A conotoxin KIIIA mutant and its preparation method and application

Abstract

The present invention provides a cone toxin KIIIA mutant having an amino acid sequence as shown in SEQ ID NO: 1: Xaa1CXaa2CXaa3Xaa4KWCXaa5DHXaa6RXaa7C*. The present invention also provides a preparation method and application of the cone toxin KIIIA mutant. This invention enables the rational design of two pairs of disulfide bond analogs of cone toxins targeting the Nav1.7 channel, significantly improving the efficiency of cone toxin synthesis and reducing synthesis costs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a cone snail toxin KIIIA mutant, a preparation method and an application thereof. Background Art

[0002] Voltage-gated sodium (Nav) channels are a class of transmembrane glycoprotein complexes that underlie the generation and propagation of action potentials in excitable cells, such as cardiac muscle, skeletal muscle, and neurons. They are also important therapeutic targets for diseases such as epilepsy, arrhythmias, and neuropathic pain. Nav is composed of two subunits, α and β, each with distinct functions. The α subunit is a single chain that folds to form four highly homologous repeating domains. Each repeating domain contains six transmembrane segments, S1-S6. These four homologous domains surround a central hollow ion pore. The α subunit is encoded by ten different genes and expressed in different excitable tissues. Based on the sequence of the α subunit, sodium channels can be divided into nine subtypes, ranging from 1.1 to 1.9. Different subtypes are typically distributed in different regions and exert distinct roles and functions. Among them, Nav1.7 is widely distributed in peripheral sensory neurons and is encoded by SCN9A. It is involved in the generation of action potentials and the transmission of pain signals. Loss-of-function mutations in this gene lead to congenital insensitivity to pain, while gain-of-function mutations can cause diseases such as erythromelalgia and paroxysmal extreme pain disorder, which are characterized by severe paroxysmal pain. Targeting Nav1.7 through specific inhibition may have the potential to treat chronic pain. Non-selective Nav inhibitors, such as the local anesthetic lidocaine, the antiarrhythmic drug mexiletine, and the antiepileptic drug carbamazepine, have been used clinically to treat chronic pain, but they all show limited efficacy and large side effects. The acquisition of selective Nav1.7 inhibitors is expected to develop effective analgesics with fewer adverse reactions. Therefore, Nav1.7 has become an important target for the development of analgesic drugs.

[0003] The cone snail is a marine invertebrate commonly found near coral reefs in equatorial waters. Its venom contains hundreds of different components, which are called conotoxins. The main components are some active polypeptide compounds with high specificity for different ion channels and neuroreceptors. It includes different amino acids and targets a specific nerve channel or receptor. Generally speaking, most cone snail toxins are rich in disulfide bonds. According to the amino acid sequence of the conserved region and the cysteine ​​skeleton, cone snail toxins can be divided into multiple superfamilies. The cone snail toxins that interact with Nav channels mainly come from the M- and O-superfamilies. The cone snail toxins in the M-superfamily that act on Na ion channels are mainly μ-CTX. μ-Conotoxin is a selective blocker of sodium channels, generally composed of 17-26 amino acid residues, with the C-terminus amidated, rich in positive charges, and a unique cysteine ​​skeleton.

[0004] Among the cone snail toxins that act on Nav1.7, μ-conotoxin KIIIA has a high potential for drug development. KIIIA is derived from Conus kinoshitai, which is native to the Indo-Pacific region. It consists of 16 amino acids and contains three pairs of disulfide bonds. Wild-type KIIIA has strong inhibitory activity against Nav1.2, Nav1.4, Nav1.6 and Nav1.7, but poor selectivity. Keith K. Khoo et al. found that the main oxidative folding product of wild-type KIIIA is Disulfide bonds. Tiffany S. Han et al. first investigated the activity of three mutants compared to wild-type KIIIA by deleting three disulfide bonds through alanine mutations, resulting in two disulfide analogs with comparable activity and simplified synthesis. Mingming Zhang et al. first proposed constructing KIIIA analogs by deleting one disulfide bond and then investigated the activity differences of analogs substituted with nine amino acids varying in size, charge, or hydrophobicity at position 7. Annelies Van Der Haegen et al. identified Trp8 as a key residue in the pharmacophore. Replacing Trp8 resulted in more selective inhibitors of neuronal sodium channels against Nav1.2-1.6. Because the formation of three disulfide bonds requires a three-step oxidation process, the experimental process is cumbersome and difficult to achieve. Further studies exploring the activity of deleting a disulfide bond and mutating other sites using this linkage are currently lacking. Furthermore, while several groups worldwide have conducted mutation studies on amino acids in non-conserved regions of KIIIA, systematic studies examining the inhibitory potential of KIIIA analogs against Nav1.7 remain lacking. At present, the binding mode and mechanism of action of KIIIA on the analgesic target Nav1.7 are still unclear, and related research is still blank, which hinders its in-depth research and development and utilization. Summary of the Invention

[0005] The present invention analyzes the interaction between cone snail toxin KIIIA and voltage-gated sodium channel 1.7 through computer simulation, designs a series of KIIIA mutants, and obtains inhibitors targeting voltage-gated sodium channels with improved activity and selectivity and reduced synthesis difficulty by modifying μ cone snail toxin KIIIA. This solves the problems of cumbersome synthesis steps, poor activity and selectivity of cone snail toxins targeting voltage-gated sodium channels in the prior art.

[0006] The present invention aims to provide a series of mutants of cone snail toxin KIIIA containing only two pairs of disulfide bonds that have a strong inhibitory effect on voltage-gated sodium ion channel 1.7. The mutants are short peptides consisting of 16 amino acids, and the sequence thereof is SEQ ID NO: 1:

[0007] Xaa1CXaa2CXaa3Xaa4KWCXaa5DHXaa6RXaa7C*

[0008] wherein Xaa1 is selected from any one of A, R, N, S, H, T, Cit, Dap or Dab;

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

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

[0011] Xaa4 is selected from any one of S, T, Dab or Dapa;

[0012] Xaa5 is selected from any one of R, S, V, L, A, Dapa or F;

[0013] Xaa6 is selected from any one of S, R, Dab or Dapa;

[0014] Xaa7 is selected from any one of A, D, S, R, T, N, H, Dab or Dap;

[0015] “*” indicates amidation at the C-terminus;

[0016] The amino acid sequence formed when Xaa1 is A, Xaa2 is N, Xaa3 is S, Xaa4 is S, Xaa5 is R, Xaa6 is S, and Xaa7 is A is not included. Structure-activity relationship analysis shows that the introduction of positively charged amino acids into the above sequence is beneficial to improving the activity of the polypeptide.

[0017] In one embodiment according to the present invention, 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.

[0018] In one embodiment of 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, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32; preferably, selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 Any one of NO:5.

[0019] The present invention also provides a method for preparing the above-mentioned cone snail toxin KIIIA mutant, comprising:

[0020] 1) synthesizing a crude peptide using Fmoc solid-phase synthesis, and subjecting the C-terminus to amidation; wherein the cysteines at positions 2 and 9 are protected with a first thiol protecting group, and the cysteines at positions 4 and 16 are protected with a second thiol protecting group, wherein the first thiol protecting group and the second thiol protecting group are different and independently selected from one of trityl (Trt) and acetamidomethyl (Acm);

[0021] 2) After disulfide bond folding based on the specific groups of the first thiol protecting group and the second thiol protecting group, purification is performed.

[0022] In one embodiment according to the present invention, step 2) further comprises:

[0023] First, cysteine ​​with a trityl (Trt) thiol protecting group was oxidized by air oxidation; then, cysteine ​​with an acetamidomethyl (Acm) thiol protecting group was oxidized;

[0024] The oxidation of cysteine ​​with acetamidomethyl (Acm) as a sulfhydryl protecting group is carried out by a method comprising the following steps:

[0025] The crude peptide after air oxidation was dissolved in a reaction solvent, which was a mixture of water, acetonitrile and TFA in a volume ratio of 5:5:0.01. An iodine-acetonitrile solution with an iodine concentration of 5 mg / mL was then added. After stirring for sufficient reaction in a closed environment at 28 degrees Celsius, an ascorbic acid aqueous solution was slowly added to neutralize excess iodine. The peptide was then separated by HPLC.

[0026] The present invention also provides the use of the above-mentioned cone snail toxin KIIIA mutant in the preparation of analgesic drugs.

[0027] In one embodiment according to the present invention, the analgesic drug is a drug for treating chronic pain.

[0028] The present invention further provides an analgesic drug, characterized in that it comprises the above-mentioned cone snail toxin KIIIA mutant.

[0029] The beneficial effects of the above technical solution of the present invention are as follows:

[0030] The present invention achieves for the first time the rational design of two pairs of disulfide bond analogs of conotoxin targeting the Nav1.7 channel, significantly improving the efficiency of conotoxin synthesis and reducing synthesis costs. In vitro experiments were conducted to determine the blocking strength of various KIIIA mutants on NaV1.7 ion channel currents, demonstrating a good inhibitory effect. The present invention also conducted in vivo animal experiments to determine the analgesic activity of the mutant KIIIA [A1R, S5Dapa] containing two pairs of disulfide bonds in a formalin-induced biphasic pain model, demonstrating a good analgesic effect. This suggests that it may have the potential to treat chronic pain by inhibiting and targeting Nav1.7. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The inhibitory activity of KIIIA and KIIIA analogs with one disulfide bond deleted against Nav1.7 at a concentration of 1 μM;

[0032] Figure 2 It is a KIIIA[C1A,C15A] structure;

[0033] Figure 3 This is the binding mode of the complex between cone snail toxin KIIIA [C1A, C15A] and Nav1.7;

[0034] Figure 4 The 150 ns molecular dynamics simulation results of KIIIA[C1A, C15A] and Nav1.7 under cell membrane conditions;

[0035] Figure 5 Schematic diagram of the structure of the KIIIA mutant after partial deletion of a pair of disulfide bonds;

[0036] Figure 6 Schematic diagram of the synthesis process of KIIIA mutants after deleting a pair of disulfide bonds, taking A15R as an example;

[0037] Figure 7 This is a comparison of the blocking ability of μ-KIIIA analogs at a concentration of 1 μM on Nav1.7;

[0038] Figure 8 The diagram shows the binding pattern between the 15th position analog of KIIIA [C1A, C15A] and Nav1.7;

[0039] Figure 9 This is the inhibitory concentration effect curve of KIIIA analogs on Nav1.7;

[0040] Figure 10 This is a graph showing the experimental results of SEQ ID NO: 2 and SEQ ID NO: 3 in the formalin-induced biphasic pain model. DETAILED DESCRIPTION

[0041] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0042] Example 1 Computer-aided design of mutants

[0043] The disulfide bonds at positions 1 and 15 of the wild-type μ cone snail toxin KIIIA peptide chain were deleted to obtain the amino acid sequence μ cone snail toxin KIIIA [C1A, C15A] with activity similar to that of the wild-type toxin. The inhibitory activity of KIIIA and KIIIA analogs with a pair of disulfide bonds deleted against Nav1.7 at a concentration of 1 μM is shown in the following figure: Figure 1 shown.

[0044] 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 a model of the complex of KIIIA[C1A,C15A] and Nav1.7. The structure of KIIIA[C1A,C15A] is shown in Figure 2 shown.

[0045] Modeller (version 9v14) software was used to generate a three-dimensional structural model of the target protein using the homology modeling method in the LINUX system environment. The model with the lowest DOPE score was selected as the optimal target structure model for further structural optimization by molecular dynamics. Figure 3 shown.

[0046] The original data of KIIIA[C1A,C15A] were generated using modeller software, and the subsequent analogue data were also generated from this. The unnatural amino acid analogues were obtained by manually modifying the side chain of KIIIA in PyMOL. In the dynamic simulation using AMBER16 software, the Lipid14 force field was used for lipids, and the ff14SB force field was used for proteins and peptides. Since AMBER could not recognize the name CYS of cysteine ​​and thus could not generate disulfide bonds, CYS was changed to CYX and the command to connect the disulfide bonds was manually added. Then, the size distance complex was added around the complex. The octahedral water box is selected, the water molecule position is TIP3P, and Na ions are added to the whole system to neutralize the system and keep it stable. After the parameter calculation is completed, the energy optimization of the whole system is performed. First, when the solute constraint force is Constrained optimization was performed for 100 ps, ​​followed by 2000 steps of steepest descent optimization and 3000 steps of conjugate gradient optimization. After the first round of optimization, the position constraints were removed. The MD simulation process included heating and equilibration. The entire system was heated under isovolumetric conditions for 100 ps, ​​gradually heating the temperature from 50 K to 300 K. The solute binding force was Subsequently, a 250 ns MD simulation was performed under the boundary conditions of temperature and pressure maintained at 300 K and 1 atm, respectively. The time step used in the MD simulation was 2 fs, and all bonds involving hydrogen atoms were kept at their standard lengths using the SHAKE algorithm. The software VMD (http: / / www.ks.uiuc.edu / ) was used to analyze the motion trajectory after the MD simulation and calculate the RMSD value of the obtained conformation. The results of the 150 ns molecular dynamics simulation of KIIIA[C1A, C15A] and Nav1.7 under cell membrane conditions are shown in Figure 2. Figure 4 shown.

[0047] Chemical synthesis of mutants of Example 2;

[0048] The mutant was chemically synthesized using the Fmoc solid-phase method. The C-terminus of KIIIA was amidated using Rink-Amide resin, with the synthesis direction from C-terminus to N-terminus. The resin loading was 0.631 mmol / g, and the equivalent weight was 0.2 mmol. The amino acid and condensing agent were added at 4 times the resin loading, and the activating agent was added at 8 times the resin loading. First, 0.317 g of resin was weighed and added to a solid-phase reaction tube. The resin was activated by swelling it with a 1:2 volume ratio of dichloromethane (DCM) and N,N-dimethylformamide (DMF) at room temperature overnight or on a constant temperature shaker for 2 hours to fully expose the active groups. The activated resin was then reacted with a 20% piperidine / DMF solution for 30 minutes to remove the Fmoc protecting groups from the RAM resin, exposing the amino groups. The resin was then washed three times with DCM and DMF, respectively. A small amount of resin was tested using the ninhydrin assay, indicating complete removal of the Fmoc protecting groups. Next, add the first amino acid counting from the C-terminus, dissolve in DMF, use 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) as a condensing agent, and activate the carboxyl groups on the amino acids with N,N-diisopropylethylamine (DIPEA). The coupling reaction is carried out at room temperature on a shaker for 1 hour. A small amount of resin is aspirated using a capillary tube and tested with the ninhydrin assay. If the resin does not turn blue, the coupling reaction is complete. The resin is then washed three times with DCM and DMF. Repeat these steps for each amino acid. The feed ratio, synthesis method, and detection method for the unnatural amino acids are the same as above. The sulfhydryl protecting group selected for cysteine ​​at positions 2 and 9 is trityl (Trt), while the sulfhydryl protecting group selected for cysteine ​​at positions 4 and 16 is acetamidomethyl (Acm). Finally, the resin peptide SEQ ID NO: 1Xaa1CXaa2CXaa3Xaa4KWCXaa5DHXaa6RXaa7C* was obtained.

[0049] Take half of the resin peptide and add 20% piperidine / DMF solution to react for 30 minutes to remove the Fmoc protecting group on the last amino acid. Take a small amount of resin and use the ninhydrin detection method to detect that the resin turns blue, indicating that the Fmoc protecting group is completely removed. Use DCM and DMF to wash the resin 3 times. After the resin is drained, add 20mL of lysis solution (trifluoroacetic acid: triisopropylsilane: water = 9:0.5:0.5) and react in a shaker at room temperature for 3 hours to cut the resin. After the cutting is completed, use DCM to wash the resin, collect the filtrate, and use a rotary evaporator equipped with an alkali tower to remove trifluoroacetic acid under reduced pressure. Add 6-8 times the volume of ice ether to precipitate the polypeptide, centrifuge the suspension for 5 minutes, discard the supernatant, and obtain a white paste solid. Take an appropriate amount of solid and dissolve it in water. ESI-MS detection confirms whether the crude peptide synthesis is successful. The verified solution is freeze-dried using a freeze dryer to obtain a white solid powder. The structure of the KIIIA mutant after partial deletion of a pair of disulfide bonds is as follows Figure 5 shown.

[0050] Example 3 Folding of disulfide bonds

[0051] 1. Oxidation of the first disulfide bond:

[0052] The Trt protecting group is removed during the cleavage process, exposing the sulfhydryl group. Air oxidation is used to naturally form disulfide bonds. 50 mg of the crude peptide 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. The solution is transferred to a 250 mL eggplant-shaped flask, and the pH of the solution is adjusted to 8. A magnetic stirrer is placed in the flask, stirring the mixture on a magnetic stirrer. The reaction is allowed to proceed at room temperature for 24-48 hours. Reaction progress is monitored by ESI-MS. After completion, HPLC separation and analysis are performed. The target peak is collected and lyophilized to yield a white solid powder.

[0053] The specific purification process is:

[0054] The solution was filtered through a 0.22 μm organic microporous membrane using Buffer A (90% water, 10% acetonitrile, 0.05% trifluoroacetic acid) and Buffer B (10% water, 90% acetonitrile, 0.05% trifluoroacetic acid) as eluents. The elution gradient was Buffer A 100% to 95% over 6 min; 95% to 80% over 14 min; and 80% to 60% over 20 min. The eluent flow rate was 6 mL / min, and dual wavelength detection at 214 nm and 280 nm was used. The product peaks were collected and analyzed by ESI-MS. The verified solution was lyophilized using a freeze dryer to obtain a white solid powder.

[0055] 2. Oxidation of the second disulfide bond:

[0056] Weigh 10 mg of the above 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 appear brownish yellow; in a 50 mL eggplant-shaped flask, stir the reaction for 2-3 hours in a closed environment at 28 degrees Celsius, and use ESI-MS to detect the reaction progress. After the reaction is complete, slowly add ascorbic acid aqueous solution (5 mg / mL) to neutralize the excess iodine in the solution to make the solution colorless and clear. Then perform HPLC separation and analysis. The separation conditions are consistent with the separation conditions for the first pair of disulfide bond oxidation. The target peak collected is the final product of the formation of two pairs of disulfide bonds. After freeze-drying, the final product is a white solid powder. The specific process of peptide solid-phase chemical synthesis and oxidative folding is as follows. Figure 6 shown.

[0057] Example 4 Electrophysiological activity test

[0058] 1. Cell culture (taking Nav1.7 as an example):

[0059] HEK293 cells stably expressing Nav1.7 channels were cultured at 37°C in a 5% CO2 incubator using basal F12 medium supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin, and 500 μg / mL G418 to prepare complete medium. The cells were allowed to adhere to the culture flask for 2-3 days. Microscopic observation revealed that the cell density reached approximately 80%. The flask was removed from the incubator, the medium discarded, and the cells were rinsed with PBS. The cells were then digested with 2.5 mg / mL trypsin for 3 minutes. The cells were then centrifuged at 800 rpm, 100 rcf, for 3 minutes. The supernatant was discarded, and the cells were resuspended in complete medium. The cell suspension was then plated at a low density on a 12 mm diameter coverslip. Electrophysiological recordings were performed within 48 hours.

[0060] The other Nav isoforms were cultured by cells stably expressing their respective channels, and the culture method and electrophysiological recording method were the same as Nav1.7.

[0061] 2. Electrophysiological recording:

[0062] Whole-cell patch clamp recordings were performed. Data were acquired using a MultiClamp 700A amplifier and pCLAMP 10.6 software. Glass electrodes were pulled using a P-97 electrode puller and polished using a heat polisher using an MF-830 polisher. Noise was filtered at one-fifth the sampling frequency. The extracellular solution used (mM) consisted of: NaCl, 137; KCl, 4; CaCl₂, 1.8; MgCl₂, 1; HEPES, 10; glucose, 10; pH 7.4 (titrated by NaOH). The intracellular solution (mM) consisted of: CsCl, 130; MgCl₂, 5; EGTA, 5; HEPES, 10; Tris-ATP, 4; pH 7.2 (titrated by KOH). After filling the electrode with the internal solution, the inlet resistance was between 2 and 4 MΩ. Cells were transferred to a perfusion tank, and the extracellular solution was perfused using a gravity-fed perfusion system. After the whole-cell recording mode is established, the cell is clamped at -80mV and then depolarized to -10mV with a 10ms square wave to obtain the NaV1.7 ion channel current. This procedure is repeated every 10 seconds. The maximum current induced by the square wave is detected, and after it stabilizes, the test compound is perfused. When the reaction stabilizes, the blocking strength is calculated. The blocking ability of each μ-KIIIA analog at a concentration of 1μM on NaV1.7 is shown in the figure. Figure 7 shown.

[0063] Interaction mode with KIIIA[C1A,C15A] and Nav1.7 ( Figure 8 Compared with the [A15R]KIIIA[C1A,C15A] mutant, the R15 side chain is respectively connected to the D476 ( Figure 8 Left) and D727( Figure 8 Left) Multiple salt bridges and hydrogen bonds are formed. Activity test results show that the [A15R]KIIIA[C1A,C15A] mutant has enhanced activity. The binding mode between the 15th position analog of KIIIA[C1A,C15A] and Nav1.7 is shown in the figure. Figure 8 shown.

[0064] Designing double or triple mutations by combining single mutations with activity-enhancing effects is an effective strategy to significantly improve the inhibitory activity of cone snail toxins. We designed the second-generation double mutants, and we performed chemical solid-phase synthesis and activity testing on all analogs. By measuring the inhibitory activity of the second-generation analogs of μ-conotoxin KIIIA on Nav1.7 under different concentration conditions, the measured data were processed using Clampfit software, and the response value was divided by the control value to obtain the response rate. GraphPad Prism was used for fitting analysis, and the fitting equation of the dose curve was: % inhibition rate = 100 / (1+10^((LogIC50-X)*nH))), where X is the concentration of cone snail toxin, nH is the Hill coefficient, and IC50 is the half-maximal inhibitory concentration. The inhibitory concentration effect curve of KIIIA analogs on Nav1.7 is shown in the figure. Figure 9 As shown. Wherein Figure A is compound SEQ ID NO: 2, IC 50 =190.91±37.60nM; Figure B is compound SEQ ID NO: 3, IC 50 =110.75±20.42nM

[0065] The modification method, amino acid sequence and inhibitory activity against Nav1.7 at a concentration of 1 μM of the KIIIA analog after deleting a pair of disulfide bonds are shown in Table 1; the inhibition rate of the double mutant against each Nav subtype at a concentration of 1 μM is shown in Table 2.

[0066] Table 1. Modification methods, amino acid sequences and activities of KIIIA analogs after deletion of a pair of disulfide bonds

[0067]

[0068]

[0069] Table 2 Inhibition rate of double mutants on each Nav subtype (1 μM)

[0070]

[0071] Example 5 Animal in vivo activity test

[0072] A formalin-induced biphasic pain model was used, and C57BL / 6J mice (20-25g) were selected. Before the experiment began, the mice were transferred to a transparent organic observation box to acclimate for 1 hour. The experiment set up four groups: a model control group, a positive control morphine group, a drug group 1 (50μg / kg), and a drug group 2 (50μg / kg). The corresponding compound was injected intraperitoneally and the animals were placed in a transparent observation box. 20 minutes after administration, 20μL of 5% formalin was injected subcutaneously in the left hind paw of the mouse. The mouse was then transferred to a transparent observation box and videotaped. After the experiment, the video was played back and the total duration of the mouse's pain response during the first stage of pain (0-5 minutes) and the second stage of pain (15-45 minutes) was recorded. The specific pain response was manifested as lifting, extending, and licking the injected paw. The experimental results of the formalin-induced biphasic pain model are shown in the figure. The drug administration group 1 was administered with compound SEQ ID NO: 2 at a concentration of 50 ug / kg; the drug administration group 2 was administered with SEQ ID NO: 3 at a concentration of 50 ug / kg; the positive control was morphine at a concentration of 2.5 mg / kg.

[0073] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention. Sequence Listing <110> Qingdao Institute of Marine Biomedicine <120> A conotoxin KIIIA mutant and its preparation method and application <160> 32 <170> SIPOSequenceListing 1.0 <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> MUTAGEN <222> (1)..(1) <223> Any one selected from A, R, N, S, H, T, Cit, Dap or Dab <220> <221> MUTAGEN <222> (3) <223> Any one selected from N, H, R, Dab or Dap <220> <221> MUTAGEN <222> (5)..(5) <223> S, Dab or Dapa <220> <221> MUTAGEN <222> (6) <223> Any one selected from S, T, Dab or Dap <220> <221> MUTAGEN <222> (10)..(10) <223> Any one selected from R, S, V, L, A, Dapa or F <220> <221> MUTAGEN <222> (13)..(13) <223> Any one selected from S, R, Dab or Dapa <220> <221> MUTAGEN <222> (15)..(15) <223> Any one selected from A, D, S, R, T, N, H, Dab or Dap <220> <221> DISULFID <222> (2)..(9) <220> <221> DISULFID <222> (4)..(16) <220> <221> BLOCKED <222> (16)..(16) <223> C-terminal amidation <220> <221> UNSURE <222> (1)..(1) <223> The 'Xaa' at location 1 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (3) <223> The 'Xaa' at location 3 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (5)..(5) <223> The 'Xaa' at location 5 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (6)..(6) <223> The 'Xaa' at location 6 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (10)..(10) <223> The 'Xaa' at location 10 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (13)..(13) <223> The 'Xaa' at location 13 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (15)..(15) <223> The 'Xaa' at location 15 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (1)..(1) <223> The 'Xaa' at location 1 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (3)..(3) <223> The 'Xaa' at location 3 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (5)..(5) <223> The 'Xaa' at location 5 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (6)..(6) <223> The 'Xaa' at location 6 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (10)..(10) <223> The 'Xaa' at location 10 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (13)..(13) <223> The 'Xaa' at location 13 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (15)..(15) <223> The 'Xaa' at location 15 stands for Gln, Arg, Pro, or Leu. <400> 1 Xaa Cys Xaa Cys Xaa Xaa Lys Trp Cys Xaa Asp His Xaa Arg Xaa Cys 1 5 10 15 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> MUTAGEN <222> (5)..(5) <223> Dapa <220> <221> BLOCKED <222> (16)..(16) <223> Amidation occurs <220> <221> UNSURE <222> (5)..(5) <223> The 'Xaa' at location 5 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (5)..(5) <223> The 'Xaa' at location 5 stands for Gln, Arg, Pro, or Leu. <400> 2 Arg Cys Asn Cys Xaa Ser Lys Trp Cys Arg Asp His Ser Arg Ala Cys 1 5 10 15 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> BLOCKED <222> (3) <223> Xaa is Dab <220> <221> BLOCKED <222> (16)..(16) <223> Amidation occurs <220> <221> UNSURE <222> (3)..(3) <223> The 'Xaa' at location 3 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (3)..(3) <223> The 'Xaa' at location 3 stands for Gln, Arg, Pro, or Leu.<s <400> 3 Ala Cys Xaa Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg Arg Cys 1 5 10 15 [[ID=z0]]<210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> BLOCKED <222> (5)..(5) <223> Xaa is Dapa <2z0> <221> BLOCKED <222> (16)..(16) <223> The C-terminus is amidated <220> <221> UNSURE <222> (5)..(5) <223> The 'Xaa' at location 5 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (5)..(5) <223> The 'Xaa' at location 5 stands for Gln, Arg, Pro, or Leu. <400> 4 Note: There seems to be an inconsistent tag `<210>` which is changed to `<z0>` in the translation for better readability as it's likely an error in the original. If this is not allowed, please let me know and I'll adjust accordingly. Ala Cys Asn Cys Xaa Ser Lys Trp Cys Arg Asp His Ser Arg Arg Cys 1 5 10 15 <210> 5 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> BLOCKED <222> (16)..(16) <223> C-terminal amidation <220> <221> MUTAGEN <222> (3) <223> Xaa is Dab <220> <221> UNSURE <222> (3) <223> The 'Xaa' at location 3 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (3) <223> The 'Xaa' at location 3 stands for Gln, Arg, Pro, or Leu. <400> 5 Arg Cys Xaa Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg Ala Cys 1 5 10 15 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> MUTAGEN <222> (3) <223> Xaa1 is Dab <220> <221> MUTAGEN <222> (6) <223> Xaa2 is Dapa <220> <221> BLOCKED <222> (16)..(16) <223> C-terminal amidation <220> <221> UNSURE <222> (6) <223> The 'Xaa' at location 6 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (6) <223> The 'Xaa' at location 6 stands for Gln, Arg, Pro, or Leu. <400> 6 Ala Cys Asp Cys Ser Xaa Lys Trp Cys Arg Asp His Ser Arg Ala Cys 1 5 10 15 <210> 7 <211> 16 <212> PRT <213> Artificial Sequence <400> 7 Arg Cys Asn Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg Arg Cys 1 5 10 15 <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <400> 8 Ala Cys Asn Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg Arg Cys 1 5 10 15 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <400> 9 Arg Cys Asn Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg Ala Cys 1 5 10 15 <210> 10 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> MUTAGEN <222> (5)..(5) <223> Xaa for Dapa <220> <221> BLOCKED <222> (16)..(16) <223> C-terminal amidation <220> <221> UNSURE <222> (5)..(5) <223> The 'Xaa' at location 5 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (5)..(5) <223> The 'Xaa' at location 5 stands for Gln, Arg, Pro, or Leu. <400> 10 Ala Cys Asn Cys Xaa Ser Lys Trp Cys Arg Asp His Ser Arg Ala Cys 1 5 10 15 <210> 11 <211> 16 <212> PRT <213> Artificial Sequence <400> 11 Ala Cys Asn Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg Asn Cys 1 5 10 15 <210> 12 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> MUTAGEN <222> (15)..(15) <223> Xaa is Dab <220> <221> BLOCKED <222> (16)..(16) <223> C-terminal amidation <220> <221> UNSURE <222> (15)..(15) <223> The 'Xaa' at location 15 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (15)..(15) <223> The 'Xaa' at location 15 stands for Gln, Arg, Pro, or Leu. <400> 12 Ala Cys Asn Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg Xaa Cys 1 5 10 15 <210> 13 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> MUTAGEN <222> (3) <223> Xaa is Dab <220> <221> BLOCKED <222> (16)..(16) <223> C-terminal amidation <220> <221> UNSURE <222> (3) <223> The 'Xaa' at location 3 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (3) <223> The 'Xaa' at location 3 stands for Gln, Arg, Pro, or Leu. <400> 13 Ala Cys Xaa Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg Ala Cys 1 5 10 15 <210> 14 <211> 16 <212> PRT <213> Artificial Sequence <400> 14 Ala Cys Arg Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg Ala Cys 1 5 10 15 <210> 15 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> MUTAGEN <222> (5)..(5) <223> Xaa is Dab <220> <221> BLOCKED <222> (16)..(16) <223> C-terminal amidation <220> <221> UNSURE <222> (5)..(5) <223> The 'Xaa' at location 5 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (5)..(5) <223> The 'Xaa' at location 5 stands for Gln, Arg, Pro, or Leu. <400> 15 Ala Cys Asn Cys Xaa Ser Lys Trp Cys Arg Asp His Ser Arg Ala Cys 1 5 10 15 <210> 16 <211> 16 <212> PRT <213> Artificial Sequence <400> 16 Ala Cys Asn Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg Ser Cys 1 5 10 15 <210> 17 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> MUTAGEN <222> (13)..(13) <223> Xaa for Dapa <220> <221> BLOCKED <222> (16)..(16) <223> C-terminal amidation <220> <221> UNSURE <222> (13)..(13) <223> The 'Xaa' at location 13 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (13)..(13) <223> The 'Xaa' at location 13 stands for Gln, Arg, Pro, or Leu. <400> 17 Ala Cys Asn Cys Ser Ser Lys Trp Cys Arg Asp His Xaa Arg Ala Cys 1 5 10 15 <210> 18 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> MUTAGEN <222> (1)..(1) <223> Xaa is Dab <220> <221> BLOCKED <222> (16)..(16) <223> C-terminal amidation <220> <221> UNSURE <222> (1)..(1) <223> The 'Xaa' at location 1 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (1)..(1) <223> The 'Xaa' at location 1 stands for Gln, Arg, Pro, or Leu. <400> 18 Xaa Cys Asn Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg Ala Cys 1 5 10 15 <210> 19 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> MUTAGEN <222> (13)..(13) <223> Xaa is Dab <220> <221> BLOCKED <222> (16)..(16) <223> C-terminal amidation <220> <221> UNSURE <222> (13)..(13) <223> The 'Xaa' at location 13 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (13)..(13) <223> 位置13处的“Xaa”代表Gln、Arg、Pro或Leu。 <400> 19 丙氨酸 半胱氨酸 天冬酰胺 半胱氨酸 丝氨酸 丝氨酸 赖氨酸 色氨酸 半胱氨酸 精氨酸 天冬氨酸 组氨酸 Xaa 精氨酸 丙氨酸 半胱氨酸 1 5 10 15 <210> 20 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> MUTAGEN <222> (6)..(6) <223> Xaa为Dapa <220> <221> BLOCKED <222> (16)..(16) <223> C末端发生酰胺化 <220> <221> UNSURE <222> (6)..(6) <223> 位置6处的“Xaa”代表Gln、Arg、Pro或Leu。 <220> <221> UNSURE <222> (6)..(6) <223> 位置6处的“Xaa”代表Gln、Arg、Pro或Leu。 <400> 20 丙氨酸 半胱氨酸 天冬酰胺 半胱氨酸 丝氨酸 Xaa 赖氨酸 色氨酸 半胱氨酸 精氨酸 天冬氨酸 组氨酸 丝氨酸 精氨酸 丙氨酸 半胱氨酸 1 5 10 15 <210> 21 <211> 16 <212> PRT <213> Artificial Sequence <400> twenty one Ala Cys His Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg Ala Cys 1 5 10 15 <210> twenty two <211> 16 <212> PRT <213> Artificial Sequence <220> <221> MUTAGEN <222> (15)..(15) <223> Xaa for Dapa <220> <221> BLOCKED <222> (16)..(16) <223> C-terminal amidation <220> <221> UNSURE <222> (15)..(15) <223> The 'Xaa' at location 15 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (15)..(15) <223> The 'Xaa' at location 15 stands for Gln, Arg, Pro, or Leu. <400> twenty two Ala Cys Asn Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg Xaa Cys 1 5 10 15 <210> twenty three <211> 16 <212> PRT <213> Artificial Sequence <400> twenty three Ala Cys Asn Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg His Cys 1 5 10 15 <210> twenty four <211> 16 <212> PRT <213> Artificial Sequence <400> twenty four Asn Cys Asn Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg Ala Cys 1 5 10 15 <210> 25 <211> 16 <212> PRT <213> Artificial Sequence <400> 25 His Cys Asn Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg Ala Cys 1 5 10 15 <210> 26 <211> 15 <212> PRT <213> Artificial Sequence <400> 26 Cys Asn Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg Ala Cys 1 5 10 15 <210> 27 <211> 16 <212> PRT <213> Artificial Sequence <400> 27 Ala Cys Asn Cys Ser Ser Lys Trp Cys Ala Asp His Ser Arg Ala Cys 1 5 10 15 <210> 28 <211> 16 <212> PRT <213> Artificial Sequence <400> 28 Ala Cys Asn Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg Asp Cys 1 5 10 15 <210> 29 <211> 16 <212> PRT <213> Artificial Sequence <400> 29 Ala Cys Asn Cys Ser Ser Lys Trp Cys Leu Asp His Ser Arg Ala Cys 1 5 10 15 <210> 30 <211> 16 <212> PRT <213> Artificial Sequence <400> 30 Ser Cys Asn Cys Ser Ser Lys Trp Cys Arg Asp His Ser Arg Ala Cys 1 5 10 15 <210> 31 <211> 16 <212> PRT <213> Artificial Sequence <400> 31 Ala Cys Asn Cys Ser Ser Lys Trp Cys Phe Asp His Ser Arg Ala Cys 1 5 10 15 <210> 32 <211> 16 <212> PRT <213> Artificial Sequence <400> 32 Ala Cys Asn Cys Ser Ser Lys Trp Cys Val Asp His Ser Arg Ala Cys 1 5 10 15

Claims

1. A cone snail toxin KIIIA mutant, characterized in that: 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, and SEQ ID NO: 8; SEQ ID NO:2:RCNCDapaSKWCRDHSRAC*; SEQ ID NO:3:ACDabCSSKWCRDHSRRC*; SEQ ID NO:4:ACNCDapaSKWCRDHSRRC*; SEQ ID NO:5: RCDabCSSKWCRDHSRAC*; SEQ ID NO:6:ACDabCSDapaKWCRDHSRAC* SEQ ID NO:7:RCNCSSKWCRDHSRRC* SEQ ID NO:8: ACNCSSKWCRDHSRRC*; Among them, * indicates that the C-terminus is amidated; 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.

2. The method for preparing the cone snail toxin KIIIA mutant according to claim 1, wherein include: 1) synthesizing a crude peptide using Fmoc solid-phase synthesis, and subjecting the C-terminus to amidation; wherein the cysteines at positions 2 and 9 are protected with a first thiol protecting group, and the cysteines at positions 4 and 16 are protected with a second thiol protecting group, wherein the first thiol protecting group and the second thiol protecting group are different and independently selected from one of trityl (Trt) and acetamidomethyl (Acm); 2) performing disulfide bond oxidation based on the specific groups of the first thiol protecting group and the second thiol protecting group, and then purifying the product.

3. The preparation method according to claim 2, wherein Step 2) also includes: First, cysteine ​​with a trityl (Trt) thiol protecting group was oxidized by air oxidation; then, cysteine ​​with an acetamidomethyl (Acm) thiol protecting group was oxidized; The oxidation of cysteine ​​with acetamidomethyl (Acm) as a sulfhydryl protecting group is carried out by a method comprising the following steps: The crude peptide after air oxidation was dissolved in a reaction solvent, which was a mixture of water, acetonitrile and TFA in a volume ratio of 5:5:0.

01. An iodine-acetonitrile solution with an iodine concentration of 5 mg / mL was then added. After stirring for sufficient reaction in a closed environment at 28 degrees Celsius, an ascorbic acid aqueous solution was slowly added to neutralize excess iodine. The peptide was then separated by HPLC.

4. Use of the cone snail toxin KIIIA mutant as claimed in claim 1 in the preparation of analgesic drugs.

5. The use according to claim 4, characterized in that The analgesic drug is a drug used to treat chronic pain, neuralgia, cancer pain, herpes pain or diabetic pain.

6. An analgesic drug, characterized in that: The invention comprises the cone snail toxin KIIIA mutant according to claim 1.

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