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] This invention relates to the field of biopharmaceutical technology, and in particular to μ-conotoxin peptides, pharmaceutical compositions thereof, and applications. Background Technology

[0002] Conotoxins (CTX) are mostly cysteine-rich neuropeptide toxins composed of 7-50 amino acid residues. Conotoxins (peptides) are classified into different gene families based on the similarity of their precursor protein endoplasmic reticulum signal peptide sequences and cysteine ​​patterns. To date, all known conotoxins can be divided into 17 superfamilies: A, C, D, S, M, I1, I2, I3, J, L, O1, O2, O3, P, T, V, and Y. Conotoxins (peptides) can also be classified into various pharmacological families based on their receptor target sites, such as α, ω, μ, and δ. Each superfamily can be further divided into families (subtypes) based on receptor target type, such as α, αA, κA (A-superfamily), ω, δ, κ, μO (O-superfamily), and μ, φ, KM (M-superfamily). Natural μ-conotoxin (μ-CnIIIC) is a conotoxin peptide composed of 22 amino acid residues. It has analgesic, muscle-reducing, and anesthetic properties and is an effective antagonist of voltage-gated Nav1.4 sodium channels.

[0003] Voltage-gated sodium channels play a crucial role in cellular electroexcitability by regulating the influx of sodium ions. Nine distinct subtypes (Nav1.1–1.9) have been identified in mammals, each with varying distributions in vivo. Neuronal subtypes, particularly those related to pain perception, have attracted significant attention. Studies have reported that μ-CnIIIC participates in regulating Nav1.4 sodium channel-related diseases, such as pain syndromes, arrhythmias, and muscle paralysis. However, naturally occurring μ-CnIIIC exhibits low activity, leading to high costs in practical production. Furthermore, the presence of three disulfide bonds in the μ-CnIIIC amino acid sequence necessitates ensuring correct linkage formation during peptide synthesis or recombination to maintain the peptide's conformation and activity, further increasing the cost of peptide synthesis. Therefore, a highly active and low-cost conotoxin peptide is urgently needed to reduce production costs. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a μ-conotoxin peptide, its pharmaceutical composition, and its applications. This 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 reduces muscle contraction, reduces or eliminates the formation of wrinkles on the surface of human skin, and can also be used for muscle relaxation and analgesia.

[0005] This invention is the first to discover that replacing the 17th amino acid of wild-type μ-conotoxin (μ-CnIIIC) with serine (Ser) and the 6th amino acid with tyrosine (Tyr), while deleting the first amino acid at the N-terminus of the original sequence and replacing the second amino acid with D-arginine (d-Arg), can further enhance the activity of μ-CnIIIC.

[0006] This invention provides a μ-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 aforementioned μ-conotoxin peptide.

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

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

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

[0011] The present invention also provides the use of the aforementioned μ-conotoxin peptide in the preparation of drugs for the treatment or prevention of diseases related to sodium ion channels.

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

[0013] The present invention also provides the use of the aforementioned μ-type cone snail toxin peptide in the preparation of drugs for treating or preventing pain.

[0014] The present invention also provides the application of the aforementioned μ-type cone snail toxin peptide in the preparation of anesthetic drugs.

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

[0016] The present invention also provides a pharmaceutical composition comprising the aforementioned μ-type cone snail toxin peptide.

[0017] In some embodiments, the dosage form of the pharmaceutical composition is any one of tablets, capsules, pills, solutions, absorbents, and ointments.

[0018] In summary, compared with the prior art, the present invention achieves the following technical effects: (1) The μ-type cone snail toxin peptide [Ser] disclosed in this invention 17 Tyr 6 Compared to wild-type μ-CnIIIC, dR-μ-CnIIIC exhibits more than 10 times higher animal activity. It can specifically block Nav1.4 channels, 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, with highly active anesthetic effects.

[0019] (2) The amino acid sequence composition of the μ-type cone snail toxin peptide of the present invention is reduced by 1 position compared with the wild type μ-CnIIIC, which can further reduce the synthesis cost. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This invention proposes a μ-type conotoxin peptide [Ser] 17 Tyr 6 Schematic diagram of the synthesis process of ]-dR-μ-CnIIIC; Figure 2 This is the chromatogram of wild-type μ-cono toxin μ-CnIIIC in Example 1 of the present invention; Figure 3 This is the mass spectrum of wild-type μ-cono toxin μ-CnIIIC from Example 1 of the present invention; Figure 4 The μ-type conotoxin peptide [Ser] of Example 1 of this invention 17 Tyr 6 Chromatogram of ]-dR-μ-CnIIIC; Figure 5 The μ-type conotoxin peptide [Ser] of Example 1 of this invention 17 Tyr 6 Mass spectrum of ]-dR-μ-CnIIIC. Detailed Implementation

[0022] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0023] In this invention, the term "μ-CnIIIC" refers to wild-type μ-conotoxin; The Chinese meaning of the English abbreviation of this invention: “dR” refers to D-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 ethyl 2-oxime cyanoacetate; "TFA" refers to trifluoroacetic acid; "Fmoc" refers to 9-fluorenemethyloxycarbonyl. "MS" refers to mass spectrometry. This invention is the first to discover a novel μ-conotoxin peptide [Ser] by replacing the 17th amino acid of wild-type conotoxin (μ-CnIIIC) with serine (Ser) and the 6th amino acid with tyrosine (Tyr), while simultaneously deleting the first amino acid at the N-terminus of the original sequence and replacing the second amino acid with D-arginine (d-Arg). A solid-phase peptide synthesis technique was used to synthesize the analog, followed by liquid-phase oxidation to form disulfide bonds. Finally, the peptide was purified by high-performance liquid chromatography and lyophilized to obtain the novel conotoxin peptide [Ser]. 17 Tyr 6 The addition of ]-dR-μ-CnIIIC can further enhance the activity of wild-type μ-CnIIIC. The μ-type conotoxin peptide [Ser 17 Tyr 6 The synthesis process of ]-dR-μ-CnIIIC is as follows Figure 1 As shown.

[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 6 Synthesis of ]-dR-μ-CnIIIC (1) Preparation of Fmoc-Cys(Trt)-Rink AM resin ① Weigh 1.332 g of Rink Amide AM resin (containing 1% DVB crosslinking agent, 100~200 mesh, 0.64 mmol / g) (0.85 mmol) and add it to a 60 mL peptide solid-phase reactor. Add 15 mL of DCM solution to the reactor. Set the shaking speed of the shaker to 550 r / min and shake for 45 min. Drain the solution and add DMF solution to wash the resin twice. The washing solvent volume is 15 mL / time and the washing time is 3 min / time. The shaking speed of the shaker is 500 r / min.

[0027] ② After washing, 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 and 25℃ for 5 min, then drain the solution. Next, add another 15 mL of 20% piperidine / DMF solution to the resin and shake at 500 r / min and 25℃ for 15 min, then drain the solution. Wash the resin 5 times with DMF solution (15 mL / 3 min / wash).

[0028] ③ Weigh 0.996 g Fmoc-Cys(Trt)-OH (2.0 eq, 1.7 mmol) and 0.241 g Oxyma (2 eq, 1.7 mmol) into a 50 mL beaker, add 10 mL of DMF solution to dissolve, add 0.263 mL of condensing agent DIC (2 eq, 1.7 mmol) to the amino acid solution to activate the reaction for 5 min, and then add it to the above-mentioned deprotected resin. Shake at 500 r / min and 25℃ for 1 h. After the reaction is completed, wash the resin 5 times with DMF solution (15 mL / 3 min / time).

[0029] ④ Peptide chain elongation Following the sequence composition, steps ② and ③ were repeated until the last amino acid coupling was completed. The Fmoc protecting group was removed, and the resin was washed 5 times with DMF solution (15 mL / 3 min / time). Then, the resin was washed alternately with DCM × 5 times (15 mL / 3 min / time) and MeOH × 5 times (15 mL / 3 min / time) until the resin was in a shrunken state. The resin was then placed in a vacuum drying oven and dried at 25°C to constant weight, yielding 5.977 g of peptide resin, with a yield of 98.1%.

[0030] ⑤ Pyrolysis Weigh 5.977 g of the dried resin obtained in step ④ above. Add freshly prepared and pre-cooled K reagent lysis buffer at a ratio of 15 mL lysis buffer per gram of peptide resin. React at 300 r / min, 25℃, and in the dark for 3 h. After the reaction is complete, slowly add the lysis buffer to pre-cooled MTBE solution at a ratio of 1:10 (v / v) of lysis buffer / methyl tert-butyl ether. A white precipitate forms. Centrifuge at 500 rpm, discard the supernatant, add fresh MTBE solution, shake, centrifuge, discard the supernatant, and repeat the centrifugation process 5 times. Collect the sludge-like white precipitate, and vacuum dry at 25℃ to constant weight. Finally, 1.998 g of white solid crude peptide is obtained, with a yield of 96.5%.

[0031] ⑥ Cycloning Weigh 0.100 g of the white solid crude peptide obtained in step ⑥ above, add 100 mL of disodium hydrogen phosphate / guanidine hydrochloride buffer solution, adjust the pH to 7.83, and react with the mixture under open stirring at room temperature for 24 h. Monitor the reaction progress by HPLC. After the reaction is complete, it can be directly purified by HPLC.

[0032] ⑦ Preparative HPLC is used for peptide purification. The cyclization reaction solution from step ⑥ above was directly injected into the sample, and the sample purification was completed according to the gradient elution program in Table 1. The mobile phase A was 80% acetonitrile / water (containing 0.1% TFA), and the mobile phase B was water (containing 0.1% TFA). The detection wavelength was 220 nm, the flow rate was 10 mL / min, and the column specifications were 20×250 mm, 10 µm, and 120 A.

[0033] Table 1 Purification and elution procedure for crude product cyclization solution

[0034] The collected fractions were analyzed by MS and HPLC, the target fractions were combined, and the final product was obtained by freeze drying. 17 Tyr 6 The total yield of 38.5 mg of ]-dR-μ-CnIIIC was 38.5%, with an HPLC purity of 98.783%.

[0035] MS results show: [M+2H] 2+ =1221.5251, molecular weight correct. The sample obtained above will be used in the experiment of Example 2. The chromatogram of wild-type μ-CnIIIC is as follows. Figure 2 As shown, the mass spectrum of wild-type μ-CnIIIC is as follows: Figure 3 As shown. μ-type conotoxin peptide [Ser 17 Tyr 6 The chromatogram of ]-dR-μ-CnIIIC is as follows Figure 4 As shown, μ-type conotoxin peptide [Ser 17 Tyr 6 The mass spectrum of ]-dR-μ-CnIIIC is as follows Figure 5 As shown.

[0036] [Ser] prepared in Example 1 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.

[0037] Example 2 Wild-type μ-CnIIIC and [Ser 17 Tyr 6 Bioactivity assay of ]-dR-μ-CnIIIC The mice used in the experiments of this invention were adult male Kunming mice, which were purchased from the Lanzhou Veterinary Research Institute of the Chinese Academy of Sciences. The animal experiments were approved by the Ethics Committee of Lanzhou Peptide Valley Research Institute.

[0038] Mice were allowed free access to food and water for one week prior to testing. Intramuscular injection of 20 μL was administered into the right anterior tibial muscle group of mice using a 50 μL microsyringe (30 G needle). An equal volume of physiological saline was injected as a negative control, and wild-type μ-CnIIIC was used as a positive control. The experimental setup included a concentration gradient of 50 μM. The activity of the sample was assessed by observing the onset time and duration of drug effect after injection, as well as the behavior of the mice after injection, including toe clenching (inhibition of muscle contraction), leg dragging / paralysis (anesthetic effect), and death behavior.

[0039] The mouse toe-clamping (inhibition of muscle contraction) behavior was analyzed using the mouse toe abduction scoring test (DAS). Mice were suspended by their tails to elicit a characteristic shock response in terms of hind limb extension and abduction. The mice were then injected with saline, wild-type μ-CnIIIC, and different concentrations of μ-conotoxin peptide [Ser] into the right anterior tibial muscle group. 17 Tyr 6After ]-dR-μ-CnIIIC, the degree of toe abduction in the left and right hind limbs was measured as a function of time. The behavior of the mice was evaluated according to a 5-point scale (0 indicates normal, 1 indicates only 2 toes (index and middle toes) are brought together, 2 indicates 3 toes (index, middle, and little toes) are brought together; 3 indicates 4 toes except the ring toe are brought together; 4 indicates the maximum reduction in toe abduction and leg extension).

[0040] The dragging / paralysis (anesthetic effect) behavior in mice was observed by injecting the right tibialis anterior muscle group into the mice and then placing them in a new environment to stimulate their exploratory instincts. In the early stages of the dragging / paralysis effect, the mice crawled forward with their lower body close to the ground; in the later stages, the entire body became paralyzed. Toe clenching and dragging / paralysis are two separate behaviors in mice, and they generally occur simultaneously.

[0041] The experimental results are shown in Table 2: Table 2 Wild-type μ-CnIIIC and [Ser 17 Tyr 6 Animal experiment results of ]-dR-μ-CnIIIC (50 μM)

[0042] Note 1: " / " indicates that the mice were asymptomatic after drug injection, so the onset time and duration of action could not be calculated. Table 2 shows that after intramuscular injection of 50 μM wild-type μ-CnIIIC into the hind leg of mice, the mice exhibited toe-clamping behavior, indicating that wild-type μ-CnIIIC at a concentration of 50 μM exhibited inhibitory activity against muscle contraction. However, injection of the same concentration of [Ser...] 17 Tyr 6 Following administration of ]-dR-μ-CnIIIC, mice exhibited lethal behaviors due to excessive dosage within a short period. The effect was immediate, with mice displaying obvious leg-dragging and toe-clamping behaviors. Five minutes later, the mice became paralyzed and immobile, eventually dying 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 exhibits higher activity compared to wild-type μ-CnIIIC.

[0043] To further explore [Ser 17 Tyr 6 The activity of ]-dR-μ-CnIIIC will [Ser 17 Tyr 6The concentration of ]-dR-μ-CnIIIC was reduced to 25 μM, and the results are shown in Table 3.

[0044] Table 3 [Ser] 17 Tyr 6 Animal experimental results of ]-dR-μ-CnIIIC (25 μM)

[0045] Table 3 shows the results of intramuscular injection of 25 μM [Ser] in the hind leg of mice. 17 Tyr 6 Eight minutes after inoculation with dR-μ-CnIIIC, mice exhibited typical muscle paralysis behaviors such as leg dragging and toe clenching, lasting for more than 240 minutes. [Ser] 17 Tyr 6 [-dR-μ-CnIIIC] still exhibited inhibitory activity against muscle contraction and paralysis at low concentrations, demonstrating high biological activity. Furthermore, experimental mice fully recovered normal activity after 24 hours, and no long-term toxicity or irreversible damage was observed, suggesting that [Ser]... 17 Tyr 6 ]-dR-μ-CnIIIC maintains effective pharmacological effects while exhibiting good safety.

[0046] Subsequently, mice were treated with μ-CnIIIC at two concentrations of 100 μM and 500 μM. The onset time, duration of action, and toe-clamping (inhibition of muscle contraction) behavior of the mice after injection were observed. The results are shown in Table 4.

[0047] Table 4. Animal experimental results of wild-type μ-CnIIIC at concentrations greater than 50 μM.

[0048] Note: 1. " / " indicates that the mice were asymptomatic after the drug injection, so the onset time and duration of action could not be calculated.

[0049] Table 4 shows that mice injected with an equal volume of physiological saline at various concentrations exhibited normal behavior. The results indicate that mice only died after intramuscular injection of 500 μM wild-type μ-CnIIIC in their calf muscles, suggesting that μ-type conotoxin peptide [Ser 17 Tyr 6 The activity of ]-dR-μ-CnIIIC is more than 10 times that of wild-type μ-CnIIIC.

[0050] In summary, compared with wild-type μ-CnIIIC, μ-conotoxin peptide [Ser 17 Tyr 6]-dR-μ-CnIIIC exhibits high activity, increased by at least 10-fold, further demonstrating that [Ser 17 Tyr 6 The high activity of ]-dR-μ-CnIIIC and its ability to specifically block Nav1.4 channels, reduce or inhibit muscle contraction, and have analgesic and anesthetic effects, can be used in the preparation of drugs for the treatment or prevention of diseases related to Nav1.4 sodium ion channels, or in the preparation of cosmetics for eliminating wrinkles, or in the preparation of anesthetic drugs, or in the preparation of drugs for the treatment or prevention of pain.

[0051] 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 principles of the present invention should be included within the protection scope of the present invention.

[0052] sequence list SEQ ID NO.1 Three characters: d-Arg- Cys-Cys-Asn- Tyr -Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg- Ser -His-Ala-Arg-Cys-Cys-NH2 Single character: r CCN Y PKGCSSKWCR S HARCC-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 Single character: (X represents pyroglutamic acid) XGCCNGPKGCSSKWCRDHARCC-NH2.

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. A polynucleotide encoding the μ-type conotoxin peptide as described in claim 1.

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

4. An expression carrier, characterized in that, It includes the nucleic acid construct as described in claim 3.

5. A fusion protein, characterized in that, It contains the μ-type conotoxin peptide as described in claim 1.

6. The use of the μ-type cone snail toxin peptide according to claim 1 in the preparation of drugs for treating or preventing diseases related to sodium ion channels.

7. The use of the μ-type cone snail venom peptide according to claim 1 in the preparation of drugs for treating or preventing pain.

8. The use of the μ-type cone snail toxin peptide according to claim 1 in the preparation of anesthetic drugs.

9. The application of the μ-type cone snail toxin peptide according to claim 1 in the preparation of cosmetics.

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

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