Polypeptide ligand and derivative of E3 ubiquitin ligase as well as preparation method and application of polypeptide ligand and derivative

Abstract

The invention relates to a polypeptide ligand and a derivative of E3 ubiquitin ligase as well as a preparation method and application of the polypeptide ligand and the derivative, and relates to the technical field of biological medicines. The polypeptide ligand of the E3 ubiquitin ligase is characterized in that the amino acid sequence of the polypeptide ligand is shown as any one of SEQ ID NO: 1-3, and at least one disulfide bond is formed between cysteine residues in the amino acid sequence of the polypeptide ligand; the polypeptide ligand is combined with the E3 ubiquitin ligase RNF43 and / or the E3 ubiquitin ligase ZNRF3. The polypeptide ligand provided by the invention can be used for flexibly and modularly constructing various bifunctional molecules, such as small-size bifunctional molecules or bifunctional molecules with components which cannot be obtained through expression, so as to degrade disease-related membrane proteins, such as PD-L1, EGFR and the like, and has the potential of disease treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to polypeptide ligands and derivatives of E3 ubiquitin ligases, and preparation methods and applications thereof. Background Art

[0002] Targeted protein degradation (TPD) is a promising new drug entity. Using the endogenous ubiquitin-proteasome pathway or autophagy pathway, TPD can selectively remove disease-related proteins. Compared with traditional drugs such as small molecule inhibitors and antibodies, TPD can improve drug efficacy and expand druggable targets.

[0003] Proteins located on the cell membrane (hereinafter referred to as membrane proteins) play an important role in regulating cell functions and are important targets for the treatment of cancer, autoimmune diseases, etc. More than 60% of FDA-approved drugs act on such proteins. The development of targeted protein degradation technology for membrane proteins has received increasing attention in the TPD field in recent years.

[0004] At present, a feasible strategy is to use bifunctional molecules that can simultaneously bind to E3 ubiquitin ligase RNF43 or ZNRF3 and membrane protein targets, so that the membrane protein targets are spatially close to RNF43 / ZNRF3, and then ubiquitinated and degraded. Based on this strategy, three bifunctional molecules, AbTAC (NO: PCT / US20 / 58328), PROTAB (Nature 2022, 610, 182-189) and ROTAC (NO: EP 22 194 882.1), have been reported. The part responsible for binding to RNF43 or ZNRF3 in these bifunctional molecules is an antibody or protein. However, antibodies or proteins have the problems of easy aggregation, high production cost, poor stability, and poor tissue or solid tumor penetration, which seriously limits the application potential of the corresponding bifunctional molecules. Compared with proteins, peptides have better tissue penetration, lower immunogenicity and production cost; compared with small molecules, peptides have better selectivity, activity and lower off-target effects. In view of this, the present invention provides polypeptide ligands and derivatives of E3 ubiquitin ligase and preparation methods and applications thereof. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide polypeptide ligands, derivatives and preparation methods and applications of E3 ubiquitin ligases, and to develop polypeptide ligands of E3 enzyme RNF43 / ZNRF3 for the development of membrane protein degradation molecules.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] In the first aspect, a polypeptide ligand of an E3 ubiquitin ligase, the amino acid sequence of the polypeptide ligand being as shown in any one of SEQ ID NOs: 1-3, and at least one disulfide bond being formed between the cysteine ​​residues in the amino acid sequence of the polypeptide ligand; the polypeptide ligand binds to the E3 ubiquitin ligase RNF43 and / or the E3 ubiquitin ligase ZNRF3.

[0008] An amino acid sequence having more than 80% identity to an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 3, or an amino acid sequence having identity to an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 3 by deletion, substitution, insertion and / or addition of one or more amino acid residues. The polypeptide ligand binds to RNF43 / ZNRF3 with a binding affinity of an affinity constant (KD) less than or equal to 1 mM.

[0009] Based on the above technical solution, the present invention can also be improved as follows.

[0010] Furthermore, in the amino acid sequence of the polypeptide ligand from the amino terminus to the carboxylic acid terminus, the first cysteine ​​residue forms a disulfide bond with the third cysteine ​​residue, the second cysteine ​​residue forms a disulfide bond with the fourth cysteine ​​residue, the fifth cysteine ​​residue forms a disulfide bond with the sixth cysteine ​​residue, and the seventh cysteine ​​residue forms a disulfide bond with the eighth cysteine ​​residue.

[0011] Further, the polypeptide ligand includes any one of the following structures:

[0012]

[0013] Various Represents a disulfide bond.

[0014] In a second aspect, a polypeptide ligand derivative of an E3 ubiquitin ligase, the polypeptide ligand derivative comprising a product in which at least one of a fatty acid molecule, a polyethylene glycol molecule, a polysaccharide molecule, a fluorescent molecule, a biotin molecule, a phosphate group, an acetyl group, a methylene group, a polypeptide, or a drug molecule having a disease therapeutic effect is connected to at least one position of a side chain of lysine, arginine, aspartic acid, glutamic acid, cysteine, serine, threonine, tryptophan, tyrosine, or histidine, a terminal alpha-amino group, or a main chain peptide bond in the amino acid sequence of the polypeptide ligand of the E3 ubiquitin ligase, or a product in which the disulfide bond of the polypeptide ligand of the E3 ubiquitin ligase is replaced by at least one of a thioether bond, an ether bond, a triazole, a carbon-carbon single bond, and a carbon-carbon double bond, or a pharmaceutically acceptable salt of the polypeptide ligand of the E3 ubiquitin ligase.

[0015] In a third aspect, a method for preparing a polypeptide ligand of an E3 ubiquitin ligase comprises the following steps:

[0016] (1) using a standard N-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis method, sequentially coupling side chain protected amino acid residues or side chain unprotected amino acid residues to extend the peptide chain, using a cleavage reagent to cleave the resin to obtain a main chain linear crude peptide; using reversed-phase high performance liquid chromatography to purify the main chain linear crude peptide to obtain a main chain linear peptide;

[0017] (2) pairing the cysteine ​​residues of the main chain linear peptide through a liquid phase oxidative folding reaction to obtain a multi-disulfide bond cyclic peptide product; and purifying the multi-disulfide bond cyclic peptide product using high performance liquid chromatography to obtain a polypeptide ligand.

[0018] Furthermore, step (2) comprises the following specific steps: firstly dissolving the main chain linear peptide in water to obtain a main chain linear peptide solution with a concentration of 5-20 μM; then sequentially adding 10 times the equivalent of reduced glutathione and 10 times the equivalent of oxidized glutathione to the main chain linear peptide solution, adjusting the pH of the reaction solution to between 7 and 8 after dissolution, reacting for 15-60 minutes, monitoring the reaction progress by analytical reverse HPLC, and determining whether the product has the correct molecular weight by mass spectrometry; finally separating and purifying the main product with the correct molecular weight by semi-preparative reverse HPLC, and obtaining the polypeptide ligand after freeze-drying.

[0019] In a fourth aspect, the bifunctional molecule comprises three parts: a first part, which is a polypeptide, aptamer, antibody, or small molecule that can bind to a cell surface protein; a second part, which is one or more RNF43 / ZNRF3 polypeptides or pharmaceutically acceptable salts thereof; and a third part, a flexible linker connecting the first part and the second part via a covalent chemical bond. The linker allows amino acids of any type and length or other chemical structural groups of similar length to be used as the connecting part.

[0020] A bifunctional molecule, wherein the structure of the bifunctional molecule is shown in the following formula (I) or (II):

[0021] ALB(Ⅰ),CLB(Ⅱ);

[0022] In formula (I), A is a group that binds to the group of PD-L1; in formula (II), C is a group that binds to the group of EGFR; in formula (I) and formula (II), L is a coupling group; in formula (I) and formula (II), B is the polypeptide ligand of the E3 ubiquitin ligase or the polypeptide ligand derivative of the E3 ubiquitin ligase.

[0023] Further, in formula (I), A is BMS-57 or a nanobody having an amino acid sequence as shown in SEQ ID NO: 4; the structural formula of the BMS-57 is as follows:

[0024]

[0025] In formula (II), C is any one of a nanobody having an amino acid sequence as shown in SEQ ID NO: 5 and an affibody having an amino acid sequence as shown in SEQ ID NO: 6.

[0026] Furthermore, in formula (I) and formula (II), L is any of the following:

[0027] -(GGGGS) n -、 Formula - (GGGGS) n -and The value range of n is 1-50.

[0028] In a fifth aspect, the application of bifunctional molecules is to use the bifunctional molecule of formula (I) to prepare a drug for reducing the level of programmed death receptor-ligand 1 (PD-L1) in cells, or to use the bifunctional molecule of formula (II) to prepare a drug for reducing the level of epidermal growth factor receptor (EGFR) in cells.

[0029] The beneficial effects of the present invention are as follows: the present invention provides a polypeptide ligand of E3 ubiquitin ligase (RNF43 / ZNRF3), which is simple and fast to synthesize, and can be chemically modified to further improve physical and chemical properties, such as stability, activity, etc. In addition, the polypeptide ligand has a smaller molecular weight than monoclonal antibodies or recombinant proteins; the above characteristics enable the polypeptide ligand provided by the present invention to flexibly and modularly construct a variety of bifunctional molecules, such as small-sized bifunctional molecules (molecular weight less than 10 kDa) or bifunctional molecules with non-expressible components (such as mirror peptides or cyclic peptides) to degrade disease-related membrane proteins, such as PD-L1, EGFR, etc., and have the potential for disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a high performance liquid chromatogram of the linear sequence of the polypeptide ligand P1 of Example 1; the upper figure is a high performance liquid chromatogram of the purified product, and the lower figure is a high performance liquid chromatogram of the unpurified crude peptide;

[0031] Figure 2 is the mass spectrum of the linear sequence of the polypeptide ligand P1 of Example 1;

[0032] Figure 3 1 is a high performance liquid chromatogram of the polypeptide ligand P1 after disulfide bond cyclization in Example 1; the upper figure is a high performance liquid chromatogram of the purified product, and the lower figure is a high performance liquid chromatogram after the cyclization reaction is completed;

[0033] Figure 4 This is the mass spectrum of the polypeptide ligand P1 after disulfide bond cyclization in Example 1;

[0034] Figure 5 It is a high performance liquid chromatogram of the linear sequence of the polypeptide ligand P2 of Example 2; wherein the upper figure is a high performance liquid chromatogram of the purified product, and the lower figure is a high performance liquid chromatogram of the unpurified crude peptide;

[0035] Figure 6 is the mass spectrum of the linear sequence of the polypeptide ligand P2 of Example 2;

[0036] Figure 7 : is a high performance liquid chromatogram of the polypeptide ligand P2 after disulfide bond cyclization in Example 2; wherein the upper figure is a high performance liquid chromatogram of the purified product, and the lower figure is a high performance liquid chromatogram after the cyclization reaction is completed;

[0037] Figure 8 This is the mass spectrum of the polypeptide ligand P2 after disulfide bond cyclization in Example 2;

[0038] Fig. 9 The figure is a high performance liquid chromatogram of the linear sequence of the polypeptide ligand P3 of Example 3; the upper figure is a high performance liquid chromatogram of the purified product, and the lower figure is a high performance liquid chromatogram of the unpurified crude peptide;

[0039] Fig.10 is the mass spectrum of the linear sequence of the polypeptide ligand P3 of Example 3;

[0040] Fig.11 : is a high performance liquid chromatogram of the polypeptide ligand P3 after disulfide bond cyclization in Example 3; wherein the upper figure is a high performance liquid chromatogram of the purified product, and the lower figure is a high performance liquid chromatogram after the cyclization reaction is completed;

[0041] Fig.12 This is the mass spectrum of the polypeptide ligand P3 after disulfide bond cyclization in Example 3;

[0042] Fig.13 This is a graph showing the binding force of polypeptide ligands P1-P3 to RNF43 / ZNRF3 determined by ITC in Example 5;

[0043] Fig.14 Schematic diagram of the structure of bifunctional molecules D1-D4;

[0044] Fig.15 This is a graph showing the results of the PD-L1 degradation experiment in Example 8;

[0045] Fig.16 The figure is a graph showing the experimental results of PD-L1 degradation by D1-D4 in Example 9; the left figure is a graph showing the experimental results of PD-L1 degradation by D1 and D2, and the right figure is a graph showing the experimental results of PD-L1 degradation by D3 and D4;

[0046] Fig.17The figure is a graph showing the experimental results of using flow cytometry to analyze the clearance of PD-L1 on the surface of wild-type T24 cells by D1 in Example 10; the left figure is a flow histogram at different D1 concentrations, and the right figure is a quantitative analysis result of three repeated experiments;

[0047] Fig.18 Schematic diagram of the structure of bifunctional molecules D5-D7;

[0048] Fig.19 This is a diagram of the EGFR degradation experimental results in Example 13. DETAILED DESCRIPTION

[0049] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0050] Sources of materials and reagents: All reagents used in the present invention were of analytical or chemical purity.

[0051] Example 1: Peptide ligand P1 of RNF43 / ZNRF3 and its synthesis method.

[0052] The amino acid sequence and disulfide bond composition of P1 are as follows:

[0053]

[0054] Represents a disulfide bond.

[0055] The preparation method of P1 involved in this embodiment comprises the following steps:

[0056] Step 1: Synthesize Linear P1:

[0057] 1a. Weigh 446.4 mg (0.25 mmol) of Rink AM amide resin with a degree of substitution of 0.56 mmol / g, add 10 mL of N,N-dimethylformamide (DMF) / dichloromethane solution (DCM) to the resin to swell the resin. The volume ratio of the added solution is: DMF:DCM=1:1. The swelling time is 10 minutes. Use a diaphragm pump as a power source to drain the swollen product to obtain a swollen resin.

[0058] 1b. Fmoc-Arg(Pbf)-OH was condensed on the resin using the standard Fmoc solid phase peptide synthesis method (4 equivalents of amino acids, 4 equivalents of 1,3-diisopropylcarbodiimide (DIC), 4 equivalents of ethyl cyano(hydroxyimino)acetate (Oxyma), 89°C, 1.5 minutes, repeated twice, and the equivalents of other raw materials added in the following preparation were all molar equivalents of the resin). After the reaction, the resin was washed three times with DMF, DCM, and DMF respectively.

[0059] 1c. Add a DMF solution containing 20% ​​(volume fraction) piperidine and 0.1 M Oxyma to the resin, react at 90° C. for 1 minute to remove the Fmoc protecting group, and then wash with DMF, DCM, and DMF three times each in sequence.

[0060] 1d. Repeat the operations described in 1b and 1c to condense Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Met-OH, Fmoc-Asp(OMpe)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-His(Trt) on the resin in sequence. -OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-L eu-OH, Fmoc-Cys(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH , Fmoc-Met-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, F moc-Phe-OH, Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Dmb-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OMpe)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Cys(Trt)-OH, F moc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ile-OH.

[0061] In step 1d, the amino acid after proline and the first to twenty-ninth amino acids in the amino acid sequence from left to right need to be condensed twice, and the condensation temperature of Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH is 50° C. and the condensation time is 10 minutes.

[0062] 1e. After the amino acid condensation is completed, the resin is washed three times with DCM, and the DCM solvent is removed under reduced pressure to obtain a dry resin.

[0063] 1f, add 20mL of pre-configured cutting reagent (a mixture of trifluoroacetic acid, phenol, water, thioanisole and 1,2-ethanedithiol, trifluoroacetic acid: water: phenol: thioanisole: 1,2-ethanedithiol, volume ratio = 82:5:5:5:3) to the dry value obtained in step 1e, react at room temperature for 2.5 hours, collect the filtrate in a centrifuge tube after the polypeptide chain is cleaved from the resin, and concentrate the cutting solution by nitrogen bubbling method; finally, after the cutting solution is concentrated to less than 10mL, add 40mL of icy ether for sedimentation, centrifuge with a low-speed centrifuge (3500rpm), and allow the crude peptide to settle to the bottom; after removing the supernatant, add icy ether again, ultrasonically form a crude peptide suspension, and the small molecule impurities removed are dissolved in the icy ether, and finally removed by centrifugation; after completing twice, the solid sediment is placed in a cool place for air-drying to obtain a solid crude peptide.

[0064] 1g, take a small amount of crude peptide and dissolve it in a mixed solution of water and acetonitrile (volume ratio of 3:1), and use analytical reverse high performance liquid chromatography (RP-HPLC) for analysis after passing through the membrane; the analysis gradient is 20%-60% acetonitrile concentration, and the time is 30 minutes; after chromatographic analysis, the main peak is identified by ESI-MS to verify the correctness of linear P1; after correct verification, the solid crude peptide is separated and purified by semi-preparative RP-HPLC (semi-preparative gradient is 20%-50% acetonitrile concentration, time is 30 minutes), the correct product peak solution is collected and placed in a freeze dryer for freeze drying to obtain a white flocculent product.

[0065] in, Figure 1 is a high performance liquid chromatogram of the linear sequence of the polypeptide ligand P1 of Example 1; Figure 2 This is the mass spectrum of the linear sequence of the polypeptide ligand P1 in Example 1.

[0066] Step 2: Liquid phase oxidation folding:

[0067] The linear P1 powder was dissolved in 100 mL of water at a concentration of 10 μM, and then 10 times the equivalent of reduced glutathione and 10 times the equivalent of oxidized glutathione were added respectively; after the reactants were fully dissolved by ultrasound, the pH of the reaction solution was adjusted to between 7 and 8, and the reaction was carried out for 30 minutes. The reaction progress was monitored by analytical reverse HPLC, and mass spectrometry was used to determine whether the product had the correct molecular weight; finally, the main product with the correct molecular weight was separated and purified by semi-preparative reverse HPLC, and the final product P1 was obtained after freeze-drying.

[0068] Figure 3 This is a high performance liquid chromatogram of the polypeptide ligand P1 after disulfide bond cyclization in Example 1; Figure 4This is the mass spectrum of the polypeptide ligand P1 after disulfide bond cyclization in Example 1.

[0069] Example 2: The polypeptide ligand P2 of RNF43 / ZNRF3 and its synthesis method.

[0070] The amino acid sequence and disulfide bond composition of P2 are as follows:

[0071]

[0072] Represents a disulfide bond.

[0073] P2 was synthesized by a method similar to that of P1, which will not be repeated here.

[0074] Figure 5 is a high performance liquid chromatogram of the linear sequence of the polypeptide ligand P2 of Example 2; Figure 6 is the mass spectrum of the linear sequence of the polypeptide ligand P2 of Example 2; Figure 7 This is a high performance liquid chromatogram of the polypeptide ligand P2 after disulfide bond cyclization in Example 2; Figure 8 This is the mass spectrum of the polypeptide ligand P2 after disulfide bond cyclization in Example 2.

[0075] Example 3: Peptide ligand P3 of RNF43 / ZNRF3 and its synthesis method.

[0076] The amino acid sequence and disulfide bond composition of P3 are as follows:

[0077]

[0078] Represents a disulfide bond.

[0079] P3 was synthesized by a method similar to that of P1, which will not be repeated here.

[0080] Fig. 9 is a high performance liquid chromatogram of the linear sequence of the polypeptide ligand P3 of Example 3; Fig.10 is the mass spectrum of the linear sequence of the polypeptide ligand P3 of Example 3; Fig.11 This is a high performance liquid chromatogram of the polypeptide ligand P3 after disulfide bond cyclization in Example 3; Fig.12 This is the mass spectrum of the polypeptide ligand P3 after disulfide bond cyclization in Example 3.

[0081] Example 4: Binding ability of polypeptide ligands P1-P3 to RNF43 / ZNRF3.

[0082] The determination of the binding force of the polypeptide ligands P1-P3 to RNF43 / ZNRF3 involved in this embodiment comprises the following steps:

[0083] Step 1: Obtain RNF43 / ZNRF3 extracellular domain:

[0084] 1a. The plasmid encoding SEQ ID NO: 7 (RNF43) and SEQ ID NO: 8 (ZNRF3) (the vector is pcDNA 3.1, purchased from Nanjing GenScript Biotechnology Co., Ltd.) was transformed into Escherichia coli competent DH5α (purchased from Shenzhen Kangti Life Science Technology Co., Ltd.) using the heat stimulation transformation method. The competent cells after transformation were spread on an agar plate containing ampicillin antibiotics and incubated in a 37°C incubator for 16 hours. The selected monoclonal clone was placed in 5 mL LB culture medium for shake flask culture, and after 16 hours, it was transferred to 300 mL LB culture medium for culture. After 16 hours, the cells were harvested by centrifugation, and then the plasmid was extracted using a plasmid extraction kit (purchased from Nanjing GenScript Biotechnology Co., Ltd.).

[0085] 1b. HEK 293F cells (purchased from the Chinese Academy of Sciences Cell Bank) were cultured in 400 mL SMM 293-CD1 medium at 37°C and 5% carbon dioxide. The cell density was 4 × 10 6 When cells were transfected, the RNF43 / ZNRF3 plasmid was transfected into the cells. The transfection method was as follows: 400 micrograms of RNF43 / ZNRF3 plasmid and 1600 micrograms of PEI 400 were dissolved in 12 mL of normal saline, vortexed thoroughly, and allowed to stand for 5 minutes. The plasmid solution was then added to the PEI solution, inverted 10 times to mix thoroughly, allowed to stand for 20 minutes, and then added to the cells. After continuing to culture for 6 days, the supernatant was collected by centrifugation. 5 mL of Ni Smart Beads (purchased from Tiandirenhe) was added to the supernatant and incubated in a shaker (150 rpm, 30 minutes). The beads were washed with 25 mL of 1x PBS buffer and eluted with 10 mL of 1x PBS buffer containing 400 mM imidazole. After the eluate was concentrated, it was separated and purified by size exclusion chromatography to obtain the final product.

[0086] Step 2: Isothermal Titration Calorimetry (ITC) to determine binding capacity:

[0087] 2a. Use size exclusion chromatography to place the peptide and RNF43 / ZNRF3 extracellular domain in the same buffer solution.

[0088] 2b. The titration was performed at room temperature using a MicroCal PEAQ-ITC system (Malvern). The peptide concentration was 200 μM, the RNF43 / ZNRF3 concentration was 20 μM, and the peptide solution was dripped into the RNF43 / ZNRF3 solution 19 times, with an interval of 120 seconds between each drip.

[0089] MicroCal PEAQ-ITC analysis software was used for data analysis. Fig.13 shown by Fig.13 It can be seen that P1-P3 can bind to RNF43 / ZNRF3 with strong binding force, or selectively bind to RNF43. Among them, the binding force of P1 to RNF3 and ZNRF3 is 146±7.26nM and 477±52.7nM, the binding force of P2 to RNF3 and ZNRF3 is 2.04±0.19μM and 5.70±1.78μM, and the binding force of P3 to RNF43 is 104±13.8nM, and it does not bind to ZNRF3.

[0090] Example 5: The polypeptide ligand P1 is used to construct bifunctional molecules D1 and D2 targeting PD-L1.

[0091] The polypeptide ligand P1 involved in this embodiment is used to construct bifunctional molecules D1 and D2 targeting PD-L1, comprising the following steps:

[0092] Step 1: Obtain the PD-L1-targeting nanoantibody KN035 with a Sortase A enzyme recognition motif:

[0093] The Escherichia coli host strain BL21 (DE3) (purchased from Nanjing Novozyme Biotechnology Co., Ltd.) was selected, and the plasmid encoding the amino acid sequence shown in SEQ ID NO: 9 (the vector is pet-22b, purchased from Nanjing GenScript Biotechnology Co., Ltd.) was transformed into the host, and the transformed Escherichia coli was spread on an agar plate containing the corresponding antibiotics. After overnight incubation, a single clone was selected, and the selected single clone was placed in 5 mL LB medium for shake flask culture. After 16 hours, it was transferred to 800 mL LB medium and cultured to OD600 of about 0.6, and then IPTG (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) was added to induce protein expression. After the induction, the bacteria were collected by centrifugation, and the cells were lysed by a high-pressure disruptor. After lysis, the supernatant was collected by centrifugation, and then AKTA protein purification instrument was used to purify the protein through HisTrap TM A 5 mL column (purchased from Stefan Biotech Co., Ltd.) was used to further improve the purity of the protein and remove some DNA impurities, and the protein was concentrated to the target concentration of approximately 1 mM using an ultrafiltration device.

[0094] Step 2: Synthesize the GGG tripeptide-modified P1 analogs P4 or P5 using a similar synthesis method as P1:

[0095] The GGG tripeptide in the above P4 is modified at the amino terminus of P1, and the amino acid sequence and disulfide bond group are as follows:

[0096]

[0097] The GGG tripeptide modification in P5 is in P1 S58K The side chain amino group, amino acid sequence and disulfide bond group of lysine at position 5 and 8 are as follows:

[0098]

[0099] Step 3: Sortase A enzymatically connects and synthesizes bifunctional molecules D1 and D2 targeting PD-L1:

[0100] KN035 was diluted to 0.2 mM with reaction buffer (50 mM HEPES, 150 mM NaCl, 1 mM CaCl2, pH 7.0), followed by addition of Sortase A (purchased from Shanghai Biotech Biotechnology Co., Ltd., 5 μM) and P4 or P5 (1 mM), and incubated at room temperature for 30 min. 10 times the reaction volume of 50 mM Tris pH 7.4, 500 mM NaCl buffer solution was added for dilution, and then purified by HisTrap using an AKTA protein purifier. TM 5mL column (purchased from Si Tuo Fan Biotechnology Co., Ltd.) and imidazole concentration gradient elution were used to separate and purify D1 or D2. The structural diagram of D1-D2 is shown in the figure Fig.14 .

[0101] Example 6: The polypeptide ligand P1 is used to construct the bifunctional molecules D3 and D4 targeting PD-L1.

[0102] The polypeptide ligand P1 involved in this embodiment is used to construct bifunctional molecules D3 and D4 targeting PD-L1, comprising the following steps:

[0103] Step 1: Synthesis of BMS-57 with LPET-thioester at the carboxylic acid end:

[0104] 1a. A linear peptide was synthesized using an Fmoc solid phase synthesis method similar to P1. The linear peptide structure is shown below:

[0105]

[0106] 1b. Dissolve the linear peptide in a buffer solution of 0.2M NaHPO4, 6M Gn-HCl, pH 8.0 at a concentration of 0.1 mM. Incubate at room temperature for 30 minutes to allow the chloroacetyl group to react with the side chain sulfhydryl group of the cysteine ​​residue to achieve cyclization.

[0107] 1c. Adjust the pH of the reaction solution in the previous step to about 2.3 with aqueous hydrochloric acid solution, then place the reaction solution in an ice-salt bath at a temperature of minus 15°C, add 10 times the equivalent of sodium nitrite, and stir the reaction for 30 minutes. Add 50 times the equivalent of methyl thioglycolate (MTG), adjust the pH to 5.4, and continue the reaction at room temperature for 30 minutes to convert the carboxylic acid terminal hydrazide group into an MTG thioester group to obtain BMS-57-LPET-MTG. The product was identified and purified by reversed-phase high performance liquid chromatography and mass spectrometry.

[0108] Step 2: Using a Sortase A enzymatic method similar to that in Example 5, the bifunctional molecules D3 and D4 targeting PD-L1 were connected and synthesized:

[0109] Briefly, BMS-57-LPET-MTG was dissolved in reaction buffer (50 mM HEPES, 150 mM NaCl, 1 mM CaCl2, pH 7.0) to a concentration of 0.2 mM, followed by addition of Sortase A (5 μM) and P4 or P5 (0.24 mM) and incubation at room temperature overnight. After the reaction, separation and purification were performed using reversed-phase high-performance liquid chromatography. The structural diagram of D3-D4 is shown in Figure 1. Fig.14 .

[0110] Example 7: Characterization of cellular activity of bifunctional molecules D1-D4.

[0111] The cell activity characterization of the bifunctional molecules D1-D4 involved in this embodiment includes the following steps:

[0112] Step 1: Construction of RNF43 overexpressing T24 cell line (purchased from the Cell Bank of the Chinese Academy of Sciences).

[0113] Step 2: Inoculate approximately 4 × 10 cells per well of a 24-well plate. 4 T24 cells were cultured overnight at 37°C in a cell culture incubator. When the cells reached 40% to 50% confluence, 500 μL of culture medium containing 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, and 10 μM of the bifunctional molecule prepared in Example 5 or 6 was placed in each well and incubated for 24 hours. Total protein was extracted using cell lysate, and after quantification by BCA protein quantification, the PD-L1 protein content was quantitatively analyzed using immunoblot analysis to test the degradation ability of D1-D4 on PD-L1; the results are shown in Fig.15 As shown by Fig.15 It can be seen that D1-D4 can achieve more than 80% degradation of PD-L1.

[0114] Example 8: Determination of the half maximal degradation concentration (DC 50 ).

[0115] The same experimental method as in Example 8 was used to perform D1-D4 DC 50 The DC50 values ​​were determined by three replicate experiments, wherein the concentration gradients of D1 and D2 were 0.00064 nM, 0.0032 nM, 0.016 nM, 0.08 nM, 0.4 nM and 2 nM; the concentration gradients of D3 and D4 were 0.032 nM, 0.16 nM, 8 nM, 40 nM, 200 nM and 1000 nM. The DC50 values ​​were calculated based on the results of three replicate experiments. The results are shown in Fig.16 As shown, the DC of D1-D4 50 The values ​​were 0.045 nM, 0.061 nM, 34 nM and 73 nM, respectively.

[0116] Example 9: Flow cytometry analysis was used to show that D1 reduces the level of PD-L1 on the surface of wild-type T24 cells.

[0117] Wild-type T24 cells were evenly seeded in a 12-well plate and cultured overnight at 37°C in a cell culture incubator. When the cells reached 40% to 50% confluence, D1 was added with final concentrations of 0.01nM, 0.1nM, 1nM, 10nM, 100nM and 1000nM, respectively. After 24 hours of treatment in the cell culture incubator, the culture medium was aspirated, the cells were washed with PBS, digested with Versene, collected and centrifuged, and finally the cells were reselected with phenol red-free, HEPES-containing DMEM. After washing the cell suspension once with PBS buffer containing 1% FBS, PE dye-modified PDL1 antibody was added and incubated on ice in the dark for 20 minutes. After washing, the cells were sorted by flow cytometry in sequence. The total number of sorting was set to 30,000 and the detection channel was set to the PE channel. The results are shown in Fig.17 As shown, D1 can significantly reduce PD-L1 on the surface of T24 cells.

[0118] Example 10: The polypeptide ligand P1 was used to construct the bifunctional molecule D5-D6 targeting EGFR.

[0119] The polypeptide ligand P1 involved in this embodiment is used to construct the bifunctional molecule D5-D6 targeting EGFR, comprising the following steps:

[0120] Step 1: The nanobody expression and purification method described in Example 6 was used to obtain the nanobody 9G8 with the Sortase A enzyme recognition sequence LPETG at the carboxylic acid terminal, and the amino acid sequence is shown in SEQ ID NO: 10.

[0121] Step 2: Using the Sortase A enzymatic reaction method described in Example 6, 9G8 was connected to P1 or P1 S58K to obtain D5 or D6, the structure of which is as follows: Fig.18 shown.

[0122] Example 12: The polypeptide ligand P1 was used to construct the bifunctional molecule D7 targeting EGFR.

[0123] The polypeptide ligand P1 involved in this embodiment is used to construct a bifunctional molecule D7 targeting EGFR, comprising the following steps:

[0124] Step 1: Prepare the P1 derivative P7 with LPETGG hexapeptide at the carboxylic acid end using the standard Fmoc peptide solid phase synthesis method described in Example 1. The amino acid sequence and disulfide bond composition of P7 are as follows:

[0125]

[0126] Step 2: The standard Fmoc polypeptide solid phase synthesis method described in Example 1 was used to prepare the EGFR affibody shown in SEQ ID NO: 11.

[0127] Step 3: P7 is linked to the EGFR affinity body using a Sortase A enzymatic reaction method similar to that described in Example 6 to obtain D7, the structure of which is as follows: Fig.18 shown.

[0128] Example 13: Characterization of cellular activity of bifunctional molecules D5-D7.

[0129] The cell activity characterization of the bifunctional molecules D5-D7 involved in this embodiment includes the following steps:

[0130] Approximately 4 × 10 cells were evenly seeded per well of a 24-well plate. 4 T24 cells (purchased from the cell bank of the Chinese Academy of Sciences) were cultured in a cell culture incubator at 37°C overnight. When the cells reached 40% to 50% confluence, 500 μL of culture medium containing 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, and 10 μM of the bifunctional molecule prepared in Example 9 or 10 was placed in each well plate and incubated for 24 hours. Total protein was extracted using cell lysate, and after quantification by BCA protein quantification, the EGFR protein content was quantitatively analyzed using immunoblot analysis to test the degradation ability of D5-D7 on EGFR. The results are shown in FIG. Fig.17 As shown by Fig.19 It can be seen that D5-D7 can achieve efficient degradation of EGFR, and the maximum degradation efficiency is greater than 70%.

[0131] In summary, the present invention provides a polypeptide ligand of E3 ubiquitin ligase (RNF43 / ZNRF3), which is simple and fast to synthesize, and can be chemically modified to further improve the physicochemical properties, such as stability, activity, etc. In addition, the polypeptide ligand has a smaller molecular weight than monoclonal antibodies or recombinant proteins; the above characteristics enable the polypeptide ligand provided by the present invention to flexibly and modularly construct a variety of bifunctional molecules, such as small-sized bifunctional molecules (molecular weight less than 10 kDa) or bifunctional molecules with non-expressible components (such as mirror peptides or cyclic peptides) to degrade disease-related membrane proteins, such as PD-L1, EGFR, etc., and have the potential for disease treatment.

[0132] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A polypeptide ligand for an E3 ubiquitin ligase, characterized in that: The amino acid sequence of the polypeptide ligand is shown in any one of SEQ ID NOs: 1-3, and at least one disulfide bond is formed between the cysteine ​​residues in the amino acid sequence of the polypeptide ligand; the polypeptide ligand binds to the E3 ubiquitin ligase RNF43 and / or the E3 ubiquitin ligase ZNRF3.

2. The polypeptide ligand of E3 ubiquitin ligase according to claim 1, characterized in that The amino acid sequence of the polypeptide ligand is from the amino terminal to the carboxylic acid terminal, the first cysteine ​​residue forms a disulfide bond with the third cysteine ​​residue, the second cysteine ​​residue forms a disulfide bond with the fourth cysteine ​​residue, the fifth cysteine ​​residue forms a disulfide bond with the sixth cysteine ​​residue, and the seventh cysteine ​​residue forms a disulfide bond with the eighth cysteine ​​residue.

3. The polypeptide ligand of E3 ubiquitin ligase according to claim 1, characterized in that The polypeptide ligand includes any one of the following structures: Various Represents a disulfide bond.

4. A polypeptide ligand derivative of an E3 ubiquitin ligase, characterized in that: The polypeptide ligand derivative includes a product in which at least one of the following is connected to the side chain of lysine, arginine, aspartic acid, glutamic acid, cysteine, serine, threonine, tryptophan, tyrosine, histidine, the terminal alpha-amino group, and the main chain peptide bond in at least one position in the amino acid sequence of the polypeptide ligand of the E3 ubiquitin ligase according to any one of claims 1 to 3: a fatty acid molecule, a polyethylene glycol molecule, a polysaccharide molecule, a fluorescent molecule, a biotin molecule, a phosphate group, an acetyl group, a methylene group, a polypeptide, and a drug molecule having a disease therapeutic effect; or a product in which the disulfide bond of the polypeptide ligand of the E3 ubiquitin ligase is replaced by at least one of a thioether bond, an ether bond, a triazole, a carbon-carbon single bond, and a carbon-carbon double bond; or a pharmaceutically acceptable salt of the polypeptide ligand of the E3 ubiquitin ligase.

5. A method for preparing a polypeptide ligand for an E3 ubiquitin ligase according to any one of claims 1 to 3, characterized in that: The steps include: (1) using a standard N-fluorenylmethoxycarbonyl solid phase peptide synthesis method, sequentially coupling side chain protected amino acid residues or side chain unprotected amino acid residues to extend the peptide chain, using a cleavage reagent to cleave the resin to obtain a main chain linear crude peptide; using reverse phase high performance liquid chromatography to purify the main chain linear crude peptide to obtain a main chain linear peptide; (2) pairing the cysteine ​​residues of the main chain linear peptide by liquid phase oxidative folding reaction to obtain a multi-disulfide bond cyclic peptide product; The multi-disulfide bond cyclic peptide product is purified by high performance liquid chromatography to obtain a polypeptide ligand.

6. The method for preparing the polypeptide ligand of E3 ubiquitin ligase according to claim 5, characterized in that: Step (2) comprises the following specific steps: firstly dissolving the main chain linear peptide in water to obtain a main chain linear peptide solution with a concentration of 5-20 μM; then adding 10 times the equivalent of reduced glutathione and 10 times the equivalent of oxidized glutathione to the main chain linear peptide solution in sequence, adjusting the pH of the reaction solution to between 7 and 8 after dissolution, reacting for 15-60 minutes, monitoring the reaction progress by analytical reverse HPLC, and determining whether the product has the correct molecular weight by mass spectrometry; finally separating and purifying the main product with the correct molecular weight by semi-preparative reverse HPLC, and obtaining the polypeptide ligand after freeze-drying.

7. A bifunctional molecule, characterized in that The structure of the bifunctional molecule is shown in the following formula (I) or (II): ALB(Ⅰ),CLB(Ⅱ); In formula (I), A is a group that binds to the group of PD-L1; in formula (II), C is a group that binds to the group of EGFR; in formula (I) and (II), L is a coupling group; in formula (I) and (II), B is the polypeptide ligand of the E3 ubiquitin ligase according to any one of claims 1 to 3 or the polypeptide ligand derivative of the E3 ubiquitin ligase according to claim 4.

8. The bifunctional molecule according to claim 7, characterized in that In formula (I), A is BMS-57 or a Nanobody with an amino acid sequence as shown in SEQ ID NO: 4; The structural formula of BMS-57 is shown below: In formula (II), C is any one of a nanobody having an amino acid sequence as shown in SEQ ID NO: 5 and an affibody having an amino acid sequence as shown in SEQ ID NO:

6.

9. The bifunctional molecule according to claim 7, characterized in that In formula (I) and formula (II), L is any of the following: Formula - (GGGGS) n -and The value range of n is 1-50.

10. Use of bifunctional molecules, characterized in that The bifunctional molecule of formula (I) as described in any one of claims 7 to 9 is used to prepare a drug for reducing the level of programmed cell death receptor-ligand 1, or the bifunctional molecule of formula (II) as described in any one of claims 7 to 9 is used to prepare a drug for reducing the level of epidermal growth factor receptor.

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