A μ-type conotoxin peptide and its encoded polynucleotide and uses

Abstract

This invention discloses a μ-conotoxin peptide, its encoded polynucleotide, and its uses. The amino acid sequence of the μ-conotoxin peptide is shown in SEQ ID NO.1. This invention provides a novel μ-conotoxin peptide [Ser 17 Tyr 5 ]‑dR‑μ‑CnIIIC, compared to wild-type μ‑CnIIIC, [Ser 17 Tyr 5 The animal activity of ]-dR-μ-CnIIIC increased more than 10 times, while [Ser 17 Tyr 5 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.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a μ-type cone snail toxin peptide, its encoded polynucleotide, and its uses. Background Technology

[0002] Calcium channels are widely distributed transmembrane proteins, mainly found in excitatory cells, that regulate extracellular calcium ions (Ca). 2 +) enter the cytoplasm. They are present in various Ca2+... 2 Calcium channels play a crucial role in voltage-dependent physiological activities, such as hormone and neurotransmitter release, muscle contraction, and cell signal transduction. Based on their voltage sensitivity, calcium channels can be classified into low-voltage activated calcium channels (LVA) or T-type calcium channels (Cav3.x) and high-voltage activated calcium channels (HVA). HVA calcium channels are further divided into L-type (Cav1.x), P / Q-type (Cav2.1), N-type (Cav2.2), and R-type (Cav2.3). Cav1.x channels are primarily involved in muscle contraction and cell secretion, while Cav2.x channels are involved in neurotransmitter release and regulation of neuronal excitation. Calcium channels consist of a primary functional α subunit and auxiliary functional β, γ, and δ subunits.

[0003] Cone snails are venomous animals widely distributed in tropical oceans, and their venom contains abundant polypeptide ion channel active substances. μ-CnIIIC cone snail venom can specifically block voltage-sensitive calcium ion channels and participate in the regulation of Nav1.4 sodium channel-related diseases, such as pain syndromes, arrhythmias, and muscle paralysis, making it an important natural resource for developing polypeptide drugs. However, μ-CnIIIC peptides suffer from low activity and high production costs. Furthermore, its amino acid sequence contains three disulfide bonds, which increase the difficulty of peptide synthesis. During production, it is crucial to ensure the correct connection of these disulfide bonds to maintain the peptide's conformation and activity while controlling costs. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a μ-conotoxin peptide, its encoded polynucleotide, and its uses. This invention aims to provide a μ-conotoxin peptide (named [Ser]) with higher activity than wild-type μ-conotoxin. 17 Tyr 5 [-dR-μ-CnIIIC] can specifically block Nav1.4 channels and reduce muscle contraction in mice. The [Ser] of this invention... 17 Tyr 5 ]-dR-μ-CnIIIC can be used for muscle relaxation and pain relief. In addition, it can be used to reduce or eliminate the formation of wrinkles on the surface of human skin.

[0005] This invention provides a μ-conotoxin peptide, the amino acid sequence of which is shown in SEQ ID NO.1, and the amino acid sequence contains three pairs of disulfide bonds located at Cys. 2 -Cys 14 Cys 3 -Cys 20 and Cys 9 -Cys 21 The position is identical to the disulfide bond structure of the natural μ-type conotoxin peptide.

[0006] 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 5th 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.

[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, and one or more control sequences operably linked thereto that guide the production of peptides in an expression host.

[0009] The present invention also provides a transformed cell wherein the nucleic acid construct described herein has been transformed.

[0010] The present invention also provides a pharmaceutical composition comprising the aforementioned μ-conotoxin peptide and a pharmaceutically acceptable carrier.

[0011] In some embodiments, the dosage form of the pharmaceutical composition is any one of tablets, capsules, pills, solutions, absorbents, and ointments.

[0012] 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.

[0013] In some embodiments, the sodium ion channel-related disease is any one of epilepsy, arrhythmia, muscle paralysis, tonic-clonic syndrome, or autism spectrum disorder.

[0014] 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.

[0015] The present invention also provides the application of the aforementioned μ-type cone snail toxin peptide in the preparation of anesthetic drugs.

[0016] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0017] (1) This invention provides a novel μ-type cone snail toxin peptide [Ser 17 Tyr 5 ]-dR-μ-CnIIIC, compared to wild-type μ-CnIIIC, [Ser 17 Tyr 5 The animal activity of ]-dR-μ-CnIIIC increased more than 10-fold, while [Ser 17 Tyr 5 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 of cone snail toxin peptides.

[0018] (2) The μ-type cone snail toxin peptide [Ser] of the present invention 17 Tyr 5 ]-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 soothe muscles and relieve pain, and has good anesthetic activity. Attached Figure Description

[0019] 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.

[0020] Figure 1 The μ-type cone snail toxin peptide [Ser] proposed in this invention 17 Tyr 5 Schematic diagram of the synthesis process of ]-dR-μ-CnIIIC;

[0021] Figure 2 This is the chromatogram of wild-type μ-cono toxin μ-CnIIIC in Example 1 of the present invention;

[0022] Figure 3 This is the mass spectrum of wild-type μ-cono toxin μ-CnIIIC in Example 1 of the present invention;

[0023] Figure 4 In Example 1 of this invention, the μ-type conotoxin peptide [Ser] 17 Tyr 5 Chromatogram of ]-dR-μ-CnIIIC;

[0024] Figure 5 In Example 1 of this invention, the μ-type conotoxin peptide [Ser] 17 Tyr5 Mass spectrum of ]-dR-μ-CnIIIC. Detailed Implementation

[0025] 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.

[0026] In this invention, the term "μ-CnIIIC" refers to wild-type μ-conotoxin;

[0027] The Chinese meanings of the English abbreviations are as follows:

[0028] “dR” refers to D-arginine;

[0029] “S” refers to serine;

[0030] “Y” refers to tyrosine;

[0031] "DCM" refers to dichloromethane;

[0032] "DIC" refers to N,N-diisopropylcarbodiimide;

[0033] "DMF" refers to N,N-dimethylformamide;

[0034] "HPLC" refers to High Performance Liquid Chromatography;

[0035] “MeOH” refers to methanol;

[0036] "MTBE" refers to methyl tert-butyl ether;

[0037] “Oxyma” refers to ethyl 2-oxime cyanoacetate;

[0038] "TFA" refers to trifluoroacetic acid;

[0039] "Fmoc" refers to 9-fluorenemethyloxycarbonyl.

[0040] "MS" refers to mass spectrometry.

[0041] This invention is the first to discover that replacing the 17th amino acid of wild-type cone snail toxin (μ-CnIIIC) with serine (Ser) and the 5th 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), can further enhance the activity of μ-CnIIIC. The analogue was synthesized using solid-phase peptide synthesis technology, and disulfide bond formation was achieved through liquid-phase oxidation. Finally, the novel cone snail toxin peptide [Ser] was obtained through high-performance liquid chromatography purification and lyophilization. 17 Tyr 5 ]-dR-μ-CnIIIC, the synthesis process is as follows Figure 1 As shown.

[0042] The μ-type conotoxin peptide [Ser] of this invention 17 Tyr 5 The amino acid sequence of ]-dR-μ-CnIIIC is shown in SEQ ID NO.1. This invention utilizes the μ-type conotoxin peptide [Ser] 17 Tyr 5 The amino acid sequence ]-dR-μ-CnIIIC allows for the determination of the μ-type conotoxin peptide [Ser] without inventive effort. 17 Tyr 5 The nucleotide sequence of ]-dR-μ-CnIIIC, therefore, encodes the μ-conotoxin peptide [Ser 17 Tyr 5 The 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 5 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 5 All of these are within the protection scope of this invention.

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

[0044] Reagent K: lysis buffer, prepared according to the volume ratio of TFA: phenol: water: anisole: ethylene dithiol = 82.5: 5: 5: 5: 2.5.

[0045] Example 1 μ-type conotoxin peptide [Ser 17 Tyr 5 Synthesis of ]-dR-μ-CnIIIC

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

[0047] ① Weigh 1.501g of Rink Amide MBHA resin (containing 1% DVB crosslinking agent, 100-200 mesh, 0.68mmol / g) (1.02mmol) and add it to a 60mL peptide solid-phase reactor. Add 10mL of DCM solution to the reactor. Set the shaking speed of the shaker to 550r / min and shake for 45min. Drain the solution and add DMF solution to wash the resin twice. The washing solvent volume is 15mL / time, and the washing time is 3min / time. The shaking speed of the shaker is 500r / min.

[0048] 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 rpm and 25°C for 5 min, then drain the solution. Next, add another 15 mL of 20% piperidine / DMF solution to the resin and shake at 500 rpm and 25°C for 15 min, then drain the solution. Wash the resin 5 times with DMF solution (15 mL / 3 min / wash).

[0049] ③ Weigh 1.195g Fmoc-Cys(Trt)-OH (2.0eq, 2.04mmol) and 0.290g Oxyma (2eq, 2.04mmol) into a 50mL beaker, add 7mL DMF solution to dissolve, add 0.316mL condensing agent DIC (2eq, 2.04mmol) to the amino acid solution to activate the reaction for 5min, then add it to the above-mentioned deprotected resin, and shake at 500r / min and 25℃ for 1h; after the reaction is completed, wash the resin 5 times with DMF solution (15mL / 3min / time).

[0050] ④ Peptide chain elongation

[0051] 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 6.573 g of peptide resin, with a yield of 94.9%.

[0052] ⑤ Pyrolysis

[0053] Weigh 6.573 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 rpm and 25 °C 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 °C to constant weight. Finally, 2.006 g of white solid crude peptide is obtained, with a yield of 89.9%.

[0054] ⑥ Cycloning

[0055] 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 7.96, and stir the mixture at room temperature for 24 h. Monitor the reaction progress by HPLC. After the reaction is complete, it can be directly purified by HPLC.

[0056] ⑦ Preparative HPLC is used for peptide purification.

[0057] The cyclization reaction solution from step ⑥ above was directly injected into the sample, and the sample was purified 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.

[0058] Table 1 Purification and elution procedure for crude product cyclization solution

[0059]

[0060] 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 5 The total yield of ]-dR-μ-CnIIIC was 39.1 mg, with a yield of 39.1% and an HPLC purity of 98.236%.

[0061] MS results show: [M+2H] 2+ =1192.5120, molecular weight correct. The sample obtained above will be used in the experiment of Example 2. The chromatogram of wild-type μ-CnIIIC is shown below. 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 5The chromatogram of ]-dR-μ-CnIIIC is as follows Figure 4 As shown, μ-type conotoxin peptide [Ser 17 Tyr 5 The mass spectrum of ]-dR-μ-CnIIIC is as follows Figure 5 As shown.

[0062] [Ser] prepared in Example 1 17 Tyr 5 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.

[0063] Example 2 Wild-type μ-CnIIIC and [Ser 17 Tyr 5 Bioactivity assay of ]-dR-μ-CnIIIC

[0064] The mice used in this invention were adult male Kunming mice, purchased from the Lanzhou Veterinary Research Institute of the Chinese Academy of Sciences. The animal experiments were approved by the Ethics Committee of the Lanzhou Peptide Valley Research Institute. 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 the mice using a 50 μL microsyringe (30G 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 concentration gradients were 25 and 50 μM. The activity of the sample was assessed by observing the onset time and duration of the drug's 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.

[0065] 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 5 After ]-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).

[0066] 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.

[0067] The experimental results are shown in Table 2:

[0068] Table 2 Wild-type μ-CnIIIC and [Ser 17 Tyr 5 Results of experiments on ]-dR-μ-CnIIIC mice (50μM)

[0069]

[0070] Note 1: " / " indicates that the mice were asymptomatic after the drug injection, so the onset time and duration of action could not be calculated.

[0071] 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 5 Following administration of ]-dR-μ-CnIIIC, mice exhibited lethal behavior due to a short-term overdose. Specifically, mice showed leg-dragging behavior 8 minutes after injection, followed by paralysis and death 2 minutes later, indicating that [Ser 17 Tyr 5 ]-dR-μ-CnIIIC exhibits higher activity compared to wild-type μ-CnIIIC.

[0072] To further explore [Ser 17 Tyr 5 The activity of ]-dR-μ-CnIIIC will [Ser 17 Tyr 5 The concentration of ]-dR-μ-CnIIIC was reduced to 25 μM, and the results are shown in Table 3.

[0073] Table 3 [Ser] 17 Tyr 5 Animal experimental results of ]-dR-μ-CnIIIC (25μM)

[0074]

[0075] Table 3 shows the results of intramuscular injection of 25 μM [Ser] in the hind leg of mice. 17Tyr 5 After 11 minutes of treatment with dR-μ-CnIIIC, mice exhibited significant leg-dragging behavior that lasted for more than 24 hours. This indicates that [Ser...] 17 Tyr 5 ]-dR-μ-CnIIIC still exhibits activity in inhibiting muscle contraction and paralysis at low concentrations, and shows high activity.

[0076] 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.

[0077] Table 4. Animal experimental results of wild-type μ-CnIIIC at concentrations greater than 50 μM.

[0078]

[0079] Note 1: " / " indicates that the mice were asymptomatic after the drug injection, so the onset time and duration of action could not be calculated.

[0080] Table 4 shows that mice injected with an equal volume of saline at each concentration appeared normal. Death only occurred after intramuscular injection of 500 μM wild-type μ-CnIIIC into the calf muscles, further illustrating the efficacy of μ-conotoxin peptide [Ser...]. 17 Tyr 5 The activity of ]-dR-μ-CnIIIC is more than 10 times that of wild-type μ-CnIIIC.

[0081] In summary, compared with wild-type μ-CnIIIC, μ-conotoxin peptide [Ser 17 Tyr 5 ]-dR-μ-CnIIIC exhibits high activity, with an increase of at least 10-fold, indicating that [Ser 17 Tyr 5 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.

[0082] 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.

[0083]

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; The structure of the μ-type conotoxin peptide is as follows: ； The amino acid sequence contains three pairs of disulfide bonds, which are located in Cys. 2 -Cys 14 Cys 3 -Cys 20 and Cys 9 -Cys 21 Location.

2. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the μ-type conotoxin peptide of claim 1 and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 2, characterized in that, The dosage form of the pharmaceutical composition is any one of tablets, capsules, pills, solutions, and ointments.

4. 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; The sodium ion channel-related disease is tonic-clonic syndrome.

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

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