AI-design-based analgesic active peptides CSK and CSC as well as preparation method and application thereof

Abstract

The invention discloses analgesic active peptides CSK and CSC designed based on AI as well as a preparation method and application thereof, relates to the technical field of biological medicines and the field of biological active peptides, and in particular relates to the analgesic active peptides CSK and CSC designed based on AI as well as the preparation method and application thereof in preparation of analgesic drugs and cosmetics. The analgesic active peptide CSK or CSC can be mixed with a pharmaceutically acceptable carrier to prepare clinically acceptable injections and oral preparations for preparing analgesic drugs. The analgesic active peptides CSK and CSC provided by the invention have better analgesic activity, the preparation method has simple steps, and the yield is higher. The invention has important guiding significance and practical value for clinical transformation and industrialization of conotoxin.

Description

Technical Field

[0001] The present invention relates to the fields of biomedicine technology and bioactive peptides, and particularly relates to an analgesic active peptide CSK and CSC designed based on AI, a preparation method thereof, and applications in the preparation of analgesic drugs and cosmetics. Background Art

[0002] Conotoxin (CTX) is a peptide toxin secreted by cone snails. It generally has a small molecular weight, is rich in disulfide bonds, and has good structural stability. Such toxins can exert various physiological activities such as muscle relaxation, paralysis, and analgesia by specifically acting on targets such as ion channels, acetylcholine receptors, and G protein-coupled presynaptic receptors, and are ideal molecules for the development of polypeptide drugs. Among them, μ-type conotoxin is one of the most intensively studied conotoxins, generally consisting of 16-26 amino acids and targeting voltage-gated sodium channels. Research shows that μ-KIIIA and μ-CnIIIC in such toxins can exert non-addictive analgesic effects by inhibiting Na V 1.7 and have relatively low cardiotoxicity, showing the potential for development as non-addictive analgesics, but there are deficiencies such as low blocking efficiency. Therefore, obtaining conotoxin active peptides with higher analgesic activity is an important research direction to promote their clinical transformation. Based on the previous computational simulation results and an independently designed AI prediction model, the present invention designed conotoxin mutants CSK and CSC with significant analgesic activity and relatively low cardiotoxicity using μ-KIIIA and μ-CnIIIC as skeletons. Summary of the Invention

[0003] The primary object of the present invention is to provide two analgesic active peptides CSK and CSC designed based on AI, and their amino acid sequences are shown as SEQ ID NO.1 and SEQ ID NO.2.

[0004] The analgesic active peptides CSK and CSC of the present invention are obtained by mutating the 10th position of μ-KIIIA and the 20th position of μ-CnIIIC respectively.

[0005] The conservative amino acid residues with analgesic activity of the analgesic active peptide CSK of the present invention include five cysteine residues at the 1st, 2nd, 9th, 15th, and 16th positions at the N-terminus.

[0006] The conservative amino acid residues with analgesic activity of the analgesic active peptide CSC of the present invention include six cysteine residues at the 3rd, 4th, 10th, 15th, 21st, and 22nd positions at the N-terminus.

[0007] The present invention further provides a preparation method for the above-mentioned analgesic active peptides CSK and CSC designed based on AI.

[0008] The analgesic active peptides CSK and CSC of the present invention are obtained by expression using genetic engineering techniques or by chemical synthesis techniques, and specifically include the following steps:

[0009] (1) Recombine the DNA encoding the analgesic active peptides CSK and CSC into an expression vector;

[0010] (2) Transform the host cell with the recombinant expression vector of step (1);

[0011] (3) Cultivate the transformed host cell of step (2) under induction expression conditions;

[0012] (4) Harvest and purify the obtained expression product.

[0013] The expression vectors described in step (1) are: Escherichia coli vectors and eukaryotic expression vectors.

[0014] The host cells described in step (2) include prokaryotic cells and eukaryotic cells. The prokaryotic cells are Escherichia coli or Bacillus subtilis, and the eukaryotic cells are yeast cells, insect cells or mammalian cells.

[0015] The induction expression conditions described in step (3) are: Add the recombinant plasmid with correct sequencing to the medium containing the corresponding antibiotic, and add an inducer (such as IPTG) to induce the expression of the target protein.

[0016] The purification methods described in step (4) include methods such as salting-out precipitation, ultrafiltration, ion exchange chromatography, hydrophobic interaction chromatography and gel filtration, etc., to separate and purify the required expression product from the cell lysate and culture solution. During the separation and purification process of the expression product, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA) or Western blotting can be used to detect the presence of the expression product and biomass spectrometry to detect the molecular size.

[0017] The present invention also provides in vivo analgesic biological activity experiments for the analgesic active peptides CSK and CSC. The experimental results show that the analgesic active peptides CSK and CSC of the present invention have obvious analgesic activity and can be used to prepare analgesics.

[0018] The present invention also provides the application of the analgesic active peptides CSK and CSC in the preparation of analgesic drugs. The analgesic active peptide of the present invention or a pharmaceutical composition containing the active peptide can be used as a therapeutic agent for treating various chronic pain-related diseases of a specific type of human body. The therapeutically effective dose of the pharmaceutical composition of the present invention generally should be determined by a clinician according to the nature, severity and sensitivity adaptability of the disease, as well as many factors such as the administration route, etc., in accordance with the principle of individualization.

[0019] The present invention also provides a clinically acceptable dosage form prepared by mixing the analgesic active peptides CSK and CSC with a pharmaceutically acceptable carrier, such as an injection, an oral preparation, a transdermal absorption preparation, and a mucosal absorption preparation. And the pharmaceutical composition of the present invention is administered by various administration routes, especially parenteral routes such as intravenous, intramuscular, intra-articular, intraperitoneal, intranasal, intradermal, subcutaneous, etc.

[0020] The present invention also provides the use of the analgesic active peptides CSK and CSC in the preparation of cosmetics. In addition to being used in the preparation of analgesic drugs, the analgesic active peptides CSK and CSC of the present invention can also be used in cosmetics. By relieving pain and reducing inflammation, skin discomfort such as pain, itching or inflammation is alleviated, thereby improving the soothing and repairing effects of the product.

[0021] Advantages of the present invention:

[0022] The analgesic polypeptide of the present invention is designed and obtained based on the site-directed mutation of conotoxin. The conotoxin mutants CSK and CSC are prepared by genetic engineering technology. The results of in vivo experiments in animals show that the analgesic activities of the two are significantly improved compared with the corresponding wild-type conotoxins. Cell-level experiments find that the inhibitory activity of CSC on myoblasts is significantly reduced, and it has the potential to further develop highly effective, low-toxic and non-addictive analgesic drugs. At the same time, the preparation method described in the present invention has simple steps and high yield. In summary, the present invention has important guiding significance and practical value for the clinical transformation and industrialization of conotoxin. Description of the Drawings

[0023] Figure 1 It is the separation and purification map of CSK in Example 2 of the present invention. Wherein A is metal ion chelating chromatography; B is SDS-PAGE analysis; wherein buffer B: Tris-HCl + 80 mM imidazole, pH 7.5; buffer C: Tris-HCl + 150 mM imidazole, pH 7.5; buffer D: 0.05 M EDTA.

[0024] Figure 2 It is the separation and purification map of CSC in Example 2 of the present invention. Wherein A is metal ion chelating chromatography; B is SDS-PAGE analysis; wherein buffer B: Tris-HCl + 80 mM imidazole, pH 7.5; buffer C: Tris-HCl + 150 mM imidazole, pH 7.5; buffer D: 0.05 M EDTA.

[0025] Figure 3 It is the effect of CSK and CSC in the formalin model in Example 3 of the present invention. Compared with the control group #### P < 0.0001; compared with formalin ββββP < 0.0001; *P < 0.05, **P < 0.01, ***P < 0.001 compared with wild-type μ-conotoxin; compared with the morphine group && P < 0.01, &&& P < 0.001, &&&& P < 0.0001.

[0026] Figure 4 This is the effect of CSK and CSC in the hot plate model in Example 3 of the present invention. Among them, A is the effect of CSK in the hot plate model; B is the effect of CSC in the hot plate model. *P < 0.05, **P < 0.01, ***P < 0.001 compared with wild-type μ-conotoxin.

[0027] Figure 5 This is the inhibitory effect of CSK and CSC on myocardial enzymes LDH and CK in Example 4 of the present invention. Among them, A is the inhibitory effect of CSK and CSC on CK; B is the inhibitory effect of CSK and CSC on LDH. Detailed implementation manners

[0028] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

[0029] The terms "μ-KIIIA" and "μ-CnIIIC" in the present invention both refer to wild-type μ-conotoxin. Among them, the amino acid sequence of μ-KIIIA is shown in SEQ ID NO.3, and the amino acid sequence of μ-CnIIIC is shown in SEQ ID NO.4.

[0030] Example 1: Construction of analgesic active peptide CSK and CSC genes

[0031] This example describes the construction strategy and basic method for expressing and obtaining the analgesic active peptide CSK and CSC genes of the present invention: Using genetic engineering technology, according to the amino acid sequence and gene sequence of μ-KIIIA, the tenth amino acid residue R (arginine) is converted to E (glutamic acid) to obtain the analgesic active peptide CSK; according to the amino acid sequence and gene sequence of μ-CnIIIC, the twentieth amino acid residue R (arginine) is converted to W (tryptophan) to obtain the analgesic active peptide CSC.

[0032] Example 2: Obtaining of analgesic active peptide CSK and CSC

[0033] 1. Construction of analgesic active peptide CSK and CSC:

[0034] According to the analgesic active peptides CSK and CSC, corresponding oligonucleotide primers were designed (Table 1). At the same time, the restriction endonuclease cleavage site sequences were added to the 5′ ends of the above oligonucleotide primers respectively for the implementation of gene recombination. Using the CSK and CSC genes obtained in Example 1 as templates, PCR amplification was carried out. The reaction conditions were: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 63°C for 30 s, extension at 72°C for 5 s, final extension at 72°C for 5 min, and 30 cycles. The products were detected by agarose gel electrophoresis and the nucleic acid fragments were recovered by gel extraction. After double digestion with the above-mentioned restriction endonucleases, they were recombinantly ligated with the plasmid that was also double digested under the action of T4 DNA ligase, and heat-transformed into Escherichia coli competent cells DH5α. After screening positive transformants through colony PCR and restriction endonuclease digestion verification, they were submitted to a biotechnology service company for DNA sequencing. The results showed that the expression recombinant vectors of the analgesic active peptides CSK and CSC were successfully constructed by the above genetic engineering method.

[0035] Table 1 Gene primer sequences

[0036]

[0037] 2. Obtaining of analgesic active peptides CSK and CSC:

[0038] The positive recombinant plasmid was heat-transformed into Escherichia coli BL21(λDE3). Then, a single colony was picked from the LB solid plate and inoculated into 4 mL of LB liquid medium (containing 50 μg / mL ampicillin), and cultured overnight with shaking at 37°C and 220 rpm. The overnight culture was inoculated into 400 mL of fresh LB medium containing 50 μg / mL ampicillin at an inoculation amount of 1%, and cultured with shaking at 37°C and 220 rpm for 4 h. An inducer, isopropyl β-D-thiogalactoside (IPTG) with a final concentration of 0.2 mM, was added, and the culture was continued with shaking at 25°C and 220 rpm for about 10 h. The fermentation was terminated, and the cells were collected by centrifugation at 3500 rpm and 4°C for 20 min. The cells were resuspended with 1 mL of lysis buffer (0.1 M PBS, 0.15 M NaCl, 50 mM imidazole), sonicated, and after completion of sonication, centrifuged at 12,000 rpm and 4°C for 20 min to obtain the supernatant. The precipitate was resonicated once according to the above steps, and the two supernatants were combined and directly loaded onto a metal ion chelating chromatography column pre-equilibrated with Tris-HCl (pH 8.0). After being washed with two different stages of pH buffer for 5 column volumes respectively, 150 mM imidazole (pH 7.5) was used for elution and the eluate was harvested. The obtained product was verified for its purity by 15% SDS-PAGE. The protein sample was detected by HPLC. Chromatographic conditions: Thermo C18, 4.6*250 mm, 5 μm, 130A; mobile phase A: 0.05 mol / L sodium dihydrogen phosphate (adjusted to pH 2.5 with phosphoric acid); mobile phase B: acetonitrile; flow rate: 1.0 mL / min; column temperature: 40°C; detection wavelength: 220 nm; injection volume: 20 μL; the results were calculated by the area normalization method. From the column chromatography chromatogram and SDS-PAGE pattern ( Figure 1 and Figure 2 ), it was shown that the recombinant protein was highly expressed and reached electrophoretic purity. For the expression product obtained above, enterokinase was used to utilize the self-cleaving protein in the vector to obtain the analgesic active peptides CSK and CSC without any tags. The structural characteristics of the expression product encoded by the recombinant expression plasmid were the analgesic active peptides CSK and CSC. The protein sample was detected for its concentration according to the instructions of the BCA protein concentration kit, and the concentration of the protein sample was calculated based on the obtained standard curve. The yield was 2.1 mg / L of the fermentation broth.

[0039] Example 3: In vivo bioactivity determination of CSK and CSC

[0040] In this example, the in vivo analgesic activities of CSK and CSC were determined through the mouse formalin model and the mouse hot plate pain model. Adult male Kunming mice weighing 18 g - 22 g were selected and randomly grouped, with 8 mice in each group.

[0041] 1. Mouse formalin model: Mice in each group were administered by tail vein injection with 0.1 mL / 10 g of normal saline (control group), morphine (2.0 mg / kg) (positive control group), μ-KIIIA (1.0 mg / kg), μ-CnIIIC (1.0 mg / kg), CSK (1.0 mg / kg), and CSC (1.0 mg / kg). 30 minutes later, 20 μL of 2.5% formalin solution was subcutaneously injected into the left hind paw. After injection, all mice were immediately transferred to a transparent box, and a mirror was placed at a 45° angle below to fully observe the animals' paws. The mice were observed for one hour, and the frequencies of licking, contracting, and shaking the paws within every 5 minutes were recorded to illustrate nociception.

[0042] The results showed that plantar formalin injection produced spontaneous pain responses in the control group, accompanied by licking, contracting, and shaking behaviors. After using morphine, CSK, and CSC, these nociceptive behaviors and emotional responses were alleviated. CSK and CSC had significant inhibitory effects on pain in both phase I (0 min - 5 min, an immediate phase that could cause spontaneous paw withdrawal, licking, and shaking behavior responses lasting about 5 min) and phase II (15 min - 40 min, experiencing a quiescent phase of about 10 min - 15 min and then an ongoing response phase of 15 min - 40 min) of the formalin model. For phase I pain, compared with the control group, the pain inhibition rates of CSK and CSC were 68.55% and 75.78% respectively, showing no significant difference compared with the wild-type polypeptide; for phase II pain, the pain inhibition rates of CSK and CSC were 65.34% and 61.90% respectively, showing a significant increase compared with the wild-type. CSK and CSC showed good analgesic effects on inflammatory pain ( Figure 3 ).

[0043] 2. Mouse hot plate model: Ensure that the mice are in a quiet state without obvious anxiety or discomfort. Mice in each group were administered by tail vein injection with 0.1 mL / 10 g of normal saline (control group), morphine (2.0 mg / kg) (positive control group), μ-KIIIA (1.0 mg / kg), μ-CnIIIC (1.0 mg / kg), CSK (1.0 mg / kg), and CSC (1.0 mg / kg). The time when the mice showed behaviors such as licking the paws or jumping on the hot plate was recorded at 0 min, 15 min, 30 min, 45 min, 60 min, and 90 min after administration.

[0044] The results showed that the hot plate produced spontaneous pain responses in the control group, accompanied by pain behaviors such as foot licking or jumping. After using morphine, CSK, and CSC, the above behaviors were alleviated. Both CSK and CSC significantly increased the pain tolerance time of mice caused by the hot plate. When comparing μ-KIIIA and μ-CnIIIC respectively, the onset speed of CSK and CSC became faster. During the period from 0 min to 15 min, the pain tolerance time of mice increased by 1.57 times and 1.45 times respectively. From the perspective of the persistence of analgesic activity, CSK and CSC had relatively stable effects after onset. CSK and CSC showed good analgesic effects on acute pain( Figure 4 ).

[0045] Example 4: Cardiotoxicity evaluation of CSK and CSC

[0046] Detection of myocardial enzyme activities in mouse serum: Mice in each group were intravenously injected with morphine (2.0 mg / kg), μ-KIIIA (1.0 mg / kg), μ-CnIIIC (1.0 mg / kg), CSK (1.0 mg / kg), and CSC (1.0 mg / kg) through the tail vein once a day for 7 consecutive days. 1 h after the last administration, orbital venous blood was collected into a 1 mL centrifuge tube, stored at room temperature for 2 h, then stored at 4°C for 1 h, and then centrifuged at low temperature to collect the supernatant. The activity levels of lactate dehydrogenase (LDH) and creatine kinase (CK) in mouse serum were detected using an ELISA kit: Prepare the standard product according to the instructions. Add 50 μL of sample diluent to the blank well, add 50 μL of standard products with different concentrations to the standard wells respectively, add 10 μL of samples from different groups to the sample wells, and then supplement the sample diluent to 50 μL. Add 100 μL of horseradish peroxidase (HRP) to each well, without adding to the blank well. Seal the plate with a sealing film and incubate at 37°C in the dark for 1 h. Add 350 μL of washing solution diluted by an appropriate multiple to each well for washing, let it stand for 30 s and then discard, and then gently pat on the filter paper to remove the residual liquid, repeating the operation five times. Add 50 μL of chromogenic solution A and an equal amount of chromogenic solution B to each well in sequence, shake and mix evenly, and develop color at 37°C in the dark for 15 min. Then add 50 μL of stop solution to each well to terminate the reaction. Zero with the blank well, and measure the absorbance values of each well at a wavelength of 450 nm within 15 min after adding the stop solution. The results showed that there were no significant differences in the inhibition of the two myocardial enzymes by CSK and CSC compared with μ-KIIIA (1.0 mg / kg) and μ-CnIIIC (1.0 mg / kg) respectively, indicating that both maintained a relatively low cardiotoxicity of the wild type( Figure 5 ).

Claims

1. AI-designed analgesic active peptides CSK and CSC, characterized in that: The amino acid sequence of CSK is shown in SEQ ID NO.1, and the amino acid sequence of CSC is shown in SEQ ID NO.

2.

2. The analgesic active peptides CSK and CSC designed based on AI according to claim 1, characterized in that: CSK and CSC were obtained by mutating the 10th position of μ-KIIIA and the 20th position of μ-CnIIIC, respectively.

3. The analgesic active peptides CSK and CSC designed based on AI according to claim 1, characterized in that: The conserved amino acid residues for analgesic activity of CSK include five cysteine ​​residues at the 1st, 2nd, 9th, 15th and 16th positions at the N-terminus; the conserved amino acid residues for analgesic activity of CSC include six cysteine ​​residues at the 3rd, 4th, 10th, 15th, 21st and 22nd positions at the N-terminus.

4. Genes encoding the AI-designed analgesic active peptides CSK and CSC according to claim 1.

5. The method for preparing the analgesic active peptides CSK and CSC based on AI design according to claim 1, characterized in that: It is obtained by expression using genetic engineering technology or by chemical synthesis technology.

6. The preparation method according to claim 5, characterized in that: The following steps are involved: (1) Recombining the analgesic peptides CSK and CSC encoding DNA into an expression vector; (2) transforming a host cell with the recombinant expression vector of step (1); (3) culturing the transformed host cell of step (2) under inducing expression conditions; (4) Harvest and purify the resulting expression product.

7. The preparation method according to claim 6, characterized in that: The host cells in step (2) include prokaryotic cells and eukaryotic cells. The prokaryotic cells are Escherichia coli or Bacillus subtilis, and the eukaryotic cells are yeast cells, insect cells or mammalian cells.

8. A pharmaceutical composition, characterized in that Comprising the analgesic active peptides CSK and / or CSC designed based on AI according to claim 1.

9. The pharmaceutical composition according to claim 8, characterized in that The AI-designed analgesic active peptides CSK and / or CSC are mixed with a pharmaceutically acceptable carrier to prepare clinically acceptable injections, oral preparations, transdermal absorption preparations, and mucosal absorption preparations.

10. Use of the AI-designed analgesic active peptides CSK and CSC according to any one of claims 1 to 3, or the pharmaceutical composition according to claim 8 or 9 in the preparation of analgesic drugs or cosmetics.

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