An activatable blood-brain barrier penetrating macrocyclic targeted protein degradation chimera, and methods of making and using the same

Abstract

This invention discloses a chimeric macrocyclic target protein degradation agent capable of activating blood-brain barrier penetration, its preparation method, and its application. The chimeric structure is ROS-VHL-Linker1-TP-Linker2-cTP-Alk. F -2; where ROS is a reactive oxygen species response group, VHL is an E3 ubiquitin ligase ligand, TP is a targeting peptide, and cTP-Alk F -2 is a blood-brain barrier-penetrating macrocyclic peptide; Linker1 is a linker for connecting VHL and TP; Linker2 is a linker for connecting TP and cTP-Alk. F -2 linker. This invention utilizes pituitary-mediated transcytosis mediated by aging blood-brain barrier upregulation to achieve efficient intrabrain delivery and selective degradation of pathologically activated DAPK1 for the treatment of neurodegenerative diseases such as Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a chimeric macrocyclic target protein degradation agent that can activate blood-brain barrier penetration, its preparation method, and its application. Background Technology

[0002] Targeted protein degradation technology, especially protein degradation-targeting chimeras (PROTACs), provides a disruptive new approach to overcome the limitations of traditional small molecule inhibitors. PROTACs can recruit E3 ubiquitin ligases to target proteins, induce ubiquitination of target proteins through the proximity effect, and then achieve target protein degradation via the proteasome pathway, thus completely eliminating pathogenic proteins rather than merely occupying protein active sites. However, the clinical translation of PROTACs into central nervous system diseases is still severely limited by multiple factors: (1) large molecular weight and poor cell membrane permeability; (2) insufficient selectivity for pathological protein subtypes; (3) low blood-brain barrier (BBB) ​​penetration efficiency, especially with a significant decrease in delivery efficiency in the aging brain.

[0003] Cell-penetrating peptides (CPPs) hold promise as drug delivery carriers, but they suffer from drawbacks such as lack of cell type specificity, high concentration toxicity, and rapid blood clearance. While traditional cyclization strategies can enhance stability, their targeting specificity has not met expectations. More critically, existing BBB-based transdermal penetration strategies using cationic cell-penetrating peptides severely hinder their clinical translation due to their non-specific membrane adsorption properties and potential neurotoxicity.

[0004] Significant pathological remodeling of the blood-brain barrier occurs during aging, with an upregulation of pituitary-mediated transcytosis. This provides an endogenous advantage for developing novel delivery systems adapted to the aging BBB transport pathway. However, current mainstream receptor-mediated transcytosis delivery systems cannot effectively utilize this pathological feature, exhibiting insufficient delivery efficiency in elderly individuals with a high incidence of Alzheimer's disease (AD). Therefore, there is an urgent need to develop novel therapeutic strategies that can simultaneously adapt to the pathological features of the aging BBB, achieve efficient central delivery, and precisely and selectively degrade pathological proteins. Summary of the Invention

[0005] The main objective of this invention is to provide a blood-brain barrier-permeable macrocyclic targeted chimera, its preparation method, and its application, aiming to solve at least one technical problem in the prior art.

[0006] To achieve the above objectives, the present invention provides a chimeric macrocyclic target protein degradation inhibitor that can activate blood-brain barrier penetration, with the structural formula ROS-VHL-Linker1-TP-Linker2-cTP-Alk. F -2; Wherein, ROS is a reactive oxygen species responsive group, VHL is an E3 ubiquitin ligase ligand, TP is a targeting peptide, and cTP-Alk is a target peptide. F -2 is a blood-brain barrier-penetrating macrocyclic peptide; Linker1 is a linker for connecting VHL and TP; Linker2 is a linker for connecting TP and cTP-Alk. F -2 connector.

[0007] Furthermore, the cTP-Alk F The structural formula for -2 is shown in Equation 1:

[0008] Formula 1.

[0009] Formula 1 contains a perfluoroalkyl-modified cyclic framework, which can efficiently penetrate the aging blood-brain barrier and target neurons.

[0010] Furthermore, the ROS is a phenylboronic acid group. The phenylboronic acid group forms a reversible shield by linking to the hydroxyproline domain in the E3 ubiquitin ligand via triphosgene.

[0011] Furthermore, the structural formula of the VHL is shown in Formula 2, Formula 3, or Formula 4:

[0012] Formula 2

[0013] Formula 3

[0014] Formula 4.

[0015] Furthermore, the TP is a DAPK1 targeting peptide, which is a recognition peptide derived from the GluN2B subunit, and its amino acid sequence contains KKNRKLRRQHSY. This peptide specifically binds to DAPK1 in its activated conformation (i.e., dephosphorylated state), thereby achieving precise targeted degradation of pathologically activated DAPK1.

[0016] Furthermore, the structural formula of the TP is shown in Formula 5, Formula 6, Formula 7, Formula 8 or Formula 9:

[0017] Formula 5

[0018] Formula 6

[0019] Formula 7

[0020] Formula 8

[0021] Formula 9.

[0022] Furthermore, Linker2 is selected from 6-aminohexanoic acid (Ahx); Linker1 is selected from any one of the following flexible alkyl chain linkers and rigid cyclic linkers: Flexible alkyl chain linkers: C3, C5, C7, C9; Rigid cyclic linkers: cis-cyclohexane, trans-cyclohexane.

[0023] Studies have shown that the stereoconfiguration and rigidity of linkers play a decisive role in the spatial distance and orientation required for the assembly of functional ternary complexes. Overly flexible linkers are detrimental to the stable formation of the E3-linker-target protein; while cis-cyclohexane linkers tend to exhibit spatially distorted conformations, increasing steric hindrance and disrupting the spatial orientation matching between the target protein and the E3 ubiquitin ligase. Trans-cyclohexane rigid linkers exhibit optimal degradation activity.

[0024] Furthermore, the structural formula of the blood-brain barrier-penetrating macrocyclic targeting protein degradation chimera is shown in Formula 10:

[0025] Formula 10.

[0026] This invention also provides a method for preparing the above-mentioned blood-brain barrier-penetrating macrocyclic targeting protein degradation chimera, comprising the following steps: (1) TP-Linker2-cTP-Alk was prepared using the standard solid-phase peptide synthesis method. F -2; (2) Modify ROS on VHL, and then connect it to TP-Linker2-cTP-Alk via Linker1. F -2 coupling.

[0027] This invention also provides the application of the above-mentioned blood-brain barrier-activating macrocyclic targeting protein degradation chimera in the preparation of drugs for treating neurodegenerative diseases. The neurodegenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS).

[0028] This invention also provides the application of the above-mentioned blood-brain barrier-penetrating macrocyclic targeting protein degradation chimera in the preparation of a drug delivery carrier. When used as a carrier, the drug is linked to cTP-Alk. F-2, the drug includes, but is not limited to, any one of fluorescent markers (such as Cy5), chemotherapy drugs, and neuroprotective agents.

[0029] Design concept and principle of the invention: This invention focuses on two major challenges in AD treatment: aging blood-brain barrier (BBB) ​​delivery and selective degradation of pathological protein subtypes. Existing technologies have significant shortcomings in both areas. Traditional strategies rely on endogenous receptor-mediated transcytosis, but with age, the transport mechanism of brain endothelial cells shifts to pituitary-mediated transport, leading to a sharp decrease in delivery efficiency in older individuals. Since the elderly are a high-risk group for AD, this creates a fundamental contradiction. Current PROTACs cannot distinguish between pathological and physiologically homeostatic target proteins, posing a risk of "off-target degradation." DAPK1 participates in important physiological functions such as synaptic plasticity; non-selective degradation would pose serious safety risks.

[0030] Taking DAPK1 targeting peptide as an example, the systematic design scheme is as follows: 1. BBB Shuttle Module: Employing a "macrocyclic + perfluorinated aliphatic crosslinking modification" strategy. The blood-brain barrier-penetrating macrocyclic peptide possesses conformational stability and membrane permeability. The high electronegativity, strong dipole interaction, and amphiphilicity of the fluorine atom promote binding to lipid alkyl chains, triggering pitot-mediated endocytosis. This pathway is independent of endogenous receptors and can still efficiently penetrate the BBB in aging individuals, addressing the problem of age-dependent delivery failure.

[0031] 2. Targeted Ligand Selection: A recognition peptide derived from the GluN2B subunit is used to specifically bind to the dephosphorylated activated state of DAPK1. In the pathological state of AD, DAPK1 is abnormally activated and presents in a dephosphorylated conformation; in the physiological homeostatic state, it is in a phosphorylation-inhibited state. This peptide precisely targets the pathological activated state through conformational selectivity without interfering with the function of the physiological homeostatic state.

[0032] 3. Linker optimization: Through systematic screening of different flexible and rigid linkers, it was found that the trans-cyclohexane rigid linker optimally matches the spatial distance and orientation required for the assembly of the ternary complex. Molecular dynamics simulations confirmed that this linker enables PROTAC to stably bind to both DAPK1 and E3 ligases simultaneously, conferring optimal degradation activity.

[0033] 4. Conditional Activation Design: This design utilizes phenylboronic acid groups to reversibly shield hydroxyproline in the VHL ligand. Based on the specifically elevated ROS levels in AD neurons, it achieves targeted release of active PROTAC within diseased neurons, selectively degrading pathogenic DAPK1, reducing systemic nonspecific protein degradation, and improving therapeutic specificity and safety. The activation condition is a reactive environment rich in reactive oxygen species (ROS), preferably a pathological microenvironment with H2O2 concentration ≥100 μM, such as the high ROS microenvironment in Alzheimer's diseased neurons.

[0034] Based on the above modular design, this invention constructs a macrocyclic targeted chimera that combines four major functions: "highly efficient penetration of the aging BBB, selective identification of pathology, conditional activation of the lesion microenvironment, and multi-pathway pathological intervention," providing a novel technical solution for AD treatment.

[0035] The beneficial effects of this invention are reflected in: (1) Overcoming the age-related blood-brain barrier delivery bottleneck: Existing technologies rely on endogenous receptor-mediated transcytosis, which leads to decreased delivery efficiency as receptors are downregulated with age. This invention employs a "macrocyclization + perfluorinated aliphatic crosslinking" design to trigger pituitary-mediated endocytosis pathway, which does not rely on endogenous receptors and still efficiently penetrates the blood-brain barrier and targets neurons in aging individuals. Experiments have shown that the accumulation in the brains of aging mice is 2.01 times that of young mice, solving the problem of age-dependent delivery failure.

[0036] (2) Achieving selective degradation of pathological subtypes: Existing PROTACs cannot distinguish between pathological and physiological homeostatic target proteins, easily disrupting normal function; this invention uses the GluN2B conformation-selective recognition peptide, specifically binding to dephosphorylated activated DAPK1, but not binding to phosphorylated physiological homeostatic proteins. Experiments have confirmed that it can selectively degrade 62% of activated DAPK1, while the level of phosphorylated DAPK1 remains unchanged, achieving pathological selective degradation.

[0037] (3) Achieving conditional activation of the lesion microenvironment: Existing PROTACs are activated in systemic circulation, which easily leads to degradation of non-target tissues; the present invention introduces a phenylboronic acid ROS-responsive group into the VHL ligand, which is activated only in the high ROS microenvironment of AD lesions. Experiments have confirmed that it can be activated by 100 μM H2O2 to release active degrading agents, achieving lesion microenvironment-dependent activation and reducing the risk of off-target effects throughout the body.

[0038] (4) Achieving multi-target pathological intervention: Existing Aβ monoclonal antibodies only target Aβ and cannot block the Aβ-Tau pathogenic cascade; this invention selectively degrades DAPK1 and simultaneously regulates two pathways: APP metabolism and Tau phosphorylation. Experiments have shown that after treatment, APP expression, Tau phosphorylation, and Aβ42 oligomer deposition all decreased significantly, while the morphological integrity of hippocampal neurons was maintained, achieving a single-target degradation-multi-pathway regulation effect.

[0039] (5) Excellent safety and cognitive protection: Existing cationic penetrating peptides pose a risk of neurotoxicity; the perfluorinated aliphatic cross-linked ring structure of this invention has conformational flexibility, adapting to cell membrane structures and avoiding membrane damage. Experiments have confirmed that the blood biochemical indicators of the treatment group were normal, with no key hematological abnormalities; Morris water maze and nesting experiments showed significant improvement in spatial memory and autonomous behavior, demonstrating excellent safety and cognitive protection effects.

[0040] (6) Controllable preparation and support for precise characterization: The mature Fmoc solid-phase peptide synthesis strategy is adopted, with clear parameters, high reproducibility, and easy large-scale production; the purity of all chimeric structures is confirmed by HRMS and HPLC, and Cy5 fluorescent labeling is supported, which meets the requirements of in vitro characterization and in vivo tracing, providing reliable support for pharmaceutical research. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the structure of BmTAC of the present invention.

[0042] Figure 2 This is the synthetic route diagram for the cTP-AlkF-2 macrocyclic peptide.

[0043] Figure 3 Synthetic route diagram for phenylboronic acid-modified VHL ligands.

[0044] Figure 4 This is a synthetic route diagram for the PROTAC molecule.

[0045] Figure 5 This is a schematic diagram of the structure of the VHL-C3-DAPK1 molecule.

[0046] Figure 6 This is a schematic diagram of the structure of the VHL-C5-DAPK1 molecule.

[0047] Figure 7 This is a schematic diagram of the structure of the VHL-C7-DAPK1 molecule.

[0048] Figure 8 This is a schematic diagram of the structure of the VHL-C9-DAPK1 molecule.

[0049] Figure 9 This is a schematic diagram of the structure of the VHL-c-Cy-DAPK1 molecule.

[0050] Figure 10 This is a schematic diagram of the structure of the VHL-t-Cy-DAPK1 molecule.

[0051] Figure 11 The HPLC and HRMS characterization diagrams of VHL-C3-DAPK1 are shown.

[0052] Figure 12 The HPLC and HRMS characterization diagrams of VHL-C5-DAPK1 are shown.

[0053] Figure 13 The HPLC and HRMS characterization diagrams for VHL-C7-DAPK1 are shown.

[0054] Figure 14 The HPLC and HRMS characterization diagrams for VHL-C9-DAPK1 are shown.

[0055] Figure 15 HPLC and HRMS characterization of VHL-c-Cy-DAPK1.

[0056] Figure 16 The HPLC and HRMS characterization diagrams of VHL-t-Cy-DAPK1 are shown.

[0057] Figure 17A -B stands for PVtCD-cTP-Alk F HPLC and HRMS characterization chromatograms of -2.

[0058] Figure 18 This is an HPLC monitoring chromatogram of ROS-responsive release of BmTAC.

[0059] Figure 19 This image shows a comparison of BmTAC accumulation in the brain tissues of aging and young mice.

[0060] Figure 20 Western blot analysis of BmTAC degradation of DAPK1.

[0061] Figure 21 This is a diagram showing the proteomic analysis of brain tissue from APP / PS1 mice after BmTAC treatment.

[0062] Figure 22 For PVtCD-TP and PVtCD-cTP-Alk F HPLC quantitative results of serum stability at -2.

[0063] Figure 23 Comparison of intracellular uptake fluorescence imaging in hCMEC / D3 cells of brain vascular endothelium.

[0064] Figure 24 Confocal comparison of blood-brain barrier penetration capabilities. Detailed Implementation

[0065] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0066] Unless otherwise specified, the raw materials, reagents or devices used in the following embodiments can be obtained from conventional commercial sources or by existing known methods; unless otherwise specified, the methods used in the embodiments of the present invention are methods mastered by those skilled in the art.

[0067] Example 1 Linker1 linker type screening and PROTAC molecular library construction: I. DAPK1, as a classic serine / threonine kinase, is abnormally overactivated in the pathological state of Alzheimer's disease (AD). To construct an efficient PROTAC molecular library, a series of linkers with gradient flexibility and rigidity were designed and synthesized, as shown in Table 1.

[0068] Table 1

[0069] II. Using the different Linker1 linkers in Table 1, synthesize PROTAC molecules using standard solid-phase peptide synthesis (SPPS) technology, namely VHL (E3 ubiquitin ligase ligand) - Linker1 (linker) - TP (targeting peptide): TP uses the DAPK1 targeting peptide, the structural formula of which is as follows:

[0070] The structural formula of VHL is as follows:

[0071] See the synthetic route for PROTAC molecules. Figure 4 The synthesis method is as follows: (1) Preparation of DAPK1 targeting peptide: Take 300 mg of Rink Amide-MBHA resin and place it in N,N-dimethylformamide (DMF) to swell for 30 minutes.

[0072] Deprotection solution: 2% DBU, 5% piperazine, 93% DMF (volume fraction); use this solution to remove the Fmoc protecting group.

[0073] Amino acid coupling solution: Fmoc-protected amino acids, HATU, and DIPEA were prepared at a molar ratio of 3:3:6 relative to the resin and dissolved in DMF. Each amino acid was coupled twice, with each coupling reaction carried out for 1 hour under a nitrogen atmosphere. Peptide sequence: KKNRNKLRRQHSY.

[0074] (2) VHL coupling: The resin with peptides was mixed with linker1 (1.5 equivalents), VHL ligand (2 equivalents), and DIPEA (4 equivalents) in DMF and treated. The mixture was reacted at room temperature under nitrogen protection for 2 hours, and then the resin was washed with DMF and dichloromethane (DCM) in sequence.

[0075] (3) Pyrolysis and complete deprotection: The resin was treated with a mixed lysis buffer (95% trifluoroacetic acid (TFA) + 2.5% triisopropylsilane (TIS) + 2.5% water) for 2 hours. The lysis buffer was concentrated under reduced pressure, and ice-cold diethyl ether was added to precipitate the crude peptide. The crude peptide product was dissolved in an acetonitrile / water mixed solvent and purified by semi-preparative high-performance liquid chromatography.

[0076] This section uses six linker 1s (flexible alkyl chain linkers: C3, C5, C7, C9; rigid cyclic linkers: cis-cyclohexane, trans-cyclohexane) to prepare six PROTAC molecules, denoted as VHL-C3-DAPK1, VHL-C5-DAPK1, VHL-C7-DAPK1, VHL-C9-DAPK1, VHL-c-Cy-DAPK1, and VHL-t-Cy-DAPK1, respectively.

[0077] III. Structural verification of each PROTAC molecule using HPLC and HRMS: The structural diagrams of each PROTAC molecule, namely VHL-C3-DAPK1, VHL-C5-DAPK1, VHL-C7-DAPK1, VHL-C9-DAPK1, VHL-c-Cy-DAPK1, and VHL-t-Cy-DAPK1, are shown below. Figure 5-10 As shown, the HPLC and HRMS characterization diagrams are as follows: Figure 11-16 As shown in Table 2: Table 2

[0078] Example 2 Screening for optimal PROTAC molecular degradation activity To assess the degradation activity of PROTAC molecules under pathological conditions, an Aβ42 oligomer-induced SH-SY5Y neuron pathological model was constructed.

[0079] Experimental methods: (1) Preparation of Aβ42 oligomers: 1 mg of Aβ42 peptide was dissolved in HFIP and incubated at room temperature overnight. The peptide membrane was lyophilized and dissolved in DMSO to prepare 5 mM stock solution. The solution was diluted to 100 μM with pre-cooled PBS and incubated at 4 °C for 24 h to form oligomers. Cy5-labeled Aβ42 oligomers were prepared simultaneously.

[0080] (2) Molecular dynamics simulations were performed using Gromacs 2020.22 software.

[0081] (3) Western blot assay: Cells / brain tissues were lysed with RIPA containing PMSF, centrifuged at 10,000 rpm for 15 min at 4 ℃, and protein was quantified by BCA; samples were boiled to denature, electrophoresed by 6%~12% gradient SDS-PAGE, and transferred to PVDF; blocked with 5% skim milk for 2 h, incubated with DAPK primary antibody (1:1000) overnight at 4 ℃, incubated with secondary antibody (1:5000) at room temperature for 1 h, ECL luminescence, and ImageJ grayscale quantification to detect changes in DAPK expression.

[0082] Experimental results: Aβ42 oligomers not only significantly reduced cell viability but also induced upregulation of DAPK1 protein expression and decreased autophosphorylation levels. This lead PROTAC molecule can simultaneously form stable binding with both DAPK1 and E3, achieving a half-maximal degradation concentration (DCS). 50 The concentration was 42.43 μM, and a significant hook effect was observed at 100 μM, suggesting that it can exert the best target protein degradation activity in the medium concentration range.

[0083] VHL-t-Cy-DAPK1 was the optimal degradation molecule. At a concentration of 50 μM, it significantly downregulated DAPK1 by 57%. Western blot analyses of each PROTAC molecule are shown in Table 3.

[0084] Table 3

[0085] Example 3 cTP-Alk F -2 macrocyclic peptides enhance the brain cell penetration ability of DAPK1-targeting peptides. Although VHL-t-Cy-DAPK1 has been identified as a candidate degradation molecule, its half-maximal degradation concentration (DC50) remains as high as 42 μM. This phenomenon is not solely due to the efficiency of target binding affinity formation, but rather to delivery limitations caused by the inherent physicochemical properties of the peptide backbone.

[0086] This embodiment utilizes the macrocyclic peptide cTP-Alk F The DAPK1 targeting peptide was structurally modified using a coupling modification strategy, leveraging the high membrane penetration capability and targeted recognition characteristics of -2, and then fluorescently labeled with Cy5 dye.

[0087] cTP-Alk F The structural formula for -2 is as follows:

[0088] I. Construction of Couplers: (1) Conjugation of macrocyclic peptides The linear peptide DAPK1-Tet1 (sequence: KKNRNKLRRQHSY-Ahx-CHLNILSTLWKYRC) was prepared using the standard solid-phase peptide synthesis (SPPS) method. Ahx, representing 6-aminohexanoic acid, served as linker 2. The resulting peptide resin was treated with a mixture of trifluoroacetic acid (TFA), triisopropylsilane (TIS), and water (95:2.5:2.5 v / v) for 2 hours to complete cleavage and deprotection. After cold ether precipitation to obtain the crude linear peptide (denoted as DAPK1-Tet1), alkylation and macrocyclization reactions were performed, as detailed below: 1.1) Alkylation of linear peptides: The crude linear peptide was dissolved in a DMF / NH4HCO3 mixed solution with bromoacetamide to achieve a final concentration of 1 mM for the linear peptide, 2 mM for the bromoacetamide, and 50 mM for the NH4HCO3. The mixture was stirred at room temperature for 24 h, and the crude peptide was precipitated using ice-cold diethyl ether.

[0089] 1.2) Macrocyclization reaction: The specific method is described in the published patent (publication number: CN121494933A).

[0090] This step ultimately yields DAPK1-cTP-Alk. F -2.

[0091] (2) Preparation of Cy5 dye 2.1) Preparation of 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indole-1-onium 1.59 g (10.00 mmol) of 2,3,3-trimethyl-3H-indole was weighed and completely dissolved in 14 mL of acetonitrile (ACN). Then, 5.82 g (30 mmol) of 6-bromohexanoic acid was added. The reaction system was sealed and refluxed in an oil bath at 130 °C with stirring for 48 h. After the reaction was complete, the mixture was allowed to cool naturally to room temperature, and the acetonitrile solvent was removed by rotary evaporation under reduced pressure. 20 mL of ethyl acetate was added to the solid residue, and the mixture was washed with stirring at room temperature for 1 h. The solid filter cake was collected by vacuum filtration and dried to obtain 2.32 g of a pale pink solid compound, namely 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indole-1-onium, with a molar yield of 84.67%. This compound is a precursor of the Cy5 fluorescent chromophore, and the terminal free carboxyl group can be used for covalent labeling of peptides.

[0092] 2.2) Preparation of near-infrared fluorescent Cy5 dye 120.0 mg (0.34 mmol) of 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indole-1-onium and 41.65 mg (0.17 mmol) of N-(3-(phenylamino)allyl)aniline hydrochloride were dissolved together in 5 mL of pyridine and reacted under reflux at 90 °C for 1 h. After cooling to room temperature, the reaction solution was concentrated under reduced pressure to remove the pyridine solvent. The crude product was purified by silica gel column chromatography with dichloromethane:methanol = 10:1 (v / v). The target fraction was collected and evaporated to dryness to obtain 185.42 mg of solid Cy5 fluorescent dye, with a yield of 82.38%. This Cy5 dye retains the terminal carboxyl group and can undergo amide condensation with the amino group of the polypeptide, and can be used to trace the cross-blood-brain barrier and neuronal uptake behavior of molecules at the cellular and in vivo levels.

[0093] (3) The Cy5 dye was coupled to DAPK1-cTP-Alk using solid-state technology. FThe N-terminus of -2 results in a conjugate denoted as Cy5-DAPK1-cTP-Alk. F -2; The conjugate is denoted as Cy5-DAPK1 by coupling Cy5 dye to the N-terminus of the DAPK1 targeting peptide; The conjugate is denoted as Cy5-DAPK1 by coupling Cy5 dye to the linear peptide DAPK1-Tet1.

[0094] II. Study on the uptake and multicellular spheroid permeability of brain endothelial cells Experimental methods: (1) Qualitative and quantitative analysis of cellular uptake: hCMEC / D3 brain microvascular endothelial cells and SH-SY5Y neurons were taken at 2×10 5 Cells were cultured in a single dish for 24 h, then incubated with 5 μM Cy5-labeled peptide for 4 h. After washing with PBS, the nuclei were stained with Hoechst and observed qualitatively under a fluorescence microscope. Single-cell suspensions were prepared by digestion and the fluorescence internalization intensity was quantified by flow cytometry.

[0095] (2) Identification of endocytosis pathway: hCMEC / D3 cells were pretreated with genistein, cytochalasin D and chlorpromazine for 2 h, respectively, and then incubated with Cy5-labeled peptides for 4 h. The uptake of each group of cells was compared by flow cytometry to identify the main transmembrane endocytosis pathway of peptides.

[0096] (3) In vitro blood-brain barrier permeation model: A multilayer cell sphere model simulating the blood-brain barrier (bionic BBB barrier sphere model) was constructed with an outer layer of hCMEC / D3 and an inner layer of SH-SY5Y. The sphere was co-incubated with 5 μM Cy5 peptide for 4 h, and the peptide permeation depth was observed by confocal layer-by-layer scanning.

[0097] Experimental results: See Figure 24 .

[0098] via cTP-Alk F The -2-modified DAPK1 targeting peptide exhibited significantly higher fluorescence signal intensity in brain endothelial cells than the unmodified DAPK1 targeting peptide and its linear conjugate, suggesting that cyclization modification and conjugation strategies can effectively enhance the uptake efficiency of peptides by brain endothelial cells. (See also...) Figure 23 Flow cytometry results showed Cy5-DAPK1-cTP-Alk F The fluorescence intensity at -2 was 16.6 times that of Cy5-DAPK1 and 5.6 times that of Cy5-DAPK1-TP. This confirms the cTP-Alk... F -2 Macrocyclic peptides have significant advantages in enhancing the efficiency of peptide transmembrane uptake.

[0099] The identification results of the endocytosis pathway indicate that the conjugate is mainly internalized into brain endothelial cells through the pituitary-mediated endocytosis pathway and the macropinocytosis pathway.

[0100] Confocal microscopy imaging results show cTP-Alk F -2 conjugates can efficiently cross the endothelial layer and accumulate in the core region of central neurons, while linear peptides remain confined to the peripheral endothelial layer.

[0101] The above results indicate that, in the biomimetic BBB barrier sphere model, macrocyclic peptides endow the conjugates with stronger neuronal targeting ability and permeability.

[0102] Example 4 ROS-responsive BmTAC-mediated targeted degradation of DAPK1 To address the core issue that the uptake mechanism of macrocyclic peptide conjugates easily leads to off-target distribution and premature degradation in vivo, thereby reducing therapeutic efficacy and weakening target specificity, this embodiment utilizes phenylboronic acid (a ROS-responsive group) to reversibly shield the hydroxyproline domain in the VHL ligand. Through a microenvironment-dependent activation mechanism, the active PROTAC is targeted and released within the diseased neurons, selectively degrading the pathogenic DAPK1 while minimizing systemic nonspecific protein degradation, thus improving the specificity and safety of the treatment.

[0103] I. Preparation of BmTAC The structural formula of BmTAC is as follows: Figure 1 As shown, the synthesis method is as follows: First, hydroxymethylphenylboronic acid (ROS-responsive group) was linked to a VHL ligand via triphosgene to prepare phenylboronic acid-modified VHL (i.e., ROS-VHL). Then, following the method in Example 1, trans-cyclohexane was used as linker 1 to connect ROS-VHL with DAPK1-cTP-Alk obtained in Example 3. F -2 was coupled, and the crude product was finally purified by C18 reversed-phase semi-preparative HPLC gradient elution, freeze-dried, and BmTAC was obtained, denoted as PVtCD-cTP-Alk. F -2; In addition, ROS-VHL was coupled with the linear peptide DAPK1-Tet1, and the resulting conjugate was denoted as PVtCD-TP.

[0104] The BmTAC structure is ROS-VHL-Linker1-TP-Linker2-cTP-Alk. F -2; including VHL, TP (targeting peptide), and cTP-Alk F The structure of -2 is shown in Examples 1 and 3, with linker 1 being trans-cyclohexane and linker 2 being 6-aminohexanoic acid. The product was verified by HPLC and HRMS (see Figure 17), and the molecular ion peak was consistent with the theoretical value.

[0105] For the preparation process of ROS-VHL, please refer to [link / reference needed]. Figure 3The method is as follows: (1) Feeding and activation: Take 234.14 mg (1.0 mmol) of compound 1 and 129.3 mg (1.0 mmol) of N,N-diisopropylethylamine (DIPEA), add 10 mL of anhydrous dichloromethane (DCM) to dissolve them completely, and purge with nitrogen gas throughout the process to prevent water vapor from entering; separately weigh 178.0 mg (0.6 mmol) of triphosgene, dissolve it in 3 mL of anhydrous DCM to prepare a dropping solution, and slowly add it dropwise to the above mixture under stirring. After the addition is completed, stir at room temperature for 30 min to activate the amino group in compound 1 into a carbonyl chloride active intermediate by triphosgene.

[0106] (2) Coupling to urea: Prepare an anhydrous DCM solution containing 530.7 mg (1.0 mmol) of compound 2 (an amino functional building block containing phenylboronic acid) and 129.3 mg (1.0 mmol) of DIPEA. Add the solution dropwise to the activated reaction system and stir continuously at room temperature for 24 h. Carbonyl chloride undergoes an addition reaction with the amino group of compound 2, and the VHL core is linked to the phenylboronic acid functional fragment through the urea bond.

[0107] (3) Purification to obtain compound 3: After the reaction, the solvent was removed by vacuum concentration. The crude product was purified by silica gel column chromatography using dichloromethane / methanol (volume ratio 10:1) as eluent. The target component was collected and evaporated to dryness to obtain a total of 690.5 mg of yellow solid intermediate compound 3. The yield of this step was 88.6%. The side chain of compound 4 has an acid-sensitive protecting group, so it cannot be directly coupled to the polypeptide for the time being.

[0108] (4) Acid deprotection: The obtained compound 3 was dissolved in a trifluoroacetic acid (TFA) / dichloromethane mixed solution (volume ratio 1:3), and stirred at room temperature for 1 h to remove all acid-sensitive side chain protecting groups on the molecule, exposing active sites that can undergo condensation reaction with the polypeptide.

[0109] (5) Reverse-phase purification and lyophilization: After deprotection, the solvent was removed by concentration. The crude product was purified by C18 reverse-phase column chromatography with acetonitrile-water system as the mobile phase and elution with a linear gradient of acetonitrile volume fraction of 10% to 90%. The target product components were collected and lyophilized to finally obtain 345.5 mg of solid phenylboronic acid modified VHL degradation ligand (compound 4). The total yield of the two-step synthesis was 65.0%.

[0110] II. ROS Response Activation Verification Experimental methods: Cell ROS detection: SH-SY5Y cells 1×10 4 Cells / well were plated, Aβ42 was treated for 6 h, and DCFH-DA probe was incubated at 37 ℃ in the dark for 30 min to quantitatively detect intracellular oxidative stress levels.

[0111] The BmTAC prepared in this experiment was incubated in PBS buffer containing 100 μM H2O2, and the release process of active PROTAC was monitored by high performance liquid chromatography (HPLC).

[0112] Experimental results: The release of the active product increased in a time-dependent manner with increasing incubation time. (See [link to relevant documentation]). Figure 18 The released product was confirmed by MS, and its molecular weight was consistent with the expected activity of the prodrug, demonstrating that the prodrug can be controlled and activated under ROS stimulation.

[0113] The BmTAC prepared in this embodiment has three functions: the macrocyclic peptide can efficiently penetrate the blood-brain barrier, the VHL ligand mediates the degradation of the DAPK1 target protein, the Cy5 near-infrared fluorescence enables in vivo and in vitro drug distribution tracking, and the intramolecular alkylamide linker has in vivo biodegradable properties. It is suitable for the treatment and visual efficacy evaluation of neurological diseases related to DAPK1 overexpression in the central nervous system.

[0114] III. BmTAC's ability to degrade DAPK1 To detect changes in ROS levels in nerve cells after treatment with Aβ42 oligomers, intracellular ROS levels were measured using a ROS-specific fluorescent probe. To investigate whether the DAPK1 degradation mediated by this conjugate is dependent on the proteasome pathway, neuronal cells were pretreated with the proteasome-specific inhibitor MG132, followed by Western blotting to detect DAPK1 protein expression levels.

[0115] Experimental methods: Cellular immunofluorescence: SH-SY5Y cells were plated for 24 h and set up Aβ model group, peptide drug administration group, and MG132 proteasome inhibition control group. After co-incubation for 24 h, cells were fixed with 4% paraformaldehyde and blocked permeabilized. DAPK primary antibody was incubated at 4 ℃ for 24 h, followed by fluorescent secondary antibody incubation in the dark for 1 h. DAPI was used to stain the nuclei, and the intracellular distribution of DAPK was observed under a fluorescence microscope.

[0116] SH-SY5Y cells were pretreated with MG132 for 4 h, and then treated with Aβ oligomers and the target peptide for 24 h. Western blotting was used to detect DAPK protein expression and verify the PROTAC-mediated target protein degradation function.

[0117] SH-SY5Y cells were treated with 20 μM Aβ42 oligomers for 6 h, and intracellular reactive oxygen species were detected using DCF probes. The time-dependent changes in the degradation of DAPK1 by the conjugates were analyzed by Western blotting.

[0118] Experimental results: See Figure 20The ROS fluorescence intensity in neurons of the Aβ42-treated group was significantly higher than that of the control group, indicating the successful construction of a ROS-enriched microenvironment under AD pathological conditions. BmTAC (10 μM) induced significant DAPK1 degradation, reducing total DAPK1 levels by 62%. The level of phosphorylated DAPK1 (p-DAPK1) remained essentially unchanged, confirming that the conjugate selectively degraded the dephosphorylated activated kinase form. Time-dependent degradation kinetics further confirmed the gradual accumulation of the active degradative agent within cells, with the degradation effect gradually increasing with prolonged incubation time.

[0119] Example 5 Age-dependent brain tissue accumulation and neuronal targeting capabilities of Cy5-labeled peptides Based on the effective degradation activity of BmTAC against DAPK1 at the cellular level, its in vivo therapeutic potential was further systematically evaluated in a mouse model.

[0120] Near-infrared in vivo fluorescence imaging: Cy5-labeled peptides were prepared into a 100 μM working solution, and 100 μL was injected into the tail vein of each C57BL / 6 mouse; mice were anesthetized at different time points, and in vivo imaging was performed using the Maestro™ EX imaging system to track peptide enrichment in the brain and distribution throughout the body. See [link to documentation]. Figure 19 .

[0121] Serum stability evaluation: PVtCD-TP, PVtCD-cTP-Alk F -2 The peptide was incorporated into phosphate-buffered saline (PBS) containing 25% fetal bovine serum (FBS) and incubated at 37°C. Aliquots were collected at preset time points, and the reaction was immediately terminated by adding pre-cooled acetonitrile solution containing trichloroacetic acid to precipitate the protein. After centrifugation (12,000 rpm, 5 min), the supernatant was collected, and the remaining peptide amount was monitored by HPLC over incubation time.

[0122] Experimental results: Aβ42 oligomers not only significantly reduced cell viability but also induced upregulation of DAPK1 protein expression and decreased autophosphorylation levels; serum stability experiments showed that the macrocyclic structure conferred serum stability, effectively resisting protease degradation. (See [link to relevant documentation]). Figure 22 .

[0123] Example 6 Long-term treatment, behavioral and proteomic analysis of APP / PS1 transgenic mice Establishment of an acute AD model using Aβ42 oligomers: C57BL / 6 mice were anesthetized with sodium pentobarbital and injected with 3 μL of 1 mg / mL Aβ42 oligomer solution into the hippocampus via stereotaxic instrument. After one week of routine feeding, intervention was performed via tail vein administration. At the end of treatment, hippocampal tissue was harvested, and DAPK expression in the brain tissue was detected by Western blotting.

[0124] Long-term dosing regimen: 12-month-old APP / PS1 transgenic AD mice were selected, and the drug-treated group received intravenous injections of the peptide a total of 5 times. Nine days before the last dose, a full set of behavioral tests were performed. Mice were sacrificed at the experimental endpoint, and brain tissue was collected for protein quantification and pathological analysis. (See below) Figure 21 .

[0125] Behavioral assessment (1) Morris water maze: The pool is divided into four quadrants. Titanium dioxide is added to shield the underwater fixed platform. The water temperature is 21℃ and the water surface is 1.5 cm higher than the platform. The positioning and navigation training is carried out for 6 consecutive days. The escape latency within 60 seconds is recorded. The animal tracking system counts the time spent in each quadrant and evaluates the spatial learning and memory ability.

[0126] (2) Nesting behavior test: Mice were housed in individual cages with sterile paper sheets spread in the cages. The nesting status was recorded by video for 24 hours to evaluate the autonomous cognitive behavior.

[0127] Brain tissue DIA proteomics: Total protein was extracted from hippocampal tissue using the SDC lysis method and quantified by BCA; protein was reduced and alkylated, digested with trypsin, and peptides were desalted and quantified; data were acquired by Astral mass spectrometry in DIA mode after liquid chromatography separation; protein identification and quantification were completed, and GO and KEGG enrichment analysis was performed to analyze changes in brain signaling pathways after peptide intervention.

[0128] Experimental results: BmTAC treatment significantly shortened the escape latency of mice, prolonged the time spent in the target quadrant, and increased the number of platform crossings, suggesting that the spatial learning and memory abilities of mice were significantly improved, and that it could also enhance the nesting quality and autonomous behavior of mice.

[0129] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A chimera that can activate blood-brain barrier-penetrating macrocyclic targeting protein degradation, characterized in that, Its structural formula is ROS-VHL-Linker1-TP-Linker2-cTP-Alk F -2; Wherein, ROS is a reactive oxygen species responsive group, VHL is an E3 ubiquitin ligase ligand, TP is a targeting peptide, and cTP-Alk is a target peptide. F -2 is a blood-brain barrier-penetrating macrocyclic peptide; Linker1 is a linker for connecting VHL and TP; Linker2 is a linker for connecting TP and cTP-Alk. F -2 connector.

2. The blood-brain barrier-penetrating macrocyclic targeting protein degradation chimera as described in claim 1, characterized in that, The cTP-Alk F The structural formula for -2 is shown in Equation 1: Formula 1.

3. The blood-brain barrier-penetrating macrocyclic targeting protein degradation chimera as described in claim 1 or 2, characterized in that, The ROS is a phenylboronic acid group.

4. The blood-brain barrier-penetrating macrocyclic targeting protein degradation chimera as described in claim 1 or 2, characterized in that, The structural formula of the VHL is shown in Equation 2, Equation 3 or Equation 4: Formula 2 Formula 3 Formula 4.

5. The blood-brain barrier-penetrating macrocyclic targeting protein degradation chimera as described in claim 1 or 2, characterized in that, The TP is a DAPK1 targeting peptide, which is a recognition peptide derived from the GluN2B subunit, and its amino acid sequence contains KKNRKLRRQHSY.

6. The blood-brain barrier-penetrating macrocyclic targeting protein degradation chimera as described in claim 1 or 2, characterized in that, The structural formula of the TP is shown in Equation 5, Equation 6, Equation 7, Equation 8 or Equation 9: Formula 5 Formula 6 Formula 7 Formula 8 Formula 9.

7. The blood-brain barrier-penetrating macrocyclic targeting protein degradation chimera as described in claim 1 or 2, characterized in that, The Linker2 is selected from 6-aminohexanoic acid; the Linker1 is selected from any one of the following flexible alkyl chain linkers and rigid cyclic linkers: Flexible alkyl chain linkers: C3, C5, C7, C9; Rigid cyclic linkers: cis-cyclohexane, trans-cyclohexane.

8. The blood-brain barrier-penetrating macrocyclic targeting protein degradation chimera as described in claim 1, characterized in that, Its structural formula is shown in Equation 10: Formula 10.

9. The method for preparing the blood-brain barrier-penetrating macrocyclic targeting protein degradation chimera as described in any one of claims 1 to 8, characterized in that, Includes the following steps: (1) TP-Linker2-cTP-Alk was prepared using the standard solid-phase peptide synthesis method. F -2; (2) Modify ROS on VHL, and then connect it to TP-Linker2-cTP-Alk via Linker1. F -2 coupling.

10. The use of the blood-brain barrier-penetrating macrocyclic target protein degradation chimera as described in any one of claims 1 to 8 in the preparation of a drug for treating neurodegenerative diseases or in the preparation of a drug delivery carrier.

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