Polypeptide degradation agent targeting ANP32B protein as well as preparation method and application of polypeptide degradation agent

Abstract

The invention discloses a polypeptide degradation agent targeting ANP32B protein as well as a preparation method and application of the polypeptide degradation agent, and belongs to the technical field of tumor targeted therapy. The polypeptide degradation agent targeting the ANP32B protein is obtained by modifying an ANP32B protein targeting binding ligand (the sequence of the ANP32B protein targeting binding ligand is shown as SEQ ID NO.1-SEQ ID NO.4 in a table 1); wherein R6 (RRRRRRR) is a cationic cell membrane penetrating peptide, and is connected with a polypeptide ligand (IAP (OH) YI) of the E3 ubiquitin ligase VHL through Linker (Acp). Experiments prove that the polypeptide degradation agent targeting the ANP32B protein has the capability of remarkably inhibiting the activity of breast cancer cells MDA-MB-231, can degrade target proteins in the cells and influence the cell cycles of the breast cancer cells MDA-MB-231, can be used for preparing anti-tumor drugs, has a good application prospect in preparation of drugs targeting human breast cancer cells, and has a wide application prospect in preparation of drugs targeting human breast cancer cells. The polypeptide can be used in another important field of polypeptide PROTAC drug development.

Description

Technical Field

[0001] This invention relates to the field of tumor targeted therapy technology, specifically to a peptide degrader targeting ANP32B protein, its preparation method, and its application. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide and one of the major threats to women's health. Despite continuous improvements in breast cancer diagnosis and treatment, its incidence continues to rise. Current clinical challenges include limited treatment options for specific subtypes (such as triple-negative breast cancer), significant drug resistance issues, and poor prognosis for patients with advanced-stage disease. Therefore, developing novel, highly effective, and low-toxicity breast cancer drugs targeting new targets, especially those addressing resistance mechanisms and refractory subtypes, is of significant clinical importance for overcoming current treatment bottlenecks and improving patient survival outcomes.

[0003] Recent studies have revealed that the acidic nuclear phosphoprotein 32 (ANP32) family of proteins may be associated with the development, progression, and prognosis of malignant tumors. Three members (ANP32A, ANP32B, and ANP32E) are conserved in vertebrates and collectively participate in biological processes such as chromatin regulation, caspase activation, protein phosphatase inhibition, and intracellular transport. ANP32B possesses unique structural features; its acidic amino acid-rich region and leucine repeat sequence enable it to mediate nuclear transport, chromatin remodeling, and gene expression regulation. Studies have shown that ANP32B is identified as a poor prognostic indicator in breast cancer, and its pro-cancer mechanism involves a core driving role in cell proliferation. Using gene knockout models and RNAi silencing technology, ANP32B-deficient mice exhibit incomplete tissue development and limited embryonic fibroblast proliferation, continuing to show proliferative defects even after oncogene-induced immortalization. Further clinical cohort analysis revealed that ANP32B protein expression was significantly higher in malignant breast cancer tissue than in adjacent normal tissue, and was positively correlated with pathological grade. Mechanistic studies showed that ANP32B mediates a pro-proliferative effect by regulating AKT phosphorylation levels; knockdown of ANP32B induced G1 phase arrest and significantly inhibited cancer cell growth in vitro and in vivo. In summary, ANP32B drives tumorigenesis through multiple mechanisms, especially as a key pro-proliferative factor in breast cancer, highlighting its importance as a potential therapeutic target.

[0004] While ANP32B protein can serve as a target for breast cancer treatment drugs, the following challenges exist in drug design: The ANP32B protein structure itself lacks a suitable binding pocket for chemical drug molecules, making it difficult to design ANP32B protein inhibitors based on pocket structure models, as is the case with kinase inhibitors. ANP32B protein can form complexes with other proteins, and the protein-protein interaction interface becomes a potential drug binding site, competitively inhibiting the binding of other proteins to the ANP32B protein molecule, thus limiting protein activity. However, the surface area of ​​protein-protein interaction interfaces is often relatively large (approximately 1000-3000 Å). 2 Furthermore, the relatively flat interface makes it difficult for small molecules to bind to proteins, resulting in low affinity and poor drug-like properties. Therefore, there is an urgent need for an inhibitor that can target and degrade the ANP32B protein. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a peptide degrader targeting ANP32B protein, its preparation method and application, so as to solve the technical problem that it is difficult for drugs developed from ANP32B protein to exert tumor-inhibiting activity.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention discloses a polypeptide degrader targeting ANP32B protein, wherein the polypeptide degrader contains a membrane-penetrating peptide and an ANP32B protein targeting ligand at its N-terminus, and a polypeptide ligand motif of E3 ubiquitin ligase VHL at its C-terminus, and the ANP32B protein targeting ligand and the polypeptide ligand of E3 ubiquitin ligase VHL are linked by a linker. The amino acid sequences of the ANP32B protein targeting and binding ligand are shown in SEQ ID NO.1 to SEQ ID NO.4.

[0007] Preferably, the general structural formula of the peptide degrader targeting the ANP32B protein is:

[0008] Among them, R1 is an arginine or tryptophan side chain, R2 is a tyrosine, tryptophan or arginine side chain, R3 is a histidine, threonine, lysine or arginine side chain, R4 is a lysine, tryptophan or isoleucine side chain, and R5 is a tryptophan, arginine or glutamine side chain.

[0009] Preferably, when R3 is a histidine side chain, R1 is an arginine side chain, R2 is a tyrosine side chain, R4 is a lysine side chain, and R5 is a tryptophan side chain, and the amino acid sequence of the peptide degrader targeting ANP32B protein is shown in SEQ ID NO.5.

[0010] Preferably, when R3 is a threonine side chain, R1 is an arginine side chain, R2 is a tryptophan side chain, R4 is a lysine side chain, and R5 is an arginine side chain. The amino acid sequence of the peptide degrader targeting the ANP32B protein is shown in SEQ ID NO.6.

[0011] Preferably, when R3 is a lysine side chain, R1 is an arginine side chain, R2 is a tyrosine side chain, R4 is a tryptophan side chain, and R5 is a glutamine side chain. The amino acid sequence of the peptide degrader targeting the ANP32B protein is shown in SEQ ID NO.7.

[0012] Preferably, when R3 is an arginine side chain, R1 is a tryptophan side chain, R2 is an arginine side chain, R4 isoleucine side chain, and R5 is a glutamine side chain, and the amino acid sequence of the peptide degrader targeting ANP32B protein is shown in SEQ ID NO.8.

[0013] In a second aspect, the present invention discloses a method for synthesizing a peptide degrader targeting ANP32B protein, wherein a peptide degrader targeting ANP32B protein is prepared by an artificial solid-phase synthesis method based on FMOC amino acids.

[0014] A third aspect of the present invention discloses the application of the aforementioned peptide degrader targeting ANP32B protein in the preparation of antitumor drug formulations.

[0015] Preferably, the antitumor drug is a drug for human breast cancer tumor cells.

[0016] Preferably, the concentration of the peptide degrader targeting the ANP32B protein is less than 200 μM.

[0017] Preferably, the concentration of the peptide degrader targeting the ANP32B protein is 3~200 μM.

[0018] A fourth aspect of the present invention discloses an anti-breast cancer composition comprising other active ingredients having anti-breast cancer activity, a peptide degrader targeting the ANP32B protein, and one or more pharmaceutically acceptable excipients.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a peptide degrader targeting ANP32B protein. The ANP32B protein-protein interaction interface serves as the ligand binding site, and a five-amino acid-long peptide acts as the ANP32B protein-targeting ligand. This five-amino acid-long peptide exhibits a strong binding affinity to ANP32B. Based on this structure, an E3 ubiquitin ligase ligand structure is added to the linker to construct a PROTAC molecule that can degrade ANP32B protein via the ubiquitin-protease system. This ANP32B-targeting peptide degrader can simultaneously bind to both ANP32B and the E3 ubiquitin ligase ligand, forming an ANP32B-degrader-E3 complex. This ubiquitinates ANP32B protein, which is then recognized and degraded by the ubiquitin-protease system, thereby reducing the intracellular ANP32B protein content and inhibiting its function in tumor cells, thus inhibiting the proliferation of breast cancer cells. Considering the large molecular weight and poor membrane permeability of peptide drugs, making it difficult for them to enter the cell membrane and exert their effects, a membrane-penetrating peptide was further introduced into the N-segment to increase the peptide's membrane permeability. Experiments demonstrated that the half-maximal inhibitory concentration (IC50) of this peptide degrader targeting ANP32B protein was 46–67 μM. At the maximum treatment concentration, it could inhibit tumor cell proliferation by less than 10%, achieving tumor proliferation inhibition through ANP32B degradation. It exhibited significant inhibitory activity against MDA-MB-231 breast cancer cells, degrading target proteins in cells and affecting their cell cycle. This makes it suitable for the preparation of anti-tumor drugs and shows promising application prospects in the development of drugs targeting human breast cancer cells, representing another important area in the development of peptide-based PROTAC drugs.

[0020] The method for synthesizing a peptide degrader targeting ANP32B protein provided by this invention is prepared through chemical synthesis. The technology is mature, easy to prepare in large quantities, and has good stability. It has potential application prospects for the development of the currently limited number of peptide-based PROTAC degraders targeting breast cancer. Attached Figure Description

[0021] Figure 1 The graph shows the change of RMSD during molecular dynamics simulation after the ligand sequences (i.e., P1~P4) targeting the ANP32B protein in the degradation agent of the present invention bind to the ANP32B protein to form a stable complex. Figure 2 The graph shows the change in MM / GBSA binding energy during molecular dynamics simulation after the ligand sequences (i.e., P1~P4) targeting the ANP32B protein in the degradation agent of the present invention bind to the ANP32B protein to form a stable complex. Figure 3The diagram shows the interaction interface between the ligand and receptor and the key interacting amino acid residues after the ligand sequence (i.e., P1~P4) targeting the ANP32B protein in the degradation agent of the present invention binds to the ANP32B protein to form a stable complex. Figure 4 shows the results of the MST investigation of the affinity of P1~P4 for protein ANP32B in vitro; where A represents P1 and P2, and B represents P3 and P4. Figure 5 The figure shows the results of the study on the degradation ability of cpPROTAC1~cpPROTAC4 on ANP32B protein in human breast cancer cells MDA-MB-231 using Western blotting technology. Figure 6 The figure shows the results of the investigation on the inhibitory effect of cpPROTAC1~cpPROTAC4 of the present invention on the proliferation of human breast cancer cells MDA-MB-231. Figure 7 The image shows the cell cycle results of human breast cancer cells MDA-MB-231 investigated by cpPRAOTAC1~cpPROTAC4 of the present invention; the left side is a flow cytometry plot, and the right side is a statistical plot of cell cycle distribution results. Figure 8 The figure shows the results of the toxic side effects study of cpPROTAC1~cpPROTAC4 of the present invention based on the proliferation of non-tumor cells NIH 3T3. Detailed Implementation

[0022] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0023] Unless otherwise specified, in this article, "Ac" in polypeptide structures represents the acetyl group, "Acp" represents 6-aminocaproic acid, "Linker" represents a linker, and "NH2" represents an amino group. In the amino acid sequence, R represents arginine, Y represents tyrosine, H represents histidine, K represents lysine, W represents tryptophan, T represents threonine, Q represents glutamine, I represents isoleucine, A represents alanine, and P(OH) represents L-hydroxyproline.

[0024] This invention provides a peptide degrader targeting ANP32B protein, which is formed by linking an ANP32B protein targeting ligand (amino acid sequence shown in SEQ ID NO.1~SEQ ID NO.4 in Table 1) and a peptide ligand of E3 ubiquitin ligase VHL (amino acid sequence: IAP(OH)YI-NH2) via a linker (6-aminocaproic acid, Acp), and then modifying the N-terminus with a cationic cell membrane permeability peptide (amino acid sequence: RRRRRR). The amino acid sequence of the peptide degrader targeting ANP32B protein is shown in SEQ ID NO.5~SEQ ID NO.8 in Table 1.

[0025] Table 1 Amino acid sequence listing

[0026] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0027] The experimental drugs and reagents used in the following examples include: Rink Amide-MBHA resin (Bai Ling Wei), Fmoc protected amino acids, piperidine (Sinopharm Group), DMF (Comeo Chemical Reagent), DCM (Comeo Chemical Reagent), HATU (Bide Pharmaceutical), HOBt (Bide Pharmaceutical), DIPEA (Bide Pharmaceutical), PyBop (Bide Pharmaceutical), TFA (Aladdin), TIS (Aladdin), 1,3-dimethoxybenzene (Aladdin), DODT (Aladdin), chromatographic acetonitrile (Aladdin), PBS buffer (CORNING), L-15 (Kaiji Biotechnology), penicillin-streptomycin solution (double antibody) (MCE), fetal bovine serum (NEWZERUM), Trypsin (CORNING), DMSO (MP Biomedicals), Monolith NT 115 protein labeling kit (NanoTemper), MTT (Solarbio), HEPES (supplemented with 0.1% Pluronic F127, 1 mM TECP) buffer, and cell cycle kit (Elabscience). The cells used in the experiments included MDA-MB-231 (human breast cancer cells). All other raw materials used, unless otherwise specified, were conventional commercially available products of standard specifications in the art. Standard instruments and equipment in the art were used. Experimental methods in the following examples, unless otherwise specified, were generally performed under standard conditions or as recommended by the manufacturer.

[0028] I. Molecular Dynamics Simulation Investigation To verify that the ligands (P1-P4; amino acid sequences shown in Table 1, SEQ ID NO.1-SEQ ID NO.4) in the peptide degrader of this invention continuously and stably interact with the protein during binding, molecular dynamics simulation experiments were conducted. The binding stability of the complex formed by the ANP32B protein and the ligands (P1-P4) was evaluated using Desmond (v53011) software. Specifically, the complex structure was imported into the System Builder module, and solvation was performed using the TIP3P water model and OPLS3 force field. To ensure the complex was completely encapsulated in the simulated solvent environment, we set the orthogonal box shape, the boundary condition parameters of the buffer calculation method (10 Å), and selected optimized volume. Na+ was added. + To simulate physiological saline conditions, the complex was then kinetically simulated using the Molecular Dynamics module with default parameters. The simulation duration was set to 100 ns, and trajectory frames were recorded at 100 ps intervals. Finally, the output trajectory file of the kinetic simulation was analyzed using the Simulation Interaction Diagram function. The fluctuation range of the structure was examined by calculating the root mean square deviation (RMSD) of the structure in all trajectory frames. Figure 1 Furthermore, structures in the trajectory were extracted at 10 ns intervals, and the MM / GBSA algorithm was used to calculate the change in protein-ligand complex binding energy during the kinetic simulation. Figure 2 This allows for verification of the stability of the complex, enabling subsequent biological experiments.

[0029] MD results analysis revealed that the RMSD fluctuations of the four complexes stabilized within the range of 2.5 Å in the later stages of the simulation or for most of the simulation time. MM / GBSA calculations indicated that the binding energy between the peptide and protein was within the range of 60–80 kcal / mol, which is considered ideal. Further analysis of protein-peptide interactions revealed that the stability of the complex was significantly improved when the peptide contained both long-chain basic amino acids such as lysine and arginine, and aromatic heterocyclic amino acids such as tyrosine and tryptophan. Figure 3As shown, P1 to P4 all satisfy the above structural characteristics and can effectively bind to the hydrophobic groove of the ANP32B protein. The binding mode is as follows: aromatic amino acids can form a key pi-pi stacking interaction with the amino acid HIS92 deep in the hydrophobic groove of the protein, thus making HIS92 a binding anchor point; long side-chain basic amino acids can bind to ASP119 or GLU70 at the edge of the groove through hydrogen bonds, salt bridges and other interactions to form a charge-complementary interface, like a "claw structure" tightly gripping the edge of the groove, significantly reducing the conformational perturbation of the peptide.

[0030] II. Synthesis of Polypeptides 1. Synthesis of ANP32B protein-targeted binding ligands 1) Synthesis of ANP32B protein targeting and binding ligand P1

[0031] Add 50.0 mg (0.32 mmol / g, 0.016 mmol) of Rink Amide-Resin resin to a centrifuge column, add 5 mL of DMF / DCM (1:1) to swell the resin for 1 h, and then filter under reduced pressure and discard the filtrate. At room temperature, add 2 mL of a solution containing 20% ​​piperidine / DMF to the centrifuge column for 10 minutes to remove the FMOC protecting group. Filter the filtrate under reduced pressure, repeating this process twice to completely remove the Fmoc protecting group at the N-terminus of the Rink Amide-MBHA resin. After filtration, thoroughly wash the resin with DCM / DMF. The washing method is as follows: add 2 mL of DMF to the centrifuge column, tighten the cap, invert the column 5 times, then open the cap and the bottom seal, filter under reduced pressure, and repeat three times. Then switch to DCM for washing, repeating 3 times, and finally switch to DMF for washing three times. Fmoc-protected L-amino acids (0.08 mmol) were reacted with 42.1 mg (0.08 mmol) of Fmoc-Trp(Boc)-OH (CAS:143824-78-6) in a DMF environment containing HATU (30.3 mg, 0.08 mmol), HOBt (12.2 mg, 0.08 mmol) and DIPEA (37.6 μL, 0.08 mmol). After the reaction was completed, the mixture was filtered to obtain intermediate 1a.

[0032]

[0033] Intermediate 1a was washed with DMF / DCM, filtered, and then subjected to deFMOC protection by adding 2 mL of DMF containing 20% ​​piperidine at room temperature for 10 minutes. After filtration, it was thoroughly washed with DCM / DMF, and the process was repeated twice. Subsequently, it was reacted with 37.5 mg (0.08 mmol) of Fmoc-Lys(Boc)-OH (CAS:71989-26-9) in DMF containing HATU, HOBt, and DIPEA. After the reaction was completed, it was filtered to obtain intermediate 1b.

[0034]

[0035] Intermediate 1b was washed with DMF / DCM, filtered, and then deprotected with DMF containing 20% ​​piperidine. After filtration, it was thoroughly washed with DCM / DMF. Then, it was reacted with 49.6 mg (0.08 mmol) of Fmoc-His(Trt)-OH (CAS:109425-51-6) in DMF containing HATU, HOBt, and DIPEA. After the reaction was complete, it was filtered to obtain intermediate 1c.

[0036]

[0037] Intermediate 1c was washed with DMF / DCM, filtered, and then subjected to deFMOC protection by adding 2 mL of DMF containing 20% ​​piperidine at room temperature for 10 minutes. After filtration, it was thoroughly washed with DCM / DMF, and the process was repeated twice. Subsequently, it was reacted with 36.8 mg (0.08 mmol) of Fmoc-Tyr(tBu)-OH (CAS:71989-38-3) in DMF containing HATU, HOBt, and DIPEA. After the reaction was completed, it was filtered to obtain intermediate 1d.

[0038]

[0039] Intermediate 1d was washed with DMF / DCM, filtered, and then subjected to deFMOC protection by adding 2 mL of DMF containing 20% ​​piperidine at room temperature for 10 minutes. After filtration, it was thoroughly washed with DCM / DMF, and the process was repeated twice. Subsequently, it was reacted with 51.9 mg (0.08 mmol) of Fmoc-Arg(Pbf)-OH (CAS:154445-77-9) in DMF containing HATU, HOBt, and DIPEA. After the reaction was completed, it was filtered to obtain intermediate 1e.

[0040]

[0041] Intermediate 1e was washed with DMF / DCM, filtered, and then deprotected with DMF containing 20% ​​piperidine, followed by thorough washing with DCM / DMF. Next, 88 μL of DIPEA was added to 2 mL of DCM, mixed well, and then 48 μL of acid anhydride was added to the solution. After thorough mixing, the mixture was poured into a centrifuge tube for acetylation capping. The reaction was carried out at room temperature for 20 minutes, repeated twice. Then, TFA / TIS / H2O / DODT / 1,3-dimethoxybenzene (volume ratio 90:2.5:2.5:2.5:2.5) was added, and the reaction was carried out at room temperature for 3 hours for lysis. The ether was pre-cooled at -80°C. After lysis, the resin tube was rinsed with DCM, and the filtrate was collected into a 50 mL centrifuge tube. After evaporation to approximately 2 mL, approximately 20 mL of anhydrous refrigerated ether was added, precipitating a solid. The solid was centrifuged at low temperature for 15 minutes at 12000 rpm, and the supernatant was discarded to obtain the crude peptide. The crude peptide was purified by linear gradient elution on a Shimadzu LC-2030 using a Shim-pack Scepter C18-120 (5 μm, 10 mm × 250 mm) to obtain product P1 (amino acid sequence: Ac-RYHKW-NH2). Purity analysis was performed using a Shimadzu LC-2030C3D Plus equipped with a PDA detector using a Shim-pack Scepter C18-120 (5 μm, 4.6 mm × 250 mm). The purity of the purified peptide was >90.0% at 254 nm.

[0042] The synthesis methods for ANP32B protein-targeting ligands P2, P3, and P4 are the same as those for ANP32B protein-targeting ligand P1, except that the amino acid sequence RYHKW is replaced with amino acids RWTKR, RYKWQ, or WRRIQ sequentially linked from the C-terminus.

[0043] 2. Synthesis of peptide degraders targeting ANP32B protein 1) Synthesis of cpPROTAC1, a peptide degrader targeting ANP32B protein

[0044] Rink Amide-Resin (50.0 mg, 0.32 mmol / g, 0.016 mmol) was swollen in 5 mL of DMF / DCM (1:1), filtered, and then DMF containing 20% ​​piperidine was added to remove the FMOC protecting group, exposing the primary amine group. After washing with DCM / DMF, it was reacted with 28.27 mg (0.08 mmol) of Fmoc-Ile-OH (CAS:71989-23-6) in a DMF environment containing HATU, HOBt, and DIPEA. After the reaction was complete, it was filtered to obtain intermediate 5a.

[0045]

[0046] Intermediate 5a was washed with DMF / DCM, filtered, and then deprotected with DMF containing 20% ​​piperidine. After filtration, it was thoroughly washed with DCM / DMF. Then, it was reacted with 36.8 mg (0.08 mmol) of Fmoc-Tyr(tBu)-OH (CAS:71989-38-3) in DMF containing HATU, HOBt, and DIPEA. After the reaction was complete, it was filtered to obtain intermediate 5b.

[0047]

[0048] Intermediate 5b was washed with DMF / DCM, filtered, and then deprotected with DMF containing 20% ​​piperidine. After filtration, it was thoroughly washed with DCM / DMF. Then, it was reacted with 32.8 mg (0.08 mmol) of Fmoc-Hyp(tBu)-OH (CAS:122996-47-8) in DMF containing HATU, HOBt, and DIPEA. After the reaction was complete, it was filtered to obtain intermediate 5c.

[0049]

[0050] Intermediate 5c was washed with DMF / DCM, filtered, and then deprotected with DMF containing 20% ​​piperidine. After filtration, it was thoroughly washed with DCM / DMF. Then, it was reacted with 32.8 mg (0.08 mmol) of Fmoc-Ala-OH (CAS:122996-47-8) in DMF containing HATU, HOBt, and DIPEA. After the reaction was complete, it was filtered to obtain intermediate 5d.

[0051]

[0052] Intermediate 5d was washed with DMF / DCM, filtered, and then deprotected with DMF containing 20% ​​piperidine. After filtration, it was thoroughly washed with DCM / DMF. Then, it was reacted with 28.27 mg (0.08 mmol) of Fmoc-Ile-OH (CAS:71989-23-6) in DMF containing HATU, HOBt, and DIPEA. After the reaction was complete, it was filtered to obtain intermediate 5e.

[0053]

[0054] Intermediate 5e was washed with DMF / DCM, filtered, and then deprotected with DMF containing 20% ​​piperidine. After filtration, it was thoroughly washed with DCM / DMF. Then, it was reacted with 28.3 mg (0.08 mmol) of Fmoc-Ile-OH (CAS:88574-06-5) in DMF containing HATU, HOBt, and DIPEA. After the reaction was complete, it was filtered to obtain intermediate 5f.

[0055]

[0056] Intermediate 5f was washed with DMF / DCM, filtered, and then deprotected with DMF containing 20% ​​piperidine. After filtration, it was thoroughly washed with DCM / DMF. Then, it was reacted with 42.1 mg (0.08 mmol) of Fmoc-Trp(Boc)-OH (CAS:143824-78-6) in DMF containing HATU, HOBt, and DIPEA. After the reaction was complete, it was filtered to obtain 5 g of intermediate.

[0057]

[0058] 5 g of the intermediate was washed with DMF / DCM, filtered, and then DMF containing 20% ​​piperidine was added to remove the FMOC protecting group. After filtration, it was thoroughly washed with DCM / DMF. Then, it was reacted with 37.5 mg (0.08 mmol) of Fmoc-Lys(Boc)-OH (CAS:71989-26-9) in DMF containing HATU, HOBt, and DIPEA. After the reaction was complete, it was filtered to obtain the intermediate 5 h.

[0059]

[0060] Intermediate 5h was washed with DMF / DCM, filtered, and then deprotected with DMF containing 20% ​​piperidine. After filtration, it was thoroughly washed with DCM / DMF. Then, it was reacted with 49.6 mg (0.08 mmol) of Fmoc-His(Trt)-OH (CAS:109425-51-6) in DMF containing HATU, HOBt, and DIPEA. After the reaction was complete, it was filtered to obtain intermediate 5i.

[0061]

[0062] Intermediate 5i was washed with DMF / DCM, filtered, and then deprotected with DMF containing 20% ​​piperidine. After filtration, it was thoroughly washed with DCM / DMF. Then, it was reacted with 36.8 mg (0.08 mmol) of Fmoc-Tyr(tBu)-OH (CAS:71989-38-3) in DMF containing HATU, HOBt, and DIPEA. After the reaction was complete, it was filtered to obtain intermediate 5j.

[0063]

[0064] Intermediate 5j was washed with DMF / DCM, filtered, and then deprotected with DMF containing 20% ​​piperidine. After filtration, it was thoroughly washed with DCM / DMF. Then, it was reacted with 36.8 mg (0.08 mmol) of Fmoc-Arg(Pbf)-OH (CAS:71989-38-3) in DMF containing HATU, HOBt, and DIPEA. After the reaction was complete, it was filtered to obtain intermediate 5k. This process was repeated 6 times to continue linking 6 arginine residues, yielding intermediate 5l.

[0065]

[0066] Intermediate 5L was washed with DMF / DCM, filtered, and then DMF containing 20% ​​piperidine was added to remove the FMOC protecting group. After filtration, it was thoroughly washed with DCM / DMF. Then, 88 μL of LIPEA was added to 2 mL of DCM, mixed well, and then 48 μL of acid anhydride was added to the solution. After thorough mixing, the solution was poured into the peptide for acetylation capping. The reaction was repeated twice for 20 minutes. Next, TFA / TIS / H2O / DODT / 1,3-dimethoxybenzene (volume ratio 90:2.5:2.5:2.5:2.5) was added, and the reaction was carried out at room temperature for 3 h for lysis. After lysis, the resin tube was rinsed with DCM, the filtrate was collected, and after a certain amount evaporated, anhydrous refluxed ether was added to precipitate the solid. The solid was centrifuged at low temperature for 15 min at 12000 rpm, and the supernatant was discarded to obtain the crude peptide. The crude peptide was purified by linear gradient elution using a Shim-pack Scepter C18-120 (5 μm, 10 mm × 250 mm) on a Shimadzu LC-2030 to obtain the product cpPROTAC1 (amino acid sequence shown in SEQ ID NO. 5 in Table 1). Purity analysis was performed using a Shimadzu LC-2030C 3D Plus equipped with a PDA detector using a Shim-pack Scepter C18-120 (5 μm, 4.6 mm × 250 mm). The purity of the purified peptide was >90.0% at 254 nm.

[0067] 2) Synthesis of cpPROTAC2, a peptide degrader targeting ANP32B protein

[0068] Intermediate 5f (synthetic method as described in cpPROTAC1) was washed with DMF / DCM, filtered, and then subjected to deFMOC protection by adding 2 mL of DMF containing 20% ​​piperidine at room temperature for 10 minutes. After filtration, it was thoroughly washed with DCM / DMF, and the process was repeated twice. Then, it was reacted with 51.9 mg (0.08 mmol) of Fmoc-Arg(Pbf)-OH (CAS: 154445-77-9) in DMF containing HATU, HOBt, and DIPEA. After the reaction was complete, it was filtered to obtain intermediate 6a.

[0069]

[0070] Intermediate 6a was washed with DMF / DCM, filtered, and then subjected to deFMOC protection by adding 2 mL of DMF containing 20% ​​piperidine at room temperature for 10 minutes. After filtration, it was thoroughly washed with DCM / DMF, and the process was repeated twice. Subsequently, it was reacted with 37.5 mg (0.08 mmol) of Fmoc-Lys(Boc)-OH (CAS:71989-26-9) in DMF containing HATU, HOBt, and DIPEA. After the reaction was completed, it was filtered to obtain intermediate 6b.

[0071]

[0072] Intermediate 6b was washed with DMF / DCM, filtered, and then subjected to deFMOC protection by adding 2 mL of DMF containing 20% ​​piperidine at room temperature for 10 minutes. After filtration, it was thoroughly washed with DCM / DMF, and the process was repeated twice. Subsequently, it was reacted with 31.8 mg (0.08 mmol) of Fmoc-Thr(tBu)-OH (CAS:71989-35-0) in DMF containing HATU, HOBt, and DIPEA. After the reaction was completed, it was filtered to obtain intermediate 6c.

[0073]

[0074] Intermediate 6c was washed with DMF / DCM, filtered, and then subjected to deFMOC protection by adding 2 mL of DMF containing 20% ​​piperidine at room temperature for 10 minutes. After filtration, it was thoroughly washed with DCM / DMF, and the process was repeated twice. Subsequently, it was reacted with 42.1 mg (0.08 mmol) of Fmoc-Trp(Boc)-OH (CAS:143824-78-6) in DMF containing HATU, HOBt, and DIPEA. After the reaction was completed, it was filtered to obtain intermediate 6d.

[0075]

[0076] Intermediate 6d was washed with DMF / DCM, filtered, and then subjected to deFMOC protection by adding 2 mL of DMF containing 20% ​​piperidine at room temperature for 10 minutes. After filtration, it was thoroughly washed with DCM / DMF, and the process was repeated twice. Subsequently, it was reacted with 51.9 mg (0.08 mmol) of Fmoc-Arg(Pbf)-OH (CAS:154445-77-9) in DMF containing HATU, HOBt, and DIPEA. After the reaction was completed, it was filtered to obtain intermediate 6e.

[0077] After the above amino acid linkage is completed, the subsequent processing methods, such as linking the transmembrane peptide, N-terminal acetylation, and cleavage, are the same as those described in the preparation method of cpPROTAC1. The ligand polypeptide cpPTORAC2 is obtained (amino acid sequence shown as SEQ ID NO.6 in Table 1).

[0078] 3) Synthesis of cpPROTAC3, a peptide degrader targeting ANP32B protein

[0079] Intermediate 5f (synthetic method as described in cpPROTAC1) was washed with DMF / DCM, filtered, and then subjected to deFMOC protection by adding 2 mL of DMF containing 20% ​​piperidine at room temperature for 10 minutes. After filtration, it was thoroughly washed with DCM / DMF, and this process was repeated twice. Then, it was reacted with 48.86 mg (0.08 mmol) of Fmoc-Gln(Trt)-OH (CAS:132327-80-1) in DMF containing HATU, HOBt, and DIPEA. After the reaction was complete, it was filtered to obtain intermediate 7a.

[0080]

[0081] Intermediate 7a was washed with DMF / DCM, filtered, and then subjected to deFMOC protection by adding 2 mL of DMF containing 20% ​​piperidine at room temperature for 10 minutes. After filtration, it was thoroughly washed with DCM / DMF, and the process was repeated twice. Subsequently, it was reacted with 42.1 mg (0.08 mmol) of Fmoc-Trp(Boc)-OH (CAS:143824-78-6) in DMF containing HATU, HOBt, and DIPEA. After the reaction was completed, it was filtered to obtain intermediate 7b.

[0082]

[0083] Intermediate 7b was washed with DMF / DCM, filtered, and then subjected to deFMOC protection by adding 2 mL of DMF containing 20% ​​piperidine at room temperature for 10 minutes. After filtration, it was thoroughly washed with DCM / DMF, and the process was repeated twice. Subsequently, it was reacted with 37.5 mg (0.08 mmol) of Fmoc-Lys(Boc)-OH (CAS:71989-26-9) in DMF containing HATU, HOBt, and DIPEA. After the reaction was completed, it was filtered to obtain intermediate 7c.

[0084]

[0085] After completing the above amino acid linkage, subsequent linkages of tyrosine, arginine, transmembrane peptides, acetylation, and cleavage are performed as described in the preparation method of cpPROTAC1. The ligand polypeptide cpPROTAC3 is obtained (amino acid sequence shown in SEQ ID NO. 7 in Table 1).

[0086] 4) Synthesis of cpPROTAC4, a peptide degrader targeting ANP32B protein.

[0087] Intermediate 7a (synthesized according to the method described for cpPROTAC3) was washed with DMF / DCM, filtered, and then deprotected with DMF containing 20% ​​piperidine. After filtration, it was thoroughly washed with DCM / DMF. Then, it was reacted with 28.27 mg (0.08 mmol) of Fmoc-Ile-OH (CAS:71989-23-6) in DMF containing HATU, HOBt, and DIPEA. After the reaction was complete, it was filtered to obtain intermediate 8a.

[0088]

[0089] Intermediate 8a was washed with DMF / DCM, filtered, and then deprotected with DMF containing 20% ​​piperidine. After filtration, it was thoroughly washed with DCM / DMF. Then, it was reacted with 36.8 mg (0.08 mmol) of Fmoc-Arg(Pbf)-OH (CAS:71989-38-3) in DMF containing HATU, HOBt, and DIPEA. After the reaction was complete, it was filtered to obtain intermediate 8b. This process was repeated to obtain intermediate 8c.

[0090]

[0091] Intermediate 8c was washed with DMF / DCM, filtered, and then subjected to deFMOC protection by adding 2 mL of DMF containing 20% ​​piperidine at room temperature for 10 minutes. After filtration, it was thoroughly washed with DCM / DMF, and the process was repeated twice. Then, it was reacted with 42.1 mg (0.08 mmol) of Fmoc-Trp(Boc)-OH (CAS:143824-78-6) in DMF containing HATU, HOBt, and DIPEA. After the reaction was complete, it was filtered to obtain intermediate 8d.

[0092] After the above amino acid linkage is completed, the subsequent processing methods such as linkage of the transmembrane peptide, N-terminal acetylation and cleavage are the same as those for the preparation of cpPROTAC1, to obtain the ligand polypeptide cpPTORAC4 (amino acid sequence shown in SEQ ID NO.8 in Table 1).

[0093] III. Micro-thermophoresis (MST) The interaction between peptide ligands and ANP32B was studied using microthermophoresis. ANP32B protein (purchased from GenScript) was analyzed using Monolithic Histogram. Tag Labeling Kit RED tris NTA second-generation dye labeling. Various concentrations of peptide ligand candidates (0.1 nM–100 μM) were titrated with labeled ANP32B (125 nM) in HEPES buffer (supplemented with 0.1% Pluronic F127, 1 mM TECP). Samples were aspirated into capillaries and then measured in an MST instrument. Data were processed using MO Affinity Analysis software (NanoTemper Technologies GmbH, Munich, Germany) to analyze binding constants.

[0094] The MST method was used to detect the interaction between the above-mentioned peptide ligand candidates and the ANP32B protein. The results are shown in Figure 4. It can be seen that peptides P1–P4 have different degrees of affinity for ANP32B. Steady-state analysis was performed based on the recorded data. The dissociation constants (Kd) calculated from the fitted saturation binding curves were 2.01 nM (P1), 11.3 nM (P2), 12.0 nM (P3), and 1.21 nM (P4), respectively. These results indicate that nanomolar-level peptides have a very strong binding affinity to ANP32B. Therefore, these sequences were further designed as peptide-based PROTAC degraders.

[0095] IV. Western blot analysis of the degradation level of intracellular ANP32B protein by peptide degrading agents Based on the protein affinity of P1-P4, the peptide structure was further derivatized to obtain peptide degraders cpPROTAC1-cpPROTAC4, which exerted their intracellular protein degradation capabilities. To investigate the degradation ability of the peptide degraders on intracellular proteins, the intracellular ANP32B protein content was evaluated after co-incubation with the drugs. MDA-MB-231 cells were used in the experiment and divided into two groups: a control group without drug incubation and a drug group treated with the degraders. The control group was cultured in Leibovitz's L-15 complete medium containing 0.5% (v / v) triple-distilled water (containing 10% fetal bovine serum and 1% penicillin antibiotics); the drug group was cultured in complete medium containing different concentrations (6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM) of the peptide degraders, with the degraders dissolved in triple-distilled water to prepare a stock solution. MDA-MB-231 cells were cultured in serum-free complete medium at 3 × 10⁻⁶ cells / year.5 Cells were seeded at a density of 100 cells / well in 6-well plates and incubated overnight for starvation treatment. The culture medium was then discarded, and the medium described above (control group / drug group) was added. Cells were incubated at 37°C with 5% CO2 for 48 h. After incubation, cells were washed with ice-cold PBS, and RIPA lysis buffer containing the protease inhibitor PMSF was added to each well for cell lysis on ice. The lysis buffer was centrifuged at 12000 g for 10 min at 4°C to extract total cell protein. The protein content of the supernatant was determined using a BCA protein quantification kit. Equal amounts of the collected protein samples were then analyzed by SDS-PAGE. PAGE protein blotting separates proteins of equal concentration.

[0096] The results are as follows Figure 5 As shown, compared with the control group that was not incubated with the degradation agent, the level of ANP32B protein in the cells of the drug group decreased significantly after the drug concentration was higher than 12.5~25 μM. This indicates that the peptide degradation agent of the present invention can degrade intracellular ANP32B protein. It also proves that the ligand sequence of the peptide degradation agent of the present invention that targets the ANP32B protein can achieve targeting of ANP32B protein, laying the foundation for designing other derivatives that target this protein.

[0097] V. Experiment on Anti-breast cancer tumor cell proliferation activity 1. Set up a blank group, a control group, and an experimental group. The blank group was given only Leibovitz's L-15 complete medium, while the control and experimental groups were given an equal volume of cell suspension (logarithmic growth phase human breast cancer cells MDA-MB-231 were used for the experiment, at a concentration of 1×10⁻⁶). 4 180 μL of cells per well were seeded into a 96-well plate and incubated overnight at 37°C in a 5% CO2 cell culture incubator.

[0098] 2. Prepare a concentration of 2×10 -2 The stock solutions of peptide degradation agents (cpPROTAC1~cpPROTAC4) were prepared at mol / L. When preparing for the experiment, the stock solutions were serially diluted with culture medium to a total of five concentrations (12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM).

[0099] 3. Add 20 μL of diluted peptide degradation agent stock solution to each well in the drug treatment group, and add an equal volume of culture medium to the blank group and control group. Incubate at 37℃ in a 5% CO2 incubator for 48 h. Remove the 96-well plate and add 22 μL of MTT (5% concentration) to each well, then incubate at 37℃ in a 5% CO2 incubator for 4 h. Discard the supernatant from the wells and add 150 μL of DMSO. Shake in the dark for 15 min to fully dissolve the crystals. Measure the absorbance of each well at 490 nm using a multi-mode microplate reader. Calculate the proliferation inhibition rate (IR) according to the inhibition rate-absorbance value formula, and then calculate the IC50 based on the inhibition rate of each concentration. 50 The value was used to characterize the antiproliferative activity of the compound against cells.

[0100]

[0101] The MTT method was used to detect the effect of peptide degrading agents on cell proliferation, and the measured OD value was directly proportional to cell viability. Figure 6 The results showed that after 48 h of treatment with the peptide degrading agent, compared with the Control (0 μM) group, the peptide degrading agent inhibited cell proliferation in a concentration-dependent manner, especially at concentrations higher than 100 μM, where the cell proliferation rate was less than 10%, and the half-maximal inhibitory concentration (IC50) was lower than 10%. 50 The concentrations were 46.1 μM (cpPROTAC1), 67.51 μM (cpPROTAC2), 58.21 μM (cpPROTAC3), and 58.47 μM (cpPROTAC4), respectively.

[0102] VI. Cell cycle experiments to investigate the effects of degradation agents on the cell cycle MDA-MB-231 cells in logarithmic growth phase were used for experiments. Seeding and drug administration procedures were the same as for Western blotting. After co-incubating the cells with the drug for 48 h, the six-well plates were removed, digested with trypsin, and centrifuged at 300 g for 5 min at 4°C. The supernatant was discarded, and the cells were washed twice with PBS. The cell pellet was gently mixed with 1 mL of pre-chilled 70% ethanol, fixed overnight at -20°C, and then centrifuged at 300 g for 5 min at 4°C to precipitate the cells. The supernatant was discarded, and the cells were washed twice with PBS. 100 μL of RNase A was added, and the cells were incubated at 37°C for 30 min. Then, 400 μL of propidium iodide staining solution was added to each cell sample, and the mixture was gently mixed. The cells were incubated at 4°C in the dark for 30 min, mixed, and then placed on ice. The samples were analyzed using a NovoCyte Flow Cytometer within 1 h. The data were analyzed using Novoexpress.

[0103] The effects of CPP1-CPP4 on the cell cycle distribution of MDA-MB-231 cells are as follows: Figure 7As shown, the four peptide degrading agents described in this invention significantly increased the number of cells in the G0 / G1 phase, while decreasing the proportion of cells in the S and G2 / M phases, indicating that the degrading agents arrested cells in the G1 phase. In particular, the proportion of cells in the G1 phase showed a concentration-dependent increase with increasing drug concentration. This is consistent with the literature (Yang S, Zhou L, Reilly PT, Shen SM, He P, Zhu XN, Li CX, Wang LS, Mak TW, Chen GQ, Yu Y. ANP32B deficiency impairs proliferation and suppressestumor progression by regulating AKT phosphorylation. Cell Death Dis. 2016 Feb4;7(2):e2082.) which reported that reducing ANP32B protein expression through gene knockout resulted in cell arrest in the G1 phase. This indicates that this invention achieves a reduction in ANP32B protein content at the protein level rather than the gene level, while achieving an equivalent effect.

[0104] VII. Investigation of Drug Toxicity and Side Effects Non-tumor cells, NIH 3T3 cells (embryonic fibroblasts), in the logarithmic growth phase were used for experiments, and the experimental methods were the same as those for the anti-breast cancer tumor cell proliferation activity experiment.

[0105] The MTT method was used to detect the effect of peptide degrading agents on the survival and growth of non-tumor cells, and the measured OD value was proportional to cell activity. Figure 8 The results showed that, at the same administered concentration, only cpPRAOTAC1 had an inhibitory effect on cell proliferation (cell survival rate of about 60%) at the highest administered concentration (200 μM). The other drugs did not show significant concentration-dependent inhibition of cell proliferation at any concentration, indicating that the degrading agents would not exert their effect in cells that do not highly express ANP32B protein, thus avoiding toxic side effects on normal cells.

[0106] In summary, the four degradative agents P1-P4 obtained by further structural derivatization using P1-P4 as lead compounds all exhibited good inhibitory activity against the MDA-MB-231 breast cancer cell line, arresting the cells in the G0 / G1 phase and dose-dependently degrading ANP32B protein.

[0107] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A peptide degrader targeting ANP32B protein, characterized in that, The peptide degrader contains a membrane-penetrating peptide and an ANP32B protein-targeting ligand at its N-terminus, and a peptide ligand motif of the E3 ubiquitin ligase VHL at its C-terminus. The ANP32B protein-targeting ligand and the peptide ligand of the E3 ubiquitin ligase VHL are linked by a linker. The amino acid sequences of the ANP32B protein targeting and binding ligand are shown in SEQ ID NO.1 to SEQ ID NO.

4.

2. The peptide degrader targeting ANP32B protein according to claim 1, characterized in that, The general structural formula of the peptide degrader targeting the ANP32B protein is: Among them, R1 is an arginine or tryptophan side chain, R2 is a tyrosine, tryptophan or arginine side chain, R3 is a histidine, threonine, lysine or arginine side chain, R4 is a lysine, tryptophan or isoleucine side chain, and R5 is a tryptophan, arginine or glutamine side chain.

3. A peptide degrader targeting ANP32B protein according to claim 2, characterized in that, When R3 is a histidine side chain, R1 is an arginine side chain, R2 is a tyrosine side chain, R4 is a lysine side chain, and R5 is a tryptophan side chain. The amino acid sequence of the peptide degrader targeting the ANP32B protein is shown in SEQ ID NO.

5.

4. A peptide degrader targeting ANP32B protein according to claim 2, characterized in that, When R3 is a threonine side chain, R1 is an arginine side chain, R2 is a tryptophan side chain, R4 is a lysine side chain, and R5 is an arginine side chain. The amino acid sequence of the peptide degrader targeting the ANP32B protein is shown in SEQ ID NO.

6.

5. A peptide degrader targeting ANP32B protein according to claim 2, characterized in that, When R3 is a lysine side chain, R1 is an arginine side chain, R2 is a tyrosine side chain, R4 is a tryptophan side chain, and R5 is a glutamine side chain. The amino acid sequence of the peptide degrader targeting the ANP32B protein is shown in SEQ ID NO.

7.

6. A peptide degrader targeting ANP32B protein according to claim 2, characterized in that, When R3 is an arginine side chain, R1 is a tryptophan side chain, R2 is an arginine side chain, R4 isoleucine side chain, and R5 is a glutamine side chain. The amino acid sequence of the peptide degrader targeting ANP32B protein is shown in SEQ ID NO.

8.

7. A method for synthesizing a polypeptide degrader targeting ANP32B protein as described in any one of claims 1 to 6, characterized in that, A peptide degrader targeting ANP32B protein was prepared by an artificial solid-phase synthesis method based on FMOC amino acids.

8. The use of the peptide degrader targeting ANP32B protein as described in any one of claims 1 to 6 in the preparation of antitumor drug formulations.

9. The application according to claim 8, characterized in that, The anti-tumor drug is a drug for human breast cancer tumor cells.

10. An anti-breast cancer composition, characterized in that, It includes other active ingredients with anti-breast cancer activity, a peptide degrader targeting ANP32B protein as described in any one of claims 1 to 6, and one or more pharmaceutically acceptable excipients.

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