Mu-type conotoxin peptide [Asn17, Arg5]-dR-mu-CnIIIC and application thereof

Abstract

The invention discloses a [mu] type conotoxin peptide [Asn17, Arg5]-dR-[mu]-CnIIIC and an application thereof. The amino acid sequence of the [mu] type conotoxin peptide [Asn17, Arg5]-dR-[mu]-CnIIIC is as shown in SEQ ID NO. 1. Compared with the wild type [mu]-CnIIIC, the [mu]-type conotoxin peptide [Asn17, Arg5]-dR-[mu]-CnIIIC provided by the invention has the advantages that the animal activity is improved by 10 times, meanwhile, the amino acid sequence composition of the [Asn17, Arg5]-dR-[mu]-CnIIIC is reduced by 1 site compared with the wild type [mu]-CnIIIC, and the synthesis cost can be further reduced.

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 5 ]-dR-μ-CnIIIC and its applications. Background Art

[0002] Conotoxins (CTX) are mostly cysteine ​​(Cys)-rich neuropeptide toxins composed of 7 to 50 amino acid residues. Conotoxins are divided into different gene families according to the similarity of the endoplasmic reticulum signal peptide sequence of their precursor proteins and the cysteine ​​pattern. Each superfamily of conotoxins can be divided into families (subtypes) such as α, αA, κA (A-superfamily), ω, δ, κ, μO (O-superfamily), μ, ψ, κM (M-superfamily) according to the receptor target type. In recent years, the μ-conotoxin (μ-CnIIIC) family has attracted increasing attention from biochemists, pharmacologists and neurobiologists. This type of toxin can highly specifically block voltage-gated Na + However, natural μ-CnIIIC generally has the defects of low stability and activity, and the chemical synthesis of μ-conotoxin is challenging due to its complex structure and multiple disulfide bonds. Summary of the invention

[0003] In view of the defects in the prior art, the present invention discloses a μ-type conotoxin peptide [Asn 17 ,Arg 5 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 present invention provides a μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC, the μ-type conotoxin peptide [Asn 17 ,Arg 5 ]The amino acid sequence of -dR-μ-CnIIIC is shown in SEQ ID NO.1.

[0005] The present invention first discovered that replacing the 17th amino acid of the wild-type μ-conotoxin (μ-CnIIIC) with asparagine (Asn), the 5th 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. The amino acid sequence contains three pairs of disulfide bonds, and the disulfide bonds are located at Cys 2 -Cys 14 、Cys 3 -Cys 20 and Cys 9 -Cys 21 The position is the same as the disulfide bond structure of the natural μ-type conotoxin peptide.

[0006] The present invention also provides a method for encoding the μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC DNA molecule.

[0007] The present invention also provides a vector comprising the DNA molecule.

[0008] In some embodiments, the vector is an expression vector.

[0009] The present invention also provides a host cell, wherein the host cell comprises the μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC, the described DNA molecule or the described vector.

[0010] The present invention also provides the μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC in the preparation of drugs for treating or preventing diseases related to sodium ion channels.

[0011] In some embodiments, the sodium ion channel-related disease is any one of autism spectrum disorder, myotonia, arrhythmia, epilepsy, and muscle paralysis.

[0012] The present invention also provides the μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC in the preparation of drugs for treating or preventing pain.

[0013] The present invention also provides the μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC in the preparation of anesthetic drugs.

[0014] The present invention also provides the μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC in the preparation of cosmetics.

[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 5 ]-dR-μ-CnIIIC, compared with wild-type μ-CnIIIC, the activity of animals increased by 10 times, and [Asn 17 ,Arg 5 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 5 ]-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 5 ]-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 5 ]-dR-μ-CnIIIC chromatogram;

[0023] Figure 5 The μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-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 5 ]-dR-μ-CnIIIC" means that the first pyroglutamic acid at the N-terminus of the wild-type μ-conotoxin is deleted, the second amino acid is replaced by D-arginine, and the 17th amino acid is replaced by an L-polar uncharged amino acid, and the 5th amino acid is replaced by an L-basic amino acid, forming a new high-activity cono peptide with a sequence length reduced by 1 position compared with the wild-type μ-conotoxin.

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

[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 first discovered that replacing the 17th amino acid of the wild-type μ-conotoxin (μ-CnIIIC) with asparagine (Asn), the 5th 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. The analog was synthesized by solid phase peptide synthesis technology, the disulfide bond was formed by liquid phase oxidation, and finally purified by high performance liquid chromatography and freeze-dried to obtain a novel μ-conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC, the μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC synthesis process is as follows Figure 1 Compared with wild-type μ-CnIIIC, μ-conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC increased its animal activity by 10 times, while [Asn 17 ,Arg 5 The amino acid sequence of ]-dR-μ-CnIIIC is reduced by one position compared with the wild-type μ-CnIIIC, which can further reduce the synthesis cost. 17 ,Arg 5 ]-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, as well as having an anesthetic effect.

[0042] The μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC amino acid sequence is shown in SEQ ID NO.1, and the μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC amino acid sequence, can be obtained without creative work encoding the μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC nucleotide sequence, thus encoding the μ-type conotoxin peptide [Asn17 ,Arg 5 The nucleotide sequence of the μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-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 5 ]-dR-μ-CnIIIC, all are within the protection scope of the present invention.

[0043] Reagent K: lysis solution, prepared according to the volume ratio of TFA:phenol:water:thioanisole:ethanedithiol=82.5:5:5:5:2.5.

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

[0045] Example 1 μ-type conotoxin peptide [Asn 17 ,Arg 5 Synthesis of ]-dR-μ-CnIIIC

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

[0047] ① Weigh 2.000g (1.34mmol) of Rink Amide MBHA resin (containing 1% DVB cross-linker, 100-200 mesh, 0.67mmol / g) and add it to a 60mL peptide solid phase reactor, add 20mL 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 20mL / time, the washing time is 3min / time, and the shaking speed is 500r / min.

[0048] ② After washing, drain the solvent, add 20 mL of 20% piperidine / DMF solution to the reactor resin to remove the resin Fmoc protecting group, shake at 500 r / min and 25°C for 5 min, and drain the solution; then add 20 mL of 20% piperidine / DMF solution to the resin again, shake at 500 r / min and 25°C for 15 min, and drain the solution. Wash the resin with DMF solution (20 mL / 3 min / time) 5 times.

[0049] ③ Weigh 1.962g Fmoc-Cys(Trt)-OH (2.5eq, 3.35mmol) and 0.476g Oxyma (3eq, 3.35mmol) into a 50mL beaker, add 15mL DMF solution to dissolve, add 0.518mL condensing agent DIC (3eq, 3.35mmol) 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 (20mL / 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 (20 mL / 3 min / time). The resin was then washed alternately according to the following procedure: DCM × 5 times (20 mL / 3 min / time), MeOH × 5 times (20 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, 9.606 g of peptide resin was obtained with a yield of 98.5%.

[0052] ⑤ Cracking

[0053] Weigh 9.606g 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 3.060g of white solid crude peptide with a yield of 95.0%.

[0054] ⑥Cyclization

[0055] Weigh 0.151 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 7.82, stir the mixture in an open air 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] The fractions were collected for MS and HPLC analysis, and the target fractions were combined and freeze-dried to obtain the final [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC totaled 50.1 mg, yield 33.2%, HPLC purity 98.559%. MS results showed: [M+2H] 2+ =1203.0450, 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 ,Arg 5 ]-dR-μ-CnIIIC chromatogram Figure 4 As shown, μ-conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC mass spectrum Figure 5 shown.

[0061] Example 1 Preparation of [Asn 17 ,Arg 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.

[0062] Example 2 Wild-type μ-CnIIIC and [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC biological activity assay

[0063] The mice used in this example were adult male Kunming mice purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Sciences, and the animal experiments were approved by the Ethics Committee of Lanzhou Peptide Valley Research Institute.

[0064] 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.

[0065] 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 ,Arg 5 ]-dR-μ-CnIIIC, the degree of toe abduction of the left and right hind limbs was measured as a function of time, and whether there was corresponding behavior was observed based on 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).

[0066] 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.

[0067] The experimental results are summarized in Tables 2-3 in the order of increasing sample injection concentration:

[0068] Table 2 Wild-type μ-CnIIIC and [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC mouse experimental results (50μM)

[0069]

[0070] Note: 1. “ / ” means that the mice were asymptomatic after drug injection, so the onset time and duration of the effect could not be calculated;

[0071] Table 3 Animal experimental results of wild-type μ-CnIIIC at concentrations greater than 50 μM

[0072]

[0073] Note: 1. “ / ” means that the mice were asymptomatic after drug injection, so the onset time and duration of the effect could not be calculated;

[0074] 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 5 After injection of [Asn]-dR-μ-CnIIIC, mice showed lethal behaviors caused by overdose in a short period of time. The mice showed obvious leg dragging behavior 5 minutes after injection and died 2 minutes later. 17 ,Arg 5 ]-dR-μ-CnIIIC has higher activity than wild-type μ-CnIIIC.

[0075] The results in Table 3 show that mice injected with equal volumes of saline at different concentrations behaved normally, and mice died only after 500 μM wild-type μ-CnIIIC was injected into the calf muscle. This further illustrates that the μ-conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC has 10 times the activity of wild-type μ-CnIIIC.

[0076] In summary, compared with wild-type μ-CnIIIC, μ-conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC has a higher activity, which is 10 times higher. It can inhibit muscle contraction and cause anesthesia in mice at low concentrations, indicating that [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC can reduce or inhibit muscle contraction, has analgesic and anesthetic effects, and 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.

[0077] 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.

[0078]

Claims

1. A μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC, characterized in that The μ-type conotoxin peptide [Asn 17 ,Arg 5 ]The amino acid sequence of -dR-μ-CnIIIC is shown in SEQ ID NO.

1.

2. Encoding the μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC DNA molecule.

3. A vector comprising the DNA molecule according to claim 2.

4. The carrier according to claim 3, characterized in that The vector is an expression vector.

5. A host cell, characterized in that The host cell contains the μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC, the DNA molecule according to claim 2, or the vector according to any one of claims 3 to 4.

6. The μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC in the preparation of drugs for treating or preventing diseases related to sodium ion channels.

7. The use according to claim 6, characterized in that: The sodium ion channel-related disease is any one of autism spectrum disorder, myotonia, arrhythmia, epilepsy, and muscle paralysis.

8. The μ-conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC in the preparation of drugs for treating or preventing pain.

9. The μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC in the preparation of anesthetic drugs.

10. The μ-type conotoxin peptide [Asn 17 ,Arg 5 ]-dR-μ-CnIIIC in the preparation of cosmetics.

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