Animal toxin polypeptides and uses thereof

Abstract

The present application relates to polypeptides, in particular to an animal toxin polypeptide and application thereof. The polypeptide is identified by RNA sequencing of centipede transcriptome, and bioinformatics screening and Motif scanning analysis of translated protein sequence. Experimental results show that SM-8 shows strong analgesic effect in various pain models, including hot plate test, acetic acid writhing test, formalin inflammatory pain model and migraine model, and the analgesic effect is equivalent to that of butorphanol tartrate and sumatriptan. Molecular mechanism research shows that SM-8 can regulate the expression of pain-related genes. Preliminary safety evaluation by mouse open field test shows that the polypeptide does not cause obvious toxic side effects, has good safety, and has good prospects for development into a new polypeptide analgesic drug.

Description

Technical Field

[0001] This invention relates to polypeptides, specifically to an animal toxin polypeptide and its applications. Background Technology

[0002] Studies have found that the global prevalence of chronic pain is close to 22.9%. Currently, the main analgesics used clinically are opioids, nonsteroidal anti-inflammatory drugs (NSAIDs), and some antidepressants and anticonvulsants. NSAIDs are antipyretic analgesics that also typically have anti-inflammatory and antirheumatic effects, and are widely used clinically, for example, for postoperative acute pain and inflammatory pain. Opioids act on the pain centers in the brain and brainstem, activating the inhibitory function of the descending pain regulation system. These drugs have a strong analgesic effect and are mainly used to treat moderate to severe pain such as cancer pain. Other adjunctive analgesics, such as antidepressants and anticonvulsants, are not pain-treating drugs themselves, but can be used in combination with analgesics in clinical practice to improve the analgesic effect, especially for pain caused by nerve damage. Opioid analgesics play an irreplaceable role in pain relief, but they have many side effects, such as nausea, vomiting, drowsiness, and dizziness. More seriously, they can sedate the central nervous system and inhibit the respiratory rhythm generated by the brainstem, easily leading to respiratory distress and even death. In addition, opioids pose serious risks of addiction and abuse. Data shows that 32% of opioid users may abuse them, and 23% may become addicted. The WHO estimates that approximately 15 million people worldwide are addicted to opioids, and 69,000 people die from opioid overdoses annually. Despite the widespread safety and addictive risks associated with opioids, the clinical need for analgesia continues to grow. Surveys indicate that the global analgesic market reached $71 billion in 2019 and may reach $92 billion by 2027, while the domestic Chinese analgesic market was relatively small at approximately $530 million in 2017. NSAIDs, another major analgesic, also have various side effects such as gastrointestinal bleeding, myocardial infarction, and stroke, and cannot be used by patients with cardiovascular diseases or diabetes. Therefore, the current analgesic market still has significant clinical demand, and the development of a new, safe, and non-addictive non-opioid analgesic has enormous market potential.

[0003] Peptide toxins have attracted increasing attention due to their excellent specificity to specific molecular targets and their extreme biomedical and pharmacological activities. For example, ziconotide, approved by the FDA in 2004 for the treatment of severe chronic pain, was originally isolated from the venom of the cone snail (Conus magus) and contains 25 amino acids and three pairs of disulfide bonds. As a selective Cav2.2 channel antagonist, ziconotide blocks Ca2+ channels. 2+Ziconotide infuses and inhibits the release of excitatory neurotransmitters from afferent nerve endings, and its analgesic efficacy is 1000 times that of opioids. However, the special administration method of intrathecal injection and a series of adverse reactions in the central nervous system limit the widespread use of ziconotide.

[0004] The main component of centipede venom is polypeptide toxins, which possess a variety of biomedical and pharmacological activities. Currently, approximately 50 centipede venom components have been reported to exhibit ion channel activity, antibacterial activity, platelet aggregation activity, anticoagulant activity, phospholipase A2 activity, and trypsin inhibition. Most centipede toxins are neurotoxins that act on ion channels, interfering with the prey's central nervous system, causing pain or paralysis for effective hunting. Centipede peptides are characterized by novel structures, high efficacy, and strong specificity, showing broad application prospects in disease diagnosis and treatment. SsmTX-I is a polypeptide isolated from the venom of *Scolopendra subspinipes*. Electrophysiological studies show that this peptide can selectively block Kv2.1 channels without affecting other channels. In acetic acid-induced writhing models and thermal pain experiments, its analgesic effect is comparable to morphine, while in formalin-induced foot-licking experiments, its analgesic effect is significantly superior to morphine. However, this polypeptide sequence consists of 37 amino acids, has a long peptide chain, and contains two pairs of disulfide bonds, making its structure very complex.

[0005] Although various centipede peptides have been discovered, their analgesic effects or adverse reactions still cannot meet clinical needs. Therefore, there is an urgent need to develop a novel peptide with good analgesic effects. Summary of the Invention

[0006] Purpose of the invention: Based on high-throughput sequencing of the transcriptome of *Scolopendra subspinipes*, this invention uses a combination of computer simulation screening and in vitro and in vivo efficacy verification to screen a series of novel analgesic peptides.

[0007] Technical solution: An animal toxin polypeptide containing a type I structure from the N-terminus to the C-terminus; Arg-Arg-X1-Ala-Glu-Thr-Ile-Lys-Val-X2-Arg-Lys-Lys-Glu-Arg Formula I in, In Formula I, the "-" between amino acid residues represents a peptide bond, the C-terminus of the polypeptide is a free carboxyl group or amide, and X1 and X2 are any amino acids.

[0008] Preferably, X1 is selected from any one of His, Arg, Lys, Phe, and Tyr; X2 is selected from any one of Leu, Arg, His, Met, Trp, and Tyr.

[0009] Preferably, any of the above-mentioned polypeptides is selected from any one of the following amino acid sequences: SEQ ID NO: 1-SEQ ID NO: 10.

[0010] A nucleic acid molecule that encodes any of the aforementioned polypeptides.

[0011] An expression vector containing the aforementioned nucleic acid molecules.

[0012] A host cell containing the aforementioned expression vector.

[0013] A pharmaceutical composition comprising any of the above-mentioned polypeptides and their pharmaceutically acceptable salts, and a pharmaceutically acceptable carrier.

[0014] Preferably, the dosage form of the pharmaceutical composition is any one of the following: injection, lyophilized powder for injection, gel, cream, ointment, patch, spray, and suppository.

[0015] Preferably, the use of any of the above-described polypeptides or any of the above-described pharmaceutical compositions in the preparation of a pain-relieving drug.

[0016] Preferably, the pain includes any one or more of the following: migraine, trigeminal neuralgia, postoperative pain, bone and joint pain, nutritional deficiency pain, inflammatory pain, hypothyroidism pain, Guillain-Barré syndrome pain, poisoning pain, drug-induced pain, and tumor lesion pain.

[0017] Beneficial Effects: The peptides of this invention are based on a centipede transcriptome database obtained from RNA sequencing of centipede tissue. Further protein analysis was conducted on the translated protein sequences. After sequencing and translating the centipede tissue RNA, preliminary screening of the protein sequences was performed using Motif Scan. Ultimately, a series of peptides SM-8 and their analogues (such as SEQ ID NO: 1-SEQ ID NO: 10) were obtained, exhibiting significant analgesic activity, as detailed below: The SM-8 polypeptide is composed of 15 amino acids and does not contain disulfide bonds. It has a strong analgesic effect in various classic analgesic animal models, such as the mouse hot plate test, mouse acetic acid writhing test, mouse formalin inflammatory pain test, and mouse migraine test. Its efficacy is comparable to that of the clinically marketed opioid analgesic butorphanol tartrate and the first-line migraine analgesic sumatriptan.

[0018] Pain is a very serious clinical problem that negatively impacts the quality of life for patients and their families. However, existing analgesics cannot specifically address different types of pain and have adverse effects with long-term use, such as opioid addiction, which can lead to drug abuse. The peptide of this invention is a novel non-opioid analgesic. Preliminary safety evaluations using mouse open field experiments showed no significant toxic side effects. Furthermore, the metabolites of this peptide are mainly peptides and amino acids, suggesting an inherent safety advantage, and it is unlikely to cause drug addiction or other toxic side effects in clinical practice. Compared to ziconopeptide, the only currently marketed animal toxin peptide, this peptide does not require the complex intrathecal infusion method, thus improving patient compliance.

[0019] Furthermore, ziconopeptide is composed of 25 amino acids and contains 3 pairs of disulfide bonds in its peptide sequence, making its preparation process complex and greatly increasing production costs. In contrast, the polypeptide of this invention is composed of 15 amino acids and does not contain disulfide bonds, so its production process is simple and is very conducive to automated large-scale production. This can greatly reduce drug production costs and alleviate the medical burden on patients, and is expected to serve as a safe and effective new type of analgesic drug. Attached Figure Description

[0020] The following is a brief description of the accompanying drawings, which are used to illustrate the exemplary embodiments disclosed herein and are not intended to limit those embodiments.

[0021] Figure 1 The mass spectrometry pattern of peptide SM-8 in Example 1 is shown.

[0022] Figure 2 The HPLC chemical purity chromatogram of peptide SM-8 in Example 1 is shown.

[0023] Figure 3 The pain threshold latency in mice after administration in the mouse hot plate test model in Example 2 is shown. The vertical axis represents the pain threshold latency, i.e., the time when mice exhibit a licking hind paw or jumping response during the hot plate test, and the horizontal axis represents the different groups. The results are shown as pain threshold latency (±SD). Graphpad, one-way ANOVA (n=8). Treatment group vs. negative control group: ****p<0.0001; **p<0.01; *p<0.05; ns: no statistical significance. Butorphanol tartrate was administered at 5 mg / kg·d·qd, intraperitoneally; SM-8 was administered at 10 mg / kg·d·qd and 20 mg / kg·d·qd, intraperitoneally.

[0024] Figure 4The changes in the number of writhing movements in mice after administration in the acetic acid writhing model in Example 3 are shown. The vertical axis represents the number of writhing movements in mice, and the horizontal axis represents the different groups. The results are shown as the number of writhing movements in mice (±SD). Graphpad, one-way ANOVA (n=8). Drug administration group vs. model group: ****p<0.0001. SM-8 was administered at 10 mg / kg·d·qd and 20 mg / kg·d·qd, intraperitoneally.

[0025] Figure 5 The changes in paw-licking time in mice after administration in the formalin-induced inflammatory pain model in Example 4 are shown. The vertical axis represents mouse paw-licking, and the horizontal axis represents different groups. Results are shown as mouse paw-licking time (±SD). Graphpad, one-way ANOVA (n=8). Treatment group vs. model group: ****p<0.0001, *p<0.05. SM-8 was administered at 5 mg / kg·d·qd and 20 mg / kg·d·qd, intraperitoneally.

[0026] Figure 6 This paper illustrates the changes in the expression levels of pain-related genes in mice after drug administration, detected by real-time quantitative PCR (qPCR) using RNA samples extracted from a formalin-induced inflammatory pain model in mice, as shown in Example 5. The vertical axis represents the relative expression levels of relevant pain genes in mice, and the horizontal axis represents different groups. A represents c-fos; B represents CGRP; C represents PACAP; and D represents PENK. The results show the relative expression levels of relevant pain genes (±SD). Graphpad analysis, one-way ANOVA (n=8). Drug administration group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05. SM-8 was administered intraperitoneally at 5 mg / kg·d·qd and 20 mg / kg·d·qd.

[0027] Figure 7 The pain threshold latency in mice after administration in a mouse migraine model in Example 6 is shown. The vertical axis represents the total number of head scratches in mice within 2 hours after administration, and the horizontal axis represents the different groups. Results are shown as the number of head scratches per hour (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. negative control group: ****p<0.0001; administration group vs. model group: ****p<0.0001; ***p<0.001. Sumatriptan was administered intraperitoneally at 1 mg / kg·d·qd; SM-8 was administered intraperitoneally at 15 mg / kg·d·qd, 20 mg / kg·d·qd, and 25 mg / kg·d·qd.

[0028] Figure 8This paper illustrates the changes in the expression levels of pain-related genes in mice after drug administration, detected by real-time quantitative PCR (qPCR) using RNA samples from the trigeminal ganglion and brainstem tissues extracted in a formalin-induced inflammatory pain model in mice, as shown in Example 7. The vertical axis represents the relative expression levels of relevant pain genes in mice, and the horizontal axis represents different groups. AC represents CGRP, SP, and c-fos in the trigeminal ganglion tissue, respectively; DF represents CGRP, SP, and c-fos in the brainstem tissue, respectively. The results show the relative expression levels of relevant pain genes (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. negative control group: ****p<0.0001; ***p<0.001; **p<0.01; Drug administration group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01. Sumatriptan is administered intraperitoneally at 1 mg / kg / day qd; SM-8 is administered intraperitoneally at 15 mg / kg / day qdd, 20 mg / kg / day qd, and 25 mg / kg / day qd.

[0029] Figure 9 The changes in spontaneous movement behavior of mice after administration in the mouse open field model in Example 8 are shown. The vertical axis represents the number of times mice entered the central region, the time mice spent in the central region, and the distance mice traveled in the central region; the horizontal axis represents the different groups. The results are shown as the number of times mice entered the central region (A), the time mice spent in the central region (B), and the distance mice traveled in the central region (C) (±SD). Graphpad, one-way ANOVA (n=8). SM-8 was administered at 20 mg / kg·d·qd, 40 mg / kg·d·qd, and 80 mg / kg·d·qd, via intraperitoneal injection.

[0030] Figure 10 The effect of peptide SM-8 on the Rack1 / PLCγ / PIP2 / IP3 signaling pathway in Example 9 is shown. (A) The figure shows the interaction between peptide SM-8 and Rack1 protein detected by Far-Western Blotting; (B) The figure shows the standard curve of IP3 concentration and absorbance (450 nm) detected by ELISA; (C) The figure shows the effect of peptide SM-8 on the release concentration of IP3 in DRG cells after stimulation with iomycin. (±SD). Graphpad, one-way ANOVA (n=3). Model group vs. negative control group: ****p<0.0001; drug-treated group vs. model group: ****p<0.0001.

[0031] Figure 11 Example 10 illustrates the detection of the effect of peptide SM-8 on Ca using Western blotting. 2+Inhibition of the CaMKIIα / CREB signaling pathway. The protein bands represent, in order, the phosphorylation levels of CaMKIIα and CREB, and GAPDH, with GAPDH serving as an internal control protein.

[0032] Figure 12 The results of molecular docking (interaction force analysis) between peptide SM-8 and target protein Rack1 in Example 11 are shown. Detailed Implementation

[0034] When used in this article, the site numbering of peptides is in the order from the N-terminus to the C-terminus. For example, the first amino acid starting from the N-terminus of the peptide is the first amino acid, the second amino acid starting from the N-terminus of the peptide is the second amino acid, the third amino acid starting from the N-terminus of the peptide is the third amino acid, and so on.

[0035] Unless otherwise defined, as is commonly understood in the art, the left-hand end of an amino acid sequence or polypeptide sequence is an N-terminus / amino terminus, and the right-hand end is a C-terminus / carboxyl terminus. As used herein, the term "prodrug" refers to all molecules that are converted into the polypeptides of the present invention through reactions such as oxidation, reduction, and hydrolysis under physiological conditions in vivo, for example, in the presence of enzymes, gastric acid, etc.

[0036] All natural amino acids, except glycine, contain a chiral carbon atom. Unless otherwise specified, the optically active amino acids described in this invention are all L-configured. As is customary, the structures of peptides in this document are written with the amino terminus (N-terminus) on the left and the carboxyl terminus (C-terminus) on the right.

[0037] The present invention provides a polynucleotide that encodes the polypeptide described herein.

[0038] As used herein, “polynucleotide” refers to a polymer of nucleotides (nucleotides or deoxynucleotides) of any length. The term refers to the primary structure of a molecule. Thus, it includes both double-stranded and single-stranded RNA and double-stranded and single-stranded DNA. It also includes polynucleotides modified by methylation and / or end-capping, as well as unmodified polypeptide nucleotides. Polynucleotides described herein are not necessarily obtained by physical methods and can be produced in any way, including through chemical synthesis or DNA replication, reverse transcription, or transcription.

[0039] This invention provides an expression vector capable of expressing the polynucleotides described herein.

[0040] The present invention provides a host cell containing the polynucleotide or expression vector described in the present invention.

[0041] As used herein, “host cell” refers to the cell that is the recipient of the expression vector or other transformed genetic fragment. These include the original transfected cell and its progeny. Host cells include prokaryotic, yeast, or other eukaryotic cells.

[0042] As used herein, the term "pharmaceutical composition" refers to a mixture of the peptide of the present invention with other chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. The pharmaceutical composition facilitates the administration of the peptide to a living organism. Various methods of administering the peptide exist in the art, including but not limited to subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal, transpulmonary, ocular, and topical application.

[0043] In this invention, the pharmaceutical composition can be formulated into a dosage form suitable for administration to a subject via the desired route of administration. The dosage forms include, but are not limited to, tablets, capsules, pouches, pills, lozenges, powders, syrups, infusions, suspensions, solutions, emulsions, transdermal patches, suppositories, inhalers, creams, ointments, lotions, pastes, sprays, lyophilized products, injections, and gels.

[0044] As used herein, the term "pharmaceutically acceptable salt" includes both acid addition salts and base addition salts. Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include, but are not limited to: acetates, adipates, aspartates, benzoates, benzenesulfonates, bicarbonates / carbonates, hydrogen sulfates / sulfates, borates, camphor sulfonates, citrates, cyclohexylamine sulfonates, ethanedisulfonates, formates, fumarates, gluconate, glucuronates, glucuronates, hexafluorophosphates, 2-(4-hydroxybenzyl)benzoates, hydrochlorides / chlorides, hydrobromines / bromines, hydroiodides / iodides, 2-hydroxyethanesulfonates, lactates, malates, maleates, malonates, methanesulfonates, methyl sulfates, naphthalates, 2-naphthalenesulfonates, nicotinates, nitrates, orotates, oxalates, hexadecates, phosphates / hydrogen phosphates / dihydrogen phosphates, pyroglutamates, gluconate, stearates, salicylates, tannins, tartrates, toluenesulfonates, and trifluoroacetates. Suitable base addition salts are formed by bases that form non-toxic salts. Examples include, but are not limited to: aluminum, arginine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts. They can also form hemisalts of acids and bases, such as hemisulfates and hemicalcium salts. For a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0045] The term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, relating to carrying or delivering the peptides of the present invention within or to a subject, enabling them to perform their intended function. Each salt or carrier must be "acceptable" in the sense of compatibility with other components of the formulation and not harmful to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth gum; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate. Esters and ethyl lauryl ester; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginate; pyrogen-free raw water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solution; diluents; granulators; lubricants; binders; disintegrants; wetting agents; emulsifiers; colorants; release agents; coating agents; sweeteners; flavoring agents; preservatives; antioxidants; plasticizers; gelling agents; thickeners; hardeners; setting agents; suspending agents; surfactants; humectants; carriers; stabilizers; and other non-toxic, compatible substances used in pharmaceutical preparations, or any combination thereof.

[0046] In some embodiments, the prepared medicament for treating pain is administered via methods including subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal administration, transpulmonary administration, ocular administration, or local administration. In a preferred embodiment, the prepared medicament for treating pain is administered via subcutaneous injection.

[0047] In some embodiments, the pain includes migraine, trigeminal neuralgia, postoperative pain, bone and joint pain, infection pain, nutritional deficiency pain, inflammatory pain, chronic alcoholism pain, hypothyroidism pain, autoimmune disease pain, Guillain-Barré syndrome pain, poisoning pain, drug-induced pain, tumor lesion pain, genetic disease pain, and contusion pain.

[0048] The present invention further provides the use of the polypeptide, polynucleotide, expression vector, host cell, or pharmaceutical composition described herein for the treatment of pain.

[0049] In some embodiments, the polypeptide or pharmaceutical composition is administered via a route including subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal administration, transpulmonary administration, ocular administration, or topical administration. In a preferred embodiment, the polypeptide or pharmaceutical composition is administered via subcutaneous injection.

[0050] Example 1, Synthesis of SM-8 polypeptide: Using Wang resin (Xi'an Lanxiao Technology New Materials Co., Ltd.) with a substitution degree of 1.0 mmol / g as the starting resin, the synthesis scale was 1 mmol. The resin was weighed and added to a peptide solid-phase reactor (Nanjing Luanyu Chemical Glass Instrument Co., Ltd.), and swollen for 30 minutes under nitrogen purging with dichloromethane, followed by washing twice with DMF (dimethylformamide). Fmoc-Arg(pbf)-OH (1.5 equivalents), HoBt (1.5 equivalents), and DMAP (0.15 equivalents) were weighed, dissolved in DMF, and the swollen resin was added under nitrogen purging. DIC (1.5 equivalents) was then slowly added dropwise, and the reaction was carried out at room temperature for 1-3 hours. The resin was washed three times with DMF, approximately 3 minutes each time. The resin was then washed alternately with dichloromethane and methanol, and finally, the shrunken and dried peptide resin intermediate was obtained by vacuum drying. The resin substitution degree of the C-terminal amino acid coupling was detected using a UV spectrophotometer, the synthesis scale was recalculated, and the resin was swollen under the same conditions as described above. The N-terminal Fmoc group of the protected amino acid was removed twice with 20% piperidine / DMF solution, each reaction lasting 10 minutes. After the deprotection reaction, the resin was washed six times with DMF, approximately 3 minutes each time. Following the conventional principle of chemically synthesized peptides, amino acids are coupled one by one from the C-terminus to the N-terminus according to the peptide sequence. The corresponding protected amino acid (Chengdu Zhengyuan Biochemical Technology Co., Ltd.) and condensation reagents (HoBt, DIC) (Suzhou Haofan Biotechnology Co., Ltd.) were weighed out, and the reaction was carried out for approximately 1 hour. After the condensation reaction, the resin was washed three times with DMF. This condensation cycle (deprotection-washing-condensation-washing) was then performed, i.e., 20% piperidine / DMF solution was added to the reaction vessel again for deprotection reaction, followed by DMF washing and then the addition of the protected amino acid and the corresponding condensation reagent for condensation reaction. The condensation cycle method was used to couple the protected amino acid one by one to the N-terminus until all amino acids of the peptide were coupled. The Fmoc groups at the N-terminus of the peptide were removed using a 20% piperidine / DMF solution. The resin was then washed alternately with dichloromethane and methanol, and finally dried under vacuum to obtain a shrunken peptide resin intermediate. The peptide resin was weighed, and a lysis buffer was prepared at a ratio of 6-10 mL of lysis buffer per gram of peptide resin. The lysis buffer formulation was TFA:EDT:TIS:H2O = 90:5:2.5:2.5. The lysis buffer was added to the peptide resin, and the lysis reaction was carried out for approximately 3-4 hours. The filtrate was collected, and the filtrate was precipitated with frozen methyl tert-butyl ether (MTBE), using 6-10 times the volume of the filtrate. The precipitate was thoroughly stirred, centrifuged in separate cups to obtain a crude peptide precipitate. This precipitate was then washed with MTBE and centrifuged again. The precipitate was then vacuum dried overnight to obtain the crude SM-8 peptide sample. The crude peptide was dissolved, filtered through a filter membrane, and then purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The qualified fractions were combined and finally lyophilized to obtain the lyophilized pure peptide SM-8. The purity analysis of peptide SM-8 by mass spectrometry and HPLC is shown below. Figure 1 and Figure 2 As shown, the synthesized peptides confirmed to meet the quality requirements of this experimental study. Peptide SM-8 and its analogues can also be synthesized by commercial companies.

[0051] The peptide sequences involved in the embodiments of the present invention are shown in Table 1.

[0052] Table 1. Peptide sequences .

[0053] Example 2: Evaluation of the in vivo analgesic activity of peptide SM-8 using a mouse hot plate test: The hot plate test is a classic model for evaluating the central analgesic activity of analgesics. Mice have hairless hind paws with high sensitivity. When mice are placed on a constant-temperature hot plate at 55 ± 0.50°C, the heat stimulus is transmitted to the central nervous system via afferent nerves, and then via efferent nerves, causing the mice to lick their hind paws and jump. These physiological responses are used as indicators of pain response in mice. The time from the moment the mouse's hind paw comes into contact with the constant-temperature hot plate until the mouse exhibits a pain response is called the hot plate pain threshold. Mice were randomly divided into groups, and the hot plate pain threshold of each group was recorded and analyzed using a timer. Differences were then analyzed between the groups and the control group to determine the in vivo analgesic activity of the peptide SM-8.

[0054] The specific operating steps are as follows: To prevent individual differences in mice from affecting the experimental results, Kunming mice (Changzhou Cavens Laboratory Animal Co., Ltd.) weighing 20±2g were purchased before the experiment. The indoor temperature was maintained at 20~24℃. The mice were acclimatized to the new environment for about one week. The mice were randomly divided into groups and treated according to the dosing regimen in Table 2. At 0, 30, 60, 90, and 120 minutes after administration, the mice were placed on a thermostatic metal plate at a temperature of 55±0.5℃. The time from when the mouse touched the metal plate to when it exhibited a licking of its hind paw or a jumping response was used as the pain threshold. The baseline pain threshold of the mice was recorded. Each mouse was tested at least twice, with an interval of more than 10 minutes between the two tests. The average value was taken as the final pain threshold, and the results were expressed as Mean±SD.

[0055] like Figure 3As shown, compared with the negative control group injected with physiological saline, the positive drug butorphanol tartrate group showed significant analgesic activity at all test time points within 30-120 minutes after administration (P<0.05); the low-dose peptide SM-8 group (10 mg / kg) showed significant analgesic activity at the 60-minute time point compared with the negative control group (P<0.05); the high-dose peptide SM-8 group (20 mg / kg) showed significant analgesic activity at both the 30 and 60-minute time points compared with the negative control group (P<0.05). The analgesic activity was comparable to that of the marketed analgesic opioid drug butorphanol tartrate. In fact, at the 60-minute time point, the pain threshold curve of the high-dose peptide SM-8 group was slightly higher than that of the butorphanol tartrate group, indicating that peptide SM-8 has strong analgesic activity and can be further developed.

[0056] Table 2. Dosing regimen for analgesic activity of peptide SM-8 in mouse hot plate test .

[0057] Example 3: Evaluation of the in vivo analgesic activity of peptide SM-8 using the mouse acetic acid writhing test: The acetic acid writhing test in mice is a classic analgesic pharmacodynamic screening model, belonging to the "chemical stimulation method." Acetic acid, as a strong chemical irritant, directly stimulates and damages the serosal membranes within the abdominal cavity, particularly the peritoneum. Acetic acid stimulation leads to the release of large amounts of pain-inducing inflammatory mediators in local tissues. These inflammatory mediators, along with acetic acid itself, directly act on pain receptors in the abdominal organs, activating sensory nerve fibers. The pain signals generated by this stimulation are transmitted to the central nervous system via nerves, and mice exhibit a characteristic, stereotyped pain behavior response called "writhing." Typical writhing behaviors include: abdominal muscle contraction, trunk twisting (body elongation, hip elevation), hind limb extension, and back arching. This can be seen as a "struggling" movement made by mice to relieve severe abdominal pain. It primarily represents indications with therapeutic potential for inflammatory pain and visceral colic (such as dysmenorrhea and gastrointestinal spasms).

[0058] SPF-grade Kunming mice (Changzhou Cavens Laboratory Animal Co., Ltd.), weighing 20±2g, were housed in an SPF-grade animal facility and acclimatized to the environment for about one week. Mice were weighed and randomly grouped. Following the dosing regimen shown in Table 3, the corresponding drug dilution was injected into the left abdominal cavity of each group. Ten minutes later, a freshly prepared 0.80% acetic acid solution (0.1mL / 10g) was injected into the right abdominal cavity. Five minutes later, the number of writhing responses (body extension, abdominal retraction, hind leg extension, and hip elevation) in each mouse within 15 minutes was observed and recorded.

[0059] like Figure 4As shown, compared with the model group, the number of writhing movements in mice in the low-dose group (10 mg / kg) and the high-dose group (20 mg / kg) of peptide SM-8 was significantly less than that in the model group, and the difference was statistically significant (P<0.0001). This indicates that peptide SM-8 has significant efficacy in treating inflammatory pain and visceral pain and has broad development prospects.

[0060] Table 3. Dosing regimen for analgesic activity of peptide SM-8 in mouse acetic acid writhing test .

[0061] Example 4: Evaluation of the in vivo analgesic activity of peptide SM-8 using a mouse formalin assay. The formalin model is a classic model of inflammatory pain. A specific concentration of formalin solution is injected into a specific site on the animal, such as the sole of the foot, to induce persistent pain through chemical stimulation. This pain model is easy to perform and allows for the evaluation of the analgesic effects of different drugs on inflammatory pain. The most famous and unique feature of the formalin experiment is its clear biphasic pain response. Phase I (acute / neurogenic phase) begins immediately after injection, directly stimulating the local nociceptive receptors (C-fibers and Aδ-fibers). The pain in this phase is primarily driven by direct excitation of peripheral nerves, resembling an acute, transient tissue injury pain, lasting approximately 5-10 minutes. Phase II (inflammatory / central sensitization phase) follows Phase I with a brief quiescent period (5-15 minutes), after which Phase II begins and lasts 20-40 minutes. Phase II is a persistent pain sustained by inflammatory factors and accompanied by alterations in central nervous system function.

[0062] SPF-grade Kunming mice (Changzhou Cavens Laboratory Animal Co., Ltd.), weighing 20±2g, were randomly assigned to groups after acclimatization. Administered the drugs according to the protocol shown in Table 4 30 minutes before the experiment, and the administration time was recorded. Ten minutes after administration, each group of mice underwent a subcutaneous injection of 0.02 mL of pre-diluted 2.5% formalin solution into the right hind paw toe to induce the formalin model. Only one mouse was observed at a time, and the cumulative time spent licking, biting, and shaking the hind paw was recorded using a timer within 0-5 min and 15-40 min after formalin injection.

[0063] like Figure 5As shown, compared with the model group, both the low-dose group (5 mg / kg) and the high-dose group (20 mg / kg) of peptide SM-8 significantly reduced the time mice spent licking their paws in both of the above-mentioned pain zones (P<0.05), indicating that peptide SM-8 has significant analgesic activity in the mouse formalin model, and that this analgesic activity is dose-dependent. Detailed analysis revealed that peptide SM-8 has stronger analgesic activity in the phase I neuropathic pain zone. This model can well simulate postoperative pain, arthritis pain, or other persistent pain caused by tissue inflammation in humans, revealing the broad development prospects of peptide SM-8 in these indications.

[0064] Table 4. Dosage of peptide SM-8 in analgesic activity assay in a mouse formalin-induced inflammatory pain model .

[0065] Example 5: Real-time quantitative PCR (qPCR) experiment to detect the effect of peptide SM-8 on the expression level of pain-related genes in formalin-induced inflammatory pain model mice: Spinal cord tissue was extracted from mice in each group of Example 4 above, and the mRNA expression levels of c-fos (immediate early gene product), CGRP (calcitonin gene-related peptide), PACAP (pituitary adenylate cyclase activating peptide), and PENK (proenkephalin) were detected to further elucidate the mechanism of action of peptide SM-8 at the molecular level. The c-fos gene is rapidly expressed after strong neuronal activation, and therefore it is widely used as a marker of neuronal activation. Formalin stimulation induces the release of CGRP from sensory nerve endings. CGRP causes vasodilation and extravasation of plasma proteins, leading to neurogenic inflammation, thereby exacerbating and maintaining pain (peripheral sensitization). The detection of increased release or synthesis indicates activation of the peripheral sensitization mechanism. The role of PACAP may be more complex than that of CGRP; it not only plays a role in pain but also participates in stress and mood regulation. PENK is a precursor protein of endogenous opioid peptides—methionine enkephalin and leucine enkephalin. Upregulation of PENK expression was detected in the spinal cord or related brain regions, indicating that the body is trying to suppress pain on its own, which is evidence of a negative feedback regulation mechanism.

[0066] After recording the number of times mice in the negative control group, model group, and high-dose SM-8 group (20 mg / kg) of Example 4 licked their paws, the mice were euthanized and fixed to a dissecting board. The skin on the back of the mice was carefully cut open with surgical scissors and a scalpel, and the spine was cut and separated. The spinal cord tissue was blown out from one end of the spine using a syringe filled with physiological saline. 50-100 mg of frozen spinal cord tissue was placed in a pre-chilled grinding tube, 1 mL of TRIzol reagent was added, and the tissue was thoroughly ground and homogenized using a tissue homogenizer until no obvious tissue blocks were visible. The entire process was performed on ice to prevent RNA degradation. RNA samples were separated using the chloroform method and finally precipitated with isopropanol. The RNA precipitate was washed with pre-chilled 75% ethanol (prepared with DEPC water), and the RNA precipitate was air-dried in a clean bench for 2-5 minutes. Depending on the amount of precipitate, 20-50 μL of RNase-free water or TE buffer was added, and the mixture was gently blown or placed in a 55-60°C water bath to aid dissolution. RNA concentration and purity were determined by reverse transcription of RNA into cDNA using the Hifair® AdvanceFast First-Strand cDNA Synthesis Kit (11149ES60, Yeasen, Shanghai, China). cDNA was amplified using the Blastaq™ 2X Qpcr-MasterMix Kit (Cat#G891, abm, Canada) and quantitative PCR was performed using Quantstudio3 (ABI, USA). GAPDH was used as an endogenous normalization control.

[0067] like Figure 6 China A- Figure 6 As shown in Figure C, the mRNA expression levels of c-fos, CGRP, and PACAP in the spinal cord tissue of the model group mice were significantly increased compared with those of the negative control group (P<0.05); Figure 6 As shown in Figure D, the expression level of PENK in the spinal cord tissue of the model group mice was significantly decreased (P<0.05), indicating that the mouse formalin model was successfully established. Compared with the model group, after treatment with the high-dose peptide SM-8 (20 mg / kg), the mRNA expression levels of c-fos, CGRP, and PACAP in the spinal cord tissue of mice were significantly downregulated (P<0.05), while the expression level of PENK was significantly increased (P<0.05), reaching normal levels. These results reveal the significant analgesic activity of peptide SM-8 against mouse formalin at the molecular level.

[0068] Example 6: Evaluation of the in vivo analgesic activity of peptide SM-8 in a mouse migraine model: A migraine model can be established by subcutaneously injecting nitroglycerin (NTG) into mice. This is based on the principle that nitroglycerin metabolizes in vivo to produce nitric oxide (NO). NO is a recognized migraine trigger, capable of dilating cerebral blood vessels, activating the trigeminal neurovascular system, and inducing central sensitization, thus mimicking many pathophysiological features of human migraines. In the migraine model, the "head-scratching" behavior in mice is interpreted as a spontaneous pain-related behavior. It mimics the involuntary actions of pressing the temples or feeling discomfort in the head during a migraine attack in human patients. The underlying mechanism is that nitroglycerin-induced activation of the trigeminal neurovascular system and neurogenic inflammation lead to an itching, pain, or discomfort sensation in the intracranial and pericranial regions (such as the meninges), thereby triggering the head-scratching response.

[0069] SPF-grade Kunming mice (Changzhou Cavens Laboratory Animal Co., Ltd.) were acclimatized for about one week and then randomly divided into groups. Nitroglycerin was diluted with physiological saline. The modeling group received a single subcutaneous injection of 10 mg / kg of nitroglycerin per mouse, while the blank control group received physiological saline as an excipient. About one hour after modeling, the mice were administered the drug according to the dosing regimen shown in Table 5 (one-time dosing in total), and the number of times the mice scratched their heads in each group was recorded within 0-2 hours after administration.

[0070] like Figure 7 As shown, within 2 hours of administration, compared with the negative control group, the total number of head scratches in the model group (injected with nitroglycerin) was significantly increased (P<0.0001); compared with the model group, the total number of head scratches in the positive drug sumatriptan group, peptide SM-8 (15 mg / kg), peptide SM-8 (20 mg / kg) and peptide SM-8 (25 mg / kg) was significantly reduced (P<0.0001), and the number of head scratches was comparable to that of normal mice without modeling. Peptide SM-8 can relieve migraine symptoms after administration, and its migraine treatment activity is comparable to that of sumatriptan, a first-line marketed migraine drug, indicating that peptide SM-8 has a good prospect for clinical translational application.

[0071] Table 5. Dosage of peptide SM-8 in analgesic activity assay in a mouse migraine model .

[0072] Example 7: Real-time quantitative PCR (qPCR) experiment to detect the effect of peptide SM-8 on the expression levels of related genes in a mouse migraine model: When stimulated by internal or external factors, nerve endings around the blood vessels of the dura mater are activated, releasing neuropeptides (such as CGRP and SP) that lead to neurogenic inflammation, vasodilation, and pain signals. These signals are transmitted to the central nervous system via the trigeminal nerve and are ultimately perceived by the brain as headache. Calcitonin gene-related peptide (CGRP) is the most critical signaling molecule in migraines. Released by activated trigeminal nerve endings, it causes strong vasodilation, promotes mast cell degranulation, exacerbates neurogenic inflammation, and directly sensitizes the pain pathway in the central nervous system. Substance P (SP) is mainly associated with the early stages of neurogenic inflammation. It increases vascular permeability, leading to plasma protein extravasation (neurogenic plasma extravasation), causing tissue edema, and activating immune cells. Furthermore, the expression of the immediate early gene (c-fos) is a marker of neuronal activation. In migraine models, the massive firing of neurons along the pain pathway induces rapid expression of the c-Fos gene. Detecting the changes in the levels of these molecular markers in different experimental groups of a mouse acute migraine model can further illustrate the analgesic potential of peptide SM-8 in the migraine model.

[0073] After administering the drug and recording the total number of head scratches within 2 hours, the mice in each group of Example 5 were euthanized. RNA samples were extracted from the trigeminal ganglion and brainstem tissue of each mouse as described in Example 5. Real-time quantitative PCR (qPCR) experiments were performed on the RNA samples of each group to detect the changes in the mRNA expression levels of CGRP, SP, and c-fos in the trigeminal ganglion and brainstem tissues, respectively. Figure 8 As shown, the mRNA expression levels of c-fos, CGRP, and SP in the trigeminal ganglion and brainstem tissues of the model group mice were significantly increased compared with those of the negative control group (P<0.05), indicating that the mouse migraine model was successfully established. Compared with the model group, the mRNA expression levels of c-fos, CGRP, and SP in the trigeminal ganglion and brainstem tissues of mice were significantly downregulated after treatment with the positive drug sumatriptan (1 mg / kg), peptide SM-8 (15 mg / kg), peptide SM-8 (20 mg / kg), and peptide SM-8 (25 mg / kg) (P<0.05). These results further reveal the significant analgesic activity of peptide SM-8 in the mouse migraine model at the molecular level, and its activity is comparable to that of the first-line positive drug sumatriptan, showing the broad application and development prospects of peptide SM-8.

[0074] Example 8: Evaluation of the safety of peptide SM-8 using a mouse open field assay: The Open Field Test (OFT) is a classic behavioral experiment for studying the voluntary movement of mice. By observing the number of times a mouse enters the central area, the duration of its stay, and the distance it moves, the effects of drugs on the voluntary movement of mice can be analyzed, and the side effects of drugs can be explored.

[0075] SPF-grade Kunming mice (Changzhou Cavens Laboratory Animal Co., Ltd.) were acclimatized for approximately one week. The mice were then randomly divided into groups and administered the drugs according to the regimen shown in Table 6. Spontaneous movement behavior was assessed at 0, 1, 2, and 24 hours after drug administration. Specifically, at each time point, the mice were gently placed facing the same direction in any corner of an open field box (typically a square box (e.g., 40cm x 40cm x 40cm) or a circular box (30-100cm in diameter)). The mice were immediately removed from the experimental area, avoiding eye contact or noise. Video recording and software tracking were immediately initiated. The experimenters should observe the mice from outside the room or in a concealed location, monitoring the process via a monitor. Recording was stopped after the set experimental time (10-30 minutes). After all experiments were completed, the recorded videos were analyzed using behavioral analysis software (Anymaze, Beijing Zhongshidichuang Technology Co., Ltd.). Various indicators of spontaneous movement were extracted (number of times the mouse entered the central area, duration of stay in the central area, and distance traveled in the central area), and the data were statistically analyzed.

[0076] like Figure 9 As shown, after injection of peptide SM-8, compared with the negative control group, the number of times mice entered the central region, the time mice stayed in the central region, and the distance mice moved in the central region were not significantly different (P>0.05) in the treatment group mice. This indicates that peptide SM-8 has no significant effect on the spontaneous movement behavior of mice, revealing the preliminary safety of peptide SM-8, and it can be further developed and transformed as a candidate analgesic lead compound.

[0077] Table 6. Dosage table for safety evaluation of peptide SM-8 in a mouse open field model .

[0078] Example 9, Effect of peptide SM-8 on the Rack1 / PLCγ / PIP2 / IP3 signaling pathway: The scaffold protein Rack1 recruits and activates the key enzyme PLCγ to the cell membrane. Messenger generation: Activated PLCγ "cleaves" PIP2 on the membrane into two messengers: IP3 and DAG. IP3, acting as a key messenger, rapidly diffuses into the cytoplasm and binds to IP3 receptors on the endoplasmic reticulum. This opens calcium channels, releasing large amounts of calcium ions stored in the endoplasmic reticulum into the cytoplasm, leading to a sharp increase in intracellular calcium ion concentration. This amplifies pain signals, alters neuronal excitability, and ultimately causes hyperalgesia and atypical pain.

[0079] Far-Western blotting was used to study the interaction between peptide SM-8 and Rack1 protein. Specifically, a sufficient number of DRG cells (Nanjing Saihongrui Biotechnology Co., Ltd.) were cultured and washed with pre-chilled PBS. RIPA lysis buffer containing a protease inhibitor was added, and the cells were lysed on ice for 30 minutes. The cells were centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant (total protein solution) was carefully aspirated to prepare an appropriate concentration of SDS-PAGE gel to ensure good separation of Rack1 protein. The gel was loaded and electrophoresis was performed. The experimental groups included DRG cell proteins and BSA (bovine serum albumin as a negative control). After electrophoresis, the proteins were transferred to a PVDF membrane. The membrane was briefly rinsed with TBST (Tris-HCl buffer + Tween-20). The membrane was then immersed in 6M guanidine hydrochloride denaturing buffer and incubated at room temperature with shaking for 30 minutes to ensure complete denaturation of all proteins. Transfer the membrane sequentially to guanidine hydrochloride solutions of decreasing concentration (6M - 4M - 2M - 1M - 0.5M - 0M), incubating for 15-30 minutes at each concentration. The entire process is performed at 4°C to reduce protein aggregation. Finally, incubate with refolding buffer (TBS, containing 1 mM DTT) for at least 1 hour. Membrane denaturation and refolding are crucial; they remove SDS and allow proteins on the membrane (especially Rack1) to recover their native conformation, enabling them to bind to the peptide. Block the membrane with TBST solution of 5% skim milk, shaking at room temperature for 1-2 hours to block non-specific binding. Dilute the biotin-labeled peptide SM-8 with blocking buffer, and incubate the membrane with the peptide probe solution overnight at 4°C. After incubation, wash the membrane thoroughly with TBST (4-6 times, 5-10 minutes each time) to completely remove unbound peptides. Incubate the membrane with an appropriately diluted HRP-labeled streptavidin (HRP) solution at room temperature for 1 hour. Wash the membrane thoroughly again with TBST (4-6 times, 5-10 minutes each time) to remove unbound streptavidin. Mix equal volumes of ECL chemiluminescence substrate solutions A and B according to the instructions. Add the mixture to the membrane, and detect and acquire the signal using a chemiluminescence imaging system, with GAPDH as the internal control protein.

[0080] like Figure 10 As shown in Figure A, peptide SM-8 binds tightly to Rack1 protein in the total protein sample of DRG cells, but no corresponding band appears with BSA protein, indicating that peptide SM-8 has a significant interaction with Rack1 protein and has specific binding characteristics.

[0081] As mentioned above, the scaffold protein Rack1 ultimately affects the secretion and release of IP3. To verify the downstream signaling pathway following the interaction between peptide SM-8 and protein Rack1, the relationship between IP3 concentration and absorbance (450 nm) was detected using an ELISA assay. A standard curve of a four-parameter logistic function was fitted using Origin software, as shown below. Figure 10 As shown in Figure B, the fitted standard curve R² = 0.99878 can be used to calculate the concentration of IP3, as follows. Figure 10 As shown in Figure C, DRG cells were incubated in DMEM medium as a negative control group. DRG cells were stimulated with 2 μM ionomycin to establish a model. 20 μM peptide SM-8 was incubated in the modeled DRG cells. Ionomycin significantly induced the release of IP3, while incubation with peptide SM-8 significantly reduced the release of IP3. The above experimental results indicate that after peptide SM-8 interacts with Rack1, it inhibits the Rack1 / PLCγ / PIP2 / IP3 signaling pathway, which can ultimately reduce the intracellular calcium ion concentration and exert analgesic activity.

[0082] Example 10, Effects of peptide SM-8 on Ca in migraine rats 2+ Inhibition of the / CaMKⅡα / CREB signaling pathway: Phosphorylation of CaMKIIα (calmodulin kinase IIα) protein amplifies pain signals, enhancing synaptic excitability, inducing central sensitization, and linking calcium signaling to gene expression. CREB (cyclic adenosine monophosphate effector element-binding protein) is the programmer of pain persistence, initiating gene transcription related to neuronal plasticity, inflammation, and excitability. The phosphorylation of CaMKIIα and CREB together constitutes a complete pathway from electrical signals to chemical signals, and then to gene expression. They transform acute noxious stimuli into long-term functional and structural changes in the central nervous system, forming a core mechanism in the occurrence and development of chronic pain.

[0083] Brainstem tissue from Example 6 was collected, tissue proteins were extracted, and the phosphorylation levels of CaMKIIα and CREB were detected using Western blotting to study the effect of peptide SM-8 on Ca. 2+The effects of the / CaMKⅡα / CREB signaling pathway were investigated. The specific procedure was as follows: Mouse brainstem tissue was collected and washed with pre-chilled PBS. RIPA lysis buffer containing protease inhibitors was added, and lysis was performed on ice for 30 minutes. The tissue was ground in a mortar and pestle, centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant (total protein solution) was carefully aspirated to prepare an appropriate concentration of SDS-PAGE gel. The gel was loaded and subjected to electrophoresis. After electrophoresis, the protein was transferred to a PVDF membrane. The membrane was blocked with TBST solution in 5% skim milk and shaken at room temperature for 1-2 hours to block non-specific binding. The membrane was incubated with the corresponding antibody overnight at 4°C. After incubation, the membrane was thoroughly washed with TBST (4-6 times, 5-10 minutes each time) to completely remove unbound antibodies. The membrane was incubated with an appropriately diluted HRP-labeled streptavidin (HRP) solution at room temperature for 1 hour. The membrane was then washed again with TBST (4-6 times, 5-10 minutes each time). Mix equal volumes of ECL chemiluminescent substrate solutions A and B according to the instructions. Add the mixture to the membrane, and detect and acquire the signal using a chemiluminescence imaging system, with GAPDH as the internal control protein.

[0084] like Figure 11 As shown, both the positive control drug sumatriptan and the peptide SM-8 significantly inhibited the phosphorylation levels of CaMKIIα and CREB, and the inhibitory effect of peptide SM-8 was dose-dependent. p-CaMKIIα is a core molecule for inducing and maintaining central sensitization. Inhibiting it is equivalent to dismantling the "engine" that maintains central sensitization. CaMKIIα is an important upstream kinase that activates CREB. Inhibiting CaMKIIα can directly reduce CREB phosphorylation, thereby preventing the transmission of pain signals to the gene transcription level. Inhibiting p-CREB can significantly downregulate the expression of a series of pro-pain genes, such as c-Fos: a marker of sustained neuronal activation; tachykinins and inflammatory factors: such as substance P, TNF-α, IL-1β, etc., whose reduction helps to alleviate neuroinflammation and pain signal transmission.

[0085] Example 11, docking of the peptide SM-8 analog with the Rack1 protein molecule: As described in Example 9 above, the interaction between peptide SM-8 and Rack1 protein was preliminarily confirmed using Far-Western Blotting technology. In this example, the molecular docking function of Molecular Operating Environment (MOE) software will be used to obtain the key amino acid sites of peptide SM-8, and these amino acid sites will be mutated to explore the structure-activity relationship of peptide SM-8.

[0086] The structures of peptide SM-8 and protein Rack1 (UniProt ID: P63244) were imported into the molecular manipulation environment software (MOE2019.0102). The structures were preprocessed using the QuickPrep module, including hydrogen atom addition, solvation / desolvation (selected according to the specific context), and energy optimization. The Protein-Contacts module was then selected to obtain intermolecular interaction data and virtual docking scores.

[0087] Table 7 shows the virtual score results of peptide SM-8 and Rack1 protein. Specifically, the 11th Arg position of peptide SM-8 interacts ionicly with the 220th Asp position of Rack1 protein, with a bond energy of -14.69; the 11th Arg position of peptide SM-8 interacts with the 223rd Glu position of Rack1 protein via both ionic and hydrogen bonding, with a bond energy of -12.74; the 10th Leu position of peptide SM-8 interacts with the 264th Lys position of Rack1 protein via hydrogen bonding, with a bond energy of -9.40; the 3rd His position of peptide SM-8 interacts with the 201st Ser position of Rack1 protein via π-π conjugation, with a bond energy of -1.60; the 2nd Arg position of peptide SM-8 interacts with the 244th Asn position of Rack1 protein via hydrogen bonding, with a bond energy of -1.00; and the 3rd His position of peptide SM-8 interacts with the 203rd Asp position of Rack1 protein via hydrogen bonding, with a bond energy of -0.70. Figure 12 The image shows the interaction (two-dimensional structure) analysis of the peptide SM-8 and Rack1 protein, which further confirms their binding sites.

[0088] Table 7. Virtual scoring table for docking experiments of peptide SM-8 and Rack1 protein molecules. .

[0089] Note: The lower the virtual score value of the docking scoring function (i.e., the larger the negative value), the stronger the interaction between the peptide and the Rack1 protein, and the better the binding mode.

[0090] Twenty natural amino acid point mutations were performed on the distribution of key amino acid sites of peptide SM-8, namely Arg at position 2, His at position 3, Leu at position 10, and Arg at position 11, as determined by the preliminary molecular docking. The mutated peptide SM-8 analogue and protein Rack1 were imported into MOE software. The structure was preprocessed using the QuickPrep module according to the above operation, and then the Protein-Design-Residue Scan module was selected for virtual screening.

[0091] As shown in Table 8, after the Arg at positions 2 and 11 of the key amino acid site of peptide SM-8 were mutated to other natural amino acids, the relative binding affinity of the mutant peptide analog to Rack1 protein decreased, indicating that Arg at positions 2 and 11 is highly conserved and cannot be mutated to other amino acids. After the His at position 3 of the key amino acid site of peptide SM-8 was mutated to Arg, Lys, Phe, or Tyr, the relative binding affinity of the mutant peptide analog to Rack1 protein increased, indicating that in addition to His, position 3 of SM-8 can also be mutated to Arg, Lys, Phe, or Tyr. Structural analysis of these amino acids suggests that a positively charged amino acid at position 3 of SM-8 or a benzene ring structure in the side chain is more conducive to binding to Rack1 protein. After the Leu mutation at position 10 of the key amino acid site of peptide SM-8 was changed to Arg, His, Met, Trp, or Tyr, the relative binding affinity of the mutant peptide analog to Rack1 protein was increased, indicating that in addition to Leu, position 10 of SM-8 can also be mutated to Arg, His, Met, Trp, or Tyr.

[0092] Table 8. Virtual screening table of SM-8 Arg point mutation analogues and Rack1 protein molecules. .

[0093] Note: The affinity of peptide SM-8 to Rack1 protein is represented by a baseline value of 0. For other analogs, a value less than 0 indicates a stronger affinity for Rack1 protein, while a value greater than 0 indicates a weaker affinity.

Claims

1. An animal toxin polypeptide, characterized in that, The I-structure encompassing the N-terminus to the C-terminus; Arg-Arg-X1-Ala-Glu-Thr-Ile-Lys-Val-X2-Arg-Lys-Lys-Glu-Arg Formula I In Formula I, the "-" between amino acid residues represents a peptide bond, the C-terminus of the polypeptide is a free carboxyl group or amide, and X1 and X2 are any amino acids.

2. The polypeptide according to claim 1, characterized in that, X1 is selected from any one of His, Arg, Lys, Phe, and Tyr; X2 is selected from any one of Leu, Arg, His, Met, Trp, and Tyr.

3. The polypeptide according to claim 1 or 2, characterized in that, It is selected from any one of the following amino acid sequences: SEQ ID NO: 1-SEQ ID NO:

10.

4. A nucleic acid molecule, characterized in that, It encodes the polypeptide described in any one of claims 1-3.

5. An expression carrier, characterized in that, It comprises the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, It comprises the expression vector as described in claim 5.

7. A pharmaceutical composition, characterized in that, It comprises the polypeptide of any one of claims 1-3 and its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that, The dosage form of the pharmaceutical composition is any one of the following: injection, lyophilized powder for injection, gel, cream, ointment, patch, spray, and suppository.

9. The use of the polypeptide according to any one of claims 1-3 or the pharmaceutical composition according to claim 7 or 8 in the preparation of a pain-relieving medicament.

10. The application according to claim 9, characterized in that, The pain includes any one or more of the following: migraine, trigeminal neuralgia, postoperative pain, bone and joint pain, nutritional deficiency pain, inflammatory pain, hypothyroidism pain, Guillain-Barré syndrome pain, poisoning pain, drug-induced pain, and tumor-related pain.

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