A frog-derived neuropeptide and its modified peptide and application

Abstract

The present invention belongs to the field of medicinal chemistry, and specifically relates to a frog-derived neuropeptide and its modified peptide and application. The present invention provides a frog-derived neuropeptide Senegalin‑2, which is non-toxic to normal cells and exhibits a relaxing effect on rat bladder smooth muscle, indicating that it can be used to prepare drugs for relaxing bladder smooth muscle, and has application prospects in the preparation of drugs for treating overactive bladder. The present invention connects Senegalin‑2 with bradykinin to prepare a modified peptide Senegalin‑2BK, which enhances the structural stability and flexibility of the peptide, exhibits better cell safety, and low concentrations of Senegalin‑2BK significantly inhibit bradykinin, which can be used as bradykinin antagonists and is used to prepare drugs for treating diseases related to bradykinin overexpression. Senegalin‑2BK can contract the smooth muscle of the bladder and ileum, and can be used to prepare drugs for contracting the smooth muscle of the bladder or ileum.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry, and specifically relates to a frog-derived neuropeptide and a modified peptide thereof and application thereof. Background Art

[0002] As an important part of biodiversity, amphibians contain abundant bioactive peptides. These peptide molecules often have unique structures and biological functions, such as antibacterial and muscle tension regulation, providing valuable resources for drug design. Among them, frog neuropeptides, as defensive substances, are essential for the host's physiological activities such as defense against predators, inflammation and pain response. Unlike mammalian bradykinins that maintain host homeostasis through the kallikrein-kinin system (KKS), frog neuropeptides are produced by protease cleavage of precursor proteins in the frog skin granular glands.

[0003] Since the 1970s, neuropeptides have been found in different species of frogs. Frog neuropeptides are divided into bradykinin (BK), bradykinin-related peptide (BRP) and other frog neuropeptides. The sequence of frog BK is consistent with that of mammalian BK, namely RPPGFSPFR. Frog BRP mostly contains the BK sequence, usually with an extension sequence at the N-terminus or C-terminus. In addition, the BK sequence contained in BRP is usually replaced by different amino acids (such as valine and threonine), resulting in the diversification of the structure and function of BRP. There are also some natural frog neuropeptides that do not belong to the BK or BRP family. These peptides are usually composed of 8-20 amino acids and have only low similarity with known peptides in the NCBI database. Among them, the Senegal frog (Kassina senegalensis) is one of the sources of these independent neuropeptides. Neuropeptides that have been found in this frog include Senegalin, Kasstasin, and WL-8. These peptides induce changes in the tone of rat bladder or arterial smooth muscle. Some of these peptides also had inhibitory effects on microorganisms, indicating that they are neuropeptides with multiple pharmacological activities.

[0004] In recent years, scientists have discovered very few natural frog-derived neuropeptides with potential bradykinin antagonism. They belong to BRP. However, they only have antagonistic effects at high concentrations, and their physicochemical properties and toxicity remain unclear. As an inflammatory mediator, BK easily activates nociceptors, leading to symptoms such as pain and hypersensitivity. B2R, the main receptor that mediates bradykinin, is closely related to these symptoms as well as smooth muscle contraction and fluid balance. In recent years, B2R has been an ideal target for drug development, but only one drug, icaritibant, has been approved for clinical use. Therefore, designing low-toxic and highly effective bradykinin antagonists based on natural amphibian-derived peptides has gradually become a challenge in this field. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a frog-derived neuropeptide and a modified peptide and application thereof.

[0006] The present invention provides a frog-derived neuropeptide, the amino acid sequence of which is shown as SEQ ID NO.1.

[0007] The present invention provides a method for extracting the frog-derived neuropeptide, which comprises the following steps:

[0008] Step 1, collection of frog skin secretions;

[0009] Step 2, dissolving the frog skin secretion in an acid solution, centrifuging to obtain the supernatant, injecting it into a reverse phase high performance liquid chromatography system equipped with a C18 column, eluting with a mixed solution of acid, water and acetonitrile as an eluent, and collecting the obtained product.

[0010] Preferably, the acid solution is a trifluoroacetic acid solution with a volume content of 0.05%; the elution is a gradient elution, and the volume ratio of acid, water and acetonitrile in the eluent is 0.05:99.95:0 to 0.05:19.95:80.00; the acid is trifluoroacetic acid; and the collected component is a component with a retention time of 131 min.

[0011] Preferably, the flow rate of the eluent is 1 ml / min.

[0012] The present invention provides use of the frog-derived neuropeptide in preparing a drug for relaxing bladder smooth muscle; preferably, the drug for relaxing bladder smooth muscle is a drug for treating overactive bladder.

[0013] The present invention also provides a frog-derived modified neuropeptide, which is obtained by coupling the frog-derived neuropeptide and bradykinin via a linker;

[0014] Preferably, the amino acid sequence of the frog-derived modified neuropeptide is shown as SEQ ID NO.3.

[0015] The invention provides a method for preparing the frog-derived modified neuropeptide, and the method is an Fmoc solid-phase peptide synthesis method.

[0016] The present invention also provides the use of the frog-derived modified neuropeptide in the preparation of a bradykinin antagonist, wherein the bradykinin antagonist is preferably a drug for treating diseases related to bradykinin overexpression, and the drug is preferably a drug for treating cardiovascular diseases, inflammatory diseases, pain, allergies and cancer.

[0017] The present invention also provides the use of the frog-derived modified neuropeptide in preparing a drug for contracting bladder or ileum smooth muscle. The drug for contracting bladder or ileum smooth muscle is preferably a drug for treating urinary retention or constipation.

[0018] The present invention also provides a pharmaceutical composition, which is a preparation obtained by taking the frog-derived neuropeptide or the frog-derived modified neuropeptide as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients; the preparation is preferably an oral preparation or an injection preparation.

[0019] Beneficial effects: The present invention provides a frog-derived neuropeptide Senegalin-2, which is non-toxic to normal cells and exhibits a relaxant effect on rat bladder smooth muscle, indicating that it can be used to prepare a drug for relaxing bladder smooth muscle. It is well known to those skilled in the art that drugs for relaxing bladder smooth muscle can be used to treat overactive bladder. Therefore, the frog-derived neuropeptide Senegalin-2 of the present invention has broad application prospects in the preparation of drugs for treating overactive bladder (OAB).

[0020] The present invention further connects Senegalin-2 and bradykinin through GGG to prepare a modified peptide Senegalin-2BK, which enhances the structural stability and flexibility of the peptide, shows better cell safety, and low concentrations of Senegalin-2BK significantly inhibit bradykinin, and can be used to prepare BK antagonists, and applied to the preparation of drugs for treating diseases related to BK overexpression. It is well known to those skilled in the art that BK antagonists have shown broad therapeutic prospects in many fields such as cardiovascular diseases, inflammatory diseases, pain management, anti-allergic treatment, and cancer treatment.

[0021] The present invention also found that Senegalin-2BK can contract the smooth muscle of rat bladder and ileum, and can be used to prepare drugs for contracting bladder or ileum smooth muscle. It is well known to those skilled in the art that drugs for contracting bladder or ileum smooth muscle can be used to treat diseases such as urinary retention and constipation.

[0022] The frog-derived neuropeptide and its modification of the present invention help to deeply understand the structure-function relationship of the fusion peptide, provide a basis for further optimization design, and provide a new strategy for treating smooth muscle-related diseases.

[0023] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0024] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The reverse phase high performance liquid chromatography (RP-HPLC) chromatogram of the crude extract of Kassina senegalensis frog skin secretion, the peak containing Senegalin-2 is marked with a red arrow.

[0026] Figure 2 is the mass spectrum of extracted Senegalin-2.

[0027] Figure 3 For tandem mass spectrometry (MS / MS) analysis of extracted Senegalin-2, the b-ions and y-ions of the amino acids are labeled in red and blue, respectively.

[0028] Figure 4 RP-HPLC chromatogram of in vitro synthesized Senegalin-2.

[0029] Figure 5 It is the RP-HPLC chromatogram of Senegalin-2BK.

[0030] Figure 6 This is the mass spectrum of Senegalin-2 synthesized in vitro.

[0031] Figure 7 This is the mass spectrum of Senegalin-2BK.

[0032] Figure 8 Figure 1 shows the helical wheel diagram of Senegalin-2 and the color coding according to the nature of the amino acid residues: positively charged (blue), hydrophobic (yellow), uncharged polar (purple), aromatic (green) and other nonpolar / glycine (grey). The arrow indicates the direction of the hydrophobic moment.

[0033] Fig. 9 Figure 1 shows the helical wheel diagram of Senegalin-2BK and the color coding according to the nature of the amino acid residues: positively charged (blue), hydrophobic (yellow), uncharged polar (purple), aromatic (green), and other nonpolar / glycine (grey). The arrow indicates the direction of the hydrophobic moment.

[0034] Fig.10 Circular dichroism spectra of Senegalin-2 and Senegalin-2BK measured in ammonium acetate solution.

[0035] Fig.11 Circular dichroism spectra of Senegalin-2 and Senegalin-2BK measured in trifluoroacetic acid / ammonium acetate solution, respectively.

[0036] Fig.12 The hemolytic activity of Senegalin-2 and Senegalin-2BK on horse erythrocytes.

[0037] Fig.13 The effects of Senegalin-2 and Senegalin-2BK on the proliferation of normal cell line HMEC-1.

[0038] Fig.14 Figure 2 is the dose-response curve of Senegalin-2 and Senegalin-2BK on the changes of rat bladder smooth muscle tone. Error bars represent the standard error of the mean (SEM) of the data obtained from three independent experiments.

[0039] Fig.15 This is the dose-response curve of Senegalin-2 and Senegalin-2BK on the changes in rat ileal smooth muscle tension.

[0040] Fig.16 This is the dose-response curve of Senegalin-2 and Senegalin-2BK on the changes in rat uterine smooth muscle tension.

[0041] Fig.17 This is the dose-response curve of Senegalin-2 and Senegalin-2BK on the changes of rat arterial smooth muscle tension.

[0042] Fig.18 The curves of the changes in muscle strength of rat bladder smooth muscle by Senegalin-2 with or without the addition of potassium channel blockers in advance.

[0043] Fig.19 The curves of muscle strength changes of rat bladder smooth muscle by Senegalin-2BK with or without the addition of potassium channel blocker in advance.

[0044] Fig. 20 Figure 2 is a dose-response curve of bradykinin-induced bladder smooth muscle contraction in the presence or absence of pre-addition of Senegalin-2BK. DETAILED DESCRIPTION

[0045] In the following examples and experimental examples, reagents and materials not particularly described are all commercially available.

[0046] Example 1 A frog-derived neuropeptide and its extraction and synthesis method

[0047] The frog-derived neuropeptide Senegalin-2 of this example is extracted or synthesized by the following method:

[0048] (1) Collection of frog skin secretions

[0049] Transcutaneous electrical stimulation (5 V direct current; 140 ms pulse duration; 100 Hz) was applied to the dorsal skin of adult Senegal frogs ( Kassina Senegalensis , n = 5, mixed sexes) every 20 seconds for 2 minutes under the Animals (Scientific Procedures) Act 1986, Northern Ireland, Department of Health, Social Services and Public Safety, Project License PPL 2694. The dorsal skin of frogs was then rinsed with deionized water to collect secretions, which were quickly frozen in liquid nitrogen. The above procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Queen's University Belfast.

[0050] (2) Extraction and purification of frog neuropeptide Senegalin-2

[0051] 5 mg of freeze-dried frog skin secretion was dissolved in 0.05% (v / v) trifluoroacetic acid (TFA) and centrifuged. The supernatant obtained after centrifugation was injected into a reverse phase high performance liquid chromatography (RP-HPLC) system (Cambridge, UK) equipped with a C18 column, and the components were automatically collected once per minute. The separation and purification conditions were C18 chromatographic column, column temperature: 25°C, using 0.05 / 99.95 (v / v) TFA / water to 0.05 / 19.95 / 80.00 (v / v / v) TFA / water / acetonitrile to form a gradient elution in a total of 240 minutes, with a flow rate of 1 ml / min, and collecting the component with a retention time of 131 min. The amino acid sequence of Senegalin-2 is shown in Table 1.

[0052] Table 1 Amino acid sequences of bradykinin, Senegalin-2 and fusion peptide Senegalin-2BK

[0053]

[0054] (3) In vitro synthesis of Senegalin-2

[0055] According to the amino acid sequence of Senegalin-2, Senegalin-2 was synthesized using the conventional Fmoc solid phase peptide synthesis method in the art. The specific operation is as follows:

[0056] The Fmoc solid phase peptide synthesis (SPPS) was performed using a solid phase peptide synthesis reactor (Protein Technologies, USA).

[0057] Synthesis steps:

[0058] 1. The amino acids required for the peptide chain are mixed with HBTU coupling reagent to catalyze the coupling reaction and C-terminal amidation. Amino acid: HBTU: Rink amide MBHA resin required for synthesis = 2.5:2.5:1 (molar ratio). For example: to synthesize 0.15mmol of peptide, you need to weigh 0.375mmol of amino acid, 0.375mmol of HBTU, and 0.15mmol of resin.

[0059] 2. Place the Rink amide MBHA resin into the peptide synthesis reactor and soak it in DCM for 2 hours.

[0060] 3. Wash the resin with DMF and then drain it. Repeat this process four times until the resin is drained.

[0061] 4. Weigh the protected amino acid Fmoc-Asp(otbu)-OH (the first amino acid at the C-terminus) + DCM + DIEA and add them into the reactor, then place the reactor in a shaker at 30°C for reaction for 2 hours.

[0062] 5. Block with methanol solution for half an hour, then wash four times with DMF and drain.

[0063] 6. Add 20% piperidine solution to the reactor to remove the Fmoc protecting group. After the protection is removed, wash with DMF four times and then drain.

[0064] 7. Take a small amount of resin and test it using the ninhydrin method. If the resin has color, it means that the deprotection is successful.

[0065] 8. Weigh Fmoc-Gly-OH (the second amino acid at the C-terminus) + HOBT + DIC and add them to the reactor, then place the reactor in a shaker at 30° C. to react for 1 hour.

[0066] 9. Take a small amount of resin for testing, and use the ninhydrin method to test. If the resin has color, it means that the condensation is incomplete, and continue the reaction. If the resin is colorless, it means that the reaction is complete; after the reaction is complete, wash the resin four times with DMF and then drain it.

[0067] 10. Add 20% piperidine to the reactor to remove the Fmoc protecting group. After the removal of protection, wash with DMF four times and then drain.

[0068] 11. Take a small amount of resin and test it using the ninhydrin method. If the resin has color, it means that the deprotection is successful.

[0069] 12. Connect the remaining amino acids in sequence according to steps 8-11.

[0070] 13. Use a cleavage reagent (cleavage cocktail (trifluoroacetic acid / double distilled water / triisopropylsilane / ethanedithiol, volume ratio of 94 / 2 / 2 / 2)) to remove all peptide protecting groups and cleave it from the resin to obtain a crude peptide.

[0071] Purification steps:

[0072] The crude peptide was purified by RP-HPLC system. The lyophilized peptide was dissolved in buffer A (TFA / water, 0.5 / 99.95, v / v) and buffer B (TFA / water / acetonitrile, 0.5 / 19.95 / 80, v / v) at 4 mg / mL. The sample solution was vortexed and centrifuged at 5,000×g for 10 minutes. After that, the peptide was purified using CecilAdept CE4200 HPLC system (Cecil, Cambridge, UK) and RP-HPLC Jupiter C5 analytical column (250mm*4.6mm, Phenomenex, UK). Before injection, the chromatographic column was washed with buffer B and then balanced with appropriate ratios of buffer A and B. The flow rate in the liquid chromatography software was 4 mL / min, the running time was 80 min, and the detection wavelength was 214 nm. 1 mL of the sample supernatant was pumped onto the column and the peptides were eluted with a linear gradient from 100% buffer A to 100% buffer B over 80 min.

[0073] Example 2 A modified peptide Senegalin-2BK and its preparation method

[0074] The modified peptide Senegalin-2BK of this example was prepared by the following method:

[0075] The synthesis method described in step (3) of Example 1 was followed, except that the amino acid sequence was FLPFLIPVISSLISSLGGGRPPGFSPFR-NH2, as shown in Table 1.

[0076] The technical solution of the present invention is further described below through experiments. The samples tested in the following experimental examples are all prepared by the method of Example 1 or Example 2.

[0077] Experimental Example 1 Identification of frog-derived neuropeptide Senegalin-2

[0078] 1. Experimental Methods

[0079] (1) Collection of frog skin secretions

[0080] Follow the experimental steps described in step (1) of Example 1.

[0081] (2) Construction of a cDNA library from Senegal frog skin and “Shotgun” molecular cloning of the natural peptide Senegalin-2

[0082] First, polyadenylated mRNA was isolated from freeze-dried skin secretions of Rana senegalensis: mRNA DIRECT TM mRNA was isolated from freeze-dried skin secretions using the Dynal Biotech kit (UK). 5 mg of freeze-dried frog skin secretions and 1 mL of lysis / binding buffer were mixed in a 1.5 mL tube. The mixture was vortexed for 1 minute and placed on ice for 1-2 minutes. The above steps were repeated for 20 minutes. Then 250 μl of the completely resuspended Oligo(dT)25 was transferred to a 1.5 mL tube without RNase, placed on a magnetic stand for 30 seconds, and the supernatant was removed. The 1.5 mL tube was removed from the magnetic stand, and 250 μl of lysis / binding buffer was immediately added slowly to the tube wall for washing. The washed tube was then centrifuged at 18,000 × g for 5 minutes in an Eppendorf Centrifuge 5424 (Eppendorf, Germany). The centrifuged tube was placed on a magnetic stand until the solution was no longer noticeably viscous, at which point the supernatant was discarded. 500 μL of wash buffer A was slowly added at room temperature, and the wash was repeated three times. Between each wash, the tube was placed on a magnetic stand to separate the beads, and the wash buffer A was completely removed. After this, 500 μl of wash buffer B was added in the same way, and the remaining wash buffer was washed twice to remove it. After all the buffer was removed, 18 μl of elution buffer (Tris-HCl) was added to the tube and incubated at 80°C. After two minutes, immediately remove the 1.5 mL tube and place it on the magnetic stand. Remove the supernatant containing mRNA, inject it into a new RNase-free tube, and place it on ice.

[0083] Then, using mRNA Direct TM The isolated mRNA was synthesized into cDNA using a kit (Dynal Biotech, UK). Next, a cDNA library was established using the rapid amplification PCR (RACE-PCR) technique. The complete peptide nucleic acid sequence was obtained using the SMART-RACE kit (Clontech, Palo Alto, California, USA). In the 3'-RACE reaction, a nested universal primer (NUP) and a sense primer (AC-S AGC AGC AAA AGA AGA AGA AGC CAT G) were used to obtain the target sequence. -T EasyVector System (Promega, UK) was used to purify and clone the RACE products. Then, the nucleotide sequence of Senegalin-2 was analyzed using an ABI 3100 automated capillary sequencer (Applied Biosystems, Foster City, California, USA). Finally, the inferred peptides were analyzed by bioinformatics using the online tool Basic Local Alignment Search Tool (BLAST) and the sequence alignment software Clustal Omega.

[0084] (3) Determination of the amino acid sequence of Senegalin-2

[0085] Senegalin-2 was obtained according to the experimental steps described in step (2) of Example 1. Subsequently, mass-to-charge ratio determination and MS / MS fragment sequencing were performed using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF MS) (Perseptive Biosystems, Massachusetts, USA) and an LCQ Fleet ion trap mass spectrometer (Thermo Fisher Scientific, California, USA). Based on these results, the molecular weight and sequence of Senegalin-2 were analyzed and identified in combination with Proteome Discoverer software.

[0086] 2. Experimental Results

[0087] (1) Biological information of neuropeptide Senegalin-2

[0088] The results are shown in Table 2: The cDNA encoding the precursor of Senegalin-2 was cloned from the cDNA library of Kassina senegalensis frog by "Shotgun", and the precursor nucleotide sequence encoding Senegalin-2 and its corresponding translated amino acid sequence were obtained. The open reading frame first presented a highly conserved signal peptide, with a spacer rich in acidic amino acids in the middle, and finally ended with a mature peptide sequence consisting of 16 amino acids. The N-terminus of Senegalin-2 is the cleavage-specific protein convertase processing site "-lysine-arginine-" (-KR-), and its C-terminus is amidated with glycine as a donor.

[0089] Table 2 Nucleotide sequence and translated amino acid sequence of Senegalin-2

[0090]

[0091] Note: Signal peptide, mature peptide and stop codon are marked with double underline, single underline and asterisk, respectively.

[0092] (2) Nucleotide sequence of neuropeptide Senegalin-2

[0093] The results are as follows Figure 1-3 As shown: At a detection wavelength of 214 nm, the retention time of Senegalin-2 in RP-HPLC is 131 minutes. In mass spectrometry detection, the b ions and y ions of the amino acids contained in Senegalin-2 are detected respectively. The sequence of Senegalin-2 is

[0094] Phe-Leu-Pro-Phe-Leu-Ile-Pro-Val-Ile-Ser-Ser-Leu-Ile-Ser-Ser-Leu-amide, i.e. FLPFLIPVISSLISSL-NH2.

[0095] The above results indicate that the present invention provides a novel frog-derived neuropeptide, whose amino acid sequence is FLPFLIPVISSLISSL-NH2.

[0096] Experimental Example 2 Verification of Synthesized Senegalin-2 and Modified Peptide Senegalin-2BK

[0097] 1. Experimental Methods

[0098] Senegalin-2 was obtained according to the experimental steps described in step (3) of Example 1, and Senegalin-2BK was obtained according to the preparation method described in Example 2. The two polypeptides were determined by RP-HPLC and mass spectrometer (Waters, USA), respectively.

[0099] The RP-HPLC determination method is: C18 column, column temperature: 25°C, using 0.05 / 99.95 (v / v) TFA / water to 0.05 / 19.95 / 80.00 (v / v / v) TFA / water / acetonitrile to form a gradient elution peptide in a total of 240 minutes, with a flow rate of 1 ml / min.

[0100] The mass spectrometer is measured as follows: The internal mass calibration of the instrument using known standards can determine the measurement accuracy to ±0.1%. 2 μL of sample is added to the sample plate and dried. Then 1 μL of 40 mM α-cyano-4-hydroxycinnamic acid (CHCA) solution is added to the sample and the sample is allowed to air dry. The sample is then loaded into the mass spectrometer. The ions that absorb the instrument laser move in the electric field, causing the detector to receive a signal. The detector displays the mass-to-charge ratio (m / z) of different ion fragments based on the arrival time of the ions.

[0101] 2. Experimental Results

[0102] RP-HPLC results are as follows Figure 4 , 5 As shown: At a detection wavelength of 214 nm, the retention times of Senegalin-2 and Senegalin-2BK in RP-HPLC were 17.042 minutes and 16.446 minutes, respectively, with a purity of over 95%. Figure 6 , 7 As shown: In mass spectrometry detection, the [M+2H]2+ mass-to-charge ratio (m / z) ion peak is +2-valent Senegalin-2 with 2 hydrogen ions. And the [M+3H]3+ m / z ion peak is +3-valent Senegalin-2BK with 3 hydrogen ions. After measurement, the molecular weights of Senegalin-2 and Senegalin-2BK are 1745 and 2958, respectively, which is consistent with the sequence calculation results.

[0103] The above results show that the present invention successfully prepared Senegalin-2 and Senegalin-2BK using the Fmoc solid phase peptide synthesis method.

[0104] Experimental Example 3 Structural Analysis of Senegalin-2 and Senegalin-2BK

[0105] 1. Experimental Methods

[0106] The physicochemical properties of the peptides were predicted by Heliquest (online access address: https: / / heliquest.ipmc.cnrs.fr / cgi-bin / ComputParams.py, access date: May 14, 2024). The peptide samples were dissolved in 10mM ammonium acetate buffer and 50 / 50 (v / v) trifluoroethanol (TFE) / 10mM ammonium acetate buffer at a concentration of 50μM. The analysis was performed using a JASCO J815 polarimeter (JASCO Inc., USA) and a quartz cuvette with an optical path of 1mm. Each sample was analyzed at 250-190nm. The scan speed was set to 100nm / min, the bandwidth was 1nm, the data spacing was 0.5nm, and the scan was repeated three times to obtain the results. Finally, the helical ratios of these peptides were determined by BestSel (access date: May 25, 2024, https: / / bestsel.elte.hu / index.php). Draw a table to compare the changes in the primary and secondary structures of the peptide before and after modification.

[0107] Circular dichroism measurement: The peptide solution was dissolved in 10mM NH4Ac solution (pH 7.4, simulating water environment) and 50% TFE / 10mM NH4Ac solution (pH 7.4, simulating microbial membrane) at a concentration of 50μM for determination of the secondary structure of the peptide. The α-helix ratio (%) of the peptide was calculated based on its circular dichroism spectrum using the online software Bestsel.

[0108] 2. Experimental Results

[0109] The results are as follows Figure 8 , 9 As shown in Table 3: Compared with Senegalin-2, Senegalin-2BK exhibits lower hydrophobicity, increased positive charge, an additional hydrophobic surface (VPFGLLGLIF), and a reduced helical ratio. Fig.10 , 11 As shown: Both peptides exhibited α-helical conformations at peaks at 192, 208 and 222 nm in the simulated microbial membrane, but appeared disordered in the simulated water environment (peak at 207 nm).

[0110] Table 3 Physicochemical properties of peptides

[0111]

[0112] The above results show that Senegalin-2 has a certain hydrophobicity and α-helical structure. Senegalin-2 is connected to bradykinin through a glycine-rich "GGG" linker to prepare the modified peptide Senegalin-2BK, which has reduced hydrophobicity, increased positive charge, and reduced α-helical ratio, which enhances the stability of the structure and has a certain local flexibility. This flexibility helps the fusion peptide better adapt to the complex environment in the body, such as passing through the cell membrane and binding to receptors.

[0113] Experimental Example 4 Safety Determination of Senegalin-2 and Senegalin-2BK

[0114] 1. Experimental Methods

[0115] (1) Red blood cell lysis test

[0116] Horse blood erythrocytes were extracted from fresh horse blood, washed repeatedly with PBS by centrifugation at 1000×g until the supernatant was clear, and then resuspended to prepare a 4% (v / v) erythrocyte suspension. The peptide was diluted with PBS to form a solution, and serial dilutions were performed, with the concentrations of the peptides set to 1, 4, 8, 16, 32, 64, 128, and 256 μM, respectively. The erythrocyte suspension was mixed with Senegalin-2, Senegalin-2BK solution, or control solution, respectively, incubated at 37°C for two hours, and then centrifuged at 900×g, the supernatant was taken, and the absorbance at 550 nm was measured using a microspectrophotometer.

[0117] Half hemolysis value (HC 50 )(Halfmaximal hemolytic concentration) calculation:

[0118] Data entry: Open Prism software and create a new data table and graph. Enter the experimental data in the data table with the concentration values ​​on the X-axis and the corresponding hemolysis percentage on the Y-axis.

[0119] Data transformation: Convert the concentration values ​​to logarithmic form to better fit the data. This is done in Prism using the "Transform concentrations(X)" option under the "Analyze" menu.

[0120] Select the analysis type: In the top menu bar of Prism, select "Analyze" > "NonlinearRegression" > "Curve Fit". In the pop-up curve fitting interface, select the Logistic model of concentration-effect relationship. Click the "Fit Curve" button to perform curve fitting. Prism automatically calculates the parameters of the fitting curve and displays them in the chart.

[0121] View the results table to find HC 50 value, which is usually marked as "EC 50 ”, represents the concentration that achieves half-maximal effect.

[0122] (2) Cytotoxicity test

[0123] Human microvascular endothelial cell line HMEC-1 (ATCC-CRL-3243) was cultured in MCDB 131 medium and seeded in 96-well plates at a density of 8000 cells per well and incubated overnight. Subsequently, peptides (Senegalin-2 or Senegalin-2BK) were added to the plate at final concentrations of 1, 5, 10 and 100 μM. The positive control was 0.1% (w / v) Triton-X-100, the negative control was PBS solution, and the growth control was an equal amount of culture medium. After incubation for 24 hours, 10 μL of MTT solution (5 mg / mL) was added to each well and incubated in the dark for 2 hours. The solution was then removed and 100 μL of DMSO solution was added. After shaking the plate for 10 minutes, the optical density (OD) at λ = 570 nm was measured using a Synergy HT microspectrophotometer. To evaluate the effect of peptides on cell proliferation, cell viability (%) was calculated as follows: cell viability (%) = [A(sample) - A(positive control)] / [A(growth control) - A(positive control)] × 100%. A represents the plate absorbance OD value. Error bars represent the mean ± standard error (SEM) of three replicates, ns indicates no significant difference, **** indicates p ≤ 0.0001.

[0124] 2. Experimental Results

[0125] Hemolysis test results Fig.12 As shown in Table 4: For Senegalin-2, its HC 50 23.19μM. Senegalin-2BK HC 50 It is 85.09μM.

[0126] Cytotoxicity test results Fig.13 Shown: Both peptides showed negligible cytotoxicity against the HMEC-1 cell line in the MTT assay.

[0127] Table 4 Peptide HC against horse erythrocytes 50 value

[0128]

[0129] The above results show that Senegalin-2 and Senegalin-2BK have no cytotoxicity to normal vascular endothelial cells. Senegalin-2 can cause a high proportion of hemolysis at high concentrations, but combined with the data of Experimental Example 5, it can be seen that Senegalin-2 did not induce red blood cell lysis when used in smooth muscle tension experiments at therapeutic concentrations; compared with Senegalin-2, Senegalin-2BK exhibits lower hemolytic activity and is safer than Senegalin-2, suitable for oral or intravenous injection.

[0130] Experimental Example 5 Determination of the pharmacological activity of Senegalin-2 and Senegalin-2BK on smooth muscle

[0131] 1. Experimental Methods

[0132] (1) Preparation of Krebs solution

[0133] Krebs solution was used to simulate the ionic environment in rats. First, MgCl2 (1.1 mmol), KCl (4.7 mmol), NaHCO3 (25 mmol), NaH2PO4 (1.15 mmol), NaCl (118 mmol), CaCl2 (2.5 mmol) and glucose (5.6 mmol) were mixed with double distilled water in a flask by rotation. Before use, a mixed gas of 95% O2 and 5% CO2 was passed into the solution for 10 minutes and preheated to 37°C in a water bath.

[0134] (2) Preparation of peptide solution

[0135] Weigh pure peptide Senegalin-2 or Senegalin-2BK and dissolve it in Krebs solution to make a concentration of 10 -3 Then, the stock solution was diluted 10 times with Krebs solution to obtain 10 -4 , 10 -5 , 10 -6 , 10 -7 When using, add 20 μL of peptide solution to a total volume of 2 mL of rat organ water bath to make the final peptide dilution 100 times.

[0136] (3) Preparation of isolated rat tissue

[0137] Adult Wistar rats of 12 weeks old and weighing about 200-250g were selected for the experiment. According to the British animal research guidelines and with permission, the rats were killed by carbon dioxide asphyxiation. The rats were placed supine on the operating table and the abdomen was cut along the midline. Then the bladder, ileum and uterus were carefully removed with forceps. The tail of the rat was carefully peeled off, other tissues were removed, and a certain length of the tail artery was removed with surgical scissors under a dissecting microscope. After the organs were removed, they were immediately immersed in ice-cold Krebs solution with 95% O2 and 5% CO2.

[0138] (4) Organizational processing

[0139] For the bladder, the bladder was cut from the bladder neck to the top of the bladder to remove the inner urethral epithelial lining. Under a dissecting microscope, the bladder was cut into parallel muscle bundles (2 mm wide and 10 mm long). The two ends of the smooth muscle strip were fixed with hooks of 0.2 mm in diameter and mounted longitudinally in an organ bath, applying a basic tension of 0.5 g.

[0140] For the proximal tail artery of rats, a 2 mm wide vascular ring was cut. Then, both ends of the tail artery were connected to a triangular hook and gently rubbed to remove the endothelial lining with the hook. The proximal tail artery with the hook was immersed in an organ bath, and a basal tension of 0.5 g was applied by an isotonic contraction device.

[0141] For the ileum, a 1 cm long ileum segment was first cut and its lumen was opened. Then, both ends of the cut section were tied with a triangular hook with a diameter of 0.2 mm and immersed longitudinally in an organ bath. Finally, a basic tension of 0.5 g was applied by an isotonic contraction device.

[0142] For the uterus, the uterine horns were first cut and placed in ice-cold Krebs solution filled with 95% O2 and 5% CO2. The uterine horns were further cut in half and each half was placed in an organ bath. The uterine horn smooth muscle strips were perfused with Krebs solution at 37°C for 10 minutes and then gradually tensioned to 0.5 g.

[0143] (5) Installation of isotonic contraction recording equipment and determination of the pharmacological activity of peptides on smooth muscle

[0144] The isotonic contraction equipment consists of an organ bath, a peristaltic pump, a personal control computer, and a tension change detection system. The prepared organ is placed in a 2 mL organ bath and perfused with 37 ° C Krebs solution. Under a basic tension of 0.5 g to 1 g, the organ is equilibrated with Krebs solution for 10-15 minutes, and then replaced with an organ water bath formed by continuous aeration of 95% O2 and 5% CO2 at a rate of 2 mL / min. Then, 20 μL of peptide solution is added to the organ bath containing the bladder, ileum, or uterus to observe the changes. For the rat tail artery, 20 μL of 10-3M phenylephrine is added after equilibration, and then the peptide solution is added 10 minutes later. At the same time, the organ begins to produce tension changes. The basic muscle tension is set to 0.5 g. Every 10 minutes, a concentration of peptide solution is added to the bladder muscle. Between different doses of peptide, the muscle strips need to be washed for 5 minutes and equilibrated for 5 minutes. The changes in smooth muscle tension are recorded and stored by a PowerLab system with a force sensor (AD Instruments Pty Ltd.).

[0145] (6) Determination of the effects of ion channels on peptides

[0146] In order to study the ion pathway through which the peptide affects smooth muscle tension, 20 μL of potassium channel blocker aminopyridine (concentration 10 -3 M). The subsequent steps are consistent with the experimental method 5 of this experimental example.

[0147] (7) Determination of the effects of frog-derived modified neuropeptides on bradykinin-induced smooth muscle responses

[0148] 10 minutes before bradykinin was added to the organ bath, 20 μL of a final concentration of 10 -7 The subsequent steps were consistent with the experimental method 5 of this experimental example.

[0149] Data were analyzed using t-tests and mean and standard error of responses were reported. Dose-response curves were constructed by a "best fit" algorithm. Changes in tension were plotted as contraction or relaxation of smooth muscle in muscle strips according to the concentration of each peptide.

[0150] 2. Experimental Results

[0151] (1) Pharmacological activities of Senegalin-2 and Senegalin-2BK on rat bladder, uterus, artery and ileum smooth muscle

[0152] The results are as follows Figure 14-17 As shown, Senegalin-2 is expressed as EC 50 The concentration of 17.94 nM relaxes the bladder smooth muscle, has a slight relaxing effect on the ileum, and has negligible effects on the arteries and uterus. The prior art (AminoAcids (2013) 44: 1347–1355, DOI: 10.1007 / s00726-013-1470-8) discloses another frog-derived neuropeptide, Senegalin, which induces contraction of rat bladder smooth muscle, which is opposite to the effect of Senegalin-2 of the present invention.

[0153] Unlike Senegalin-2, Senegalin-2BK induces smooth muscle contraction in the bladder and ileum of rats, EC 50 The values ​​were 2.84 μM and 56.64 nM, respectively. Both peptides had negligible effects on arterial and uterine smooth muscle.

[0154] (2) Study on the mechanism of action of Senegalin-2 and Senegalin-2BK on bladder smooth muscle

[0155] The results are as follows Fig.18 , 19 As shown: Aminopyridine inhibited the effects of Senegalin-2 and Senegalin-2BK on bladder smooth muscle.-5 At a concentration of 1.44 M, aminopyridine had a more significant inhibitory effect on Senegalin-2 and Senegalin-2BK. These data suggest that Senegalin-2 and Senegalin-2BK may relax bladder smooth muscle through potassium channels.

[0156] (3) Effect of Senegalin-2BK on the activity of bradykinin on rat bladder smooth muscle

[0157] The results are as follows Fig. 20 Shown: Effect of bradykinin on EC of rat bladder smooth muscle 50 2.38×10 -7 M, in advance with 10 -7 After incubation with 10 μM of Senegalin-2BK, the contraction of bladder smooth muscle induced by bradykinin was inhibited. The inhibitory effect of Senegalin-2BK increased with the increase of bradykinin concentration from 10 -9 M increased to 10 -6 M, but when bradykinin concentration exceeds 10 -6 M, the inhibitory effect weakened.

[0158] The above results show that Senegalin-2 exhibits a relaxant effect on rat bladder smooth muscle, suggesting its potential as a drug for the preparation of OAB treatment. Potassium ion channels play a key role in mediating the effects of Senegalin-2 on the bladder. Low concentrations of Senegalin-2BK significantly inhibit bradykinin and can be used as a low-toxic BK antagonist, showing promising therapeutic potential in maintaining blood pressure stability and reducing inflammation. Senegalin-2BK contracted the smooth muscle of the rat bladder and ileum, indicating its potential for the preparation of lead compounds for the treatment of urinary retention and constipation.

[0159] Through the above embodiments and experimental examples, it can be seen that the present invention provides a frog-derived neuropeptide Senegalin-2, which is non-toxic to normal cells and exhibits a relaxing effect on rat bladder smooth muscle, indicating that it can be used to prepare drugs for relaxing bladder smooth muscle, and has broad application prospects in the preparation of drugs for treating OAB. The present invention connects Senegalin-2 and bradykinin through GGG to prepare a modified peptide Senegalin-2BK, which enhances the structural stability and flexibility of the peptide, shows better cell safety, and low concentrations of Senegalin-2BK significantly inhibit bradykinin, which can be used as a BK antagonist, and is used to prepare drugs for treating diseases related to BK overexpression, showing good prospects in cardiovascular diseases, inflammatory diseases, pain management, anti-allergic treatment and cancer treatment. In addition, the present invention also found that Senegalin-2BK can contract the smooth muscle of rat bladder and ileum, can be used to prepare drugs for contracting bladder or ileum smooth muscle, and has application prospects in treating diseases such as urinary retention or constipation. This frog-derived neuropeptide and its modification help to deeply understand the structure-function relationship of fusion peptides, provide a basis for further optimization of design, and provide a new strategy for the treatment of smooth muscle-related diseases.

Claims

1. Use of a frog-derived neuropeptide having an amino acid sequence as shown in SEQ ID NO.1 in the preparation of a drug for relaxing bladder smooth muscle; the drug for relaxing bladder smooth muscle is a drug for treating overactive bladder.

2. A frog-derived modified neuropeptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

3.

3. The method for preparing the frog-derived modified neuropeptide according to claim 2, characterized in that: The preparation method is Fmoc solid phase peptide synthesis method.

4. Use of the frog-derived modified neuropeptide according to claim 2 in the preparation of a drug for contracting bladder or ileum smooth muscle, wherein the drug for contracting bladder or ileum smooth muscle is a drug for treating urinary retention or constipation.

5. A pharmaceutical composition, characterized in that The invention is a preparation obtained by taking the frog-derived modified neuropeptide as claimed in claim 2 as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

6. The pharmaceutical composition according to claim 5, characterized in that: The preparation is an oral preparation or an injection preparation.

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