Mu-type conotoxin peptide [Ser17, Tyr11]-dR-Mu-CnIIIC and application thereof

Abstract

The invention discloses a [mu] type conotoxin peptide [Ser17, Tyr11]-dR-[mu]-CnIIIC and an application thereof. The amino acid sequence of the mu-type conotoxin peptide is as shown in SEQ ID NO. 1. Compared with wild type mu-CnIIIC and [Ser17, Tyr11]-dR-mu-CnIIIC, the animal activity of the mu type conotoxin peptide disclosed by the invention is improved by 20 times, and meanwhile, the amino acid sequence composition of the [Ser17, Tyr11]-dR-mu-CnIIIC is reduced by 1 site compared with the wild type mu-CnIIIC, so that the synthesis cost can be further reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biopharmacy, in particular to a μ-conotoxin peptide [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC and application thereof. BACKGROUND

[0002] Conotoxin is from the venom of a gastropod mollusk conus growing in the tropical ocean, which is complex in composition and has numerous families. The action site of μ-conotoxin (μ-CnIIIC) family is a sodium ion channel, which is a muscle-type sodium ion channel inhibitor. Although μ-CnIIIC and classic sodium channel antagonists tetrodotoxin and saxitoxin both act on sodium channel receptor site 1, it selectively blocks muscle-type sodium channels without blocking nerve-type sodium channels. μ-CnIIIC can be used as a specific ligand for exploring biochemical mechanisms by selectively blocking sodium conduction, and is an excellent tool for studying sodium ion channels. Natural μ-CnIIIC is composed of 22 amino acids, can reduce muscle contraction, analgesia, and is widely used in medicine, chemical industry, and can smooth facial fine lines as a cosmetic.

[0003] However, natural μ-CnIIIC has the defects of low stability and activity, and the cost of polypeptide synthesis is high in the production industry, and μ-CnIIIC peptide as a drug has potential limitations. SUMMARY

[0004] In view of the defects in the prior art, the present application provides a μ-conotoxin peptide [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC and application thereof. The present application provides a μ-conotoxin peptide with higher activity than wild-type μ-conotoxin, which can be used for specific blocking of Nav1.4 channel, reducing muscle contraction, reducing or eliminating the formation of wrinkles on the surface of human skin, and also can be used for muscle relaxation and analgesia.

[0005] The present application provides a μ-conotoxin peptide, and the amino acid sequence of the μ-conotoxin peptide is shown in SEQ ID NO. 1. The present application first finds that replacing the 17th amino acid of wild-type μ-conotoxin (μ-CnIIIC) with serine (Ser), replacing the 11th amino acid with tyrosine (Tyr), and deleting the 1st amino acid at the N-terminal of the original sequence and replacing the 2nd amino acid with D-arginine (d-Arg) can further improve the activity of μ-CnIIIC.

[0006] Furthermore, the μ-type cone snail toxin peptide also includes a product obtained by chemically modifying one or more sites at the ends of the main chain or side chain of the polypeptide shown in SEQ ID NO.1, including amino, carboxyl, thiol, phenolic hydroxyl, imidazole, guanidinyl, indole, and methylthio groups.

[0007] This invention also provides the application of μ-conotoxin peptide in the preparation of anesthetic drugs.

[0008] This invention also provides the application of μ-conotoxin peptides in the preparation of drugs for the prevention or treatment of pain.

[0009] This invention also provides the use of μ-conotoxin peptides in the preparation of drugs for the treatment or prevention of diseases related to sodium ion channels.

[0010] Furthermore, the sodium ion channel-related diseases are any one of muscle paralysis, tonic-clonic syndrome, arrhythmia, epilepsy, or autism spectrum disorder.

[0011] The present invention also provides a composition comprising the aforementioned μ-type cone snail toxin peptide.

[0012] Furthermore, the composition is a pharmaceutical composition or a health product.

[0013] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients and / or additives.

[0014] Furthermore, the dosage form of the pharmaceutical composition is a liquid dosage form, a solid dosage form, or a semi-solid dosage form.

[0015] In summary, compared with the prior art, the present invention achieves the following technical effects: The μ-type conotoxin peptide [Ser] of the present invention 17 Tyr 11 ]-dR-μ-CnIIIC, compared to wild-type μ-CnIIIC, [Ser 17 Tyr 11 The animal activity of ]-dR-μ-CnIIIC increased 20-fold, while [Ser 17 Tyr 11 The amino acid sequence of ]-dR-μ-CnIIIC is reduced by one position compared to wild-type μ-CnIIIC, which can further reduce the synthesis cost.

[0016] The μ-type conotoxin peptide [Ser] of the present invention 17 Tyr 11 ]-dR-μ-CnIIIC can 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, and has an anesthetic effect. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required to be used in the embodiments will be briefly introduced as follows. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. Other related drawings can also be obtained by those skilled in the art without creative labor on the basis of the drawings.

[0018] Figure 1 The μ-conotoxin peptide [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC synthesized in the embodiment 1 of the present application; Figure 2 The chromatogram of the wild-type μ-conotoxin μ-CnIIIC in the embodiment 1 of the present application; Figure 3 The mass spectrum of the wild-type μ-conotoxin μ-CnIIIC in the embodiment 1 of the present application; Figure 4 The chromatogram of the μ-conotoxin peptide [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC in the embodiment 1 of the present application; Figure 5 The mass spectrum of the μ-conotoxin peptide [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC in the embodiment 1 of the present application. DETAILED DESCRIPTION

[0019] In order to make the person skilled in the art better understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should belong to the scope of protection of the present application.

[0020] The term "μ-CnIIIC" in the present application refers to the wild-type μ-conotoxin; "[Ser 17 , Tyr 11 ]-dR-μ-CnIIIC" refers to The Chinese meaning of the English abbreviation: "d-R" refers to D-type arginine; "S" refers to serine; "Y" refers to tyrosine; “DCM” refers to dichloromethane; “DIC” refers to N,N-diisopropylcarbodiimide; “DMF” refers to N,N-dimethylformamide; “HPLC” refers to high performance liquid chromatography; “MeOH” refers to methanol; “MTBE” refers to methyl tert-butyl ether; “Oxyma” refers to 2-hydroxy-5-norbornene-2-carboxylic acid ethyl ester; “TFA” refers to trifluoroacetic acid; “Fmoc” refers to 9-fluorenylmethoxycarbonyl; “MS” refers to mass spectrometry; The present application replaces the 17th amino acid of wild-type mu-conotoxin (mu-CnIIIC) with serine (Ser) and the 11th amino acid with tyrosine (Tyr), 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), to form a new high-activity conotoxin peptide with 1 less amino acid than the wild-type mu-conotoxin sequence.

[0021] The present application completes the synthesis of the analog by solid-phase peptide synthesis technology, forms the disulfide bond by liquid-phase oxidation, and finally purifies and lyophilizes by high-performance liquid chromatography to obtain the new mu-conotoxin peptide, and the synthesis flow is shown in Figure 1 Compared with the wild-type mu-CnIIIC, the animal activity of the mu-conotoxin peptide [Ser 17 , Tyr 11 ]-dR-mu-CnIIIC is increased by 20 times, and the amino acid sequence composition of [Ser 17 , Tyr 11 ]-dR-mu-CnIIIC is reduced by 1 compared with the wild-type mu-CnIIIC, which can further reduce the synthesis cost.

[0022] The amino acid sequence of the mu-conotoxin peptide [Ser 17 , Tyr 11 ]-dR-mu-CnIIIC according to the present application is shown in SEQ ID NO. 1, and the nucleotide sequence encoding the mu-conotoxin peptide [Ser 17 , Tyr 11 ]-dR-mu-CnIIIC can be obtained without making creative labor, so the nucleotide sequence encoding the mu-conotoxin peptide [Ser 17 , Tyr 11 ]-dR-mu-CnIIIC is obtained, and therefore, the nucleotide sequence encoding the mu-conotoxin peptide [Ser 17 , Tyr 11The nucleotide sequence of ]-dR-μ-CnIIIC also falls within the scope of protection of this invention. By encoding the μ-type conotoxin peptide [Ser 17 Tyr 11 The nucleotide sequence of ]-dR-μ-CnIIIC is inserted into a vector or strain for fermentation to produce the μ-type cone snail toxin peptide [Ser 17 Tyr 11 All of these are within the protection scope of this invention.

[0023] The μ-type conotoxin peptide [Ser 17 Tyr 11 ]-dR-μ-CnIIIC is used to prepare pharmaceutical compositions, or the μ-type conotoxin peptide [Ser 17 Tyr 11 The use of ]-dR-μ-CnIIIC in the preparation of medicaments for the treatment or prevention of pain, or the μ-type conotoxin peptide [Ser 17 Tyr 11 The use of ]-dR-μ-CnIIIC in the preparation of drugs for the treatment or prevention of diseases related to Nav1.4 sodium ion channels, or the μ-type conotoxin peptide [Ser 17 Tyr 11 The application of ]-dR-μ-CnIIIC in the preparation of cosmetics is within the protection scope of this invention.

[0024] Wild-type μ-CnIIIC: Sichuan Jisheng Biopharmaceutical Co., Ltd., batch number: 2023041201-3.

[0025] Reagent K: lysis buffer, prepared according to the volume ratio of TFA: phenol: water: anisole: ethylene dithiol = 82.5: 5: 5: 5: 2.5.

[0026] Example 1 μ-type conotoxin peptide [Ser 17 Tyr 11 Synthesis of ]-dR-μ-CnIIIC (1) Preparation of Fmoc-Cys(Trt)-Rink MBHA resin ① Weigh 1.521 g (0.96 mmol) of Rink Amide AM resin (containing 1% DVB crosslinking agent, 100-200 mesh, 0.63 mmol / g) into a 60 mL peptide solid-phase reactor, add 15 mL of DCM solution to the reactor, set the shaking speed of the shaking table to 550 r / min, shake for 45 min, then drain the solution, wash the resin with DMF solution twice, the washing solvent volume is 15 mL / time, the washing time is 3 min / time, and the shaking speed of the shaking table is 500 r / min.

[0027] ② After washing is complete, drain the solvent, and add 15 mL of 20% piperidine / DMF solution to the resin in the reactor to remove the Fmoc protecting group, shake at 500 r / min for 5 min at 25°C, and drain the solution; then add another 15 mL of 20% piperidine / DMF solution to the resin, shake at 500 r / min for 15 min at 25°C, and drain the solution. Wash the resin with DMF solution (15 mL / 3 min / time) 5 times.

[0028] ③ Weigh 1.124 g of Fmoc-Cys(Trt)-OH (2.0 eq, 1.92 mmol) and 0.273 g of Oxyma (3 eq, 1.92 mmol) into a 50 mL beaker, dissolve in 10 mL of DMF solution, add 0.297 mL of condensing agent DIC (3 eq, 1.92 mmol) to the amino acid solution and activate for 5 min, then add to the above deprotected resin, shake at 500 r / min for 1 h at 25°C; after the reaction is complete, wash the resin with DMF solution (15 mL / 3 min / time) 5 times.

[0029] ④ Peptide chain extension According to the sequence composition, repeat steps ② and ③ subsequently until the coupling of the last amino acid is complete. Remove the Fmoc protecting group and wash the resin with DMF solution (15 mL / 3 min / time) 5 times. Then wash the resin alternately according to the following procedure: DCM x 5 times (15 mL / 3 min / time), MeOH x 5 times (15 mL / 3 min / time), and finally the resin is in a shrunk state, placed in a vacuum drying oven at 25°C, and dried under vacuum to a constant weight. The final peptide resin is 5.763 g with a yield of 97.3%.

[0030] ⑤ Cleavage Take the dry resin obtained in the above process ④, 5.763 g, and add the freshly prepared and pre-cooled reagent K cleavage solution according to the ratio of 15 mL of cleavage solution per gram of peptide resin. Shake at 300 r / min for 3 h at 25°C in the dark. After the reaction is completed, add the cleavage solution slowly into the pre-cooled MTBE solution at a ratio of cleavage solution / methyl tert-butyl ether = 1:10 (v / v), and a white precipitate is generated. Then centrifuge at 500 rpm / min, discard the supernatant, add new MTBE solution, shake, centrifuge, discard the supernatant, and repeat the above centrifugation process 5 times. Collect the muddy white precipitate and dry it to constant weight under vacuum at 25°C. Finally, obtain 2.139 g of white solid crude peptide with a yield of 90.2%.

[0031] ⑥ Cyclization Take the white solid crude peptide obtained in the above process ⑥, 0.100 g, and add 100 mL of sodium phosphate dibasic / guanidine hydrochloride buffer solution to adjust the pH to 7.8. Stir at room temperature for 24 h, and monitor the reaction progress by HPLC. After the reaction is completed, directly purify by HPLC.

[0032] ⑦ Purification of peptides by preparative HPLC Directly inject the cyclization reaction solution in the above process ⑥, and complete the sample purification according to the gradient elution program in Table 1, where the mobile phase A is 80% acetonitrile / water (containing 0.1% TFA), the mobile phase B is water (containing 0.1% TFA), the detection wavelength is 220 nm, the flow rate is 10 mL / min, and the column specifications are 20×250 mm, 10 µm, and 120 A.

[0033] Table 1 Elution program for purification of crude cyclization solution

[0034] Collect the fractions for MS and HPLC analysis, combine the target fractions, and freeze-dry to obtain [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC, which is 36.7 mg in total, with a yield of 36.7% and an HPLC purity of 98.600%. The MS result shows that [M+2H] 2+ = 1206.0647, which is correct in molecular weight. The sample obtained above will be used for the experiment in Example 2. The chromatogram of wild-type μ-CnIIIC is shown in Figure 2 , and the mass spectrum of wild-type μ-CnIIIC is shown in Figure 3 . The chromatogram of μ-conotoxin peptide [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC is shown in Figure 4 , and the chromatogram of μ-conotoxin peptide [Ser 17 , Tyr11 The mass spectrum of [Ser Figure 5 , Tyr

[0035] [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC prepared in Example 1 has an amino acid sequence as shown in SEQ ID NO. 1. The amino acid sequence of wild type μ-CnIIIC is shown in SEQ ID NO. 2.

[0036] Example 2 Biological activity assay of wild type μ-CnIIIC and [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC The mice used in this example are adult male Kunming mice, which are purchased from the Institute of Laboratory Animal Science, Chinese Academy of Sciences, and the animal experiments are approved by the Ethics Committee of Lanzhou Peitugu Research Institute.

[0037] The mice are allowed to eat and drink freely for one week before the test. Intramuscular injection is performed on the right tibialis anterior muscle group of the mice using a 50 μL microsyringe (30 G needle), with an injection volume of 20 μL. An equal volume of normal saline is injected as a negative control, and wild type μ-CnIIIC is injected as a positive control. The concentration gradient of the experiment is set to 25 μM and 50 μM. The activity of the sample is judged by observing the onset time and duration after injecting the drug, as well as the behavior of the mice after injection, including toe clamping (muscle contraction inhibition), dragging / paralysis (narcotic effect), and death behavior.

[0038] The toe clamping (muscle contraction inhibition) behavior of the mice is analyzed using the mouse toe spread score test (DAS). The characteristic startle response of the mice to stretch the hind limbs and the degree of spread is induced by suspending the mice by the tail. After injecting normal saline, wild type μ-CnIIIC, and different concentrations of μ-conotoxin peptide [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC into the right tibialis anterior muscle group of the mice, the degree of toe spread of the left and right hind limbs is measured as a function of time, and the corresponding behavior is observed according to a 5-point scale (0 indicates normal, 1 indicates that only 2 toes (the first and second toes) are clamped, 2 indicates that 3 toes (the first, second, and third toes) are clamped; 3 indicates that 4 toes except the fifth toe are clamped, and 4 indicates the maximum reduction of toe spread and leg stretch).

[0039] The mouse drag leg / paralysis (narcotic effect) behavior is observed by injecting the right tibialis muscle group of a mouse, placing it in a new environment to stimulate its exploratory nature, and observing the activity of the mouse; the mouse drag leg / paralysis initially appears as the lower body crawling on the ground, and the entire body is prone after the onset. Dragging legs and toe curling are two behaviors of mice, and dragging legs and toe curling generally occur at the same time.

[0040] The experimental results are shown in Table 2: Table 2 Wild type μ-CnIIIC and [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC animal experiment results (50 μM)

[0041] Note: 1, " / " indicates that the mouse has no symptoms after drug injection, so the onset time and duration cannot be counted; Table 2 shows that after injecting 50 μM concentration of wild type μ-CnIIIC into the back of the mouse's leg, the mouse showed toe curling behavior, indicating that wild type μ-CnIIIC showed muscle contraction inhibition activity at 50 μM concentration. After injecting the same concentration of [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC, the mouse showed a short-term overdose-induced lethal behavior, in which the mouse showed drag leg behavior 4 minutes after injection, and then showed death 4 minutes after being prone, indicating that [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC has higher activity than wild type μ-CnIIIC.

[0042] In order to further explore the activity of [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC, the concentration of [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC was reduced to 25 μM, and the results are shown in Table 3.

[0043] Table 3 [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC animal experiment results (25 μM)

[0044] Table 3 shows that after injecting 25 μM of [Ser 17 , Tyr 11After 8 min of [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC injection, the mice showed flaccid recumbency, obvious dragging of the legs and toe-curling behavior, which lasted for more than 120 min. This indicates that [Ser

[0045] To further explore the activity of [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC at a low concentration, the concentration of [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC was reduced to 10 μM, and the results of the animal experiment are shown in Table 4.

[0046] Table 4 Results of animal experiment of [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC (10 μM)

[0047] The results in Table 4 show that after 18 min of injection of 10 μM [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC into the hind leg muscles of the mice, the mice showed obvious dragging of the legs and toe-curling behavior, which lasted for more than 240 min. This indicates that [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC still showed the activity of inhibiting muscle contraction and paralysis at a low dose of 10 μM. At the same time, the mice remained in good survival condition during the observation period, and no obvious toxic side effects were observed, indicating that the polypeptide has good safety while exerting pharmacological effects.

[0048] Subsequently, the mice were treated with wild-type μ-CnIIIC at two concentrations of 100 μM and 500 μM, and the onset time, duration and toe-curling (inhibition of muscle contraction) behavior of the mice after injection of the drug were observed, and the results are shown in Table 5.

[0049] Table 5 Results of animal experiment of wild-type μ-CnIIIC

[0050] Note: 1 / indicates that the mice showed no symptoms after injection of the drug, so the onset time and duration cannot be calculated; The results of Table 5 show that mice injected with the same volume of normal saline at each concentration behaved normally. Death occurred only after injection of 500 μM wild-type μ-CnIIIC into the posterior calf muscle of the mice, further indicating that the activity of the μ-conotoxin peptide [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC was more than 20 times that of wild-type μ-CnIIIC.

[0051] In summary, the μ-conotoxin peptide [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC has high activity and is improved by 20 times compared to wild-type μ-CnIIIC, further indicating the high activity of [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC. The [Ser 17 , Tyr 11 ]-dR-μ-CnIIIC of the present application can specifically block the Nav1.4 channel, reduce or inhibit muscle contraction, has analgesic and anesthetic effects, and can be used for the preparation of a medicament for treating or preventing diseases related to the Nav1.4 sodium ion channel, or can be used for the preparation of a cosmetic for eliminating wrinkles, or can be used for the preparation of an anesthetic medicament, or can be used for the preparation of a medicament for treating or preventing pain.

[0052] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

[0053] SEQUENCE LISTING SEQ ID NO. 1 Three characters: d-Arg -Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys- Tyr -Ser-Lys-Trp-Cys-Arg- Ser -His-Ala-Arg-Cys-Cys-NH2 Single character: rCCNGPKGCYSKWCRSHARCC-NH2 SEQ ID NO. 2 Three characters: Pyr-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-NH2 Monomer: (X represents pyroglutamic acid) XGCCNGPKGCSSKWCRDHARCC-NH 2。

Claims

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

1.

2. The μ-type cone snail toxin peptide according to claim 1, characterized in that, The μ-type cone snail toxin peptide also includes products obtained by chemical modification of one or more sites at the ends of the main chain or side chain of the polypeptide shown in SEQ ID NO.1, including amino, carboxyl, thiol, phenolic hydroxyl, imidazole, guanidinyl, indole, and methylthio groups.

3. The use of the μ-type cone snail venom peptide according to claim 1 or 2 in the preparation of anesthetic drugs.

4. The use of the μ-type cone snail toxin peptide according to claim 1 or 2 in the preparation of drugs for the prevention or treatment of pain.

5. The use of the μ-type cone snail toxin peptide according to claim 1 or 2 in the preparation of medicaments for the treatment or prevention of diseases related to sodium ion channels.

6. The application according to claim 5, characterized in that, The sodium channel-related diseases mentioned are any one of the following: muscle paralysis, tonic-clonic syndrome, arrhythmia, epilepsy, and autism spectrum disorder.

7. A composition, characterized in that, The composition comprises the μ-type conotoxin peptide according to any one of claims 1 to 2.

8. The composition according to claim 7, characterized in that, The composition is a pharmaceutical composition or a health product.

9. The composition according to claim 8, characterized in that, The pharmaceutical composition further includes pharmaceutically acceptable excipients and / or additives.

10. The composition according to claim 8, characterized in that, The dosage form of the pharmaceutical composition is a liquid, solid, or semi-solid dosage form.

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