A polypeptide chimera targeting p53 protein stabilization, and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a polypeptide chimera targeting p53 protein stability and a preparation method and application thereof. The polypeptide chimera is a full polypeptide structure, which is connected by a USP7 recruiting domain, a p53 binding domain and a polypeptide linker containing an arginine chain, and has a general structure of A-L-B. The chimera can induce endogenous USP7 and p53 to be spatially adjacent as a molecular bridge, specifically removes the polyubiquitin chain of p53, up-regulates the p53 protein level in a concentration and time-dependent manner, prolongs the half-life of the p53 protein, activates the p21 downstream anticancer pathway, and the effect strictly depends on USP7. The chimera has high biological safety, is simple to synthesize, and provides a new strategy for the treatment of p53 function loss related tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a peptide chimera that targets and stabilizes the p53 protein, its preparation method, and its application. Background Technology

[0002] Malignant tumors are a major public health problem that seriously threatens human life and health, and the inactivation of intracellular tumor suppressor pathways is one of the core driving factors of tumor occurrence and development. Among them, the tumor suppressor protein p53, as the "genomic guardian" in cells, plays a decisive regulatory role in key physiological processes such as cell cycle arrest, DNA damage repair, apoptosis induction, and tumor cell proliferation inhibition, and is a recognized core target in the field of tumor targeted therapy.

[0003] Under physiological conditions, the expression level and activity of intracellular p53 protein are precisely regulated by the ubiquitin-proteasome system, with the dynamic balance between ubiquitination and deubiquitination being the core mechanism for maintaining p53 protein homeostasis. However, in various malignant tumor cells, even without loss-of-function mutations in the p53 gene, its protein level remains extremely low due to uncontrolled proteasome degradation caused by excessive ubiquitination, ultimately leading to complete inactivation of the p53-mediated tumor suppressor pathway and conferring a malignant phenotype of unlimited tumor cell proliferation and apoptosis resistance. Therefore, restoring the physiological homeostasis and tumor suppressor function of intracellular p53 protein by targeting the ubiquitination and degradation process has become a key research direction and core strategy in the field of targeted cancer therapy.

[0004] Currently, global drug development for restoring p53 activity mainly focuses on small molecule inhibitors that target the interaction between the E3 ubiquitin ligase MDM2 and p53. These drugs reduce p53 degradation by blocking the protein-protein interaction between MDM2 and p53, inhibiting MDM2-mediated p53 ubiquitination. However, in preclinical studies and clinical applications, these inhibitors have gradually revealed insurmountable technical defects and application limitations: First, these small molecule inhibitors generally exhibit varying degrees of off-target toxicity and clinically acquired drug resistance, severely limiting their therapeutic window and clinical application prospects; Second, the intracellular homeostasis of p53 protein is synergistically regulated by a complex intracellular ubiquitination-deubiquitination regulatory network. Simply blocking the MDM2-mediated p53 ubiquitination process is often insufficient to completely reverse the excessive degradation of p53 in tumor cells, making it difficult to fully restore p53 protein homeostasis and anti-tumor function.

[0005] Ubiquitin-specific protease 7 (USP7) is a key deubiquitinating enzyme regulating the stability of the p53 protein in cells. It occupies a central position in the p53 ubiquitination-deubiquitination regulatory network, possessing the innate ability to directly and specifically cleave the ubiquitin chain on the p53 protein and inhibit p53 proteasome degradation. However, USP7 exhibits a significant bidirectional regulation of the p53 pathway: under natural physiological conditions, in addition to directly deubiquitinizing and stabilizing p53, USP7 can also stabilize the MDM2 protein through deubiquitination modification, thereby indirectly promoting the ubiquitination and degradation of p53. This bidirectional regulatory characteristic makes it difficult for simple USP7 activators or inhibitors to achieve specific positive regulation of the p53 pathway. Therefore, how to achieve the specific and targeted recruitment of USP7 to p53 protein without interfering with the overall physiological function of USP7, so that the deubiquitination catalytic activity of USP7 can be targeted only to p53 protein and complete the specific erasure of ubiquitin chains on p53 protein, is the core technical challenge that urgently needs to be overcome in the current drug design targeting p53 stability.

[0006] In recent years, the rise of Deubiquitinase-Targeting Chimeras (DUBTAC) technology has provided a novel technical pathway for the targeted regulation of protein homeostasis. This technology constructs heterobifunctional chimeras to simultaneously achieve specific recognition of target proteins and targeted recruitment of endogenous deubiquitinases. This spatial proximity between the target protein and the deubiquitinase allows the natural catalytic activity of the deubiquitinase to actively remove ubiquitin chains from the target protein, achieving specific stabilization and functional restoration of the target protein. Compared to traditional strategies such as protein expression upregulation and ubiquitination blockade, DUBTAC technology has unique advantages, including a clear mechanism of action, strong targeting, and the ability to directly reverse excessive degradation of target proteins. However, most of the reported DUBTAC molecules are heterobifunctional small molecule structures. These small molecule DUBTACs face significant technical bottlenecks in research and application: First, the chemical synthesis routes of small molecule heterobifunctional chimeras are complex, the length and rigidity of linkers are difficult to regulate, and quality control and batch consistency during synthesis are difficult to ensure, which is not conducive to industrialization. Second, small molecule DUBTACs generally have potential cytotoxicity and in vivo metabolic toxicity, resulting in a high risk of drug development. Third, no USP7 recruitment-type DUBTAC molecules based on the all-peptide structure have been reported so far. The all-peptide structure has irreplaceable advantages in drug development compared to the small molecule structure: its in vivo metabolites are natural amino acids, with excellent biosafety and biocompatibility, and it can be flexibly and scalably prepared through mature solid-phase peptide synthesis technology or genetic engineering technology, which can effectively solve the core technical defects of small molecule DUBTACs.

[0007] In summary, current drug development targeting p53 stabilization, whether using MDM2-p53 interaction inhibitors or existing small-molecule DUBTAC technology, suffers from insurmountable technical defects and application limitations. Furthermore, the development of USP7-recruiting full-peptide DUBTACs remains a technological void. Therefore, developing a polypeptide chimera with a well-defined structure, high specificity, high biocompatibility, simple synthesis, and the ability to specifically recruit USP7 to achieve p53 protein stabilization is of significant theoretical and clinical value for the development of targeted cancer therapy. This is the core driving force and foundation for the completion of this invention. Summary of the Invention

[0008] The present invention aims to provide a peptide chimera that targets and stabilizes the p53 protein, its preparation method and application, and to provide a novel treatment strategy for tumors that have lost their anti-cancer function due to excessive ubiquitination and degradation of the p53 protein.

[0009] The objective of this invention is achieved through the following technical solution: This invention provides a full-peptide targeting chimera that targets p53 protein stability. The chimera is composed of a deubiquitinase recruitment domain A, a target protein binding domain B, and a polypeptide linker L, and has the general formula ALB. The deubiquitinase recruitment domain A specifically binds to and recruits endogenous USP7 protein, the target protein binding domain B specifically binds to p53 protein, and the polypeptide linker L is a polypeptide sequence with an arginine chain that promotes cell permeation efficiency. The chimera can act as a molecular bridge to induce spatial proximity between USP7 and p53, remove polyubiquitin chains on the p53 protein through the deubiquitinase activity of USP7, inhibit p53 protein degradation, and maintain p53 protein stability.

[0010] Furthermore, the amino acid sequence of the deubiquitinase recruitment domain A is shown in SEQ ID NO:1.

[0011] Furthermore, the amino acid sequence of the target protein binding domain B is shown in SEQ ID NO:2.

[0012] Furthermore, the polypeptide linker L contains an amino acid sequence consisting of 6 consecutive arginine residues, and the C-terminus of the polypeptide linker L is connected to a lysine residue; the C-terminus of the deubiquitinating enzyme recruitment domain A is connected to the N-terminus of the polypeptide linker L, and the C-terminus of the target protein binding domain B is connected to the free amino group of the side chain of the lysine residue in the polypeptide linker L.

[0013] Furthermore, the amino acid sequence of the full-peptide targeting chimera is shown in SEQ ID NO:3.

[0014] The present invention also provides a method for preparing the p53 protein-stabilized full-peptide targeting chimera, wherein the full-peptide targeting chimera is prepared by solid-phase peptide synthesis process.

[0015] Furthermore, the preparation method employs a solid-phase peptide synthesis process with Fmoc protection strategy, in which amino acid coupling reactions are performed sequentially from the C-terminus to the N-terminus of the chimera. After coupling, the chimera is subjected to cleavage, deprotection, and purification to obtain the full-peptide targeted chimera.

[0016] The present invention also provides the use of the described full-peptide targeting chimera in the preparation of reagents or drugs for stabilizing p53 protein.

[0017] Furthermore, the reagent or drug is used to achieve at least one of the following functions: upregulating intracellular p53 protein levels in a concentration-dependent and time-dependent manner, prolonging the intracellular half-life of p53 protein, reducing the level of the K48-linked ubiquitin chain on p53 protein, and activating downstream signaling pathways of p53 to upregulate p21 protein expression.

[0018] Furthermore, the drug is an anti-tumor drug used to treat tumors associated with p53 deficiency.

[0019] The beneficial effects of this invention are as follows: This invention pioneers a full-peptide deubiquitinating enzyme-targeting chimera targeting the USP7-p53 regulatory axis, filling the technological gap in existing technologies that lack a full-peptide USP7-recruiting DUBTAC. It fundamentally solves the core technical defects of traditional heterobifunctional small molecule DUBTACs, such as complex synthesis, difficulty in rigid linker regulation, and potential cytotoxicity. The metabolites of this invention's full-peptide chimera are natural amino acids, exhibiting excellent biocompatibility. Furthermore, by introducing an arginine-rich sequence into the linker, the chimera acquires self-penetrating membrane capabilities, allowing it to enter cells without the need for additional carriers such as liposomes. This overcomes the industry bottleneck of low cell penetration efficiency commonly found in peptide drugs, significantly improving the druggability of the molecule.

[0020] The polypeptide chimera of this invention possesses a highly specific mechanism of action, enabling precise targeted deubiquitination and stabilization of the p53 protein, effectively avoiding the risk of indirect p53 degradation caused by global USP7 regulation. This invention has been fully validated through USP7 gene knockout experiments, confirming that the stabilizing effect of this chimera on the p53 protein strictly depends on the precise recruitment of USP7, exerting its p53-stabilizing activity only in the presence of USP7. The chimera can act as a molecular bridge to induce spatial proximity between endogenous USP7 and p53, targeting only the ubiquitin chains on the p53 protein without interfering with the global regulatory network of USP7 within the cell. It exhibits strong target and pathway specificity, overcoming the technical challenge of existing USP7 targeting strategies failing to achieve targeted regulation.

[0021] The polypeptide chimera of this invention can efficiently stabilize intracellular p53 protein, exhibiting clear dose- and time-dependent effects. Experimental results confirm that the chimera of this invention can significantly upregulate intracellular p53 protein levels in a concentration- and time-dependent manner, increasing p53 protein levels to 260% of the control group at a dosage of 10 μM, and achieving optimal stabilization approximately 12 hours after administration. Simultaneously, this chimera does not alter p53 mRNA transcription levels, acting solely through post-translational regulatory mechanisms. It can significantly prolong the intracellular half-life of p53 protein, reverse the rapid degradation of p53 protein in tumor cells, and inhibit excessive p53 ubiquitination and degradation at its source by specifically clearing the K48 ubiquitin chain of p53 protein that mediates proteasome degradation.

[0022] The polypeptide chimera of this invention can effectively restore the anti-tumor biological function of p53 and has clear potential for the development of anti-tumor drugs. Experimental results confirm that the p53 protein stabilized by the chimera of this invention can perform its transcriptional regulatory function normally, significantly upregulate the expression level of the downstream cell cycle repressor protein p21, and successfully reactivate the p53 anti-tumor signaling pathway. This provides a novel treatment strategy for tumors that have lost their anti-tumor function due to excessive ubiquitination and degradation of p53 protein, while avoiding the drug resistance and off-target toxicity problems of existing MDM2-p53 interaction small molecule inhibitors.

[0023] The full-peptide chimera of this invention features a simple and easy-to-implement preparation process, which is conducive to industrialization and large-scale application. The peptide chimera of this invention can be prepared using a mature commercial solid-phase synthesis process. Compared to the complex multi-step organic synthesis required for small molecule DUBTAC, the preparation process of this invention is controllable, has simple quality control, and good batch-to-batch consistency, enabling large-scale and standardized production and demonstrating excellent prospects for industrial application. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a simulation diagram of the molecular docking between USP7NTD and EBNA1-p; where, Figure 1 A is a schematic diagram of the structure of the EBNA1-p polypeptide ligand that binds to USP7NTD. The sequence of the polypeptide is shown in SEQ ID NO:1. Figure 1 B is a three-dimensional binding pattern diagram of the docking between USP7NTD (PDBID:1YY6) and the EBNA1-p polypeptide ligand. In the diagram, the gray solid surface represents USP7NTD and the colored solid surface represents the EBNA1-p polypeptide ligand. Figure 1 C is a two-dimensional binding pattern diagram of the docking between USP7NTD and the EBNA1-p polypeptide ligand molecule, used to show the key binding amino acid residues and interaction mode between the two. Figure 2 This is a simulation diagram of the molecular docking between p53 and its polypeptide ligand; where, Figure 2 A is a schematic diagram of the structure of the polypeptide ligand that binds to the p53 protein. The sequence of the polypeptide is shown in SEQ ID NO:2. Figure 2 B is a three-dimensional binding pattern diagram of the docking between the p53 protein and its polypeptide ligand molecules. In the diagram, the gray solid surface represents the p53 protein, and the colored solid surface represents the p53 polypeptide ligand. Figure 2 C is a two-dimensional binding pattern diagram of the docking between the p53 protein and its polypeptide ligand molecule, used to show the hydrogen bond binding sites and key interacting amino acid residues. Figure 3 Schematic diagram of p53-DUBTAC design; Figure 4 This image shows the in vitro and intracellular validation of p53-DUBTAC's ability to bind to USP7; among them... Figure 4 A is the steady-state analysis curve of the binding affinity constant between p53-DUBTAC and the TRAF truncated protein of USP7, obtained by biomembrane interferometry. Figure 4 B is a Western blot result of USP7 protein in cell lysates of the control group and the p53-DUBTAC (10 μM) treatment group under temperature gradient heating conditions, used to verify the binding ability of p53-DUBTAC to USP7 in living cells. Figure 5The image shows the results of time- and concentration-dependent stabilization of p53-DUBTAC in AGS cells; among them, Figure 5 Figure A shows the Western blot results of p53 protein levels in AGS cells after treatment with p53-DUBTAC at concentration gradients of 0 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM for 24 h. Figure 5 B is the result of using ImageJ software. Figure 5 Figure A shows the grayscale quantitative analysis results of the p53 protein band in A. Figure 5 C is the Western blot result of p53 protein level in AGS cells after treatment with 10 μM p53-DUBTAC for 0 h, 4 h, 8 h, 12 h, 24 h, and 36 h. Figure 5 D is the result of using ImageJ software. Figure 5 The results of gray-scale quantitative analysis of the p53 protein band in C are shown in the figure. Figure 6 The figure shows the results of p53 detection, which indicates that p53-DUBTAC prolongs the p53 half-life in AGS cells; among them, Figure 6 A shows the agarose gel electrophoresis quality detection diagram of total RNA extracted from cells in the control group and the p53-DUBTAC treatment group. Lane 1 is the control group and lane 2 is the p53-DUBTAC treatment group. Figure 6 B is the RT-qPCR result of p53 mRNA expression in AGS cells after treatment with 10 μM p53-DUBTAC for 24 h. ns indicates no statistically significant difference between groups. Figure 6 C represents the Western blot results of p53 protein levels collected from AGS cells at 0h, 2h, 4h, 6h, and 8h after pretreatment with 10μM p53-DUBTAC for 12h and subsequent addition of cycloheximide (CHX, 20μg / mL) to block protein synthesis. Figure 6 D is the result of using ImageJ software. Figure 6 The p53 protein decay curve obtained by gray-scale quantitative analysis of the p53 protein band in C; Figure 7 The figure shows the detection results of p53-DUBTAC promoting the formation of the USP7-p53 ternary complex and reducing p53 ubiquitination levels; among them... Figure 7 A is the Western blot result of AGS cells treated with 10 μM p53-DUBTAC for 12 h, and then the proteasome inhibitor MG132 (10 μM) was added 6 h before cell collection. Immunoprecipitation was performed using anti-USP7 antibody. Figure 7B is the Western blot result of the related proteins in the complex obtained by immunoprecipitation with anti-p53 antibody for samples from the same batch; Figure 7 C is a Western blot image showing the p53 protein K48-linked ubiquitin chain level in the precipitate after AGS cells were treated under the same conditions as described above and immunoprecipitated with anti-p53 antibody. Figure 8 The image shows the USP7-dependent detection results of p53-DUBTAC-induced p53 stabilization; among them, Figure 8 Figure A shows the Western blot results of intracellular USP7 and p53 protein levels in AGS cells after treatment with 10 μM p53-DUBTAC for 24 h, representing the negative control (NC) and USP7 gene knockout (USP7KO). Figure 8 B is the result of using ImageJ software. Figure 8 The gray-scale quantitative analysis results of the p53 protein band in group A. *p<0.05 indicates statistically significant difference between groups, and ns indicates no statistically significant difference between groups. Figure 9 The image shows the results of detecting the upregulation of p53 downstream protein p21 by p53-DUBTAC; among them, Figure 9 Figure A shows the Western blot results of p53 and p21 protein levels in AGS cells after treatment with 10 μM p53-DUBTAC for 24 h. Figure 9 B is the result of using ImageJ software. Figure 9 The gray-scale quantitative analysis results of p53 and p21 protein bands in group A are shown in Figure A. *p<0.05 indicates statistically significant differences between groups. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] The present invention will be further illustrated below through examples.

[0032] The core inventive concept of this invention lies in constructing a p53-DUBTAC (p53-DUBTAC) targeting deubiquitinating enzymes that stabilize the p53 protein using a full-peptide strategy. This addresses the problems of complex synthesis, difficulty in rigid linker regulation, and potential cytotoxicity associated with traditional small-molecule DUBTACs. Simultaneously, through optimized linker design, the bottleneck of low cell penetration efficiency of peptide drugs is overcome. Ultimately, this invention achieves the specific recruitment of endogenous USP7 to the vicinity of the p53 protein, targeted removal of polyubiquitin chains on the p53 protein, inhibition of p53 proteasome degradation, and restoration of p53 protein homeostasis and downstream anti-cancer pathway activity.

[0033] Based on the above inventive concept, the present invention constructs a full-peptide targeting chimera with the general formula ALB, wherein A is the deubiquitinating enzyme USP7 recruitment domain, B is the p53 protein binding domain, and L is the peptide linker; the chimera design, synthesis, structural confirmation, in vitro and intracellular activity evaluation of the present invention are described in detail through the following examples.

[0034] Example 1: Design, chemical synthesis, and structural confirmation of a full-peptide chimera The purpose of this embodiment is to complete the rational design, molecular docking verification, chemical synthesis and structural confirmation of p53-DUBTAC, and to clarify the complete amino acid sequence of the chimera.

[0035] 1.1 Screening and molecular docking verification of USP7 binding ligands This invention selects USP7 as the recruitment target of deubiquitinating enzyme. USP7 is a natural regulator of p53. By utilizing its natural affinity for ligands, the steric hindrance caused by forced recruitment can be minimized. Moreover, the selected ligands bind to the non-catalytic domain of USP7, which does not affect the deubiquitinating enzyme activity of USP7 itself.

[0036] In this embodiment, an EBNA1-derived polypeptide capable of binding with high affinity to the TRAF domain of USP7 was selected as the USP7 recruitment domain. Its amino acid sequence is shown in SEQ ID NO:1: DPGEGPSTGP. Molecular docking simulations were performed using MOE (Molecular Operating Environment) software to verify the binding affinity and binding mode between the polypeptide and USP7NTD (PDBID: 1YY6).

[0037] Molecular docking results showed that the docking simulation score S-value of the peptide with USP7NTD was -10.1829, indicating that the two can form a stable complex; the three-dimensional binding mode is as follows: Figure 1 As shown in Figure B, the polypeptide represented by SEQ ID NO:1 can bind tightly to a shallow, groove-like hydrophobic pocket on the surface of the USP7TRAF domain; two-dimensional binding mode analysis is as follows. Figure 1 As shown in C, residues 441-448 of the polypeptide are key amino acids that bind to USP7, and proline at position 450 is in the solvent-exposed region, thus determining that a polypeptide linker is attached to the C-terminus of this ligand.

[0038] 1.2 Screening and molecular docking verification of p53 binding ligands In this embodiment, a polypeptide capable of binding to the p53 core domain with high affinity was selected as the target protein binding domain. Its amino acid sequence is shown in SEQ ID NO:2: AEDEDIEW, and the polypeptide structure is as follows: Figure 2 As shown in Figure A, the MOE software was also used to simulate the molecular docking of the peptide and p53 protein to verify their binding stability and binding mode.

[0039] Molecular docking results showed that the docking simulation score S-value of the polypeptide with p53 protein was -9.2833, verifying the stability of their binding; the three-dimensional binding mode was as follows: Figure 2 As shown in Figure B, the polypeptide represented by SEQ ID NO:2 can bind tightly to shallow grooves on the surface of the p53 core domain; two-dimensional interaction analysis is as follows. Figure 2As shown in Figure C, this polypeptide can form key hydrogen bonds with Lys164 and Lys291 of the p53 protein, and multiple hydrogen bonds with Ser269, Thr102, and Asn268, exhibiting excellent binding stability. The docking results also show that the C-terminus of the polypeptide is mainly exposed to the solvent environment, thus confirming the optimal method for linking the polypeptide linker to the C-terminus of this p53 binding ligand.

[0040] 1.3 Linker design and improvement of cell penetration To address the technical bottlenecks of large molecular weight and low cell penetration efficiency commonly found in peptide drugs, this invention introduces the arginine-rich RRRRRR (R6) sequence as a core linker. This sequence not only serves as a physical bridge to connect two functional domains but also endows the entire chimeric molecule with self-penetrating membrane capabilities, allowing it to autonomously enter cells and exert its effects without the need for transfection reagents such as liposomes. To achieve effective coupling of the two ligands and avoid the steric hindrance of the binding site, this embodiment employs the following connection strategy: the C-terminus of the USP7 binding ligand shown in SEQ ID NO:1 is connected to the N-terminus of the R6 linker; a lysine residue (Lys, K) is introduced at the C-terminus of the R6 linker as a connection fulcrum; and the free amino group of this lysine side chain is used to connect to the C-terminus of the p53 binding ligand shown in SEQ ID NO:2.

[0041] 1.4 Determination and Synthesis of the Final Chimeric Molecule Based on the above rational design and docking verification, the p53-DUBTAC, a stable full-peptide chimeric targeting the p53 protein, was finally synthesized. Its complete amino acid sequence is shown in SEQ ID NO:3: N-DPGEGPSTGP-RRRRRRK-WEIEDEDEA-N. The molecular design diagram is shown below. Figure 3 As shown in the figure. This design ensures that the chimera possesses dual target-specific recognition capabilities for both USP7 and p53, while also exhibiting good cell penetration efficiency. All peptide sequences used in this embodiment were synthesized by Sangon Biotech (Shanghai) Co., Ltd. using solid-phase peptide synthesis (SPPS) technology, and purified by high-performance liquid chromatography (HPLC) and identified by mass spectrometry to ensure peptide purity ≥95%, meeting the requirements for subsequent cell and in vitro experiments.

[0042] Example 2: In vitro and intracellular validation of the binding ability of p53-DUBTAC to USP7 The purpose of this embodiment is to verify the binding affinity of p53-DUBTAC to the USP7 protein in vitro, as well as its ability to target and bind to endogenous USP7 in living cells.

[0043] 2.1 In vitro binding affinity determination based on biomembrane interferometry The in vitro binding kinetics and affinity of p53-DUBTAC to the truncated USP7TRAF protein were detected using biomembrane layer interferometry (BLI).

[0044] Experimental methods: A streptavidin (SA) sensor was used. First, biotin-labeled USP7TRAF truncated protein was immobilized on the sensor surface, and PBST buffer was set as the detection system. The concentration gradient of p53-DUBTAC was set at 50 nM, 500 nM, 1000 nM, 10000 nM, 25000 nM, and 50000 nM. Baseline equilibration, binding, and dissociation experiments were performed sequentially, and binding and dissociation signals were collected and data were fitted.

[0045] Experimental results: Steady-state analysis curves are as follows Figure 4 As shown in Figure A, the equilibrium dissociation constant KD value of p53-DUBTAC and the truncated USP7TRAF protein was 7.1 ± 2.8 μM, calculated by a 1:1 binding model. This indicates that the two have good in vitro binding affinity and can effectively recruit the USP7 protein.

[0046] 2.2 Validation of Intracellular Target Binding Based on Cell Thermal Migration Assay The cell thermal migration assay (CETSA) was used to verify whether p53-DUBTAC could penetrate the cell membrane and specifically bind to the endogenous USP7 protein in living cells.

[0047] Experimental methods: Human gastric cancer AGS cells were seeded in cell culture dishes. After cell attachment, 10 μM p53-DUBTAC (drug treatment group) or an equal volume of DMSO (control group) was added, and the cells were incubated in a cell culture incubator for 12 hours. After incubation, the cells were collected and divided into 6 equal parts. The cells were heated for 3 minutes at temperature gradients of 42℃, 43.5℃, 45℃, 46.5℃, 48℃, and 49.5℃, respectively. After heating, the cells were lysed, and the expression level of USP7 protein in each group of cells was detected by Western blot.

[0048] Experimental results: such as Figure 4 As shown in Figure B, in the control group, USP7 protein rapidly underwent thermal denaturation and precipitation with increasing heating temperature, resulting in a significant decrease in detectable protein levels. In contrast, the p53-DUBTAC treatment group showed significantly enhanced thermal stability of USP7 protein, with detectable USP7 protein levels at the same temperature gradient being significantly higher than in the control group. These results confirm that p53-DUBTAC can effectively penetrate cell membranes and enter living cells, specifically binding to endogenous USP7 protein within the cells, thereby enhancing the thermal stability of USP7 protein.

[0049] Example 3: Verification of the stabilizing effect of p53-DUBTAC on intracellular p53 protein levels The purpose of this embodiment is to verify the upregulation effect of p53-DUBTAC on intracellular p53 protein and to clarify the concentration- and time-dependent nature of its effect.

[0050] 3.1 Concentration-dependent upregulation of p53 protein levels Experimental Methods: Human gastric cancer AGS cells in logarithmic growth phase were seeded at an appropriate density in 6-well cell culture plates. When the cells adhered and grew to approximately 70% confluence, the medium was replaced with fresh medium containing different concentrations of p53-DUBTAC. The drug concentrations were set at 0 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM, with 3 replicates for each concentration. After drug administration, the cells were incubated in a cell culture incubator for 24 hours. After incubation, the medium was discarded, and the cells were washed twice with pre-cooled PBS. Total cell protein was extracted by adding RIPA cell lysis buffer. After protein quantification using the BCA method, the expression level of intracellular p53 protein was detected by Western blot. GAPDH was used as an internal control protein, and the gray values ​​of protein bands were quantitatively analyzed using ImageJ software.

[0051] Experimental results: such as Figure 5 As shown in Figure A, with increasing p53-DUBTAC concentration, the band intensity of p53 protein in AGS cells showed a significant increasing trend; ImageJ quantitative analysis results are as follows. Figure 5 As shown in Figure B, when the drug concentration was 10 μM, the p53 protein level reached its peak, approximately 260% of that in the control group (0 μM group). These experimental results confirm that p53-DUBTAC can significantly increase the intracellular p53 protein level in a concentration-dependent manner, achieving effective stabilization of the p53 protein.

[0052] 3.2 Time-dependent upregulation of p53 protein levels Experimental Methods: AGS cells in the logarithmic growth phase were seeded at an appropriate density in 6-well cell culture plates. When the cells adhered and grew to approximately 70% confluence, the medium was replaced with fresh medium containing 10 μM p53-DUBTAC. Cell samples were collected at 0 h, 4 h, 8 h, 12 h, 24 h, and 36 h after drug administration, with three replicates at each time point. Total cellular protein was extracted and quantified using the same method described above. The expression changes of p53 protein in cells at each time point were detected by Western blot. GAPDH was used as an internal control protein, and the gray values ​​of protein bands were quantitatively analyzed using ImageJ software.

[0053] Experimental results: such as Figure 5As shown in Figure C, the p53 protein level initially increased and then slowly decreased with prolonged p53-DUBTAC administration time; ImageJ quantitative analysis results are as follows. Figure 5 As shown in Figure D, intracellular p53 protein levels significantly increased 4 hours after administration. The stabilizing effect of p53 protein reached its peak between 8 and 12 hours post-administration, approximately 240% of the initial value (0h group). Subsequently, although p53 protein levels decreased slightly between 24 and 36 hours, they remained significantly higher than the control group. These experimental results confirm that the stabilizing effect of p53-DUBTAC on p53 protein is significantly time-dependent, with the optimal stabilizing effect achieved approximately 12 hours after administration.

[0054] Example 4: Verification of the mechanism by which p53-DUBTAC stabilizes p53 protein The purpose of this embodiment is to verify the role of p53-DUBTAC in upregulating p53 protein levels and to clarify its mechanism of action in extending the half-life of p53 protein through post-translational regulation.

[0055] 4.1 Detection of p53 mRNA transcription level To rule out that the increased p53 protein level was due to enhanced gene transcription, this experiment used RT-qPCR to detect changes in the expression level of p53 mRNA in cells after p53-DUBTAC treatment.

[0056] Experimental methods: AGS cells were seeded in 6-well cell culture plates. After cell attachment, 10 μM p53-DUBTAC (drug treatment group) or an equal volume of DMSO (control group) was added and incubated for 24 hours. After incubation, total RNA was extracted from cells in each group using an RNA extraction kit (Sparkjadem, China). The integrity of total RNA was detected by agarose gel electrophoresis. Subsequently, total RNA was reverse transcribed into cDNA using a reverse transcription kit (Vazyme, China). The relative expression level of p53 mRNA was detected by real-time quantitative PCR. GAPDH was used as an internal reference gene.

[0057] Experimental results: Total RNA was detected by agarose gel electrophoresis as follows: Figure 6 As shown in Figure A, the total RNA bands extracted from both the control group and the drug-treated group were clear, intact, and showed no significant degradation; the RT-qPCR results are as follows. Figure 6 As shown in Figure B, there was no significant difference in p53 mRNA levels between the p53-DUBTAC treatment group and the control group (ns, p>0.05). These experimental results confirm that the p53-DUBTAC-induced upregulation of p53 protein levels does not occur at the transcriptional level, but rather through post-translational regulatory mechanisms.

[0058] 4.2 Detection of p53 protein half-life To verify whether p53-DUBTAC improves the stability of p53 protein by slowing down the protein degradation rate, this experiment used the protein synthesis inhibitor cyclohexylimide (CHX) to block the synthesis of new protein and examined the effect of p53-DUBTAC on the intracellular half-life of p53 protein.

[0059] Experimental Methods: AGS cells were seeded in 6-well cell culture plates. After cell adhesion, 10 μM p53-DUBTAC (drug treatment group) or an equal volume of DMSO (control group) was added for pretreatment for 12 hours. After pretreatment, CHX with a final concentration of 20 μg / mL was added to the cell culture medium of each group to block the synthesis of new intracellular proteins. Cell samples were collected at 0 h, 2 h, 4 h, 6 h, and 8 h after CHX addition, with 3 replicates at each time point. Total cell protein was extracted and quantified using the same method. The content of residual p53 protein in cells at each time point was detected by Western blot. GAPDH was used as an internal control protein. ImageJ software was used to quantitatively analyze the gray values ​​of protein bands and plot the p53 protein decay curve.

[0060] Experimental results: Western blot detection results are as follows Figure 5 As shown in Figure C, in the control group, the p53 protein degraded extremely rapidly after CHX treatment, with a short half-life; while in the p53-DUBTAC treatment group, the degradation rate of p53 protein was significantly slowed down; the decay curves quantitatively plotted by ImageJ are shown below. Figure 5 As shown in Figure D, p53-DUBTAC treatment significantly prolonged the intracellular half-life of p53 protein. These experimental results confirm that p53-DUBTAC significantly enhances the intracellular stability of p53 protein by inhibiting its proteasome degradation pathway.

[0061] Example 5: Formation of p53-DUBTAC-mediated ternary complex and verification of its deubiquitination function The purpose of this embodiment is to verify whether p53-DUBTAC can act as a molecular bridge to simultaneously bind USP7 and p53 in cells and form a ternary complex, and whether this complex can achieve specific deubiquitination of p53 protein.

[0062] 5.1 Verification of the formation of the intracellular USP7-chimera-p53 ternary complex The ability of p53-DUBTAC to mediate the interaction between USP7 and p53 in living cells was verified by co-immunoprecipitation (Co-IP) assay.

[0063] Experimental Methods: AGS cells were seeded in cell culture dishes. After the cells reached a suitable confluence, 10 μM p53-DUBTAC (drug-treated group) or an equal volume of DMSO (control group) was added for 12 hours. Six hours before cell collection, 10 μM of the proteasome inhibitor MG132 was added to the cell culture medium of each group to avoid degradation of p53 protein. After incubation, cells were collected, and total cell protein was extracted by adding pre-cooled non-denaturing cell lysis buffer. A portion of the protein sample was used as the input control, and the remaining protein sample was divided into two parts. USP7 antibody (Proteintech, China) and p53 antibody (CST, USA) were added to the two parts, respectively, and the samples were incubated overnight at 4°C with slow rotation. The next day, protein A / G agarose microspheres were added and incubated at 4°C for 4 hours. The microspheres were washed 5 times with pre-cooled lysis buffer, and the samples were denatured by boiling in protein loading buffer. The protein content in the immunoprecipitation complex was detected by Western blot.

[0064] Experimental results: The results of immunoprecipitation detection using USP7 antibody are as follows: Figure 7 As shown in Figure A, a significantly enhanced p53 protein band was detected in the immunoprecipitation complex of the p53-DUBTAC treated group, while only a very weak p53 protein signal was detected in the control group; the results of immunoprecipitation using p53 antibody are as follows. Figure 7 As shown in Figure B, a significantly enhanced USP7 protein band was detected in the immunoprecipitation complex of the p53-DUBTAC-treated group, while only a very weak USP7 protein signal was detected in the control group. These experimental results strongly confirm that p53-DUBTAC can act as a molecular bridge, simultaneously binding endogenous USP7 and p53 proteins in living cells, inducing the formation of a stable USP7-chimera-p53 ternary complex.

[0065] 5.2 Detection of p53 protein K48 ubiquitination level To verify whether the p53-DUBTAC-mediated ternary complex can specifically remove the K48-linked ubiquitin chain on the p53 protein that mediates proteasome degradation through the deubiquitinating enzyme activity of USP7, this experiment used immunoprecipitation combined with Western blot to detect the K48 ubiquitination level of the p53 protein.

[0066] Experimental methods: AGS cells were treated and total protein was extracted using the same method described in 5.1 above. A portion of the protein sample was used as an input control, and the remaining protein sample was immunoprecipitated with p53 antibody. The immunoprecipitation procedure was the same as in 5.1. The level of K48 ubiquitination on the immunoprecipitated p53 protein was detected by Western blot using a K48-linkage specific ubiquitin antibody.

[0067] Experimental results: such as Figure 7 As shown in Figure C, in the control group, a large number of K48-linked ubiquitin chains were detected in the p53 protein obtained by immunoprecipitation, appearing as high-molecular-weight diffuse bands; while in the p53-DUBTAC treatment group, the K48-linked ubiquitin chain signal on the p53 protein was significantly weakened. These experimental results confirm that p53-DUBTAC can successfully recruit endogenous USP7 to the vicinity of the p53 protein, utilizing the deubiquitinating enzyme activity of USP7 to specifically remove the K48-linked ubiquitin chains on the p53 protein that mediate proteasome degradation, thereby preventing the proteasome degradation of p53.

[0068] Example 6: Validation of the USP7 dependence of p53-DUBTAC on the stabilization of p53 protein The purpose of this embodiment is to verify whether the stabilizing effect of p53-DUBTAC on p53 protein specifically depends on the presence of endogenous USP7.

[0069] Experimental Methods: AGS cell lines with USP7 gene knockout (USP7KO) were constructed using CRISPR-Cas9 technology, and a negative control (NC) cell line was also set up. AGS cells from the NC group and the USP7KO group were seeded into 6-well cell culture plates. After cell attachment, 10 μM p53-DUBTAC was added and incubated for 24 hours. A control group without drug administration was also set up. Each group had 3 replicates. After incubation, cells were collected, and total cellular protein was extracted. The expression levels of USP7 and p53 proteins in each group were detected by Western blot. GAPDH was used as an internal reference protein, and the gray values ​​of protein bands were quantitatively analyzed using ImageJ software.

[0070] Experimental results: Western blot detection results are as follows Figure 8 As shown in Figure A, in the NC group cells, the p53 protein band intensity was significantly enhanced after the addition of p53-DUBTAC; while in the USP7KO group cells, although the baseline level of p53 protein changed due to USP7 gene knockout, the p53 protein level did not show a significant increase after the addition of p53-DUBTAC; ImageJ quantitative analysis results are as follows. Figure 8 As shown in Figure B, in the NC group, the increase in p53 protein levels after p53-DUBTAC administration was statistically significant (*p<0.05), while in the USP7KO group, there was no significant difference in p53 protein levels before and after administration (ns, p>0.05). These experimental results strongly confirm that the stabilizing effect of p53-DUBTAC on p53 protein is entirely dependent on the presence of intracellular USP7 protein. When the recruitment target USP7 is absent, this chimera will lose its deubiquitination and stabilizing function of p53.

[0071] Example 7: Verification of the activation effect of p53-DUBTAC on downstream tumor suppressor pathways of p53 The purpose of this embodiment is to verify whether the p53 protein stabilized by p53-DUBTAC can restore its biological function and activate downstream tumor suppressor signaling pathways.

[0072] p53 is a key tumor suppressor protein, and its downstream target gene p21 is an important cell cycle repressor. Upregulation of p21 expression is a core marker of p53 pathway activation. In this study, Western blot was used to detect changes in the expression levels of p53 and its downstream target protein p21 in cells after p53-DUBTAC treatment.

[0073] Experimental methods: AGS cells were seeded in 6-well cell culture plates. After cell adhesion, 10 μM p53-DUBTAC (drug-treated group) or an equal volume of DMSO (control group) was added and incubated for 24 hours. After incubation, cells were collected, total cell protein was extracted, and the expression levels of p53 protein and its downstream target protein p21 were detected simultaneously by Western blot. GAPDH was used as an internal reference protein, and the gray values ​​of protein bands were quantitatively analyzed using ImageJ software.

[0074] Experimental results: Western blot detection results are as follows Figure 9 As shown in Figure A, in the p53-DUBTAC treatment group, the intracellular p53 protein level was significantly increased, and the band intensity of its downstream target protein p21 was also significantly enhanced; ImageJ quantitative analysis results are as follows. Figure 9 As shown in Figure B, the p53 protein level in the p53-DUBTAC treatment group increased to approximately 180% of that in the control group, and the p21 protein level simultaneously increased to approximately 195% of that in the control group, with both increases showing statistically significant differences (*p<0.05). These experimental results confirm that the p53-DUBTAC described in this invention can not only effectively stabilize intracellular p53 protein but also successfully restore the biological function of p53, significantly upregulate the expression of its downstream tumor suppressor protein p21, and activate the p53-mediated tumor suppressor signaling pathway.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A full-peptide targeting chimera that targets and stabilizes the p53 protein, characterized in that, The chimera is composed of a deubiquitinase recruitment domain A, a target protein binding domain B, and a polypeptide linker L, and has the general formula ALB. The deubiquitinase recruitment domain A specifically binds to and recruits endogenous USP7 protein, the target protein binding domain B specifically binds to p53 protein, and the polypeptide linker L is a polypeptide sequence with an arginine chain that promotes cell permeation efficiency. The chimera can act as a molecular bridge to induce spatial proximity between USP7 and p53, remove polyubiquitin chains on the p53 protein through the deubiquitinase activity of USP7, inhibit p53 protein degradation, and maintain p53 protein stability.

2. The full-peptide targeted chimera according to claim 1, characterized in that, The amino acid sequence of the deubiquitinase recruitment domain A is shown in SEQ ID NO:

1.

3. The full-peptide targeted chimera according to claim 1 or 2, characterized in that, The amino acid sequence of the target protein binding domain B is shown in SEQ ID NO:

2.

4. The full-peptide targeted chimera according to claim 3, characterized in that, The polypeptide linker L contains an amino acid sequence consisting of 6 consecutive arginine residues, and the C-terminus of the polypeptide linker L is connected to a lysine residue; the C-terminus of the deubiquitinating enzyme recruitment domain A is connected to the N-terminus of the polypeptide linker L, and the C-terminus of the target protein binding domain B is connected to the free amino group of the side chain of the lysine residue in the polypeptide linker L.

5. The full-peptide targeted chimera according to claim 4, characterized in that, The amino acid sequence of the full-peptide targeted chimera is shown in SEQ ID NO:

3.

6. A method for preparing a p53 protein-stabilized full-peptide targeting chimera according to any one of claims 1 to 5, characterized in that, The full-peptide targeted chimera was prepared using a solid-phase peptide synthesis process.

7. The preparation method according to claim 6, characterized in that, The preparation method employs a solid-phase polypeptide synthesis process with Fmoc protection strategy, in which amino acid coupling reactions are performed sequentially from the C-terminus to the N-terminus of the chimera. After coupling, the chimera is subjected to cleavage, deprotection, and purification to obtain the full-peptide targeted chimera.

8. Use of the full-peptide targeting chimera according to any one of claims 1 to 5 in the preparation of reagents or drugs for stabilizing p53 protein.

9. The application according to claim 8, characterized in that, The reagent or drug is used to achieve at least one of the following functions: upregulating intracellular p53 protein levels in a concentration-dependent and time-dependent manner, prolonging the intracellular half-life of p53 protein, reducing the level of the K48-linked ubiquitin chain on p53 protein, and activating downstream signaling pathways of p53 to upregulate p21 protein expression.

10. The application according to claim 8 or 9, characterized in that, The drug is an anti-tumor drug used to treat tumors associated with p53 deficiency.

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