A method for constructing a phage display library of polypeptides containing thiazoline groups

Abstract

This invention, entitled "A Method for Constructing a Phage-Displayed Cyclic Peptide Library Containing a Thiazoline Group," belongs to the field of biomedical technology. The key technical points are a method for constructing a phage-displayed cyclic peptide library containing a thiazoline group, comprising the following steps: constructing a DNA library, wherein the DNA library at least encodes an enzyme restriction site and a peptide library, and the backbone sequence of the peptide library contains the amino acid sequence CX9C, where C represents cysteine ​​and X represents a random amino acid; infecting host cells displaying the DNA library with helper phages to obtain a progeny phage library displaying the peptides; digesting the progeny phage library with a proteolytic enzyme; and cyclizing the digested progeny phage library by co-incubating it with 6-chloromethyl-2-cyanopyridine to obtain a phage-displayed cyclic peptide library containing a thiazoline group. This method can be used to screen high-affinity peptide molecules targeting various proteins and has high drug development potential and medical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically the field of polypeptide cyclization technology, and more specifically to a method for constructing a phage-displayed polypeptide library containing a thiazoline group. Background Technology

[0002] For understanding the technical content of this invention: Many heterocyclic compounds exist in natural products; for example, thiazoline groups are commonly found in metabolites produced by microorganisms such as streptococci and actinomycetes. Products containing thiazoline groups are widely available and possess potential pharmacological activities, such as antitumor, antibacterial, and anti-inflammatory effects. Thiazoline, as an important pharmacologically active group, plays a crucial role in the efficacy of many drugs. Bleomycin, lagazole, and vitamin B1 all contain similar azole structures, demonstrating the significant role of heterocyclic compounds in regulating physiological processes. Due to their unique structural characteristics, peptides containing thiazoline heterocycles have become highly attractive lead compounds in drug development, and bioactive peptide derivatives containing thiazoline groups have been extensively studied (Dahiya, R. “Natural Bioactive Thiazole-Based Peptides from Marine Resources: Structuraland Pharmacological Aspects.” Journal of Marine Drugs vol. 6, 18 (2020):329.). In ribosomal peptide natural products, the thiazoline ring is formed by the dehydration of the thiazole group on the cysteine ​​side chain of the ribosomally synthesized peptide chain and the carbonyl group of the preceding amino acid (Just-Baringo, X. “Chiral thiazoline and thiazole building blocks for the synthesis of peptide-derived natural products.” Journal of Current Topics In Medicinal Chemistry vol. 14,10(2014):1244-1256.). The nitrogen atom on the heterocyclic structure is a strong hydrogen bond acceptor, making it an ideal carrier for intramolecular hydrogen bonding and potentially directly involved in the binding process of biological targets. Integrating flat thiazole structural units into peptides and their polypeptide backbones allows for direct regulation of their physical properties, such as conformational kinetics; it also enhances metabolic stability and resistance to protease degradation (C. Cai. “Macrocyclization of bioactive peptides with internal thiazolemotifs via palladium-catalyzed CH olefination.” Journal of Chemical Communications vol. 58, 31 (2022): 4861-4864.). In recent years, researchers have developed various methods for the synthesis and functionalization of peptides containing thiazoline (thiazole) groups, such as solid-phase synthesis using orthogonal protecting groups, cyclization reactions of cysteine ​​with cyanopyridine reagents, one-pot multi-component condensation strategies, heterologous expression and engineering of heterocyclases based on RiPP gene clusters, and chemical-enzymatic combination strategies. These methods not only simplify the synthetic steps of complex cyclic peptides but also yield candidate drug molecules with good cell permeability and metabolic stability.

[0003] Phage display technology is a method for displaying polypeptide sequences on the surface of phage coat proteins. Developed over thirty years, it has been widely applied in biotechnology fields such as antibodies and polypeptide display. The core of phage display lies in constructing and analyzing the polypeptide library. A polypeptide library is a diverse population of phage clones, each containing randomly inserted exogenous DNA fragments, thus displaying different polypeptides on the phage surface. This technology can be used to analyze massive amounts of samples and perform affinity screening for targets. This method boasts high transformation efficiency and rapid screening speed; typically, 3-5 rounds of bioscreening are sufficient to obtain candidate polypeptide molecules with the highest binding potential, thereby discovering high-affinity polypeptide ligands. Therefore, it has become a powerful tool for discovering novel polypeptide or protein drugs using phage display technology. However, current phage display techniques are mainly limited to displaying polypeptide sequences composed of 20 natural amino acids. Among these, cyclic peptides have become a research hotspot due to their superior conformational stability and targeting affinity compared to linear peptides. To enrich the diversity of peptide types and structures displayed by bacteriophages, researchers have conducted a series of studies. For example, George P. Smith constructed cyclic peptides by forming disulfide bonds between the thiol groups of cysteine ​​residues on the peptide chain (Smith, George P., and Valery A. Petrenko. “Phage Display.” Chemicalreviews vol. 97,2 (1997): 391-410), achieving a transformation from linear peptides to cyclic peptides while constraining the peptide conformation. Another example is the covalent linkage of two cysteine ​​residues using the reactivity of nucleophilic thiols with small organic molecule linkers. Studies have shown that constructing monocyclic or bicyclic peptides by linking cysteine ​​residues with small organic molecules is possible. These methods, which modify the peptide chain, are of great significance for further expanding the diversity of peptide types displayed by bacteriophages.

[0004] Relevant patent documents retrieved: The country of origin for this publication is China, publication number CN118324860A, publication date July 12, 2024, entitled "A Method for Constructing a Phage Display Cyclic Peptide Library Based on a Natural Cyclic Peptide Backbone," which discloses a method for constructing a phage display cyclic peptide library based on a natural cyclic peptide backbone, wherein the cyclic peptide contains X. p CC-X n -CX m -CX q The amino acid sequence shown is provided, where p = 0-10, q = 0-10, n = 4 and m = 4-16, and X is any one of 20 natural amino acids. The cyclic peptide library contains cyclic peptides having two disulfide bonds. This invention utilizes a strategy of fixing the position of cysteine ​​residues and embedding random amino acids based on a conserved cone snail toxicone backbone to generate phages that can display cyclic peptides, thereby targeting more target proteins and screening for cyclic peptide ligands with high affinity and high selectivity.

[0005] The publication, titled "A Phage Cyclic Peptide Library with Biphenyl-Dihydrothiazole Linkage and Screening Method," published in China (CN121518459A, February 13, 2026), discloses a phage cyclic peptide library with biphenyl-dihydrothiazole linkage and a screening method. This method involves peptide cyclization and a strategy for discovering high-affinity cyclic peptide ligands for proteins. First, a novel ethoxylated molecule is synthesized and reacted with a linear peptide containing cysteine ​​residues at its N-terminus and in the chain to synthesize a cyclic peptide. Based on the rigid biphenyl structure of this molecule and the high biocompatibility of the reaction, a phage cyclic peptide library with biphenyl-dihydrothiazole linkage is obtained through post-translational modification. High-affinity cyclic peptide ligands are then screened for target proteins, providing more possibilities for the discovery and development of cyclic peptide compounds in drug development.

[0006] Relevant non-patent literature retrieved: The paper, titled "Study on Peptide Ligand Screening Based on Phage Display Cyclic Peptide Libraries," by Zhang Shilong, Xiamen University, and published on April 1, 2022, utilizes the rapid, specific, and efficient reaction of 1,2-aminothiol with 2-((alkylthio)(aryl)methylene)malonium under biocompatible conditions to design and synthesize two molecules, 2Cl-Ac and Rotor. The 2Cl-Ac molecule contains three active functional groups: 2-((alkylthio)(aryl)methylene)malonium and two chlorine atoms. The Rotor molecule has 2-((alkylthio)(aryl)methylene)malonium functional groups and chlorine atoms at both ends, with a molecular rotor-type fluorescent group in the middle. Introducing these two molecules into the post-translational chemical modification of phage display peptide libraries resulted in the construction of different types of phage display cyclic peptide libraries, enriching the construction methods of phage display cyclic peptide libraries, increasing library diversity, and endowing phage libraries with more functions.

[0007] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: (1) The inability to introduce thiazoline heterocyclic structures into phage surface peptides: Existing methods for constructing phage-display cyclic peptide libraries (such as disulfide cyclization in CN118324860A and biphenyl-dihydrothiazol linkage in CN121518459A) do not introduce thiazoline groups into the peptide backbone. Thiazoline rings have unique physicochemical properties (such as strong hydrogen bond acceptors and conformational rigidity), which can significantly improve the metabolic stability and target affinity of peptides. However, existing technologies lack effective means to introduce this group at the phage display level, which limits drug development. Furthermore, the cyclization in CN118324860A depends on the random pairing or complete substitution of cysteine ​​residues within the peptide, lacking precise exposure and site-directed cyclization of N-terminal cysteine ​​residues.

[0008] (2) Existing chemical modification methods have poor biocompatibility and are prone to damage phage activity: Existing technologies such as the relevant non-patent literature “Study on peptide ligand screening based on phage display cyclic peptide library” and patent literature CN121518459A both rely on chemical cyclization. The reaction conditions used (such as high temperature, organic solvents, metal catalysts, etc.) will cause significant loss of phage activity, resulting in a reduction in library capacity.

[0009] In summary, existing technologies have not solved the defects such as lack of structure, poor biocompatibility, difficulty in site-directed cyclization, and limited drug-likeness. Summary of the Invention

[0010] The purpose of this invention is to provide: This invention discloses a method for constructing a phage-displayed polypeptide library containing a thiazoline group, and related technologies, aiming to address technical problems in existing technologies such as structural deficiencies, poor biocompatibility, difficulties in site-directed cyclization, and limited drug-likeness. This invention introduces thiazoline into phage-displayed polypeptide chains, constructs libraries, and performs affinity screening of target proteins, which is of great significance for discovering active polypeptide lead compounds and guiding the development of drugs for treating related diseases.

[0011] Terminology Explanation: To provide clarity, the relevant terms, core concepts, and technical solutions will be systematically defined and explained below.

[0012] Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0013] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, unless specifically stated otherwise, the singular is used to include the plural, and vice versa. It should also be noted that unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0014] Definitions of standard terms can be found in the references “Modern Molecular Biology (5th Edition), Higher Education Press, authors: Zhu Yuxian, Li Yi, Zheng Xiaofeng and Guo Hongwei, 2019-06-19” and “Genetic Engineering, Higher Education Press, 2013-08-01”.

[0015] Unless otherwise specified, conventional methods within the scope of the art, such as primer design, PCR, electroporation, phage infection, centrifugation, mixing, phage titer determination, HPLC, mass spectrometry, etc., shall be used.

[0016] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0017] The term "phage display" as used in this article refers to the technique of cloning the coding gene of a polypeptide or protein into an appropriate position in the structural gene of a phage coat protein, so that the exogenous polypeptide or protein is fused with the coat protein and expressed and displayed on the surface of the phage.

[0018] The term "GusA enzyme" as used in this article refers to a β-glucuronidase that specifically recognizes the GGVTPS sequence and cleaves peptide bonds at its C-terminus.

[0019] The term "6-chloromethyl-2-cyanopyridine" as used in this article refers to: [the structure is C7H5C]. l The organic small molecules of N2 are available from companies such as Shanghai Yuanye Biotechnology Co., Ltd., product number: S93377.

[0020] The term "thiazoline" as used in this article refers to a five-membered heterocyclic structure containing sulfur and nitrogen, formed by cyclization and dehydration of the thiol and amino groups of cysteine ​​with a cyanopyridine reagent.

[0021] The term "TCEP solution" as used in this article refers to tris(2-chloroethyl) phosphate, also known as trichloroethyl phosphate, which is an organophosphate compound containing chlorine and phosphorus. It appears as a colorless or slightly yellow transparent oily liquid with a density of 1.420-1.431 g / cm³. 3 It has a boiling point of 194℃ (1.33kPa), is soluble in water and a variety of organic solvents, and exhibits hydrolytic stability.

[0022] The term "NNK coding" used in this article refers to: N representing the four bases A / T / G / C, and K representing the two bases G / T, for a total of 32 codons that encode 20 natural amino acids.

[0023] In a first aspect, the present invention provides: a method for constructing a phage-displayed cyclic peptide library containing a thiazoline group, comprising the following steps: S1. Construct a DNA library, wherein the DNA library encodes at least an enzyme restriction site and a polypeptide library, and the backbone sequence of the polypeptide library contains the amino acid sequence CX9C, where C represents cysteine ​​and X represents a random amino acid. S2. Use helper phages to infect host cells displaying the DNA library to obtain a progeny phage library displaying peptides; S3. The progeny phage library is digested with a proteolytic enzyme, wherein the proteolytic enzyme specifically recognizes the enzyme recognition site. S4. The progeny phage library after enzyme digestion is co-incubated with 6-chloromethyl-2-cyanopyridine to cyclize, thereby obtaining the phage display library containing thiazoline group cyclic peptides.

[0024] The backbone sequence of the polypeptide library, from the N-terminus to the C-terminus, contains the amino acid sequence CX9C. In CX9C, X is a random amino acid selected from 20 natural amino acids. The library capacity is approximately 5.16 × 10⁻⁶. 7 .

[0025] Furthermore, the peptide library sequence can be: from the N-terminus to the C-terminus, X m -C-X9-CX n Where X can be C, and m and n can be independently selected from 0 or any integer; when one of X in X9 is cysteine ​​C, the backbone sequence is as follows: X m -CX z -CX r -CX n z and r are integers from 0 to 8 and z + r = 8; understandably, X can also be represented by the number of C, which can be 2, 3, 4, 5, 6, 7, 8 or 9.

[0026] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred embodiment is as described in the first aspect, wherein the enzyme cleavage site is a GusA enzyme recognition site, and the proteolytic enzyme is a GusA enzyme.

[0027] Further, the amino acid sequence of the GusA enzyme is shown in SEQ ID NO.1, SEQ ID NO.1: MAKVKTIPGLNNLWEQTLGNSDICVAVLDGLVDSSHPCFVGADLSQIPTLVNPNPIKEGLMYQHGTHVASVIFGQHGSPVEGIAPQCKGLIIPIFCDKERKLSQLDLSRAIEQAVQAGAHIINISGGQLTDYGEADDWLQQAVALCQQHNVLIVAAAGNNGCDCLHVPAALPAALAVGAMNDNGKPIDYSNWGSAYQTQGILAPGEKILGAKTGGGIIELSGTSFATPIVTGVAALLLSLQKQWGEQPNPQKVRAALLKSATPCDFPELEDKARCLVGKLNILGAIEKFKGG, and the GusA enzyme cleavage recognition site is GGVTPS (SEQ ID NO.2). This technical solution not only solves the technical problem of how to specifically expose N-terminal cysteine, but also provides clear enzymatic tools and recognition sequences, ensuring the high efficiency and specificity of enzyme digestion.

[0028] The second preferred embodiment is as described in the first aspect, wherein the X in CX9C is encoded by the NNK degenerate codon.

[0029] Furthermore, the DNA library contains at least: 5'-GGTGGAGTCACACCAAGTTGTNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKGCA-3' (SEQ ID NO.3). This technical solution, while solving the problem of cyclic peptide library construction, further defines the specific library sequence, which is beneficial to improving library diversity.

[0030] A third preferred embodiment: As described in the first aspect, the step S4 of cyclizing the enzymatically digested progeny phage library with 6-chloromethyl-2-cyanopyridine further includes: The digested progeny phage library was first mixed with TCEP solution and incubated, followed by co-incubation with 6-chloromethyl-2-cyanopyridine to carry out the cyclization reaction. TCEP is a mild and efficient thiol reducing agent that can reduce oxidized disulfide bonds that may exist in linear peptide libraries to free thiol groups (-SH), thereby improving the activity state of the thiol groups of the two cysteine ​​residues in the subsequent cyclization reaction and increasing the reaction efficiency.

[0031] The fourth preferred embodiment: As described in the first aspect, the co-incubation conditions in step S4 are as follows: a final concentration of 6-chloromethyl-2-cyanopyridine of 0.3-0.8 mM, a reaction temperature of 35℃-39℃, and a reaction time of 1-3 hours. This technical solution, based on solving the problem of introducing a thiazoline ring through chemical modification, further optimizes the reaction concentration and time, ensuring high conversion rates while maximizing the protection of phage activity.

[0032] Furthermore, co-incubation can be carried out in a buffer solution with a pH of 7.2–7.6.

[0033] Preferably, the co-incubation conditions in step S4 are as follows: in a buffer solution at pH 7.4, the final concentration of 6-chloromethyl-2-cyanopyridine is 0.5 mM, the reaction temperature is 37°C, and the reaction time is 2 hours.

[0034] In some embodiments, the DNA library is a circular plasmid, and the vector for the circular plasmid is pCANTAB 5E.

[0035] In some embodiments, the host cell is a chassis cell, which is selected from at least one of Escherichia coli, yeast, filamentous fungi, tobacco, CHO, etc. The host cell is preferably Escherichia coli TG1.

[0036] In some embodiments, the helper phage is M13KO7.

[0037] In some embodiments, the method for introducing a DNA library into a host cell can be electroporation.

[0038] Secondly, the present invention provides a cyclic peptide library containing a thiazoline group, which is constructed by the above-described construction method.

[0039] The library size of this cyclic peptide library is approximately 5.16 × 10⁻⁶. 7 This ensures that the library has sufficient diversity to cover a large sequence space and increases the probability of screening high-affinity ligands.

[0040] Because of the introduction of thiazoline groups, the cyclic peptides in the cyclic peptide library remain stable in reducing environments (such as intracellular and blood) and are not easily destroyed by reducing agents such as glutathione.

[0041] Thirdly, the present invention provides the application of a cyclic peptide library containing a thiazoline group in screening peptides targeting target proteins.

[0042] In some embodiments, the target protein includes, but is not limited to, at least one of the following: ion channel proteins, G protein-coupled receptors (GPCRs), transcription factors, kinases, phosphatases, etc.

[0043] For example, the target protein is the Keap1 protein, which can screen for cyclic peptides with nanomolar affinity (Ki as low as 47.4 nM).

[0044] Examples 1-5 of this invention at least support the protection scope of "method for constructing a phage display library of cyclic peptides containing thiazoline groups".

[0045] The "method for constructing a phage-displayed cyclic peptide library containing a thiazoline group" is summarized from the foregoing explanation and / or the corresponding experimental steps in Examples 1-5. Therefore, those skilled in the art can reasonably presume that the "method for constructing a phage-displayed cyclic peptide library containing a thiazoline group," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it based on existing technology and conventional technical means and common knowledge, should all fall within the protection scope of the "method for constructing a phage-displayed cyclic peptide library containing a thiazoline group."

[0046] Examples 1-5 of this invention at least support the protection scope of "cyclic peptide libraries containing thiazoline groups".

[0047] The term "cyclic peptide library containing thiazoline groups" is a generalization derived from the foregoing explanation and / or the corresponding cyclic peptide libraries containing thiazoline groups obtained in Examples 1-5. Therefore, those skilled in the art can reasonably presume that the "cyclic peptide library containing thiazoline groups," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of the "cyclic peptide library containing thiazoline groups."

[0048] Example 6 of this invention at least supports the protection scope of "the application of cyclic peptide libraries containing thiazoline groups in screening peptides targeting target proteins".

[0049] The application of "the cyclic peptide library containing thiazoline groups in screening peptides targeting target proteins" is summarized from the foregoing explanation and / or the screening of high-affinity cyclic peptides for the target protein Keap1 in Example 6. Therefore, those skilled in the art can reasonably presume that "the application of the cyclic peptide library containing thiazoline groups in screening peptides targeting target proteins," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of "the application of the cyclic peptide library containing thiazoline groups in screening peptides targeting target proteins."

[0050] The present invention has at least the following beneficial effects: Compared with existing technologies, this invention provides a method for constructing a phage-displayed cyclic peptide library containing a thiazoline group, which has better technical effects, specifically in the following aspects: (1) This invention is the first to combine precise cleavage of proteolytic enzymes with one-step chemical modification of 6-chloromethyl-2-cyanopyridine. By precisely cleaving the proteolytic enzymes to expose phage cysteine ​​residues, and then using the small organic molecule 6-chloromethyl-2-cyanopyridine as a bridging structure for cyclization, a cyclic peptide library containing a thiazoline structure was successfully constructed on the phage surface under mild conditions. Experimental tests showed no significant loss in phage titer before and after modification, and the library capacity reached 5.16 × 10⁻⁶. 7 This significantly enriches the diversity of phage-displayed peptide libraries, which can be used to screen various target proteins to obtain high-affinity peptide molecules.

[0051] In summary, this method enables the introduction of thiazoline groups into the polypeptide backbone and solves the biocompatibility problem that exists when introducing thiazoline groups into polypeptide chains using traditional chemical methods. It achieves modification of the polypeptide backbone, overcomes the limitation of existing technologies that can only modify polypeptide side chains, expands the types of polypeptides that can be displayed by bacteriophages, and is of great significance for the early discovery of polypeptide lead compounds. It also has high drug development potential and medical application value. Attached Figure Description

[0052] Figure 1 This is a schematic diagram illustrating the incubation process of the model peptide with GusA hydrolase in Example 1 of this invention.

[0053] Figure 2 The chromatograms are of the model peptide in Example 1 of this invention before and after treatment with GusA hydrolase, wherein, Figure 2 Figure A is the chromatogram before the reaction. Figure 2 Figure B is the chromatogram after the reaction.

[0054] Figure 3 The above are the mass spectrometry characterization results of the model peptide in Example 1 of this invention before and after treatment with GusA hydrolase, wherein... Figure 3 Figure A shows the mass spectrum before processing. Figure 3 Figure B shows the processed mass spectrum.

[0055] Figure 4 This is a chromatogram of the model peptide in Example 2 of the present invention before and after reaction with 6-chloromethyl-2-cyanopyridine.

[0056] Figure 5 This is a mass spectrometry characterization of the model peptide in Example 2 of the present invention before and after the reaction with 6-chloromethyl-2-cyanopyridine, wherein, Figure 5 Figure A shows the mass spectrum before the reaction. Figure 5 Figure B is the mass spectrum after the reaction.

[0057] Figure 6 This invention relates to the chemical structure of a cyclic peptide containing a thiazoline group formed by modifying the 6-chloromethyl-2-cyanopyridine model peptide in Example 2 of this invention.

[0058] Figure 7 This is a DNA electrophoresis image of the DNA library introduced by the PCR method in Example 3 of the present invention.

[0059] Figure 8 The sticky ends are generated after the linear DNA library in Example 3 of this invention is digested with Sfi I enzyme.

[0060] Figure 9 In Example 3 of this invention, the T4 ligase circularizes the linear DNA library into a circular plasmid.

[0061] Figure 10 This describes the generation and assembly of the phage library of the polypeptides shown in Example 4 of the present invention.

[0062] Figure 11 This shows the colony growth after gradient dilution plating in Example 4 of the present invention.

[0063] Figure 12 This is the phage library containing a thiazoline-containing polypeptide constructed in Example 5 of the present invention.

[0064] Figure 13 This describes the screening process for target proteins in the phage library containing thiazoline-containing polypeptides constructed in Example 5 of this invention.

[0065] Figure 14 The high-enrichment polypeptide sequence obtained by screening the phage cyclic peptide library containing a thiazoline structure constructed in Example 5 of this invention for the target protein Keap1.

[0066] Figure 15 Cyclic peptide 1 in Example 6 of this invention ( Figure 15 Figure A), Cyclic peptide 2 ( Figure 15 Figure B), Cyclic peptide 3 ( Figure 15 (Figure C), Cyclic peptide 4 ( Figure 15 The structure of the middle D diagram.

[0067] Figure 16 Cyclic peptide 1 in Example 6 of this invention ( Figure 16 Figure A), Cyclic peptide 2 ( Figure 16 Figure B), Cyclic peptide 3 ( Figure 16 (Figure C), Cyclic peptide 4 ( Figure 16 The competition curve in Figure D). Detailed Implementation

[0068] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents. Experimental methods in the following embodiments that do not specify specific conditions are generally carried out under conventional conditions or according to the conditions and methods recommended by the raw material or product manufacturer.

[0069] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0070] Data analysis and statistical analysis are performed using professional data processing software.

[0071] The experimental design for displaying a cyclopeptide library containing a thiazoline structure using bacteriophages in this invention includes: (1) obtaining proteolytic enzymes through genome mining, verifying enzyme recognition sites, and determining the substrate generalization and enzyme digestion efficiency of the GusA enzyme; (2) introducing enzyme digestion sites and random amino acids using NNK-encoded PCR, and constructing a DNA library by enzyme digestion and ligation; (3) electroporating the DNA into host cells (chassis cells TG1), and obtaining progeny bacteriophages displaying the peptide library through helper phage infection; verifying the cyclization ability of the model peptide with 6-chloromethyl-2-cyanopyridine and the product yield; and incubating the GusA enzyme after exposing cysteine ​​with small molecules to form a cyclopeptide library containing a thiazoline structure. Specific steps are detailed in the following examples.

[0072] Example 1: Discovery of proteolytic enzymes and analysis of enzyme cleavage sites (1) Discovery of proteolytic enzymes: The specific process is as follows: By comparing a series of gene clusters, analysis revealed a high degree of local similarity between the substrates of the proteolytic enzyme (GusA enzyme, sequence shown in SEQ ID NO.1). This similarity was identified as the enzyme's recognition sequence (restriction site shown in SEQ ID NO.2, SEQ ID NO.2: GGVTPS). Based on this analysis, a model peptide was designed to verify the above hypothesis. Subsequently, experiments were conducted to investigate the enzyme's enzymatic heterogeneity and enzymatic efficiency.

[0073] The final concentration of GusA was fixed at 1 μM, and the final concentration of the model peptide was 200 μM. Both were incubated for different times in Tris buffer (50 mM, pH 7.4). After treatment, the enzyme was characterized by chromatography and mass spectrometry to determine the cleavage heterogeneity of GusA and the effect of terminal amino acids on cleavage efficiency. Taking the model peptide WEGGVTPSC (SEQ ID NO.4) as an example, the principle is as follows: Figure 1 As shown, the chromatographic and mass spectrometric characterization results are as follows: Figure 2 and Figure 3 As shown in the figure. The results indicate that GusA treatment can break the amide bond between serine (S) and cysteine ​​(C), exposing the N-terminal cysteine, and under this concentration, it can be completely hydrolyzed within 2 hours.

[0074] Example 2: Reactivity analysis of model peptides with the small organic molecule 6-chloromethyl-2-cyanopyridine This embodiment investigates the reactivity of the model peptide with the small organic molecule 6-chloromethyl-2-cyanopyridine. The specific process is as follows: (1) The model peptide (SEQ ID NO.5: CGSAKGCGW-NH2) was designed and purified to obtain high purity for subsequent use.

[0075] (2) 500 μM 6-chloromethyl-2-cyanopyridine (purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number: S93377) and 500 μM model peptide were reacted in Tris buffer at 37℃ for 2 hours. The reaction product was separated by HPLC and characterized by mass spectrometry. The HPLC separation conditions were as follows: column: C18 reversed-phase column (4.6×250 mm, 5 μm); column temperature: 40℃; mobile phase A: water, mobile phase B: acetonitrile; flow rate: 1.0 mL / min; detection wavelength: 280 nm; gradient elution program: 0-5 min, B phase maintained at 5%; 5-40 min, B phase linearly increased from 5% to 95%; 40-45 min, B phase maintained at 95%; 45-46 min, B phase decreased to 5%; 46-50 min, B phase maintained at 5% for column equilibration. Mass spectrometry characterization conditions: LC-MS2020 mass spectrometry (ESI) was used for detection.

[0076] Chromatographic results as follows Figure 4 As shown, the mass spectrometry characterization results are as follows: Figure 5 As shown in the figure. The results indicate that 6-chloromethyl-2-cyanopyridine, acting as a bridging structure, can cyclize the model peptide into a cyclic peptide containing a thiazoline structure, the molecular structure of which is shown in the figure. Figure 6 As shown.

[0077] Example 3: A DNA library was constructed and host cells displaying the DNA library were infected using helper phages to obtain a progeny phage library displaying peptides. In this embodiment, a DNA library containing the GGVTPSCX9C sequence was constructed using the pCANTAB 5E-antibody phage as a template. This library was electroporated into chassis cells (TG1), and library diversity was measured. After infection with helper phages, progeny phages were collected to obtain a linear polypeptide library. The specific process is as follows: (1) PCR to obtain linear phage vectors Using the phage particle pCANTB 5E-antibody as a template, PCR was performed using the primers in Table 1, the expansion system in Table 2, and the amplification program in Table 3. Table 1

[0078] Note: "NNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNK" indicates the location of the library.

[0079] Table 2

[0080] Table 3

[0081] Note: Repeat steps 2-4 30 times.

[0082] 5 μL of PCR amplification product was taken for electrophoresis verification, and the results are as follows: Figure 7 As shown. The validated product was mixed with 400 μL QG buffer, passed through a DNA extraction purification column, and then eluted with 30 μL MQ water and quantified.

[0083] (2) Sticky ends were obtained by Sfi I enzyme digestion. The Sfi I enzyme digestion system is shown in Table 4.

[0084] Table 4

[0085] Sfi I enzyme digestion procedure: incubate at 50 ℃ for 12 h, and then perform in a PCR instrument.

[0086] Take 5 μL of the enzyme digestion product and perform electrophoresis to verify whether the enzyme digestion was complete. Use the DNA library before digestion as a control. The results are as follows: Figure 8 As shown. The validated product was mixed with 400 μL QG buffer, passed through a DNA extraction purification column, and the DNA was recovered by elution with 30 μL MQ water and quantified.

[0087] (3) T4 ligase cyclizes linear vectors as shown in Table 5.

[0088] Table 5

[0089] T4 ligase ligation procedure: incubate at 16 ℃ for 12 h, then perform in a PCR instrument.

[0090] 5 μL of the ligation product was subjected to electrophoresis to verify the circularization effect. A DNA library before ligation (i.e., the Sfi I digested substrate) was used as a control. The results are as follows: Figure 9 As shown. The validated product was mixed with 400 μL QG buffer, passed through a DNA extraction purification column, and then eluted with 30 μL MQ water and quantified.

[0091] A small amount of the T4 ligase ligation product was transformed into TG1 competent cells, and single clones were picked for sequencing to verify that the plasmid had been circularized, thus confirming the acquisition of the target DNA library.

[0092] Example 4 In this embodiment, the constructed phage plasmid pCANTAB 5E-GusAsite-CX9C-P3 (a DNA library plasmid containing the GGVTPSCX9C sequence) was electroporated into chassis cells (TG1), and library diversity was subsequently measured. After infection with helper phages, progeny phages were collected to obtain a linear polypeptide library, such as... Figure 10 As shown. The specific implementation process is as follows: (1) Transfection of DNA library plasmids containing GGVTPSCX9C Strawberry strain TG1 was streaked onto solid medium to obtain single colonies. A single colony was picked and inoculated into 5 mL of liquid medium and incubated overnight on a shaker (37℃, 220 rpm) to obtain a saturated concentration. 5 mL of the bacterial suspension was transferred to an Erlenmeyer flask containing 400 mL of 2YT liquid medium and incubated on a shaker (37℃, 220 rpm) until the OD600 value reached 0.3. The suspension was then incubated on ice for 15 min, centrifuged for 15 min at 220 rpm using a pre-chilled centrifuge, and the supernatant was discarded. The bacterial pellet was resuspended in 45 mL of MQ water, centrifuged at 220 rpm for 15 min, and the supernatant was discarded. The bacterial pellet was then resuspended in 10% glycerol, centrifuged at 220 rpm for 15 min, and the supernatant was discarded. Resuspend the bacterial pellet in 10% glycerol, centrifuge at 220 rpm for 15 min, and discard the supernatant. Resuspend the pellet in 3-5 mL of 10% glycerol.

[0093] The recombinant plasmid pCANTAB 5E-GusAsite-CX9C-P3 was added to the prepared competent TG1 chassis cells, gently mixed, and then transferred to an electroporation cuvette. Electroporation was performed using an electroporator with the parameters set (200 Ω, 2.5 kV). Immediately after electroporation, the cells were resuspended in SOC medium and cultured at 37°C with shaking at 220 rpm for 1 hour. The bacterial culture was then evenly spread onto LB-Amp agar plates using a spreading stick and incubated statically at 37°C for 16-18 hours. The cells were collected, and half a volume of 80% glycerol was added. The cells were then stored at -80°C for later use.

[0094] Library capacity assessment was performed on the above peptide library: the constructed peptide library was serially diluted, and the diluted samples were plated on LB-Amp solid medium plates for titer determination. The library diversity was calculated based on the number of colonies on the plates. Total library capacity (CFU) = number of colonies on plate × (1 / dilution factor) × (1000 / plate volume) × total library volume. The dilution factor was 10. -1 10 -2 10 -3 Colony growth at that time Figure 11 As shown, the dilution factor is 10. -3 The colony count was 43, which, after calculation, is 43 × 10⁻⁶. 3 ×12×1000 / 10=5.16×10 7 CFU, library diversity is 5.16 × 10 7 CFU.

[0095] (2) Amplification of the phage library The TG1 cells containing the phage plasmid library preserved in glycerol were seeded into 100 mL of 2×YT liquid medium and the initial OD was adjusted. 600 =0.1, and incubated at 37 ℃ with shaking until OD 600 =0.4-0.6. Add 20 times the number of helper phage M13KO7 as the host cells, incubate at 37°C for 1 hour, centrifuge at 3500 rpm for 10 minutes, and discard the supernatant. Resuspend the pellet in 100 mL of fresh 2×YT liquid medium and incubate at 30°C overnight to obtain a phage culture medium displaying the GGVTPSCX9C peptide library.

[0096] (3) Collecting phage libraries Centrifuge the phage culture at 8000 rpm for 15 minutes, collect the supernatant, and add 1 / 4 volume of 20% PEG8000-NaCl solution (containing 2.5 M NaCl). After incubating on ice for 4-6 hours, centrifuge at 11000 rpm for 25 minutes, discard the supernatant, collect the precipitate, and dissolve it in PBS. Centrifuge again at 8000 rpm for 15 minutes, collect the supernatant, and add 1 / 4 volume of 20% PEG8000-NaCl solution. After incubating on ice for 2 hours, centrifuge at 11000 rpm for 25 minutes, collect the precipitate again, and dissolve it in PBS to obtain the amplified progeny phage library.

[0097] Example 5 In vitro modification (1) In vitro modification to construct a phage display library containing thiazoline cyclic peptides The progeny phage library described in Example 4 was treated with GusA enzyme (final concentration 1 μM) for 2 hours to expose the N-terminal cysteine. 97 μL of the digested progeny phage library was added to a 1.5 mL low-adsorption centrifuge tube, followed by TCEP solution to a final concentration of 1 mM. After mixing, the tube was incubated on a centrifuge for 30 minutes. 6-chloromethyl-2-cyanopyridine was added to a final concentration of 0.5 mM, and the tube was incubated on a centrifuge for 2 hours. 1 / 4 volume of 20% PEG8000-NaCl solution was added, and the tube was incubated on ice for 2 hours. The tube was centrifuged at 11000 rpm for 10 min, the supernatant was discarded, and the precipitate was resuspended in 200 μL of 1×PBS buffer to obtain a phage library containing thiazoline cyclic peptides. Figure 12 As shown.

[0098] (2) Screening experiments were conducted on the target protein (Keap1) affinity of the thiazoline cyclic peptide library, such as... Figure 13 As shown. The following instructions use Keap1 as an example.

[0099] Take two portions of streptavidin magnetic beads and place them in two separate 1.5 mL low-adsorption centrifuge tubes. Add 20 μg of Keap1 protein to one tube as the experimental group, and add an equal volume of PBS to the other tube as the control group. Incubate both groups in a vortex mixer at room temperature for 15-20 minutes.

[0100] Place two centrifuge tubes on a magnetic rack and remove the supernatant after the magnetic beads separate. Resuspend the beads in 1 mL of binding buffer and wash three times. Resuspend the beads in 300 μL of binding buffer and 150 μL of blocking buffer and incubate at room temperature for 2 hours to block the magnetic beads.

[0101] Meanwhile, 100 μL of phage library containing thiazoline cyclic peptide, 300 μL of binding buffer, and 150 μL of locking buffer were mixed and placed on a vortex mixer and incubated at room temperature for 2 hours to perform blocking.

[0102] The sealed phage library was divided into two portions, and each portion was mixed with the sealed magnetic beads from the experimental group and the control group in centrifuge tubes and incubated at room temperature for 30 minutes.

[0103] Place the centrifuge tube on a magnetic rack and wait for the magnetic beads to separate. Remove the supernatant after separation. Add 1 mL of washing buffer to resuspend and rinse the magnetic beads. Place the tube on the magnetic rack again and wait for the beads to separate. Remove the supernatant after separation. Repeat the washing process 9 times. Then wash twice with binding buffer.

[0104] Add 300 μL of Elution buffer to resuspend the magnetic beads and incubate at room temperature for 5-8 minutes. Place the centrifuge tube on a magnetic rack and, after the magnetic beads separate, collect the supernatant and transfer it to a 1.5 mL centrifuge tube containing 40 μL of Neutralization buffer. Mix well to obtain the phage library for the first round of screening.

[0105] The phage libraries obtained from the previous round of screening were used in the next round of screening, with the amount of the target protein Keap1 gradually reduced. The titers of the acid-eluted phage libraries were then determined.

[0106] After four rounds of screening, the enrichment levels for the target protein Keap1 were: 20.23 in the second round, 50.91 in the third round, and 115.25 in the fourth round.

[0107] High-throughput sequencing results: The highly enriched cyclic peptide sequences obtained from the above cyclic peptide library for Keap1 screening are as follows: Figure 14 As shown (only a portion is displayed).

[0108] Example 6: Validation of Cyclic Peptide Sequence Cyclic peptides 1-4 obtained in Example 5 are the cyclic peptides to be tested, and their structures are as follows: Figure 15 As shown.

[0109] Prepare the reaction mixture: Keap1 protein (final concentration 800 nM), fluorescently labeled peptide (FITC-β-Ala-DEETGEF, final concentration 20 nM), and gradient concentrations of the target cyclic peptide, with PBS added to a final volume of 150 μL. Mix well and incubate at room temperature for 10 min. Transfer 130 μL of the mixture to a black 96-well microplate and measure the fluorescence polarization values ​​(excitation wavelength 485 nm, emission wavelength 535 nm) using a BioTek Synergy Neo2 microplate reader. Perform three independent experiments for each sample, and use the average value for data fitting.

[0110] The competition curve was fitted using Origin software according to the following formula: .

[0111] Where x is the concentration of the analyte cyclic peptide, Y is the polarization value, A1 is the maximum polarization value when only the protein binds to the fluorescent peptide, A2 is the polarization value when the analyte cyclic peptide competes with the fluorescent peptide, and EC50 is the half-maximal effective concentration. The competition curves for cyclic peptides 1-4 are shown below. Figure 16 As shown.

[0112] The suppression constant Ki is calculated using the following formula:

[0113] Among them, [L] 50 [P] represents the concentration of the fluorescent peptide at 50% inhibition, [P]0 represents the initial protein concentration, and KD represents the equilibrium dissociation constant between the fluorescent peptide and the protein. The Ki values ​​for cyclic peptide 1, cyclic peptide 2, cyclic peptide 3, and cyclic peptide 4 are 58.3±9.4 nM, 47.4±8.5 nM, 3.75±0.86 μM, and 7.92±0.91 μM, respectively.

[0114] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for constructing a phage-displayed cyclic peptide library containing a thiazoline group, characterized in that, Includes the following steps: S1. Construct a DNA library, wherein the DNA library encodes at least an enzyme restriction site and a polypeptide library, and the backbone sequence of the polypeptide library contains the amino acid sequence CX9C, where C represents cysteine ​​and X represents a random amino acid. S2. Use helper phages to infect host cells containing the DNA library to obtain a progeny phage library displaying polypeptides; S3. The progeny phage library is digested with a proteolytic enzyme, wherein the proteolytic enzyme specifically recognizes the enzyme recognition site. S4. The progeny phage library after enzyme digestion is co-incubated with 6-chloromethyl-2-cyanopyridine to cyclize, thereby obtaining the phage display library containing thiazoline group cyclic peptides.

2. The construction method according to claim 1, characterized in that, The enzyme cleavage site is the GusA enzyme recognition site, and the proteolytic enzyme is the GusA enzyme.

3. The construction method according to claim 2, characterized in that, The GusA enzyme cleavage recognition site is GGVTPS, and the amino acid sequence of the GusA enzyme is shown in SEQ ID NO.

1.

4. The construction method according to claim 1, characterized in that, The X in CX9C is encoded by the NNK degenerate codon.

5. The construction method according to claim 1, characterized in that, The DNA library contains at least the sequence shown in SEQ ID NO.

3.

6. The construction method according to claim 1, characterized in that, The step S4, which involves co-incubating the enzyme-digested progeny phage library with 6-chloromethyl-2-cyanopyridine for cyclization, further includes: The progeny phage library digested by enzymes was first mixed with TCEP solution and incubated, and then 6-chloromethyl-2-cyanopyridine was added for co-incubation to carry out the cyclization reaction.

7. The construction method according to claim 1, characterized in that, The co-incubation conditions in step S4 are as follows: the final concentration of 6-chloromethyl-2-cyanopyridine is 0.3-0.8 mM, the reaction temperature is 35℃-39℃, and the reaction time is 1-3 hours.

8. The construction method according to claim 1, characterized in that, The DNA library is a circular plasmid, and the vector for the circular plasmid is pCANTAB 5E; and / or The host cell was Escherichia coli TG1.

9. A cyclic peptide library containing a thiazoline group, characterized in that, It is constructed by the construction method of any one of claims 1-8.

10. The use of the thiazoline-containing cyclic peptide library of claim 9 in screening peptides targeting target proteins.

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