Targeting chimera based on cationic polymer protein hydrolysis as well as preparation and application of targeting chimera

Abstract

The invention discloses a cationic polymer-based proteolysis targeting chimera as well as preparation and application thereof, and belongs to the fields of targeting protein degradation and nanotechnology. According to the chimera, a cationic polymer is used as a connecting arm to connect a target protein ligand and an E3 ubiquitin ligase ligand to form a ternary complex, so that efficient degradation of target protein is realized. By adjusting the ligand proportion, chimeras with different degradation efficiencies can be synthesized, and accurate regulation and control are realized. The pH-responsive nano-drug obtained by hydrolyzing the targeting chimera by using the targeting protein can respond to an acidic tumor microenvironment, specifically release the chimera, and improve the targeting property and the treatment effect of the drug. Compared with the traditional PROTACs, the compound has higher bioavailability and tumor permeation effect; the preparation method of the nano-drug is simple and easy to implement, provides possibility for preparation of high-performance nano-drugs, shows potential in preparation of anti-tumor cell drugs, provides new thoughts and methods for tumor treatment, and has important clinical application value.

Description

Technical Field

[0001] The invention relates to the field of targeted protein degradation and nanotechnology, and in particular to a cationic polymer-based protein hydrolysis targeted chimera and the preparation and application thereof. Background Art

[0002] As an innovative drug form, PROTACs degrade pathogenic proteins in an event-driven cyclic manner compared to traditional small molecule drugs, solving the toxicity and drug resistance problems caused by traditional small molecule drugs inhibiting pathogenic proteins through long-term high-concentration occupancy-driven modes; at the same time, PROTACs can regulate those undruggable targets that lack classic hydrophobic drug binding pockets or strongly bind to endogenous molecules, which is difficult for traditional small molecule drugs to do; by applying PROTACs, the selectivity of closely related proteins can be improved, so that the target protein can be treated more accurately; therefore, PROTACs have broad application prospects in the field of disease treatment, especially cancer treatment. PROTACs is a bifunctional targeted compound, mainly composed of three parts: target protein ligand, E3 ubiquitin ligase ligand and linker. This special design enables PROTACs to simultaneously recognize and bind to the target protein and E3 ubiquitin ligase, thereby shortening the spatial distance between the two. The mechanism of PROTACs targeted degradation of target proteins is as follows: PROTACs recognizes and binds to the target protein at one end and binds to the E3 ubiquitin ligase at the other end. Under the action of the connecting arm, the target protein and the E3 ubiquitin ligase are brought close enough so that the E3 ubiquitin ligase can catalyze the ubiquitination process of the target protein. Subsequently, the surface of the target protein is marked with polyubiquitination and then recognized and degraded by the proteasome.

[0003] Although PROTACs have many advantages, their composition and structural characteristics also bring some challenges. Since PROTACs usually have a large molecular weight and most of their components are highly hydrophobic and difficult to be taken up by cells, their bioavailability is poor, which greatly limits the clinical application of PROTACs. In addition, it is difficult to screen the ratio of the two ligands during the synthesis of PROTACs, and a large number of screening of the types and lengths of the intermediate connecting arms are required. This limits the efficiency of PROTACs synthesis and screening of active degradation compounds.

[0004] Although pH-activated stimuli-responsive nanomedicines have great potential in tumor treatment, they still face many problems, such as material selection and design, drug release control, tumor penetration and targeting, and clinical transformation and supervision. Although polyester materials such as PEG-PCL have been approved by the FDA for use as drug carriers, their long-term biocompatibility and possible toxic reactions still need to be considered in practical applications, and further research and optimization of the chemical structure of the material are needed to improve its biosafety and stability. The acidity of the tumor microenvironment may vary depending on individual differences and tumor types. Current pH-responsive materials often have a narrow response threshold, which limits their applicability in complex tumor environments. Therefore, it is necessary to design nanomedicines with a wider pH response range to ensure that drugs can be effectively released in different tumor environments. Existing pH-activated nanomedicines often have a "burst release" phenomenon when releasing drugs, that is, a large amount of drugs are quickly released after reaching the pH response threshold, resulting in a rapid decrease in drug concentration, affecting the therapeutic effect. It is necessary to develop nanomedicines with more sophisticated release control mechanisms to achieve sustained and stable drug release; during the preparation and storage process, the stability of nanomedicines may be affected by a variety of factors, such as light, temperature, humidity, etc., and it is necessary to optimize the preparation process and storage conditions to ensure that nanomedicines maintain stable performance during preparation, storage and use. Tumor tissues often have complex vascular structures and dense stroma, which makes it difficult for nanomedicines to effectively penetrate deep into the tumor. It is necessary to develop nano drug carriers with better permeability, such as by optimizing the shape, size, surface properties, etc. of the carrier to improve its penetration into tumor tissues; although pH-activatable nano drugs can respond to the acidic characteristics of the tumor microenvironment, their targeting accuracy still needs to be considered in practical applications. It is necessary to combine advanced targeting technology and imaging technology, such as molecular imaging, optical imaging, etc., to improve the targeting accuracy and therapeutic effect of nano drugs. Summary of the invention

[0005] In view of the poor bioavailability of existing PROTACs, the difficulty in screening the ratio of two ligands during synthesis, and the need to screen a large number of intermediate linker arm types and lengths, which limits the efficiency of PROTACs synthesis and screening of active degradation compounds, as well as the technical status quo of pH-activatable nanomedicines in material selection, drug release control, tumor penetration and targeting, the present invention aims to provide a cationic polymer proteolysis-targeted chimera, a nano-targeted protein degradation drug, and its preparation and application, so as to address the existing limitations of small molecule PROTACs.

[0006] The present invention is achieved through the following technical solutions: The present invention provides a cationic polymer-based proteolysis targeting chimera, wherein the proteolysis targeting chimera comprises a cationic polymer, a target protein ligand and an E3 ubiquitin ligase ligand; The target protein ligand is used to target the target protein; The E3 ubiquitin ligase ligand is used to target the E3 ubiquitin ligase; The cationic polymer serves as a connecting arm between the target protein ligand and the E3 ubiquitin ligase ligand, coupling the target protein ligand and the E3 ubiquitin ligase ligand.

[0007] The cationic polymer is any one of linear or branched polyethyleneimine, dendritic polyamidoamine (PAMAM) and functionalized multi-arm PEG; The targeting protein ligand is a ligand targeting hexokinase-2, pyruvate dehydrogenase kinase, bromoprotein domain 4 (BRD4) or indoleamine-2,3-dioxygenase (IDO); The E3 ubiquitin ligase ligand is any one of cereblon protein (CRBN), angiostatin tumor suppressor protein (VHL), mouse double minute gene 2 protein (MDM2) and cell inhibitor of apoptosis protein (cIAP) ligand.

[0008] The linear or branched polyethyleneimine is a linear or branched polyethyleneimine with a molecular weight of 1000-40000 Da; The targeting protein ligand is any one or more combinations of lonidamine, 3-bromopyruvic acid, JQ-1, NLG919 and dichloroacetic acid; The E3 ubiquitin ligase ligand is any one of thalidomide, lenalidomide, pomalidomide and VHL-032.

[0009] The linear or branched polyethyleneimine is a linear or branched polyethyleneimine with a molecular weight of 1000-40000 Da; The ligand targeting hexokinase-2 is any one of lonidamine and 3-bromopyruvate, the ligand targeting pyruvate dehydrogenase kinase is dichloroacetic acid, the ligand targeting BRD4 is JQ1, and the ligand targeting IDO is NLG919; The E3 ubiquitin ligase ligand is any one of thalidomide, lenalidomide, pomalidomide and VHL-032.

[0010] Furthermore, the polyethyleneimine compound is a branched polyethyleneimine with a molecular weight of 1800 Da, and the specific structure is as follows: ; The present invention discloses a method for preparing the above-mentioned cationic polymer-based proteolysis targeting chimera, comprising: S1, after the cationic polymer and the E3 ubiquitin ligase ligand are dissolved and mixed, the mixture is evacuated and filled with nitrogen for protection, and a catalyst dissolved in the same solvent is added dropwise for reaction to obtain a reaction solution 1; S2, cooling the reaction solution 1 obtained in S1, and adding the targeted protein ligand and the catalyst dissolved in the same solvent respectively to react, so as to obtain the reaction solution 2; S3, the reaction solution 2 obtained in S2 is concentrated, precipitated, centrifuged, and dialyzed for purification to obtain a cationic polymer-based protein hydrolysis targeting chimera.

[0011] In S1, the molar ratio of the cationic polymer to the E3 ubiquitin ligase ligand is 1:1-3, the catalyst used is O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) or N,N-diisopropylethylamine (DIPEA), the amount of the catalyst added is 1-3 times the molar amount of the E3 ubiquitin ligase ligand, the reaction temperature is 25°C-100°C, and the reaction time is 10 h-24 h.

[0012] In S2, the molar ratio of the targeting protein ligand to the cationic polymer is 1-3:1, the catalyst used is O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), the amount of the catalyst added is 1-3 times the molar amount of the targeting protein ligand, the reaction temperature is 25°C-38°C, and the reaction time is 5 h-24 h.

[0013] In S3, the precipitate is precipitated with anhydrous ether precooled at 4°C for 8 min~12 min.

[0014] The present invention provides a pH-responsive targeted protein degradation nanomedicine, wherein the pH-responsive targeted protein degradation nanomedicine is obtained by connecting the cationic polymer-based protein hydrolysis targeting chimera to an amphiphilic polymer via a pH-responsive arm; The pH-responsive linker is any one of cis-aconitic anhydride and 2-propionic acid-3-methylmaleic anhydride; The amphiphilic polymer is any one of polyethylene glycol-polycaprolactone, polyethylene glycol-polylactic acid and polyethylene glycol-polylactic acid-glycolic acid copolymer.

[0015] Furthermore, the PEG-PCL polymer is a PEG-PCL polymer with a molecular weight of 2000-1000000 Da.

[0016] The present invention provides a method for preparing the pH-responsive targeted protein degradation nano-drug, comprising dissolving the cationic polymer-based protein hydrolysis targeting chimera, the amphiphilic polymer and the pH-responsive arm respectively and then mixing them evenly to obtain an engineered pH-activatable nano-protein hydrolysis targeting chimera; dissolving and mixing the engineered pH-activatable nano-protein hydrolysis targeting chimera and the amphiphilic polymer, dropping them into ultrapure water, stirring, and dialyzing to obtain the pH-responsive targeted protein degradation nano-drug; The molar ratio of the cationic polymer-based proteolysis targeting chimera, the amphiphilic polymer and the pH-responsive arm is 1:1.2-2; The molar ratio of the engineered pH-activatable nanoprotein hydrolysis targeting chimera to the amphiphilic polymer is 1:1-2.

[0017] Furthermore, the dissolving solvent is N,N-dimethylformamide or dimethyl sulfoxide.

[0018] The application of the cationic polymer-based protein hydrolysis targeted chimera or the pH-responsive targeted protein degradation nanomedicine in the preparation of anti-tumor cell drugs.

[0019] Compared with the prior art, the present invention has the following technical effects: The present invention provides a novel cationic polymer-based proteolysis targeting chimera, which combines a cationic polymer, a target protein ligand and an E3 ubiquitin ligase ligand to form a novel targeted protein degradation strategy, wherein the cationic polymer is used as a connecting arm, and the target protein ligand and the E3 ubiquitin ligase ligand are modified on the free amino group of the cationic polymer, and can bind to the cell membrane with a negative charge on the surface, and then enter the cell through endocytosis, and escape from the lysosome, so that the proteolysis targeting chimera can more effectively approach and degrade the target protein. The proteolysis targeting chimera is connected to the target protein at one end and to the E3 ubiquitin ligase at the other end to form a ternary complex, and ubiquitin is transferred to the surface of the target protein, and the target protein is polyubiquitinated and marked, and then the target protein is recognized and degraded by the proteasome.

[0020] Furthermore, by adjusting the ratio of the target protein ligand to the E3 ubiquitin ligase ligand, protein hydrolysis targeting chimeras with different degradation efficiencies can be synthesized, thereby achieving precise regulation of the target protein; polyethyleneimine acts as a linker between the target protein ligand and the E3 ubiquitin ligase. The surface of polyethyleneimine carries a large number of free amino groups. By changing the ratio between the two ligands, cationic polymer-based protein hydrolysis targeting chimeras modified with different ratios of ligands can be synthesized to improve the degradation efficiency of the target protein; the spatial distance range of the amino groups on the surface of polyethyleneimine is large, and there is no need to screen a large number of linkers. It is only necessary to change the feed ratio of the two ligands to achieve the synthesis of polymers with targeted degradation activity, saving the time of traditional small molecule PROTACs linker screening.

[0021] The method for preparing a cationic polymer-based proteolysis targeting chimera disclosed in the present invention has simple steps, convenient operation, and is relatively easy to achieve industrial production. Polyethyleneimine, target protein ligand, and E3 ubiquitin ligase ligand are prepared by chemical synthesis, which is simple and relatively easy to prepare; there is no need to screen a large number of connecting arms, and a polymer with targeted degradation activity can be synthesized by only changing the feed ratio of two ligands, which greatly saves time and cost.

[0022] The present invention discloses a pH-responsive targeted protein degradation nano-drug, which is connected to an amphiphilic polymer through a pH-responsive arm to achieve precise release under a specific pH environment. The tumor microenvironment presents an acidic pH. The nano-drug can respond to the acidic tumor microenvironment and release an engineered pH-activated nano-protein hydrolysis targeting chimera to degrade the target protein in the tumor. Compared with traditional small molecule PROTACs, the pH-responsive nano-drug prepared by the present invention using the advantages of nanomedicine has higher bioavailability and tumor penetration; it can be specifically released in an acidic tumor microenvironment, thereby improving the targeting and therapeutic effect of the drug.

[0023] The method for preparing a pH-responsive targeted protein degradation nanomedicine disclosed in the present invention is simple and easy to implement, and provides the possibility for preparing high-performance nanomedicines. The cationic polymer-based protein hydrolysis targeting chimera is prepared into an engineered pH-activatable nano protein hydrolysis targeting chimera by utilizing the advantages of nanomedicine, so that the cationic polymer-based protein hydrolysis targeting chimera is specifically released in an acidic tumor microenvironment, which not only improves the bioavailability of the drug, but also improves the tumor penetration of the drug.

[0024] The application provided by the present invention, the application of cationic polymer-based protein hydrolysis targeting chimeras and pH-responsive nano protein hydrolysis targeting chimeras in the preparation of anti-tumor cell drugs, especially shows potential in inhibiting tumor metastasis and inducing tumor cell pyroptosis, provides new ideas and methods for tumor treatment, and has important scientific significance and clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The present invention is a PEI-based PROTACs chemical targeted degradation of HK-2, wherein A is a schematic diagram of PEI-based PROTACs synthesis; B is a UV scanning spectrum of LND, THA, and PEI-based PROTACs, with a wavelength scanning range of 230 nm to 350 nm; C is a Western blot detection of the degradation effect of PEI-based PROTACs with different LND and THA ratios; D is a quantitative result of PEI-based PROTACs degradation with different LND and THA ratios; E is the degradation effect of CT26 cells treated with PEI-based PROTACs at different concentrations for 6 h; F is a quantitative result of the concentration-dependent degradation effect of PEI-based PROTACs; G is the degradation mechanism of HK-2 by PEI-based PROTACs; H is the quantification of the HK-2 degradation mechanism by PEI-based PROTACs; I is a Western blot detection result; J is the quantification of the effect of PEI-based PROTACs on polyubiquitin after treating CT26 cells; Figure 2 It is the Fourier transform infrared scanning spectrum of free LND, free 4-F-THA and PEI-based PROTACs of the present invention, and the infrared wavelength scanning range is 400 nm to 4000 nm; Figure 3 The WB analysis of the effect of PEI-based PROTACs on HK-2 degradation in CT26 cells using different ratios of PEI, LND and THA in the present invention is performed, wherein A is the WB analysis of the ratio of PEI to the targeting probe of 1:0.5; B is the quantitative result of the ratio of PEI to the targeting probe of 1:0.5; C is the WB analysis of the ratio of PEI to the targeting probe of 1:1; D is the quantitative result of the ratio of PEI to the targeting probe of 1:1; E is the WB analysis of the ratio of PEI to the targeting probe of 1:1.5; F is the quantitative result of the ratio of PEI to the targeting probe of 1:1.5; Figure 4 The present invention is based on PEG 5k -PCL 5k -H NMR spectrum of PROTACs of CAA-PEI; Figure 5 The present invention is based on PEG 5k -PCL 5k -H NMR spectrum of PROTACs of SA-PEI; Figure 6Figure 2 is the preparation and acid response characteristics of the engineered nano-PROTACs of the present invention, wherein A is a schematic diagram of the preparation of nano-PROTACs and sNano-PROTACs; B is the particle size of nano-PROTACs and sNano-PROTACs in ultrapure water detected by DLS; C is a TEM photo of nano-PROTACs and sNano-PROTACs in PBS at pH = 7.4 and PBS at pH = 6.5, scale: 100 nm; D is the change in particle size of nano-PROTACs and sNano-PROTACs in PBS at pH = 7.4 and PBS at pH = 6.5 detected by DLS at 37 ° C after incubation for 0 h, 4 h, and 24 h; E is the Zeta potential of nano-PROTACs and sNano-PROTACs in ultrapure water; F is the change in particle size of Nano-PROTACs and sNano-PROTACs in PBS at pH = 7.4 and PBS at pH = 6.5 at 37 ° C for 0 h, 3 h, 6 h h after zeta potential change; G is the release of PEI-based PROTACs from Nano-PROTACs and sNano-PROTACs in PBS at pH = 6.5 at 37 °C; H is the particle stability test of Nano-PROTACs and sNano-PROTACs in PBS at pH = 7.4 and ultrapure water within 7 days; Figure 7Figure 3 is a schematic diagram of the pH-activatable Nano-PROTACs of the present invention that induce HK-2 degradation and reprogram glycolysis, wherein A is a schematic diagram of the mechanism by which Nano-PROTACs release PEI-based PROTACs and degrade HK-2; B is the time dependence of Nano-PROTACs-induced HK-2 degradation; C is the time dependence of sNano-PROTACs-induced HK-2 degradation; D is the quantitative result of the time dependence of the HK-2 degradation effect of Nano-PROTACs and sNano-PROTACs; E is the change in ECAR of CT26 cells after treatment with PEI-based PROTACs, Nano-PROTACs and sNano-PROTACs for 12 h; F is the quantification of glycolysis after incubation of CT26 cells with PEI-based PROTACs, Nano-PROTACs and sNano-PROTACs. (G) Quantification of glycolytic capacity of CT26 cells after incubation with PEI-based PROTACs, Nano-PROTACs and sNano-PROTACs; H is the quantification of glycolytic reserve after incubation with PEI-based PROTACs, Nano-PROTACs and sNano-PROTACs; I is the co-localization between Ce6-labeled Nano-PROTACs and Mito-tracker, CT26 cells were pretreated with Ce6-labeled Nano-PROTACs for 3 hours, scale bar: 10 μm; J is the co-localization quantification between Ce6-labeled Nano-PROTACs and Mito-tracker; K is the tumor sphere permeability of Nano-PROTACs and sNano-PROTACs in pH 6.5 and pH 7.4 environments, CT26 tumor spheres were incubated with Ce6-labeled Nano-PROTACs and Ce6-labeled sNano-PROTACs for 24 hours; Figure 8Figure 3 is the in vivo antitumor ability of Nano-PROTACs of the present invention; A is the antitumor and biosafety treatment scheme of PEI-based PROTACs, Nano-PROTACs and sNano-PROTACs in CT26 tumor-bearing mice; B is the relative tumor volume of CT26 tumor-bearing mice treated with PEI-based PROTACs, Nano-PROTACs and sNano-PROTACs at the same dose (LND: 5 mg / kg, THA: 5 mg / kg) for 5 times; C is the relative body weight detection of CT26 tumor-bearing mice; D is a photo of CT26 tumor-bearing mice; E is H&E staining (scale bar: 50 μm) and immunofluorescence staining of CT26 tumors (green: TUNEL, caspase and PCNA, blue: cell nucleus, scale bar: 50 μm; F is the quantitative immunofluorescence staining of PCNA; G is the quantitative immunofluorescence staining of active caspase 3; H is the immunofluorescence staining of CT26 tumors (green: HK-2, blue: cell nucleus, scale bar: 50 μm); I is the quantification of HK-2 by immunofluorescence staining; J is the protein blot analysis in CT26 tumor lysate; K is the quantification of HK-2 degradation effect; L is the protein blot analysis in CT26 tumor lysate; M is the quantification of active caspase 3; N is the protein blot analysis in CT26 tumor lysate; O is the quantification of GSDME-N; Fig. 9 Figure 2 is the anti-tumor efficacy of Nano-PROTACs of the present invention in 4T1 orthotopic tumor mice, wherein A is a schematic diagram of the treatment process; B is the change in tumor volume of mice receiving each group of treatment; C is the weight growth curve of mice receiving each group of treatment; D is the orthotopic tumor image of mice receiving each group of treatment; E is H&E staining (scale bar: 50 μm) and immunofluorescence staining (green: TUNEL, caspase 3 and PCNA, blue: cell nucleus, scale bar: 50 μm) of 4T1 tumor; F is the quantification of immunofluorescence staining of PCNA; G is the quantification of immunofluorescence staining of active caspase 3; H is the immunofluorescence staining of 4T1 tumor (green: HK-2, blue: cell nucleus, scale bar: 50 μm); I is the quantification of immunofluorescence staining of HK-2; J is Western blot detection; K is the quantification of HK-2 degradation effect in 4T1 tumor lysate; L is Western blot detection; M is the active caspase in 4T1 tumor lysate 3, quantification; N, Western blot detection; O, quantification of GSDME-N in 4T1 tumor lysate; Fig.10The inhibitory effect of Nano-PROTACs of the present invention on tumor metastasis, wherein A is a schematic diagram of the treatment process, 4T1 orthotopic tumor mice were treated with PBS, PEI-based PROTACs, sNano-PROTACs and Nano-PROTACs at the indicated time points, B is an in vivo bioluminescent image of lung metastatic nodules in 4T1 orthotopic tumor mice treated with the indicated treatment methods; C is a quantitative analysis; D is an in vitro bioluminescent image of lung metastatic nodules in 4T1 orthotopic tumor mice treated with the indicated treatment methods; E is a quantitative analysis; F is a representative image of lungs isolated from 4T1 orthotopic tumor mice treated with the indicated treatment methods; G is H&E staining of lung tissues of 4T1 orthotopic tumor mice treated with the indicated treatment methods, scale bar: 200 μm; Fig.11 The concentration dependence of the proteolysis targeting chimera based on the cationic polymer of the present invention on the degradation of HK-2 protein in 4T1 cells; Fig.12 The cytotoxicity assay of the cationic polymer-based proteolysis targeting chimera on 4T1 cells of the present invention; Fig.13 The present invention is based on the concentration-dependent detection of the degradation of pyruvate dehydrogenase kinase 1 (PDK1) in 4T1 cells by cationic polymer proteolysis targeting chimeras. DETAILED DESCRIPTION

[0026] In order to make the main purpose, technical key and main function of the present invention clearer, a detailed description will be given in conjunction with the accompanying drawings and embodiments. It should be noted that the following description is only used to explain the present invention, is only a part of the embodiments of the present invention, and is not used to limit the present invention.

[0027] Example 1 This example provides a cationic polymer-based proteolysis targeting chimera. The preparation process and related characterization results are shown in the attached Figure 1-3 shown.

[0028] The specific preparation process of the cationic polymer-based proteolysis targeting chimera is as follows: The cationic polymer used in this embodiment is a branched polyethyleneimine with a molecular weight of 1800 Da, and the specific structure is as follows: .

[0029] After mixing branched polyethyleneimine (1 mmol) with a molecular weight of 1800 Da and the E3 ubiquitin ligase ligand thalidomide (1.2 mmol), the mixture was evacuated and filled with nitrogen for protection, and the diluted catalyst O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (3 mmol) was added dropwise, and the mixture was reacted at 90 °C for 12 h. After cooling, lonidamine (LND, 1.2 mmol) and HATU (1.2 mmol) diluted with DMF were added dropwise, and the mixture was reacted at 37 °C for 6 h. The mixture was concentrated and precipitated with anhydrous ether precooled at 4 °C for 10 min. The mixture was centrifuged in a centrifuge at 4 °C and 10,000 rpm for 10 min to obtain the final product of cationic polymer-based proteolysis targeting chimeras (PEI-based PROTACs), and the reaction yield was 95%.

[0030] The cationic polymer proteolysis targeting chimera prepared in this example was further characterized.

[0031] By the attached Figure 1-3 The data show that the PEI-based PROTACs synthesized in this example have obvious ultraviolet absorption peaks at 278 nm and 237 nm, indicating that LND and 4-F-THA have been successfully bound to PEI or its derivatives to form PEI-based PROTACs; Fourier transform infrared spectrometer scans the infrared spectrum of free LND, free 4-F-THA, and PEI-based PROTACs. There is a carboxyl group in LND, and the carbon-oxygen double bond stretching vibration peak of the carboxyl group in the infrared is located at 1700 cm -1 The stretching vibration peak of fluorobenzene in 4-F-THA is located at 1250 cm -1 The nitrogen-hydrogen stretching vibration peak of the free amino group of PEI is located at 3450 cm -1 In the infrared scanning spectrum of PEI-based PROTACs, there are characteristic peaks of LND and PEI, and the fluorobenzene stretching vibration peak in 4-F-THA disappears. The above results prove that the amino group of PEI polymer successfully connects LND and 4-F-THA, indicating the successful synthesis of PEI-based PROTACs.

[0032] The ratio of HK-2 to CRBN targeting module was optimized to achieve effective degradation of HK-2. When the mass ratio was 1:1, the HK-2:CRBN targeting module showed better degradation efficiency than other ratios, achieving 46.8% HK-2 degradation. PEI-based PROTACs had a concentration-dependent effect on HK-2 degradation. The proteasome inhibitor MG132 effectively inhibited the degradation of HK-2 in CT26 cells induced by PEI-based PROTACs, and the HK-2 degradation rate increased to 97.9%, indicating that PEI-based PROTACs degraded HK-2 in a manner dependent on the ubiquitin proteasome system (UPS). CT26 cells treated with PEI-based PROTACs showed more polyubiquitination than the PBS group, indicating that PEI-based PROTACs degraded HK-2 in a manner dependent on the ubiquitin proteasome system (UPS). CT26 cells treated with PEI-based PROTACs showed more polyubiquitination than the PBS group, further confirming its degradation mechanism.

[0033] Example 2 This example provides the preparation of a pH-activatable engineered nanoparticle-type selective hexokinase 2 nanoprotein hydrolysis targeting chimera based on Example 1, wherein PEI-based PROTACs are covalently grafted onto the main chain of an amphiphilic block polymer via an acid-cleavable bond, and both Nano-PROTACs and sNano-PROTACs nanoparticles are prepared by self-assembly of PEG-PCL-CAA / SA-PEI-based PROTACs copolymers using a nanoprecipitation method. The specific preparation process and related characterization are shown in the attached Figure 4-6 shown.

[0034] FDA-approved poly(ethylene glycol)-poly(ε-caprolactone) copolymer (PEG-PCL) was selected as the amphiphilic polymer, and pH-activatable nano-PROTACs (Nano-PROTACs) were synthesized by binding PEI-based PROTACs to PEG-PCL diblock polymers via acid-detachable cis-aconitic anhydride (CAA) bonds. The specific steps were as follows: the cationic polymer-based proteolysis targeting chimera (1 mmol) of Example 1 above, PEG-PCL diblock polymer with a molecular weight of 10,000 Da (1 mmol) and cis-aconitic anhydride (CAA) were mixed. (1.2mmol) were dissolved in N,N-dimethylformamide and mixed evenly to obtain an engineered pH-activatable nanoprotein hydrolysis targeting chimera (100mg); the engineered pH-activatable nanoprotein hydrolysis targeting chimera (1mmol) and PEG-PCL diblock polymer (1mmol) were dissolved in N,N-dimethylformamide and mixed evenly, dropped into ultrapure water with stirring, and dialyzed to obtain pH-responsive targeted protein degradation nanodrugs (Nano-PROTACs).

[0035] As a negative control, pH-stable nano-PROTACs (sNano-PROTACs) were synthesized by conjugating PEI-based PROTACs to PEG-PCL via acid-stable succinic anhydride bonds, and their structures were verified by H NMR spectroscopy. Figure 4-5 shown.

[0036] By the attached Figure 4-5 The data show that this example successfully constructed an engineered pH-activatable proteolysis targeting chimera and a non-pH-sensitive proteolysis targeting chimera.

[0037] By the attached Figure 6 The data shows that Figure 6In the figure, A is a schematic diagram of the preparation of nano-PROTACs and sNano-PROTACs; B is the particle size of nano-PROTACs and sNano-PROTACs in ultrapure water detected by DLS; C is a TEM photo of nano-PROTACs and sNano-PROTACs in PBS at pH = 7.4 and PBS at pH = 6.5, scale: 100 nm; D is the change in particle size of nano-PROTACs and sNano-PROTACs in PBS at pH = 7.4 and PBS at pH = 6.5 detected by DLS at 37 ° C after incubation for 0 h, 4 h, and 24 h; E is the Zeta potential of nano-PROTACs and sNano-PROTACs in ultrapure water; F is the change in zeta potential of Nano-PROTACs and sNano-PROTACs in PBS at pH = 7.4 and PBS at pH = 6.5 at 37 ° C after incubation for 0 h, 3 h, and 6 h; G is the change in zeta potential of Nano-PROTACs and sNano-PROTACs in PBS at pH = 7.4 and PBS at pH = 6.5 at 37 ° C. ℃ is the release of PEI-based PROTACs from Nano-PROTACs and sNano-PROTACs in PBS at pH = 6.5; H is the particle stability test of Nano-PROTACs and sNano-PROTACs in PBS at pH = 7.4 and ultrapure water within 7 days.

[0038] Morphology observation and particle size analysis: Dynamic light scattering (DLS) showed that at pH = 7.4, the diameter of Nano-PROTACs was about 24.70 ± 2.00 nm, and the diameter of sNano-PROTACs was about 25.00 ± 1.61 nm, which was consistent with the transmission electron microscopy (TEM) images. Both Nano-PROTACs and sNano-PROTACs were monodisperse spherical nanoparticles with similar particle sizes and were both at the nanometer level. pH responsiveness: At pH 6.5, Nano-PROTACs nanoparticles disintegrated due to acid-induced bond cleavage, while the morphology of sNano-PROTACs did not change significantly compared with pH 7.4, indicating that Nano-PROTACs are pH responsive, while sNano-PROTACs are relatively stable; Zeta potential analysis: At pH = 7.4, the zeta potentials of Nano-PROTACs and sNano-PROTACs were both negative. At pH 6.5, due to the acid-induced dissociation of PEI-based PROTAC, the zeta potential of Nano-PROTACs reversed from negative to positive, while the zeta potential of sNano-PROTACs was almost unaffected, further confirming the pH responsiveness of Nano-PROTACs.

[0039] Drug release properties: At pH 6.5, more than 43.6% of PEI-based PROTACs were sharply released from Nano-PROTACs. In contrast, under acidic conditions, very little release was detected in the sNano-PROTACs group, indicating that Nano-PROTACs have the ability to release drugs in acidic environments.

[0040] Stability analysis: Both Nano-PROTACs and sNano-PROTACs can maintain stability for more than 7 days in a physiological environment, indicating that both nanoparticles have good stability and are suitable for long-term applications.

[0041] In summary, Nano-PROTACs and sNano-PROTACs are both monodisperse spherical nanoparticles with similar particle sizes. However, Nano-PROTACs have significant pH responsiveness and can dissolve and release drugs in acidic environments, while sNano-PROTACs are relatively stable. In addition, both nanoparticles can maintain long-term stability in physiological environments. These characteristics make Nano-PROTACs and sNano-PROTACs have potential application value in drug delivery and targeted therapy.

[0042] Example 3 In this example, based on Example 1-2, PEI-based PROTACs can induce HK-2 degradation through the UPS pathway, further explore whether pH-activated nano-PROTACs can also achieve ubiquitinated protein degradation, and examine the in vitro reprogramming of glycolysis by nano-PROTACs. For details, see the attached Figure 7 shown.

[0043] By the attached Figure 7 The data shows that Figure 7In the figure, A is a schematic diagram of the mechanism by which Nano-PROTACs release PEI-based PROTACs and degrade HK-2; B is the time dependence of Nano-PROTACs-induced HK-2 degradation; C is the time dependence of sNano-PROTACs-induced HK-2 degradation; D is the quantitative result of the time dependence of the HK-2 degradation effect of Nano-PROTACs and sNano-PROTACs; E is the change of ECAR in CT26 cells after treatment with PEI-based PROTACs, Nano-PROTACs and sNano-PROTACs for 12 h; F is the quantification of glycolysis after CT26 cells were incubated with PEI-based PROTACs, Nano-PROTACs and sNano-PROTACs. (G) Quantification of glycolytic capacity of CT26 cells after incubation with PEI-based PROTACs, Nano-PROTACs and sNano-PROTACs; H is the quantification of glycolytic reserve after incubation of CT26 cells with PEI-based PROTACs, Nano-PROTACs and sNano-PROTACs; I is the colocalization between Ce6-labeled Nano-PROTACs and Mito-tracker, CT26 cells were pretreated with Ce6-labeled Nano-PROTACs for 3 hours, scale bar: 10 μm; J is the colocalization quantification between Ce6-labeled Nano-PROTACs and Mito-tracker; K is the tumor sphere permeability of Nano-PROTACs and sNano-PROTACs in pH 6.5 and pH 7.4 environments, CT26 tumor spheres were incubated with Ce6-labeled Nano-PROTACs and Ce6-labeled sNano-PROTACs for 24 hours.

[0044] Nano-PROTACs induce HK-2 degradation: After CT26 cells were incubated with Nano-PROTACs for 24 hours, the expression of HK-2 was significantly downregulated to 54.9%, indicating that Nano-PROTACs can effectively induce HK-2 degradation. In contrast, sNano-PROTACs did not show any effect on HK-2 degradation within the same period of time.

[0045] Analysis of mitochondrial bioenergetic profile: The mitochondrial bioenergetic profile in CT26 cells after different treatments was studied using the Seahorse XF96 extracellular flux analyzer. Compared with PBS and sNano-PROTACs, Nano-PROTACs significantly reduced the extracellular acidification rate (ECAR), which reflects the change in glycolytic activity. Specifically, after Nano-PROTACs treatment, key indicators of glycolysis (such as ECAR after induced glycolysis, maximum glycolytic capacity, and glycolytic reserve) were significantly reduced, indicating that Nano-PROTACs can inhibit glycolytic activity.

[0046] Intracellular behavior and mitochondrial targeting ability of Nano-PROTACs: The intracellular behavior and mitochondrial targeting ability of Nano-PROTACs were studied by confocal laser scanning microscopy (CLSM). Ce6-labeled nano-PROTACs achieved mitochondrial co-localization after incubation with CT26 cells for 3 hours, indicating that Nano-PROTACs have mitochondrial targeting ability. The fluorescence of Nano-PROTACs and mitochondria is almost consistent in cells, further confirming the mitochondrial targeting of Nano-PROTACs.

[0047] Tumor permeability of Nano-PROTACs: At pH 6.5, the permeability performance of Nano-PROTACs in CT26 tumor spheres was better than that of sNano-PROTACs, indicating that Nano-PROTACs have better tumor permeability in an acidic environment.

[0048] In summary, Nano-PROTACs showed significant HK-2 degradation ability and glycolysis inhibition effect in in vitro experiments. Mitochondrial bioenergetic spectrum analysis confirmed that Nano-PROTACs can inhibit the glycolytic activity of tumor cells. In addition, Nano-PROTACs also showed good mitochondrial targeting ability and tumor permeability, especially in acidic environments. These characteristics make Nano-PROTACs have potential application value in tumor treatment and energy metabolism regulation.

[0049] Example 4 This example, based on Example 1-2, investigates the anti-tumor effect of Nano-PROTACs in the CT26 tumor model, and uses the CT26 tumor model to study the anti-tumor effect of Nano-PROTACs. CT26 cells were subcutaneously injected into the back of mice. When the tumor size reached about 50 mm³, starting from day 0, Nano-PROTACs were intravenously injected every two days (days 2, 4, 6, 8, 10 and 12), and different treatment groups (Nano-PROTACs, sNano-PROTACs, PEI-based PROTACs and saline treatment groups) were set up. For specific experimental procedures and experimental results, please refer to the attached Figure 8 shown.

[0050] By the attached Figure 8 The data shows that Figure 8 In the figure, A is the treatment scheme of PEI-based PROTACs, Nano-PROTACs and sNano-PROTACs for antitumor and biosafety of CT26 tumor-bearing mice; B is the relative tumor volume of CT26 tumor-bearing mice after 5 treatments with PEI-based PROTACs, Nano-PROTACs and sNano-PROTACs at the same dose (LND: 5 mg / kg, THA: 5 mg / kg); C is the relative body weight detection of CT26 tumor-bearing mice; D is a photo of CT26 tumor-bearing mice; E is H&E staining (scale bar: 50 μm) and immunofluorescence staining of CT26 tumors (green: TUNEL, caspase and PCNA, blue: cell nucleus, scale bar: 50 μm; F is the quantification of immunofluorescence staining of PCNA; G is the quantification of immunofluorescence staining of active caspase 3; H is the immunofluorescence staining of CT26 tumors (green: HK-2, blue: cell nucleus, scale bar: 50 μm); I is the quantification of HK-2 by immunofluorescence staining; J is the protein blot analysis in CT26 tumor lysate; K is the quantification of HK-2 degradation effect; L is the protein blot analysis in CT26 tumor lysate; M is the quantification of active caspase 3; N is the protein blot analysis in CT26 tumor lysate; O is the quantification of GSDME-N.

[0051] Changes in tumor volume over time: Comparing the changes in tumor volume in each group, Nano-PROTACs achieved a tumor eradication rate of 79.2%, which was significantly higher than the saline treatment group (0%) and PEI-based PROTACs (24.9%), which intuitively shows the advantages of Nano-PROTACs in inhibiting tumor growth. The growth rate of tumor volume in the Nano-PROTACs group was significantly slower than that in other groups, indicating its strong anti-tumor effect.

[0052] Mouse weight changes: There was no significant difference in body weight changes between the Nano-PROTACs group and other groups, indicating that Nano-PROTACs have lower systemic toxicity.

[0053] In vivo tumor photos: The photos show that the Nano-PROTACs group had significantly reduced tumor size, further confirming its anti-tumor effect.

[0054] H&E staining and immunofluorescence staining: H&E staining showed a large number of cell apoptosis and necrosis after Nano-PROTACs treatment. PCNA immunofluorescence staining was used to evaluate cell proliferation, while TUNEL assay was used to detect cell apoptosis. These results confirmed the effect of Nano-PROTACs in inducing tumor cell pyroptosis.

[0055] Quantitative analysis of PCNA and TUNEL staining: Quantitative analysis can further confirm the inhibitory effect of Nano-PROTACs on tumor cell proliferation and induction of cell apoptosis.

[0056] HK-2 expression: Nano-PROTACs caused a stronger downregulation of HK-2 expression by 77.9%, while sNano-PROTACs did not affect HK-2 expression in CT26 tumors because PEI-based PROTACs did not separate from sNano-PROTACs and could not perform protein degradation in vivo, indicating that Nano-PROTACs can effectively degrade HK-2 protein in vivo.

[0057] Caspase 3 expression and gasdermin cleavage: Compared with the saline group, Nano-PROTACs upregulated the expression of activated caspase 3 by 3.0 times and GSDME-N fragment by 1.4 times, indicating that Nano-PROTACs achieve anti-tumor effects by inducing cell pyroptosis.

[0058] In summary, Nano-PROTACs showed a strong anti-tumor effect in the CT26 tumor model and significantly inhibited tumor growth; Nano-PROTACs induced tumor cell pyroptosis, leading to a large number of cell apoptosis and necrosis; Nano-PROTACs could effectively degrade HK-2 protein in vivo; Nano-PROTACs upregulated the expression of activated caspase 3 and GSDME-N fragments, further confirming its mechanism of achieving anti-tumor effect by inducing cell pyroptosis; Nano-PROTACs had low systemic toxicity and had no significant effect on mouse body weight.

[0059] Example 5 In this example, based on Example 1-2, in order to further verify the selectivity of pH-activatable engineered nanoparticles and investigate the in vivo anti-tumor efficacy of HK-2 degraders, a 4T1 orthotopic tumor-bearing mouse model was constructed by injecting luciferase-labeled 4T1 cells into the chest pads of BALB / c mice. 3 The mice were divided into four groups (PBS, PEI-based PROTACs, sNano-PROTACs, and Nano-PROTACs). The treatment plan and related test results are shown in the attached Fig. 9 shown.

[0060] By the attached Fig. 9 The data shows that Fig. 9 In the figure, A is a schematic diagram of the treatment process; B is the change of tumor volume of mice receiving each group of treatment; C is the weight growth curve of mice receiving each group of treatment; D is the in situ tumor image of mice receiving each group of treatment; E is H&E staining (scale bar: 50 μm) and immunofluorescence staining (green: TUNEL, caspase 3 and PCNA, blue: cell nucleus, scale bar: 50 μm) of 4T1 tumor; F is the quantification of immunofluorescence staining of PCNA; G is the quantification of immunofluorescence staining of active caspase 3; H is the immunofluorescence staining of 4T1 tumor (green: HK-2, blue: cell nucleus, scale bar: 50 μm); I is the quantification of immunofluorescence staining of HK-2; J is Western blot detection; K is the quantification of HK-2 degradation effect in 4T1 tumor lysate; L is Western blot detection; M is the quantification of active caspase in 4T1 tumor lysate 3; N is Western blot detection; O is the quantification of GSDME-N in 4T1 tumor lysate.

[0061] Tumor inhibitory effect: The tumor inhibitory effect of Nano-PROTACs was significantly better than that of other groups, achieving a tumor elimination rate of 61.2%.

[0062] Changes in mouse weight: There was no significant difference in mouse weight between the Nano-PROTACs group and other groups, indicating that Nano-PROTACs has excellent biosafety.

[0063] Histopathological examination: H&E staining showed a large amount of cell apoptosis and necrosis after Nano-PROTACs treatment.

[0064] PCNA immunofluorescence staining and TUNEL assay further confirmed Nano-PROTACs-induced cell apoptosis.

[0065] HK-2 expression: Nano-PROTACs caused a greater decrease in HK-2 expression, reaching 84.8%.

[0066] Caspase 3 expression and gasdermin cleavage: Nano-PROTACs upregulated the expression of activated caspase 3 by 2.8-fold and GSDME-N fragment by 1.7-fold, indicating that Nano-PROTACs achieved anti-tumor effects by inducing tumor cell pyroptosis.

[0067] In summary, Nano-PROTACs achieved a broad spectrum of anti-tumor effects by inducing pyroptosis of tumor cells and exhibited excellent biosafety. These results provide a strong experimental basis for Nano-PROTACs as potential anti-tumor drugs.

[0068] Example 6 Based on Example 1-2, this example further investigated the inhibition of tumor lung metastasis by Nano-PROTACs. Inhibition of glycolysis is essential for reprogramming the tumor microenvironment, which is closely related to tumor metastasis. Therefore, we evaluated the effect of Nano-PROTACs on the anti-tumor metastasis ability in vivo by monitoring lung metastasis using a 4T1 orthotopic tumor mouse model. When the tumor volume reached 50 mm 3 For follow-up treatment, the mice were divided into three groups: PEI-based PROTACs group, sNano-PROTACs group and Nano-PROTACs group. The mice were injected intravenously once every two days for a total of five times. Lung metastasis: observed in vivo and in vitro by bioluminescence imaging. The number of lung tumor nodules: the degree of lung metastasis was visually evaluated by dissecting the mice and observing the number of tumor nodules on the surface of their lungs. For specific treatment plans and results, please refer to the attached Fig.10 shown.

[0069] By the attached Fig.10 The data shows that Fig.10 In the figure, A is a schematic diagram of the treatment process, and 4T1 orthotopic tumor mice were treated with PBS, PEI-based PROTACs, sNano-PROTACs and Nano-PROTACs at the indicated time points. B is an in vivo bioluminescent image of metastatic nodules in the lungs of 4T1 orthotopic tumor mice treated with the indicated treatment methods; C is a quantitative analysis; D is an in vitro bioluminescent image of metastatic nodules in the lungs of 4T1 orthotopic tumor mice treated with the indicated treatment methods; E is a quantitative analysis; F is a representative image of lungs isolated from 4T1 orthotopic tumor mice treated with the indicated treatment methods; G is H&E staining of lung tissues of 4T1 orthotopic tumor mice treated with the indicated treatment methods, scale bar: 200 μm.

[0070] Bioluminescence imaging results: The bioluminescence intensity of the lungs of mice treated with Nano-PROTACs was lower than that of mice treated with PBS and PEI-based PROTACs, indicating that Nano-PROTACs exhibited an effective inhibitory effect on lung metastasis.

[0071] Number of lung tumor nodules: The number of tumor nodules on the lung surface of mice treated with Nano-PROTACs was less than that of mice treated with PBS or PEI-based PROTACs, further confirming the inhibitory effect of Nano-PROTACs on tumor lung metastasis.

[0072] In summary, Nano-PROTACs have a significant inhibitory effect on tumor lung metastasis. By inhibiting glycolysis, Nano-PROTACs can reprogram the tumor microenvironment, thereby reducing the risk of tumor metastasis. This provides a strong experimental basis for Nano-PROTACs as a potential anti-tumor metastasis drug. In the future, the mechanism of action of Nano-PROTACs will be further studied, and its application prospects in clinical practice will be explored. Example 7 This embodiment provides a cationic polymer-based proteolysis targeting chimera, and the specific preparation process is as follows: polyethyleneimine with a molecular weight of 1800 Da is selected as the middle connecting arm, thalidomide is selected as the ligand of E3 ubiquitin ligase, and 3-bromopyruvate is selected as the ligand of HK-2.

[0073] (1) Dissolve polyethyleneimine (1 mmol) and 4-fluoro-thalidomide (1 mmol) in DMF (2 mL), mix, evacuate, and use N 2 Balloon protection; (2) DIPEA (3 mmol) diluted with DMF was added dropwise and reacted at 90 °C for 12 h; (3) After the reaction mixture was cooled, 3-bromopyruvic acid (1.2 mmol) and HATU (1.2 mmol) diluted with DMF were added dropwise and reacted at 37 °C for 6 h. (4) The synthesis results were verified by thin layer chromatography. The reaction product was then concentrated and dropped into anhydrous ether precooled at 4 °C for precipitation for 10 min. The product was centrifuged at 4 °C and 10,000 rpm for 10 min to obtain a pure cationic polymer-based protein hydrolysis targeting chimera (3-bromopyruvate).

[0074] CT26 cells were seeded in a 6-well plate and cultured in a cell culture incubator until the cell confluence reached 30%. The cationic polymer-based proteolysis targeting chimera (3-bromopyruvate) was prepared at different concentrations and incubated with the CT26 tumor cells for 9 h. The degradation of HK-2 in CT26 tumor cells by the cationic polymer-based proteolysis targeting chimera (3-bromopyruvate) was detected by Western blot protein content detection. The experimental results are shown in the attached Fig.11 shown.

[0075] By the attached Fig.11 The data show that the degradation of HK-2 by the cationic polymer-based proteolysis targeting chimera (3-bromopyruvate) is concentration-dependent. As the concentration of the degrading agent increases, the degree of HK-2 protein degradation also gradually increases, indicating that the degrading agent has a good HK-2 degradation effect. This result verifies the effectiveness of the cationic polymer-based proteolysis targeting chimera combinatorial library constructed in this example, as well as the application potential of the degrading agent in the degradation of specific proteins.

[0076] This example successfully constructed a proteolysis-targeted chimera combinatorial library based on a cationic polymer (polyethyleneimine), and synthesized a degrader for HK-2 protein. Western blot detection verified that the degradation of HK-2 protein by the degrader was concentration-dependent, indicating that it had a good degradation effect. This study provides new ideas and methods for the development of new proteolysis-targeted chimeras, and has potential application value in fields such as tumor treatment.

[0077] Example 8 This example aims to detect the killing effect of a cationic polymer-based proteolysis targeting chimera (with 3-bromopyruvate as ligand) on CT26 tumor cells and compare it with free 3-bromopyruvate (3-BP).

[0078] CT26 tumor cells were seeded in 96-well plates and cultured until the cell confluence reached 30%. The cationic polymer-based proteolysis targeting chimera (3-bromopyruvate) and free 3-BP were prepared into a series of concentration gradients (0.0001 μg / mL, 0.001 μg / mL, 0.005 μg / mL, 0.1 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL) and incubated with CT26 tumor cells for 24 h. After the incubation, the medium containing the drug was discarded, and the prepared 500 μg / mL MTT solution was added to the cells. After culturing in a cell culture incubator for 3 h, the MTT solution was discarded, and 150 μL DMSO solution was added to each well. After being placed on a shaker for 10 min, the ultraviolet absorption wavelength was detected at 490 nm using an enzyme reader. The test results are shown in the attached Fig.12 shown.

[0079] By the attached Fig.12 The data show that for both drugs, with the increase in concentration, the cell viability showed a trend of first increasing and then decreasing; at lower concentrations (0.0001 μg / mL to 0.005 μg / mL), the cell viability of PEI-Tha-3BP was higher than that of 3-BP, which was because the cationic polymer carrier promoted the cellular uptake of the drug or enhanced its biological activity; at higher concentrations (such as 1 μg / mL to 10 μg / mL), the cell viability of PEI-Tha-3BP was significantly lower than that of 3-BP, indicating that its cell killing effect was stronger; compared with free 3-BP, the cationic polymer-based proteolysis targeting chimera (PEI-Tha-3BP) had a stronger cell killing effect at higher concentrations, which was because the cationic polymer carrier not only increased the intracellular concentration of the drug, but also enhanced the drug's toxicity to tumor cells.

[0080] The experimental results of this example show that the cationic polymer-based proteolysis targeting chimera (PEI-Tha-3BP) has a strong killing effect on CT26 tumor cells at a higher concentration, and it is concentration-dependent. Compared with free 3-BP, PEI-Tha-3BP shows superior performance in anti-tumor, providing a strong experimental basis for the development of new anti-tumor drugs.

[0081] Example 9 In this example, polyethyleneimine with a molecular weight of 1800 Da was selected as the intermediate linker, thalidomide was selected as the ligand of E3 ubiquitin ligase, and dichloroacetic acid was selected as the ligand of pyruvate dehydrogenase kinase to construct a cationic polymer-based protein hydrolysis targeting chimera, and its degradation effect on PDK1 was detected at the same time.

[0082] The preparation method is as follows: (1) Dissolve polyethyleneimine (1 mmol) and 4-fluoro-thalidomide (1 mmol) in DMF (2 mL), mix, evacuate, and use N 2 Balloon protection; (2) Add N,N-diisopropylethylamine (DIPEA) (3 mmol) diluted with DMF dropwise and react at 90 °C for 12 h; (3) After the reaction mixture was cooled, dichloroacetic acid (1.2 mmol) and HATU (1.2 mmol) diluted with DMF were added dropwise and reacted at 37 °C for 6 h. (4) The synthesis results were verified by thin layer chromatography. The reaction product was then concentrated and dropped into anhydrous ether precooled at 4 °C for precipitation for 10 min. The product was centrifuged at 4 °C and 10,000 rpm for 10 min to obtain a pure cationic polymer-based protein hydrolysis targeting chimera (dichloroacetic acid).

[0083] 4T1 tumor cells were seeded in 6-well plates and cultured in a cell culture incubator until the cell confluence reached 30%. The cationic polymer-based proteolysis targeting chimera (dichloroacetic acid) was prepared into different concentrations (such as 0, 0.01, 0.1, 10, 20 μM) and incubated with CT26 tumor cells for 9 h. The degradation of PDK1 in CT26 tumor cells by the cationic polymer-based proteolysis targeting chimera (dichloroacetic acid) was detected by Western blot protein content detection. The experimental results are shown in the attached Fig.13 shown.

[0084] By the attached Fig.13 The data show that as the drug concentration increases, the protein level of PDK1 gradually decreases, while the protein level of β-actin remains stable. At different concentrations (0.01, 0.1, 1, 10, 20 μM), the degree of PDK1 degradation is concentration-dependent. In particular, at 20 μM, 58.8% of PDK1 can be degraded. This indicates that the cationic polymer-based proteolysis targeting chimera (dichloroacetic acid) has a good degradation effect on PDK1 in 4T1 tumor cells.

[0085] This example successfully constructed a cationic polymer-based proteolysis-targeted chimera and tested its degradation effect on PDK1 in 4T1 tumor cells. The experimental results showed that the drug can specifically degrade PDK1 and exhibit concentration dependence, providing a strong experimental basis and potential clinical application prospects for the further development of proteolysis-targeted chimeras for tumor cells.

[0086] The above embodiments are only preferred implementations of the present invention, and their descriptions are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that for those skilled in the art, local modifications and improvements can be made to the invention in different ways without departing from the principles and concepts of the invention. The protection scope of the present invention shall be based on the claims and shall not be limited by the above specific implementations. All implementations within its scope are subject to the constraints of the present invention and belong to the protection scope of the present invention.

Claims

1. A cationic polymer-based proteolysis targeting chimera, characterized in that The targeted proteolysis targeting chimera comprises a cationic polymer, a target protein ligand and an E3 ubiquitin ligase ligand; The target protein ligand is used to target the target protein; The E3 ubiquitin ligase ligand is used to target the E3 ubiquitin ligase; The cationic polymer serves as a connecting arm between the target protein ligand and the E3 ubiquitin ligase ligand, coupling the target protein ligand and the E3 ubiquitin ligase ligand.

2. The cationic polymer-based proteolysis targeting chimera according to claim 1, characterized in that: The cationic polymer is any one of linear or branched polyethyleneimine, dendritic polyamidoamine and functionalized multi-arm PEG; The targeting protein ligand is a ligand targeting hexokinase-2, pyruvate dehydrogenase kinase, bromoprotein domain 4 or indoleamine-2,3-dioxygenase; The E3 ubiquitin ligase ligand is any one of the ligands of cereblon protein, angiostatin tumor suppressor protein, mouse double minute gene 2 protein and cell apoptosis inhibitor protein.

3. The cationic polymer-based proteolysis targeting chimera according to claim 2, characterized in that: The linear or branched polyethyleneimine is a linear or branched polyethyleneimine with a molecular weight of 1000-40000 Da; The ligand targeting hexokinase-2 is any one of lonidamine and 3-bromopyruvate, the ligand targeting pyruvate dehydrogenase kinase is dichloroacetic acid, the ligand targeting BRD4 is JQ1, and the ligand targeting IDO is NLG919; The E3 ubiquitin ligase ligand is any one of thalidomide, lenalidomide, pomalidomide and VHL-032.

4. The method for preparing a cationic polymer-based proteolysis targeting chimera according to any one of claims 1 to 3, characterized in that: include: S1, after the cationic polymer and the E3 ubiquitin ligase ligand are dissolved and mixed, the mixture is evacuated and filled with nitrogen for protection, and a catalyst dissolved in the same solvent is added dropwise for reaction to obtain a reaction solution 1; S2, cooling the reaction solution 1 obtained in S1, and adding the targeted protein ligand and the catalyst dissolved in the same solvent respectively to react, so as to obtain the reaction solution 2; S3, the reaction solution 2 obtained in S2 is concentrated, precipitated, centrifuged, and dialyzed for purification to obtain a cationic polymer-based protein hydrolysis targeting chimera.

5. The method for preparing a cationic polymer-based proteolysis targeting chimera according to claim 4, characterized in that: In S1, the molar ratio of the cationic polymer to the E3 ubiquitin ligase ligand is 1:1~3, the catalyst used is O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate or N,N-diisopropylethylamine, the amount of the catalyst added is 1~3 times the molar amount of the E3 ubiquitin ligase ligand, the reaction temperature is 25°C~100°C, and the reaction time is 10h~24h.

6. The method for preparing a cationic polymer-based proteolysis targeting chimera according to claim 4, characterized in that: In S2, the molar ratio of the targeting protein ligand to the cationic polymer is 1-3:1, the catalyst used is O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, the amount of the catalyst added is 1-3 times the molar amount of the targeting protein ligand, the reaction temperature is 25°C-38°C, and the reaction time is 6 h-24 h.

7. The method for preparing a cationic polymer-based proteolysis targeting chimera according to claim 4, characterized in that: In S3, the precipitate is precipitated with anhydrous ether precooled at 4°C for 8-12 minutes.

8. A pH-responsive targeted protein degradation nanodrug, characterized in that: The pH-responsive targeted protein degradation nanomedicine is obtained by connecting the cationic polymer-based proteolysis targeting chimera described in any one of claims 1 to 3 to an amphiphilic polymer through a pH-responsive arm; The pH-responsive linker is any one of cis-aconitic anhydride and 2-propionic acid-3-methylmaleic anhydride; The amphiphilic polymer is any one of polyethylene glycol-polycaprolactone, polyethylene glycol-polylactic acid and polyethylene glycol-polylactic acid-glycolic acid copolymer.

9. The method for preparing the pH-responsive targeted protein degradation nanomedicine according to claim 8, characterized in that: The cationic polymer-based protein hydrolysis targeting chimera, the amphiphilic polymer and the pH-responsive arm according to any one of claims 1 to 3 are dissolved and mixed uniformly to obtain an engineered pH-activatable nano protein hydrolysis targeting chimera; the engineered pH-activatable nano protein hydrolysis targeting chimera and the amphiphilic polymer are dissolved and mixed uniformly, dripped into ultrapure water, stirred, and dialyzed to obtain a pH-responsive targeted protein degradation nano drug; The molar ratio of the cationic polymer-based protein hydrolysis targeting chimera, the amphiphilic polymer and the pH response arm is 1:1.2-2, and the molar ratio of the engineered pH-activatable nano protein hydrolysis targeting chimera to the amphiphilic polymer is 1:1-2.

10. Use of the cationic polymer-based protein hydrolysis targeting chimera according to any one of claims 1 to 3, or the pH-responsive targeted protein degradation nanomedicine according to claim 8 in the preparation of drugs against tumor cells or metabolism-related diseases.

Images