Novel mu-type conotoxin peptide as well as pharmaceutical composition and application thereof

Abstract

The invention discloses a novel 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 wild type mu-CnIIIC, the animal activity of the [d-Arg2, Gly17]-dR-mu-CnIIIC provided by the invention is improved by more than 20 times, and meanwhile, the [d-Arg2, Gly17]-dR-mu-CnIIIC increases the number of basic amino acids compared with the wild type mu-CnIIIC, so that the hydrophilicity of the sequence and the membrane permeability can be further improved.

Description

Technical Field

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

[0002] Conotoxins are a class of marine biotoxins with unique pharmacological activity. Seven types have been discovered so far. All of them are small peptides with 16 to 22 amino acids in size, with three pairs of disulfide bonds formed within the molecule. They highly specifically block voltage-gated Na + Channels, and can even distinguish different subtypes of Na+ channels. Natural μ-conotoxin (μ-CnIIIC) is one of the conotoxin peptides composed of 22 amino acid residues. It is an effective antagonist of the voltage-gated Nav1.4 sodium channel and has a wide range of clinical application value, such as pain management, treatment of neurological diseases, etc. It can also be used as a potential drug to participate in the development of new drugs and provide an effective molecular template for the design of new drugs targeting sodium ion channels. In addition, the μ-CnIIIC peptide can also be used in the beauty industry to smooth out fine lines on the face. However, the biological activity and stability of natural μ-CnIIIC are not high, and there are limitations on its use as a drug. Therefore, it is urgent to develop a new μ-conotoxin with high activity and simple synthesis. Summary of the invention

[0003] In view of the defects in the prior art, the present invention proposes a novel μ-conotoxin peptide, a pharmaceutical composition and use thereof. The μ-conotoxin peptide of the present invention has higher biological activity than wild-type μ-conotoxin, and the synthesis method is simple.

[0004] The present invention first adds a D-arginine (d-Arg) at the C-terminus of the wild-type μ-conotoxin (μ-CnIIIC), deletes the pyroglutamic acid (Pyr) at the first position, and replaces the amino acid at the 17th position with glycine (Gly) and the amino acid at the second position with D-arginine (d-Arg), which can further improve the activity of μ-CnIIIC.

[0005] The present invention provides a novel μ-type conotoxin peptide, named [d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC, the amino acid sequence of the μ-type conotoxin peptide is shown in SEQ ID NO.1. 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 polynucleotide encoding the μ-type conotoxin peptide.

[0007] The present invention also provides an expression vector comprising the polynucleotide.

[0008] The present invention also provides a recombinant strain comprising the expression vector.

[0009] The present invention also provides application of the μ-type conotoxin peptide in preparing medicine for treating or preventing pain.

[0010] The present invention also provides application of the μ-type conotoxin peptide in preparing anesthetic drugs.

[0011] The present invention also provides application of the μ-type conotoxin peptide in preparing cosmetics.

[0012] 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.

[0013] Furthermore, the sodium ion channel-related disease is any one of epilepsy, arrhythmia, muscle paralysis, myotonia, and autism spectrum disorder.

[0014] The present invention also provides a pharmaceutical composition comprising the μ-type conotoxin peptide.

[0015] Furthermore, the dosage form of the pharmaceutical composition is any one of tablets, capsules, pills, solutions, absorbents, and ointments.

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

[0017] (1) A novel μ-type conotoxin peptide [d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC, compared with wild-type μ-CnIIIC, the activity of animals increased by more than 20 times, and [d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC increases the number of basic amino acids compared to the wild-type μ-CnIIIC, which can further improve the hydrophilicity and membrane permeability of the sequence and reduce potential toxicity and side effects.

[0018] (2) [d-Arg 2 ,Ser 17]-dR-μ-CnIIIC can specifically block Nav1.4 channels, reducing muscle excitability and contractility. Blocking muscle contraction can relax facial muscles, reduce dynamic wrinkles caused by facial expressions, and help prevent and reduce the formation of wrinkles. [d-Arg 2 ,Ser 17 ]-dR-μ-CnIIIC can reduce nerve conduction, thereby producing a local anesthetic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] Figure 1 The present invention proposes a novel μ-type conotoxin peptide [d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC synthesis process diagram;

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

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

[0023] Figure 4 The novel μ-type conotoxin peptide [d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC chromatogram;

[0024] Figure 5 Example 1 of the present invention: a novel μ-type conotoxin peptide [d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC mass spectrum. DETAILED DESCRIPTION

[0025] 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.

[0026] The term "μ-CnIIIC" in the present invention refers to the wild-type μ-conotoxin;

[0027] "[d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC" refers to the addition of a D-arginine (d-Arg) at the C-terminus of the wild-type μ-conotoxin (μ-CnIIIC), the deletion of the pyroglutamic acid (Pyr) at the first position, the replacement of the amino acid at the 17th position with glycine (Gly), and the replacement of the amino acid at the second position with D-arginine (d-Arg), to form a conopeptide with higher activity than the wild-type μ-conotoxin;

[0028] The Chinese meaning of the English abbreviation:

[0029] “dR” refers to D-arginine;

[0030] “G” refers to glycine;

[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 invention extends the sequence of wild-type μ-conotoxin (μ-CnIIIC), adds a D-arginine (d-Arg) at the C-terminus, replaces the 17th amino acid with glycine (Gly), deletes the 1st amino acid, and replaces the 2nd amino acid with the basic amino acid D-arginine (D-Arg), thereby improving the activity of wild-type μ-CnIIIC. 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 the μ-conotoxin peptide [d-Arg2 ,Gly 17 ]-dR-μ-CnIIIC, the synthesis process is as follows Figure 1 shown.

[0042] Reagent K: lysis solution, prepared according to the volume ratio of TFA:phenol:water:thioanisole:ethanedithiol=82.5:5:5:5:2.5.

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

[0044] Example 1 μ-type conotoxin peptide [d-Arg 2 ,Gly 17 Synthesis of ]-dR-μ-CnIIIC

[0045] (1) Preparation of Fmoc-d-Arg(Pbf)-Rink AM resin

[0046] ① Weigh 1.00g (0.64mmol) of Rink Amide AM resin (containing 1% DVB cross-linker, 100-200 mesh, 0.64mmol / g) and add it to a 20mL peptide solid phase reactor, add 10mL 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 10mL / time, the washing time is 3min / time, and the shaking speed is 500r / min.

[0047] ② After washing, drain the solvent, add 10mL of 20% piperidine / DMF solution to the reactor resin to remove the Fmoc protecting group of the resin, shake at 500r / min and 25℃ for 5min, and drain the solution; then add 10mL 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 (10mL / 3min / time) 5 times.

[0048] ③ Weigh 0.830g Fmoc-Arg(Pbf)-OH (2.0eq, 1.28mmol) and 0.182g Oxyma (2eq, 1.28mmol) into a 50mL beaker, add 10mL DMF solution to dissolve, add condensation agent DIC (2eq, 1.28mmol) 0.198mL 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 (10mL / 3min / time).

[0049] ④ Peptide chain extension

[0050] 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 (10 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, 3.745 g of peptide resin was obtained with a yield of 93.3%.

[0051] ⑤ Cracking

[0052] Weigh 3.745g 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, vacuum dry at 25℃ to constant weight, and finally obtain 0.756g of white solid crude peptide with a yield of 94.2%.

[0053] ⑥Cyclization

[0054] 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 7.88, 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.

[0055] ⑦Preparative HPLC for peptide purification

[0056] 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.

[0057] Table 1 Purification and elution procedures of crude cyclization solution

[0058]

[0059] The fractions were collected for MS and HPLC analysis, and the target fractions were combined and freeze-dried to obtain 35.4 mg of [d-Arg2, Gly17]-dR-μ-CnIIIC in total, with a yield of 35.4% and a HPLC purity of 97.881%. The MS results showed: [M+2H] 2+ =1231.5398, 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 [d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC chromatogram Figure 4 As shown, μ-conotoxin peptide [d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC mass spectrum Figure 5 shown.

[0060] Example 1 Preparation of [d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC amino acid sequence is shown in SEQ ID NO.1, and the amino acid sequence of wild-type μ-CnIIIC is shown in SEQ ID NO.2.

[0061] Example 2 Wild-type μ-CnIIIC and [d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC biological activity assay

[0062] 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. The mice were free to eat and drink water one week before the test. A 50 μL microsyringe (30G needle) was used to inject intramuscularly into the right tibialis anterior muscle group of the mouse, and the injection volume was 20 μL. An equal volume of saline was injected as a negative control, and the wild type μ-CnIIIC was used as a positive control. The experiment set a concentration gradient of 25 and 50 μM. The activity of the sample was judged by observing the onset time, duration, and behavior of the mice after injection, including toes together (inhibiting muscle contraction), dragging legs / paralysis (anesthetic effect), and death behavior.

[0063] 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 muscles of the mice were injected with physiological saline, wild-type μ-CnIIIC, different concentrations of μ-conotoxin peptide [d-Arg 2 ,Gly 17 ]-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).

[0064] 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.

[0065] The experimental results are shown in Table 2:

[0066] Table 2 Wild-type μ-CnIIIC and [d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC Animal Experiment Results (50μM)

[0067]

[0068] Note: 1. “ / ” means that the mice were asymptomatic after drug injection, so the onset time and duration of the effect could not be calculated;

[0069] 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. 2 ,Gly 17 After injection of [d-Arg]-dR-μ-CnIIIC, mice showed lethal behaviors caused by overdose in a short period of time. The mice showed obvious leg dragging and toe-pulling behaviors 2 minutes after injection, and died 7 minutes after injection. 2 ,Gly 17 ]-dR-μ-CnIIIC has higher activity than wild-type μ-CnIIIC.

[0070] To further explore [d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC activity, [d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC concentration was reduced to 25 μM, and the results are shown in Table 3.

[0071] Table 3 [d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC Animal Experiment Results (25μM)

[0072]

[0073] The results in Table 3 show that after injection of 25 μM [d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC 1 minute later, mice showed obvious leg-dragging and toe-pulling behaviors, which lasted for more than 24 hours. 2 ,Gly 17 ]-dR-μ-CnIIIC still exhibited the activity of inhibiting muscle contraction and paralysis at low concentrations and showed high activity.

[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 4.

[0075] Table 4 Animal experimental results of wild-type μ-CnIIIC at concentrations greater than 50 μM

[0076]

[0077] Note: 1. “ / ” means that the mice were asymptomatic after drug injection, so the onset time and duration of the effect could not be calculated;

[0078] In Table 4, mice injected with an equal volume of saline showed normal behavior, and mice died only after 500 μM wild-type μ-CnIIIC was injected into the calf muscle, further indicating that the μ-conotoxin peptide [d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC has an activity more than 20 times that of wild-type μ-CnIIIC.

[0079] In summary, the present invention first discovered that extending the sequence of wild-type μ-conotoxin (μ-CnIIIC), adding a D-arginine (d-Arg) at the C-terminus, replacing the 17th amino acid with glycine (Gly), deleting the 1st amino acid, and replacing the 2nd amino acid with the basic amino acid D-arginine (d-Arg) can produce μ-CnIIIC with different activities. The synthesis of the analogue was completed by solid phase peptide synthesis technology, the formation of disulfide bonds was completed by liquid phase oxidation, and finally the novel μ-conotoxin peptide [d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC. Compared with wild-type μ-CnIIIC, [d-Arg 2 ,Gly 17 ]-dR-μ-CnIIIC has a biological activity that is increased by at least 20 times, and can specifically block the Nav1.4 channel, reduce or inhibit muscle contraction, and has analgesic and anesthetic effects.

[0080] 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.

[0081] Sequence Listing

[0082] SEQ ID NO.1

[0083] Three characters:

[0084] d-Arg -Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg- Gly -His-Ala-Arg-Cys-Cys- d-Arg -NH2

[0085] Single character:

[0086] r CCNGPKGCSSKWCR S HARCC r -NH2

[0087] SEQ ID NO.2

[0088] Three characters:

[0089] Pyr-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-NH2

[0090] Single character: (X represents pyroglutamic acid)

[0091] XGCCNGPKGCSSKWCRDHARCC-NH2

Claims

1. A new 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. An expression vector comprising the polynucleotide of claim 2.

4. A recombinant strain comprising the expression vector according to claim 3.

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

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

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

8. 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.

9. The use according to claim 8, characterized in that: The sodium ion channel-related disease is any one of epilepsy, arrhythmia, muscle paralysis, myotonia, and autism spectrum disorder.

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

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