Nectin-4 targeted cyclic peptide and its preparation method and application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a cyclic peptide targeting Nectin-4, and a preparation method and application thereof. Specifically, the present invention provides an isolated polypeptide or a pharmaceutically acceptable salt thereof, wherein the polypeptide is a cyclic peptide with cysteine ​​at both ends; the polypeptide has an amino acid sequence shown in SEQ ID NO: 56. The target of the polypeptide sequence of the present invention is Nectin-4, and it has the potential to be used as a polypeptide drug for preventing and / or treating cancers with abnormal, excessive, or uncontrolled expression of Nectin-4.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a nectin-4 targeted cyclic peptide and a preparation method and application thereof. Background Art

[0002] To date, small molecule drugs and biomacromolecule drugs have been highly sought after in the early research and development of drugs. Although they each have many advantages as drug molecules, such as small molecule drugs can be taken orally and can penetrate the cell membrane to act on intracellular targets, and biomacromolecule drugs have higher specificity, the application of small molecule drugs and biomacromolecule drugs is still limited. Small molecule drugs can only interact with targets with rigid and hydrophobic patches or grooves. Moreover, the binding regions of small molecule drugs on target proteins are usually highly conserved, and this low selectivity inevitably leads to many side effects. How to screen and develop kinase inhibitors with high selectivity for specific targets has always been an important problem in the development of small molecule drugs. Macromolecule drugs essentially cannot pass through the cell membrane, so their scope of use is limited to cell membranes or extracellular targets. In addition, macromolecule drugs are mainly produced by cell culture methods, which makes it difficult to avoid heterogeneity caused by post-translational modifications and other derivatizations. Finally, macromolecule drugs cannot be taken orally, and the usual means of administration is intravenous administration. However, macromolecule drugs are easily degraded and neutralized in the bloodstream, and even cause serious immunogenicity.

[0003] The structure and functional properties of peptide molecules are between small molecules and large molecules. In recent years, peptide molecules have attracted more and more attention, especially cyclic peptides, which have brought hope to the development of new drugs. As the name suggests, cyclic peptides are polypeptide chains with a ring structure, usually composed of 5 to 14 amino acids, with a molecular weight of about 500 to 2000 Da. Currently, more than 40 cyclic peptide drugs have been approved by FDA / EMA for marketing. Cyclic peptides have a wide range of biological activities, such as antibacterial, antitumor, antifungal, antiviral, anti-inflammatory, etc.

[0004] Cyclic peptide drugs are ring-structured peptide drugs formed by a cyclization reaction connecting two atoms. Cyclization promotes intramolecular hydrogen bonding within the ring structure and reduces the external hydrogen bonding ability of the molecule, thereby reducing the polarity of the molecule. Compared with acyclic precursors, cyclization improves the membrane permeability of the compound. The synthesis of cyclic peptide molecules is an important part of cyclic peptide drug development. Rapid identification and screening of target binding candidates from cyclic peptide libraries is crucial to the development of cyclic peptide molecular drugs.

[0005] Currently, the screening methods for cyclopeptide drugs can be divided into two categories: in vitro screening and in vivo screening. In vitro screening is mainly based on the binding affinity between cyclopeptides and target proteins, and peptides with higher affinity are selected through continuous screening steps. The more widely used screening technologies include phage display technology and mRNA display screening technology.

[0006] Phage display technology is to insert different exogenous genes into phage vectors respectively. The exogenous genes are expressed along with the expression of the coat protein, so that the polypeptide or protein is displayed on the phage surface in the form of a fusion protein, thereby maintaining the relative spatial structure and biological activity. Then the linear library can be connected to the reactant by its own cysteine ​​(Cys) to form a cyclization. It is then screened with a specific target protein.

[0007] mRNA display is another effective in vitro screening method. Its principle is that the newly translated peptide fragments of ribosomes in the in vitro translation system (PURExpress) are covalently linked to the corresponding mRNA through puromycin; then they are cyclized by chemical means to establish a polypeptide-mRNA library, and finally screened and amplified. The FIT (flexible in vitro translation) system can incorporate a variety of non-protein amino acids through modified ribozymes. During the screening process, mRNA is covalently bound to the polypeptide or protein it encodes to form an mRNA-protein fusion, so that it can be selected from a large polypeptide library (10 13 ~10 15 ) to screen for peptides and proteins with specific biological functions. The basic principle of this method is to use the protein synthesis inhibitor puromycin to form mRNA-protein fusions, thereby achieving the combination of gene phenotype (RNA) and protein phenotype (polypeptide). Puromycin is an antibiotic that simulates the terminal aminoacyl group of tRNA. It is connected to the 3' end of the mRNA molecule. When the translation of mRNA is completed, puromycin enters the A site of the ribosome to accept the nascent peptide, inhibit protein translation, and form a stable amide bond between the polypeptide and the O-methyltyrosine of puromycin to generate a peptidyl puromycin complex, so that the 3' end of the mRNA and the carboxyl end of the protein are covalently linked together through the puromycin molecule. After the fusion peptide is fixed on the carrier, the fusion peptide that failed to bind is eluted, and the fusion peptide that can bind to the target protein is amplified and sequenced by PCR. According to the DNA sequence, the structure of the peptide is compared, and after multiple cycles, a high-affinity cyclic peptide molecule is optimized.

[0008] Nectin-4 is a 55.5 kDa protein composed of 510 amino acids and has three typical domains of the nectin family. Nectin-4 contains a signal peptide at the N-terminus. Nectin-4 interacts with adhesion junction protein (afadin) through the conserved Gly-His-Leu-Val motif at the C-terminus. The poliovirus receptor-related molecule 4 (PVRL4) gene contains 9 exons. Exons 1, 2, and 3 encode the signal peptide, V-type1, and C2-type1 loops, respectively. Exons 4 and 5 encode the C2-type2 loop. The transmembrane region is encoded by exon 6, and exons 7, 8, and 9 encode the cytoplasmic region.

[0009] In solid tumors, nectin-4 can promote tumor proliferation and migration by activating the P13K / Akt pathway (Takai et al., 2008a, b). Nectin-4 has two interesting characteristics: on the one hand, as a cell surface receptor that mediates the entry of measles virus into cells, nectin-4 has an inducible and efficient internalization effect, which can mediate the efficient internalization of antibody drugs; on the other hand, nectin-4 has limited overexpression in healthy tissues of adults, but is overexpressed in malignant tumor cells. This differential expression pattern can achieve targeted drug therapy and minimize the risk of adverse events. In view of these two characteristics, nectin-4 has become an induced target for targeted therapy, and its huge clinical potential has also been confirmed in the development of its targeted PDC drug. Summary of the invention

[0010] In view of the above problems, the present invention provides a cyclic peptide targeting Nectin-4 and its preparation method and application. The target of the polypeptide sequence of the present invention is Nectin-4, which has the potential to be used as a polypeptide drug for preventing and / or treating cancers with abnormal, excessive or uncontrolled Nectin-4 expression.

[0011] In a first aspect, the present invention provides an isolated polypeptide or a pharmaceutically acceptable salt thereof. The polypeptide is a linear peptide; the polypeptide has an amino acid sequence as shown in SEQ ID NO: 11; or, the polypeptide has an amino acid sequence as shown in SEQ ID NO: 21; or, the polypeptide has an amino acid sequence as shown in SEQ ID NO: 56.

[0012] Preferably, the polypeptide targets Nectin-4 protein. For example, the Nectin-4 protein has the amino acid sequence shown in SEQ ID NO:58.

[0013] In a second aspect, the present invention provides an isolated polypeptide or a pharmaceutically acceptable salt thereof. The polypeptide is a cyclic peptide with cysteine ​​residues at both ends; the polypeptide has an amino acid sequence as shown in SEQ ID NO: 11; or, the polypeptide has an amino acid sequence as shown in SEQ ID NO: 21; or, the polypeptide has an amino acid sequence as shown in SEQ ID NO: 56.

[0014] Preferably, the polypeptide is a targeting peptide having a ring structure targeting Nectin-4 protein. For example, the Nectin-4 protein has an amino acid sequence shown in SEQ ID NO:58.

[0015] Preferably, the cysteines at both ends of the polypeptide serve as cyclization sites, which form a ring structure through α,α'-dibromo-m-xylene.

[0016] Preferably, the polypeptide is a single-ring peptide.

[0017] In a third aspect, the present invention provides a method for preparing an isolated polypeptide or a pharmaceutically acceptable salt thereof. The preparation method comprises:

[0018] Preparation of mRNA-puromycin conjugates;

[0019] performing in vitro translation on the mRNA-puromycin conjugate to generate a linear mRNA-puromycin-polypeptide;

[0020] Cyclizing the linear mRNA-puromycin-polypeptide to prepare an mRNA-cyclic peptide compound;

[0021] Reverse transcribing the mRNA-cyclic peptide compound to synthesize a cDNA / mRNA-cyclic peptide complex;

[0022] Performing cyclic peptide screening on the cDNA / mRNA-cyclic peptide complex and eluting cyclic peptides having affinity with the GST-Nectin 4 target protein;

[0023] The eluted cyclic peptide was amplified by PCR;

[0024] The PCR amplification product was used as a template for the DNA library, and the structure of the polypeptide was determined by cDNA sequencing.

[0025] In some technical solutions, conventional molecular biological techniques are used to construct a cDNA library; RNA polymerase is used to transcribe the DNA library into an mRNA library; and then the splint method is used to connect puromycin to mRNA to prepare an mRNA-puromycin conjugate.

[0026] Preferably, the steps of preparing mRNA-puromycin conjugate by connecting puromycin to mRNA using the splint method include:

[0027] The puromycin linker is phosphorylated; the purotoxin linker includes purotoxin and an RNA linker sequence connected to the purotoxin, wherein the RNA linker sequence has the nucleotide sequence shown in SEQ ID NO: 5; and the puromycin molecule is connected to each mRNA through the RNA linker sequence through a splint to achieve the connection between the mRNA library and the puromycin linker; wherein the splint has the nucleotide sequence shown in SEQ ID NO: 6; the 5' end of the splint is a sequence complementary to the purotoxin, and the 3' end is a sequence complementary to the mRNA library.

[0028] In a fourth aspect, the present invention provides a use of the polypeptide in the preparation of a drug for preventing and / or treating tumors or cancers with abnormal, excessive or uncontrolled expression of Nectin-4.

[0029] In a fifth aspect, the present invention provides a use of the polypeptide in preparing a detection reagent or a kit for detecting Nectin-4 protein.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The target of the polypeptide sequence of the present invention is Nectin-4, and it has the potential to be used as a polypeptide drug for preventing and / or treating cancers with abnormal, excessive or uncontrolled Nectin-4 expression.

[0032] 2. Compared with phage display technology, the present invention utilizes mRNA display technology using in vitro transcription and translation (IVTT), avoiding the need for E. coli transfection (which limits the size of the phage display library to 10 8 ~10 9 sequences), a larger compound library (10 12 ~10 13 sequence). BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a full-field gel electrophoresis diagram of a DNA library; the first column is the molecular marker (marker), and the second and third columns are samples (sample);

[0034] Figure 2 This is the surface plasmon resonance test result of the cyclic peptide numbered cand3 and Nectin-4 protein;

[0035] Figure 3 This is the surface plasmon resonance test result of the cyclic peptide numbered cand48 and Nectin-4 protein;

[0036] Figure 4This is the surface plasmon resonance detection result of the cyclic peptide numbered cand13 and Nectin-4 protein. DETAILED DESCRIPTION

[0037] The present invention is further described by the following embodiments. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention.

[0038] The present invention belongs to the field of drug discovery, and specifically relates to a cyclic peptide targeting Nectin-4 screened based on mRNA display technology and its application. The present invention provides a brand-new polypeptide sequence, which can specifically bind to the target protein Nectin-4, wherein CXXXXXXXXXXXXC is the site that specifically binds to the target protein Nectin-4. The first and last C (cysteine) are the sites for polypeptide ring formation. These two sites are the common identical sequences of the polypeptide sequence to be protected, and the rest are target sequences. A ring is formed between the first cysteine ​​and the last cysteine. It is mainly by means of the ring-forming substance α,α′-dibromo-m-xylene (α,α`-dibromo-m-xylene), which is combined with the -SH group of cysteine ​​to form a ring. The present invention also shows a method for screening polypeptides targeting Nectin-4 based on mRNA display technology. In addition, the present invention applies the polypeptide sequence to the screening of polypeptide conjugate drugs (PDC drugs) and the verification of the mRNA display technology polypeptide screening platform.

[0039] The following examples are further listed to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific values ​​​​exemplified below.

[0040] Experimental Materials

[0041] Table 1

[0042]

[0043] Example 1: Construction of DNA library

[0044] Conventional molecular biology techniques were used to construct cDNA libraries.

[0045] DNA library strand (SEQ ID NO: 1):

[0046] 5'TAATACGACTCACTATAGGGTTAACTTTAGTAAGGAGGACAGCTAAATGTGCNNSNNSNNSNNSNNSNNSNNSNNNSNNSNNSNNSTGCGGCTCCGGTAGCTTAGGCCACCATCACCATCACCACCGGCTATAGGTAGCTAG3'

[0047] Design the following three primers:

[0048] AA63949-F (SEQ ID NO: 2):

[0049] 5'TAATACGACTCACTATAGGGTTAACTTTAGTAAGGAGGACAGCTAAATGTGC3'

[0050] AA63949-F1 (SEQ ID NO:3):

[0051] 5'GTAAGGAGGACAGCTAAATGTGCNNSNNSNNSNNSNNSNNSNNSNNNSNNSNNSNNSTGCGGCTCCGGTAGCTTAGG3'

[0052] AA63949-R (SEQ ID NO:4):

[0053] 5'CTAGCTACCTATAGCCGGTGGTGATGGTGATGGTGGCCTAAGCTACCGGAGCCGCA3'

[0054] Table 2

[0055]

[0056] PCR amplification was performed using the PCR reaction system for preparing a DNA library shown in Table 2. Each component was weighed and mixed evenly according to the PCR reaction system. Among them, the primers were prepared into a solution using nuclease-free water.

[0057] Table 3

[0058]

[0059] The PCR reaction was performed for the expansion of the DNA library according to the PCR operation program for preparing the DNA library shown in Table 3. After completion, nucleic acid gel verification and gel recovery were performed. Figure 1 This is the full-field gel electrophoresis of the DNA library. The target band is 144 bp. The DNA library was obtained by gel recovery using the UE DNA gel recovery kit.

[0060] Example 2: In vitro transcription of mRNA

[0061] RNA polymerase is used to transcribe the DNA library into an mRNA library. Specifically, the cDNA library is used as a template and HiScribe ® T7 Quick High Yield RNA Synthesis Kit was used to perform in vitro transcription of RNA to prepare RNA library. Specific operations were performed according to the instructions of the kit.

[0062] Table 4

[0063]

[0064] According to the instructions of the kit, prepare the transcription reaction solution shown in Table 4 for RNA in vitro transcription and incubate at 37°C for 16 hours. Then, add nuclease-free water to 50 μL of the transcription reaction solution, add 2 μL of deoxyribonuclease (DNase) to remove the DNA template, and continue to incubate at 37°C for 15 minutes. In practice, the volume of other components of the transcription reaction solution can be adjusted proportionally according to the amount of template. For example, when the amount of template is 2000 ng, the volume of other components can be increased proportionally.

[0065] Example 3: Purification of mRNA

[0066] Using Monarch ® RNA Cleanup Kit was used to purify mRNA. The specific operation was performed according to the instruction manual. Before purification, anhydrous ethanol was added to RNA Cleanup Wash Buffer, and then centrifuged at room temperature (16000×g). The purification of mRNA includes the following steps:

[0067] Step (1) Add 100 μL of RNA Cleanup Binding Buffer to 50 μL of RNA sample to be purified and mix gently. If the volume of RNA sample is less than 50 μL, add nuclease-free water to make up to 50 μL.

[0068] Step (2) Add 150 μL of anhydrous ethanol to the solution in step (1) and mix gently. Do not vortex.

[0069] Step (3) Transfer the above sample to the adsorption column, centrifuge for 30 seconds, and discard the supernatant.

[0070] Step (4) Add 500 μL of RNA Cleanup Wash Buffer to the product of step (2), centrifuge for 30 seconds, and discard the supernatant.

[0071] Step (5) Repeat step (4) for a total of 2 rinses.

[0072] Step (6) Transfer the product of step (5) to a new tube, open the lid and let it dry for 5 minutes at room temperature, add 10 μL of nuclease-free water, let it stand at room temperature for 3 minutes, and centrifuge for 3 minutes. Then transfer the liquid at the bottom of the tube back into the adsorption column, let it stand at room temperature for 3 minutes, and centrifuge for 3 minutes. Take 1 μL and quantify it with a micro-UV spectrophotometer, and freeze the remaining sample at -80℃. Since the obtained RNA is extremely unstable, it is recommended to proceed to the next step as soon as possible. If storage is required, store it at -80℃.

[0073] Example 4: Connecting puromycin to mRNA to prepare mRNA-puromycin conjugate

[0074] Puromycin is an antibiotic. tRNA delivers amino acids to the ribosome during translation. Puromycin is an aminoacylated tRNA analog that can serve as a linker between mRNA and peptide.

[0075] First, a puromycin linker having an RNA linker sequence was prepared.

[0076] RNA linker sequence (SEQ ID NO:5):

[0077] 5'AAA AAA AAA AAA AAA AAA AAA AAA AAA CC3'

[0078] Preparation of Puromycin Linker:

[0079]

[0080] Step 1, 1.3 g (2.56 mmol) of intermediate 1 was dissolved in 12 mL of acetonitrile (ACN), and then 950 mg (2.82 mmol) of Fmoc-OSu was added at room temperature under nitrogen protection, and the resulting mixture was stirred and reacted for 2 hours at room temperature under nitrogen protection. During this period, LC-MS was used to detect and identify the product intermediate 2 ([M+H]: 694). After the reaction was completed, 1.2 g of intermediate 2 (purity 49.37%) was obtained by filtration and centrifugation (decompression state).

[0081] Step 2, 400 mg of intermediate 2 (0.58 mmol) was dissolved in 5 mL of pyridine under nitrogen protection, 236 mg of DMTrCl (0.696 mmol) was added, and the resulting mixture was stirred at room temperature for 16 hours. During this period, LC-MS was used to detect and identify the product intermediate 3 ([M+H]: 996). After the reaction was completed, the intermediate 3 was purified from the crude product by liquid chromatography-silica gel chromatography column method (with a concentration gradient of dichloromethane-methanol as the mobile phase, with a volume fraction of 0-2%, 2%-4%, 4%-6%, 6%-8%;).

[0082] Step 3, 280 mg of intermediate 3 (0.28 mmol) was dissolved in 3 ml of dichloromethane (DCM) under nitrogen protection, and then 108 mg of DIEA (0.84 mmol) and 33 mg of dihydrofuran-2,5-dione (0.84 mmol) were added, and the resulting mixture was stirred at room temperature for 16 hours. During this period, LC-MS was used to detect and identify the product intermediate 4 ([M+H]: 1096). Under Prep-HPLC (Welch Xtimate C18 150×30mm×5um water (0.5% TEA)-MeCN, 5-95%, 30 mL / min), intermediate 4 was purified to obtain 60 mg of intermediate 4 with a purity of 100%.

[0083] Step 4, 50 mg of intermediate 4 (0.021 mmol) was dissolved in 3 mL of CAN, and then 20 mg of HATU (0.052 mmol) was added. Then 20 μL of DIEA was added to the reaction solution. After all the drugs were dissolved, 250 mg of lcaa-CPG (1000 Å) was added to the reaction solution, and then stirred at room temperature for 2 hours. After the reaction, CPG was washed with acetonitrile, and 0.5 mL of CAP A (acetic anhydride: tetrahydrofuran=1:9, v / v) and 0.5 mL of CAP B (n-methylimidazole: pyridine: acetonitrile= 15:10:75, v / v / v) were added to the washed CPG, and the reaction was stirred at room temperature for 1 hour. The mixture was filtered and centrifuged, and washed with CAN three times. The pure product of intermediate 5 (210 mg) was obtained by lyophilization.

[0084] Step 5, weigh 30 mg of intermediate 5, and then use KA H-8 solid phase synthesizer to synthesize intermediate 6 (puromycin linker). The solid phase synthesizer synthesizes the puromycin linker through four steps (Detritylation, Coupling, Capping and Oxidation). After the solid phase synthesis reaction is completed, an equal volume of ammonia water is added to each batch of synthesized CPG, and then incubated at 55°C for 8 hours; then centrifuge and collect the supernatant, and wash the CPG with sterile water; freeze-dry the obtained supernatant to obtain the crude product of the puromycin linker. The obtained puromycin linker is purified under the following conditions: RP-IP-HPLC (WATERS 489, 3767) (column: XBridge BET C18 2.5μm. RP-IP-HPLC liquid phase method: mobile phase A: 2% HFIP+0.1%TEA+50 mL MeOH (DD water), mobile phase B: MeOH). Finally, the solid pure product of puromycin linker (purity: 98.691%) was obtained by freeze drying.

[0085] The puromycin linker is then phosphorylated to introduce a phosphate group at the end (5' end) of the puromycin linker.

[0086] Table 5

[0087]

[0088] Prepare the phosphorylation system as shown in Table 5. The puromycin linker is prepared into a solution with nuclease-free water. The role of ATP is to provide phosphate. Mix the components according to the above phosphorylation system, incubate in a 37°C water bath for 2 hours, and then incubate at 75°C for 10 minutes to inactivate the T4 PNK enzyme (T4 polynucleotide kinase). In actual situations, the volume of each component of the phosphorylation system can be adjusted proportionally according to the amount of the purotoxin linker. After the incubation is completed, cool on ice to obtain the phosphorylated purotoxin linker.

[0089] Finally, the phosphorylated purotoxin linker is connected to the mRNA library by the splint method. The puromycin molecule is connected to each mRNA through the RNA linker sequence. The specific operation of the splint method can be referred to the literature [Keefe, ADProtein selection using mRNA display. Curr. Protoc. Mol. Biol. 2001, 24.5.1-24.5.34], [Pamela A. Barendt, et al. Streamlined protocol for mRNA display.ACS Combinatorial Science. 2013, 15, 77-81]. The 5' end of the splint is a sequence complementary to the purotoxin (≥10 bp), and the 3' end is a sequence complementary to the mRNA library (≥10 bp).

[0090] Splint sequence (SEQ ID NO:6):

[0091] 5'TTT TTT TTT TT CTA GCT ACC TA3'

[0092] Table 6

[0093]

[0094] The ligation system of puromycin linker and mRNA is shown in Table 6. The phosphorylated puromycin linker, mRNA library, splint sequence, and RNase inhibitor were mixed, first heated at 95°C for 2 minutes, then T4 ligase 10×reaction buffer was added, vortexed and placed on ice for 10 minutes. After that, it was returned to room temperature, T4 DNA ligase was added, and the reaction was allowed to proceed at 16°C overnight to obtain mRNA-purine toxin. The volume of each component of the ligation system can be adjusted proportionally as needed. For example, the total volume of the ligation system can be set to 100 μL.

[0095] Example 5: Purification of mRNA-puromycin conjugate

[0096] The purification of mRNA-purine toxin conjugates was performed as described above. Before purification, anhydrous ethanol was added to the RNA Cleanup Wash Buffer and centrifuged at room temperature (16,000 × g).

[0097] Example 6: In vitro translation of mRNA-puromycin conjugate to generate linear mRNA-puromycin-peptide

[0098] The in vitro translation of mRNA-puromycin conjugate was performed using the PURExpress kit. The in vitro translation was performed by ribosome translation to generate the corresponding peptide. The specific operation of in vitro translation was performed according to the kit instructions.

[0099] Table 7

[0100]

[0101] According to the instructions of the PURExpress kit, the operating system shown in Table 7 was prepared. Among them, solution A and solution B of the PURExpress kit were melted on ice before use and can be divided into small tubes to avoid repeated freezing and thawing. The final template concentration is between 1 mg / L and 40 mg / L for better results. The reaction system can be expanded as needed. Mix the operating system and start the reaction. The reaction system was incubated in a 37℃ water bath for 2~4 hours. After the incubation time, the test tube was placed on ice to terminate the reaction. 2.5 µL of the obtained reaction solution (translated protein) was taken out for silver staining experiment verification, and the remaining reaction solution was used in the next step or frozen in a -20℃ refrigerator for later use.

[0102] Example 7: Purification of linear mRNA-puromycin-polypeptide and cyclization of the linear polypeptide

[0103] 7.1 Purification of mRNA-Puromycin-Peptide

[0104] Reagents:

[0105] 1 M TCEP (Tris(2-carboxyethyl)phosphine, CAS: 5961-85-3), which is a thiol reducing agent and is stored at -20°C;

[0106] SSC solution: 0.15 M sodium chloride, 0.015 M sodium citrate dihydrate, adjust pH to 7.0, store at 4°C;

[0107] 3.5× SSC solution: 0.525 M sodium chloride, 0.0525 M sodium citrate dihydrate, adjust pH to 7.0, store at 4°C;

[0108] Oligo(dT) binding buffer: 3.5×SSC, 5 mM EDTA, 0.2% SDS, add TCEP to a final concentration of 0.5 mM before use, and store at 4°C.

[0109] Specific operation: Use Oligo(dT)-magnetic beads for purification. Use DEPC water to make up the volume of the mRNA-puromycin-peptide sample to be purified to 100 μL for use. Take out the magnetic beads in advance and restore them to room temperature. After mixing evenly, take out 50 μL, wash them twice with 100 μL of Oligo(dT) binding buffer, and then resuspend the magnetic beads with 100 μL of Oligo(dT) binding buffer. Add 100 μL of the sample to be purified to the magnetic beads and stir gently for 5 minutes to allow them to bind. After adsorption on the magnetic stand, remove the supernatant and take a sample for silver staining. The magnetic beads are used for subsequent circularization.

[0110] 7.2 Cyclization of linear peptides to prepare mRNA-cyclic peptide compounds

[0111] Reagents:

[0112] Cyclization buffer: 20 mM Tris-HCl (pH 7.8), 0.66 M NaCl, 3 mM α,α′-dibromo-m-xylene (dissolved in acetonitrile, Sigma-Aldrich), 33% acetonitrile (v / v), 0.5 mM TCEP;

[0113] Oligo(dT) wash buffer (0.5×SSC): 0.075 M sodium chloride, 0.0075 M sodium citrate, adjust pH to 7.0, and store at 4°C.

[0114] Experimental steps:

[0115] Add 100 μL of cyclization buffer to the magnetic beads containing mRNA-puromycin-peptide, mix well, and incubate at room temperature (25°C) and 100 rpm for 30 minutes to cyclize the linear peptide. After the cyclization operation is completed, adsorb the supernatant on the magnetic stand and discard it. Wash the Oligo(dT)-magnetic beads / mRNA-puromycin-peptide twice with 50 μL of Oligo(dT) wash buffer. In the first wash, 5 mM of mercaptoethanol was added to the buffer to inactivate the cyclization agent; 0.5 mM of TCEP was added as a reducing agent during the second wash. Adsorb the supernatant on the magnetic stand and discard it. Elute once with 50 μL of DEPC water (sample for silver staining detection); then elute 7 times with 100 μL of DEPC water, collect the eluate into a new 1 mL centrifuge tube, concentrate it with a 3KDa ultrafiltration tube, and store it at -80°C for later use.

[0116] Example 8: Reverse transcription to form cDNA / mRNA-cyclic peptide complex

[0117] Using SuperScript for RT-PCR ® III First-Strand Synthesis System Kit is used for reverse transcription to synthesize cDNA / mRNA-cyclic peptide complex. Since mRNA-cyclic peptide compounds have unstable characteristics, the synthesis of cDNA / mRNA-cyclic peptide complex by reverse transcription can not only protect the mRNA-cyclic peptide compounds, but also facilitate subsequent sequencing. RNA / primer mixed system and cDNA synthesis system are configured separately. The sequence of reverse transcription primer is the same as that of splint.

[0118] Table 8

[0119]

[0120] Table 9

[0121]

[0122] Mix the components of the RNA / primer mixture according to Table 8, incubate at 65°C for 5 minutes, and then place on ice for more than 1 minute. Mix the components of the cDNA synthesis system according to Table 9. Mix 10 μL of the cDNA synthesis system and 10 μL of the RNA / primer mixture, and incubate at 50°C for 50 minutes. After the incubation, place on ice to cool down for later use. The volume of the reverse transcription system can be adjusted proportionally according to the actual situation.

[0123] Example 9: Purification of cDNA / mRNA-cyclic peptide complexes using Ni-NTA magnetic beads

[0124] Reagents:

[0125] Ni-NTA magnetic beads can bind to peptides containing six His tags and be used to purify them;

[0126] Denaturing Ni-NTA binding buffer: 100 mM NaH2PO4, 10 mM Tris-HCl, 6 M guanidine hydrochloride, 0.2% Triton X-100, pH adjusted to 8.0, add 5 mM mercaptoethanol before use, store at 4°C;

[0127] Ni-NTA wash buffer: 100 mM NaH2PO4, 300 mM NaCl, 0.2% Triton X-100, pH adjusted to 8.0, add 5 mM mercaptoethanol before use, store at 4°C;

[0128] Ni-NTA elution buffer: 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 0.2% Triton X-100, pH adjusted to 8.0, add 5 mM mercaptoethanol before use, store at 4°C;

[0129] Replacement buffer: 0.1% Triton X-100 aqueous solution, precooled at 4°C;

[0130] Selection buffer: 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 4 mM MgCl2, 0.25% Triton X-100, stored at 4°C.

[0131] Ni-NTA agarose pretreatment: Take 50 μL of Ni-NTA magnetic beads, wash twice with 50 μL of enzyme-free water, and then wash twice with 50 μL of Denaturing Ni-NTA binding buffer for later use.

[0132] Specific steps for purification of cDNA / mRNA-cyclic peptide complex:

[0133] Step 1: Transfer the cDNA / mRNA-cyclic peptide complex obtained by reverse transcription to the treated Ni-NTA magnetic beads. In order to transfer the cDNA / mRNA-cyclic peptide complex as much as possible, take 100 μL of Denaturing Ni-NTA binding buffer to rinse the centrifuge tube containing the cDNA / mRNA-cyclic peptide complex and transfer the rinse solution to the magnetic beads.

[0134] Step 2: Cover the lid and incubate at 4°C with shaking for 1 hour. After incubation, adsorb on the magnetic stand and discard the supernatant. Add 50 μL of Ni-NTA wash buffer to the magnetic beads and wash once.

[0135] Step 3: Add 50 μL of Ni-NTA elution buffer to the chromatography column. Incubate the Ni-NTA elution buffer and agarose for 5 minutes before elution to allow for full contact. Then collect the eluted Ni-NTA elution buffer (take a sample for silver staining identification), then elute 5 times with 100 μL of Ni-NTA elution buffer, and pool the eluted Ni-NTA elution buffer.

[0136] Step 4: Combine the Ni-NTA elution buffer obtained in the previous step and concentrate by ultrafiltration to replace the buffer (0.1% Triton X-100 aqueous solution, precooled at 4°C).

[0137] Example 10: Cyclic peptide screening of cDNA / mRNA-cyclic peptide complex

[0138] Reagents:

[0139] GST beads wash buffer: 125 mM Tris-HCl (pH 8.0), 150 mM NaCl, stored at 4°C;

[0140] GSH elution buffer: 250 mM Tris-HCl (pH 9.0), 500 mM NaCl, 100 mM reduced glutathione, 1% Triton X-100, prepared and used immediately, filtered and sterilized;

[0141] Selection buffer: 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 4 mM MgCl2, 0.25% Triton X-100, stored at 4°C;

[0142] 10 g / L BSA protein solution, stored at -20°C;

[0143] Dialysis buffer: 0.1% Triton X-100 aqueous solution, precooled at 4°C;

[0144] Purified GST-Nectin 4 protein and GST protein (negative control).

[0145] Experimental operation

[0146] 10.1 GST beads pretreatment

[0147] Step 1: Take two 1.5 mL centrifuge tubes, add 50 μL of GST magnetic beads to each tube, then add 50 μL of GST beads wash buffer to each tube, gently blow and mix, then place the centrifuge tubes on the magnetic rack, remove the supernatant, and repeat three times (be careful not to suck up the beads).

[0148] Step 2: Add 1 mL of GST beads wash buffer mixed with 10 μM GST or 10 μM GST-Nectin-4 target protein to the GST beads centrifuge tube washed above, mix well, and rotate and incubate at 4°C for 1 hour to allow the GST beads to fully bind to the GST or GST-Nectin-4 target protein; after incubation, place the centrifuge tube on a magnetic rack, remove the supernatant, wash once with 50 μL of GST beads wash buffer, wash once with 50 μL of Selection buffer, and place on ice for later use. The centrifuge tube containing GST is used as a negative control.

[0149] 10.2 Cyclic Peptide Screening

[0150] Step 1: Redissolve the cDNA / mRNA-cyclic peptide complex obtained in the previous step, add it to the pre-treated GST magnetic beads, then add BSA with a final concentration of 0.1 g / L (BSA stabilizes the peptide), and then rotate and incubate the centrifuge tube at 4°C for 1 hour. The purpose of this step is to remove the cyclic peptide that has affinity with the GST protein.

[0151] Step 2: After the incubation, place the above centrifuge tube on a magnetic stand, use a pipette to transfer the supernatant to the magnetic beads treated with the GST-nectin-4 target protein, rotate the centrifuge tube at 4°C for 1 hour, then transfer the supernatant to a new centrifuge tube by magnetic stand adsorption and store at -20°C for later use; then wash the magnetic beads with 50 μL of Selection buffer (wash once), and transfer the supernatant to a new centrifuge tube by magnetic stand adsorption for later use. The purpose of this step is to allow the GST-Nectin 4 target protein to fully bind to the affinity cyclic peptide.

[0152] Step 3: Use GSH elution buffer to elute the proteins bound to the magnetic beads. Ensure that the contact time between GSH elution buffer and beads is more than 5 minutes each time. First, elute once with a volume of 50 μL (sample for silver staining identification), then elute 5 times with a volume of 50 μL, and absorb and collect the supernatant using the magnetic bead method; then concentrate and replace the buffer with an ultrafiltration tube. The purpose of this step is to elute the cyclic peptide with affinity to the GST-Nectin 4 target protein in preparation for the next sequencing.

[0153] The supernatant of each step of the reaction solution was taken for silver staining to determine the amount of protein. The detailed operation steps of silver staining refer to the instructions of the kit.

[0154] Example 11: PCR amplification

[0155] Primer pairs for PCR amplification

[0156] Forward primer (SEQ ID NO:7):

[0157] 5'TAATACGACTCACTATAGGGTTAACTTTAGTAAGGAGG3'

[0158] Reverse Primer (SEQ ID NO:8):

[0159] 5'CTAGCTACCTATAGCCGGTGGTGATG3'

[0160] Different volumes (10, 20, 30 μL, etc.) of the buffer-replaced screening eluate were used for PCR reaction. The total volume of the PCR reaction was 50 μL.

[0161] Table 10

[0162]

[0163] Table 11

[0164]

[0165] The PCR amplification system was configured according to Table 10. The dosage gradient of different DNA templates and the annealing temperature were optimized to find the best PCR amplification parameters. The PCR amplification results were detected by nucleic acid gel, and the best PCR operating conditions were determined based on the brightness of the DNA bands. The best operating conditions are shown in Table 11.

[0166] Example 12: Cyclic Peptide Sequencing

[0167] Amplification, enrichment and sequencing: The cDNA of the bound peptide is amplified by PCR and then used as a template for a DNA library. Finally, the structure of the peptide is determined by sequencing the cDNA.

[0168] 12.1 Cloning

[0169] Using TOPO ® The DNA product obtained above was cloned using TA Cloning® Kit. Specific operations were performed according to the instructions.

[0170] Table 12

[0171]

[0172] After mixing according to the cloning system in Table 12, the mixture was reacted at room temperature for 5 minutes, and the obtained cloning products were used for subsequent transformation.

[0173] 12.2 Conversion

[0174] 100 µL of freshly prepared or -20°C competent DH5α cells were placed on ice and gently suspended after complete thawing. 6 µL of cloned product was added, gently mixed, and placed on ice for 25 minutes. Heat shock was performed in a 42°C water bath for 45 seconds and placed on ice for 5 minutes. Then 800 µL of LB culture medium was added and cultured at 37°C and 220 rpm for 60 minutes. The bacteria were then spread on a plate containing antibiotics (Kana + / Amp + ) on LB agar plates, place the plates upright at 37°C for 1 hour, and after the inoculated liquid is absorbed into the agar, turn the plates upside down and culture overnight.

[0175] 12.3 Sequencing

[0176] Pick a single colony grown overnight and inoculate it into 1 mL of culture medium containing antibiotics (Kana + / Amp + ) in LB liquid medium, at 37°C, 220 rpm for 4 hours, and then sent for sequencing.

[0177] Table 13

[0178]

[0179]

[0180] The results of amino acid sequencing are shown in Table 13. In the above sequence, the first C (cysteine) and the last C are the sites of polypeptide ring formation. These two sites are common sequences of all polypeptide sequences, and the rest are specific sequences. The first cysteine ​​and the last cysteine ​​form a ring with the help of α,α′-dibromo-m-xylene (α,α`-dibromo-m-xylene). The principle is that α,α′-dibromo-m-xylene and the -SH group of cysteine ​​can combine to form a ring.

[0181] Example 13: Verification of binding ability of polypeptide sequences

[0182] The above-mentioned cyclic peptide and Nectin-4 protein were subjected to computer-aided drug design (CADD) simulation calculations, and three peptides (Cand 48 / Cand 13 / Cand 3) with strong binding affinity to Nectin-4 protein in the simulation calculation were selected and the corresponding cyclic peptide sequences were obtained by chemical synthesis. The binding affinity of the chemically synthesized macrocyclic peptide to the target protein Nectin4 was verified by surface plasmon resonance (SPR) experiment. For specific operations, please refer to [Xiao Liu, Tai An, Dongdong Li et al., Structure of the heterophilic interaction between the nectin-like 4 and nectin-like 1 molecules. PNAS 2018, 116(6), 2068-2077.].

[0183] Nectin4 protein (SEQ ID NO:58)

[0184] MPLSLGAEMWGPEAWLLLLLLLASFTGRCPAGELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAE GSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSAS

[0185] Table 14

[0186]

[0187] Combined with Table 14 and Figures 2~4 , it can be seen that the macrocyclic peptide cand48 (K d =7.7×10 -7 M) and cand3 (K d =5.6×10 -7M) has obvious binding to the target protein Nectin4, while the macrocyclic peptide cand13 has a very weak binding to the target protein Nectin4.

Claims

1. An isolated polypeptide or a pharmaceutically acceptable salt thereof, characterized in that: The polypeptide is a cyclic peptide with cysteine ​​residues at both ends; the polypeptide has an amino acid sequence as shown in SEQ ID NO:

56.

2. The isolated polypeptide or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The polypeptide is a targeting peptide with a ring structure and targets the Nectin-4 protein.

3. The isolated polypeptide or pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: The cysteines at both ends of the polypeptide serve as ring-forming sites, which form a ring structure through α,α'-dibromo-m-xylene.

4. The isolated polypeptide or pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: The polypeptide is a single-ring peptide.

5. Use of the isolated polypeptide or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a detection reagent or a kit for detecting Nectin-4 protein.

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