A mutant of conotoxin KIIIA for inhibiting voltage-gated sodium channel 1.4, a preparation method thereof, and an application thereof

Abstract

The present invention provides a conotoxin KIIIA mutant with an amino acid sequence of SEQ ID NO: 1: Xaa1CXaa2CSXaa3KWCXaa4DHXaa5RXaa6C*, where "*" represents C-terminal amidation. The present invention also provides a preparation method and applications thereof. The present invention not only retains the activity, conformation and stability of the wild polypeptide, but is also easier to synthesize compared to the wild polypeptide.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a mutant of conotoxin KIIIA for inhibiting voltage-gated sodium channel 1.4. Background Art

[0002] Voltage-gated sodium (Nav) channels (voltage-gated sodium channels, VGSCs) are a class of large voltage-dependent transmembrane proteins that regulate the rising phase of the action potential in excitable cells. VGSCs consist of one α subunit (pore) and one or more β subunits (β1-β4). To date, nine mammalian VGSC α subunits (Nav1.1-Nav1.9) have been identified, each with different tissue distributions, biophysical properties, and sensitivities to tetrodotoxin (TTX). There are two main mechanisms by which toxins act on VGSCs: (1) changing ion permeability by blocking the pore with the toxin itself; (2) binding to an allosteric coupling site on the channel through the toxin, causing a conformational change at this site, altering the gating properties of the channel, and changing the open, closed, and inactivated balance of the channel. Nav1.4 is mainly distributed in skeletal muscle cells, and overexcitation can cause muscle diseases such as congenital myasthenia and congenital myotonia. Inhibiting Nav1.4 can relax muscles and paralyze facial muscles to achieve an anti-wrinkle effect. The research by Gregory N. Filatov et al. found that regulating the voltage dependence of Nav1.4 and Nav1.5 inactivation contributes to the electrical inexcitability of muscle fibers in an animal model of critical illness myopathy, thus contributing to the treatment of critical illness myopathy. The experimental results of the Mohammad-Reza Ghovanloo research group showed that Nav1.4 is crucial for skeletal muscle contraction, and the presence of a saturating level of CBD reduces skeletal muscle contraction and has therapeutic value for myotonia. Considering the different important roles played by each Nav subtype, how to improve the selectivity of inhibitors for different sodium channel subtypes has become the most critical scientific issue in the research and development of targeted Na ion channel analgesic drugs and other potential drugs. In 2017, the young scientist team of Yan Ning at Tsinghua University resolved the cryo-EM structure of EeNav1.4 from the electric eel, making a great contribution to the milestone of the structural analysis of mammalian sodium channels and providing conditions for studying the binding mode and structure-activity relationship of μ-conotoxin.

[0003] A class of bioactive polypeptides extracted from the venom of the marine carnivorous mollusk Conus, rich in disulfide bonds, small in molecular weight, can specifically act on ion channels and receptors, and have high subtype selectivity. Such short peptides are called conotoxins (CTx). Among them, μ-conotoxin can specifically act on each subunit of VGSCs. By binding to the subunits, it can change the structure and function of the Nav channel. μ-KIIIA is derived from Conus kinoshitai produced in the Indo-Pacific region, consists of 16 amino acids, contains three pairs of disulfide bonds, and wild-type KIIIA has strong inhibitory activity against multiple Nav subtypes including Nav1.4. The research on μ-CTX mainly uses the solid-phase synthesis method. The formation of three pairs of disulfide bonds requires three-step oxidation, and the experimental process is cumbersome and difficult to succeed. Currently, there is no efficient synthesis method for conotoxins with three pairs of disulfide bonds. Generally speaking, μ-conotoxin is of great significance for in-depth study of the structure and function of Nav channels and the development of drugs for Nav channel-related diseases.

[0004] In the research on conotoxins containing three pairs of disulfide bonds, Tiffany S. Han et al. first explored the activities of three mutants and the wild-type KIIIA by alanine mutation to delete three pairs of disulfide bonds respectively, and obtained two-pair disulfide bond analogues with equivalent activities and simplified synthesis steps; the Keith K. Khoo research group designed and synthesized 6 truncated μ-KIIIA analogues, which contain a single lactam bridge at different positions. The helicity of these lactam analogues was analyzed by nuclear magnetic resonance spectroscopy, and their activities against mammalian VGSC subtypes Nav1.1 to 1.7 were tested. Thereafter, the research group also determined the KIIIA disulfide connectivity by direct mass spectrometry collision-induced dissociation cleavage of disulfide-bonded polypeptides. The results showed that the preferred disulfide bond pattern for in vitro folding of KIIIA is 1-5 / 2-4 / 3-6, but other disulfide isomers are also effective sodium channel blockers. Existing conotoxins targeting Nav1.4 mostly contain three pairs of disulfide bonds, with complex synthesis steps, high synthesis costs, low yields, and high activities against other Nav subtypes. These factors have greatly hindered the further development and utilization of conotoxins targeting Nav1.4. Summary of the Invention

[0005] The purpose of the present invention is to provide a conotoxin KIIIA mutant containing only two pairs of disulfide bonds, and this mutant has a strong inhibitory effect on voltage-gated sodium channel 1.4.

[0006] The present invention provides a conotoxin KIIIA mutant for specifically inhibiting voltage-gated sodium ion passage through 1.4, and its amino acid sequence is SEQ ID NO:1

[0007] Xaa1CXaa2CSXaa3KWCXaa4DHXaa5RXaa6C*

[0008] Wherein, Xaa1 is selected from any one of A, R or Dapa;

[0009] Xaa2 is selected from any one of N, R, Dab or Dapa;

[0010] Xaa3 is selected from any one of S, T or Dab;

[0011] Xaa4 is selected from any one of R or V;

[0012] Xaa5 is selected from any one of S or R;

[0013] Xaa6 is selected from any one of A, R or T;

[0014] "*" indicates amidation at the C-terminus;

[0015] It does not include the amino acid sequence formed when Xaa1 is A, Xaa2 is N, Xaa3 is S, Xaa4 is R, Xaa5 is S and Xaa6 is A;

[0016] Dapa is L-2,3-diaminopropionic acid; Dab is L-2,4-diaminobutyric acid.

[0017] In one embodiment according to the present invention, the cysteine residues at the 2nd and 9th positions form a disulfide bond; the cysteine residues at the 4th and 16th positions form a disulfide bond.

[0018] In one embodiment according to the present invention, the amino acid sequence is selected from any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19;

[0019] Preferably, it is selected from any one of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO:17.

[0020] The present invention also provides a method for preparing the above-mentioned conotoxin KIIIA mutant, comprising:

[0021] 1) Synthesizing a resin peptide by Fmoc solid-phase synthesis method, and amidating the C-terminus of the resin peptide;

[0022] Among them, cysteines at the 2nd and 9th positions are protected with a first thiol protecting group, and cysteines at the 4th and 16th positions are protected with a second thiol protecting group. The first thiol protecting group and the second thiol protecting group are different and each independently selected from one of triphenylmethyl (Trt) or acetamidomethyl (Acm);

[0023] 2) Cleaving the resin peptide to obtain a linear peptide;

[0024] 3) After performing disulfide bond folding based on the specific groups of the first thiol protecting group and the second thiol protecting group respectively, purifying to obtain the product.

[0025] In one embodiment according to the present invention, step 2) further includes:

[0026] First, oxidize cysteine with triphenylmethyl (Trt) as the thiol protecting group by air oxidation method; then, oxidize cysteine with acetamidomethyl (Acm) as the thiol protecting group by iodine oxidation method.

[0027] In one embodiment according to the present invention, the iodine oxidation method is carried out by a method including the following steps:

[0028] Dissolve the crudely peptide after air oxidation in a reaction solvent, which is a mixture of water, acetonitrile and TFA in a volume ratio of 5:5:0.01, and then add an iodine-acetonitrile solution; stir thoroughly in a sealed environment at 28°C for reaction, and then slowly add an ascorbic acid aqueous solution to neutralize the excessive iodine, and separate by HPLC to obtain the product.

[0029] The present invention also provides the application of the above-mentioned conotoxin KIIIA mutant in the preparation of a preparation for inhibiting voltage-gated sodium channel 1.4. Preferably, the preparation is a muscle relaxant drug or an anti-wrinkle agent.

[0030] The present invention further provides a preparation for inhibiting voltage-gated sodium channel 1.4, which contains the above-mentioned conotoxin KIIIA mutant.

[0031] The beneficial effects of the above technical solutions of the present invention are as follows:

[0032] The present invention has realized for the first time the rational design of two pairs of disulfide bond analogs of conotoxin targeting Nav1.4 channel, greatly improving the synthesis efficiency of conotoxin and reducing the synthesis cost.

[0033] The blocking intensity of mutants of KIIIA on Nav1.4 ion channel current was measured by in vitro experiments, and the results showed that the mutants of KIIIA provided by the present invention had good targeted inhibitory effects on Nav1.4 ion channels and could be used to prepare muscle relaxant drugs or as aesthetic anti-wrinkle agents. Description of the Drawings

[0034] Figure 1 Schematic diagram of the inhibitory activity of the KIIIA analog KIIIA[C1A,C9A] and KIIIA analogs with one pair of disulfide bonds deleted at a concentration of 1 μM on Nav1.4;

[0035] Figure 2 Schematic diagram of the structure of KIIIA[C1A,C15A];

[0036] Figure 3 Schematic diagram of the binding mode of the conotoxin KIIIA[C1A,C15A] to Nav1.4;

[0037] Figure 4 Binding mode diagram between analogs at positions 1 and 15 of KIIIA[C1A,C15A] and Nav1.4;

[0038] Figure 5 Specific flow chart of solid-phase chemical synthesis and oxidative folding of polypeptides;

[0039] Figure 6 Blocking ability diagram of various μ-KIIIA analogs at a concentration of 1 μM on Nav1.4;

[0040] Figure 7 Representative current trace diagram under the inhibition of 1 μM of KIIIA analog on Nav1.4;

[0041] Figure 8 Inhibitory concentration-effect curve of [S13R]KIIIA[C1A,C15A] on Nav1.4; where, IC 50 = 130.24 ± 14.69 nM. Detailed Embodiments

[0042] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the drawings and specific embodiments.

[0043] Example 1 Mechanism of Action and Molecular Dynamics Study of KIIIA

[0044] The binding mode of KIIIA to each Nav subtype can be obtained by homology modeling using Nav1.2 / KIIIA (PDB ID: 6J8E) as a template. Then, molecular dynamics (MD) simulation was used to further optimize the binding mode of KIIIA analogs on each subtype. After the MD simulation-optimized complex model was subjected to energy calculations (including MMGBSA and Umbrella sampling methods), the optimal binding mode of KIIIA analogs was further determined. Finally, the binding mode of KIIIA and its analogs was verified by site-directed mutagenesis (or published site-directed mutagenesis data). The disulfide bonds at different positions of the wild-type μ-conotoxin KIIIA peptide chain were deleted to obtain the amino acid sequence μ-conotoxin KIIIA[C1A,C15A] with activity similar to that of the wild-type toxin. The inhibitory activities of KIIIA analog KIIIA[C1A,C9A] and KIIIA analogs with one pair of disulfide bonds deleted respectively at a concentration of 1 μM against Nav1.4 are as Figure 1 shown.

[0045] Among them, KIIIA-1 is KIIIA[C1A,C15A]. The sequence KIIIA[C1A,C15A] was selected, and the crystal structure of the complex of μ-conotoxin KIIIA and Nav1.2 (PDB ID: 6J8E) was used as a template to construct the complex model of KIIIA[C1A,C15A] and Nav1.4. The structure of KIIIA[C1A,C15A] is as Figure 2 shown.

[0046] Since molecular docking cannot consider the flexibility of polypeptides and proteins and solvent effects, it is necessary to perform MD optimization on the complex model obtained by docking. The MD simulation was carried out using AMBER16 software. The force field was selected as ff14SB, the water molecule model was selected as TIP3P, and Na+ / Cl- ions were added to the whole system to neutralize the system. After the system was established, energy optimization was performed on the whole system to remove van der Waals collisions. First, it was constrained optimization. After the first round of energy optimization was completed, the constraint force on the solute molecule was cancelled, and unconstrained optimization was performed. Then MD simulation was carried out, which was divided into two processes: heating and equilibration. All hydrogen bonds used the SHAKE algorithm, and the experimental step size during the heating process and the equilibration process was set to 2 fs. The PME method was used to handle long-range electrostatic interactions. The VMD software was used to analyze the MD trajectory. The PYMOL software was used for analysis. The binding mode of the complex of conotoxin KIIIA[C1A,C15A] and Nav1.4 is as Figure 3 shown.

[0047] Example 2 Structure optimization and structure-activity relationship of KIIIA

[0048] After determining the binding mode of conotoxin and its related analogs to the receptor, the key amino acid residues affecting the activity and selectivity of conotoxin are analyzed, and side chain replacement or design is carried out based on traditional medicinal chemistry knowledge to improve the activity and selectivity for specific receptor subtypes. Taking the mutation at position 15 as an example, compared with the interaction mode of KIIIA[C1A,C15A] and Nav1.4 ( Figure 4 B therein), the R15 side chain in the [A15R]KIIIA[C1A,C15A] mutant forms multiple salt bridges and hydrogen bond interactions with D476 ( Figure 4 A therein) and D727 ( Figure 4 C therein) in the receptor respectively. The activity test results show that the activity of the [A15R]KIIIA[C1A,C15A] mutant is enhanced. There is a hydrogen bond interaction between the T15 side chain and serine at position 13 in the [A15T]KIIIA[C1A,C15A] mutant ( Figure 4 D therein), which strengthens the structural stability of conotoxin itself and weakens its binding ability to the receptor protein to a certain extent. The activity test results show that the activity of the [A15T]KIIIA[C1A,C15A] mutant is slightly weakened; while the mutation of [A1R]KIIIA[C1A,C15A] ( Figure 4 F therein) does not have an obvious impact on the binding mode between the complexes. The binding mode between the analogs at positions 1 and 15 of KIIIA[C1A,C15A] and Nav1.4 is as shown in Figure 4 .

[0049] Calculations are performed on the amino acids at specific positions to obtain variants with potentially improved activity and selectivity. The specific structure optimization methods are shown in Table 1 for subsequent activity tests.

[0050] Table 1 Mutation sites and sequences of KIIIA analogs targeting Nav1.4

[0051]

[0052]

[0053] Example 3 Synthesis method of conotoxin analogs

[0054] 1. Orthogonal protection synthesis method

[0055] The synthesis of KIIIA analogs mainly adopts the strategy of orthogonal protection. Since it contains 2 pairs of disulfide bonds, in order to avoid the formation of disulfide bond isomers, the strategy of orthogonal protection is adopted during the synthesis. Trt and Acm are respectively used to protect the 2 pairs of cysteines in the sequence. Ammonium bicarbonate and iodine are respectively used for oxidation during the oxidation process. Finally, the obtained product needs to be verified by MS, purified and analyzed by HPLC, etc.

[0056] 2. Specific operation steps:

[0057] 1) Resin peptide synthesis:

[0058] The mutant was chemically synthesized in full by the Fmoc solid-phase synthesis method, and the C-terminus of KIIIA was amidated. Rink-Amide resin was used, and the synthesis direction was from the C-terminus to the N-terminus. The resin loading was 0.661 mmol / g, the equivalent was selected as 0.2 mmol for amino acids, and the equivalent of the condensing agent was fed according to 4 times the molar amount of the resin loading, and the equivalent of the activator was fed according to 8 times the molar amount of the resin loading. First, 0.303 g of resin was weighed and added to a solid-phase reaction tube. The resin was swollen overnight at room temperature or reacted in a constant-temperature shaker for 2 h with a 1:1 volume ratio of dichloromethane (DCM) and N,N-dimethylformamide (DMF) to activate the resin and fully expose the active groups. The activated resin was added to a 20% piperidine / DMF solution and reacted for 30 min to remove the Fmoc protecting group on the RAM resin, exposing its free amino group. The resin was washed 3-5 times with DCM and DMF respectively. A small amount of resin was taken and detected by the ninhydrin detection method. If the resin turned blue, it indicated that the Fmoc protecting group was completely removed. Subsequently, the first amino acid counted from the C-terminus was added and dissolved with DMF. 6-Chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) was used as the condensing agent, and N,N-diisopropylethylamine (DIPEA) was used to activate the carboxyl group on the amino acid. The coupling reaction was carried out at room temperature in a shaker for 1 h. A small amount of resin was aspirated with a capillary tube and detected by the ninhydrin detection method. If the resin did not turn blue, it indicated that the coupling reaction was complete. Then the resin was washed 3 times with DCM and DMF in sequence. The above steps were repeated to add each amino acid in turn. The feeding ratio, synthesis method, and detection method of non-natural amino acids were the same as the above method.

[0059] 2) Cleavage of resin peptide to obtain linear peptide:

[0060] Half of the resin peptide was taken and added to a 20% piperidine / DMF solution and reacted for 30 min to remove the Fmoc protecting group on the last amino acid. A small amount of resin was taken and detected by the ninhydrin detection method. If the resin turned blue, it indicated that the Fmoc protecting group was completely removed. The resin was washed 3 times with DCM and DMF. After the resin was dried by suction, 20 ml of cleavage solution (trifluoroacetic acid: triisopropylsilane: water = 9:0.5:0.5) was added and reacted in a shaker at room temperature for 3 h to cleave the resin. After the cleavage was completed, the resin was washed with DCM, and the filtrate was collected. Trifluoroacetic acid was removed under reduced pressure using a rotary evaporator equipped with an alkali tower. 6-8 times the volume of ice-cold diethyl ether was added to precipitate the polypeptide. The suspension was centrifuged for 5 min, and the supernatant was discarded to obtain a white paste-like solid. An appropriate amount of the solid was dissolved in water, and ESI-MS was used to detect and confirm whether the synthesis of the crude peptide was successful. The verified solution was freeze-dried by a freeze-dryer to obtain a white solid powder.

[0061] 3) Oxidation of linear peptides:

[0062] For example, the thiol protecting groups selected for cysteine at positions 2 and 9 are trityl (Trt). For air oxidation, 50 mg of the crude peptide solid powder obtained in the previous step is dissolved in 150 mL of 0.2 M ammonium bicarbonate aqueous solution at a concentration of 0.2 mg / mL, transferred to a 250 mL eggplant-shaped flask, and the pH of the solution is adjusted to 8. A magnetic stirrer bar is added to the eggplant-shaped flask and placed on a magnetic stirrer for stirring. The reaction is carried out at room temperature for 24 - 48 h, and the reaction progress is detected by ESI-MS. After the reaction is completed, HPLC separation and analysis are carried out. After collecting the target peak, a white solid powder is obtained using a freeze dryer.

[0063] The specific purification process is as follows:

[0064] The solution is filtered through a 0.22 μm organic microporous filter membrane. The eluents are Buffer A (90% water, 10% acetonitrile, 0.05% trifluoroacetic acid) and Buffer B (10% water, 90% acetonitrile, 0.05% trifluoroacetic acid). The elution gradient is from 100% Buffer A to 60% in 40 min. The flow rate of the eluent is 6 mL / min. Detection is carried out at two wavelengths of 214 nm and 280 nm. The product peak is collected and ESI-MS detection is carried out. The verified solution is freeze-dried using a freeze dryer to obtain a white solid powder.

[0065] For cysteine at positions 4 and 16, the thiol protecting group selected is acetamidomethyl (Acm), and iodine oxidation is used:

[0066] Weigh 10 mg of the above-mentioned white solid powder and dissolve it in 10 mL of reaction solvent (water:acetonitrile:TFA = 5:5:0.01, V / V). Add 3 mL of iodine / acetonitrile solution (5 mg / mL) to make the solution show a brownish-yellow color. In a 50 mL eggplant-shaped flask, stir and react in a sealed environment at 28 °C for 2 - 3 h. Then, detect the reaction progress by ESI-MS. After the reaction is complete, slowly add an ascorbic acid aqueous solution (5 mg / mL) to neutralize the excess iodine in the solution and make the solution colorless and clear. Then, carry out HPLC separation and analysis. The eluents are Buffer A (90% water, 10% acetonitrile, 0.05% trifluoroacetic acid) and Buffer B (10% water, 90% acetonitrile, 0.05% trifluoroacetic acid). The elution gradient is from 100% Buffer A to 60% in 40 min. The flow rate of the eluent is 3 mL / min. Detection is carried out at two wavelengths of 214 nm and 280 nm. The target peak collected is the final product with two pairs of disulfide bonds formed. After freeze-drying, the final product white solid powder is obtained.

[0067] The specific process of solid-phase chemical synthesis and oxidative folding of polypeptides is as Figure 5 shown.

[0068] Example 4 Electrophysiological Activity Test

[0069] 1. Solution Preparation

[0070] The extracellular fluid (mM) is: NaCl, 137; KCl, 4; CaCl2, 1.8; MgCl2, 1; HEPES, 10; glucose 10; pH 7.4 (titrated with NaOH).

[0071] The intracellular fluid (mM) is: CsCl, 130; MgCl2, 5; EGTA 5; HEPES, 10; Tris-ATP 4; pH 7.2 (titrated with KOH). The intracellular fluid is stored in small batches at -80 °C in a refrigerator and thawed on the day of the experiment.

[0072] 2. Cell Culture

[0073] CHO cells stably expressing Nav1.4 channels are cultured at 37 °C in a 5% CO2 incubator. The basal F12 medium is supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin, and 500 μg / mL of G418 to prepare a complete medium. The cells are allowed to adhere and grow in a culture flask for 2 - 3 days. Under a microscope, when the cell density reaches about 80%, the culture flask is taken out of the incubator, the medium is discarded, and the cells are rinsed with PBS. The cells are digested with 2.5 mg / mL of trypsin for 3 min, and the digestion is terminated by adding 2 mL of the complete medium. After centrifugation at 800 rpm and 100 rcf for 3 min, the supernatant is discarded, and the cells are resuspended in the complete medium. The cell suspension is seeded at a low density on a coverslip with a diameter of 12 mm. Electrophysiological recordings are performed within 48 h.

[0074] Other Nav subtypes are cultured from cells stably expressing their respective channels. The culture method and the electrophysiological recording method are the same as those for Nav1.4.

[0075] 3. Operating Procedures:

[0076] 1) Check that the input voltage of P97 is set to 220 V, and turn on the power; select the desired program (0 - 99), and press ENTER to enter;

[0077] 2) Load the glass micropipette and cover it with the protective cover; press Pull to draw.

[0078] 3) Add the internal solution to the glass electrode. Rotate the micromanipulator so that the tip of the electrode and the cell appear in the same field of view under the microscope. Apply positive pressure before inserting the electrode into the solution. After inserting the electrode into the solution, a square wave appears on the Clampex interface. Slowly lower the electrode until it contacts the cell. When the Ra value increases and the square wave on the computer side decreases, apply negative pressure. After successful sealing, perform compensation. Apply negative pressure again to rupture the membrane. Clamp the cell at -80 mV, and then depolarize it to -10 mV with a 10-ms square wave to obtain the ion channel current.

[0079] 4) This procedure is repeated every 10 seconds.

[0080] 5) Detect the maximum current induced by the square wave. After it stabilizes, perfuse the test compound. When the response stabilizes, calculate the blocking intensity. Stop perfusing the compound and wash it out with the extracellular solution.

[0081] All experiments were carried out at room temperature. Each cell served as its own control. The blocking ability of various μ-KIIIA analogs at a concentration of 1 μM on Nav1.4 is as Figure 6 shown.

[0082] 4. Experimental Results

[0083] Representative current traces under the inhibition of Nav1.4 by 1 μM of KIIIA analogs are as Figure 7 shown, where Figure 7 A in Figure 7 is SEQ ID NO.2;

[0084] Designing double mutants or triple mutants by combining single mutations with enhanced activity is an effective strategy to significantly improve the inhibitory activity of conotoxins. The second-generation double mutants were designed, and all analogs were subjected to chemical solid-phase synthesis and activity testing. The activity test results are shown in Tables 2 and 3.

[0085] Table 2. Modification methods, amino acid sequences, and activities of KIIIA analogs after deleting a pair of disulfide bonds

[0086]

[0087]

[0088] Table 3. Inhibition rates of KIIIA analogs on various Nav subtypes (1 μM)

[0089]

[0090]

[0091] By measuring the inhibitory activity of the second-generation analog of μ-conotoxin KIIIA against Nav1.4 under different concentration conditions, the measured data was processed using Clampfit software, and the response rate was obtained by dividing the response value by the control value. Fitting analysis was performed using GraphPad Prism, and the fitting equation for the dose curve was: % inhibition rate = 100 / (1 + 10^((LogIC50 - X) * nH))), where X is the concentration of conotoxin and nH is the Hill coefficient, and IC 50 is the half-maximal inhibitory concentration. The inhibitory concentration-effect curve of the KIIIA analog with the sequence SEQ ID NO:2 against Nav1.4 is as shown in Figure 8 . IC 50 = 130.24 ± 14.69 nM.

[0092] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A mutant of conotoxin KIIIA for inhibiting voltage-gated sodium ions through 1.4, characterized in that, In the amino acid sequence of the mutant, the cysteine residues at positions 2 and 9 form a disulfide bond, and the cysteine residues at positions 4 and 16 form a disulfide bond. The specific sequences are: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:

13.

2. The conotoxin KIIIA mutant for inhibiting voltage-gated sodium ions from passing through 1.4 according to claim 1, wherein the amino acid sequence of the mutant is any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:

6.

3. The method for preparing the conotoxin KIIIA mutant according to any one of claims 1-2, the method comprising the following steps: 1) Synthesize resin peptides using the Fmoc solid-phase synthesis method, and amidate the C-terminus of the resin peptides; wherein, The cysteines at positions 2 and 9 are protected with a first mercapto protecting group, and the cysteines at positions 4 and 16 are protected with a second mercapto protecting group. The first mercapto protecting group and the second mercapto protecting group are different and each independently selected from one of triphenylmethyl (Trt) or acetamidomethyl (Acm); 2) Cleaving the resin peptide to obtain a linear peptide; 3) Performing disulfide bond folding based on the specific groups of the first mercapto protecting group and the second mercapto protecting group, and purifying to obtain the product.

4. The preparation method according to claim 3, characterized in that, Step 2) further includes: First, oxidizing the cysteine with triphenylmethyl (Trt) as the mercapto protecting group by air oxidation method; then, oxidizing the cysteine with acetamidomethyl (Acm) as the mercapto protecting group by iodine oxidation method.

5. The preparation method according to claim 4, characterized in that, The iodine oxidation method is carried out by a method including the following steps: Dissolving the crude peptide after air oxidation in a reaction solvent, which is a mixture of water, acetonitrile and TFA in a volume ratio of 5:5:0.01, and then adding an iodine-acetonitrile solution; After stirring and reacting fully in a sealed environment at 28°C, an aqueous solution of ascorbic acid is slowly added to neutralize the excessive iodine, and separation by HPLC gives the product.

6. The application of the conotoxin KIIIA mutant according to any one of claims 1-2 in the preparation of a preparation for inhibiting voltage-gated sodium channel 1.

4.

7. The application according to claim 6, characterized in that The preparation is a muscle relaxant drug or an anti-wrinkle agent.

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