Analgesic polypeptide SaTX-I and application thereof

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to analgesic polypeptide SaTX-I and application thereof. The invention provides analgesic polypeptide SaTX-I, and the amino acid sequence of the analgesic polypeptide SaTX-I is as shown in SEQ ID NO. 1. The analgesic polypeptide SaTX-I provided by the invention can act on NaV1.7 channels of related species including human beings, remarkably inhibits the current intensity of the NaV1.7 sodium ion channels, reduces the induction of an organism (human or animal) to pain to a certain extent, improves the resistance of the organism to the pain, can be used as a resource to be developed into a potential analgesic drug, and has a wide application prospect. The medicine is applied to human and other animals to relieve pains. Secondly, the analgesic polypeptide SaTX-I provided by the invention can act on a NaV1.7 sodium ion channel in a targeting manner, can be applied to research on related ion channel diseases and can be used as a potential therapeutic drug for related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to an analgesic polypeptide SaTX-I and an application thereof. Background Art

[0002] Among the various receptors associated with pain, voltage-gated sodium channel (Na V Voltage-gated sodium channels were discovered early on and are associated with a variety of pain diseases. There are many types of voltage-gated sodium channels, which are named Na V 1.1~Na V 1.9, these receptors are distributed in different tissues and organs of the human body, and perform different functions, such as Na+ expressed in the central nervous system (CNS). V 1.1. Na expressed in skeletal muscle V 1.4. Na expressed in the heart V 1.5, Na V 1.6 Na V 1.7 Na V 1.8 and Na V 1.9 There are many distributions in the dorsal root ganglion (DRG), many of which are related to pain, such as Na distributed on dorsal root ganglion cells and trigeminal neurons. V 1.7 Na V 1.8 and Na V 1.9.

[0003] Voltage-sensitive sodium channel Na V 1.7 is involved in the generation and transmission of pain signals, and all knockout mice encode Na V The gene SCN9A of the 1.7 channel will make mice lose the perception of thermal pain, mechanical pain, inflammatory pain, etc., but it has no effect on motor function, cognitive state, sympathetic nervous system and gastrointestinal system. V Compounds with Nav1.7 activity may be used to treat chronic pain, so specific inhibitors targeting Nav1.7 channels are expected to be developed into new analgesic drugs. V 1.7 The research on screening specific inhibitors for pain targets has become a hot topic in the field of pain, but so far there are few inhibitors with strong activity and good specificity. V 1.7 Inhibitors and polypeptides with strong analgesic activity are particularly important in this field. Summary of the invention

[0004] The purpose of the present invention is to provide an analgesic polypeptide SaTX-I and its application, targeting Na V 1.7 Sodium ion channel, inhibiting Na V 1.7 Sodium ion channels reduce the body's sensitivity to pain and increase the body's resistance to pain. They can be developed as resources into potential analgesics, applied to the research of related ion channel diseases and as potential therapeutic drugs for related diseases.

[0005] The present invention provides an analgesic polypeptide SaTX-I, whose amino acid sequence is shown in SEQ ID NO.1.

[0006] The present invention also provides the use of the analgesic polypeptide SaTX-I described in the above technical solution in the preparation of analgesic products.

[0007] Preferably, the analgesic product comprises a thermal analgesic product and / or a sore pain analgesic product.

[0008] The present invention also provides the use of the analgesic polypeptide SaTX-I described in the above technical solution in the preparation of a product that improves the body's resistance to pain.

[0009] Preferably, the pain includes thermal pain and / or soreness.

[0010] The present invention also provides an analgesic drug, which includes the analgesic polypeptide SaTX-I described in the above technical solution.

[0011] Preferably, the concentration of the analgesic polypeptide SaTX-I in the analgesic drug is ≥ 0.3 μM.

[0012] The present invention also provides the analgesic polypeptide SaTX-I or analgesic drug described in the above technical solution in the preparation of Na V 1.7 Application of ion channel inhibitors.

[0013] The present invention also provides a Na V 1.7 ion channel inhibitor, the NaV1.7 ion channel inhibitor includes the analgesic polypeptide SaTX-I or analgesic drug described in the above technical solution.

[0014] The present invention also provides the analgesic polypeptide SaTX-I or analgesic drug or Na V 1.7 Ion channel inhibitors in the preparation of Na V 1.7 Application of the product in sodium ion channel related diseases.

[0015] Beneficial effects:

[0016] The present invention provides an analgesic polypeptide SaTX-I, the amino acid sequence of which is shown in SEQ ID NO.1. The analgesic polypeptide SaTX-I provided by the present invention can act on the Na of relevant species including humans. V 1.7 channels, significantly inhibiting Na V 1.7 The current intensity of the sodium ion channel can reduce the body's (human and animal) sensitivity to pain to a certain extent, improve the body's resistance to pain, and can be used as a resource to develop into a potential analgesic drug for use in humans and other animals to relieve pain. V 1.7 Sodium ion channels can be applied to the study of related ion channel diseases and serve as potential therapeutic drugs for related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0018] Figure 1 is a simulated diagram of the tertiary structure of the SaTX-I polypeptide in Example 1;

[0019] Figure 2 The effects of SaTX-I peptide on different NaV channels (Na V 1.1、Na V 1.4 Na V 1.5 and Na V 1.7) inhibition (voltage-current mode recording results);

[0020] Figure 3 The effects of different concentrations of SaTX-I peptide on different NaV channels (Na V 1.1、Na V 1.4 Na V 1.5 and Na V 1.7) inhibition rate;

[0021] Figure 4 The half-inhibitory concentration of SaTX-I peptide (0.3 μM) on Na V 1.7 Current-voltage relationship curve of the channel; where A is the normalized current statistics at different voltages, B is the normalized conductance statistics at different voltages, and Bath represents the external solution;

[0022] Figure 5 The pain-relieving effect of SaTX-I polypeptide on mice; A is the effect on thermal pain, B is the effect on soreness, NS indicates no significant difference, *P<0.05, **P<0.01, ***P<0.0001. DETAILED DESCRIPTION

[0023] The present invention provides an analgesic polypeptide SaTX-I, whose amino acid sequence is shown in SEQ ID NO.1.

[0024] The inventors of the present invention have discovered a SaTX-I polypeptide with an amino acid sequence as shown in SEQ ID NO.1. Based on electrophysiological experiments, it was found that the SaTX-I polypeptide is an effective method for relieving Na V 1.7 Key chemicals that induce pain can target the receptor Na V 1.7, for Na V 1.7 produces significant inhibition, reduces the perception of pain in humans and animals, so that humans and animals can relieve pain through the SaTX-I polypeptide in painful conditions, and can be used to improve the resistance of animals to pain.

[0025] In view of the effects of the analgesic polypeptide SaTX-I provided by the present invention, the use of the analgesic polypeptide SaTX-I in the preparation of analgesic products and / or products that improve the body's resistance to pain also falls within the protection scope of the present invention.

[0026] In the present invention, as an embodiment, the analgesic product includes a thermal analgesic product and / or an acid analgesic product. As an embodiment, the acid analgesic product includes an acetic analgesic product. As an embodiment, the pain includes thermal pain and / or acid pain; as an embodiment, the thermal pain includes heat-induced acute pain; as an embodiment, the acid pain includes acetic acid-induced acute pain.

[0027] The present invention also provides an analgesic drug, which includes the analgesic polypeptide SaTX-I described in the above technical solution.

[0028] In the present invention, as one embodiment, the concentration of the analgesic polypeptide SaTX-I in the analgesic drug is ≥0.3 μM; as another embodiment, the concentration of the analgesic polypeptide SaTX-I in the analgesic drug is 0.3-2 μM; as another embodiment, the concentration of the analgesic polypeptide SaTX-I in the analgesic drug is 0.4-1 μM; as another embodiment, the concentration of the analgesic polypeptide SaTX-I in the analgesic drug is 0.7 μM. In a specific embodiment of the present invention, any value can be taken within the range of ≥0.3 μM, such as 0.3 μM, 0.35 μM, 0.4 μM, 0.42 μM, 0.5 μM, 0.58 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.5 μM, 1.8 μM or 2 μM, etc.

[0029] The present invention does not specifically limit the dosage form of the analgesic drug, and conventional dosage forms in the art can be used. As an embodiment, the analgesic drug of the present invention can be used in humans and animals to relieve pain, overcome acute and chronic pain in a short period of time, etc. by oral administration or injection. As an embodiment, when used by injection, the solvent of the analgesic drug is physiological saline.

[0030] The present invention also provides the analgesic polypeptide SaTX-I or analgesic drug described in the above technical solution in the preparation of Na V 1.7 Application of ion channel inhibitors.

[0031] The present invention also provides a Na V 1.7 Ion channel inhibitors, the Na V 1.7 Ion channel inhibitors include the analgesic polypeptide SaTX-I or analgesic drugs described in the above technical solution.

[0032] In the present invention, as an embodiment, the Na V 1.7 The concentration of the analgesic polypeptide SaTX-I in the ion channel inhibitor is ≥ 0.3 μM; as another embodiment, the Na V 1.7 The concentration of the analgesic polypeptide SaTX-I in the ion channel inhibitor is 0.3-2 μM; as another embodiment, the Na V 1.7 The concentration of the analgesic polypeptide SaTX-I in the ion channel inhibitor is 0.4 to 1 μM; as another embodiment, the Na V 1.7 The concentration of the analgesic polypeptide SaTX-I in the ion channel inhibitor is 0.7 μM. In a specific embodiment of the present invention, any value can be taken within the range of ≥0.3 μM, such as 0.3 μM, 0.35 μM, 0.4 μM, 0.42 μM, 0.5 μM, 0.58 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.5 μM, 1.8 μM or 2 μM, etc.

[0033] Na V 1.7 is a type of pain-related voltage-gated sodium ion channel, which is widely distributed in the dorsal root ganglion (DRG), is related to nociceptive pain perception, and is an important target for the treatment of pain. The present invention verifies the pain-relieving effect of SaTX-I polypeptide on mice through a mouse pain model experiment. V Whole-cell patch clamp experiments were performed on HEK293T cells expressing 1.7-channel plasmids. The results showed that SaTX-I peptide could inhibit the activity of mammalian Na V The current strength of 1.7 does not change hNa V1.7 Channel activation voltage, fast activation, fast inactivation and other properties.

[0034] The present invention also provides the analgesic polypeptide SaTX-I or analgesic drug or the Na V 1.7 Ion channel inhibitors in the preparation of Na V 1.7 Application in products for treating diseases related to sodium ion channels. In the present invention, as an embodiment, the Na V 1.7 Ion channel-related diseases include chronic pain.

[0035] In order to further illustrate the present invention, an analgesic polypeptide SaTX-I and its application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] 1. The inventors discovered a new bird-catching spider, Anubis clavipes, measured its transcriptome and proteome, synthesized the toxin sequence obtained by analyzing these data, and obtained toxin molecules with biological activity through polypeptide renaturation and separation and purification, and then performed toxin target screening, and finally screened out a polypeptide, which was named SaTX-I, and its specific amino acid sequence was TCQKWMWTCDSKRKC CEGMVCKLWCKKIL (SEQ ID NO.1).

[0038] 2. The tertiary structure of SaTX-I obtained in step 1 of AlphaFold simulation shows that the six cysteines in SaTX-I form three pairs of disulfide bonds, connected in the form of C1-C4, C2-C5, and C3-C6, which is the inhibitory cystine knot (ICK) model ( Figure 1 ).

[0039] Example 2

[0040] Electrophysiological experiments on the targeting effect of SaTX-I peptide on pain-related receptors

[0041] 1. The SaTX-I polypeptide in Example 1 was obtained by artificial synthesis.

[0042] 2. Preparation of plasmid:

[0043] (1)Na V 1.1 Preparation of plasmid: Na was obtained by artificial synthesis V 1.1 protein coding gene, and inserted it between HindIII and EcoR I of pcDNA3.1(+) vector to obtain the coding gene of NaV 1.1 protein plasmid; among them, Na V 1.1 The amino acid sequence of the protein is accessed as: NCBI Reference Sequence: NP_001300926.1, encoding Na V 1.1 The nucleotide sequence accession information of the protein is: Mus musculus Scn1a (Gene ID: 20265);

[0044] (2)Na V 1.4 Plasmid preparation: Na was obtained by artificial synthesis V 1.4 protein coding gene, and inserted it between HindIII and EcoR I of pcDNA3.1(+) vector to obtain the coding gene of Na V 1.4 protein plasmid; among them, Na V 1.4 The amino acid sequence of the protein is accessed as: NCBI Reference Sequence: NP_001390570.1, encoding Na V 1.4 The accession information of the nucleotide sequence of the protein is: GeneID: 110880;

[0045] (3)Na V 1.5 Plasmid preparation: Na was obtained by artificial synthesis V 1.5 protein coding gene, and inserted it between HindIII and EcoR I of pcDNA3.1(+) vector to obtain the coding gene of Na V 1.5 protein plasmid; among them, Na V The amino acid sequence of protein 1.5 is accessed as: NCBI Reference Sequence: NP_001240789.1, encoding Na V The accession information of the nucleotide sequence of 1.5 protein is: GeneID: 20271;

[0046] (4)Na V 1.7 Plasmid preparation: Na was obtained by artificial synthesis V 1.7 protein coding gene, and inserted it between HindIII and EcoR I of pcDNA3.1(+) vector to obtain the coding gene of Na V 1.7 protein plasmid; among them, Na V The amino acid sequence of protein 1.7 is accessed as: NCBI Reference Sequence: NP_001277603.1, encoding Na V The accession information of the nucleotide sequence of 1.7 protein is: GeneID: 20274;

[0047] (5) Preparation of EGFP plasmid: The EGFP protein coding gene was obtained by artificial synthesis and inserted between HindIII and EcoR I of the pcDNA3.1(+) vector to obtain a plasmid encoding the EGFP protein; the amino acid sequence of the EGFP protein was accessed at GenBank: AAK15492.1.

[0048] 3. HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture. HEK293T cells were cultured in a 3.5 cm diameter culture dish. When the cell density reached 60% to 70%, Lipofectamine 3000 transfection reagent was used for transfection. The specific steps are as follows: 200 μL of reduced serum medium Opti-MEM 200 μL was added to the transfection system tube A. TM , 6μL Lipofectamine TM 3000 reagent; add 200μL reduced serum medium in transfection system, 6μL P3000 reagent, 5μg plasmid containing the corresponding target protein obtained in step 2, and 0.5μg EGFP plasmid to the transfection system tube B in turn, mix well and let stand for 3 minutes, then add the transfection system of tube B to the transfection system of tube A, mix well and let stand for 15 minutes. Carefully drop the transfection system into the culture dish, and after 24 hours, digest the cells and spread them on a 3.5cm culture dish with a round glass slide and place it in an incubator.

[0049] 4. After the cells in step 3 adhere to the wall, conduct electrophysiological experiments, specifically whole-cell patch clamp experiments: Under voltage-gated sodium channel experimental conditions and recording mode, use a HEKA EPC10 amplifier with PatchMaster software to measure voltage-gated sodium channels. Glass electrodes are used for cell-attached recordings, with an input resistance of 3-5MΩ. The perfusion tube relies on gravity to operate, and real-time data is sent back to the amplifier for processing via PatchMaster, and current changes are recorded in real time. The results of the whole-cell recording mode show that the transfected Na V The clamping voltage of HEK293T cells with 1.7 plasmid was -80mV, the duration was 50ms; the stimulation voltage was 0mV, the duration was 100ms; the transfected Na V The clamping voltage of HEK293T cells with 1.5 plasmid was -80mV, the duration was 50ms; the stimulation voltage was 0mV, the duration was 100ms; the transfected Na VThe clamping voltage of HEK293T cells transfected with 1.4 plasmid was -120mV, the duration was 50ms; the stimulation voltage was 0mV, the duration was 100ms; the clamping voltage of HEK293T cells transfected with NaV1.1 plasmid was -80mV, the duration was 50ms; the stimulation voltage was 0mV, the duration was 100ms;

[0050] 5. Based on the conclusion of step 4, after the cell transfection in step 3 is completed, the transfection system is carefully dripped into the culture dish. After 24 hours, the cells are digested and spread on a 3.5 cm culture dish covered with a round glass slide. The cells transfected with the corresponding target protein particles are randomly divided into treatment groups with different concentrations of SaTX-I polypeptide (0.1 μM, 0.2 μM, 0.4 μM, 0.7 μM, 1 μM and 2 μM). The corresponding concentration of SaTX-I polypeptide is added to the culture dish, placed in an incubator, and a treatment group without adding SaTX-I polypeptide is set as a control; after the cells adhere to the wall, electrophysiological experiments are performed according to the operation in step 4. The voltage-current (IV) mode was used for recording in the whole-cell sodium channel test mode. The clamping voltage was maintained at -80 mV, and the activation voltage was increased from -80 mV by 10 mV each time to obtain the activation current under different voltages (-80 to 10 mV, with an increase of 10 mV each time); wherein, the recording external solution composition was: 140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 11.1 mM D-glucose, 10 mM HEPES (NaOH adjusted pH 7.3-7.4); the recording internal solution composition was: 130 mM KCl, 15 mM NaCl, 0.37 mM CaCl2, 1 mM MgCl2, 1 mM MgATP, 1 mM EGTA, 10 mM HEPES (KOH adjusted pH 7.2-7.3).

[0051] Electrophysiological results showed that SaTX-I peptide has a certain selectivity in inhibiting NaV channels, especially in inhibiting Na V 1.7 channels, and for Na V The inhibition of the channel showed a concentration-dependent relationship ( Figure 2 and Figure 3 ). SaTX-I polypeptide has an effect on Na V The half-inhibitory concentration of 1.1 is 0.65 μM, and the half-inhibitory concentration of Na V The half-inhibitory concentration of 1.4 is 0.60 μM, and the half-inhibitory concentration of Na V The half-inhibitory concentration of 1.5 is 2.3 μM, and the half-inhibitory concentration of Na V The half-inhibitory concentration of 1.7 is 0.32 μM. Under the stimulation of the half-inhibitory concentration of SaTX-I peptide, Na V 1.7 The current morphology and kinetic properties of the channel, such as fast activation and fast inactivation, did not change significantly ( Figure 4), the stimulation of SaTX-I peptide will not cause the delay of opening and closing of NaV1.7 channels, nor will it change the maximum activation voltage, indicating that the activation and closing of the channels will not be greatly affected at the cellular level, but the inhibitory effect is achieved by reducing the current intensity when the channels are activated, thereby reducing the influx of intracellular sodium ions, reducing cell excitability, and cutting off the conduction of pain signals, thereby achieving the purpose of analgesia.

[0052] Example 2

[0053] Analgesic activity of SaTX-I

[0054] This example uses C57BL / 6J mice, which were kept in the clean-grade breeding room of experimental animals at the Kunming Institute of Zoology. Before the experiment, sufficient food, clean water and suitable living space were maintained, and the experiment was conducted in an animal experiment laboratory at 25°C. Animal ethics approval number: IACUA-RE-2023-06-009.

[0055] Tail immersion model: The experimental mice were randomly divided into a negative control group, a positive control group, and an experimental group treated with different concentrations of SaTX-I peptide. The tail tips of the mice in each treatment group were placed in hot water at 55±0.5℃ 30 minutes after intraperitoneal injection of the drug. The time required for the tail of the mouse to leave the water was recorded. The results are shown in Figure 5 As shown in A; the negative control group was intraperitoneally injected with 100 μL of normal saline; the positive control group was intraperitoneally injected with 100 μL of 5 mg / kg morphine; the experimental groups treated with different concentrations of SaTX-I polypeptide were intraperitoneally injected with 100 μL of SaTX-I polypeptide dissolved in normal saline (the injection concentrations of SaTX-I polypeptide were 0.2 mg / kg, 1 mg / kg and 5 mg / kg), with 6 mice (C57BL / 6J, 20-25 g) in each treatment group.

[0056] Acid writhing model: The experimental mice were randomly divided into a negative control group, a positive control group and an experimental group treated with different concentrations of SaTX-I peptide. 200 μL of 0.8% acetic acid (vol / vol) was injected intraperitoneally 30 minutes after the injection of the drug. The mice were placed in an uncovered observation box of 20 cm×20 cm×15 cm. The number of licking the abdomen, writhing, and abdominal twitching of the mice within 30 minutes was recorded. The results are shown in Table 1. Figure 5 As shown in B; the negative control group was intraperitoneally injected with 100 μL of normal saline; the positive control group was intraperitoneally injected with 100 μL of 5 mg / kg morphine; the experimental groups treated with different concentrations of SaTX-I polypeptide were intraperitoneally injected with 100 μL of SaTX-I polypeptide dissolved in normal saline (the injection concentration of SaTX-I polypeptide was 0.2 mg / kg, 1 mg / kg and 5 mg / kg), with 6 mice (C57BL / 6J, 20-25 g) in each treatment group.

[0057] according to Figure 5 It can be seen that SaTX-I polypeptide treatment can enhance the tolerance of mice to acute pain induced by heat and acetic acid, and the number of pain behaviors in mice is significantly reduced. SaTX-I polypeptide has a concentration-dependent inhibitory effect on heat pain and soreness. Intraperitoneal injection of SaTX-I polypeptide into mice can help mice relieve pain.

[0058] Based on the above, it can be seen that SaTX-I peptide can improve the resistance of mice to pain and can be used as a resource to develop into a potential analgesic drug for use in humans and other animals to relieve pain. V 1.7, can be applied to the study of related ion channel diseases and as potential therapeutic drugs.

[0059] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An analgesic polypeptide SaTX-I, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. Use of the analgesic polypeptide SaTX-I according to claim 1 in the preparation of analgesic products.

3. The use according to claim 1, characterized in that: The analgesic products include thermal analgesic products and / or sore pain analgesic products.

4. Use of the analgesic polypeptide SaTX-I according to claim 1 in the preparation of a product for improving the body's resistance to pain.

5. The use according to claim 4, characterized in that: The pain includes heat pain and / or soreness.

6. An analgesic drug, characterized in that: The analgesic drug includes the analgesic polypeptide SaTX-I according to claim 1.

7. The analgesic drug according to claim 6, characterized in that The concentration of the analgesic polypeptide SaTX-I in the analgesic drug is ≥0.3 μM.

8. The analgesic polypeptide SaTX-I according to claim 1 or the analgesic drug according to claim 6 or 7 in the preparation of Na V 1.7 Application of ion channel inhibitors.

9. A Na V 1.7 Ion channel inhibitor, characterized in that The Na V 1.7 Ion channel inhibitors include the analgesic polypeptide SaTX-I described in claim 1 or the analgesic drug described in claim 6.

10. The analgesic polypeptide SaTX-I according to claim 1 or the analgesic drug according to claim 6 or 7 or the Na according to claim 9 V 1.7 Ion channel inhibitors in the preparation of Na V 1.7 Application of the product in sodium ion channel related diseases.

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