Mu-type conotoxin peptide mutant as well as pharmaceutical composition and application thereof

Abstract

The invention discloses a mu-type conotoxin peptide mutant as well as a pharmaceutical composition and application thereof. The amino acid sequence of the mu-type conotoxin peptide mutant is as shown in SEQ ID NO. 1. Compared with a wild type mu-type conotoxin peptide, the mu-type conotoxin peptide [d-Arg2, Ser17]-dR-mu-CnIIIC disclosed by the invention has higher activity, the animal activity is improved by more than 20 times, and muscle contraction can be effectively reduced by specifically blocking a Nav1.4 channel. In addition, the compound can be used as a non-invasive anti-wrinkle treatment means to reduce or eliminate the formation of wrinkles on the human skin surface, and has a wide application prospect in the fields of medical treatment and beauty.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a μ-type conotoxin peptide mutant, a pharmaceutical composition and use thereof. Background Art

[0002] The natural μ-conotoxin μ-CnIIIC peptide is a polypeptide toxin extracted from the venom of the marine mollusk cone snail, belonging to the conotoxin family. The μ-CnIIIC peptide mainly acts on voltage-gated sodium channels (VGSCs), especially the Nav1.4 subtype. Nav1.4 is mainly expressed in skeletal muscle and regulates muscle contraction. By blocking these sodium channels, the μ-CnIIIC peptide can inhibit the influx of sodium ions into nerve and muscle cells, thereby interfering with nerve signal transduction and muscle excitation. Since the μ-CnIIIC peptide can specifically block sodium channels, it also has significant analgesic effects, mainly by interfering with the signal transduction of pain-transmitting neurons, and has broad application prospects as an analgesic. In addition to its potential pharmaceutical applications, the μ-CnIIIC peptide has also been used in cosmetic dermatology to smooth out fine lines on the face. The natural μ-CnIIIC peptide exhibits significant biological activity, including analgesic effects and potential as a tool for neuroscience research, by specifically blocking sodium channels. These properties have made it of great interest in biomedical research and potential clinical applications.

[0003] However, the activity of natural μ-CnIIIC peptide is low, and high-dose use is prone to side effects, and the treatment cost is high. Therefore, it is urgent to provide a highly active and low-cost μ-CnIIIC peptide to reduce the drug dose required by users and further expand its clinical application range. Summary of the invention

[0004] In view of the defects in the prior art, the present invention proposes a μ-conotoxin peptide mutant, its pharmaceutical composition and use. The present invention provides a μ-conotoxin peptide with higher biological activity than wild-type μ-conotoxin, with a wide range of clinical applications, and can be used to specifically block Nav1.4 channels, reduce muscle contraction, reduce or eliminate the formation of wrinkles on the surface of human skin, and can also be used for muscle relaxation and analgesia.

[0005] The present invention discovers for the first time that adding a D-arginine (d-Arg) to the C-terminus of the wild-type μ-conotoxin (μ-CnIIIC), deleting the pyroglutamic acid (Pyr) at the first position, and replacing the amino acid at the 17th position with serine (Ser) and the amino acid at the second position with D-arginine (d-Arg) can further improve the activity of μ-CnIIIC.

[0006] The present invention provides a μ-type conotoxin peptide mutant, named [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC, the amino acid sequence of the μ-type conotoxin peptide mutant is shown in SEQ ID NO.1. The amino acid sequence contains three pairs of disulfide bonds, and the disulfide bonds are located at Cys 2 -Cys 14 、Cys 3 -Cys 20 and Cys 9 -Cys 21 The position is the same as the disulfide bond structure of the natural μ-type conotoxin peptide.

[0007] The present invention also provides a nucleotide sequence, which is any one of the following:

[0008] i) encoding the μ-type conotoxin peptide mutant;

[0009] ii) a nucleotide sequence complementary to i).

[0010] The present invention also provides an expression vector, which contains the nucleotide sequence.

[0011] The present invention also provides a recombinant strain, which comprises the expression vector.

[0012] The present invention also provides a pharmaceutical composition, which comprises the μ-type conotoxin peptide mutant.

[0013] In some embodiments, the dosage form of the pharmaceutical composition is any one of a tablet, a capsule, a pill, a solution, an absorbent, and an ointment.

[0014] The present invention also provides the use of the μ-type conotoxin peptide mutant in preparing drugs for treating or preventing diseases related to sodium ion channels.

[0015] In some embodiments, the sodium ion channel-related disease is any one of epilepsy, arrhythmia, muscle paralysis, myotonia, and autism spectrum disorder.

[0016] The present invention also provides the use of the μ-type conotoxin peptide mutant in preparing medicine for treating or preventing pain.

[0017] The present invention also provides application of the μ-type conotoxin peptide mutant in preparing anesthetic drugs.

[0018] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0019] 1. A μ-type conotoxin peptide mutant [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC, compared to wild-type μ-CnIIIC, [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC animal activity was increased by more than 20 times, and the desired therapeutic effect could be achieved at a lower concentration, enhancing target selectivity.

[0020] 2. [d-Arg 2 ,Ser 17 The increase in the number of basic amino acids in ]-dR-μ-CnIIIC can improve the hydrophilicity and membrane permeability of the sequence. Increasing the hydrophilicity of the peptide sequence can increase its solubility in the body. Enhancing membrane permeability allows the conotoxin peptide to more effectively pass through the cell membrane and enter the cell, thereby reaching its target, and can reduce the accumulation of conotoxin peptides in non-target tissues, thereby reducing potential toxicity and side effects.

[0021] 3. [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC can specifically block Nav1.4 channels, reducing muscle excitability and contractility. Blocking muscle contraction can relax facial muscles, reduce dynamic wrinkles caused by facial expressions, and help prevent and reduce the formation of wrinkles. [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC can reduce nerve conduction, thereby producing a local anesthetic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 The figure is a schematic diagram of the synthesis process of the μ-type conotoxin peptide mutant disclosed in the present invention;

[0024] Figure 2 This is a chromatogram of the wild-type μ-conotoxin μ-CnIIIC in Example 1 of the present invention;

[0025] Figure 3 This is the mass spectrum of the wild-type μ-conotoxin μ-CnIIIC in Example 1 of the present invention;

[0026] Figure 4 In Example 1 of the present invention, [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC chromatogram;

[0027] Figure 5 In Example 1 of the present invention, [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC mass spectrum. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0029] The term "μ-CnIIIC" in the present invention refers to the wild-type μ-conotoxin;

[0030] "[d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC" refers to the addition of a D-arginine (d-Arg) at the C-terminus of the wild-type μ-conotoxin (μ-CnIIIC), the deletion of the pyroglutamic acid (Pyr) at the first position, the replacement of the amino acid at the 17th position with serine (Ser), and the replacement of the amino acid at the second position with D-arginine (d-Arg), to form a conopeptide with higher activity than the wild-type μ-conotoxin;

[0031] The Chinese meaning of the English abbreviation:

[0032] “dR” refers to D-arginine;

[0033] “S” refers to serine;

[0034] “DCM” means dichloromethane;

[0035] “DIC” refers to N,N-diisopropylcarbodiimide;

[0036] "DMF" means N,N-dimethylformamide;

[0037] “HPLC” means high performance liquid chromatography;

[0038] “MeOH” means methanol;

[0039] “MTBE” means methyl tert-butyl ether;

[0040] "Oxyma" means ethyl 2-oximecyanoacetate;

[0041] “TFA” means trifluoroacetic acid;

[0042] "Fmoc" means 9-fluorenylmethoxycarbonyl;

[0043] "MS" refers to mass spectrometry.

[0044] The present invention first discovered that extending the sequence of the wild-type μ-conotoxin (μ-CnIIIC), adding a D-arginine (d-Arg) at the C-terminus, replacing the 17th amino acid with serine (Ser), deleting the 1st amino acid, and replacing the 2nd amino acid with the basic amino acid D-arginine (D-Arg) can produce μ-CnIIIC with different activities. The analogues were synthesized by solid-phase peptide synthesis technology, the disulfide bond was formed by liquid-phase oxidation, and finally purified by high-performance liquid chromatography and freeze-dried to obtain a novel μ-conotoxin peptide [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC, the synthesis process is as follows Figure 1 shown.

[0045] The μ-type conotoxin peptide [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC has an animal activity that is more than 20 times higher than that of the wild-type μ-CnIIIC. Retaining μ-CnIIIC can specifically block the Nav1.4 channel, reduce muscle contraction, reduce or eliminate the formation of wrinkles on the human skin surface, and can also be used for muscle relaxation, analgesia, and anesthesia.

[0046] Reagent K: lysis solution, prepared according to the volume ratio of TFA:phenol:water:thioanisole:ethanedithiol=82.5:5:5:5:2.5.

[0047] Wild-type μ-CnIIIC: Sichuan Jisheng Biotechnology Co., Ltd., batch number: 2023041201-3.

[0048] Example 1 μ-type conotoxin peptide [d-Arg 2 ,Ser 17 Synthesis of ]-dR-μ-CnIIIC

[0049] (1) Preparation of Fmoc-d-Arg(Pbf)-Rink AM resin

[0050] ① Weigh 2.00g (1.28mmol) of Rink Amide AM resin (containing 1% DVB cross-linker, 100-200 mesh, 0.64mmol / g) and add it to a 60mL peptide solid phase reactor, add 15mL of DCM solution to the reactor, set the shaking speed to 550r / min, shake for 45min, drain the solution, add DMF solution to wash the resin twice, the washing solvent volume is 15mL / time, the washing time is 3min / time, and the shaking speed is 500r / min.

[0051] ② After washing, drain the solvent, add 15mL of 20% piperidine / DMF solution to the reactor resin to remove the resin Fmoc protecting group, shake at 500r / min and 25℃ for 5min, and drain the solution; then add 15mL of 20% piperidine / DMF solution to the resin again, shake at 500r / min and 25℃ for 15min, and drain the solution. Wash the resin with DMF solution (15mL / 3min / time) for 5 times.

[0052] ③ Weigh 1.660g Fmoc-Arg(Pbf)-OH (2.0eq, 2.56mmol) and 0.363g Oxyma (2eq, 2.56mmol) into a 50mL beaker, add 10mL DMF solution to dissolve, add 0.396mL condensing agent DIC (2eq, 2.56mmol) to the amino acid solution for activation reaction for 5min, then add to the above deprotected resin, shake at 500r / min and 25℃ for 1h; after the reaction is completed, wash the resin with DMF solution (15mL / 3min / time) for 5 times.

[0053] ④ Peptide chain extension

[0054] According to the sequence composition, steps ② and ③ were repeated until the last amino acid was coupled. The Fmoc protecting group was removed and the resin was washed 5 times with DMF solution (15 mL / 3 min / time). The resin was then washed alternately according to the following procedure: DCM × 5 times (15 mL / 3 min / time), MeOH × 5 times (15 mL / 3 min / time). The resin was finally in a shrunk state and placed in a vacuum drying oven at 25 ° C to constant weight. Finally, 7.495 g of peptide resin was obtained with a yield of 94.1%.

[0055] ⑤ Cracking

[0056] Weigh 7.495g of the dry resin obtained in the above process ④, add the pre-prepared pre-cooled K reagent lysis solution according to the ratio of 15mL lysis solution per gram of peptide resin, and shake at 300r / min 25℃ to avoid light for 3h. After the reaction is completed, the lysis solution is slowly added dropwise to the pre-cooled MTBE solution according to the ratio of lysis solution / methyl tert-butyl ether = 1:10 (v / v), and a white precipitate is generated. Then centrifuge at 500rpm / min, discard the supernatant, add new MTBE solution, shake, centrifuge, discard the supernatant, repeat the above centrifugation process 5 times, collect the sludge-like white precipitate, vacuum dry at 25℃ to constant weight, and finally obtain 3.029g of white solid crude peptide with a yield of 95.1%.

[0057] ⑥Cyclization

[0058] Weigh 0.100 g of the crude white solid peptide obtained in step ⑥ above, add 100 mL of sodium hydrogen phosphate / guanidine hydrochloride buffer solution, adjust the pH to 7.82, stir the mixture in an open air at room temperature for 24 h, and monitor the reaction progress by HPLC. After the reaction is completed, HPLC purification can be performed directly.

[0059] ⑦Preparative HPLC for peptide purification

[0060] The cyclization reaction liquid of the above process ⑥ was directly injected, and the sample purification was completed according to the gradient elution program in Table 1, wherein mobile phase A: 80% acetonitrile / water (containing 0.1% TFA), mobile phase B: water (containing 0.1% TFA); detection wavelength: 220nm; flow rate: 10mL / min; chromatographic column specifications: 20×250mm, 10μm, 120A.

[0061] Table 1 Purification and elution procedures of crude cyclization solution

[0062]

[0063] The fractions were collected for MS and HPLC analysis, and the target fractions were combined and freeze-dried to obtain the final [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC totaled 36.3 mg, with a yield of 36.3%, HPLC purity of 99.217%, and MS results showed: [M+2H] 2+ =1246.5422, the molecular weight is correct. The sample obtained above will be used in the experiment of Example 2. The chromatogram of wild type μ-CnIIIC is shown in Figure 2 As shown, the mass spectrum of wild-type μ-CnIIIC is shown Figure 3 As shown. μ-type conotoxin peptide [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC chromatogram Figure 4As shown, μ-conotoxin peptide [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC mass spectrum Figure 5 shown.

[0064] Example 1 Preparation of [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC amino acid sequence is shown in SEQ ID NO.1, and the amino acid sequence of wild-type μ-CnIIIC is shown in SEQ ID NO.2.

[0065] Example 2 Wild-type μ-CnIIIC and [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC biological activity assay

[0066] The mice used in the experiment of the present invention are adult male Kunming mice, which were purchased from the Lanzhou Veterinary Research Institute of the Chinese Academy of Sciences, and the animal experiments were approved by the Ethics Committee of the Lanzhou Peptide Valley Research Institute. The mice were free to eat and drink water one week before the test. A 50 μL microsyringe (30G needle) was used to inject intramuscularly into the right tibialis anterior muscle group of the mouse, and the injection volume was 20 μL. An equal volume of saline was injected as a negative control, and the wild type μ-CnIIIC was used as a positive control. The experiment set a concentration gradient of 25 and 50 μM. The activity of the sample was judged by observing the onset time, duration, and behavior of the mice after injection, including toes together (inhibiting muscle contraction), dragging legs / paralysis (anesthetic effect), and death behavior.

[0067] The toe abduction score (DAS) test was used to analyze the toe abduction behavior of mice. The mice were suspended by their tails to stimulate the characteristic startle response of the mice to extend their hind limbs and abduct them. The right tibialis anterior muscles of the mice were injected with physiological saline, wild-type μ-CnIIIC, different concentrations of μ-conotoxin peptide [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC, the degree of toe abduction of the left and right hind limbs was measured as a function of time, and whether there was corresponding behavior was observed based on a 5-point scale (0 means normal, 1 means only two toes (index and middle toe) are together, 2 means three toes (index, middle and little toe) are together; 3 means all four toes except the ring toe are together, and 4 means the maximum reduction of toe abduction and leg extension).

[0068] The behavior of leg dragging / paralysis (anesthetic effect) in mice is tested by injecting the drug into the right tibialis anterior muscle group of mice, placing them in a new environment to stimulate their exploratory nature, and observing the activity of the mice; the initial onset of leg dragging / paralysis in mice is characterized by the lower body crawling on the ground, and the whole body lying down in the later stage of the onset. Toes together and leg dragging / paralysis are two behaviors of mice, and leg dragging and toes together usually occur at the same time.

[0069] The experimental results are shown in Table 2:

[0070] Table 2 Wild-type μ-CnIIIC and [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC Animal Experiment Results (50μM)

[0071]

[0072] Note: 1. “ / ” means that the mice were asymptomatic after drug injection, so the onset time and duration of the effect could not be calculated;

[0073] The results in Table 2 show that after the mouse calf muscle was injected with 50 μM concentration of wild-type μ-CnIIIC, the mouse showed toe-closing behavior, indicating that wild-type μ-CnIIIC showed inhibitory muscle contraction activity at a concentration of 50 μM. 2 ,Ser 17 After injection of [d-Arg]-dR-μ-CnIIIC, mice showed lethal behaviors caused by excessive doses in a short period of time. The mice showed obvious leg dragging and toe-pulling behaviors 3 minutes after injection. The mice became paralyzed and motionless 5 minutes after injection, and died 7 minutes later. 2 ,Ser 17 ]-dR-μ-CnIIIC has higher activity than wild-type μ-CnIIIC.

[0074] To further explore [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC activity, [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC concentration was reduced to 25 μM, and the results are shown in Table 3.

[0075] Table 3 [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC Animal Experiment Results (25μM)

[0076]

[0077] The results in Table 3 show that after injection of 25 μM [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC 1 minute later, the mice showed obvious leg-dragging behavior, which lasted for 30 minutes and eventually died due to excessive dose, indicating that [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC still exhibited the activity of inhibiting muscle contraction and paralysis at a low concentration (25 μM) and showed high activity.

[0078] Subsequently, mice were treated with wild-type μ-CnIIIC at two concentrations, 100 μM and 500 μM, and the onset time and duration of the drug after injection as well as the toe-closing (inhibiting muscle contraction) behavior of the mice after injection were observed. The results are shown in Table 4.

[0079] Table 4 Animal experimental results of wild-type μ-CnIIIC at concentrations greater than 50 μM

[0080]

[0081] Note: 1. “ / ” means that the mice were asymptomatic after drug injection, so the onset time and duration of the effect could not be calculated;

[0082] The mice in the group injected with an equal volume of saline in Table 4 showed normal performance. The results showed that the mice died only after 500 μM wild-type μ-CnIIIC was injected into the calf muscle. This further illustrates that the μ-conotoxin peptide [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC has an activity more than 20 times that of wild-type μ-CnIIIC.

[0083] In summary, the present invention first discovered that extending the sequence of the wild-type μ-conotoxin (μ-CnIIIC), adding a D-arginine (d-Arg) at the C-terminus, replacing the 17th amino acid with serine (Ser), deleting the 1st amino acid, and replacing the 2nd amino acid with the basic amino acid D-arginine (d-Arg) can produce μ-CnIIIC with different activities. The synthesis of the analogue was completed by solid phase peptide synthesis technology, the formation of disulfide bonds was completed by liquid phase oxidation, and finally the novel μ-conotoxin peptide [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC, the μ-type conotoxin peptide [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC synthesis process is as follows Figure 1Compared with wild-type μ-CnIIIC, μ-conotoxin peptide [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC increased its animal activity by more than 20 times.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0085] Sequence Listing

[0086] SEQ ID NO.1

[0087] Three characters:

[0088] d-Arg -Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg- Ser -His-Ala-Arg-Cys-Cys- d-Arg -NH2

[0089] Single character:

[0090] r CCNGPKGCSSKWCR S HARCC r -NH2

[0091] SEQ ID NO.2

[0092] Three characters:

[0093] Pyr-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-NH2

[0094] Single character: (X represents pyroglutamic acid)

[0095] XGCCNGPKGCSSKWCRDHARCC-NH2.

Claims

1. A μ-type conotoxin peptide mutant, characterized in that: The amino acid sequence of the μ-type conotoxin peptide mutant is shown in SEQ ID NO.

1.

2. A nucleotide sequence, characterized in that The nucleotide sequence is any one of the following: i) encoding the μ-type conotoxin peptide mutant according to claim 1; ii) a nucleotide sequence complementary to i).

3. An expression vector, characterized in that: The expression vector comprises the nucleotide sequence of claim 2.

4. A recombinant strain, characterized in that: The recombinant strain comprises the expression vector according to claim 3.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the μ-type conotoxin peptide mutant according to claim 1.

6. The pharmaceutical composition according to claim 5, characterized in that The dosage form of the pharmaceutical composition is any one of tablets, capsules, pills, solutions, absorbents and ointments.

7. Use of the μ-type conotoxin peptide mutant according to claim 1 in the preparation of drugs for treating or preventing diseases related to sodium ion channels.

8. The use according to claim 7, characterized in that: The sodium ion channel-related disease is any one of epilepsy, arrhythmia, muscle paralysis, myotonia, and autism spectrum disorder.

9. Use of the μ-type conotoxin peptide mutant according to claim 1 in the preparation of a drug for treating or preventing pain.

10. Use of the μ-type conotoxin peptide mutant according to claim 1 in the preparation of anesthetic drugs.

Images