A leech-derived polypeptide with both analgesic and antithrombotic functions, its precursor protein, and its application

Abstract

The present invention provides a leech-derived polypeptide having both analgesic and antithrombotic functions, a precursor protein thereof, and applications thereof, belonging to the technical field of functional peptides. The leech-derived polypeptide provided by the present invention has an amino acid sequence as shown in SEQ ID NO: 1. The leech-derived polypeptide provided by the present invention can inhibit the activity of proteases such as elastase, FXIIa, and kallikrein, and has good anticoagulant effects, while also having good analgesic and antithrombotic effects. The polypeptide has the characteristics of a simple structure, strong analgesic and antithrombotic activities, and can be used as an analgesic and antithrombotic drug.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional peptides, and in particular relates to a leech-derived polypeptide having both analgesic and anti-thrombotic functions, a precursor protein thereof, and applications thereof. Background Art

[0002] Elastase belongs to the chymotrypsin family of serine proteases. Recent studies have shown that elastase is involved in the development of various types of pain. Mechanical wounds that cause nociceptive pain contain large amounts of elastase, which may contribute to the development of nociceptive pain. Elastase induces inflammatory pain responses by activating protease-activated receptor 2 (PAR2) and transient receptor potential vanilloid 4 (TRPV4). Deficiency of the neutrophil elastase inhibitor gene, SerpinA3N, in mice enhances neuropathic pain sensitivity in a nerve injury model. Neutrophil elastase knockout or the use of elastase inhibitors significantly suppresses neuropathic pain responses in mice. Elastase participates in the development of dysfunctional pain responses by hydrolyzing elastin and activating PAR2. In summary, elastase is a key molecule in pain perception, and its specific inhibitors could be used to alleviate pain responses caused by various causes. Known elastase inhibitors include sivelestat sodium, which is currently approved for clinical use. Although it has promising neuroprotective effects and reduces inflammatory responses, it is a Western medicine and may have toxic side effects with long-term use.

[0003] Thrombosis is also a life-threatening disease. Cardiovascular diseases caused by thrombosis significantly impact quality of life. Methods for restoring vascular patency include manual and mechanical methods, specifically balloon catheterization and surgical embolectomy, which place significant physical and psychological burdens on patients. Antithrombotic therapies also include thrombolytics, antiplatelet drugs, and anticoagulants. Thrombolytics remove established clots, causing minimal damage to the body and making them more readily accepted by patients. Common antithrombotic drugs fall into two main categories: antiplatelet drugs, represented by aspirin, clopidogrel, and ticagrelor; and anticoagulants, represented by warfarin, dabigatran, and rivaroxaban. These drugs are collectively referred to as antithrombotic drugs. While these drugs offer rapid thrombolysis, they can also be quite irritating to the gastrointestinal tract and can easily lead to drug resistance after two to four weeks of combined use. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a leech-derived polypeptide having both analgesic and anti-thrombotic functions.

[0005] The present invention provides a leech-derived polypeptide having both analgesic and anti-thrombotic functions, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0006] The present invention provides a precursor protein of the leech-derived polypeptide, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0007] The present invention provides a gene encoding the precursor protein, the nucleotide sequence of which is shown in SEQ ID NO: 3.

[0008] The present invention provides an analgesic and / or anticoagulant drug comprising the leech-derived polypeptide and auxiliary materials.

[0009] The present invention provides an application of the leech-derived polypeptide in the preparation of anticoagulant and / or analgesic medicines or preparations.

[0010] Preferably, the anticoagulant comprises one or more of the following: anti-elastase activity, anti-coagulation factor FXIIa activity and anti-kallikrein activity.

[0011] Preferably, the analgesia includes inhibiting pain caused by inflammatory response.

[0012] Preferably, the inflammatory response is induced by elastase.

[0013] Preferably, the concentration of the leech-derived polypeptide in the drug is not less than 10 μM.

[0014] The present invention provides the use of the leech-derived polypeptide in the preparation of anti-thrombotic drugs.

[0015] The present invention provides a leech-derived polypeptide having both analgesic and antithrombotic functions, the amino acid sequence of which is shown in SEQ ID NO: 1. The precursor peptide of the leech-derived polypeptide contains 76 amino acids, and the mature peptide after removing the signal peptide contains 57 amino acids, with a molecular weight of 6061.6 and an isoelectric point of 4.07. The leech-derived polypeptide Poeciguamerin provided by the present invention can inhibit the activity of proteases such as elastase, FXIIa, and kallikrein, and has a good anticoagulant effect. At the same time, the leech-derived polypeptide can effectively inhibit the pain induced by elastase and has a good analgesic effect. In addition, experiments were conducted on a thrombosis mouse model, and it was found that the leech-derived polypeptide can effectively prevent and treat the formation of thrombi. It can be seen that the leech-derived polypeptide provided by the present invention has the characteristics of simple structure, strong analgesic and antithrombotic activity, and can be used in the preparation of analgesic and antithrombotic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1These are the results of the isolation and purification of the elastase inhibitor Poeciguamerin from Hirudo philadelphica, wherein A is the result of separation of Hirudo philadelphica saliva by Sephadex G-50 dextran gel filtration chromatography, B is the result of elastase activity determination of the fractions separated by Sephadex G-50 dextran gel filtration chromatography, C is the result of reversed-phase high-pressure liquid chromatography separation, and D is the result of screening the elastase activity inhibition of the elastase inhibitor Poeciguamerin;

[0017] Figure 2 The results of the anticoagulant activity test of the elastase inhibitor Poeciguamerin in the Philippine leech are shown in Figure 1, where A is elastase, B is coagulation factor FXIIa, C is kallikrein, D is coagulation factor FXa, E is trypsin, and F is thrombin.

[0018] Figure 3 The inhibition constants of Poeciguamerin from Hirudo philippinosus against elastase, coagulation factor FXIIa and kallikrein are determined, where A is elastase, B is coagulation factor FXIIa and C is kallikrein.

[0019] Figure 4 The results show that Poeciguamerin from Hirudo philippinosus inhibits pain induced by elastase.

[0020] Figure 5 Figure 3 is the inhibitory effect of Poeciguamerin on FeCl3-induced carotid artery thrombosis. A is the in vivo imaging result of the antithrombotic effect in the carotid artery thrombosis mouse model. B is the bar graph of antithrombotic activity. DETAILED DESCRIPTION

[0021] The present invention provides a leech-derived polypeptide having both analgesic and anti-thrombotic functions, the amino acid sequence of which is shown in SEQ ID NO: 1 (VDEKAEVTDDLCGDKTCSGAQVCQNDACVCSPVRCMI MCPNGFKLDENGCEYPCSCA).

[0022] In the present invention, the source of the leech-derived polypeptide is obtained by isolating it from the saliva of the leech or by artificial synthesis. The method for isolating the saliva of the leech preferably includes collecting the saliva of the leech, separating the saliva by size exclusion chromatography, screening the components with the activity of inhibiting elastase for re-separation by reversed-phase high-performance liquid chromatography, and identifying the components with high activity of inhibiting elastase by mass spectrometry to obtain the leech-derived polypeptide. The conditions for the size exclusion chromatography separation are preferably to dissolve the freeze-dried saliva sample in 0.1M PB buffer and load it on a equilibrated Sephadex G-50 gel filtration column; elute with PB, connect a fully automatic fraction collector for sample collection, set the flow rate to 0.3mL / min, and collect one tube every 10min; use a spectrophotometer to measure the absorbance values at 280nm and 215nm for every other tube. The PB buffer is preferably a Na2HPO4-NaH2PO4 solution with a pH value of 6.0. Before loading, filtration with a 0.22 μm membrane is preferably performed. The specifications of the Sephadex G-50 gel filtration column are preferably 100×2.6 cm, purchased from GE Health. The conditions for the reversed-phase high-performance liquid chromatography re-separation are preferably: the chromatographic column is RP-HPLC C8 (Sepax C8, 30×0.46 cm), the amount of protein loaded each time is about 0.2 mg, the elution system consists of liquid A and liquid B, the flow rate is 0.7 mL / min, and the flow rate of liquid B increases by 1% per minute for gradient elution. The visible / ultraviolet detector detects the light absorption value of the sample at 280 / 215 nm, and each absorption peak sample is collected as one unit. Liquid A is double distilled water containing 0.1% trifluoroacetic acid. Liquid B is acetonitrile containing 0.1% trifluoroacetic acid.

[0023] The present invention provides a precursor protein of the leech-derived polypeptide, the amino acid sequence of which is shown in SEQ ID NO: 2 (MKIAILLSLFLATLLVVRAVDEKAEVTDDLCGDKTCSGAQVCQ NDACVCSPVRCMIMCPNGFKLDENGCEYPCSCA), and the N-terminus comprises a signal peptide MKIAILLSLFLATLLVVRA (SEQ ID NO: 4).

[0024] The present invention provides a gene encoding the precursor protein, the nucleotide sequence of which is shown in SEQ ID NO: 3 (ATGAAGATTGCAATCCTTTTGAGCCTTTTCCTCGCAACACTTCT TGTTGTCCGGGCAGTCGATGAAAAAGCAGAAGTAACCGATGATCTTTGCGGGGACAAGACATGTTCAGGAGCGCAAGTTTGTCAAAACGACGCATGCGTTTGCAGTCCGGTGAGATGCATGATCATGTGTCCGAATGGATTCAAGTTGGATGAAAATGGATGTGAATATCCTTGTTCCTGTGCTTAG).

[0025] The present invention provides an analgesic and / or anticoagulant drug comprising the leech-derived polypeptide and an excipient. The present invention does not specifically limit the type of the excipient; any excipient known in the art can be used. The present invention does not specifically limit the preparation method of the drug; any method known in the art for preparing protein or polypeptide drugs can be used.

[0026] The present invention provides an application of the leech-derived polypeptide in the preparation of anticoagulant and / or analgesic medicines or preparations.

[0027] In the present invention, the anticoagulant preferably includes one or more of the following: anti-elastase activity, anti-coagulation factor FXIIa activity, and anti-kallikrein activity. Experiments have shown that the leech-derived polypeptide has significant inhibitory effects on elastase, coagulation factor FXIIa, and kallikrein, but has no significant inhibitory effect on coagulation factor FXa, trypsin, and thrombin. Inhibition constant measurements showed that the leech-derived polypeptide has inhibition constants of 262.4 nM, 10.72 μM, and 18.97 μM for elastase, coagulation factor FXIIa, and kallikrein, respectively. The concentration of the leech-derived polypeptide in the drug is preferably no less than 10 μM.

[0028] In the present invention, the analgesic preferably includes suppressing pain caused by an inflammatory response. The inflammatory response is preferably induced by elastase. In the examples, elastase-induced plantar pain in the hind limbs of mice was tested, with the duration of paw licking being used as a pain indicator. The results showed that the elastase-induced paw licking duration was significantly reduced after treatment, indicating that the leech-derived polypeptide can significantly inhibit the elastase-induced pain response in mice, providing a basis for the preparation of analgesic drugs.

[0029] The present invention provides the use of the leech-derived polypeptide in the preparation of anti-thrombotic drugs.

[0030] In an example of the present invention, the antithrombotic effect of a leech-derived polypeptide was tested in a ferric chloride-induced carotid artery thrombosis model. The results showed that the leech-derived polypeptide significantly inhibited the formation of ferric chloride-induced carotid artery thrombosis at concentrations of 1.25 and 2.5 mg / kg. Therefore, it can be seen that the leech-derived polypeptide has an antithrombotic effect.

[0031] The following is a detailed description of the leech-derived polypeptide with both analgesic and antithrombotic functions and its precursor protein and applications provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1

[0033] Method for Isolating and Purifying Poeciguamerin, a Functional Peptide from Hirudo philadelphica

[0034] A multifunctional electromagnetic massager was placed close to the corners of the mouth of the Philippine leech for 5 to 10 seconds. The stimulation voltage was 6V and the power was 2.5 to 345Hz. After stimulation, a colorless viscous liquid was seen overflowing from the mouth. The saliva was repeatedly rinsed with saliva stimulation solution (150mM NaCl, 1mML-Arginine). The rinse solution was collected, centrifuged (4℃, 12000g, 1h), freeze-dried at low pressure, and stored at -80℃ for later use.

[0035] After the washing solution is lyophilized into powder, the elastase inhibitor is separated and purified according to the following procedure. After each purification step, an elastase chromogenic substrate test is performed to determine the purified peak where the target protein is located.

[0036] (1) Sephadex G-50 dextran gel filtration chromatography

[0037] The lyophilized sample was dissolved in 0.1 M PB (Na₂HPO₄-NaH₂PO₄, pH 6.0) buffer and filtered through a 0.22 μm filter. 5 mL (approximately 200 mg protein) was loaded onto a Sephadex G-50 gel filtration column (100 × 2.6 cm, GE Health) equilibrated with the same buffer according to the manufacturer's instructions. Elution was performed using the same PB buffer. Samples were collected using an automated fraction collector at a flow rate of 0.3 mL / min, with one tube collected every 10 minutes. Absorbance was measured at 280 nm and 215 nm for every other tube using a spectrophotometer. Peak fractions were collected and integrated based on the absorbance values. Sample activity was assayed using an elastase chromogenic substrate assay. In a 96-well plate, 3 μL of sample was mixed with 5 μL of 0.1 mg / mL elastase in 52 μL of buffer (100 mM NaCl, 50 mM Tris-HCl, pH 8.0, and 5 mM CaCl₂). After standing at room temperature for 5 minutes, add 35 μL of buffer and 5 μL of 0.1 mg / mL elastase substrate mixture to a final volume of 100 μL. Detect OD 405 Measure continuously for 20 min at 47 s intervals. After activity detection, freeze-dry and set aside.

[0038] turn out, Figure 1 Peak 2 in A has elastase activity ( Figure 1 Peaks 1 and 3 have weak elastic protease activity, but their activity is weaker than that of peak 2.

[0039] (2) Reverse phase high pressure liquid chromatography

[0040] The above-mentioned protein peak freeze-dried sample with the activity of inhibiting elastase was dissolved in double distilled water, centrifuged (12000g, 20min), and the supernatant was taken. After filtering through a 0.22μm filter, separation and purification was carried out on a liquid chromatograph. The chromatographic column used was RP-HPLC C8 (Sepax C8, 30×0.46cm). The protein loading was about 0.2mg at each time. The elution system consisted of liquid A (double distilled water containing 0.1% trifluoroacetic acid) and liquid B (acetonitrile containing 0.1% trifluoroacetic acid). The flow rate was 0.7mL / min, and the liquid B flow rate increased by 1% per minute for gradient elution. The sample 280 / 215nm light absorption value was detected by a visible / ultraviolet detector. Each absorption peak sample was collected as a unit. After the freeze-dried sample was redissolved, the inhibitory effect of each component on elastase was measured according to the above method.

[0041] turn out Figure 1 Peak 6 in medium C had elastase inhibitory activity, while peaks 4, 5, and 7 and the control group showed no good inhibitory activity.

[0042] Example 2

[0043] Functional Analysis of Poeciguamerin from the Philippine Hirudo

[0044] The activity of Poeciguamerin on elastase, coagulation factor FXa, kallikrein, trypsin, coagulation factor FXIIa, and thrombin was studied using a chromogenic substrate assay. The specific assay procedure for the chromogenic substrate assay is as follows: 3 μL of sample was mixed with each enzyme in a buffer solution (100 mM NaCl, 50 mM Tris-HCl pH 8.0, 5 mM CaCl2) in a 96-well plate to a total volume of 60 μL. After standing at room temperature for 5 minutes, the enzyme substrate buffer was added to a total volume of 100 μL. The kinetics of the enzyme reaction were then measured. The concentrations of the various enzymes and substrates used are shown in Table 1:

[0045] Table 1

[0046]

[0047]

[0048] The results showed that Poeciguamerin has an inhibitory effect on elastase ( Figure 2 Middle A), coagulation factor FXIIa ( Figure 2 Middle B), kallikrein ( Figure 2 C) have obvious inhibitory effects on coagulation factor FXa ( Figure 2 D), trypsin ( Figure 2 E) and thrombin ( Figure 2 Middle F) No obvious inhibitory effect.

[0049] The inhibition constants of poeciguamerin against elastase, coagulation factor FXIIa, and kallikrein were further determined. The inhibition constant Ki value was calculated using the commonly used Dixon plot, where the inhibitor concentration is plotted on the horizontal axis and the inverse of the reaction rate is plotted on the vertical axis. The intersection of the extended lines represents the negative of the inhibitor's inhibition constant for the enzyme.

[0050] The results are as follows Figure 3 The inhibition constants of Poeciguamerin against elastase, coagulation factor FXIIa, and kallikrein are 262.4nM, 10.72μM, and 18.97μM, respectively.

[0051] Example 3

[0052] Analgesic Effects of Poeciguamerin from Hirudo philippinosus

[0053] The inhibitory effect of Poeciguamerin on elastase-induced pain was detected through in vivo experiments. The specific process was as follows: 20 μL of elastase (at a concentration of approximately 6 mg / kg) was injected intraplantarly into the right hind limb of the control group mice, and Poeciguamerin (12 mg / kg) was injected intravenously into the tail vein of the experimental group mice 10 minutes before the injection of elastase. The pain level was then evaluated by recording the total time the mice pawed within 30 minutes.

[0054] The results are as follows Figure 4 The paw licking time of the elastase-injected group was significantly longer than that of the control group, while the paw licking time induced by elastase was significantly reduced after treatment with Poeciguamerin. This indicates that Poeciguamerin can significantly inhibit the pain response of mice induced by elastase.

[0055] Example 4

[0056] Antithrombotic Effect of Poeciguamerin from Hirudo philippinarum

[0057] The antithrombotic effect of Poeciguamerin from Hirudo philadelphica was tested by establishing a ferric chloride-induced carotid artery thrombosis model. The establishment process of the ferric chloride-induced carotid artery thrombosis model is as follows:

[0058] C57BL / 6J mice were anesthetized with isoflurane and the left carotid artery was dissected and isolated. Filter paper (2×2 mm) pre-soaked in 10% (w / v) FeCl3 solution was applied to the left carotid artery to induce thrombus formation. Carotid blood flow was continuously recorded using laser Doppler flowmetry, and the duration of thrombus formation was recorded. Poeciguamerin (1.25, 2.5 mg / kg) or heparin sodium (20 mg / kg) was administered 10 minutes prior to model establishment. A saline group served as a negative control.

[0059] The results are as follows Figure 5 Poeciguamerin significantly inhibited the formation of carotid artery thrombosis induced by ferric chloride at concentrations of 1.25 and 2.5 mg / kg, and 20 mg / kg of heparin sodium was used as a positive control.

[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A leech-derived polypeptide having both analgesic and antithrombotic functions, characterized in that: The amino acid sequence is shown in SEQ ID NO:

1.

2. A precursor protein of the leech-derived polypeptide according to claim 1, characterized in that: The amino acid sequence is shown in SEQ ID NO:

2.

3. A gene encoding the precursor protein according to claim 2, characterized in that: The nucleotide sequence is shown in SEQ ID NO:

3.

4. An analgesic and / or anticoagulant drug, characterized in that: The invention comprises the leech-derived polypeptide according to claim 1 and auxiliary materials.

5. Use of the leech-derived polypeptide according to claim 1 in the preparation of anticoagulant and / or analgesic drugs or preparations.

6. The application according to claim 5, characterized in that: The anticoagulant includes one or more of the following: anti-elastase activity, anti-coagulation factor FXIIa activity and anti-kallikrein activity.

7. The use according to claim 5, characterized in that The analgesia includes suppressing pain caused by inflammatory response.

8. The application according to claim 7, characterized in that: The inflammatory response is induced by elastase.

9. The use according to any one of claims 5 to 8, characterized in that: The concentration of the leech-derived polypeptide in the medicine is not less than 10 μM.

10. Use of the leech-derived polypeptide according to claim 1 in the preparation of anti-thrombotic drugs.

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