Mu-type conotoxin peptide [Asn17, Arg6]-dR-mu-CnIIIC and application thereof

Abstract

The invention discloses a mu type conotoxin peptide [Asn17, Arg6]-dR-mu-CnIIIC and an application of the mu type conotoxin peptide [Asn17, Arg6]-dR-mu-CnIIIC. The amino acid sequence of the [mu] type conotoxin peptide [Asn17, Arg6]-dR-[mu]-CnIIIC provided by the invention is as shown in SEQ ID NO. 1. Compared with a wild type mu-type conotoxin peptide, the mu-type conotoxin peptide [Asn17, Arg6]-dR-mu-CnIIIC has higher activity, and the application of the mu-type conotoxin peptide [Asn17, Arg6]-dR-mu-CnIIIC in the fields of biological medicines and cosmetics is further expanded.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to a μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC and its applications. Background Art

[0002] μ-conotoxin (μCnIIIC) is a 22-residue cono peptide isolated from cone snails. It is an effective and long-lasting NaV1.4 channel blocker with analgesic, anesthetic and muscle relaxant properties. In addition to its potential pharmaceutical applications, it has excellent efficacy in relaxing skeletal muscle and has a rapid and immediate anti-wrinkle effect, so it is widely used in anti-wrinkle products. Studies have reported that μ-CnIIIC is involved in regulating Nav1.4 sodium channel-related diseases, such as pain syndrome, arrhythmia and muscle paralysis. The amino acid sequence of natural μ-CnIIIC contains three disulfide bonds. The presence of disulfide bonds poses a challenge to the synthesis of μ-CnIIIC peptides and the synthesis cost is high. The biological activity and stability of natural μ-CnIIIC are not high, and there are limitations on its use as a drug. Therefore, it is urgent to develop a new μ-conotoxin with high activity and simple synthesis. Summary of the invention

[0003] In view of the defects in the prior art, the present invention proposes a μ-type conotoxin peptide [Asn 17 ,Arg 6 The present invention aims to provide a μ-conotoxin peptide with higher activity than wild-type μ-conotoxin, which 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.

[0004] The invention discloses a μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC, the μ-type conotoxin peptide [Asn 17 ,Arg 6 The amino acid sequence of -dR-μ-CnIIIC is shown in SEQ ID NO. 1. The present invention firstly finds that replacing the 17th amino acid of the wild-type μ-conotoxin (μ-CnIIIC) with asparagine (Asn), the 6th amino acid with arginine (Arg), and deleting the 1st amino acid at the N-terminus of the original sequence and replacing the 2nd amino acid with D-arginine (d-Arg) can further improve the activity of μ-CnIIIC.

[0005] The present invention also provides a polynucleotide encoding the μ-type conotoxin peptide [Asn17 ,Arg 6 ]-dR-μ-CnIIIC.

[0006] The present invention also provides a nucleic acid construct comprising the polynucleotide.

[0007] The present invention also provides a recombinant expression vector comprising the nucleic acid construct.

[0008] The present invention also provides a transformed cell, comprising the recombinant expression vector.

[0009] The present invention also provides a pharmaceutical composition comprising the μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC.

[0010] Furthermore, the pharmaceutical composition also contains a pharmaceutically acceptable carrier or excipient.

[0011] The present invention also provides the μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC in the preparation of drugs for preventing or treating pain.

[0012] The present invention also provides the μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC in the preparation of anesthetic drugs.

[0013] The present invention also provides the μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC in the preparation of cosmetics.

[0014] The present invention also provides the μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC is used in the preparation of drugs for treating or preventing diseases related to sodium ion channels; the sodium ion channel-related diseases are any one of epilepsy, arrhythmia, muscle paralysis, myotonic spasm, and autism spectrum disorder.

[0015] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0016] The μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC, compared with wild-type μ-CnIIIC, [Asn 17 ,Arg6 ]-dR-μ-CnIIIC animal activity increased 10-fold, while [Asn 17 ,Arg 6 The amino acid sequence composition of ]-dR-μ-CnIIIC is reduced by 1 position compared with the wild-type μ-CnIIIC, which can further reduce the synthesis cost.

[0017] The μ-type conotoxin peptide of the present invention [Asn 17 ,Arg 6 ]-dR-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 and analgesia, and has an anesthetic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 The μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC synthesis process diagram;

[0020] Figure 2 This is a chromatogram of the wild-type μ-conotoxin μ-CnIIIC in Example 1 of the present invention;

[0021] Figure 3 This is the mass spectrum of the wild-type μ-conotoxin μ-CnIIIC in Example 1 of the present invention;

[0022] Figure 4 The μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC chromatogram;

[0023] Figure 5 The μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC mass spectrum. DETAILED DESCRIPTION

[0024] 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.

[0025] The term "μ-CnIIIC" in the present invention refers to the wild-type μ-conotoxin;

[0026] “[Asn 17 ,Arg 6 ]-dR-μ-CnIIIC" refers to a μ-conotoxin peptide in which the 17th amino acid of the wild-type μ-conotoxin (μ-CnIIIC) is replaced with asparagine (Asn), the 6th amino acid is replaced with arginine (Arg), and the 1st amino acid at the N-terminus of the original sequence is deleted, and the 2nd amino acid is replaced with D-arginine (d-Arg).

[0027] The Chinese meaning of the English abbreviation:

[0028] “dR” refers to D-arginine;

[0029] “N” refers to asparagine;

[0030] “R” refers to arginine;

[0031] “DCM” means dichloromethane;

[0032] “DIC” refers to N,N-diisopropylcarbodiimide;

[0033] "DMF" means N,N-dimethylformamide;

[0034] “HPLC” means high performance liquid chromatography;

[0035] “MeOH” means methanol;

[0036] “MTBE” means methyl tert-butyl ether;

[0037] "Oxyma" means ethyl 2-oximecyanoacetate;

[0038] “TFA” means trifluoroacetic acid;

[0039] "Fmoc" means 9-fluorenylmethoxycarbonyl;

[0040] “MS” refers to mass spectrometry;

[0041] The present invention replaces the 17th amino acid of the wild-type μ-conotoxin (μ-CnIIIC) with asparagine (Asn), and the 6th amino acid with arginine (Arg), and deletes the 1st amino acid at the N-terminus of the original sequence, and replaces the 2nd amino acid with D-arginine (d-Arg), which can further improve the activity of μ-CnIIIC. The analog is synthesized by solid phase peptide synthesis technology, and the disulfide bond is formed by liquid phase oxidation. Finally, the μ-conotoxin peptide [Asn] is purified by high performance liquid chromatography and freeze-dried. 17 ,Arg 6 ]-dR-μ-CnIIIC, the synthesis process is as follows Figure 1 shown.

[0042] The μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC amino acid sequence is shown in SEQ ID NO.1, and the μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC amino acid sequence, can be obtained without creative work encoding the μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC nucleotide sequence, thus encoding the μ-type conotoxin peptide [Asn 17 ,Arg 6 The nucleotide sequence of the μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC nucleotide sequence is inserted into a vector or strain, and fermentation is performed to produce the μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC, all are within the protection scope of the present invention.

[0043] Wild-type μ-CnIIIC: Sichuan Jisheng Biotechnology Co., Ltd., batch number: 2023041201-3.

[0044] Reagent K: lysis solution, prepared according to the volume ratio of TFA:phenol:water:thioanisole:ethanedithiol=82.5:5:5:5:2.5.

[0045] Example 1 μ-type conotoxin peptide [Asn 17 ,Arg 6 Synthesis of ]-dR-μ-CnIIIC

[0046] (1) Preparation of Fmoc-Cys(Trt)-Rink MBHA resin

[0047] ① Weigh 1.000g (0.63mmol) of Rink Amide AM resin (containing 1% DVB cross-linker, 100-200 mesh, 0.63mmol / g) and add it to a 20mL peptide solid phase reactor, add 10mL 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 10mL / time, the washing time is 3min / time, and the shaking speed is 500r / min.

[0048] ② After washing, drain the solvent, add 10mL of 20% piperidine / DMF solution to the reactor resin to remove the Fmoc protecting group of the resin, shake at 500r / min and 25℃ for 5min, and drain the solution; then add 10mL 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 (10mL / 3min / time) 5 times.

[0049] ③ Weigh 0.738g Fmoc-Cys(Trt)-OH (2.0eq, 1.26mmol) and 0.179g Oxyma (3eq, 1.26mmol) into a 50mL beaker, add 7mL DMF solution to dissolve, add 0.195mL condensing agent DIC (3eq, 1.26mmol) 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 5 times with DMF solution (10mL / 3min / time).

[0050] ④ Peptide chain extension

[0051] 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 (10 mL / 3 min / time). The resin was then washed alternately according to the following procedure: DCM × 5 times (10 mL / 3 min / time), MeOH × 5 times (10 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, 4.513 g of peptide resin was obtained with a yield of 97.1%.

[0052] ⑤ Cracking

[0053] Weigh 4.513g of the dry resin obtained in the above process ④, add the pre-prepared pre-cooled reagent K 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 1.533g of white solid crude peptide with a yield of 98.5%.

[0054] ⑥Cyclization

[0055] 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 7.96, stir and react at room temperature for 24 h, and monitor the reaction progress by HPLC. After the reaction is completed, HPLC purification can be performed directly.

[0056] ⑦Preparative HPLC for peptide purification

[0057] 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.

[0058] Table 1 Purification and elution procedures of crude cyclization solution

[0059]

[0060]

[0061] The fractions were collected for MS and HPLC analysis, and the target fractions were combined and freeze-dried to obtain the final [Asn 17 ,Arg6]-dR-μ-CnIIIC totaled 39.2 mg, yield 39.2%, HPLC purity 98.915%, MS results showed: [M+2H] 2+ =1231.5543, 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 [Asn 17 ,Arg6]-dR-μ-CnIIIC chromatogram Figure 4 As shown, μ-conotoxin peptide [Asn 17 ,Arg6]-dR-μ-CnIIIC mass spectrum is shown in Figure 5 shown.

[0062] Example 1 Preparation of [Asn 17 ,Arg6]-dR-μ-CnIIIC amino acid sequence is shown in SEQ ID NO. 1. The amino acid sequence of wild-type μ-CnIIIC is shown in SEQ ID NO. 2.

[0063] Example 2 Wild-type μ-CnIIIC and [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC biological activity assay

[0064] The mice used in this example were adult male Kunming mice purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Sciences, and the animal experiment was approved by the Ethics Committee of Lanzhou Peptide Valley Research Institute.

[0065] The mice were free to eat and drink one week before the test. A 50 μL microsyringe (30G needle) was used to intramuscularly inject the right tibialis anterior muscle group of the mice, with an injection volume of 20 μL, and an equal volume of saline was injected as a negative control, and wild-type μ-CnIIIC was used as a positive control. The minimum concentration of the experiment was set to 50 μM. The activity of the sample was judged by observing the onset time, duration, and behavior of the mice after injection, including toe closing (inhibition of muscle contraction), leg dragging / paralysis (anesthetic effect), and death behavior.

[0066] 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 reaction of extending their hind limbs and abducting them. The right tibialis anterior muscles of the mice were injected with physiological saline, wild-type μ-CnIIIC, and different concentrations of μ-conotoxin peptide [Asn 17 After ,Arg6]-dR-μ-CnIIIC, the degree of toe extension of the left and right hind limbs was measured as a function of time, and the corresponding behavior was observed based on a 5-point scale (0 means normal, 1 means only 2 toes (index and middle toes) are closed, 2 means 3 toes (index, middle and little toes) are closed; 3 means 4 toes except the ring toe are closed, and 4 means the maximum reduction of toe abduction and leg extension). Toe closing and leg dragging / paralysis are two behaviors of mice, and leg dragging and toe closing usually appear at the same time.

[0067] The behavior of leg dragging / paralysis (anesthetic effect) in mice was induced by injecting the drug into the anterior tibial muscle group of the right mice, and then placing them in a new environment to stimulate their exploratory nature, thereby observing their activity state. In the early stage of leg dragging / paralysis in mice, the lower body crawled on the ground, and in the later stage of the effect, the whole body collapsed.

[0068] The experimental results are summarized in Tables 2-3 in the order of increasing sample injection concentration:

[0069] Table 2 Wild-type μ-CnIIIC and [Asn 17 ,Arg6]-dR-μ-CnIIIC mouse experimental results (50μM)

[0070]

[0071] Note: 1. “ / ” means that the mice were asymptomatic after drug injection, so the onset time and duration of the effect could not be calculated;

[0072] Table 3 Experimental results of wild-type μ-CnIIIC mice

[0073]

[0074] Note: 1. “ / ” means that the mice were asymptomatic after drug injection, so the onset time and duration of the effect could not be calculated;

[0075] 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. 17 ,Arg 6 After injection of [Asn]-dR-μ-CnIIIC, mice showed lethal behaviors caused by excessive doses in a short period of time. The mice showed obvious leg-dragging behavior 8 minutes after injection and died 1 minute later. 17 ,Arg 6 ]-dR-μ-CnIIIC has higher activity than wild-type μ-CnIIIC.

[0076] The results in Table 3 show that mice injected with equal volumes of saline at each concentration behaved normally. Mice died only after 500 μM wild-type μ-CnIIIC was injected into the calf muscle, further indicating that μ-conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC has an activity more than 10 times that of wild-type μ-CnIIIC.

[0077] In summary, compared with wild-type μ-CnIIIC, μ-conotoxin peptide [Asn17 ,Arg 6 ]-dR-μ-CnIIIC activity increased 10-fold, indicating that [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC has high activity and can specifically block Nav1.4 channels, reduce or inhibit muscle contraction, and can be used in the preparation of drugs for treating or preventing diseases related to Nav1.4 sodium ion channels, or can be used in the preparation of cosmetics for eliminating wrinkles, or can be used in the preparation of anesthetic drugs, or can be used in the preparation of drugs for treating or preventing pain.

[0078] 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.

[0079] Sequence Listing

[0080] SEQ ID NO.1

[0081] Three characters:

[0082] d-Arg -Cys-Cys-Asn- Arg -Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg- Asn -His-Ala-Arg-Cys-Cys-NH2

[0083] Single character:

[0084] r CCN R PKGCSSKWCR N HARCC-NH2

[0085] SEQ ID NO.2

[0086] Three characters:

[0087] Pyr-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-NH2

[0088] Single character: (X represents pyroglutamic acid)

[0089] XGCCNGPKGCSSKWCRDHARCC-NH2

Claims

1. A μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC, characterized in that The μ-type conotoxin peptide [Asn 17 ,Arg 6 ]The amino acid sequence of -dR-μ-CnIIIC is shown in SEQ ID NO.

1.

2. A polynucleotide, characterized in that Encoding the μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC.

3. A nucleic acid construct, characterized in that Comprising the polynucleotide of claim 2.

4. A recombinant expression vector, characterized in that: Comprising the nucleic acid construct of claim 3.

5. A transformed cell, characterized in that: Comprising the recombinant expression vector according to claim 4.

6. A pharmaceutical composition, characterized in that The μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC.

7. The pharmaceutical composition according to claim 6, characterized in that It also contains pharmaceutically acceptable carriers or excipients.

8. The μ-conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC in the preparation of drugs for preventing or treating pain.

9. The μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC in the preparation of anesthetic drugs.

10. The μ-type conotoxin peptide [Asn 17 ,Arg 6 ]-dR-μ-CnIIIC in the preparation of cosmetics.

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