CN118791571A - Cyclic peptide and its derivatives, inhibitors, drugs, preparation methods and applications - Google Patents

Abstract

The present invention belongs to the technical field of peptides and their derivatives, and relates to cyclic peptides and their derivatives, inhibitors, drugs, preparation methods and applications. Aiming at the technical problems of low affinity, poor selectivity and weak stability of the prior art α7 nicotinic acetylcholine receptor (α7nAChR) inhibitors, the present application provides a bicyclic peptide that can be used as an α7 nicotinic acetylcholine receptor inhibitor, which has an amino acid sequence shown in SEQ ID NO:1, high affinity and strong stability. The present application also provides a bicyclic peptide derivative, in which cysteine is replaced with a non-natural amino acid diaminodiacid, which is not only more stable under redox conditions, but also has a more uniform conformation after directional ring formation, further improving the inhibitory effect. The present application also provides a method for preparing a cyclic peptide, constructs a cyclic peptide library based on phage display technology, and is used for screening α7nAChR inhibitors, which broadens the source of α7nAChR drug research and development.

Description

Cyclic peptide and its derivatives, inhibitors, drugs, preparation methods and applications

Technical Field

The present invention belongs to the technical field of peptides and their derivatives, and in particular, relates to cyclic peptides and their derivatives, inhibitors, drugs, preparation methods and applications.

Background Art

Nicotinic acetylcholine receptors (nAChRs) are a class of pentameric ligand-gated ion channels (pLGICs) that are widely distributed in the central nervous system and the peripheral nervous system. Among them, the α7 nicotinic acetylcholine receptor (α7nAChR) is the most abundant subtype in the brain. α7nAChR is composed of 5 identical subunits, can be activated by the neurotransmitter acetylcholine, has calcium ion permeability, and regulates intracellular calcium ion concentration. Abnormal function of α7nAChR receptors is related to the pathogenesis of various degenerative neurological diseases such as schizophrenia and Alzheimer's disease. α7nAChR is expressed in macrophages and various tumor cells (lung cancer, liver cancer, etc.), and is closely related to pathological processes such as inflammation and tumors, making it a very attractive drug target.

In recent years, ligands targeting α7nAChR, including small molecule compounds and bioactive peptides, have been discovered and developed as lead drugs for the treatment of neurological diseases, as well as pharmacological tools for early disease diagnosis and real-time monitoring of treatment response. Most of the small molecule compounds targeting α7nAChR are alkaloids with poor specificity, which are prone to cause neuropsychiatric side effects, including symptoms such as depression and drug addiction. Compared with small molecules, peptide ligands have the advantages of high specificity, low toxicity, easy chemical modification and relatively low preparation cost. In the past few decades, scientists have been committed to developing peptide ligands targeting α7nAChR. Most of the existing nAChR inhibitor peptide ligands are based on the known α-conotoxin. Not only are there no significant changes in sequence diversity and conformation, but the source is also very limited. Phage display technology, that is, displaying peptides or proteins on the surface of phages, is an in vitro screening technology that can directly screen the required peptides from artificially designed peptide libraries. As a basic research tool, it has become an extremely powerful means in drug discovery and development. The Chinese patent application publication number is CN113227118A, the application date is December 26, 2019, and the invention name is "Acetylcholine receptor inhibitory peptide and its use", which discloses an acetylcholine receptor inhibitory peptide and its use, using random peptide recombinant phage to screen out phages with high binding affinity to acetylcholine receptors, and screening acetylcholine receptor binding peptides through phage DNA. The development of these methods is very necessary to broaden the source of ligands for drug target membrane proteins such as α7nAChR and enrich the diversity of inhibitors.

In addition to limited sources, natural polypeptide ligands also have problems such as poor stability and conservative structure, which limit their development and application. Cyclic peptides have better drug-making advantages, making them more effective research tools and therapeutic drugs. First, cyclic peptides have high affinity with macromolecular targets. Secondly, cyclic peptides have targeted selectivity and can distinguish similar proteins with the same tertiary structure and high sequence homology. Finally, cyclic peptides are relatively stable compared with linear peptides. At present, in vitro screening methods for cyclic peptides have been developed, such as the Chinese patent application publication number CN111945231A, the application date is August 25, 2020, and the invention name is "Method for constructing a phage-displayed multi-cyclic peptide library based on disulfide bond precise pairing", which discloses a method for constructing a phage-displayed multi-cyclic peptide library based on disulfide bond precise pairing, involving cyclic peptide compounds. The disulfide bond pairing mode within the ternary cyclic peptide molecule is precisely regulated, and the ternary cyclic peptide template is fused and expressed on the phage surface to construct a phage ternary cyclic peptide library.

In addition, existing polypeptide inhibitors are all cyclized with disulfide bonds. Although disulfide bonds contribute to the conformation and rigidity of polypeptides, they are unstable in a reducing environment and in the presence of disulfide isomerase. Using diaminodiacid (DADA) to replace disulfide bonds is not only more stable under redox conditions, but also more uniform in conformation after directional cyclization. For example, the Chinese patent application publication number is CN107586265A, the application date is June 27, 2017, and the invention name is "An environmentally friendly orthogonally protected diaminodiacid compound, its preparation method and its application". When the disclosed method is used for polypeptide synthesis, carbon-sulfur bonds, carbon-carbon bonds, etc. can be easily introduced to replace the disulfide bonds in the cyclopeptide to achieve the purpose of improving the stability of the cyclopeptide.

Summary of the invention

1. Technical problem to be solved by the invention

In view of the technical problems of low affinity, poor selectivity and weak stability of the prior art α7nAChR inhibitors, the present application provides a bicyclic peptide that can be used as an α7nAChR inhibitor, which has an amino acid sequence shown in SEQ ID NO: 1, has high affinity and strong stability. The present application also provides a bicyclic peptide derivative, which is not only more stable under redox conditions, but also has a more uniform conformation after directional ring formation, further improving the inhibitory effect. The present application also provides a method for preparing a cyclic peptide, which broadens the source of α7nAChR drug research and development.

2. Technical solution

In order to achieve the above purpose, the technical solution provided is:

A cyclic peptide of the present invention has an amino acid sequence as shown in SEQ ID NO: 1; the 3rd amino acid and the 17th amino acid of the cyclic peptide are connected by an S-S bond; the 4th amino acid and the 9th amino acid of the cyclic peptide are connected by an S-S bond.

Furthermore, the 4th amino acid and the 9th amino acid of the cyclic peptide are replaced by replacement components to form a C-S bond.

Furthermore, the replacement component is a diaminodiacid; and the cyclic peptide derivative has a sequence shown in SEQ ID NO:9.

An α7nAChR inhibitor, comprising the cyclic peptide and/or the cyclic peptide derivative.

The cyclic peptide or the cyclic peptide derivative is used in the preparation of a medicine, and the medicine is used for the treatment of inflammation or chronic pain.

A medicine for treating inflammation or chronic pain, comprising the cyclic peptide and/or the cyclic peptide derivative.

Furthermore, the medicine is in the form of capsules, tablets, granules, powders, injections, or sprays.

A method for preparing the cyclic peptide comprises the following steps:

The method comprises the steps of constructing a phage display cyclic peptide library based on phage display technology to obtain the cyclic peptide library;

The method comprises the steps of using the cyclic peptide library for screening α7nAChR inhibitors to obtain several target cyclic peptides;

The step of synthesizing the most enriched target cyclic peptide among the several target cyclic peptides to obtain the cyclic peptide.

Furthermore, the sequence skeleton of the cyclic peptide library from N-terminus to C-terminus is as follows: (X) ₂ CC(X) ₄ C(X) ₇ CG, wherein X represents any amino acid and the subscript represents the number of Xs contained.

Furthermore, the several target cyclic peptides include an amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 8.

3. Beneficial effects

Compared with the existing known technology, the technical solution provided by the present invention has the following beneficial effects:

The cyclic peptide of the present invention has an amino acid sequence shown in SEQ ID NO: 1, is obtained by screening a bicyclic peptide library displayed by phage, has high affinity, an IC ₅₀ of up to 124.9 nM, strong stability, and a half-life of up to 15.87 h.

The cyclic peptide derivative of the present invention replaces cysteine with a non-natural amino acid diaminodiacid, and after directional ring formation, the conformation is more uniform, and the inhibitory effect _IC50 can be further improved to 17.11nM; it is more stable under redox conditions, and the half-life can reach 32.01h.

The cyclic peptide and its derivatives of the present invention can be used as α7nAChR inhibitors and one or more effectors for the treatment of inflammation or chronic pain.

The preparation method of the cyclic peptide of the present invention is based on phage display technology to construct a phage display cyclic peptide library, the sequence skeleton of the cyclic peptide library is (X) ₂ CC(X) ₄ C(X) ₇ CG from N-terminus to C-terminus, and then used for α7nAChR inhibitor screening, and the most enriched target cyclic peptide is synthesized. The screened cyclic peptide has better drug-making advantages, making it a more effective research tool and therapeutic drug, and broadening the source of α7nAChR drug research and development.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is the ¹ H- ¹ H NOESY spectrum of cyclic peptide KP2007.

Figure 2 is a schematic diagram of the lowest energy NMR structure of the cyclic peptide KP2007.

FIG. 3 is a concentration effect curve of the cyclic peptide derivative KP2007 in inhibiting the activity of α7nAChR.

Figure 4 is a graph showing the chromatographic analysis data of the plasma stability of cyclic peptide KP2007.

Figure 5 is a schematic diagram of the synthetic route of the cyclopeptide derivative KP2007-1 (C-S).

Figure 6 is a chromatographic purification diagram and mass spectrometry identification diagram of the cyclic peptide derivative KP2007-1 (C-S).

Figure 7 is a graph showing the concentration-effect curve of the cyclic peptide derivative KP2007-1 (C-S) in inhibiting α7nAChR.

Figure 8 is a graph showing the chromatographic analysis data of the plasma stability of the cyclic peptide derivative KP2007-1 (C-S).

Figure 9 is a graph showing the time relationship of the plasma stability of cyclic peptide KP2007 (S-S) and cyclic peptide derivative KP2007-1 (C-S).

Figure 10 is a chromatographic and mass spectrometric characterization of the cyclic peptide KP2002 (S-S) screened in the cyclic peptide library after renaturation.

Figure 11 is the chromatographic and mass spectrometric characterization of the cyclic peptide KP2003 (S-S) screened in the cyclic peptide library after renaturation.

Figure 12 is the chromatographic and mass spectrometric characterization of the cyclic peptide KP2004 (S-S) screened in the cyclic peptide library after renaturation.

Figure 13 is the chromatographic and mass spectrometric characterization of the cyclic peptide KP2005 (S-S) screened in the cyclic peptide library after renaturation.

Figure 14 is a chromatographic and mass spectrometric characterization of the cyclic peptide KP2006 (S-S) screened in the cyclic peptide library after renaturation.

Figure 15 is a chromatographic and mass spectrometric characterization of the cyclic peptide KP2007 (S-S) screened in the cyclic peptide library after renaturation.

Figure 16 is the chromatographic and mass spectrometric characterization of the cyclic peptide KP2008 (S-S) screened in the cyclic peptide library after renaturation.

Figure 17 is the chromatographic and mass spectrometric characterization of the cyclic peptide KP2009 (S-S) screened in the cyclic peptide library after renaturation.

FIG. 18 is a graph showing the concentration effect of cyclic peptides KP2002-KP2007 in inhibiting α7nAChR activity.

DETAILED DESCRIPTION

The technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Abbreviations or glossaries

nAChR (nicotinic acetylcholine receptors), nicotinic acetylcholine receptor;

Fmoc, fluorenylmethoxycarbonyl;

DIEA[N-Ethyl-N-(1-methylethyl)-2-propanamine], N,N-diisopropylethylamine;

HPLC (High Performance Liquid Chromatography), high performance liquid chromatography;

MS (Mass Spectrum), mass spectrometry;

HUTU, chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate;

DMF, N,N-dimethylformamide;

TFA, trifluoroacetic acid;

Protecting group: As used herein, the term "protecting group" is well known in the art, and suitable amino protecting groups include nonafluorenylmethoxycarbonyl (Fmoc).

Condensation Reagent: In a condensation reaction, the term "condensation reagent" refers to a reagent that activates a less reactive site and renders it more susceptible to attack by another reagent.

Example 1

Construction of 17AA phage displayed bicyclic peptide library (X) ₂ CC(X) ₄ C(X) ₇ CG.

1. Reagents, vectors, strains and instruments.

1.1. Reagents.

1.1.1. Enzyme: Dpn1 (NEB: R0176S); Bsa1 (NEB: R0535S); T4 DNA Ligase (NEB: M0202T).

1.1.2.PrimeSTAR GXL(Takara:R050A)

1.1.3. Kits: plasmid mini-extraction kit; DNA gel recovery kit; DNA recovery kit.

1.2. Carrier.

Vector: PCantab 5E’ (the base TCT at S240 position was mutated to TCG).

Note: The BSA1 restriction site of the vector was mutated.

1.3. Strain: TG1 electroporation competent.

Use plasmid to verify transformation efficiency. The pUC19 plasmid (ampicillin resistance) provided by Ang Yu Biotechnology has an electroporation efficiency of more than 0.5×10 ¹⁰ cfu/μg using TG1 competent cells. Compare the homemade competent cells with the existing competent cells to test the efficiency of the homemade competent cells.

1.4. Instruments: PCR instrument; DNA electrophoresis instrument; electrotransfer instrument.

2. Primer design.

Forward primer (SEQ ID NO: 10): TTTGGTCTCGGTGCGCCGGTGCCGTATCCGGATCCGCTG.

Reverse primer (SEQ ID NO: 11): TTTGGTCTCAGCACCACCACCGCAMNNMNNMNNMNNMNNMNNMNNGCAMNNMNNMNNMNNGCAGCAMNNMNNACCGGCCATGGCCGGCTGGGCCGCATAG, wherein N＝A/T/C/G bases; M＝A/C bases.

Note: BSA1 restriction site: GGTCTCN1; CCAGAGN5.

3. PCR reaction.

3.1. PCR reaction system (50 μL).

Table 1 Reaction system parameters

Reagents Usage Final concentration 5× PrimeSTAR GXL buffer 10μL 1× dNTP Mixture 4μL 200μM Primer1 1μL 0.2μM Primer2 1μL 0.2μM Template 1μL(100ng) PrimeSTAR GXL Polymerase 1μL 1.25U/50μL H20 Up to 50μL Up to 500μL Denaturation temperature 98℃ 10s Annealing temperature 60℃ 15s Extension temperature 68℃ 6min(1min/kb)

3.2. DNA gel identification of PCR products.

Prepare 1% agarose gel, add 1*TAE buffer, mix and heat, then add Gelred.

UV light 254 wavelength illuminates the display bands.

3.3. DNA gel recovery.

4. Enzyme digestion of PCR products (DPN1+BSA1).

4.1. Enzyme digestion system.

Table 2 Enzyme digestion system parameters

BSA1 1μL Dpn1 1μL DNA 1 μg 10×Cutsmart 5μL Total 50μL

37°C, 2h, water bath.

Save the sample and run DNA gel (1% agarose gel).

4.2. DNA purification after enzyme digestion.

5. T4 DNA enzyme ligation.

5.1. Ligation system (20 μL).

Table 3 Connection system parameters

T4 DNA ligase reaction buffer(10×) 2μL DNA 500ng T4 DNA ligase 1μL H ₂ 0 Up to 20μL

Ligate overnight at 16°C.

5.2. DNA purification.

6. Preparation of Electrocompetent Cells and Electroporation

200mL TG1 bacterial solution electrocompetent preparation

6.1. In this experiment, E. coli TG1 was selected as the competent cell. A single clone of TG1 was selected and placed in 10 mL of LB medium and cultured at 37°C and 220 rpm for 6 h.

6.2. Take 2 mL of bacterial solution and pour it into 200 mL of LB culture medium. Cultivate at 37°C and 220 rpm with shaking until OD ₆₀₀ reaches 0.6-0.7. Pour it into a pre-cooled bacteria collection bottle and place it in an ice bath for 15 minutes. Centrifuge at 4°C and 5,200 g for 10 minutes, discard the supernatant, and collect the bacteria.

6.3. Resuspend with 5 mL of pre-cooled double distilled water, then pour in 40 mL. Centrifuge at 4°C, 5,200 g for 10 min, discard the supernatant, and collect the cells.

6.4. Resuspend with 40 mL/tube of pre-cooled 10% (w/v) glycerol. Centrifuge at 4°C, 5,200 g for 10 min, discard the supernatant, and collect the cells.

6.5. Resuspend with 10 mL/tube of pre-cooled 10% (w/v) glycerol and collect into one tube. Centrifuge at 4°C, 5,200 g for 10 min, discard the supernatant and collect the cells.

6.6. Resuspend with 2 mL of pre-cooled 10% (w/v) glycerol. Dispense 100 μL of the resuspended bacterial solution into EP tubes and immediately transform using an electroporator. Alternatively, the cells can be frozen at -80°C for long-term storage.

7. Electroporate DNA into competent medium.

The EC2 program provided by Bio-Rad Instruments was used (2.5 KV, 0.2 cm electrophoresis cup).

7.1. Ice bath electric rotating cup

7.2. Take 1 μg DNA, add 100 μL competent medium, mix well, add to the electroporation cup, place on ice, and cover the electroporation cup lid.

7.3. Place the cells in the electroporator slot. After electroporation, take them out immediately and add 1 mL of SOC medium. Mix well and pour into an EP tube. Incubate at 37°C and 220 rpm for 1 hour.

7.4. Pour the electroporated bacterial solution into 1L LB liquid medium, mix well and take out 1mL of bacterial solution for use. Take out 100μL to plate first, and dilute the rest of the bacterial solution with LB medium in 10-fold gradients. Take out 100μL of each gradient and plate the solid medium plate coated with ampicillin. After culturing at 37℃ for 12h, count the logarithmic bacteria.

Peptide library capacity = 10 × gradient × 10 ³ pfu/mL.

7.5. The remaining bacterial solution was cultured at 37°C, 220 rpm, and shaken for 12 h. 40% (w/v) glycerol was added to resuspend the solution and stored at -80°C.

8. Phage preparation.

8.1. Take out the glycerol stock of phage library stored at -80℃.

8.2. Take 1 mL of glycerol bacteria and add it to 1 L of freshly sterilized LB liquid medium, add ampicillin at a final concentration of 100 μg/mL, and mix well. Incubate at 37°C with shaking at 220 rpm until the culture reaches the logarithmic growth phase (OD ₆₀₀ = 0.6-0.8). Add helper phage, incubate at 37°C for 60 minutes, and incubate on a shaker for 60 minutes.

8.3. Add kanamycin at a final concentration of 50 μg/mL and culture overnight at 30°C and 220 rpm with shaking.

8.4. Centrifuge at 10,000 g and 4°C for 15 min. Collect the supernatant in a clean and sterilized beaker. Add 1/5 volume of PEG/NaCl (20% (w/v) PEG-8000, 2.5 M NaCl) and incubate in an ice bath for 2 h.

8.5. Use a 50mL sterilized centrifuge tube to centrifuge the above liquids in batches at 4℃, 10,000g for 10min, discard the supernatant, and retain the precipitate. After all centrifugation is completed, resuspend the phage precipitate at the bottom of the centrifuge tube with 10mL PBS, and combine them in one tube after complete dissolution. Centrifuge at 4℃, 10,000g for 10min, and recover the supernatant into a new centrifuge tube. Add 1/5V volume of PEG/NaCl (20% (w/v) PEG-8000, 2.5M NaCl) to the tube and let it settle in an ice bath for 2h.

8.6. Centrifuge at 10,000g, 4℃ for 10 min, remove the supernatant, and the off-white solid at the bottom is the prepared phage.

8.7. Resuspend with 10 mL PBS, add the same volume of 40% (w/v) glycerol after complete dissolution, mix well, sterilize by filter, and store in aliquots at -80°C.

Example 2

In this example, a cyclic peptide having an amino acid sequence as shown in SEQ ID NO: 1 was prepared, and the specific steps were as follows.

1. Preparation of Phage Peptide Library

1.1. Take out the constructed phage glycerol library (17AA bicyclic peptide library) frozen at -80°C. The sequence backbone of the cyclic peptide library from N-terminus to C-terminus is as follows: (X) ₂ CC(X) ₄ C(X) ₇ CG, wherein X represents any amino acid (in the derivatives of the present invention, X can also represent a non-amino acid replacement component), and the subscript represents the number of Xs contained.

1.2. Take 1 mL of glycerol bacteria and add it to 1 L of freshly sterilized LB medium (tryptone: yeast extract: NaCl = 10 g: 5 g: 10 g), add ampicillin at a final concentration of 100 μg/mL, add glucose at a final concentration of 2% (w/v), and mix well. Incubate at 37°C, 220 rpm with shaking until the culture reaches the logarithmic growth phase (OD ₆₀₀ = 0.6-0.8). Add 20 times excess helper phage, incubate at 37°C for 60 min, and incubate on a shaker for 60 min.

1.3. Add kanamycin to a final concentration of 50 μg/mL and culture overnight at 30°C, 220 rpm with shaking.

1.4. Centrifuge at 10,000 g and 4°C for 15 min. Collect the supernatant in a clean and sterilized beaker. Add 1/5 volume of PEG/NaCl (20% (w/v) PEG-8000, 2.5 M NaCl) and incubate in an ice bath for 2 h.

1.5. Use a 50mL sterilized centrifuge tube to centrifuge the above liquids in batches at 4℃, 10,000g for 10min, discard the supernatant, and keep the precipitate. After all centrifugation is completed, resuspend the phage precipitate at the bottom of the centrifuge tube with 10mL PBS, and combine it in one tube after it is completely dissolved. Centrifuge at 4℃, 10,000g for 10min, and recover the supernatant into a new centrifuge tube. Add 1/5 volume of PEG/NaCl to the tube and let it settle in an ice bath for 2h.

1.6. Centrifuge at 10,000g, 4℃ for 10 min, remove the supernatant, and the off-white solid at the bottom is the prepared phage.

1.7. Resuspend with 10 mL PBS, add the same volume of 40% (w/v) glycerol after complete dissolution, mix well, and sterilize by filtering.

2. Targeting α7nAChR for solid phase screening

2.1. Coat the target protein overnight.

10 μg of purified α7nAChR protein was coated in a polystyrene 96-well plate (with the increase in the number of screening times, the coated protein increased from 10 μg in the first round, to 5 μg in the second round, to 2 μg in the third round). The coated protein was added to a volume of 100 μL per well using binding buffer (PBS + 0.025% (w/v) GDN (Glyco-diosgenin)), and incubated overnight at 4°C with gentle shaking at 60 rpm.

2.2. Closed.

Aspirate the liquid in the plate to remove unbound proteins.

Add 200 μL blocking buffer (1% (w/v) BSA + 0.025% (w/v) GDN, PBS) into the wells and incubate at 4°C, 60 rpm, for 2 h.

Take a 1.5 mL EP tube, add 100 μL of phage (titer: 1×10 ¹² pfu/mL) and 100 μL of blocking buffer, and incubate at 4°C, 60 rpm, for 2 h.

2.3. Co-incubation.

The liquid in the plate was removed and the blocked phages were added to the wells of the 96-well plate coated with protein.

Incubate at 4°C, 60 rpm, 2 h.

2.4. Wash the plate.

Pour off the unbound phages, invert the plate on a clean paper towel to remove the residual solution. Add 200 μL of washing buffer (PBS + 0.025% (w/v) GDN + 0.1% (v/v) Tween-20), gently shake the 96-well plate, and pipette out all the liquid, repeat 10 times.

Then add 200 μL of binding buffer (PBS + 0.025% (w/v) GDN) and gently shake the 96-well plate to absorb all the liquid, and repeat this process three times.

2.5. Elution.

The residual liquid in the wells was removed, and 200 μL of elution buffer (0.2 M Glycine-HCl, pH 2.2) was added.

The mixture was gently shaken at room temperature for 10 min, the liquid was aspirated, and 20 μL of 1 M Tris-HCl (pH 9) was added to neutralize the eluate.

2.6. Measure the titer.

2.6.1. Pick a single clone of TG1, add 15 mL to a 50 mL test tube, and culture at 4°C with shaking at 220 rpm until the logarithmic growth phase ( _OD600 value is 0.6-0.8).

2.6.2. Take 10 μL of the screened phage, add 90 μL of TG1 bacterial solution, and dilute it 5 to 6 orders of magnitude in this ratio.

2.6.3. Spread the diluted bacterial solution on solid culture medium and culture at 37℃ for 12h.

2.6.4. Counting: number of colonies on solid culture medium × dilution factor × 10 = titer (pfu/mL).

2.7. Amplify the remaining phages.

2.7.1. Add the phage to 20 mL TG1 bacterial solution (OD ₆₀₀ value is 0.6-0.8), and culture at 37°C, 220 rpm for 1 hour. Centrifuge, remove the supernatant, add LB liquid medium, resuspend, spread on solid medium, and culture at 37°C for 12 hours. Add an appropriate amount of LB liquid medium, blow off the colony, add an equal volume of 40% (w/v) glycerol, and store at -80°C.

2.7.2. Amplify phage. For details, refer to 1. Prepare phage peptide library.

2.8. Sequencing.

Pick a single clone from the colonies on the solid medium in step 2.6 and send it for sequencing. Based on the sequencing results, select the sequence with high enrichment and synthesize it.

3. Solid phase synthesis of positive peptides

3.1. Resin swelling.

3.1.1. Weigh 0.2mmol (512mg) of chlororesin and put it into a peptide synthesis tube. Add 5mL of DMF (N,N-dimethylformamide) and 5mL of DCM (dichloromethane) to the peptide synthesis tube, place it at room temperature for 30min, drain the solvent with an air pump, rinse with 10mL of DMF and drain the solvent.

The DMF and DCM involved in this example are both common reagents with a purity of 99.7%. Extracting the solvent means using an air pump to extract the solvent in the peptide synthesis tube into a suction filtration bottle.

3.2. Synthesis starts from the first amino acid on the right side (C-terminus) of the peptide sequence and moves toward the left side (N-terminus).

3.2.1. Weigh 0.8 mmol of Fmoc-Gly-OH (glycine) and place it in a 10 mL EP tube. Add 6 mL of DMF to the EP tube to dissolve it. Shake well and then add 1.6 mmol (264 μL) of DIEA to the EP tube.

3.2.2. Transfer the above mixed solution to a peptide synthesis tube, and then transfer the peptide synthesis tube to a 33°C constant temperature shaker for shaking overnight and then take out the peptide synthesis tube.

3.2.3. Cleaning: First, rinse the resin three times with DMF (10 mL each time) and drain the solvent; then rinse the resin three times with DCM (10 mL each time) and drain the solvent; finally, rinse the resin three times with DMF (10 mL each time) and drain the solvent.

3.3. Deprotection.

3.3.1. Add 10 mL of 20% piperidine solution to the peptide synthesis tube to submerge the resin, and transfer to a 33°C constant temperature shaker for 5 min.

3.3.2. Take out the peptide synthesis tube from the shaker.

3.3.3. Cleaning: Repeat the above 3.2. Cleaning steps 3.2.1.

3.4. Connect the second amino acid.

3.4.1. Weigh 0.8 mmol (468.5 mg) of Fmoc-Cys (Trt) -OH (cysteine) and 0.76 mmol (314 mg) of condensing agent (HCTU) and put them into a 10 mL EP tube. Add 6 mL of DMF to the EP tube to dissolve, shake well, and then add 1.6 mmol (264 μL) of DIEA to the EP tube.

3.4.2. Transfer the above mixed solution to a peptide synthesis tube, and then transfer the peptide synthesis tube to a 33°C constant temperature shaker and shake for 1 hour before taking out the peptide synthesis tube.

3.4.3. Cleaning: Repeat the above 3.2. Cleaning steps 3.2.1.

3.5. Peptide chain extension: According to the polypeptide sequence, continue to repeat steps 3.3. Deprotection and 3.4. Amino acid synthesis to extend the peptide chain until the last amino acid is synthesized.

3.6. Cleavage of crude peptide.

Take out the peptide synthesis tube, rinse the resin with DMF three times (10 mL each time), drain the solvent after each rinse, and then rinse the resin with DCM three times (10 mL each time), drain the solvent after each rinse (drain until the resin is dry powder). After draining, prepare a cutting reagent with a volume ratio of TFA/H2O/phenol/Tips (10 mL/500 μL/500 mg/500 μL) in a 50 mL EP tube. Transfer the cutting reagent to the above-mentioned peptide synthesis tube, put it in a 26°C constant temperature shaker for oscillation reaction for 2.5 hours, take out the peptide synthesis tube, and the solution in the tube is the peptide chain cleavage solution.

3.7. Blow dry and rinse.

3.7.2. Transfer 10 mL of peptide chain lysis solution into a 50 mL EP tube using an ear bulb and blow dry the lysis solution to less than 5 mL using nitrogen at room temperature.

3.7.3. Add 40 mL of icy ether to a 50 mL EP tube. After shaking the tube appropriately, place the tube in a centrifuge at 3500 rpm for 3 min. After centrifugation, discard the supernatant.

3.7.4. Repeat.

3.7.5. Dry at room temperature and crush after drying.

3.8. Chromatographic analysis and separation of crude peptides.

The crude peptide was analyzed for correctness using Shimadzu's HPLC and mass spectrometry. Once verified to be correct, the correct product was isolated and lyophilized.

3.9. Peptide renaturation.

Weigh 0.1M (1.6g) Tris, 2M (12g) urea, 1mM (24mg) cystine, and 1mM (10mg) cysteine into a 500mL volumetric flask, use a measuring cylinder to measure 100mL of pure water and add it to the volumetric flask, adjust the pH to 7.5-8.5, add 10mg of peptide, react at room temperature, and use Shimadzu's HPLC and mass spectrometry to analyze the correctness of the crude peptide. After verification, separate the correct product and freeze-dry it.

The chromatographic and mass spectrometric characterizations of the cyclic peptides KP2002-KP2009 screened from the cyclic peptide library after renaturation are shown in Figures 10-17.

Example 3

This example is based on a cell calcium flux experiment to test whether the cyclic peptide prepared in Example 2 inhibits the activity of α7nAChR.

1. HEK-293T cell culture

1.1. Reagents used: PBS; trypsin; DMEM culture medium.

1.2. Take 10 ⁶ HEK-293T cells, add them to a culture dish, add 4 mL of DMEM, shake gently, and culture at 37°C, 5% CO ₂ for 48 hours.

1.3. Remove all the culture medium, add 1 mL of PBS along the wall of the dish, shake gently, and then remove all the PBS.

1.4. Add 500 μL of trypsin and gently shake the culture dish so that the trypsin covers the bottom of the dish. Digest for 30 seconds and remove the trypsin.

1.5. Add 1 mL of DMEM and resuspend the cells.

1.6. Take the culture dishes, add 4 mL of DMEM culture medium to each dish, add 250 μL of cell resuspension solution, and shake gently to evenly disperse the cells.

2. Transfection of Plasmid

2.1. Reagents used: OMEM; lipo3000; p3000; main plasmid: α7-PCDNA3.1; auxiliary plasmid: RIC-PCDNA3.1; NACHO-PCDNA3.1.

2.2. Prepare solution A: OMEM 100uL+lipo3000 4μL; solution B: OMEM 100uL+p3000 4μL+2μgα7-PCDNA3.1+1μg RIC-PCDNA3.1+1μg NACHO-PCDNA3.1, and let stand for 5 minutes.

2.3. Add liquid A to liquid B and let it stand for 20 minutes.

2.4. Add the mixed solution to the culture dish and culture at 37°C, 5% CO ₂ for more than 24 hours.

3. Cell plating

The transfected cells were plated in a 96-well plate, with 6×10 ⁴ cells per well.

4. Detection of cellular calcium influx using the FLIPR instrument

4.1. Prepare calcium flux reagent:

binding buffer (Hanks solution + 0.1% BSA + 2.5 μM Probeacid, pH = 7.4).

Staining reagent: 10mL binding buffer + 10μL Fluo-4 + 5μL 20% plu.

Nicotine reagent: 1 μM nicotine (binding buffer reagent).

PNU reagent: 40 μM PNU (binding buffer reagent).

4.2. Prepare peptide reagent and dilute the peptide in a gradient manner using binding buffer.

4.3. Take out the 96-well plate, add 100 μL binding buffer to each well, wash the cells once, and remove all the liquid.

4.4. Add 50 μL of prepared dye reagent to each well, place in an incubator away from light, and let stand for 45 minutes.

4.5. Take out the 96-well plate, remove all the dye reagents, add 100 μL binding buffer, wash the cells once, remove all the liquid, and repeat twice.

4.6. Add the diluted peptide reagents one by one in order, slowly adding them while they adhere to the wall.

4.7. Detection using FLIPR instrument.

Table 4 Data related to the experiment of screening cyclic peptides to inhibit α7nAChR function

Example 4

In this example, the cyclic peptide KP2007 prepared in the example was subjected to NMR structural analysis, and the specific steps were as follows:

1. The lyophilized peptide powder was dissolved in 90% H ₂ O and 10% D ₂ O for NMR measurement.

2. All NMR experiments were performed at 298K on a Bruker Avance III 600MHz NMR spectrometer to obtain ^1H - ^1H COSY, ^1H - ^1H TOCSY and ^1H - ^1H NOESY spectra of the samples. The mixing times of TOCSY and NOESY spectra were 150ms and 500ms, respectively, as shown in Figure 1.

3. The measured data were assigned to the peaks using Sparky software and then calculated using Xplor software to obtain the three-dimensional structure of the peptide in solution.

KP2007(S-S) three-dimensional structure: After renaturation, NMR analysis of KP2007(S-S) showed only one ring conformation, C1-C4/C2-C3 (C represents cysteine), as shown in Figure 2.

Example 5

In this example, DADA was used to replace cysteine to synthesize the polypeptide derivative of KP2007. The specific steps are as follows:

1. Resin swelling

1.1. Weigh 0.2 mmol (512 mg) of chlororesin and place it into a peptide synthesis tube.

1.2. Add 5 mL of DMF (N,N-dimethylformamide) and 5 mL of DCM (dichloromethane) to the peptide synthesis tube and leave at room temperature for 30 minutes.

1.3. Use an air pump to drain the solvent.

1.4. Rinse with 10 mL of DMF and drain the solvent.

Note: The DMF and DCM involved in this example are common reagents with a purity of 99.7%. Extracting the solvent means using an air pump to extract the solvent in the peptide synthesis tube into a suction filtration bottle.

2. Synthesis starts from the first amino acid on the right side (C-terminus) of the peptide sequence and moves to the left side (N-terminus)

2.1. Weigh 0.8 mmol of Fmoc-Gly-OH (glycine) and place it in a 10 mL EP tube. Add 6 mL of DMF to the EP tube to dissolve it. Shake well and then add 1.6 mmol (264 μL) of DIEA to the EP tube.

2.2. Transfer the above mixed solution to a peptide synthesis tube, and then transfer the peptide synthesis tube to a 33°C constant temperature shaker for shaking overnight and then take out the peptide synthesis tube.

2.3. Cleaning: First, rinse the resin three times with DMF (10 mL each time) and drain the solvent; then rinse the resin three times with DCM (10 mL each time) and drain the solvent; finally, rinse the resin three times with DMF (10 mL each time) and drain the solvent.

3. Deprotection

3.1. Add 10 mL of 20% piperidine solution to the peptide synthesis tube to submerge the resin, and transfer to a 33°C constant temperature shaker for 5 min.

3.2. Take out the peptide synthesis tube from the shaker.

3.3. Cleaning: Repeat the above 2. Cleaning steps 2.3.

4. Connect the second amino acid

4.1. Weigh 0.8 mmol (468.5 mg) of Fmoc-Cys (Trt) -OH (cysteine) and 0.76 mmol (314 mg) of condensing agent (HCTU) and put them into a 10 mL EP tube. Add 6 mL of DMF to the EP tube to dissolve, shake well, and then add 1.6 mmol (264 μL) of DIEA to the EP tube.

4.2. Transfer the above mixed solution to a peptide synthesis tube, and then transfer the peptide synthesis tube to a 33°C constant temperature shaker for 1 hour and then take out the peptide synthesis tube.

4.3. Cleaning: Repeat the above 2. Cleaning steps 2.3.

5. Peptide chain extension

According to the peptide sequence, continue to repeat step 3. deprotection and step 4. amino acid synthesis to extend the peptide chain.

6. Replace Cys9 with DAAD

6.1. Weigh 0.4 mmol (228 mg) of DAAD, 0.4 mmol (208 mg) of condensing agent (Pyaop) and 0.4 mmol (54 mg) of condensing agent (HOAT) and put them into a 10 mL EP tube, add 6 mL of DMF to the EP tube to dissolve, shake well, and then add 0.8 mmol (440 μL) of N-methylmorpholine to the EP tube.

6.2. Transfer the above mixed solution to a peptide synthesis tube, and then transfer the peptide synthesis tube to a 33°C constant temperature shaker for 2 hours and then take out the peptide synthesis tube.

6.3. Cleaning: Repeat the above 2. Cleaning steps 2.3.

6.4. Repeat the above steps once.

7. Peptide chain extension

According to the peptide sequence, continue to repeat step 3. deprotection and step 4. amino acid synthesis to extend the peptide chain.

8. When receiving Cys4, remove Alloc and oall on DAAD

Weigh 0.4mmol (464mg) of tetrakistriphenylphosphine palladium and add it to a 50mL EP tube. Then add 5mL of DCM and 5mL of DMF to the EP tube to dissolve it, mix it thoroughly, then add 2mmol (248μL) of phenylsilane to the EP tube, shake it thoroughly, and transfer the solution to the peptide synthesis tube. Transfer the peptide synthesis tube to a 33℃ constant temperature shaker and shake it for 3h, then take it out and wash it.

9. Cyclization

9.1. Weigh 0.4 mmol (208 mg) of condensing agent (Pyaop) and 0.4 mmol (54 mg) of condensing agent (HOAT) and put them into a 10 mL EP tube. Add 6 mL of DMF to the EP tube to dissolve them. Shake well and then add 0.8 mmol (440 μL) of N-methylmorpholine to the EP tube.

9.2. Transfer the above mixed solution to a peptide synthesis tube, and then transfer the peptide synthesis tube to a 33°C constant temperature shaker for 2 hours and then take out the peptide synthesis tube.

9.3. Cleaning: Repeat the above 2. Cleaning steps 2.3.

9.4. Repeat the above steps once.

10. Peptide chain extension

According to the peptide sequence, continue to repeat step 3. deprotection and step 4. amino acid synthesis to extend the peptide chain until the last amino acid condensation is completed.

11. Cleavage of crude peptide

Take out the peptide synthesis tube, rinse the resin with DMF three times (10 mL each time), drain the solvent after each rinse, and then rinse the resin with DCM three times (10 mL each time), drain the solvent after each rinse (drain until the resin is dry powder). After draining, prepare a cutting reagent with a volume ratio of TFA/H2O/phenol/Tips (10mL/500μL/500mg/500μL) in a 50mL EP tube. Transfer the cutting reagent to the above-mentioned peptide synthesis tube, put it in a 26℃ constant temperature shaker for oscillation reaction for 2.5h, take out the peptide synthesis tube, and the solution in the tube is the peptide chain cleavage solution.

12. Blow dry and rinse

12.1. Transfer 10 mL of peptide chain lysis solution into a 50 mL EP tube using an ear bulb, and blow the lysis solution dry to less than 5 mL using nitrogen at room temperature.

12.2. Add 40 mL of icy ether to a 50 mL EP tube. After shaking the tube appropriately, place the tube in a centrifuge at 3500 rpm for 3 min. After centrifugation, discard the supernatant.

12.3. Repetition.

12.4. Dry at room temperature and crush after drying.

13. Chromatographic analysis and separation of crude peptides: Shimadzu's HPLC and mass spectrometry were used to analyze the crude peptides for correctness. After verification, the correct products were separated and freeze-dried.

14. Disulfide bond renaturation to obtain product KP2007-1 (C-S)

Weigh 0.1M (1.6g) Tris.HCl, 2M (12g) urea, 1mM (24mg) cystine, 1mM (10mg) cysteine into a 500mL volumetric flask, use a measuring cylinder to measure 100mL of pure water and add it to the volumetric flask, adjust the pH to 7.5-8.5, add 10mg of peptide, react at room temperature, and use Shimadzu's HPLC and mass spectrometry to analyze the correctness of the crude peptide. After verification, separate the correct product and freeze-dry it.

The concentration-effect curve of KP2007-1(CS) inhibiting α7-nAChR: IC ₅₀ =1.711±0.527×10-8M (N=3).

Example 6

In this example, the cyclic peptide having the amino acid sequence shown in SEQ ID NO: 1 and the cyclic peptide derivative having the sequence shown in SEQ ID NO: 9 (SEQ ID NO: 9: PACXEGNWXRWLRLDTCG. Note: X represents Aa, i.e., diaminodiacid) prepared in Example 2 were subjected to a plasma stability experiment to detect the half-life of the polypeptide. The specific steps are as follows:

1. Take the peptide and add it to the plasma, the total volume is 200 μL, and the peptide concentration is 400 μM. The control group is the peptide added (water/formic acid; (99.9:0.1)).

2. Incubate in a 37℃ incubator, take samples, and add 10μL of 7M guanidine hydrochloride (pH 2.0) to 20μL (at different times) to denature the protease in the plasma. Mix well and place at room temperature for 10 minutes, then store at -20℃ (waiting for chromatography analysis).

3. Add 200 μL of ice-cold ethanol/formic acid (99.9:0.1) to 30 μL of sample and place at 4°C for 30 minutes to precipitate protein. Centrifuge again at 15000g, 4°C, 20 minutes, take the supernatant, and place in a vacuum drying pump to dry for 2-3 hours to evaporate the liquid.

4. Dissolve the solid with 80 μL DMSO, add 80 μL water/formic acid (99.9:0.1) and mix well. Centrifuge at 10000 rpm for 10 min to fully dissolve.

5. Add 80 μL of liquid to a 96-well plate, and load 10 μL. Gradient 5-60%, 10 min, rinse 5 min, balance 6 min.

6. Analyze using ultra-high performance liquid chromatography (UPLC) instrument (Water Corporation).

The present invention uses phage display technology to obtain a cyclic peptide inhibitor with a different sequence and spatial conformation from the existing inhibitors. In addition, by replacing cysteine with the unnatural amino acid DADA, the stability and inhibitory effect of the peptide inhibitor are improved, which broadens the source of α7nAChRs inhibitors and provides a new idea for drug research and development.

Claims (10)

1. A cyclic peptide, characterized in that: the cyclic peptide has an amino acid sequence shown in SEQ ID NO. 1; the 3 rd amino acid and the 17 th amino acid of the cyclic peptide are connected through an S-S bond; the 4 th amino acid and the 9 th amino acid of the cyclic peptide are connected through an S-S bond.

2. A cyclic peptide derivative characterized by: the cyclic peptide of claim 1 wherein the amino acids at position 4 and 9 are replaced with a replacement member to form a C-S bond.

3. The cyclic peptide derivative according to claim 2, characterized in that: the replacement member is a diamino diacid; the cyclopeptide derivative has a sequence shown in SEQ ID NO. 9.

4. An α7nAChR inhibitor characterized by: the inhibitor comprises the cyclic peptide of claim 1 and/or the cyclic peptide derivative of any one of claims 2 or 3.

5. Use of a cyclic peptide according to claim 1 or a cyclic peptide derivative according to any one of claims 2-3 for the manufacture of a medicament, characterized in that: the medicament is useful in the treatment of inflammation or chronic pain.

6. A medicament for the treatment of inflammation or chronic pain, characterized in that: comprising a cyclic peptide according to claim 1 and/or a cyclic peptide derivative according to any one of claims 2 to 3.

7. A medicament according to claim 6, characterized in that: the medicine is in the forms of capsules, tablets, granular preparations, powder, injection and spray.

8. A method of preparing the cyclic peptide of claim 1, wherein: the method comprises the following steps:

The method comprises the steps of constructing a phage display cyclic peptide library based on phage display technology, and obtaining the cyclic peptide library;

the method comprises the steps of screening the cyclic peptide library for alpha 7nAChR inhibitors to obtain a plurality of target cyclic peptides;

synthesizing the most enriched target cyclic peptides in the target cyclic peptides to obtain the cyclic peptides.

9. The method of manufacturing according to claim 8, wherein: the sequence skeleton of the cyclic peptide library from N end to C end is as follows: (X) ₂CC(X)₄C(X)₇ CG, wherein X represents any amino acid and the subscript represents the number containing X.

10. The method of manufacturing according to claim 8, wherein: the target cyclic peptides comprise the amino acid sequences shown in any one of SEQ ID NO. 1 to SEQ ID NO. 8.