Polymer for hydrolysis of target protein and preparation method and application thereof

Abstract

The invention discloses a polymer for target protein hydrolysis and a preparation method and application thereof. The polymer is combined with a target protein ligand and an E3 ligase ligand, and the polymer can solve the delivery problems of an existing delivery system in the aspects of water solubility, pharmacokinetic property, cell membrane permeability and the like. Meanwhile, the polymer prepared by the invention can be assembled to form nanoparticles, and has natural targeting in vivo. Besides, the polymer is a multi-linker type molecule, the number and the proportion of side chain ligand molecules combined with target protein and ligand molecules recruiting E3 ligase can be flexibly adjusted, and construction of a combinatorial library for optimization is facilitated, so that the optimal protein degradation efficiency is obtained.

Description

[0001] This application is a divisional application with an application date of November 20, 2023, an application number of CN202311547234.3, and an invention name of “Polymers for target protein hydrolysis, preparation methods and uses thereof”, which is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention relates to the technical field of protein targeted degradation, and in particular to a polymer used for target protein hydrolysis and a preparation method and application thereof. Background Art

[0003] Targeted protein degradation chimeras (PROTACs) based on the ubiquitin-proteasome system are an emerging direction in the field of drug research in recent years and have received widespread attention. The intracellular ubiquitin-proteasome system is the main pathway for intracellular protein degradation (awarded the 2004 Nobel Prize in Chemistry). It is composed of ubiquitin, ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2, ubiquitin ligase E3, deubiquitinating enzymes and proteasomes. It can ubiquitinate harmful proteins ("junk proteins") in the cell and guide the protein into the proteasome ("garbage cleaning system") to be degraded into peptides or amino acids. Among them, E3 ubiquitin ligase is responsible for selectively ubiquitinating the target protein, thereby enabling it to be recognized and degraded by the proteasome, and is the key enzyme of the system.

[0004] On this basis, Professor Craig Crews of Yale University pioneered the design strategy of "double linker" molecules for targeted protein degradation chimeras (PROTAC for short), which uses the cell's own protein degradation mechanism (ubiquitin-proteasome system) to target and degrade specific intracellular proteins. Specifically, PROTAC is a "double linker" molecule consisting of a linker and two ligands (generally small molecules), one of which can bind to the target protein; and the other can bind to the E3 ubiquitin ligase. After entering the cell, PROTAC can bind to the target protein and recruit E3 ubiquitin ligase to ubiquitinate the target protein and guide it into the proteasome system for degradation. In recent years, PROTAC has become a new drug design strategy, and many PROTAC molecules have entered the clinical research stage and are progressing smoothly. It is precisely because of its good prospects that PROTAC has been reported in the form of special articles by top journals such as Nature and Science, and is called the "next blockbuster therapy" for drug treatment.

[0005] PROTAC has great potential in drug design and development due to its ultra-high activity. The advantages of PROTAC molecules are mainly manifested in: ① It has ultra-high activity. PROTAC molecules can completely degrade target proteins (rather than traditional protein inhibition mechanisms), and can cyclically degrade multiple target protein molecules, with catalytic activity. Therefore, in theory, only extremely low doses are needed to achieve extremely strong effects. ② The PROTAC strategy is particularly suitable for drug design for proteins with multiple mutants. The process of protein degradation by PROTAC molecules is an "Event-driven" behavior, that is, as long as it binds to the target protein, it can be effectively degraded, and the binding force between PROTAC molecules and target proteins is relatively low. Therefore, PROTAC is often effective for multiple mutants of its target protein. ③ The PROTAC strategy is more rational than the traditional large-scale screening of small molecule drug research and development models. It can be designed on the basis of existing small molecule inhibitors or agonists with insufficient activity, and transformed into protein degraders to exert corresponding biological activities. ④ Once PROTAC binds to the target protein in the cell, it can catalyze degradation, and the onset of action is relatively fast. Usually, the protein degradation effect occurs 15 minutes to 1 hour after administration.

[0006] However, due to its own molecular structure, PROTAC still has problems related to drug delivery and screening optimization. These problems are specifically reflected in: ① In terms of cell transport, there is a problem of difficulty in cell entry. PROTAC molecules contain two ligand molecules and a linker. Compared with traditional small molecule drugs, they are often more difficult to transport across the membrane into the cell, which restricts the full play of their pharmacological effects; ② In terms of in vivo transport, there are also some common problems with drug administration. PROTAC molecules do not conform to the drugability rules of traditional medicinal chemistry (i.e., the five rules of drug-like properties), and their solubility is generally poor, resulting in difficulties in in vivo administration and poor pharmacokinetic properties. ③ In terms of optimization and screening, there are problems with a single structural ratio and a limited ratio of target protein to E3 ligase ligand. A single fixed ratio can only be screened by adjusting the linker, which has a single means and strong restrictions, and is not conducive to the establishment of a combinatorial library for high-throughput screening and optimization.

[0007] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0008] To solve the problems of drug delivery and optimization screening of PROTAC. The present invention synthesizes a polymer that can bind and degrade target proteins in a multivalent manner, which can be delivered into cells in the form of nanoparticles to solve the delivery problem of PROTAC. The design of multiple linkers facilitates high-throughput screening and optimization. Specifically, the present invention includes the following contents.

[0009] In a first aspect, the present invention provides a polymer for hydrolyzing a target protein, wherein the polymer is combined with a target protein ligand and an E3 ligase ligand.

[0010] In certain embodiments, according to the polymer for target protein hydrolysis of the present invention, the polymer comprises a first structural unit containing a target protein ligand and a second structural unit containing an E3 ligase ligand.

[0011] In certain embodiments, according to the polymer for target protein hydrolysis of the present invention, there are 1-200 structural units between the first structural unit and the second structural unit.

[0012] In certain embodiments, according to the polymer for target protein hydrolysis of the present invention, the first structural unit and the second structural unit of the polymer are each selected from 2-(azepan-1-yl)ethyl methacrylate monomer, a small molecule compound, an amino acid, a nucleic acid molecule, a cIAP ligand, an MDM2 ligand, a Von Hippel-Lindau ligand or a cereblon ligand.

[0013] In certain embodiments, according to the polymer for target protein hydrolysis of the present invention, the target protein ligand is a group of the first structural unit, or a ligand connected through an active group; and the E3 ligase ligand is a group of the second structural unit, or a ligand connected through an active group, preferably, the active group includes but is not limited to at least one of amino, carboxyl, thiol, N-hydroxysuccinimide, maleimide, double bond, triple bond and azide.

[0014] In certain embodiments, according to the polymer for target protein hydrolysis of the present invention, the ligand contained in the first structural unit includes but is not limited to a 7-membered ring, a small molecule compound, an aptamer, a protein, a nucleic acid molecule, a cellular metabolite, a drug, a hormone, a metal ion, a sugar, a peptide, an antibody or a fragment thereof.

[0015] In certain embodiments, according to the polymer for target protein hydrolysis described in the present invention, the polymer includes but is not limited to 2-(azepan-1-yl)ethyl methacrylate-based polymers, chitosan, lactic acid and / or glycolic acid-based polymers, amino acid-based polymers, polyamidoamine-type dendrimer polymers, polypeptides or combinations thereof.

[0016] In certain embodiments, according to the polymer for target protein hydrolysis of the present invention, the polymer is selected from a random copolymer, a block polymer or a homopolymer.

[0017] In certain embodiments, according to the polymer for target protein hydrolysis of the present invention, the molecular weight of the polymer is 2000-20000. In certain embodiments, the molecular weight refers to the number average molecular weight. In certain embodiments, the molecular weight refers to the weight average molecular weight.

[0018] The second aspect of the present invention provides a method for preparing a polymer for hydrolysis of a target protein, comprising:

[0019] (1) preparing a first structural unit containing a target protein ligand;

[0020] (2) preparing structural building blocks;

[0021] (3) synthesizing a random copolymer from the first structural unit, the structural building unit and the compound containing an active group;

[0022] (4) connecting the random copolymer to a second structural unit containing an E3 ligase ligand.

[0023] The third aspect of the present invention provides use of the polymer of the present invention in degrading a target protein.

[0024] In a fourth aspect, the present invention provides a method for degrading a target protein in a cell, comprising the steps of allowing the polymer of the present invention to contact a cell and allow the polymer to enter the cell.

[0025] The fifth aspect of the present invention provides a pharmaceutical composition comprising the polymer of the present invention and a pharmaceutically acceptable carrier.

[0026] The present invention solves some basic problems of conventional PROTAC in terms of delivery. The polymer of the present invention is administered in the form of a delivery system, which is beneficial to solve the delivery problems of PROTAC molecules in terms of water solubility, pharmacokinetic properties and cell membrane permeability.

[0027] The administration of protein-degradable polymers in the form of a delivery system is conducive to solving the delivery problems of PROTAC molecules in terms of water solubility, pharmacokinetic properties, and cell membrane permeability. Protein-degradable polymers are mostly amphiphilic polymers (or with the first and second structural units as the hydrophobic part), which can be made into nanoparticles (small microparticle suspensions for intravenous injection) for administration. They have long circulation functions in the body and cell membrane permeability, which can effectively circumvent the delivery problems of traditional PROTACs. In addition, protein-degradable polymers also have passive targeting. After the protein-degradable polymer nanocarrier system is administered in vivo, it is very easy to distribute to high-metabolic disease sites such as tumors and inflammation. Protein-degradable polymers can directly target target organs, so they have unique advantages in treating diseases.

[0028] The polymer of the present invention has obvious advantages in molecular structure design and enhancing protein degradation efficiency. From the perspective of molecular structure design, conventional PROTAC is a "double linker" molecule, and the molar ratio between the two ligand molecules can usually only be 1:1, which is difficult to optimize; while the polymer of the present invention is a "multi-linker" molecule, and the number and ratio of the side chain ligand molecules that bind to the target protein and the ligand molecules that recruit the E3 ligase can be flexibly adjusted, which is conducive to the construction of a combinatorial library for optimization and obtaining the best protein degradation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A schematic diagram of the targeted protein degradation polymer designed in the present invention is shown.

[0030] Figure 2 It shows that STING signaling is activated in the synovium of RA patients. a. Representative images of mIF staining of STING (red), FAPα (green) and DAPI (blue) (n=3). Scale bar: 20μm (top), 5μm (bottom). Co-immunostaining of STING and FAPα shows that STING is highly expressed in FAPα+FLS of RA. b. Representative images of mIHC of STING and P-IRF3 (n=3). Scale bar: 100μm (top), 20μm (bottom). c. Number of FAPα+ positive cells in RA and OA synovium (%, n=3) ***P<0.001. d. Number of STING positive cells in RA and OA synovium (%, n=3) **P<0.01. e. Co-localization of STING and FAPα+ cells in RA and OA synovium (%, n=3) ***P<0.001. Histological scores showed that STING (f) and P-IRF3 (g) were highly expressed in RA synovium (n=3) *P<0.05, **P<0.01. h. Isolation, protein extraction and western blot analysis of primary synovial fibroblasts from OA and RA patients (n=3). i. Relative expression levels of STING, P-STING, P-TBK1 and P-IRF3 in RA and OA synovium (n=3) *P<0.05, **P<0.01, ***P<0.01. j. Isolation, protein extraction and western blot analysis of primary synovial fibroblasts from normal (Nor) and AIA (model) rats (n=3). k. Relative expression levels of STING, P-STING, P-TBK1 and P-IRF3 in synovium of AIA model and normal rats (n=3) *P<0.05, **P<0.01, ***P<0.001.

[0031] Figure 3Synthesis, characterization, and preliminary protein degradation of PolyTACs. a. Schematic diagram of the chemical structures of PolyTACs polymer nanoparticles and PEG-b-P(C7A70-r-E3Ln-AMA4-n) copolymer. In the nanoparticle state (pH > pHt), the relevant PolyTACs nanoprobes remain in an "inactive" state due to fluorescence quenching. In the polymer state (pH < pHt), polymer dissociation leads to fluorescence activation to an "active" state. b. PC7A and PolyTACs 1-3. c. TEM images of PolyTACs-1 / 2 are presented at higher pH 7.8 and lower pH 6.3, respectively, with a scale bar of 100 nm. d. Line graph showing the normalized fluorescence intensity of PolyTACs nanoprobes as a function of pH. The relative fluorescence intensity is normalized as (F - Fmin) / (Fmax - Fmin), where F is the fluorescence intensity of the dye-conjugated polymer solution under different pH conditions, and Fmax (Fmin) is the maximum (minimum) fluorescence intensity. e. THP1 cells treated with PolyTAC-1 (9.6 μM) and PolyTAC-2 (9.6 μM) showed a decrease in TBK1 / IRF3 phosphorylation, followed by STING degradation, while THP1 cells treated with PolyTAC-3 (9.6 μM) did not show degradation. f. Jurkat cells treated with PolyTAC-1 (9.6 μM) and PolyTAC-2 (9.6 μM) showed a decrease in TBK1 / IRF3 phosphorylation, followed by STING degradation, while Jurkat cells treated with PolyTAC-3 (9.6 μM) did not show degradation. g. Relative expression level of STING in THP-1 cells (n = 3) * compared with the NC (negative control) group, ***P < 0.001. h. Relative expression level of STING in Jurkat cells (n = 3) * P < 0.05, ***P < 0.0001 compared with the NC group. i. As the concentration of PolyTAC-1 increased within a certain concentration range (0, 2.4 μM, 4.8 μM, 7.2 μM, 9.6 μM, 12 μM, 14.4 μM), the degradation effect of PolyTAC-1 gradually enhanced with the increase in the concentration of PolyTAC1. j. Relative expression level of STING in Jurkat cells (n = 3) ****P < 0.0001.

[0032] Figure 4The effectiveness of PolyTACs in degrading STING and inhibiting the STING / IRF3 pathway in primary FLS is shown. a. In primary FLS of AIA rats treated with PolyTAC-1 (12 μM), the degradation effect of PolyTAC-1 was observed from the 12th hour. b. The relative expression level of STING in primary FLS of AIA rats (n=3) ****P<0.0001 compared with the 0h group. c. Primary FLS of AIA rats treated with PolyTAC-1 (12 μM), PolyTAC-2 (12 μM) and H-151 (2 μM) showed reduced TBK1 / IRF3 phosphorylation, followed by STING degradation. d. The relative expression levels of STING, P-STING, P-TBK1, and P-IRF3 in primary FLS of AIA rats (n=3) ***P<0.001, ***P<0.0001. e. With the increase of PolyTAC-1 concentration, within a certain concentration range (0, 2.4μM, 4.8μM, 7.2μM, 9.6μM, 12μM, 14.4μM), the degradation effect of PolyTAC-2 gradually increased with the increase of concentration. f. Relative expression levels of STING, P-STING, P-TBK1, and P-IRF3 in primary FLS of RA patients (n=3) ***P<0.001, ***P<0.0001. g. Intracellular abundance and distribution of STING (red) and P-IRF3 (green) in primary FLS of RA patients. h. Average fluorescence intensity of STING and P-IRF3 (n=4), ***P<0.0001, **P<0.01, ns (not significant) compared with the NC group. i. Primary FLS from RA patients treated with TNF-α (4 ng / ml) showed stronger activation of the STING pathway, and further treatment with PolyTAC-1 (12 μM), PolyTAC-2 (12 μM), and H-151 (2 μM) showed reduced TBK1 / IRF3 phosphorylation followed by STING degradation** compared to TNF-α treated primary FLS. In addition, when TNF-α induced cells were treated with PolyTAC-1 (12 μM), PolyTAC-2 (12 μM), and H-151 (2 μM) for 24 h, it was observed that PolyTAC-1 and PolyTAC-2 effectively degraded STING and inhibited the downstream STING / P-IRF3 signaling pathway (j).

[0033] Figure 5Exploration of the endocytosis and degradation pathways of PolyTACs is shown. ab. Cellular uptake of nanoparticles. HeLa cells were cultured until 80% confluence was reached, and then Cy3-conjugated nanoparticles were introduced at different time points. After nanoparticle treatment, the cell culture medium was replaced and lysosomes were labeled using Lyso Tracker. Subsequently, confocal microscopy was used to observe the colocalization of Cy3-labeled nanoparticles and Lyso-Tracker-labeled lysosomes. Confocal microscopy analysis of HeLa cell uptake of PolyTACs. a. PolyTACs nanoparticles were incubated with HeLa cells for different times. Lysosomes are indicated by Lyso Tracker (blue). Lysosomal localization of PolyTACs was determined by the correlation between Lyso and NP using ImageJ software, scale bar: 10 μm. b. Statistical graph. c. Intracellular abundance, distribution and colocalization of STING (red) and CRBN (blue) in primary FLS of RA patients. As indicated by the white arrows, the co-localization of STING and CRBN is shown as pink spots. Scale bar: 20 μm. d. STING (n=4) and CRBN (n=4). e. Primary FLS of RA patients treated with PolyTAC-1 (12 μM), PolyTAC-2 (12 μM) and H-151 (2 μM) showed that TBK1 / IRF3 phosphorylation was reduced with the degradation of STING, and this effect could be reversed by PS-341 (proteasome inhibitor, 7.5 nmol / l). f. Relative expression levels of STING, P-STING, P-TBK1, and P-IRF3 (n=3) ****P<0.0001 compared with the NC group #++++P<0.0001 compared with FLS treated with PolyTAC-1

[0034] Figure 6The targeted distribution characteristics of PolyTAC-1 and PolyTAC-2 in vivo and the degradation of STING improved RA pathology are shown. a. Representative IVIS images of biodistribution in AIA model rats. PolyTAC-2 exhibited targeted aggregation in vivo, and targeted aggregation of PolyTAC-1 and PolyTAC-2 was observed in the affected limb joints of AIA model rats from the 8th hour. Representative images of ankle swelling (b), HE (c), and Safranin-O / Fast Green staining (d) of Nor, AIA model, PolyTAC-1 (12 μM), PolyTAC-2 (12 μM), and control group (12 μM). e. The thickness of joint swelling was assessed every 5 days (n=6 rats per group). f. The polyarthritis index (PI) was assessed every 5 days (n=6 rats per group). Histological scores of synovial hyperplasia, cartilage destruction and bone erosion in HE (gi), n=3#Compared with the normal group, P<0.05, P<0.01, P<0.001*Compared with the AIA model, P<0.05, **P<0.01, ***P<0.001, ns (not significant). j. Representative IHC staining of STING in ankle joint sections of Nor, AIA model, PolyTAC-1 (12μM), PolyTAC-2 (12μM), and control group (12μM). k. ELISA was used to detect IFNα, IFNβ, TNFα, IL-6 and IL-17 in plasma, and it was found that PolyTAC-1 and PolyTAC-2 significantly inhibited the secretion of IFNα, IFNβ, TNFα, IL-6 and IL-17 in the AIA model induced by STING. Compared with the AIA model, n=6, *P<0.05, **P<0.01, ***P<0.001.

[0035] Figure 7 The synthetic routes of PC7A and PolyTAC-1 / 2 / 3 (with or without dyes). a. The synthetic method of 2-(azepan-1-yl)ethyl methacrylate (iii). Reagents and conditions: (I) TEA, DCM, RT, overnight. b. The steps of synthesizing PEG-Br (vi). Reagents and conditions: (II) TEA, DCM, RT, overnight. c. The steps of synthesizing PEG-PC7A-r-AMA (ix). Reagents and conditions: (III) CuBr2, TPMA, AIBN, DMF, IPA, 70°C, 48h.d, PC7A, the synthetic method of PolyTACs 1-3 and dye-coupled PolyTACs 1-3 (xii). Reagents and conditions: (IV) TEA, DCM, RT, overnight. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] Polymers for targeted protein hydrolysis

[0040] In one aspect of the present invention, a polymer for hydrolysis of a target protein is provided. The "target protein" may be any polypeptide or protein that a person skilled in the art wishes to selectively degrade in a cell or mammal, such as a human subject, and may also be referred to herein as a "protein of interest, POI". In the present invention, the selective degradation of the target protein will reduce the protein level, thereby reducing the effect of the target protein in the cell, and based on the control of the protein level, treatment or improvement of a disease or condition may be provided. The target protein includes any protein and polypeptide having a biological function or activity, such as structural, regulatory, hormonal, enzymatic, genetic, immune, storage, transport and signal transduction functions and activities.

[0041] In certain embodiments, examples of target proteins include, but are not limited to, structural proteins, receptors, enzymes, cell surface proteins, proteins associated with cellular integrated functions (including involvement in catalytic activity, epigenetic regulation, aromatase activity, motor activity, helicase activity, metabolic processes (anabolism and catabolism), antioxidant activity, proteolysis, biosynthesis), proteins with kinase activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, ligase activity, enzyme regulatory activity, signal transduction activity, structural molecule activity, binding activity, receptor activity, cell viability, membrane fusion, cell communication, regulation of biological processes, development, cell differentiation, proteins associated with stimulus response, behavioral proteins, cell adhesion proteins, proteins involved in cell death, proteins involved in transport, etc.

[0042] In the present invention, the target protein may include proteins from eukaryotic and prokaryotic organisms, including mammals, such as humans, domesticated animals, microorganisms, viruses, fungi and parasites, as well as many other targets for drug treatment. Preferably, the target protein includes but is not limited to STING, ALK, FAK, PRMT5, PLK1, etc.

[0043] In the present invention, target protein ligand refers to a ligand or part that binds to the target protein. For example, the target protein binding ligand can be any part that selectively and / or specifically binds to the target protein. The polymer for target protein hydrolysis according to the present invention can include a protein binding ligand that binds to the target protein target with sufficient binding affinity, thereby making the target protein more easily degraded or proteolyzed than when it is not bound by the polymer.

[0044] In the present invention, the target protein binding ligand may comprise or be derived from any known small molecule (or its analog or fragment) used as a regulator, promoter and / or inhibitor of protein function (e.g., any small molecule known to bind to the target protein). In certain embodiments, the target protein ligand includes a STING, ALK, FAK, PRMT5 or PLK1 binder (such as an antibody or a fragment thereof) or a small molecule compound. In a preferred embodiment, the target protein ligand may also include an active group that binds to STING, ALK, FAK, PRMT5 or PLK1.

[0045] In an exemplary embodiment, the target protein binding ligand may include a 7-membered ring, particularly a 7-membered ring containing a heteroatom, such as a nitrogen atom.

[0046] In an exemplary embodiment, the E3 ligase ligand comprises a Von Hippel-Lindau ligand or a cereblon ligand.

[0047] In certain embodiments, the polymer of the present invention includes, but is not limited to, PC7A (polymer based on 2-(azepan-1-yl)ethyl methacrylate), chitosan, random copolymers based on lactic acid and / or glycolic acid (e.g., PLGA), amino acid-based polymers (e.g., PLL), polyamidoamine dendrimers or polypeptides. In a preferred embodiment, the polymer of the present invention is PC7A, which has a structure shown in the following formula:

[0048]

[0049] In certain embodiments, the polymers of the invention have the general formula PEG-bP(C7A 70 -r-E3L n -AMA 4-n ). In a preferred embodiment, it has the structure shown below:

[0050]

[0051] In the present invention, degradation can be determined by measuring the amount of the target protein in the presence of a polymer as described herein and / or comparing it to the amount of the target protein observed in the absence of the polymer. For example, the amount of the target protein in cells that have been contacted and / or treated with a polymer as described herein can be determined. The amount can be compared to the amount of the target protein in cells that have not been contacted and / or treated with the polymer. If the amount of the target protein is reduced in cells contacted and / or treated with a polymer, it can be considered that the polymer enhances and / or promotes the degradation of the target protein.

[0052] In the present invention, the amount of the target protein can be measured using methods known in the art, for example, by immunoblotting, Western protein blot analysis and / or ELISA.

[0053] In the present invention, hydrolysis or degradation means that the amount of the target protein after administering the polymer of the present invention to the cells is reduced by at least 10%, for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, compared to not treated with the polymer.

[0054] Preparation method

[0055] In one aspect of the present invention, a method for preparing a polymer for target protein hydrolysis is provided, which comprises (1) preparing a backbone structure; (2) connecting the backbone structure to a first structural unit containing a target protein ligand and a second structural unit containing an E3 ligase ligand. In certain embodiments, the scheme comprises (1) preparing a backbone structure of a first structural unit containing a target protein ligand; (2) connecting the backbone structure to a second structural unit containing an E3 ligase ligand. In a preferred embodiment, the scheme comprises (1) preparing a first structural unit containing a target protein ligand; (2) preparing a PEG structural unit; (3) synthesizing a random copolymer PEG-PC7A-r-AMA from the first structural unit, the PEG structural unit and AMA; and (4) connecting the random copolymer to a second structural unit containing an E3 ligase ligand.

[0056] Those skilled in the art will understand that, depending on the different ligands to be connected, suitable active groups can be selected to form the desired polymer. Examples of active groups include, but are not limited to, amino (-NH2), carboxyl (-COOH), hydroxyl (-OH), carbonyl (=CO), sulfonic acid (-SO3H). In addition, aldehyde groups, ketone groups, ester groups, acyl halides, N-hydroxysuccinimide, maleimide, double bonds, triple bonds and azide can also be used to connect ligands.

[0057] The polymer of the present invention can also be responsive to different environments. In one embodiment, the polymer is a pH-sensitive polymer, which is in an aggregated state at low pH and depolymerized to a stretched state at neutral pH. In another embodiment, the polymer is a temperature-sensitive polymer.

[0058] Method for degrading target protein in cells

[0059] The present invention also discloses a method for degrading a target protein in a cell, which comprises the step of contacting the polymer described in the present invention with a cell and allowing the polymer to enter the cell. According to the present invention, when the target protein contacts the polymer described herein, for example, when the target protein contacts any one of the polymers in a cell, degradation of the target protein can occur.

[0060] In one embodiment, the method of the invention is an in vivo method. In another embodiment, the method of the invention is an in vitro method.

[0061] Pharmaceutical composition

[0062] In one aspect of the present invention, a pharmaceutical composition is provided, which comprises the polymer for hydrolyzing a target protein according to the present invention. In the composition, the polymer can be appropriately formulated so that it can enter cells and induce degradation of the target protein.

[0063] In the present invention, pharmaceutically acceptable carriers are well known to those skilled in the art, including but not limited to phosphate buffered solutions and / or saline. Pharmaceutically acceptable carriers can be aqueous solutions or non-aqueous solutions, suspensions and emulsions. In addition to the above-mentioned carrier components, the above-mentioned pharmaceutical composition can alternatively or additionally include one or more suitable other carrier components, such as diluents, buffers, flavoring agents, adhesives, surfactants, thickeners, lubricants, preservatives (including antioxidants), etc.

[0064] The pharmaceutical composition of the present invention may be present in any typical formulation of a drug compound administered to a subject, examples of which include, but are not limited to, capsules, granules, tablets, powders, lozenges, suppositories, sprays, gels, creams, ointments, sterile aqueous preparations, sterile solutions, aerosols, implants, etc. The pharmaceutical composition of the present invention may be administered to the body in a known manner. For example, it may be delivered to a tissue of interest by intramuscular injection, optionally administered via intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. Such administration may be performed via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be administered herein may vary to a great extent depending on a variety of factors, such as target cells, biological types or their tissues, the general condition of the subject to be treated, routes of administration, modes of administration, etc.

[0065] use

[0066] The present invention also provides the use of polymers for degrading target proteins. The present invention also provides the use of polymers for preparing drugs for preventing, improving and / or treating diseases of subjects in need. The disease is preferably an autoimmune disease, examples of which include but are not limited to systemic lupus erythematosus, type I diabetes, rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, myasthenia gravis or polymyositis. In a preferred embodiment, the disease is a disease associated with abnormalities in the GAS-STING immune pathway.

[0067] As used herein, the term "subject" refers to any animal (eg, mammal) including, but not limited to, humans, non-human primates, rodents, and the like, that is to receive a particular treatment.

[0068] Those skilled in the art will appreciate that the polymers described herein may also be used in combination with other drugs for the prevention, treatment and / or improvement of diseases or conditions. The other drugs may be any therapeutic agent that is beneficial to the disease, such as solid tumors or blood diseases, and there is no particular limitation on this.

[0069] Example 1

[0070] This example illustrates the polymers of the present invention using rheumatoid arthritis as an exemplary autoimmune disease.

[0071] 1. Materials and Methods

[0072] 1.1 Materials

[0073] Polyethylene glycol (PEG), tris(2-picolyl)amine (TPMA), copper bromide (CuBr2), 2-(1-azaphenyl)ethanol, methacryloyl chloride, azobisisobutyronitrile (AIBN), 2-bromoisobutyl bromide (BiBB), 2-aminoethyl methacrylate (2-aminoethyl methacrylate, AMA), 2,5-dioxopyrrolidin-1-yl 6-((2-(2,6-dioxopyrimidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)hexanoate (Thali-NHS), 2,5-difluoropyrrolidin-3-yl 6-(2S)-2-(3-amino-2-hydroxy-4-phenylbutyramido)-5-methylhexanamido)hexanoate (Besta-NHS), and fluorescent dyes.

[0074] 1.2 Methods

[0075] 1.2.1 Synthesis of PC7A and PolyTACs

[0076] 1.2.1.1 Synthesis procedure of 2-(azepan-1-yl)ethyl methacrylate (iii)

[0077] The specific synthetic route is Figure 7 As shown, 2-(azepan-1-yl) ethyl methacrylate was synthesized by one-step substitution reaction. Specifically, 2-(azepan-1-yl) ethane-1-ol (i, 14.32g, 100mmol) and triethylamine (TEA, 20.24g, 200mmol) were dissolved in dichloromethane (DCM, 200ml), and then methacryloyl chloride (ii, 12.54g, 120mmol) was added under ice bath. The mixture was stirred at room temperature overnight. The reaction was quenched by adding water. The reaction mixture was washed with H2O (50mL×3), dried with anhydrous Na2SO4 and concentrated under vacuum to obtain a light yellow liquid product iii (19.00g, yield: 90%). 1 H NMR(400MHz,Chloroform-d)δ6.14-6.04(m,1H),5.54(t,J=1.6Hz,1H),4.23(t,J=6.2H z, 2H), 2.83 (t, J = 6.2Hz, 2H), 2.76-2.68 (m, 4H), 1.97-1.88 (m, 3H), 1.71-1.50 (m, 9H).

[0078] 1.2.1.2 Synthesis procedure of PEG-Br(vi)

[0079] PEG-Br is synthesized by one-step substitution reaction. PEG 5000 (iv, 8g, 1.6mmol) is dissolved in toluene (100mL), and the mixture is then stirred at 125°C for 2 hours. Subsequently, toluene is removed under vacuum, and the remaining solid for subsequent reactions is obtained. These solids and TEA (303.57mg, 3.2mmol) are dissolved in DCM (100mL), 2-bromoisobutyl bromide (v, 441.41mg, 1.9mmol) is added dropwise, and the mixture is then stirred at room temperature overnight. Subsequently, the reaction mixture is concentrated under vacuum, the solid is redissolved with THF, then dialyzed with pure water for 2 days, and the final product PEG-Br of a white flocculated solid is obtained by freeze drying. The yield of the final product is 90% (19.00g).

[0080] 1.2.1.3 Synthesis procedure of PEG-PC7A-r-AMA

[0081] Random copolymer PEG-PC7A-r-AMA was synthesized using the classical continuous activator regeneration-reversible addition-fragmentation chain transfer polymerization (ICAR-ATRP) initiator. First, CuBr2 (178.68 μg, 0.8 μmol) and TPMA (232.28 μg, 0.8 μmol) were added. Then a mixture of isopropanol (IPA, 20 mL) and N,N-dimethylformamide (DMF, 20 ml) was added to dissolve the reactants. Finally, the incubated CuBr2 and TPMA were added. After argon was injected into the system for 30 minutes to remove oxygen, the mixture was stirred at 70 ° C for 48 hours. After polymerization, the reaction mixture was diluted with 15 mL THF and copper was removed by an Al2O3 column, and the residue was then dialyzed in distilled water and freeze-dried to obtain a yellow-white powder ix. 1 H NMR (400MHz, Chloroform-d) δ4.04 (s, 140H), 3.65 (s, 450H), 3.39 (s, 3H), 2.73 (d, J = 29.7Hz, 420H), 1.82 (m, 148H), 1.61 (s, 560H), 1.10-0.77 (m, 210H).

[0082] 1.2.1.4 Synthesis procedures of PC7A and PolyTAC (xii)

[0083] PC7A and PolyTACs were synthesized by the reaction of N-hydroxysuccinimide (NHS) with amino acids. Specifically, a fixed equivalent of 2,5-dioxopyrrolidin-1-yl 6-((2-(2,6-dioxopiperidine-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexanoate (x, Thali NHS) or 2,5-dioxopyrrolidin-1-yl-6-((2S)-2-(3-amino-2-hydroxy-4-phenylbutyramido)-5-methylhexanamido)hexanoate (xi, Besta NHS) and 1 equivalent of PEG-PC7A-r-AMA (ix) were dissolved in DCM, and 2 equivalents of TEA were added, stirred at room temperature overnight, and DCM was evaporated to obtain PC7A-PolyTAC-3 (xii, n=0, 1 or 3).

[0084] 1.2.1.5 Synthesis of fluorescent dye-conjugated PolyTACs

[0085] The synthesis of dye-conjugated PC7A and PolyTAC-1 / 2 / 3 was the same as that of PC7A and PolyTAC-1 / 2 / 3 by reacting NHS (N-hydroxysuccinimide) with amino groups. Specifically, purified PC7A or PolyTAC-1 / 2 / 3 and the corresponding dye (Cy3 or NHS or ICG-OSu) were dissolved in DCM, 2 equivalents of TEA were added, stirred at room temperature overnight, DCM was evaporated, and dye-conjugated PC7A-PolyTAC-3 was obtained by dialysis using pure water and freeze drying.

[0086] 1.2.2 Preparation of Nanoparticles

[0087] All these nanoparticles were prepared according to a typical procedure. Briefly, 10 mg of the copolymer was dissolved in 1.5 mL of tetrahydrofuran (THF). The polymer solution was added to 9 mL of Milli-Q water under ultrasonic treatment, and then ultrafiltered five times using a micro-ultrafiltration system to remove THF. Finally, the volume was fixed to 1 mL with Milli-Q water, and the concentration of the nanoparticles was 480 μM.

[0088] 1.2.3 Synthesis and preparation of control nanoparticles

[0089] The synthesis and preparation method of the control nanoparticles is as described in 1.2.1.3 and 1.2.2, except that C7A in 1.2.1.3 is replaced by EPA during the synthesis process.

[0090] 1.2.4 Transmission electron microscopy analysis

[0091] The nanoparticles were diluted with PBS (pH 7.8 or 6.3), then dropped onto a copper grid and negatively stained with 1% uranyl acetate. The samples were then observed and photographed using a transmission electron microscope (JEOL1741200EX).

[0092] 1.2.5 Particle size, shelf stability and polydispersity

[0093] The nanoparticles were diluted to 100 μg / mL with phosphate buffered saline (PBS) containing 10% fetal bovine serum (FBS) and then stored at 4°C and 37°C for different time periods. Finally, dynamic light scattering was used to detect the changes in the particle size and polydispersity coefficient of the nanoparticles under different conditions.

[0094] 1.2.6 Fluorescence activation of nanoparticles

[0095] Lengguang Technology F97Pro obtained the fluorescence activation of nanoparticle nanoprobes in different pH buffer solutions. The initial concentration of each nanoparticle was 1mg / mL and diluted to 100μg / mL with PBS of different pH values. The excitation light of the nanoprobe was 780nm and the emission spectrum was 790-900nm.

[0096] 1.2.7 Cellular uptake of nanoparticles

[0097] HeLa cells were cultured until they reached 80% confluence, and then Cy3-conjugated nanoparticles were introduced at different time points. After nanoparticle treatment, the cell culture medium was changed, and lysosomes were labeled using Lyso Tracker. Subsequently, colocalization of Cy3-labeled nanoparticles and Lyso-Tracker-labeled lysosomes was observed using confocal microscopy.

[0098] 1.2.8 Human synovial samples and cell culture

[0099] Synovial specimens were obtained from RA patients who met the 2010 ACR diagnostic criteria and underwent total knee replacement surgery, and osteoarthritis (OA) patients who met the 2010 ACR / EULAR diagnostic criteria and underwent joint replacement surgery at Peking University First Hospital. This study was approved by the Medical Ethics Committee of Peking University First Hospital.

[0100] Human primary FLS were isolated from synovial tissue of RA patients, cut into small pieces, and digested in 2mg / mL type I collagenase (C8140, Solarbio) and 0.25% trypsin-EDTA solution (T1320, Solarbio) for 4 hours. The resulting cell suspension was centrifuged and the cell pellet was resuspended in Dulbecco's modified Eagle medium (DMEM, C11995500BT, Invitrogen) containing 10% FBS (ST30-302P, Pan). After incubation for 24 hours, non-adherent cells were removed by PBS washing. FLS was used from the third to the seventh generation, and the cell type was confirmed by methods known in the art.

[0101] 1.2.9 Animals and cell culture

[0102] Male Sprague-Dawley (SD) rats, weighing 200-250 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. This study was approved by the Animal Ethics Committee of Peking University First Hospital. Rats were injected with complete Freund's adjuvant (7027, Chondrex) at the base of the tail to construct an adjuvant-induced arthritis (AIA) model, while control rats received saline injections. Clinical parameters, including joint swelling thickness, polyarthritis index (PI), and histological analysis were measured. The joint swelling thickness and PI of each posterior joint were recorded by two observers who were blind to the treatment group, and then the average was taken for each animal. Joint tissue and blood samples were collected for pathological analysis, cell culture, and inflammatory cytokine analysis. The extraction and culture protocols of primary rat FLSs were referred to previous publications, and RAFLSs were used between the third and seventh generations. Jurkat and THP-1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI medium supplemented with 10% FBS and 0.05 mM β-mercaptoethanol. All cells were grown at 37°C in 5% CO2.

[0103] 1.2.10 Hematoxylin and eosin (HE), Safranin O (SO) / Fast Green Staining

[0104] The dissected tissues were fixed in 4% formaldehyde for 48 hours and then decalcified in 10% ethylenediaminetetraacetic acid (EDTA) solution for 30 days. The specimens were embedded, cut into 7 μm paraffin sections, and stained with HE and SO dyes, respectively. Histological images were taken under a microscope (VS200, Olympus, Japan). A 4-point scale was used to evaluate synovitis, cartilage damage, and bone erosion, with 0 indicating healthy and intact tissue and 3 indicating severe arthritis. Histological scoring was performed by two independent researchers who were unaware of the groupings, and the average was then taken for analysis.

[0105] 12.2.11 Immunohistochemistry (IHC)

[0106] Paraffin-embedded samples were cut into 8 μm sections. Antigen retrieval was performed using the target retrieval solution Tris-EDTA buffer and a microwave oven for 10-15 minutes. The sections were placed in 3% H2O2 for 30 minutes to block endogenous peroxidase activity. 3% BSA was added to evenly cover the tissue to block nonspecific binding for 30 minutes. The tissue was incubated with antibodies against STING (1:100, PA5-116052, Invitrogen) and P-IRF3 (1:100, PA5-36775, Invitroen) at 4°C overnight and immunodetected using a DAB kit (G1212-200T, Servicebio).

[0107] 1.2.12 Multiplex immunohistochemistry (mIHC)

[0108] 4 μm paraffin-embedded human synovial tissue sections were dewaxed and rehydrated with a series of graded ethanol solutions, followed by antigen extraction with citrate buffer (pH 8.0). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution for 15 minutes, and nonspecific binding sites were blocked with 10% donkey serum. Tissue sections were then incubated with the first primary antibody against STING (1:100, PA5-116052, Invitrogen) at 4°C overnight, followed by incubation with the secondary antibody at room temperature for 1 hour. Slides were incubated with CY3-TSA solution (appropriately diluted with TBST) in the dark for 10 minutes. Microwave treatment was performed to remove the primary and secondary antibodies bound to the tissue by immersing the slides in EDTA antigen retrieval buffer (pH 8.0) and keeping them at sub-boiling temperature for 8 minutes, letting them stand for 8 minutes, and then repeating the sub-boiling temperature for 7 minutes. The steps were repeated for the second primary antibody FAPα+ (1:100, AP61510, Abcepta) and the secondary antibody.

[0109] 1.2.13 Immunofluorescence staining (IF)

[0110] The cells in the confocal culture dish were washed three times with PBS and fixed with 4% paraformaldehyde for 20 minutes at room temperature. After fixation, the samples were permeabilized with 0.3% Triton X-100 (Solarbio, China) in PBS for 10-15 minutes at room temperature and blocked with donkey serum for 1 hour at room temperature. After the primary antibody and the sample were washed with PBS at 4°C, the sample was further incubated with Alexa Fluor 488 or 594 secondary antibody (Abcam, China). The nuclei were stained with DAPI (Beyotime, China) for 5 minutes. Images were observed and captured by fluorescence microscopy or laser scanning confocal microscopy (ZEISS, Germany).

[0111] 1.2.14 Western blotting

[0112] The cells were washed twice with cold PBS and lysed with RIPA buffer (Beyotime, China) on ice for 30 min. The lysate was centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was collected for protein quantification using a BCA protein assay kit (Beyotime, China). Proteins were electrophoresed in 8-10% SDS-PAGE gels and transferred to PVDF membranes, which were then quantified with QuickBlock TM Blocking buffer (Beyotime, China), followed by incubation with the corresponding primary antibody at 4 ° C overnight. After washing with TBST, the membrane was incubated with a secondary antibody coupled to horseradish peroxidase (HRP). Protein bands were observed using ECL solution (Beyotime, China) and detected using an automatic chemiluminescence imaging analysis system (Syngene, England). Western blot bands were quantified by using Image J software.

[0113] 1.2.15ELISA

[0114] ELISA kits were used to assess TNF-α (HS044Ra, China) and IFN-α (HS1023-Ra, China) in rat plasma according to the manufacturer's instructions. The assay plate was read at 450 nm and 540 nm. All samples were run in duplicate.

[0115] 2.2.16 Small Animal In Vivo Imaging System

[0116] ICG-labeled PolyTAC-2 was used for IVIS spectral imaging. The cells were anesthetized with isoflurane for 5 minutes before injection. After intraperitoneal injection of labeled PolyTAC-2, the biodistribution of ICG-labeled PolyTAC-2 was tested by in vivo imaging measurements of the fluorescence intensity at 0h, 8h, 16h, 20h and 24h at a wavelength of 780nm using IVIS Lumina Series III (PerkinElmerzei).

[0117] 1.2.17 Security Assessment

[0118] Healthy mice (20-30 g) were intraperitoneally injected with a single dose of PolyTAC-1 (15 mg / kg and 75 mg / kg, respectively) and PolyTAC-2 (15 min and 75 mg / kg, respectively) to study the safety of PolyTACs in vivo.

[0119] Seven days after intraperitoneal injection, whole blood samples were collected from rats, mice were anesthetized, and organs including heart, liver, spleen, lung, and kidney were subjected to histological analysis. The blood samples were then centrifuged at 3000 rpm / min at 4°C for 30 min, and the supernatant was immediately tested. Alanine aminotransferase (ALT), aspartate aminotransferase, albumin, creatine kinase (CK), creatine kinase MB (CK-MB), lactate dehydrogenase (LDH), urea nitrogen (urea), uric acid (UA), and total protein (TP) in the obtained serum were measured on a Hitachi 7020 automatic biochemical analyzer.

[0120] 1.2.18 Statistical analysis

[0121] Quantitative results are presented as mean ± standard deviation. Student's two-sided t-test was used for statistical analysis for comparison between two groups. For multiple comparisons, two-way analysis of variance (ANOVA) was used. P values ​​were significantly different < 0.05.

[0122] 2. Results

[0123] Activation of the STING / P-IRF3 pathway in the synovium of RA patients and AIA rats

[0124] To investigate the expression level of STING in RA synovium and the activation of the STING / IRF3 pathway, we collected synovial tissues from clinical RA and OA patients. The pathogenesis and phenotypes of RA and OA are significantly different. RAFLSs maintain increased proliferation, migration, and invasion capabilities compared to OA-derived FLSs22-24, which are often used as a "disease" control to reveal the autoimmune characteristics of RA25. IF staining showed that STING was highly expressed in the synovium of RA patients ( Figure 2a). Further analysis showed that the expression of STING and FLSs marker FAPα+ in RA samples was significantly higher than that in OA samples ( Figure 2 c, 2d), the co-localization of STING and FAPα+ in RA samples reached about 61% ( Figure 2 e), indicating that STING was more highly expressed in FLSs in RA compared with the OA group. IHC showed increased fluorescence of STING and P-IRF3 in RA patients ( Figure 2 b), which is a marker of the downstream STING pathway. Further analysis showed that the expression of STING and P-IRF3 in RA was significantly higher than that in OA samples ( Figure 2 f, 2g), indicating that the STING / P-IRF3 pathway was significantly activated in FLSs. To further verify the activated STING / IRF3 pathway in FLSs, we isolated primary human FLSs and performed Western blot analysis. Figure 6 As shown, STING, P-STING, P-TBK1, and P-IRF3 were significantly upregulated in RA patients compared with OA patients ( Figure 2 h, 2i). In addition, by Western blotting, upregulation of STING, P-STING, P-TBK1, and P-IRF3 was also found in primary FLS of the AIA model ( Figure 2 j, 2k). These findings suggest high expression of STING and activation of the STING / P-IRF3 pathway in FLSs in RA synovium.

[0125] 2.2 Design of PolyTACs Nanoparticles

[0126] To degrade STING protein, PolyTACs polymer nanoparticles (PEG-bP(C7A-r-E3L)) were developed using random copolymer design. Four types of nanomaterials were screened from previous data and used for development, including PC7A and PolyTAC-1 / 2 / 3 ( Figure 3 b). PEG represents polyethylene glycol, C7A is a monomer of 2-(azepan-1-yl)ethyl methacrylate (pH-sensitive monomer), which is also a ligand of STING protein, and E3L represents E3 ubiquitin ligase ligand ( Figure 3 a) By 1HNMR confirmed the structural correctness of C7A monomer and polymer PC7A. In addition, control nanoparticles were designed and synthesized, which did not have STING binding function but had a pH transition point (pHt) similar to PC7A as a control. According to previous data, PC7A nanoparticles with 70 C7A fragments showed the strongest STING binding ability. Therefore, all PolyTACs nanoparticles selected in this study contained 70 C7A fragments. In addition, after preliminary screening, only two E3 ligase ligands-thalidomide and ubenimex were retained, and the number of E3-linked sugar ligands was less than four.

[0127] 2.3 Characterization of PolyTACs nanoparticles

[0128] PolyTAC exhibits pH-sensitive activation properties, and the pH transition point of PolyTAC is determined to be 7.1 by titration. In response to changes in environmental pH, PolyTACs transform from associated nanoparticles (NPs) to dissociated polymers ( Figure 3 a). When the environmental pH is higher than pHt, PolyTAC is a spherical particle with a size of about 30 nm, while when the environmental pH is lower than pHt, the nanoparticles dissociate into polymers and cannot be observed under a transmission electron microscope ( Figure 3 c). In addition, the pH-sensitive properties of PolyTACs were investigated using fluorescence activation analysis. Through fluorescent dye conjugation, PolyTACs nanoparticles were shown to be pH-sensitive “active / inactive” probes with high fluorescence activation rates (>50-fold between “active” and “inactive” states, Figure 3 d). Subsequently, the long-term particle size stability, potential, and polydispersity index (PDI) of PolyTACs nanoparticles were detected using dynamic light scattering. The results showed that PolyTAC could remain stable over a long period of time, but the PDI increased slightly over time, showing a wider particle size distribution.

[0129] 2.4 Initial degradation of STING protein by PolyTACs

[0130] The present invention performed a Western blot experiment to evaluate the efficacy of PolyTACs in degrading STING. THP-1 and Jurkat cells were incubated with PC7A (7.2 μM), PolyTAC-1 (7.2 μM), PolyTAC-2 (7.2 μM) and PolyTAC-3 (7.2 μM) for 20 h. The results showed that PolyTAC-1 and PolyTAC-2 were able to degrade STING, while PolyTAC-3 was not ( Figure 3eh). After co-incubation with PC7A, the degradation of STING in THP-1 and Jurkat cells was consistent with previous reports. To further evaluate the relationship between concentration and degradation efficacy, THP-1 cells were treated with PolyTAC-2 at concentrations of 0, 2.4, 4.8, 7.2, 9.6, 12, and 14.4 μM. The results showed that the degradation effect was observed at 2.4 μM, and within a certain concentration range, the concentration was positively correlated with the degradation effect ( Figure 3 i, 3j).

[0131] 2.5 PolyTACs degrade STING and inhibit the STING / P-IRF3 signaling pathway

[0132] To further evaluate the efficacy of PolyTACs in degrading STING in animal models and humans, the present invention first detected its effect on STING activation in primary FLS of AIA rats. Primary FLS were isolated from AIA models and normal rats and subjected to Western blot analysis. FLSs were treated with PolyTAC-1 (12 μM) for 0, 1, 6, 12, 18, and 24 hours, and it was found that the degradation effect was observed from 12 hours. In the range of 12-24 hours, the degradation efficiency of PolyTAC-1 increased with the increase of incubation time ( Figure 4 a, 4b).

[0133] Next, the present invention studied the role of PolyTACs in regulating the STING / P-IRF3 pathway. In this section, the STING inhibitor H-151 was selected as the positive drug control group. H-151 is a potent, selective and covalent antagonist of STING, with significant inhibitory activity both in cells and in vivo. After treatment with PolyTAC-1 (12 μM), PolyTAC-2 (12 μM) and H-151 (2 μM), the levels of P-STNG, P-TBK1 and P-IRF3 decreased, indicating that the STING / P-IRF3 pathway was inhibited by PolyTAC-1, PolyTAC2 and H-151 ( Figure 4 c, 4d).

[0134] To further investigate the potential degradation of STING by PolyTACs in human primary FLSs, primary FLs were isolated from RA patients and treated with PolyTAC-1 at concentrations of 0, 2.4, 4.8, 9.6, 12, 14.4, 19.2, and 24 μM. Figure 4e and 4f, a degradation effect was observed at 2.4 μM, and within a certain concentration range of 0-12 μM, the concentration was positively correlated with the degradation effect, which was consistent with the results of AIA rats. However, outside this range, no degradation effect was observed to increase with increasing concentration. IF staining showed that treatment with PolyTAC-1 (12 μM) and PolyTAC-2 (12 μM) led to the degradation of STING in primary RA FLSs, and both STING and P-IRF3 were reduced ( Figure 4 g, 4h). In the inflammatory cascade of RA progression, TNF plays a key proinflammatory cytokine role in promoting the activation of FLSs. Activated FLS, in turn, promote the inflammatory cycle and cartilage destruction by producing chemokines, inflammatory cytokines, and matrix metalloproteinases (MMPs). In order to study the activation of the STING signaling pathway in the inflammatory cascade of RA, the present invention stimulated primary FLS of RA patients with TNF-α (4ng / ml), and observed increased expression of STING and its downstream molecules in the pathway (including P-TBK1 and P-IRF3). These findings indicate that the inflammatory cascade of RA can further promote the activation of the STING signaling pathway. In addition, when TNF-α-induced cells were treated with PolyTAC-1 (12μM), PolyTAC-2 (12μM), and H-151 (2μM) for 24 hours, it was observed that PolyTAC-1 and PolyTAC-2 effectively degraded STING and inhibited the downstream STING / P-IRF3 signaling pathway ( Figure 4 i, 4j).

[0135] 2.6 Mechanisms of PolyTACs endocytosis and degradation pathways

[0136] PolyTACs nanoparticles exhibit good cytoplasmic effects and can escape from lysosomes to play a role in protein degradation in the cytoplasm. Figure 5 As shown in a and 5b, the colocalization of nanoparticles and lysosomes gradually increased within 12 hours, indicating that the number of nanoparticles entering the cells continued to increase during this stage. After 12 hours, the colocalization of nanoparticles and lysosomes gradually decreased, indicating that the nanoparticles were released into the cytoplasm. The temporal analysis of the colocalization of PolyTACs with lysosomes coincides with the time when PolyTACs begin to exert their protein degradation effect.

[0137] In order to further study the degradation mechanism of PolyTACs, determine the role of PolyTACs in degrading STING through intracellular proteasomes, and that the degradation of STING by PolyTACs is to degrade STING and CRBN in the form of a large complex, the present invention uses a proteasome inhibitor to block the proteasome, and then observes the changes in the degradation ability of PolyTACs protein by immunofluorescence staining and Western blotting. The present invention treats primary human FLS with PolyTAC-1, PolyTAC-2, PolyTAC-1 and PS-341, PolyTAC-2 and PS-341. Figure 5 As shown in c and 5d, PolyTAC-1 and PolyTAC-2 treatment significantly reduced the levels of STING and CRBN proteins in cells, further verifying the degradation effect of PolyTAC-1 and PolyTAC-2 on STING protein. In addition, the reduction in CRBN levels also confirmed that STING protein was degraded in the form of a large complex with CRBN. The proteasome inhibitor PS-341 can largely block the degradation of STING and CRBN proteins by PolyTAC-1 and PolyTAC-2, indicating that PolyTAC-1 or PolyTAC-2 does play a role in protein degradation through the proteasome.

[0138] With the participation of PS-341, Western blotting further verified the degradation of STING protein by PolyTAC-1 and PolyTAC-2 and their effects on the STING downstream signaling pathway. Figure 5 As shown in Figure 5e and 5f, PolyTAC-1 and PolyTAC-2 can degrade STING and inhibit the STING / IRF3 pathway, while PS-341 can reverse these effects, fully confirming the role of proteasome in protein degradation by PolyTAC-1 and PolyTAC-2.

[0139] PolyTAC inhibits STING signaling and reduces inflammation in the AIA model

[0140] In order to study the role of PolyTACs in the development and progression of RA, the present invention established the AIA model, which is a typical rheumatoid arthritis modeling method and the most widely used rat RA model. In order to study the biodistribution of PolyTACs in vivo, ICG-labeled PolyTAC-2 was constructed and injected intraperitoneally in a rat model (10 mg / kg). Using a small animal in vivo imaging system, the fluorescence intensity of ICG-labeled PolyTAC-2 in the joints at 0, 8, 16, 20 and 24 hours after administration was measured by fluorescence imaging, and fluorescent signals in arthritic joints were observed as early as 8 hours. The fluorescence aggregation intensity gradually increased to a maximum value and then showed a downward trend. The fluorescence intensity at the joints of rats in the normal group was always zero ( Figure 6 a). Comparative analysis of the fluorescence intensity of the arthritis site at 24 hours revealed that PolyTACs-2 showed significant accumulation and activation in the RA site of arthritis model mice compared with normal mice.

[0141] Next, the therapeutic potential of PolyTAC was evaluated in the AIA model. Twenty-one days after arthritis induction, AIA rats developed severe swelling in the ankles and paws. PolyTAC-1, PolyTAC-2, or control nanoparticles (control) were injected intravenously into the rats (10 mg / kg). Figure 6 b, 6e, 6f, PolyTAC-1 and PolyTAC-2 showed strong therapeutic effects after administration, significantly alleviating disease progression, and reducing joint swelling severity scores and polyarthritis index (PI). There was almost no difference in joint thickness and PI scores between the control group AIA rats and the AIA group. To further evaluate the degree of joint inflammation and cartilage destruction, synovial and cartilage histopathological examinations were performed in AIA model rats. Compared with the model group, the control nanoparticles had no effect in reducing the levels of joint inflammation and cartilage destruction, while PolyTAC-1 and PolyTAC-2 significantly reduced synovial hyperplasia and joint inflammation ( Figure 6 c), as evidenced by a decrease in histological scores ( Figure 6 gi). Safranin-O / fast green staining showed that part of the cartilage tissue disappeared in some ankle joint sections of the model group and the control group ( Figure 6 d). In contrast, almost intact and red-stained cartilage could be found in the joints of the PolyTAC-1 and PolyTAC-2 groups, indicating that PolyTAC-1 and PolyTAC-2 effectively reduced cartilage destruction, a typical pathological manifestation of RA ( Figure 6 d). In addition, IHC staining showed that PolyTAC-1 and PolyTAC-2 treatment significantly reduced the expression of STING ( Figure 6j). Immunologically, Elisa assays of inflammatory cytokines in rat plasma showed that PolyTAC-1 and PolyTAC-2 effectively reduced the secretion of TNF-α, IFNα, IFN-β, IL-6, and IL-17 levels ( Figure 6 k). These cytokines play a key role in regulating the progression of RA, and IFNα and IFNβ are downstream cytokines of the STING pathway. These findings suggest that in the AIA model, PolyTAC-1 and PolyTAC-2 can degrade STING and inhibit the STING signaling pathway and alleviate pathology.

[0142] 2.8 PolyTAC System Security

[0143] To evaluate the systemic toxicity of PolyTAC-1 and PolyTAC-2, healthy mice were treated with different doses of PolyTAC-1 and PolyTAC-2. The mice were randomly divided into a normal group, a low-dose PolyTAC-1 group (15 mg / kg), a high-dose PolyTAC-1 group (75 mg / kg), a low-dose PolyTAC-2 group, and a high-dose PolyTAC-2 group. Blood and vital organs were sampled and examined on day 7 after intraperitoneal injection. Liver function indicators, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (ALB); cardiac function indicators, including creatine kinase (CK), creatine kinase MB (CK-MB), and lactate dehydrogenase (LDH); renal function indicators, including urea nitrogen (urea) and uric acid (UA), and total protein (TP) were measured. These indicators did not show significant changes after treatment with PolyTAC-1 and PolyTAC-2. Finally, samples of the heart, liver, spleen, lung, and kidney were sectioned and stained with HE. The images showed that PolyTAC-1 and PolyTAC-2 did not cause morphological changes or inflammation in these organs. Therefore, PolyTAC-1 and PolyTAC-2 showed high biocompatibility and did not cause acute damage to major organs.

[0144] 3. Discussion

[0145] The STING pathway can be triggered by a variety of cytoplasmic dsDNA, regardless of its origin and sequence, and is thought to play a key role in autoimmune diseases. In RA patients, the expression of cytoplasmic dsDNA and cGAS in FLS and synovium is increased, and its expression is associated with the severity of rheumatoid synovitis. Increased levels of cytoplasmic dsDNA in RA FLS promote inflammatory responses through the cGAS / STING pathway, and cytoplasmic dsDNA accumulation is an important factor in FLS-mediated rheumatoid synovial inflammation. Compared with OA FLS, cGAS is overexpressed in RA-FLS. In addition, TNF-α stimulation upregulates cGAS expression in RAFLS31cGAS deficiency patients, blocks interferon response, and reduces inflammatory cell infiltration and joint swelling. In the present invention, direct evidence is provided to demonstrate that high expression of STING and activation of the STING / P-IRF3 signaling pathway are detected in RA FLS compared with OA patients. These findings suggest the potential benefits of cGAS / STING targeted therapy for RA.

[0146] The present invention designs PolyTACs, which can not only degrade STING in THP-1 cells, but also demonstrate in primary synovial fibroblasts of humans and rats that they can degrade STING and block the STING-IFN / NF-κb signaling pathway to inhibit inflammatory responses. This has also been verified in the AIA rat model. Joschka et al. demonstrated that TNF induces mitochondrial changes and blocks PINK1-mediated mitophagy induction. This TNF-dependent mitochondrial damage causes mtDNA to leak into the cytoplasmic lysate, which subsequently binds to cGAS and activates cGAS / STING / IFN signaling and subsequent ISG induction. In the present invention, it is also demonstrated that PolyTACs can still degrade STING and block the STING-IFN / NF-κb signaling pathway under the condition of TNF-α induction.

[0147] The PolyTAC design of the present invention is different from the system that uses polymer nanocarriers to deliver PROTACs, which are responsible for proteolytic activity. PolyTAC has several obvious advantages over traditional PROTACs. In the molecular design of candidate drug optimization, PolyTAC shows greater flexibility than PROTAC in structural optimization, capable of binding different ligand types and adjusting ligand ratios. The present invention demonstrates an optimized PolyTAC with 70 C7A repeating units and less than 4 THD conjugates. During the formation of the ternary complex, PolyTAC, as a multivalent macromolecule, exhibits longer persistence and stronger binding affinity with POI and E3 ligase compared to divalent PROTAC. In some cases, this polymer can even induce the condensation of multiple POIs, thereby improving the efficiency of protein degradation.

[0148] The present invention demonstrates the compatibility of synthetic PolyTACs with human and rat STING proteins. This finding is of great significance for the clinical translation of candidate drugs from laboratory research to patient care. Drug development strategies based on PolyTACs help bridge the gap between experimental animal models and human patients and address interspecies differences. In addition, PolyTACs show potential applicability to different mutants or subtypes of the target protein of interest (POI). In future clinical applications, PolyTACs are expected to treat a wider range of patients, regardless of genetic and protein changes, simplifying the drug decision-making process in clinical practice.

[0149] There are more agonists than inhibitors for STING. In the present invention, a STING agonist (PC7A) was successfully converted into a degrader (PolyTACs) by coupling it to a small molecule ligand of the E3 ligase for the treatment of another disease. This approach can be used as a general strategy for a variety of other molecules (polymers, carbohydrates, peptides) that can bind to specific proteins. Currently, a wide range of polymers have been reported as effective carriers for delivering active drug molecules into cells. In these studies, most polymers themselves generally do not exhibit pharmacological activity, probably because of their weak affinity for proteins. Interestingly, the protein degradation strategy proposed in the present invention does not need to strictly rely on high affinity. Even mild binding of PolyTACs molecules to target proteins is sufficient to induce degradation. Therefore, the PolyTACs strategy has great prospects in significantly expanding the potential of polymers in the pharmaceutical field.

[0150] In addition to its role in degrading STING protein, PolyTAC can also be used as an effective carrier for targeted delivery to the treatment site of rheumatoid arthritis (RA). In vivo imaging experiments conclusively demonstrated that PolyTAC exhibited superior site-specific enrichment and activation targeting capabilities in rheumatoid arthritis (RA) compared with non-arthritis model mice. PolyTAC is a pH-sensitive nanoparticle with unique properties. First, as an exogenous nanoparticle, PolyTAC exhibits preferential accumulation at the site of arthritis, which is characterized by excessive activation of the immune response and influx of immune-related cells. Macrophages, in particular, have the ability to phagocytize and clear foreign substances. Second, PolyTAC acts as a pH-sensitive nanoparticle with a unique pH transition point (pHt). When the surrounding pH is higher than its pHt, these nanoparticles remain intact and in an "inactive" state. However, when the pH drops below the pHt, PolyTAC dissolves into the polymer chain and enters an "active" state. The inflammatory response and high metabolic activity at the RA site create an acidic environment, which facilitates the activation of PolyTACs. The selective activation of PolyTACs at disease sites not only improves therapeutic efficacy but also minimizes systemic toxicity in vivo.

[0151] Example 2

[0152] This example shows the degradation effect of other polymer materials on the target protein, see the table below for details.

[0153] The polymers listed in the table are used as the skeleton, and the active groups (amino, carboxyl, thiol, N-hydroxysuccinimide, maleimide, double bond, triple bond or azide, etc.) on the skeleton are coupled with the target protein ligand or E3 ligase ligand to obtain the corresponding polymer, and the nanoparticles are prepared by solvent evaporation. The corresponding cells are treated with nanoparticles, and the content of the target protein and the internal reference protein after the treatment of different nanoparticles is detected by Western Blot method. The gray value of the target protein and the internal reference protein is normalized by the internal reference protein using ImageJ software, and finally compared with the control group to obtain the protein degradation efficiency value.

[0154]

[0155]

[0156]

[0157]

[0158]

[0159] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. A multi-linker polymer for target protein hydrolysis, characterized in that: The polymer has a molecular weight of 2000-40000 and comprises a first structural unit containing a target protein ligand and a second structural unit containing an E3 ligase ligand, which is selected from one of the following:

2. The multi-linker type polymer for target protein hydrolysis according to claim 1, characterized in that: The target protein ligand is a group of the first structural unit, or a ligand connected via an active group; the E3 ligase ligand is a group of the second structural unit, or a ligand connected via an active group.

3. The multi-linker type polymer for target protein hydrolysis according to claim 2, characterized in that: The active group includes at least one of an amino group, a carboxyl group, a thiol group, N-hydroxysuccinimide, a maleimide, a double bond, a triple bond and an azide.

4. The multi-linker type polymer for target protein hydrolysis according to claim 1, characterized in that: The polymer is selected from a random copolymer, a block polymer or a homopolymer.

5. The method for preparing a multi-linker type polymer for target protein hydrolysis according to any one of claims 1 to 4, characterized in that: include: (1) preparing a first structural unit containing a target protein ligand; (2) preparing structural building blocks; (3) synthesizing a random copolymer from the first structural unit, the structural building unit and the compound containing an active group; (4) connecting the random copolymer to a second structural unit containing an E3 ligase ligand.

6. Use of the linker-type polymer according to any one of claims 1 to 4 in degrading a target protein.

7. A method for degrading a target protein in a cell, characterized in that: The method comprises the steps of allowing the linker-type polymer according to any one of claims 1 to 4 to contact cells and allow the linker-type polymer to enter the cells.

8. A pharmaceutical composition, characterized in that The invention comprises the multi-joint polymer according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

9. Use of the multi-linker type polymer for target protein hydrolysis according to any one of claims 1 to 4 in the preparation of targeted protein degradation drugs.

10. The use according to claim 9, characterized in that The medicament is used to treat rheumatoid arthritis or osteoarthritis.

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