaneously injected with SCOP solution (12.5 mg kg each time) into the neck or legs of the rats. -1 , 4 times / day) for 7 consecutive days.

[0069] After each injection of SCOP solution, the corresponding drugs (1 and 4 mg kg each time) were dripped into both eyes of the rats according to the experimental groups. -1 Rats in the blank group received normal saline (15 μL each time, 4 times a day) in both eyes for 7 consecutive days. Rats in the positive control group received 0.1% sodium hyaluronate eye drops (15 μL each time, 4 times a day) for 7 consecutive days.

[0070] After the final administration, the tear secretion of rats in each group was measured using a lacrimal gland secretion test. A phenol red-soaked cotton thread was placed in the inferior fornix approximately one-third of the distance from the outer canthus of the rat's eye and removed after 60 seconds. The length (mm) of the red portion of the cotton thread was recorded to represent the rat's tear secretion.

[0071] After the tear secretion experiment, sodium fluorescein test strips were soaked in saline and set aside. Five rats were randomly selected from each group. The tip of the test strip was gently touched to the inner side of the lower eyelid and held for a short time. After three manual blinks, the rats' corneal staining was observed under cobalt blue light of a slit lamp. Each cornea was divided into four quadrants, and the corneal epithelial damage in each quadrant was scored using a four-point scoring system. The specific criteria were: absent green staining (0 point); scattered green staining (1 point); clustered green staining (2 points); and patchy green staining (3 points). The sum of the scores for the four quadrants was the rat corneal fluorescein staining score; higher scores indicated more severe corneal epithelial damage.

[0072] Compared with the normal group, the tear secretion of rats in all groups was significantly reduced (P<0.05). Compared with the model group, the tear secretion of rats in the high-dose P5 peptide group was significantly increased, and the green fluorescence in the rat corneal tissue was reduced, and the corneal fluorescein staining score was significantly reduced. The results are shown in Table 3.

[0073] Table 3. Comparison of tear secretion and corneal fluorescein staining scores in rats

[0074] Example 7 Optimization design of P5 polypeptide molecules.

[0075] In order to improve the stability of the P5 polypeptide in vivo and in vitro, and to have better activity, thereby better exerting its antagonistic effect targeting TRPV1, a single amino acid or multiple amino acids at sequence positions 3, 4, and 5 (indicated by X) of the P5 polypeptide sequence DQKNC were selected to be replaced by natural or non-natural substitutions (L-type amino acids or D-type amino acids). The specific substitutions are as follows: X3 is selected from any natural or non-natural one of Phe F, Trp W, His H, Lys K, and Arg R; X4 is any one of natural or non-natural selected from Phe F, Trp W, His H, Lys K, and Arg R; X5 is selected from any polar natural or non-natural neutral amino acid. Preferably, X5 is Asn N, Ser S, Met M; At the same time, the active peptide mutant, the modification includes at least one of glycosylation modification, phosphorylation modification, ubiquitination modification, S-nitrosylation modification, N-methylation modification, carbamoyl modification, N-acetylation modification, C-amidation modification, N-palmitoylation modification, N-myristoylation modification, PEG modification and lipidation modification.

[0076] The peptides shown in Table 2 were synthesized using the Fmoc solid-phase peptide synthesis method.

[0077] The antagonistic effect experiment targeting TRPV1 was the same as the rTRPV1 inhibitory activity verification in Example 3. The results are shown in Table 4.

[0078] Table 4. P5 amino acid substitution sequence.

[0079]

[0080] In the absence of conflict, the above embodiments and features in the embodiments may be combined with each other.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A polypeptide targeting TRPV1 or a pharmaceutically acceptable salt thereof, characterized in that: Having the amino acid sequence shown in Formula I: Asp-Gln-X 1 -X 2 -X 3 (Formula I) X 1 Any natural or non-natural one selected from Phe, Trp, His, Lys, and Arg; X 2 Any natural or non-natural one selected from Phe, Trp, His, Lys, and Arg; X 3 Any one selected from Asn, Ser, and Met, whether natural or unnatural.

2. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1, wherein: The amino acid sequence of the polypeptide is selected from any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO:

13.

3. A pharmaceutical composition, characterized in that The invention comprises at least one polypeptide according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition is formulated for topical, oral, ocular, intravenous, intravitreal, subconjunctival or subcutaneous administration.

5. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is in the form of a gel, lotion, cream, tablet, capsule, pill, solution, eye drops, spray, bandage, contact lens, depot formulation, injection, implant or sustained release formulation.

6. Use of the polypeptide according to claim 1 or an acceptable salt thereof in the preparation of a medicament for treating pain.

7. Use of the polypeptide according to claim 1 or an acceptable salt thereof in the preparation of a medicament for treating dry eye.

8. Use of the polypeptide according to claim 1 or an acceptable salt thereof in the preparation of a medicament for treating skin itching or allergy.

9. The use according to any one of claims 6 to 8, characterized in that The polypeptide is the only active ingredient.

10. Use of the polypeptide according to any one of claims 1 or 2 or a pharmaceutically acceptable salt thereof in the preparation of cosmetics.

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