μ-conotoxin peptide cniiic mutant and use thereof

Abstract

Provided are a μ-conotoxin peptide CnIIIC mutant and the use thereof. Further provided are a nucleic acid construct of the μ-conotoxin peptide CnIIIC mutant, and an expression vector and transformed cell thereof, and the use thereof. The μ-conotoxin peptide CnIIIC mutant has an enhanced transdermal penetration ability, and can effectively penetrate the skin barrier to directly act on nerves and muscle tissues in the underlying layer of skin. By means of the unique physical and chemical properties, the mutant can realize a local pharmaceutical effect within a relatively short period of time, thereby greatly improving the bioavailability of a drug.

Description

A mu-conotoxin CnIIIC mutant and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of biochemistry and molecular biology, in particular to a mu-conotoxin CnIIIC mutant and application thereof. BACKGROUND

[0002] In the 1980s, Olivera et al. first isolated conotoxin from Conus (a genus of marine mollusks), which brought a revolutionary breakthrough to the research of neuropharmacology. With the continuous development of technology, researchers found that conotoxin not only can inhibit sodium ion channels and calcium ion channels, but also can act on acetylcholine receptors, thereby effectively blocking nerve signal transmission, inhibiting muscle contraction, and ultimately achieving the effect of reducing wrinkles. The study of this mechanism provides a new direction for the application of conotoxin, especially in skin aging, wrinkle formation, etc. Conotoxin has unique functional advantages, such as small side effects, clear targeting, and sustained action, etc., so it has become the focus of attention in the fields of pharmacology and biomedical science. Especially in the field of cosmetics, conotoxin is widely studied due to its characteristics of inhibiting nerve signal transmission and relaxing muscles, and has become one of the important ingredients of anti-aging products.

[0003] At present, conotoxin applied in the field of cosmetics is mainly derived from μ-family toxins, among which the most well-known is μ-conotoxin CnIIIC. μ-conotoxin CnIIIC (skeleton: CC-C-C-CC) is a cyclic peptide composed of 22 amino acids, with a molecular weight of 2375.7 and a molecular structure containing three pairs of disulfide bonds, forming a cross structure. Due to its ring structure, the molecular dynamics radius of μ-conotoxin CnIIIC is small, and it has strong transdermal properties, especially in the areas around the eyes and other parts of the skin where wrinkles are prone to appear.

[0004] In nature, μ-conotoxin CnIIIC forms a stable three-dimensional conformation through a specific folding pattern, which endows it with biological activity. However, under artificial synthesis conditions, the cross-linking of disulfide bonds can change due to different oxidation conditions, leading to different spatial configurations of the peptide chain. Different conformations of μ-conotoxin CnIIIC may exhibit different biological activities, and some conformations may even lack biological effects. Therefore, accurate folding conformations are required for the application of conotoxins to ensure maximum efficacy. Conotoxin peptides in the cosmetics field are usually synthesized using biomimicry techniques, involving gene sequencing and artificial biomimetic synthesis, thereby retaining biological activity and efficacy while reducing most of the toxic components.

[0005] However, the balance between the bioactivity and toxicity of artificially synthesized conopod peptide variants remains difficult to control. Conformational differences may occur during synthesis, affecting biological efficacy. Furthermore, research on targeting effects on the NaV1.4 channel and its individual variability is insufficient. Therefore, developing conopod peptide variants with higher bioactivity and lower toxicity remains a current research hotspot and need in the field. Summary of the Invention

[0006] To address the deficiencies in existing technologies, this invention proposes a μ-conotoxin CnIIIC mutant and its applications. This application involves point mutations at specific sites in the peptide represented by wild-type μ-conotoxin CnIIIC (SEQ ID No. 21), resulting in a variant with higher Nav1.4 blocking activity than the wild-type conotoxin peptide. This variant can inhibit muscle contraction and paralyze nerves, ultimately reducing wrinkles and can be used in anti-wrinkle products, anesthetics, and analgesics.

[0007] This invention discloses a mutagenic conotoxin peptide CnIIIC mutant, the amino acid sequence of which is shown below:

[0008] Xaa1-Xaa2-Cys-Cys-Xaa3-Gly-Pro-Lys-Gly-Cys-Xaa4-Ser-Lys-Trp-Cys-Arg-Xaa5-His-Ala-Arg-Cys-Cys-Xaa6; (Formula I)

[0009] Where Xaa1 is any one of d-Arg, d-Lys, and d-His, or Xaa1 is missing:

[0010] Xaa2 is any one of Gly, Arg, d-Arg, Lys, and d-Lys or Xaa2 is missing;

[0011] Xaa3 is Asn or Arg;

[0012] Xaa4 is Ser or Tyr;

[0013] Xaa5 is Gly or Ser;

[0014] Xaa6 is d-Arg or Xaa6 is absent.

[0015] The above amino acids Cys, Gly, Pro, Lys, Ser, Trp, Arg, His, Ala, d-Arg, d-Lys, Asn, Tyr, Gly, d-Arg, etc. are abbreviations of amino acids, wherein "d-" means D-type amino acid.

[0016] Optionally, the C-terminus of the polypeptide of Formula I is amidated.

[0017] The C-terminus of the polypeptide of Formula I can also be represented by #, i.e.:

[0018] Xaa1-Xaa2-Cys-Cys-Xaa3-Gly-Pro-Lys-Gly-Cys-Xaa4-Ser-Lys-Trp-Cys-Arg-Xaa5-His-Ala-Arg-Cys-Cys-Xaa6#.

[0019] In some embodiments, the C-terminus of the polypeptide of Formula I can be amidated by artificial chemical synthesis, or by amidating enzyme in or outside the cell.

[0020] The amino acid sequence of the mutant of the μ-conotoxin peptide CnIIIC of the present application contains three pairs of disulfide bonds, which are located at Cys 3 -Cys 15 , Cys 4 -Cys 21 and Cys 10 -Cys 22 positions, which are the same as the disulfide bond structure of the natural μ-conotoxin peptide, and the change of amino acid at different positions does not affect the structure of the conotoxin peptide.

[0021] The present application also includes fusion polypeptides or cleavable fusion polypeptides in which other peptides / polypeptides are fused to the N-terminus and / or C-terminus of the mutant of the μ-conotoxin peptide CnIIIC of the present application. The techniques for producing fusion polypeptides are known in the art, including linking the coding sequence encoding the peptide of the present application with the coding sequence encoding the other peptides / polypeptides in the same reading frame, and the expression of the fusion polypeptide is controlled by the same promoter and terminator.

[0022] In some embodiments, the mu-conotoxin peptide CnIIIC mutant of the present application is C-terminally amidated. C-terminal amidation removes the negative charge at the C-terminus. Peptide variants with C-terminal amide are denoted with "NH2" at the C-terminus. The C-terminal amidation can be achieved by artificial chemical synthesis or by amidating enzymes, either in cells or outside cells.

[0023] The present application also provides a method for preparing the mu-conotoxin peptide CnIIIC mutant, comprising the following steps:

[0024] (1) synthesizing a linear peptide of the mu-conotoxin peptide CnIIIC mutant according to its amino acid sequence by solid phase chemical synthesis, preferably fluorenylmethyloxycarbonyl (FMOC) solid phase chemical synthesis;

[0025] (2) obtaining the mu-conotoxin peptide CnIIIC mutant by cyclization and purification of the linear peptide obtained in (1).

[0026] In some embodiments, the mu-conotoxin peptide CnIIIC mutant has an amino acid sequence as shown in any one of SEQ ID NO. 1-20.

[0027] In some embodiments, the mu-conotoxin peptide CnIIIC mutant further comprises a fluorescently labeled peptide.

[0028] The present application also provides an isolated fusion protein comprising the mu-conotoxin peptide CnIIIC mutant.

[0029] The present application also provides an isolated polynucleotide encoding the mu-conotoxin peptide CnIIIC mutant.

[0030] The present application also provides a nucleic acid construct comprising the polynucleotide.

[0031] The present application also provides a transformed cell comprising the polynucleotide or the nucleic acid construct.

[0032] The present application also provides a pharmaceutical composition comprising at least one mu-conotoxin peptide CnIIIC mutant.

[0033] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0034] The present application also provides use of the mu-conotoxin peptide CnIIIC mutant in any one of the following:

[0035] (1) use in the preparation of a medicament for treating or preventing pain;

[0036] (2) the use in the preparation of a medicament for treating or preventing a disease associated with sodium ion channels;

[0037] (3) the use in the preparation of an anesthetic medicament;

[0038] (4) the use in the preparation of a cosmetic product.

[0039] In some embodiments, the disease associated with sodium ion channels is any one of epilepsy, arrhythmia, muscle paralysis, myotonic muscular spasm, and autism spectrum disorder.

[0040] In some embodiments, the anesthetic medicament of (3) is used for surgical anesthesia or local anesthesia.

[0041] In summary, compared with the prior art, the present application achieves the following technical effects:

[0042] The present application performs point mutation on the specific site of wild-type mu-conotoxin peptide CnIIIC. Compared with the wild type, the mu-conotoxin peptide CnIIIC mutant has stronger transdermal penetration ability after optimization, can effectively penetrate the skin barrier, and directly act on the nerves and muscle tissues in the lower layer of the skin. Its unique physical and chemical properties enable it to achieve local drug efficacy in a short time, greatly improving the bioavailability of the drug. In addition, the half-inhibitory concentration of the mu-conotoxin peptide CnIIIC mutant to Nav1.4 ion channels in the resting state is significantly lower than that of the wild type, and it has stronger targeting and inhibitory activity, that is, the mutant can achieve effective Nav1.4 channel inhibition at a lower concentration, can more efficiently block the function of Nav1.4 channels, and further improve the dose benefit ratio in clinical application, reducing possible side effects. Therefore, it has significant advantages in muscle relaxation, analgesia and anesthesia applications. DETAILED DESCRIPTION

[0043] In order to enable personnel in the art to better understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should fall within the scope of protection of the present application.

[0044] Terminology

[0045] In the present application, "nucleic acid construct" is defined herein as a single- or double-stranded nucleic acid molecule, preferably an artificially constructed nucleic acid molecule. Optionally, the nucleic acid construct further comprises one or more regulatory sequences operably linked thereto. In the present application, the nucleic acid construct is used to express the mutant of conotoxin peptide CnIIIC of the μ-type to achieve efficient gene expression and protein production.

[0046] In the present application, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide that inhibits a certain protein can be inserted. For example, the vector includes a plasmid; a cosmid; a fosmid; an artificial chromosome such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or a P1-derived artificial chromosome (PAC); a phage such as a Mu phage or an M13 phage; and an animal virus, etc. The animal virus used as the vector is a retrovirus (including a lentivirus), an adenovirus, an adeno-associated virus, a herpes virus (e.g., a simplex virus), a pox virus baculovirus, a papillomavirus, a papovavirus (e.g., SV40), etc. A vector can contain various elements that control expression.

[0047] "Transformed cell" refers to a cell whose original genome or characteristics are changed by the introduction of an exogenous gene or material, thereby acquiring a specific function or characteristics. The transformed cell is generally introduced into a cell by physical, chemical, or biological methods, such as a plasmid, DNA, RNA, etc., to express a new gene product or to have a certain biological characteristics. The purpose of transformation is to perform further research, application, or production, such as protein production, gene therapy, etc. It includes many cell types such as E. coli or Bacillus subtilis, prokaryotic cells, yeast cells or Aspergillus, fungal cells, S2 Drosophila cells or Sf9, insect cells, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0048] As used herein, the term "polypeptide" or "peptide" refers to a molecule of two or more amino acids linked by peptide bonds. The terms "peptide" and "polypeptide" also refer to naturally modified peptides / polypeptides, wherein the modifications are achieved, for example, by post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. The "peptide" or "polypeptide" can also be chemically modified when mentioned herein, for example, at the N- and / or C-terminus by, for example, amidation, amination, or pegylation. Such modifications are well known in the art.

[0049] The term "amino acid" or "amino acid residue" typically refers to the building blocks of proteins, for example, an amino acid selected from the group consisting of alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C); glycine (Gly or G); histidine (His or H); lysine (Lys or K); proline (Pro or P); serine (Ser or S); tryptophan (Trp or W); tyrosine (Tyr or Y).

[0050] Peptides (at least those containing peptide bonds between amino acid residues) can be synthesized by solid phase peptide synthesis using the Fmoc strategy described in "Fmoc Solid Phase Peptide Synthesis - A Practical Approach". W. C. Chan, P. D. White, Eds., Oxford University Press, New York, 2000. Temporary N-amino protection is provided by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. All coupling and deprotection reactions are monitored by using the ninhydrin test procedure. After completion of the synthesis, the peptide is cleaved from the resin with concomitant removal of the side chain protecting groups by treatment with trifluoroacetic acid. Common scavengers are ethanedithiol, phenol, anisole and water, the precise choice depending on the component amino acids of the peptide being synthesized. After cleavage, the reaction is precipitated into methyl tert-butyl ether solution, centrifuged to obtain a linear crude peptide solid. The linear crude peptide solid is subjected to liquid phase air oxidation to obtain a crude cyclic peptide solution. Finally, the crude cyclic peptide is purified by preparative reverse phase high performance liquid chromatography and lyophilized to obtain the final product.

[0051] Reagents for peptide synthesis are generally commercially available from Chengdu Taihe Weiye, Suzhou Haofan, China. Peptide analysis can be performed by using reverse phase high performance liquid chromatography, amino acid analysis after acid hydrolysis and by electrospray (ESI) mass spectrometry.

[0052] Generally, if amino acids are connected by peptide bonds, the peptide is represented with the N-terminal amino group appearing on the left and the C-terminal carboxyl group appearing on the right. Conotoxin peptide variants according to the present application are represented in such a way.

[0053] Pharmaceutical compositions: The present application also relates to pharmaceutical compositions comprising a mutant of the mu-conotoxin CnIIIC according to the present application.

[0054] The pharmaceutical compositions can be used in research, diagnostic, palliative or therapeutic medicine for diseases or conditions associated with pain, including but not limited to:

[0055] (1) Acute pain related diseases: Traumatic pain: such as fracture, soft tissue injury, incision, burn, etc. Postoperative pain: incision site pain caused by surgery. Acute inflammatory diseases: acute appendicitis, acute cholecystitis, acute pancreatitis. Acute infectious diseases: urinary tract infection, otitis media, upper respiratory tract infection (such as tonsillitis). Acute headache: such as migraine, cluster headache, etc.

[0056] (2) Chronic pain related diseases: commonly seen in spinal diseases, herniated disc, spinal arthritis, etc. Arthritis related pain: osteoarthritis, rheumatoid arthritis; neuropathic pain, diabetic neuropathy, sciatica, trigeminal neuralgia, fibromyalgia, chronic migraine, chronic neck and shoulder pain.

[0057] (3) Neuropathic pain: peripheral neuropathy: such as diabetic neuropathy, alcoholic neuropathy; spinal cord injury.

[0058] In one embodiment, the pharmaceutical composition containing a therapeutically effective amount of the mutant of μ-conotoxin peptide CnIIIC of the present application is formulated and administered in a manner that is convenient for pharmaceutical use, taking into account the clinical condition of the individual patient, the delivery site, the method of administration, the schedule of administration and other factors known to the physician. Therefore, "effective amount" for the purpose herein is determined by the consideration of these aspects.

[0059] Mode of administration:

[0060] The pharmaceutical composition containing a therapeutically effective amount of the mutant of μ-conotoxin peptide CnIIIC of the present application can be administered by:

[0061] Parenteral administration: including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intrathecal and intra-articular injection and infusion.

[0062] Oral administration: suitable for chronic pain management.

[0063] Intracisternal administration: suitable for central nervous system related pain.

[0064] Intrathecal administration: sustained administration is achieved by slow-release preparation, reducing the frequency of administration.

[0065] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, capsule material or any type of formulation aid.

[0066] Chinese meaning of English abbreviations:

[0067] "μ-CnIIIC" refers to the wild type μ-conotoxin;

[0068] "DCM" refers to dichloromethane;

[0069] DIC refers to N,N-diisopropylcarbodiimide;

[0070] DMF refers to N,N-dimethylformamide;

[0071] HPLC refers to high performance liquid chromatography;

[0072] MeOH refers to methanol;

[0073] MTBE refers to methyl tert-butyl ether;

[0074] Oxyma refers to 2-hydroxy-5-norbornene-2-carboxylic acid ethyl ester;

[0075] TFA refers to trifluoroacetic acid;

[0076] Fmoc refers to 9-fluorenylmethoxycarbonyl;

[0077] MS refers to mass spectrometry;

[0078] IC refers to the half-inhibitory concentration value of the pharmaceutical preparation being detected. 50

[0079] K reagent: cleavage solution, prepared according to the volume ratio of TFA: phenol: water: benzyl mercaptan: ethanedithiol = 82.5:5:5:5:2.5.

[0080] The amino acid sequences of the mu-conotoxin peptide CnIIIC mutants prepared in the examples are shown in Table 1:

[0081] Table 1 Sequences of mu-conotoxin active peptides and mutants thereof

[0082] Note: Pyr refers to pyroglutamic acid.

[0083] The amino acid sequence of the wild-type conotoxin active peptide is shown in SEQ ID NO. 21, and the amino acid sequences of the conotoxin peptide mutants of the present application are shown in SEQ ID NO. 1-20. The nucleotide sequence encoding the conotoxin peptide mutants can be obtained without making creative efforts through the amino acid sequences of the conotoxin peptide mutants of the present application, and thus the nucleotide sequence encoding the conotoxin peptide mutants also falls within the scope of the present application. Fermentation production of the active peptides by inserting the nucleotide sequence encoding the conotoxin peptide mutants into a vector or a strain also falls within the scope of the present application.

[0084] Example 1 Synthesis and purification of mu-conotoxin peptide CnIIIC and variants thereof

[0085] ​The synthesis of the analogs was completed by solid-phase peptide synthesis technology, the formation of disulfide bond was completed by liquid-phase oxidation, and finally the μ-type conotoxin peptide CnIIIC analog was obtained by high performance liquid chromatography purification and freeze-drying.

[0086] Synthesis and purification of peptides SEQ ID NO. 1 to SEQ ID NO. 21:

[0087] The specific steps are as follows:

[0088] ① 1.500 g (1.005 mmol) of Rink Amide MBHA resin (containing 1% DVB crosslinking agent, 100-200 mesh, 0.67 mmol / g) was weighed into a 60 mL peptide solid-phase reactor, then 15 mL of DCM solution was added to the reactor, the shaking speed of the shaking table was set to 550 r / min, and after shaking for 45 min, the solution was drained. The resin was washed with DMF solution twice, the washing solvent volume was 15 mL / time, the washing time was 3 min / time, and the shaking speed of the shaking table was 500 r / min.

[0089] ② After washing, the solvent was drained, 15 mL of 20% piperidine / DMF solution was added to the resin in the reactor to remove the Fmoc protecting group of the resin, and the shaking reaction was carried out at 500 r / min and 25°C for 5 min, and the solution was drained. Then 15 mL of 20% piperidine / DMF solution was added to the resin, and the shaking reaction was carried out at 500 r / min and 25°C for 15 min, and the solution was drained. The resin was washed with DMF solution (15 mL / 3 min / time) for 5 times.

[0090] ③ Fmoc-protected amino acids were used as monomers, and the amino acids were sequentially connected to the resin. 1.766 g of Fmoc-protected amino acid (3 eq, 3.015 mmol), 0.428 g of Oxyma (3 eq, 3.015 mmol) was weighed into a 50 mL beaker, dissolved in 10 mL of DMF solution, 0.467 mL of condensing agent DIC (3 eq, 3.015 mmol) was added to the amino acid solution and activated for 5 min, then added to the above deprotected resin, and the shaking reaction was carried out at 500 r / min and 25°C for 1 h; after the reaction was completed, the resin was washed with DMF solution (15 mL / 3 min / time) for 5 times.

[0091] ④ Peptide chain extension

[0092] According to the sequence composition, repeat steps 2 and 3 until the last amino acid is coupled. Remove the Fmoc protecting group, and wash the resin with DMF solution (15 mL / 3 min / time) for 5 times. Then, wash the resin according to the following procedure: DCM x 5 times (15 mL / 3 min / time), MeOH x 5 times (15 mL / 3 min / time), and finally, the resin is shrunk and placed in a vacuum drying box at 25°C until the weight is constant.

[0093] 5. Cleavage

[0094] Weigh the dried resin obtained in the above process 4, and add the pre-prepared and pre-cooled K reagent cleavage solution according to the proportion of 10 mL of cleavage solution per gram of peptide resin. Shake at 300 r / min for 3 h at 25°C in the dark. After the reaction is completed, according to the proportion of cleavage solution / methyl tert-butyl ether = 1:10 (v / v), slowly drop the cleavage solution into the pre-cooled MTBE solution, and white precipitate is generated. Then, centrifuge at 500 rpm / min, discard the supernatant, add new MTBE solution, shake, centrifuge, discard the supernatant, repeat the above centrifugation process 5 times, collect the muddy white precipitate, and vacuum dry at 25°C until the weight is constant. Finally, a white solid crude peptide is obtained.

[0095] 6. Cyclization

[0096] Weigh the white solid crude peptide obtained in the above process 5, add 100 mL of sodium phosphate dibasic / guanidine hydrochloride buffer solution, adjust the pH to 8.2, and stir at room temperature for 24 h. Monitor the reaction progress by HPLC. After the reaction is completed, it can be directly purified by HPLC.

[0097] 7. Purification of the peptide by preparative HPLC

[0098] Directly inject the cyclization reaction solution in the above process 6, and complete the sample purification according to the gradient elution program in Table 2, wherein the mobile phase A is 80% acetonitrile / water (containing 0.1% TFA), the mobile phase B is water (containing 0.1% TFA), the detection wavelength is 220 nm, the flow rate is 10 mL / min, the column specifications are 20 x 250 mm, 10 μm,

[0099] Table 2 Purification elution program of crude cyclization solution

[0100] After the polypeptide is synthesized, purified and tested, it is confirmed that the polypeptide sequence shown in Table 2 is obtained, the target peptide fraction is combined, and after freeze-drying, it is used for subsequent experiments.

[0101] Example 2 Biological activity determination of μ-type conotoxin peptide CnIIIC variant

[0102] The mice used in the experiments of the present application are adult male Kunming mice, which are purchased from the Lanzhou Institute of Animal Health, Chinese Academy of Sciences. The animal experiments are approved by the Ethics Committee of Lanzhou Peitugu Research Institute.

[0103] The mice are allowed to eat and drink freely one week before the test. Intramuscular injection is performed on the right tibialis anterior muscle group of the mice using a 50 μL microsyringe (30G needle) with a volume of 20 μL. An equal volume of normal saline is injected as a negative control, and wild-type μ-CnIIIC is injected as a positive control. Multiple concentration gradients are set up: 25 μM, 50 μM, 100 μM, 250 μM, and 500 μM. The onset time, duration, and behavior of the mice after injection are observed, including toe clamping (muscle contraction inhibition), leg dragging / paralysis (narcotic effect), and death behavior to determine the activity of the sample.

[0104] The toe clamping (muscle contraction inhibition) behavior of the mice is analyzed using the mouse toe spread score test (DAS). The characteristic shock response of the mice to stretch the hind limbs and the degree of spread is triggered by suspending the mice by the tail. After injecting normal saline, wild-type μ-CnIIIC, and different concentrations of μ-type conotoxin peptide mutants into the right tibialis anterior muscle group of the mice, the toe spread of the left and right hind limbs is measured as a function of time, and scored based on a 5-point scale (0 indicates that the mouse is suddenly lifted and suspended in the air, with all 5 toes fully spread; 1 indicates that only 2 toes (the first and second toes) are clamped; 2 indicates that 3 toes (the first, second, and third toes) are clamped; 3 indicates that 4 toes are clamped except for the fifth toe; and 4 indicates the maximum reduction of toe spread and leg stretch). The leg dragging / paralysis (narcotic effect) behavior of the mice is observed by placing them in a new environment after injecting them with the drug, which triggers their natural desire to explore. In the early stage of onset, the lower body of the mouse crawls on the ground, and in the later stage, the entire body is paralyzed and cannot be moved even by pushing with the hand.

[0105] After injecting 50 μM, 100 μM, and 250 μM concentrations of wild-type μ-CnIIIC into the hind legs of multiple mice, all the mice survived and exhibited toe clamping behavior. Death only occurred after injecting 500 μM wild-type μ-CnIIIC into the hind legs of the mice. The animal experiment results of wild-type μ-CnIIIC at different concentrations are shown in Table 3.

[0106] Table 3 Animal experiment results of wild-type μ-CnIIIC Note: " / " indicates that the mice showed no symptoms after drug injection, so the onset time and duration cannot be calculated.

[0107] A plurality of mice were injected with 25 μM, 50 μM, 100 μM, 250 μM μ-CnIIIC conotoxin peptide variants, respectively, and the onset time, duration and behavior of the mice after injection were observed. The results of the μ-CnIIIC conotoxin peptide variants still active at 25 μM are summarized in Table 4.

[0108] Table 4 Animal experiment results of wild-type μ-CnIIIC and its variants at 25 μM Note: " / " indicates that the mice had no symptoms after drug injection, so the onset time and duration could not be calculated.

[0109] According to the results in Tables 3-4, after the mice were injected with 25 μM conotoxin peptides having the sequences shown in SEQ ID NO. 1-13 in the back legs, the mice exhibited obvious dragging behavior, and then died. However, the mice injected with 500 μM μ-CnIIIC in the back legs only died, which was 20 times the death concentration of the conotoxin peptides having the sequences shown in SEQ ID NO. 1-13, indicating that the activity of the conotoxin peptides having the sequences shown in SEQ ID NO. 1-13 was 20 times that of wild-type μ-CnIIIC. In addition, the mice injected with conotoxin peptides having the sequences shown in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 13 also exhibited toe clenching behavior, indicating that the conotoxin peptides having the sequences shown in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 13 also had muscle contraction inhibiting activity. After the mice were injected with 25 μM conotoxin peptides having the sequences shown in SEQ ID NO. 14-20 in the back legs, the mice exhibited toe clenching behavior, obvious dragging behavior and flaccid behavior, and eventually died due to the high dose. This indicates that the conotoxin peptides having the sequences shown in SEQ ID NO. 14-20 still exhibit muscle contraction inhibiting and paralyzing activity at low concentrations, and have higher activity than wild-type μ-CnIIIC.

[0110] Example 3 Manual patch clamp technique to detect the effect of μ-CnIIIC mutants on Nav1.4 channel current

[0111] Wild-type μ-CnIIIC can effectively block Nav1.4 ion channels. To prove the target and activity of the designed sequence, the patch clamp technique was used to determine the effect of the μ-type conotoxin peptide CnIIIC variants with higher activity in Example 2 on Nav1.4 ion channel current. The specific steps are as follows:

[0112] Obtaining of Nav1.4 cells:

[0113] The Nav1.4 cell lines were kept within 70% of the maximum density of logarithmic growth for electrophysiological experiments. All reagents were pre-warmed to 37°C before use. The old medium was discarded from the 6 cm culture dish, 1 mL PBS was added, the dish was gently shaken, the bottom of the dish was washed, then removed, followed by the addition of 1 mL trypsin, the dish was gently shaken, and the entire cells were covered. Incubate at 37°C for 2-3 min, gently blow the cells with a pipette to suspend the adherent cells. The cell suspension was transferred to a centrifuge tube and centrifuged at 1000 rpm / min for 5 min, the cell concentration was adjusted to 2x10 3 6 cells / mL, 500 μL of cell solution was inoculated into the cell climbing sheet of the 24-well plate, and the membrane patch clamp detection experiment was performed after the cells were well adhered.

[0114] The sample was configured at a concentration of 10 mM, and deionized water was used to prepare the test solution at concentrations of 1000 nM, 300 nM, 100 nM, 30 nM, 10 nM, 3 nM and 1 nM.

[0115] The whole-cell patch clamp recording experiment used Patchmaster software to collect and store Nav1.4 sodium current data on a computer through an EPC-10 amplifier. The specific test process is as follows:

[0116] (1) First, use tweezers to remove the cell climbing sheet from the cell culture dish, add extracellular fluid, and place it in the bath on the inverted microscope stage. Then use a P-1000 microelectrode puller to pull a glass microtube, fill 1 / 3 of the volume of the glass microtube (recording electrode) with intracellular fluid, and place it in the electrode holder; use a motorized micromanipulator (Scientifica-Double 1U) to contact the recording electrode to the cell surface. After the recording electrode and the cell membrane are sealed with a resistance >1 GΩ, break the membrane to form a whole-cell recording mode, and then compensate for the membrane capacitance (Cs) and series resistance (Rs) after the membrane is stable.

[0117] (2) Stimulation program:

[0118] ① The clamping voltage is -120 mV, a -120~ -10 mV, 10 mV step, 8000 ms duration square wave train stimulation is given, then step to -10 mV, time is 30 ms, finally restore to -120 mV. With membrane potential as abscissa, relative current I / Imax as ordinate, Boltzmann process I / Imax = 1 / {1+exp[(V-V1 / 2) / k]} is used for fitting, to get the steady-state inactivation curve (V1 / 2 is the condition pulse voltage when half of the channel is inactivated, k is the slope factor).

[0119] 2. Clamp at -120 mV, depolarize to 0 mV for 40 ms to elicit resting state sodium current, then step to the condition pulse voltage at which the half of the inactivated channels are inactivated (V1 / 2), for 8000 ms, repolarize to -120 mV for 30 ms, then depolarize to 0 mV for 40 ms to elicit half-inactivated state sodium current, finally return to -120 mV. Record the current every 20 s.

[0120] (3) At room temperature, record the Nav1.4 sodium channel current before drug administration. After the control current value reaches a steady state, i.e., the latest 4 current recording lines coincide, use cumulative drug administration method to add negative (extracellular fluid) and 7 drug concentrations (from low to high) in turn.

[0121] (4) Data analysis

[0122] Extract the original data Nav1.4 current peak value from PatchMaster software, and the calculation formula of current inhibition rate is as follows:

[0123] Peak current inhibition rate = (1 - Peak current compound / Peak current vehicle), calculate the mean and standard error for each concentration, and the concentration-effect relationship is fitted by Hill equation:

[0124] I = Imax · {1 / [1+(C1 / 2 / [C])h]};

[0125] Where [C] represents the drug concentration, C1 / 2 is the half-inhibitory concentration (IC50), h is the Hill coefficient, and the analysis and statistics are completed using Graphpad Prism 8.0.2 software. Tetrodotoxin is used as a positive control. The results of manual patch clamp technique for detecting the half-inhibitory concentration (IC50) of compounds on Nav1.4 ion channels are shown in Table 5.

[0126] Table 5 IC50 results of compounds on Nav1.4 ion channels detected by manual patch clamp technique

[0127] As can be seen from Table 5, compared with the half-inhibitory concentration (196.33 nM) of wild-type μ-CnIIIC on Nav1.4 ion channel in the resting state, the IC50 (8.439 nM) of the μ-CnIIIC mutant represented by SEQ ID NO. 1 prepared in the application is 23.2 times thereof, the IC50 (19.168 nM) of the conotoxin represented by SEQ ID NO. 4 is 10.2 times thereof, the IC50 (7.7464 nM) of the conotoxin represented by SEQ ID NO. 5 is 25.3 times thereof, and the IC50 (9.1439 nM) of the conotoxin represented by SEQ ID NO. 6 is 21.5 times thereof. It can be seen that the conotoxins represented by SEQ ID NO. 1, SEQ ID NO. 4-6 have stronger inhibitory activity on Nav1.4, further illustrating that the conotoxins with unchanged structure can specifically block Nav1.4 channel.

[0128] Example 4 Transdermal activity determination of wild-type μ-CnIIIC and μ-type conotoxin peptide CnIIIC variants

[0129] (1) In vitro transdermal experiment

[0130] The pig skin used in the application is the back and abdominal skin of a 1-month-old Bama miniature pig (purchased from Jingde Agricultural Products Sales Co., Ltd. in Linxi County), with a thickness of 0.8-1 mm. A vertical static diffusion cell is used to test the in vitro transdermal absorption of the conotoxin. The pig skin is cut into blocks with a size of 2 cm, and is fixed between the supply chamber and the receiving chamber of the diffusion cell (with an area of about 1 cm 2 ). PBS (pH 7.4) is added to the receiving chamber as the receiving liquid, 300 μL of PBS (pH 7.4) is added to the supply chamber, and the system is equilibrated at 32±1°C for 30 min. Then, the PBS in the supply chamber is discarded, and 300 μL of conotoxin solution (100 uM, dissolved in 20 mM HEPES, pH 5.6) is added. Wild-type μ-CnIIIC (SEQ ID NO. 21) is used as a positive control, and the transdermal experiment is performed at 32±1°C for 16 h.

[0131] (2) Extraction and quantitative analysis of polypeptides in pig skin

[0132] After the transdermal experiment, the pig skin is removed, and the stratum corneum is peeled off three times with adhesive tape to remove the residual sample on the surface of the pig skin. The pig skin is cut into small pieces, 2 mL of extraction solution (0.1% HAC) is added, and the mixture is shaken overnight at room temperature. The extraction solution is purified by a WCX solid-phase extraction cartridge (150 mg, 6 mL). After centrifugation, the supernatant is taken and loaded onto the WCX solid-phase extraction cartridge (pre-activated with 6 mL of methanol and 6 mL of water). Then, 12 mL of water and 12 mL of methanol are used for elution, and the eluate is discarded. Finally, 6 mL of 0.1% formic acid is used for elution, and the eluate is concentrated to 100 μL.

[0133] (3) Polypeptide sample quantitative analysis (HPLC determination)

[0134] The elution concentrate of the above 2) process is filtered with a 0.22 μm needle filter, transferred into a sample injection bottle, injected, and sample determination is completed according to the gradient elution program in Table 6.

[0135] wherein the mobile phase A is water (0.1% trifluoroacetic acid), the mobile phase B is acetonitrile; the detection wavelength is 210 nm; the flow rate is 1 mL / min; the column specification is InterClone ODS3 C18 (250*4.6 mm, 5 μm, ); the injection volume is 50 μL.

[0136] Table 6 Elution program

[0137] (4) Transdermal activity determination of conotoxin peptide CnIIIC and variants thereof

[0138] Using wild-type μ-CnIIIC as a positive control, the concentration of the conotoxin peptide and variants thereof in the extract is calculated according to the standard curve, and the transdermal activity determination results are shown in Table 7.

[0139] Table 7 Transdermal results of conotoxin peptide CnIIIC and variants thereof

[0140] As can be seen from Table 7, the above conotoxin peptide variants all have transdermal activity, and the conotoxin peptide shown in SEQ ID NO. 3 has better transdermal effect than the natural conotoxin peptide.

[0141] In summary, the μ-type conotoxin peptide mutants of the present application have significant advantages in terms of transdermal performance, specific targeting, muscle relaxation, anti-aging, analgesia and anesthesia, etc., and have broad clinical application prospects, especially in the treatment fields of pain management, skin anti-aging and muscle relaxation, etc.

[0142] The above only describes the preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A mutant of the μ-conotoxin peptide Cnlllc, characterized in that, The amino acid sequence of the mu-conotoxin peptide CnIIIC mutant is as follows: Xaa1-Xaa2-Cys-Cys-Xaa3-Gly-Pro-Lys-Gly-Cys-Xaa4-Ser-Lys-Trp-Cys-Arg-Xaa5-His-Ala-Arg-Cys-Cys-Xaa6; wherein, Xaa1 is any one of d-Arg, d-Lys and d-His or Xaa1 is absent; Xaa2 is any one of Gly, Arg, d-Arg, Lys and d-Lys or Xaa2 is absent; Xaa3 is Asn or Arg; Xaa4 is Ser or Tyr; Xaa5 is Gly or Ser; Xaa6 is d-Arg or Xaa6 is absent.

2. The mutant of the mu-conotoxin peptide CnIIIC according to claim 1, characterized in that The amino acid sequence of the mu-conotoxin peptide CnIIIC mutant is as shown in any one of SEQ ID NO. 1-20.

3. The mutant CnIIIC conotoxin peptide of claim 1, wherein The mu-conotoxin peptide CnIIIC mutant further comprises a fluorescently labeled peptide.

4. A separated fusion protein, characterized in that, The mu-conotoxin peptide CnIIIC mutant as claimed in any one of claims 1-3.

5. An isolated polynucleotide, comprising, The mu-conotoxin peptide CnIIIC mutant as claimed in any one of claims 1-3.

6. A nucleic acid construct, characterized in that, The polynucleotide as claimed in claim 5.

7. A transformed cell, comprising, The polynucleotide as claimed in claim 5 or the nucleic acid construct as claimed in claim 6.

8. A pharmaceutical composition, characterized by, The mu-conotoxin peptide CnIIIC mutant as claimed in any one of claims 1-3.

9. Use of the mu-conotoxin peptide CnIIIC mutant as claimed in any one of claims 1-3 in any one of: (1) the preparation of a medicament for the treatment or prevention of pain; (2) the preparation of a medicament for the treatment or prevention of a disease associated with sodium ion channels; (3) the preparation of a medicament for anesthesia; (4) the preparation of a cosmetic product.

10. Use according to claim 9, characterized in that, (2) the disease associated with sodium ion channels is any one of epilepsy, arrhythmia, muscle paralysis, myoclonus dystonia, and autism spectrum disorder.

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