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

Abstract

The invention discloses a mu-type conotoxin peptide as well as a pharmaceutical composition and application thereof. The amino acid sequence of the mu-type conotoxin peptide is shown as SEQ ID NO. 1. Compared with a wild type mu-CnIIIC, the mu-type conotoxin peptide [Ser17, Tyr6]-dR-mu-CnIIIC disclosed by the invention has the advantages that the animal activity is improved by more than 10 times, a Nav1.4 channel can be specifically blocked, muscle contraction is reduced, formation of wrinkles on the surface of human skin is reduced or eliminated, the mu-type conotoxin peptide [Ser17, Tyr6]-dR-mu-CnIIIC can also be used for relieving muscles and easing pain, and the mu-type conotoxin peptide [Ser17, Tyr6]-dR-mu-CnIIIC has a high-activity anesthesia effect.

Description

Technical Field

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

[0002] Conotoxins (conopeptide, conotoxin, CTX) are mostly cysteine-rich neuropeptide toxins composed of 7 to 50 amino acid residues. Conotoxins (peptides) are divided into different gene families according to the similarity of the endoplasmic reticulum signal peptide sequences of their precursor proteins and the cysteine ​​pattern. To date, all known conotoxins can be divided into 17 superfamilies, namely A, C, D, S, M, I1, I2, I3, J, L, O1, O2, O3, P, T, V, and Y. Conotoxins (peptides) can be divided into multiple pharmacological families according to their receptor targets, such as α, ω, μ, and δ. Each superfamily can be divided into α, αA, κA (A-superfamily), ω, δ, κ, μO (O-superfamily), μ, KM (M-superfamily) and other families (subtypes). Natural μ-conotoxin (μ-CnIIIC) is a conotoxin peptide composed of 22 amino acid residues, which has analgesic, muscle contraction-reducing and anesthetic properties and is a potent antagonist of the voltage-gated Nav1.4 sodium channel.

[0003] Voltage-gated sodium channels play a key role in the electrical excitability of cells by regulating the influx of sodium ions. In mammals, nine different subtypes (Nav1.1-1.9) have been identified, each with a different distribution in the body. Among them, neuronal subtypes, especially those associated with pain perception, have attracted much attention. Studies have reported that μ-CnIIIC is involved in regulating Nav1.4 sodium channel-related diseases, such as pain syndrome, arrhythmia, and muscle paralysis. However, the activity of natural μ-CnIIIC is low, and the cost is high in the actual production and application process. In addition, the amino acid sequence of μ-CnIIIC contains three disulfide bonds. In order to maintain the conformation of the μ-CnIIIC peptide and ensure its activity, it is necessary to ensure that the correct connection is formed during the synthesis or recombinant synthesis of the μ-CnIIIC peptide, and the cost of synthesizing the peptide is high. Therefore, a highly active conotoxin peptide with low synthesis cost is urgently needed to reduce the cost of production. Summary of the invention

[0004] In view of the defects in the prior art, the present invention proposes a μ-conotoxin peptide and its pharmaceutical composition and application. The present invention aims to provide a μ-conotoxin peptide with higher activity than wild-type μ-conotoxin, named [Ser 17 ,Tyr 6]-dR-μ-CnIIIC can specifically block the Nav1.4 channel. As an inhibitor of the Nav1.4 channel, it can 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 is the first to discover that replacing the 17th amino acid of the wild-type μ-conotoxin (μ-CnIIIC) with serine (Ser), the 6th amino acid with tyrosine (Tyr), and simultaneously 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.

[0006] The present invention provides a μ-type conotoxin peptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a polynucleotide encoding the μ-type conotoxin peptide.

[0008] The present invention also provides a nucleic acid construct comprising the polynucleotide.

[0009] The present invention also provides an expression vector comprising the nucleic acid construct.

[0010] The invention also provides a fusion protein comprising the μ-type conotoxin peptide.

[0011] The present invention also provides the use of the μ-type conotoxin peptide in preparing a drug for treating or preventing diseases related to sodium ion channels.

[0012] In some embodiments, the sodium ion channel-related disease is any one of epilepsy, arrhythmia, muscle paralysis, myotonia, and autism spectrum disorder.

[0013] The present invention also provides application of the μ-type conotoxin peptide in preparing medicine for treating or preventing pain.

[0014] The present invention also provides application of the μ-type conotoxin peptide in preparing anesthetic drugs.

[0015] The present invention also provides application of the μ-type conotoxin peptide in preparing cosmetics.

[0016] The present invention also provides a pharmaceutical composition, which comprises the μ-type conotoxin peptide.

[0017] 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.

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

[0019] (1) The μ-type conotoxin peptide [Ser 17 ,Tyr 6 ]-dR-μ-CnIIIC has an animal activity that is more than 10 times higher than that of wild-type μ-CnIIIC. It 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 a highly active anesthetic effect.

[0020] (2) The amino acid sequence composition of the μ-conotoxin peptide of the present invention is reduced by 1 position compared with the wild-type μ-CnIIIC, which can further reduce the synthesis cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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.

[0022] Figure 1 A μ-type conotoxin peptide [Ser 17 ,Tyr 6 ]-dR-μ-CnIIIC synthesis process diagram;

[0023] Figure 2 This is a chromatogram of the wild-type μ-conotoxin μ-CnIIIC in Example 1 of the present invention;

[0024] Figure 3 This is the mass spectrum of the wild-type μ-conotoxin μ-CnIIIC in Example 1 of the present invention;

[0025] Figure 4 The μ-type conotoxin peptide [Ser 17 ,Tyr 6 ]-dR-μ-CnIIIC chromatogram;

[0026] Figure 5 The μ-type conotoxin peptide [Ser 17 ,Tyr 6 ]-dR-μ-CnIIIC mass spectrum. DETAILED DESCRIPTION

[0027] 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.

[0028] The term "μ-CnIIIC" in the present invention refers to the wild-type μ-conotoxin;

[0029] The Chinese meaning of the English abbreviation of the present invention is:

[0030] “dR” refers to D-arginine;

[0031] “S” refers to serine;

[0032] “Y” refers to tyrosine;

[0033] “DCM” means dichloromethane;

[0034] “DIC” refers to N,N-diisopropylcarbodiimide;

[0035] "DMF" means N,N-dimethylformamide;

[0036] “HPLC” means high performance liquid chromatography;

[0037] “MeOH” means methanol;

[0038] “MTBE” means methyl tert-butyl ether;

[0039] "Oxyma" means ethyl 2-oximecyanoacetate;

[0040] “TFA” means trifluoroacetic acid;

[0041] "Fmoc" means 9-fluorenylmethoxycarbonyl;

[0042] “MS” refers to mass spectrometry;

[0043] The present invention is the first to discover that the 17th amino acid of the wild-type μ-conotoxin (μ-CnIIIC) is replaced by serine (Ser), the 6th amino acid is replaced by tyrosine (Tyr), and the 1st amino acid at the N-terminus of the original sequence is deleted, and the 2nd amino acid is replaced by D-arginine (d-Arg). The analog is synthesized by solid-phase peptide synthesis technology, the disulfide bond is formed by liquid-phase oxidation, and finally purified by high-performance liquid chromatography and freeze-dried to obtain a novel μ-conotoxin peptide [Ser 17 ,Tyr 6]-dR-μ-CnIIIC, which can further improve the activity of wild-type μ-CnIIIC. The μ-type conotoxin peptide [Ser 17 ,Tyr 6 ]-dR-μ-CnIIIC synthesis process is as follows Figure 1 shown.

[0044] Wild-type μ-CnIIIC: Sichuan Jisheng Biotechnology Co., Ltd., batch number: 2023041201-3.

[0045] Reagent K: lysis solution, prepared according to the volume ratio of TFA:phenol:water:thioanisole:ethanedithiol=82.5:5:5:5:2.5.

[0046] Example 1 μ-type conotoxin peptide [Ser 17 ,Tyr 6 Synthesis of ]-dR-μ-CnIIIC

[0047] (1) Preparation of Fmoc-Cys(Trt)-Rink AM resin

[0048] ① Weigh 1.332g Rink Amide AM resin (containing 1% DVB cross-linker, 100-200 mesh, 0.64mmol / g) (0.85mmol) and add it to a 60mL peptide solid phase reactor, add 15mL 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.

[0049] 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.

[0050] ③ Weigh 0.996g Fmoc-Cys(Trt)-OH (2.0eq, 1.7mmol) and 0.241g Oxyma (2eq, 1.7mmol) into a 50mL beaker, add 10mL DMF solution to dissolve, add 0.263mL condensing agent DIC (2eq, 1.7mmol) 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 (15mL / 3min / time).

[0051] ④ Peptide chain extension

[0052] 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, 5.977 g of peptide resin was obtained with a yield of 98.1%.

[0053] ⑤ Cracking

[0054] Weigh 5.977g 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, and vacuum dry at 25℃ to constant weight, and finally obtain 1.998g of white solid crude peptide with a yield of 96.5%.

[0055] ⑥Cyclization

[0056] 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 7.83, 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.

[0057] ⑦Preparative HPLC for peptide purification

[0058] 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.

[0059] Table 1 Purification and elution procedures of crude cyclization solution

[0060]

[0061] The fractions were collected for MS and HPLC analysis, and the target fractions were combined and freeze-dried to obtain the final product [Ser 17 ,Tyr 6 ]-dR-μ-CnIIIC totaled 38.5 mg, with a yield of 38.5% and a HPLC purity of 98.783%.

[0062] MS results showed: [M+2H] 2+ =1221.5251, 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 [Ser 17 ,Tyr 6 ]-dR-μ-CnIIIC chromatogram Figure 4 As shown, μ-conotoxin peptide [Ser 17 ,Tyr 6 ]-dR-μ-CnIIIC mass spectrum Figure 5 shown.

[0063] Example 1 Preparation of [Ser 17 ,Tyr 6 The amino acid sequence of ]-dR-μ-CnIIIC is shown in SEQ ID NO. 1. The amino acid sequence of wild-type μ-CnIIIC is shown in SEQ ID NO. 2.

[0064] Example 2 Wild-type μ-CnIIIC and [Ser 17 ,Tyr 6 ]-dR-μ-CnIIIC biological activity assay

[0065] 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.

[0066] 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 experimental concentration gradient 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.

[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 muscle group of mice was injected with physiological saline, wild-type μ-CnIIIC, and different concentrations of μ-conotoxin peptide [Ser 17 ,Tyr 6 ]-dR-μ-CnIIIC, the degree of toe abduction of the left and right hind limbs was measured as a function of time, and the mouse behavior was evaluated according to 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 [Ser 17 ,Tyr 6 ]-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. 17 ,Tyr 6 ]-dR-μ-CnIIIC, mice showed lethal behaviors caused by overdose in a short period of time. The mice took effect immediately after injection, and showed obvious leg dragging and toe-pulling behaviors. After 5 minutes, the mice became paralyzed and motionless, and died after 12 minutes. This indicates that [Ser 17 ,Tyr 6 ]-dR-μ-CnIIIC also has the activity of inhibiting muscle contraction, and [Ser 17 ,Tyr 6 ]-dR-μ-CnIIIC has higher activity than wild-type μ-CnIIIC.

[0074] Subsequently, mice were treated with 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 3.

[0075] Table 3 Animal experimental results of wild-type μ-CnIIIC at concentrations greater than 50 μM

[0076]

[0077]

[0078] Note: 1. “ / ” indicates that the mice were asymptomatic after drug injection, so the onset time and duration of the effect could not be calculated.

[0079] Table 3 The mice in the groups injected with equal volumes of saline at different concentrations showed normal performance. The results showed that mice died only after 500 μM wild-type μ-CnIIIC was injected into the calf muscle. This indicates that μ-conotoxin peptide [Ser 17 ,Tyr 6 ]-dR-μ-CnIIIC has an activity more than 10 times that of wild-type μ-CnIIIC.

[0080] In summary, compared with wild-type μ-CnIIIC, μ-conotoxin peptide [Ser 17 ,Tyr 6 ]-dR-μ-CnIIIC has high activity and is increased by at least 10 times, further indicating that [Ser 17 ,Tyr 6]-dR-μ-CnIIIC has high activity and can specifically block Nav1.4 channels, reduce or inhibit muscle contraction, and has analgesic and anesthetic effects. It can be used to prepare drugs for treating or preventing diseases related to Nav1.4 sodium ion channels, or can be used to prepare cosmetics for eliminating wrinkles, or can be used to prepare anesthetic drugs, or can be used to prepare drugs for treating or preventing pain.

[0081] 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.

[0082] Sequence Listing

[0083] SEQ ID NO.1

[0084] Three characters:

[0085] d-Arg- Cys-Cys-Asn- Tyr -Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg- Ser -His-Ala-Arg-Cys-Cys-NH2

[0086] Single character:

[0087] r CCN Y PKGCSSKWCR S HARCC-NH2

[0088] SEQ ID NO.2

[0089] Three characters:

[0090] Pyr-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-NH2

[0091] Single character: (X represents pyroglutamic acid)

[0092] XGCCNGPKGCSSKWCRDHARCC-NH2

Claims

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

1.

2. A polynucleotide encoding the μ-type conotoxin peptide according to claim 1.

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

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

5. A fusion protein, characterized in that The invention comprises the μ-type conotoxin peptide according to claim 1.

6. Use of the μ-conotoxin peptide according to claim 1 in the preparation of a drug for treating or preventing diseases related to sodium ion channels.

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

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

9. Use of the μ-conotoxin peptide according to claim 1 in the preparation of cosmetics.

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

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