Polypeptide-based PROTAC condensation body and preparation method and application thereof

Abstract

The invention discloses a polypeptide-based PROTAC condensation body as well as a preparation method and application of the polypeptide-based PROTAC condensation body. The polypeptide-based PROTAC aggregate comprises an HER2 (human epidermal growth factor receptor) targeting module, an EGFR (epidermal growth factor receptor) targeting module, a VHL E3 ligase targeting module and a self-assembly module. The polypeptide-based PROTAC condensation body can dynamically enrich E3 ligase VHL and promote efficient ubiquitination degradation of HER2, EGFR and downstream signal channel protein of HER2 and EGFR, the degradation efficiency of HER2 and EGFR protein can reach 92%, the degradation efficiency of downstream signal channel protein such as SOS2 and RAS can reach 80%, and in addition, the polypeptide-based PROTAC condensation body has the advantages that the polypeptide-based PROTAC condensation body can be used as an efficient ubiquitination condensation body; the polypeptide-based PROTAC condensation body has the characteristics of tumor targeting and long-acting retention, and dose-dependent tumor specific accumulation and retention can be realized through in-situ self-assembly.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a polypeptide-based PROTAC condensate, its preparation method, and its application. Background Technology

[0002] Targeted protein degradation technology is crucial for treating a variety of diseases because it directly targets abnormal proteins—core factors in disease development and progression. Many diseases, including cancer and neurodegenerative diseases, are associated with the overexpression, dysfunction, or misfolding of specific proteins. These abnormal proteins can lead to cellular dysfunction and promote disease progression.

[0003] Traditional drug therapies often only inhibit protein activity, rather than fundamentally reducing or eliminating these harmful proteins. Targeted protein degradation technology offers a novel treatment strategy by promoting the degradation of these abnormal proteins.

[0004] However, traditional targeted protein degradation technologies primarily target proteins in the cytoplasm, but are ineffective at degrading membrane proteins. While PROTAC (Proteolysis Targeting Chimeras) technology has achieved protein degradation in the cytoplasm, it still faces challenges in degrading membrane proteins. Technologies such as LYTACs (Lysosome-targeting chimeras), AbTACs (Antibody-based PROTACs), and TransTACs (Transferrin receptor-targeting chimeras) have been developed for membrane protein degradation, but they have limitations in simultaneously degrading both membrane and intracellular proteins.

[0005] Biological signal transduction is a complex and delicate process that relies on close coordination and precise regulation between cell membrane and intracellular proteins. Current protein degradation strategies mostly focus on the degradation of single membrane proteins or intracellular proteins, making it difficult to achieve simultaneous degradation of both. Furthermore, degrading multiple proteins, including target proteins and their downstream signaling pathway proteins, is even more challenging. Therefore, there is an urgent need for a revolutionary protein degradation therapy system that can redefine the norms of protein degradation and pave the way for more systematic and synergistic therapeutic approaches.

[0006] The introduction of nanotechnology has brought new opportunities for protein degradation. By activating specific transport pathways, nanotechnology has further enhanced protein degradation efficiency, not only introducing new methods but also achieving significant results. Unique protein transport carriers and cellular transport mechanisms are of great significance for the degradation of proteins and their downstream pathways. By optimizing the assembly structure, their interactions with proteins and endocytosis can be modulated, thereby achieving the synergistic degradation of multiple proteins.

[0007] Bioconcentrates, as an emerging physiological phenomenon, possess significant physiological functions such as protein loading, functional regulation, transmembrane transport, and enzyme-catalyzed reaction activation, thanks to their dynamic reversibility, compositional diversity, rapid recruitment, and adaptability. Based on this, artificial biomolecular concentrates have been designed as tools for various drugs, protein libraries, and cell signaling programming, providing a crucial structural basis for addressing the challenges of multi-target PROTACs. However, traditional highly dynamic and physiologically dependent concentrates have limitations in in vivo applications, making it difficult to simultaneously achieve precise targeting, rapid cytoplasmic entry, efficient protein recruitment, and interaction regulation.

[0008] The emergence of self-assembled condensates offers a new approach to solving these problems. Through self-assembled condensates, target proteins and their associated functional proteins can be enriched in local compartments, forming highly efficient "degradation factories." These condensates not only enable the simultaneous degradation of membrane and intracellular proteins but also optimize their interaction with target proteins by modulating their structure and composition, thereby improving degradation efficiency. Furthermore, the dynamic reversibility and adaptability of self-assembled condensates allow them to flexibly adjust to different cellular environments, further enhancing their regulatory capacity in biotransmission processes.

[0009] Therefore, there is an urgent need to provide a bioaggregate that can be assembled in situ and degraded efficiently within cells, and has significant tumor specificity and therapeutic effects. Summary of the Invention

[0010] To address the shortcomings of existing technologies and practical needs, this invention provides a polypeptide-based PROTAC condensate, its preparation method, and its applications. After entering tumor cells, the polypeptide-based PROTAC condensate triggers a coupling reaction activated by enzymes or GSH within the tumor cells. The resulting molecules possess the ability to bind to E3 ligases and target proteins, thereby performing protein ubiquitination. The critical assembly concentration of the heterobifunctional molecules decreases after the coupling reaction, leading to in-situ assembly within the cell. Simultaneously, due to the distance effect, the E3 ligase and target protein are brought closer together, satisfying the ubiquitination requirements. The long-retention assemblies formed within the cell have the potential to ubiquitinate and degrade proteins. More importantly, the self-assembly capability of the in-situ formed heterobifunctional molecules effectively resists the hook effect caused by small-molecule PROTACs under high concentration conditions. At the PROTAC molecule design level, because different target proteins and E3 ligases have different steric hindrance distances, different lengths and rigidity linkers need to be redesigned for different target proteins, which significantly increases the design cost and difficulty of PROTAC molecules for different target proteins. Utilizing assembly and its surface effects may provide new design strategies for universal PROTAC molecules. This strategy is significant for improving the specificity, selectivity, and concentration dependence of PROTAC molecules, while reducing their development cycle and cost, which will facilitate the further advancement of PROTAC technology in clinical practice.

[0011] To achieve this objective, the present invention adopts the following technical solution:

[0012] In a first aspect, the present invention provides a dynamically adaptable peptide-based PROTAC condensate, the peptide-based PROTAC condensate comprising: a HER2 targeting module, an EGFR targeting module, a VHL E3 ligase targeting module, and a self-assembly module.

[0013] The polypeptide-based PROTAC condensates prepared by this invention can efficiently degrade tumor-specific and highly efficient membrane protein complexes. The condensates are formed in situ within tumor cells, accurately identifying and degrading target proteins (such as EGFR and HER2) and their downstream signaling pathway proteins, thereby effectively inhibiting tumor cell growth and signal transduction.

[0014] Preferably, the amino acid sequence of the HER2 targeting module includes the sequence shown in SEQ ID NO.1, the EGFR targeting module includes a gefitinib derivative, the VHL E3 ligase targeting module includes a VHL ligand, and the amino acid sequence of the self-assembly module includes the sequence shown in SEQ ID NO.2.

[0015] SEQ ID NO.1: KCCYSL.

[0016] SEQ ID NO.2: YYQNNQ.

[0017] Preferably, the polypeptide-based PROTAC condensate comprises a compound as shown in formula (1);

[0018]

[0019] The HER2 targeting module described in this invention is used to specifically recognize the HER2 protein on the surface of tumor cells. The EGFR targeting module is used to recognize and bind to the EGFR protein, thereby enhancing the targeting ability of the EGFR protein. The VHL E3 ligase targeting module is used to recruit the VHL E3 ligase to the aggregate, thereby promoting the ubiquitination modification and degradation of the target protein. The self-assembly module contains an amino acid sequence that can respond to intracellular signals, thereby promoting the formation and stability of the aggregate.

[0020] Preferably, after entering tumor cells, the polypeptide-based PROTAC condensate directly internalizes the HER2 protein, its interacting proteins, and membrane components into the cytoplasm through its unique cellular uptake mechanism, forming a micron-sized condensate structure, which provides favorable conditions for subsequent protein degradation.

[0021] Preferably, the polypeptide-based PROTAC condensate can dynamically enrich E3 ligase VHL in the cytoplasm to form an optimal ternary complex, thereby achieving efficient ubiquitination and degradation of the target protein. The adaptability and dynamism of the condensate enable it to adjust its structure and function according to changes in the intracellular environment, thereby improving degradation efficiency.

[0022] In a second aspect, the present invention provides a method for preparing the polypeptide-based PROTAC condensate described in the first aspect, the method comprising: synthesizing a HER2 targeting module, an EGFR targeting module, a VHL E3 ligase targeting module and a self-assembly module using polypeptide synthesis technology; connecting each module to form a polypeptide-based PROTAC condensate precursor; and forming a polypeptide-based PROTAC condensate through in vitro or in vivo self-assembly.

[0023] Preferably, the self-assembly includes: co-incubating a peptide-based PROTAC condensate precursor, HER2-TC cells, and HER2-positive cells, forming a peptide-based PROTAC condensate on the tumor cell membrane, and the peptide-based PROTAC condensate carrying tumor cell membrane components into the tumor cell to form an intracellular peptide-based PROTAC condensate droplet.

[0024] As a preferred technical solution of the present invention, the preparation method of the polypeptide-based PROTAC condensate includes: mixing four module units, namely HER2 targeting module, EGFR targeting module, VHL E3 ligase targeting module and self-assembly module, in an appropriate buffer solution, and promoting the interaction and self-assembly process between the units by adjusting the pH value and ionic strength of the solution to form a stable polypeptide-based PROTAC condensate.

[0025] Preferably, the polypeptide-based PROTAC condensate can be administered intravenously, enabling rapid systemic distribution and efficient tumor targeting.

[0026] Preferably, the administration method involves a concentration of less than 100 μM to ensure safety and effectiveness.

[0027] Thirdly, the present invention provides the application of the polypeptide-based PROTAC condensates described in the first aspect in the degradation of proteins.

[0028] Preferably, the protein includes any one or a combination of at least two of the following: HER2 protein, EGFR protein, SOS2 protein, or RAS protein.

[0029] Fourthly, the present invention provides the use of the polypeptide-based PROTAC described in the first aspect in the preparation of products for treating tumors and / or cancer.

[0030] Preferably, the tumor includes tumors that overexpress HER2 and / or EGFR; the tumor also includes tumors with HER2 and / or EGFR mutations.

[0031] Preferably, the cancer includes breast cancer and / or stomach cancer.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) Traditional protein degradation technology mainly relies on degradation mechanisms in the cytoplasm, which has limited effect on the degradation of membrane proteins. In the degradation of membrane proteins and their downstream signaling pathways, there is currently no effective technology that can simultaneously achieve efficient degradation of membrane proteins and cytoplasmic proteins. The peptide-based PROTAC condensate of the present invention forms a dynamically reversible condensate structure through self-assembly in the cell, which can simultaneously capture membrane proteins and their downstream signaling pathway proteins, and achieve synergistic degradation of multiple proteins.

[0034] (2) Traditional protein degraders are prone to non-specific binding at high concentrations, which leads to a decrease in degradation efficiency. The dynamic structure of the polypeptide-based PROTAC condensate of the present invention can automatically adjust its conformation according to changes in the intracellular environment, so that it can still maintain high degradation efficiency at high concentrations. The degradation efficiency of HER2 and EGFR proteins can reach 92%, and the degradation efficiency of downstream signaling pathway proteins such as SOS2 and RAS can reach 80%.

[0035] (3) The polypeptide-based PROTAC condensate of the present invention has good targeting properties, can be specifically activated in tumor cells, and significantly reduces toxicity to normal cells;

[0036] (4) The polypeptide-based PROTAC condensate of the present invention exhibits excellent protein degradation effects at both the cellular and animal levels, and can effectively inhibit the proliferation and signal transduction of tumor cells, thereby inhibiting tumor growth;

[0037] (5) The polypeptide-based PROTAC condensate of the present invention exhibits good biocompatibility in vivo and does not cause obvious side effects. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the structure of a polypeptide-based PROTAC condensate;

[0039] Figure 2 The graph shows the protein binding affinity test results for peptide-PROTAC condensates.

[0040] Figure 3 Graphs showing the protein degradation effects of different concentrations of peptide-based PROTAC condensates at the cellular level;

[0041] Figure 4 This image shows the enrichment and retention effect of polypeptide-PROTAC condensates in mouse subcutaneous tumors.

[0042] Figure 5 This is a diagram showing the results of peptide-PROTAC condensates inhibiting the growth of subcutaneous tumors in mice. Detailed Implementation

[0043] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0044] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0045] Example 1

[0046] This embodiment provides a polypeptide-based PROTAC condensate, and prepares the HER2-TC structure in the polypeptide-based PROTAC by polypeptide solid-phase synthesis, and then synthesizes the complete polypeptide-based PROTAC condensate structure (hereinafter referred to as "TC Nano-PROTAC") by organic synthesis.

[0047] The steps for synthesizing the molecule using peptide solid-phase synthesis are as follows:

[0048] (1) Synthesis of the polypeptide sequence KCCYSL-YYQNNQ (hereinafter referred to as "HER2-TC")

[0049] Experimental instruments and materials:

[0050] Dimethylformamide (DMF) (Inokane, N6259-100 mL), Wang resin (1365700-43-1, Tianjin Jier Biochemical), dichloromethane (DCM) (Inokane, A32827), ninhydrin (Maclean, N790381-25 mL), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) (Inokane, A13026), hexahydropyridine (Merck, 134759), triisopropylsilane (TIS) (Aladdin, T107280-5 mL), anhydrous diethyl ether (Acros, 123990010), trifluoroacetic acid (TFA) (Inokane, A00552), N-methylmorpholine (NMM) (Inokane, A59322), methanol ( Inokai (A45780), Fmoc-glutamine (Fmoc-Gln(Trt)-OH), Fmoc-lysine (Fmoc-Lys(Boc)-OH), Fmoc-cysteine ​​(Fmoc-Cys(Trt)-OH), Fmoc-tryptophan (Fmoc-Trp(Boc)-OH), Fmoc-leucine (Fmoc-Leu-OH), Fmoc-asparagine (Fmoc-Asn(Trt)-OH), Fmoc-tyrosine (Fmoc-Tyr(Trt)-OH), (amino acid raw materials purchased from Jier Biochemical), copper acetate (Inokai, A17531), polypeptide solid-phase synthesis tubes, etc.

[0051] Preparation of experimental solutions:

[0052] Deprotection solution – a mixture of hexahydropyridine and DMF at a volume ratio of 1:4;

[0053] The reaction solution is a mixture of NMM and DMF at a volume ratio of 1:24.

[0054] The lysis buffer is a mixture of TFA, TIS and H2O, with the volume fractions of each solution being 92.5% TFA, 2.5% TIS and 2.5% H2O.

[0055] Ninhydrin test solution – one drop each of ninhydrin, vitamin C, and phenol;

[0056] Specific operation methods:

[0057] (1) Fmoc (fluorenyl methoxycarbonyl) deprotection: Weigh 0.1g of Wang resin and add it to the polypeptide solid-phase synthesis tube. Add DMF to swell for 30min. Remove the DMF and perform the Fmoc deprotection reaction with the deprotection solution. Place on a shaker for 10min. Remove the deprotection solution and wash three times with DMF and DCM. Take 10mg of Wang resin from the polypeptide solid-phase synthesis tube into a test tube and wash twice with ethanol. After the ninhydrin method shows a deep blue color, indicating a positive result, prepare to add the first amino acid (R) and proceed with the amino acid condensation reaction.

[0058] (2) Amino acid condensation: Ten equivalents of amino acids and HBTU were taken according to the amino acid sequence of HER2-TC, dissolved in 7 mL of reaction solution, and added to a peptide solid-phase synthesis tube. The mixture was stirred and reacted. After 1 h, 10 mg of Wang resin was taken from the peptide solid-phase synthesis tube and placed in a test tube. The tube was washed twice with ethanol. The ninhydrin test showed no color change, indicating a negative result and confirming the successful condensation reaction. The liquid in the peptide solid-phase synthesis tube was removed, and the tube was washed twice each with DMF and DCM to obtain the peptide resin after the first amino acid condensation.

[0059] The obtained peptide resin was subjected to the above "Fmoc deprotection-amino acid condensation" reaction steps until the reaction of the last amino acid, Boc-glycine, was completed. Then, the Dde protection of the lysine side chain was removed using 2% hydrazine hydrate, and the gefitinib derivative was linked via amide condensation. After the reaction was complete, the resin was washed three times each with DMF and DCM, and twice with methanol, and then dried under vacuum for 20 min. The synthesized peptide resin was removed from the peptide solid-phase synthesis tube and lysed in lysis buffer at room temperature for 2 h, with the lysis buffer first placed on ice for 20 min.

[0060] The crude peptide was purified using preparative reversed-phase HPLC, and the purity was >95% as determined by HPLC. The obtained pure peptide was identified by mass spectrometry (MS, electrospray), and the measured molecular weight was the same as the target molecular weight.

[0061] The final structure is as follows Figure 1As shown, the peptide sequence KCCYSL is the HER2 targeting recognition unit, YYQNNQ is the self-assembly unit, and EGFR and VHL-1 are the targeting recognition units. The obtained peptides 1 and 2 were lyophilized and stored at -20℃ for later use. The design principle of the peptide-based PROTAC condensate is as follows: When the peptide molecules of the peptide nanobioconcentrate act, the HER2 targeting recognition unit KCCYSL specifically binds to the HER2 protein, and the self-assembly unit YYQNNQ forms a condensate structure through self-assembly at this target enrichment concentration. The formation of this structure lowers the critical assembly concentration of the peptide, promoting the in-situ assembly of the peptide nanobioconcentrate on the targeted tumor cell membrane. The assembled condensate structure is dynamically reversible and can enter the cell through a unique cellular uptake mechanism. The condensate can directly interact with the cell membrane, and with the help of membrane fluidity, the condensate and its carried protein components are internalized into the cytoplasm. After entering the tumor cell, it can simultaneously bind to the target protein and E3 ligase, forming a stable ternary complex, thereby exerting the function of ubiquitinated protein degradation. Furthermore, the adaptability of condensates allows them to adapt to different cell types and pathological states, giving them wide applicability.

[0062] Example 2

[0063] The in vitro binding affinity of the polypeptide TC Nano-PROTAC prepared in Example 1 was verified by an experiment.

[0064] like Figure 2 The binding affinity between EGFR and TC-PROTAC is shown in the figure. Microscale thermophoresis (MST) revealed that the binding affinity of EGFR to TC-PROTAC is 850.2 nM. This indicates that TC-PROTAC can effectively bind to EGFR, providing a basis for the subsequent formation of the ternary complex and induction of protein degradation. The binding affinity between HER2 and VHL and the EGFR-TC-PROTAC complex is also shown. Using the same MST technique, the binding affinity of HER2 and VHL to the EGFR-TC-PROTAC complex is 1.2 μM. This demonstrates that TC-PROTAC can not only bind to EGFR but also effectively recruit HER2 and VHL into the complex, thereby promoting the formation of the ternary complex and providing the necessary interaction basis for the protein degradation process.

[0065] This experiment confirmed that TC-PROTAC can effectively bind to target proteins EGFR, HER2, and the E3 ubiquitin ligase VHL in vitro, providing an important structural basis and interaction evidence for subsequent intracellular protein degradation. This indicates that TC-PROTAC has the potential to construct ternary complexes and induce target protein degradation, laying the foundation for its application as a novel PROTAC platform in tumor therapy.

[0066] Example 3

[0067] The TC Nano-PROTAC polypeptide prepared in Example 1 was used to verify its protein degradation effect at the cellular level. Figure 3 The results of in vitro validation experiments show the efficiency of TC-PROTAC in degrading HER2 and EGFR proteins, as well as its impact on the degradation of downstream signaling pathway proteins.

[0068] TC-PROTAC exhibits dose-dependent degradation of HER2 and EGFR proteins. Experimental results show that TC-PROTAC can dose-dependently degrade HER2 and EGFR proteins within a concentration range of 10-60 μM. This indicates that TC-PROTAC can more effectively promote the degradation of target proteins at higher concentrations, providing important dosage guidance for its application in tumor therapy.

[0069] Furthermore, the polyubiquitination signals of HER2 and EGFR proteins treated with TC-PROTAC were verified using a magnetic bead pull-down experiment. The experiment revealed that HER2 and EGFR proteins treated with TC-PROTAC exhibited significant polyubiquitination signals. This further confirms the mechanism by which TC-PROTAC promotes the ubiquitination of target proteins, thereby inducing their degradation through protein degradation pathways.

[0070] It can be concluded that TC-PROTAC can effectively degrade HER2 and EGFR proteins in vitro, and its degradation efficiency is dose-dependent. Furthermore, TC-PROTAC achieves degradation by promoting the ubiquitination of target proteins, providing important experimental evidence and mechanistic support for its application as a novel PROTAC platform in tumor therapy.

[0071] Example 4

[0072] Western blot (WB) experiments were used to demonstrate the in vitro degradation of HER2 and EGFR proteins by TC-PROTAC and its effects on downstream signaling pathways.

[0073] like Figure 3As shown, TC-PROTAC significantly reduced the expression levels of HER2 and EGFR proteins, further confirming that TC-PROTAC can effectively degrade target proteins in vitro. Furthermore, it demonstrated degradation of downstream signaling pathway proteins; SOS2 and RAS are key proteins in the HER2 and EGFR signaling pathways, and their degradation indicates that TC-PROTAC can not only degrade target proteins but also affect the integrity of their downstream signaling pathways.

[0074] The inhibitory effect of TC-PROTAC on the phosphorylation levels of AKT and ERK was determined. AKT and ERK are important kinases in signaling pathways, and the reduction in their phosphorylation levels indicates that TC-PROTAC can effectively inhibit the activation of these pathways.

[0075] In summary, this experiment demonstrates that TC-PROTAC significantly inhibits the activation of signaling pathways by degrading HER2 and EGFR proteins and key proteins in their downstream signaling pathways, thus exhibiting significant anti-tumor potential in vitro.

[0076] Example 5

[0077] To investigate the tumor targeting and persistent retention of TC-PROTAC in tumor tissue.

[0078] Construction of a mouse subcutaneous tumor model: First, NCI-N87 cells (a HER2-positive human gastric cancer cell line) were subcutaneously inoculated into mice to induce tumor formation. When the tumor volume reached approximately 100 mm³ (usually 12 days post-inoculation), the mice were randomly assigned to different experimental groups for subsequent drug treatment and evaluation.

[0079] The peptide TC Nano-PROTAC (Cy) probe prepared in Example 1 was used to perform specific recognition and long-term retention experiments at the animal level. Mice were selected for the experiment. Cy7-labeled TC-PROTAC was intravenously injected into mice carrying NCI-N87 (HER2+, EGFR+) tumors. Imaging was performed using a small animal in vivo imaging system (IVIS Spectrum) to monitor its distribution and accumulation at the tumor site. The imaging results are as follows: Figure 4 As shown, the results indicate that TC-PROTAC rapidly accumulates at the tumor site within 0.5 hours after injection, continues to accumulate within four hours, and remains present for 72 hours. This demonstrates that TC-PROTAC possesses good tumor targeting and persistent retention in tumor tissue.

[0080] Example 6

[0081] Animal tumor growth inhibition experiments were conducted using the polypeptide TC Nano-PROTAC prepared in Example 1.

[0082] The animals used in the experiment were mice. Construction of the mouse subcutaneous tumor model: Subcutaneous tumors were established in mice using gastric cancer cells, with 1×10⁻⁶ cells used. 6 Two weeks after injecting NCI-N87 cells into the subcutaneous tissue of the right leg of a mouse, a tumor formed, thus obtaining a mouse subcutaneous tumor model. Figure 5 This study demonstrates the antitumor effect of TC-PROTAC in a mouse NCI-N87 tumor model. Mice were randomly divided into four groups and treated with PBS, low, medium, and high doses of TC-PROTAC, respectively. Results showed that compared with the PBS-treated group, the TC-PROTAC-treated group significantly inhibited tumor growth in a dose-dependent manner. This indicates that TC-PROTAC has a significant antitumor effect in vivo and can effectively inhibit tumor growth.

[0083] The self-assembled peptide-based PROTAC condensates provided by this invention have demonstrated significant advantages and potential in tumor therapy. Their core advantage lies in their unique degradation mechanism, enabling them to efficiently degrade target proteins and key proteins in downstream signaling pathways. Unlike traditional small-molecule inhibitors, the bioconcentrated PROTACs achieve more durable therapeutic effects by inducing protein degradation rather than simply inhibiting their function. For example, in the NCI-N87 (EGFR+) tumor model, TC-PROTAC significantly reduced the expression levels of HER2 and EGFR proteins and was able to degrade downstream signaling pathway proteins such as SOS2 and RAS. Furthermore, the bioconcentrated PROTACs also exhibit excellent intracellular delivery. Through the application of nanotechnology, they can effectively enter tumor cells and remain stable, thus exerting their effects efficiently at the tumor site. This efficient intracellular delivery capability not only improves drug targeting but also reduces toxicity to normal cells and lowers the incidence of side effects. The bioconcentrated PROTACs also demonstrate significant advantages in overcoming drug resistance. Because they induce protein degradation rather than inhibit its function, they can effectively bypass drug resistance mechanisms. This is significant for treating drug-resistant tumors, especially when traditional drug therapies are ineffective. Finally, the bioaggregate PROTAC also exhibits an anti-hook effect, meaning it maintains high degradation efficiency even at high concentrations. This property allows the drug to exert stable therapeutic effects at different concentrations, further enhancing its potential for clinical application.

[0084] In summary, the bioaggregate PROTAC of the present invention, with its efficient protein degradation ability, good targeting specificity, ability to overcome drug resistance and excellent intracellular delivery capability, shows broad application prospects in tumor treatment.

[0085] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A dynamically adaptable polypeptide-based PROTAC condensate, characterized in that, The polypeptide-based PROTAC condensate includes: a HER2 targeting module, an EGFR targeting module, a VHL E3 ligase targeting module, and a self-assembly module.

2. The polypeptide-based PROTAC condensate according to claim 1, characterized in that, The amino acid sequence of the HER2 targeting module includes the sequence shown in SEQ ID NO.1, the EGFR targeting module includes a gefitinib derivative, the VHLE3 ligase targeting module includes a VHL ligand, and the amino acid sequence of the self-assembly module includes the sequence shown in SEQ ID NO.

2.

3. The polypeptide-based PROTAC condensate according to claim 1 or 2, characterized in that, The polypeptide-based PROTAC condensate comprises compounds as shown in Formula (1); 4. A method for preparing polypeptide-based PROTAC condensates according to any one of claims 1-3, characterized in that, The preparation method includes: using peptide synthesis technology to synthesize a HER2 targeting module, an EGFR targeting module, a VHL E3 ligase targeting module, and a self-assembly module; connecting each module to form a peptide-based PROTAC condensate precursor; and forming a peptide-based PROTAC condensate through in vitro or in vivo self-assembly.

5. The preparation method according to claim 4, characterized in that, The self-assembly includes: co-incubation of a peptide-based PROTAC condensate precursor, HER2-TC cells, and HER2-positive cells, followed by the formation of a peptide-based PROTAC condensate on the tumor cell membrane. The peptide-based PROTAC condensate carries tumor cell membrane components into the tumor cell, forming an intracellular peptide-based PROTAC condensate droplet.

6. The use of the polypeptide-based PROTAC condensate according to any one of claims 1-3 in protein degradation.

7. The application according to claim 6, characterized in that, The protein includes any one or a combination of at least two of the following: HER2 protein, EGFR protein, SOS2 protein, or RAS protein.

8. Use of the polypeptide-based PROTAC condensate of any one of claims 1-3 in the preparation of products for treating tumors and / or cancer.

9. The application according to claim 8, characterized in that, The tumors include tumors that overexpress HER2 and / or EGFR; the tumors also include tumors with HER2 and / or EGFR mutations.

10. The application according to claim 8 or 9, characterized in that, The cancers mentioned include breast cancer and / or stomach cancer.

Images