Bifunctional compound and application thereof in targeted degradation of protein

Abstract

The invention relates to a bifunctional compound and application thereof in targeted degradation of protein, the compound comprises a target protein binding compound and a cell-penetrating peptide which are connected, and the target protein binding compound is a compound capable of binding with cell membrane protein or extracellular protein or a derivative thereof; the target protein binding compound and the cell-penetrating peptide are connected through a linker or directly connected. According to the invention, it is found for the first time that specific extracellular proteins or cell membrane proteins can enter cells in an endocytosis manner and are degraded through lysosomes by using cell-penetrating peptides and small molecule conjugates. The method disclosed by the invention has the characteristic of delivering bioactive macromolecules through endosome wrapping without depending on a single receptor, and the effect of degrading the target protein by using the cells can be realized as long as specific binding micromolecules of the protein to be degraded can be found and coupled with the cell-penetrating peptide; a widely applicable strategy is provided for degradation of extracellular proteins and cell membrane proteins.

Description

Technical Field

[0001] The present invention belongs to the fields of chemical biology and medicinal chemistry, and particularly relates to a bifunctional compound and its application in protein targeted degradation. Background Art

[0002] Cell membrane and extracellular proteins are an important class of drug targets related to the pathology of various diseases. In recent years, the targeted degradation of such proteins has attracted extensive attention in the fields of chemical biology and drug discovery. Traditional drug discovery methods mainly rely on inhibiting protein function, while the recently developed targeted protein degradation (TPD) technology has the ability to directly regulate protein levels, thus potentially providing a more effective method for treating diseases related to abnormal protein expression and showing great potential in the field of drug discovery. The TPD technology currently mainly relies on the endogenous ubiquitin-proteasome degradation pathway and lysosome degradation pathway in cells. Traditional proteolysis-targeting chimeras (PROTACs) consist of an E3 ligase ligand, a target protein ligand, and a "Linker" structure that covalently links the two active ligands through special design, and can simultaneously recruit the target protein and the E3 ligase to form the active form of the PROTAC ternary complex, promote the ubiquitination of the target protein through the endogenous ubiquitin-proteasome system, and then be degraded by the proteasome. Therefore, its degradation effect is mainly limited to cytoplasmic proteins. However, approximately 40% of the extracellular and membrane-associated proteins produced by protein-coding genes, as well as intact organelles and protein polymers, are usually degraded via the lysosome pathway. Among them, secreted extracellular proteins include, but are not limited to, immune effector proteins, protein aggregates, and harmful signaling factors, while membrane proteins include integrins, immune checkpoint proteins, enzymes, and ion channels, etc. Recently, researchers have also developed some novel TPD technologies specifically targeting extracellular and membrane proteins, including LYTACs, MoDE-A, PROTABs, kineTACs, etc. These engineered bifunctional degraders link the extracellular domain of the target protein to a specific lysosome-targeting receptor (LTR), thereby promoting the internalization of the cell surface target protein (POI) and its transport to the lysosome for degradation. However, the expression of such LTRs on the membrane is not the same in different cell types and tissues, thus limiting the wide application of these TPD technologies. In addition, the existence of the "hook effect" in the three-component binding model greatly limits the therapeutic window of such bifunctional degraders, thereby restricting their potential as drugs. Therefore, there is an urgent need to develop TPD technologies that do not rely on internalization receptors to degrade membrane proteins.

[0003] As is well known, the targeted protein degradation technology has made a lot of progress as an effective method for regulating protein levels. The present invention provides a novel cell-penetrating peptide (CPP)-promoted strategy for target protein degradation, CPPTACs (CPP-mediated lysosome-targeting chimeras), which uses a bifunctional compound as a molecular degrader to degrade extracellular proteins and cell membrane proteins. The bifunctional compound CPPTACs referred to in the present invention binds a target protein-binding small molecule (SMs) to different cell-penetrating peptide (CPPs) sequences through a linker, or directly binds a target protein-binding small molecule (SMs) to different cell-penetrating peptide (CPPs) sequences. CPPTACs can drive the endocytosis of cell membranes and extracellular proteins through CPPs and enter the lysosome for degradation. This degradation does not depend on a single lysosomal shuttle receptor, is not restricted by cell types, and does not have the "hook effect" commonly present in the degradation of target proteins mediated by bifunctional compounds. Therefore, it has a wider scope of application and a wider dosing window. CPPTACs provide a widely applicable strategy for the degradation of cell membranes and extracellular proteins, and thus have very good application prospects in the fields of chemical biology and medicinal chemistry. Summary of the Invention

[0004] To achieve the above object, the present invention develops a widely applicable targeted degradation strategy for extracellular proteins and membrane proteins based on CPPs.

[0005] In the first aspect of the present invention, there is provided a bifunctional compound for degrading cell membrane proteins or extracellular proteins using cell lysosomes, the bifunctional compound comprising a linked target protein-binding compound and a cell-penetrating peptide,

[0006] The target protein-binding compound is a compound or its derivative capable of binding to a cell membrane protein or an extracellular protein;

[0007] The target protein-binding compound and the cell-penetrating peptide are connected by a linker, or the target protein-binding compound and the cell-penetrating peptide are directly connected.

[0008] Furthermore, the target protein-binding compound is a compound or its derivative capable of binding to the extracellular binding domain of a cell membrane protein;

[0009] Further, the extracellular protein or cell membrane protein is selected from APOE4, PD-L1, β-amyloid protein, programmed cell death ligand 1 (i.e., PD-L1), programmed death receptor 1 (i.e., PD-1), epidermal growth factor receptor (i.e., EGFR), human epidermal growth factor receptor-2 (i.e., HER2), G protein-coupled receptor (i.e., GPCR), fibroblast growth factor receptor (i.e., FGFRs), vascular endothelial growth factor receptor family (i.e., VEGFR, VEGF represents vascular endothelial growth factor), cytotoxic T lymphocyte-associated protein 4 (i.e., CTLA4 or CTLA-4), human interleukin 5 receptor α (IL-5Rα), apolipoprotein, apolipoprotein E4 (i.e., ApoE4), β-amyloid protein, angiotensin-converting enzyme 2 (ACE2), sodium-taurocholate cotransporting polypeptide (NTCP), B7.One or more of B7, TIM-1, TNFR2, NADPH oxidase, Bcl-2 / Bax and other ligands in the apoptosis pathway, C5a receptor, HMG-CoA reductase, PDE V phosphodiesterase type, PDE IV phosphodiesterase type 4, PDE I, PDE II, PDE III, squalene cyclase inhibitor, CXCR1, CXCR2, nitric oxide synthase, cyclooxygenase 1, cyclooxygenase 2, 5HT receptor, dopamine receptor, G protein, histamine receptor, 5-lipoxygenase, serine protease, thymidylate synthase, purine nucleoside phosphorylase, glyceraldehyde-3-phosphate dehydrogenase (i.e., GAPDH), glycogen phosphorylase, carbonic anhydrase, chemokine receptor, JAK STAT, RXR and analogs, HIV-1 protease, HIV-1 integrase, influenza neuraminidase, hepatitis B reverse transcriptase, sodium channel, protein P-glycoprotein, P-glycoprotein and MRP tyrosine kinase, CD23, CD73, CD124, tyrosine kinase p56lck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-αR, ICAM1, Ca2+ channel, VCAM, VLA-4 integrin, selectin, CD40 / CD40L, neokinins and receptors, inosine monophosphate dehydrogenase, p38 MAP kinase, Ras / Raf / MEK / ERK pathway, interleukin-1 converting enzyme, caspase, HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotide formyltransferase, rhinovirus, 3C protease, herpes simplex virus-1, protease, cytomegalovirus protease, poly(ADP-ribose) polymerase, cyclin-dependent kinase, vascular endothelial growth factor, oxytocin receptor, microsomal transfer protein inhibitor, bile acid transport inhibitor, 5α reductase inhibitor, angiotensin II, glycine receptor, norepinephrine reuptake receptor, endothelin receptor, neuropeptide Y and receptor, adenosine receptor, adenosine kinase and AMP dehydrogenase, purinergic receptor, farnesyl transferase, geranyl transferase, TrkA receptor of NCF, tyrosine kinase Flk-II / KDR, vitronectin receptor, integrin receptor, Her-2 / neu, telomerase inhibition, cytosolic phospholipase A2 and EGF receptor tyrosine kinase, ecdysone 20-monooxygenase, GABA-gated chloride channel, acetylcholinesterase, voltage-sensitive sodium channel protein, calcium release channel and chloride channel, acetyl-CoA carboxylase, adenylosuccinate synthase, protoporphyrinogen oxidase and enolpyruvylshikimate phosphate synthase, and / or one or more of all variants, mutants, splice variants, indels and fusions of the above proteins.

[0010] Further, the compound or its derivative capable of binding to a cell membrane protein or an extracellular protein is selected from any one of a ligand of a cell membrane protein or an extracellular protein, Biotin, BMS-8, HU-308, Sul-COOH, PH002 or its derivative.

[0011] Further, the linker is coupled to the target protein-binding compound and the cell-penetrating peptide through chemical bonds respectively, and the chemical bonds are selected from amide bonds, ester bonds, triazoles, alkynyl groups, and C-C, C═C, or heteroatom-containing C-N, C-O, C-S, C═S, and C-P bonds.

[0012] Further, the linker comprises a chemical structure formed by coupling a compound or its derivative capable of binding to a cell membrane protein or an extracellular protein with a cell-penetrating peptide through a click chemical reaction, and the groups undergoing the click chemical reaction include any pair of a thiol group SH and maleimide MA, an azide N3 and an alkynyl group, an azide N3 and DBCO or BCN, and a tetrazine and a trans-cyclooctene or their derivatives.

[0013] Further, the linker is coupled to the C-terminus, N-terminus of the cell-penetrating peptide or to the active group on the amino acid side chain of the cell-penetrating peptide through chemical bonds respectively.

[0014] Even further, the linker is selected from

[0015] wherein, L1 and L2 are independently selected from none, -(CH2)n-, -(COC-(CH2)n)m-, -((CH2)n-COC)m-, -((CH2)n-COC-(CH2)n)m-, -(C═C-(CH2)n)m-, -((CH2)n-C═C)m-, -((CH2)n-C═C-(CH2)n)m, -(CO-(CH2)n)m-, -((CH2)n-CO)m-, -((CH2)n-CO-(CH2)n)m-, -(CONH-(CH2)n)m-, -((CH2)n-CONH)m-, -((CH2)n-CONH-(CH2)n)m-, -(NHCO-(CH2)n)m-, -((CH2)n-NHCO)m-, -((CH2)n-NHCO-(CH2)n)m-, -(OCH2CH2)n- or a combination of one or more of them;

[0016] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;

[0017] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0018] Furthermore, the linker is selected from those in which the sum of all n values and m values in L1 and L2 is 4 - 20, preferably 5 - 15.

[0019] Furthermore, the linker is selected from

[0020] Furthermore, the cell-penetrating peptide is selected from cationic cell-penetrating peptides, amphiphilic cell-penetrating peptides, hydrophobic cell-penetrating peptides, synthetic cell-penetrating peptides, or fusion peptides of cell-penetrating sequences and lysosomal sorting sequences;

[0021] Furthermore, the cell-penetrating peptide is selected from any one of the sequences shown in Table 1 or has any one of the sequences shown in Table 1 and has modifications and / or protecting groups.

[0022] Furthermore, the C-terminal α-carboxyl group of the cell-penetrating peptide is protected by an amide bond.

[0023] Furthermore, the bifunctional compound is selected from:

[0024]

[0025] Where A is selected from cell-penetrating peptides or cell-penetrating peptides whose C-terminal α-carboxyl group is protected by an amide bond. Furthermore, the bifunctional compound is selected from:

[0026]

[0027]

[0028]

[0029] The second aspect of the present invention provides a method for degrading cell membrane proteins or extracellular proteins, the method comprising the step of contacting the above-mentioned bifunctional compound with cells containing cell membrane proteins, or the method comprising the step of co-incubating the bifunctional compound with cells and extracellular proteins;

[0030] The target protein-binding compound in the bifunctional compound can specifically bind to the cell membrane protein to be degraded or the extracellular protein to be degraded.

[0031] Furthermore, the method for degrading cell membrane proteins or extracellular proteins is implemented in vitro or in vivo.

[0032] Furthermore, cell membrane proteins or extracellular proteins are degraded through the intracellular lysosome pathway.

[0033] Furthermore, cell membrane proteins or extracellular proteins enter cells through endocytosis.

[0034] Furthermore, the cells are normal cells or tumor cells.

[0035] The third aspect of the present invention provides a method for designing or preparing a compound that degrades cell membrane proteins or extracellular proteins using cell lysosomes. The design or preparation method includes the following steps:

[0036] S1) Determine the target of the cell membrane protein or extracellular protein to be degraded;

[0037] S2) Obtain a small molecule compound that can specifically bind to the target of the cell membrane protein or extracellular protein to be degraded;

[0038] S3) Connect the small molecule compound obtained in S2) with a cell-penetrating peptide to obtain a compound that can degrade cell membrane proteins or extracellular proteins using cell lysosomes;

[0039] The connection is to directly connect the small molecule compound and the cell-penetrating peptide through a chemical bond; or

[0040] Connect through a chemical bond after modifying the small molecule compound and / or the cell-penetrating peptide; or

[0041] Connect the small molecule compound and the cell-penetrating peptide through a linker by a chemical bond.

[0042] Furthermore, the chemical bond is selected from amide bond, ester bond, triazole, alkyne group, and C-C, C═C or heteroatom-containing C-N, C-O, C-S, C═S bonds.

[0043] Furthermore, the linker contains a chemical structure formed by coupling a compound or its derivative that binds to a cell membrane protein or extracellular protein with a cell-penetrating peptide through a click chemical reaction. The groups that undergo the click chemical reaction include any pair of thiol SH and maleimide MA, azide N3 and alkyne group, azide N3 and DBCO or BCN, and tetrazine and trans-cyclooctene or their derivatives.

[0044] Even further, the linker is selected from

[0045] Among them, L1 and L2 are independently selected from one or more combinations of none, -(CH2)n-, -(COC-(CH2)n)m-, -((CH2)n-COC)m-, -((CH2)n-COC-(CH2)n)m-, -(C=C-(CH2)n)m-, -((CH2)n-C=C)m-, -((CH2)n-C=C-(CH2)n)m, -(CO-(CH2)n)m-, -((CH2)n-CO)m-, -((CH2)n-CO-(CH2)n)m-, -(CONH-(CH2)n)m-, -((CH2)n-CONH)m-, -((CH2)n-CONH-(CH2)n)m-, -(NHCO-(CH2)n)m-, -((CH2)n-NHCO)m-, -((CH2)n-NHCO-(CH2)n)m-, -(OCH2CH2)n-;

[0046] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;

[0047] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0048] Furthermore, the linker is selected from those where the sum of all n values and m values in L1 and L2 is 4 - 20, preferably 5 - 15.

[0049] Furthermore, the linker is selected from

[0050] Furthermore, the cell - penetrating peptide is selected from cationic cell - penetrating peptides, amphiphilic cell - penetrating peptides, hydrophobic cell - penetrating peptides, synthetic cell - penetrating peptides or fusion peptides of cell - penetrating sequences and lysosomal sorting sequences;

[0051] Furthermore, the cell - penetrating peptide is selected from any one of the sequences shown in Table 1 or any one of the sequences shown in Table 1 with modifications and / or protecting groups.

[0052] Furthermore, the C - terminal α - carboxyl group of the cell - penetrating peptide is protected by an amide bond.

[0053] In the fourth aspect of the present invention, there is provided the use of the above - mentioned bifunctional compound in the preparation of a reagent for degrading cell membrane proteins or extracellular proteins, wherein the target protein - binding compound in the bifunctional compound can specifically bind to the cell membrane protein to be degraded or the degraded extracellular protein.

[0054] Furthermore, the reagent is for non-diagnostic and non-therapeutic purposes.

[0055] Furthermore, the reagent is for in vitro tests.

[0056] The fifth aspect of the present invention provides the use of the above-mentioned bifunctional compound in the preparation of drugs for anti-tumor, lipid-lowering, and treating neurological diseases.

[0057] The extracellular protein or cell membrane protein in the described bifunctional compound is selected from APOE4, PD-L1, β-amyloid protein, programmed death-ligand 1 (i.e., PD-L1), programmed death receptor 1 (i.e., PD-1), epidermal growth factor receptor (i.e., EGFR), human epidermal growth factor receptor-2 (i.e., HER2), G protein-coupled receptor (i.e., GPCR), fibroblast growth factor receptor (i.e., FGFRs), vascular endothelial growth factor receptor family (i.e., VEGFR, VEGF represents vascular endothelial growth factor), cytotoxic T-lymphocyte-associated protein 4 (i.e., CTLA4 or CTLA-4), human interleukin 5 receptor α (IL-5Rα), apolipoprotein, apolipoprotein E4 (i.e., ApoE4), β-amyloid protein, angiotensin-converting enzyme 2 (ACE2), sodium-taurocholate cotransporting polypeptide (NTCP), B7.one and B7, TI FR1m, TNFR2, NADPH oxidase, Bc1I Bax and other ligands in the apoptosis pathway, C5a receptor, HMG-CoA reductase, PDEⅤ phosphodiesterase type, PDEⅣ phosphodiesterase type 4, PDEⅠ, PDEⅡ, PDEⅢ, squalene cyclase inhibitor, CXCR1, CXCR2, nitric oxide synthase, cyclooxygenase 1, cyclooxygenase 2, 5HT receptor, dopamine receptor, G protein, histamine receptor, 5-lipoxygenase, serine protease-like protease, thymidylate synthase, purine nucleoside phosphorylase, glyceraldehyde-3-phosphate dehydrogenase (i.e., GAPDH), glycogen phosphorylase, carbonic anhydrase, chemokine receptor, JAW STAT, RXR and analogs, HIV1 protease, HIV1 integrase, influenza neuraminidase, hepatitis B reverse transcriptase, sodium channel, protein P-glycoprotein, P-glycoprotein and MRP tyrosine kinase, CD23, CD73, CD124, tyrosine kinase p561ck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-αR, ICAM1, Ca2+ channel, VCAM, VLA-4 integrin, selectin, CD40 / CD40L, newokinins and receptors, inosine monophosphate dehydrogenase, p38 MAP kinase, Ras / Raf / MEW / ERK pathway, interleukin-1 converting enzyme, caspase, HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotide formyltransferase, rhinovirus, 3C protease, herpes simplex virus-1, protease, cytomegalovirus protease, poly(ADP-ribose) polymerase, cyclin-dependent kinase, vascular endothelial growth factor, oxytocin receptor, microsomal transfer protein inhibitor, bile acid transport inhibitor, 5α reductase inhibitor, angiotensin 11, glycine receptor, norepinephrine reuptake receptor, endothelin receptor, neuropeptide Y and receptor, adenosine receptor, adenosine kinase and AMP dehydrogenase, purinergic receptor, farnesyl transferase, geranyl transferase, TrkA receptor of NCF, tyrosine kinase Flk-IIKDR, vitronectin receptor, integrin receptor, Her-21 sheath, telomerase inhibition, cytosolic phospholipase A2 and EGF receptor tyrosine kinase, ecdysone 20-monooxygenase, GABA-gated chloride channel, acetylcholinesterase, voltage-sensitive sodium channel protein, calcium release channel and chloride channel, acetyl-CoA carboxylase, adenylosuccinate synthetase, protoporphyrinogen oxidase and enolpyruvylshikimate phosphate synthase, one or more of the above, and / or one or more of all variants, mutants, splice variants, indels and fusions of the above proteins.

[0058] The sixth aspect of the present invention provides a preparation method of the above bifunctional compound, and the preparation method includes the following steps:

[0059] S01) Obtain a target protein-binding compound and a cell-penetrating peptide;

[0060] S02) Directly couple or couple through a linker the target protein-binding compound and the cell-penetrating peptide.

[0061] Furthermore, the step S01) also includes a step of modifying the obtained target protein-binding compound and cell-penetrating peptide.

[0062] Furthermore, the modification is such that the target protein-binding compound and the cell-penetrating peptide contain reactive functional groups capable of forming a coupling chemical bond.

[0063] Furthermore, the reactive functional groups capable of forming a coupling chemical bond are selected from any pair or its derivatives in the combination of carboxyl group and amino group, the combination of carboxyl group and hydroxyl group, the combination of azide group and alkynyl group, the combination of mercapto group SH and maleimide MA, the combination of azide N3 and alkynyl group, the combination of azide N3 and DBCO or BCN, and the combination of tetrazine and trans-cyclooctene.

[0064] The present invention connects a target protein-binding small molecule (SMs) and different cell-penetrating peptides (CPPs) through different linkers, and successfully designs a new type of CPP-mediated lysosome-targeting chimeras (CPPTACs) for specifically degrading extracellular and cell membrane proteins. After binding to the target protein, CPPTACs trigger endocytosis, resulting in the target protein being trapped in endosomal vesicles and then being delivered to lysosomes for degradation. Using this strategy, the present invention constructs bifunctional molecules targeting extracellular protein APOE4 and cell membrane proteins PD-L1, CAIX, and CB2R. As degrading agents, these molecules can effectively promote the degradation of target proteins, and the degradation efficiency of cell membrane proteins can reach 60-80%.

[0065] Beneficial effects

[0066] The present invention provides a novel technology of lysosome-targeting chimeras (CPPTACs) based on cell-penetrating peptides (CPPs). This technology provides conjugates of a target protein-binding small molecule (SMs) and different CPPs, and it is first discovered that specific extracellular proteins or cell membrane proteins can enter cells by endocytosis and be degraded by lysosomes through the use of cell-penetrating peptides CPPs. The method of the present invention has the characteristic of delivering bioactive macromolecules through endosome encapsulation in a manner independent of a single receptor. As long as a specific binding small molecule for the protein to be degraded can be found and conjugated with the cell-penetrating peptide, the effect of degrading the target protein by cells can be achieved, which provides a new and widely applicable strategy for the degradation of extracellular proteins and cell membrane proteins.

[0067] Since CPPs-mediated endocytosis and lysosomal degradation do not pass through the three-component binding model required by other heterobifunctional degraders and there is no "hook effect", CPPTACs have a simpler structural design and a wider therapeutic window.

[0068] The present invention demonstrates the ability of the bifunctional compounds involved in the present invention to regulate the levels of PD-L1, CAIX, and CB2R proteins in vitro and in vivo, which represents a simple, adaptable, and effective method for selectively degrading membrane proteins in various cellular environments and has great application potential in the fields of biological research and disease treatment. Brief Description of the Drawings

[0069] Figure 1 : Schematic diagram of the mode of action of CPPTACs-mediated degradation of cell membranes and extracellular proteins;

[0070] Figure 2 : HRMS spectrum of the compound Biotin-L1-Azide;

[0071] Figure 3 : HRMS spectrum of the compound Biotin-L1-CPP1;

[0072] Figure 4 : HPLC analysis of the compound Biotin-L1-CPP1;

[0073] Figure 5 : HRMS spectrum of the compound Biotin-L1-CPP2;

[0074] Figure 6 : HPLC analysis of the compound Biotin-L1-CPP2;

[0075] Figure 7 : HRMS spectrum of the compound Biotin-L1-CPP3;

[0076] Figure 8: HPLC analysis of compound Biotin-L1-CPP3;

[0077] Figure 9 : HRMS spectrum of compound Biotin-L1-CPP4;

[0078] Figure 10 : HPLC analysis of compound Biotin-L1-CPP4;

[0079] Figure 11 : HRMS spectrum of compound BMS-L1-Azide;

[0080] Figure 12 : HRMS spectrum of compound BMS-L2-Azide;

[0081] Figure 13 : HRMS spectrum of compound BMS-L3-Azide;

[0082] Figure 14 : HRMS spectrum of compound BMS-L1-CPP1;

[0083] Figure 15 : HPLC analysis of compound BMS-L1-CPP1;

[0084] Figure 16 : HRMS spectrum of compound BMS-L2-CPP1;

[0085] Figure 17 : HPLC analysis of compound BMS-L2-CPP1;

[0086] Figure 18 : HRMS spectrum of compound BMS-L3-CPP1;

[0087] Figure 19 : HPLC analysis of compound BMS-L3-CPP1;

[0088] Figure 20 : HRMS spectrum of compound BMS-L1-CPP2;

[0089] Figure 21 : HPLC analysis of compound BMS-L1-CPP2;

[0090] Figure 22 : HRMS spectrum of compound Sul-L4-Azide;

[0091] Figure 23 : HRMS spectrum of compound Sul-L4-CPP1;

[0092] Figure 24 HPLC analysis of compound Sul-L4-CPP1;

[0093] Figure 25 : HRMS spectrum of compound Sul-L4-CPP2;

[0094] Figure 26 : HPLC analysis of compound Sul-L4-CPP2;

[0095] Figure 27 : HRMS spectrum of compound HU-308-derived primary amine;

[0096] Figure 28 : HRMS spectrum of compound HU-CPP1;

[0097] Figure 29 : HPLC analysis of compound HU-CPP1;

[0098] Figure 30 : CPPTACs promote the degradation of extracellular proteins through the lysosomal pathway; (a) Fluorescence analysis of the cellular uptake and lysosomal degradation of different Biotin-CPP-mediated NAP-650 (red) in the presence or absence of the lysosomal proteolytic activity inhibitor chloroquine (CQ). (b, c) Fluorescence analysis of the inhibitory effect of nystatin (50 μM), CPZ (10 μg / mL), or EIPA (10 μg / mL) on the cellular uptake of Biotin-CPP1 (b) or Biotin-CPP2 (c)-mediated NAP-650 (red). (d-f) A549 cells were co-incubated with NAP-FITC (0.4 μM) and the indicated concentration of Biotin-CPP1, and fluorescence (d) and flow cytometry analysis (e) of the cellular uptake of NAP-FITC (green) were performed. (f) Quantification of the mean fluorescence intensity of NAP-FITC relative to the untreated group (n = 3 biological replicates, means ± SD). (g) Fluorescence signals showing the co-localization of NAP-FITC (green) with the early endosome marker (Rab5, red) in HeLa cells mediated by Biotin-CPP1 or Biotin-CPP2. (h) Fluorescence signals in A549 cells showing the co-localization of NAP-650 with the lysosomal marker (LysoTracker, green) mediated by Biotin-CPP1 or Biotin-CPP2 (red). Nuclei in the figures were labeled with DAPI (blue), scale bar, 10 μm.

[0099] Figure 31: CPPTACs promote the degradation of membrane-associated protein PD-L1 through the lysosomal pathway; (a-d) Western blot analysis of PD-L1 levels in MDA-MB-231 cells treated with BMS-CPP1 (a, c) or BMS-CPP2 (b, d) at the indicated concentrations or for the indicated times. (e, f) Western blot analysis of HA-PD-L1 levels in HeLa cells stably expressing HA-PD-L1 after treatment with BMS-CPP1 or BMS-CPP2 at the indicated concentrations for 8 hours. (g, h) Immunofluorescence analysis of the degradation of PD-L1 (red) on the cell membrane (g) or whole cell (h). HeLa cells stably expressing PD-L1 were treated with BMS-CPP1, BMS-8, CPP1, or a combination of BMS-8 and CPP1. The nucleus was labeled with DAPI (blue). Scale bar, 10 μm. (i) Western blot analysis of PD-L1 levels in different cell lines after treatment with 25 nM BMS-CPP1, BMS-8, or CPP1 for 8 hours. (j, k) Western blot analysis of PD-L1 levels in MDA-MB-231 cells after treatment with 25 nM BMS-CPP1 (j) or 5 nM BMS-CPP2 (k) in combination with bafilomycin A1 (100 μM) or MG132 (5 μM) for 8 hours. (l, m) Western blot analysis of the inhibitory effects of nystatin (50 μM), CPZ (10 μg / mL), or EIPA (10 μg / mL) on BMS-CPP1 (l) or BMS-CPP2 (m)-mediated PD-L1 degradation.

[0100] Figure 32 : BMS-CPP1 degrades PD-L1 in vivo; (a) Structures of BMS-L1-CPP1, BMS-L2-CPP1, and BMS-L3-CPP1. (b-c) Western blot analysis of PD-L1 levels in MDA-MB-231 cells treated with BMS-L2-CPP1 or BMS-L3-CPP1 at the indicated concentrations. (d) Schematic diagram of in vivo experiments in a B16 F10 tumor xenograft C57BL / 6J mouse model. (e, f) Western blot analysis of PD-L1 in B16 F10 xenografts after peritumoral administration of 1% DMSO / PBS, BMS-8 (2 mg / kg), or BMS-CPP1 (5 mg / kg) once daily for 2 consecutive days (n = 3 animals per group). (g) Representative immunohistochemical staining of PD-L1 in tumor tissues. Scale bar, 50 μm.

[0101] Figure 33: CPPTACs promote the degradation of membrane-associated proteins CAIX and CB2R through the lysosomal pathway. (a) Structures of Sul-CPP1 and Sul-CPP2. (b, c) Western blot analysis of CAIX levels after treating MDA-MB-231 cells with Sul-CPP1 (b) or Sul-CPP2 (c) at the indicated concentrations or for the indicated times. (d) Western blot analysis of CAIX levels after treating MDA-MB-231 cells with Sul-acid, CPP1, or Sul-CPP1 at 25 nM for 8 h. (e) Western blot analysis of CAIX levels after treating MDA-MB-231 cells with Sul-CPP1 (25 nM) in combination with bafilomycin A1 (100 μM) or MG132 (5 μM) for 8 h. (f, g) Western blot analysis of the inhibitory effects of nystatin (50 μM) or CPZ (10 μg / mL) on Sul-CPP1 (f) or Sul-CPP2 (g)-mediated CAIX degradation. (h, i) Flow cytometry analysis of Sul-CPP1-mediated CAIX degradation in MDA-MB-231 cells under CoCl2-induced hypoxic conditions. Quantitative determination of the mean fluorescence intensity of CAIX-FITC relative to the isotype group (n = 3 biological replicates, means ± SD). (j, k) Effects of Sul-CPP1 treatment on the survival of MDA-MB-231 cells under CoCl2-induced hypoxic conditions at the indicated concentrations (j) or for the indicated times (k) analyzed by CCK8. (l) Structure of Hu-CPP1. (m, n) Western blot analysis of CB2R levels after treating MDA-MB-231 (m) or HT-29 cells (n) with HU-CPP1 at the indicated concentrations for 8 h.

[0102] Figure 34 : Antitumor activity results of BMS-L1-CPP1. Among them, (a) Schematic diagram and general treatment procedure of tumor inhibition study. (b) Curve of mouse body weight change from day 6 to day 18. (c) Tumor growth curves of each group. (d) Photos of tumor tissues after being dissected on day 18. (e) Comparison of tumor weights after tumor resection. (f) Tumor metastasis in the spleen. (g-h) Flow cytometry analysis of lymphocytes for the production of T lymphocyte markers CD3, CD4, CD8 (g) and cytokine IFN-γ (h).

[0103] Figure 35 : Results of the uptake experiment of extracellular protein PD-L1-FITC. Among them, (a) BMS-CPP1 mediates the degradation of extracellular protein PD-L1; (b) PH002-CPP1 mediates the degradation of extracellular protein APOE4. Detailed implementation methods

[0104] In order to make the above objects, features and advantages of the present invention more obvious and understandable, the following provides a detailed description of the specific embodiments of the present invention, but it should not be construed as a limitation on the scope of the present invention that can be implemented.

[0105]

[0106]

[0107]

[0108] In the specific embodiments of the present invention, four different CPPs were synthesized respectively. Among them, CPP1, CPP2 and CPP3 are composed of PEN, TAT and R9 sequences respectively, while CPP4 is a fusion peptide containing both a cell-penetrating sequence and a lysosomal sorting sequence. The sequences or structures of the CPPs are as follows:

[0109] CPP1 (PEN): Alkyne-RQIKIWFQNRRMKWKK-NH2

[0110] CPP2 (TAT): Akyne-YGRKKRRQRRR-NH2

[0111] CPP3 (R9): AIkyne-RRRRRRRRR-NH2

[0112] CPP4 (CPP-LSS): Alkyne-GGrrrrrrrrrNPGY-NH2

[0113] Example 1 Synthesis of Biotin-L1-CPP(1 - 4)

[0114] Step 1. Synthesis of compound Biotin-L1-Azide

[0115]

[0116] First, 3-azidopropylamine (12.9 μL, 132 μmol) was added to a reaction flask and dissolved in 1.0 mL of anhydrous DMF. Subsequently, triethylamine (24.5 μL, 176 μmol) was added in sequence, and after stirring evenly, a DMF solution (1.0 mL) of Biotin-NHS (30.0 mg, 88 μmol) was added dropwise. After the reaction mixture was stirred at room temperature for 6 hours, the reaction progress was monitored by molecular HPLC. The retention time of the target substance Biotin-L1-Azide was t R= 8.7 min. The reaction mixture was directly fractionated and purified by preparative HPLC, and a white powder Biotin-L1-Azide (26.1 mg, 91%) was obtained after lyophilization. HRMS (ESI) m / z: calcd. for C 13 H 23 O2N6S[M+H] + 327.1598, found 327.1597; calcd. for C 13 H 22 O2N6NaS[M+Na] + 349.1417, found 349.1416.( Figure 2 )

[0117] Step 2. Synthesis of compound Biotin-CPP(1-4)

[0118]

[0119] Biotin-L1-CPP1. CPP1-Alkyne (5.0 mg, 2.14 μmol), Biotin-L1-Azide (2.1 mg, 6.43 μmol), CuSO4·5H2O (0.534 mg, 2.14 μmol) and NaVc (2.54 mg, 12.8 μmol) were successively added into a reaction flask and dissolved with a mixed solution of DMF / H2O (500 μL). The reaction mixture was stirred at room temperature for 6 hours, and the reaction progress was monitored by analytical HPLC. The retention time of the target substance Biotin-L1-CPP1 was t R = 9.05 min( Figure 4 ). The reaction mixture was directly fractionated and purified by preparative HPLC, and a white powder Biotin-L1-CPP1 (3.71 mg, 65%) was obtained after lyophilization. HRMS (ESI) m / z: calcd. for C 123 H 200 N 41 O 22 S2[M+3H] 3+ 889.1744, found 889.1730.( Figure 3 )

[0120]

[0121] Biotin-L1-CPP2. CPP2-Alkyne (5.0 mg, 3.02 μmol), Biotin-L1-Azide (2.96 mg, 9.1 μmol), CuSO4·5H2O (0.755 mg, 3.02 μmol) and NaVc (3.6 mg, 18.2 μmol) were successively added to a reaction flask and dissolved with a DMF / H2O (600 μL) mixed solution. After stirring the reaction mixture at room temperature for 6 hours, the reaction progress was monitored by analytical HPLC. The retention time of the target substance Biotin-L1-CPP2 was t R = 6.49 min( Figure 6 ). The reaction mixture was directly fractionated and purified by preparative HPLC, and after freeze-drying, white powder Biotin-L1-CPP2 (3.7 mg, 62%) was obtained. HRMS (ESI) m / z: calcd. for C 83 H 151 N 39 O 16 S[M+4H] 4+ 495.5480, found 495.5476.( Figure 5 )

[0122]

[0123] Biotin-L2-CPP3. CPP3-Alkyne (5.0 mg, 3.3 μmol), Biotin-L1-Azide (3.23 mg, 9.9 μmol), CuSO4·5H2O (0.823 mg, 3.3 μmol) and NaVc (3.92 mg, 19.8 μmol) were successively added to a reaction flask and dissolved with a DMF / H2O / ACN (800 μμL) mixed solution. After stirring the reaction mixture at room temperature for 6 hours, the reaction progress was monitored by analytical HPLC. The retention time of the target substance Biotin-L1-CPP3 was t R = 6.19 min( Figure 8 ). The reaction mixture was directly fractionated and purified by preparative HPLC, and after freeze-drying, white powder Biotin-L1-CPP3 (4.0 mg, 66%) was obtained. HRMS (ESI) m / z: calcd. for C 73 H 142 N 43 O 12 S[M+3H] 3+ 615.0514, found 615.0503.( Figure 7 )

[0124]

[0125] Biotin-L1-CPP4. CPP4-Alkyne (5.0 mg, 2.424 μmol), Biotin-L1-Azide (1.58 mg, 4.849 μmol), a catalytic amount of CuI, and DIEA (20.0 μL, 121.2 μmol) were successively added to a reaction flask and dissolved in 500 μμL of DMF. After the reaction mixture was stirred at room temperature for 6 hours, the reaction progress was monitored by analytical HPLC. The retention time of the target substance Biotin-L1-CPP4 was t R = 6.40 min( Figure 10 ). The reaction mixture was directly fractionated and purified by preparative HPLC, and white powder Biotin-L1-CPP4 (4.8 mg, 83%) was obtained after lyophilization. HRMS (ESI) m / z: calcd. for C 97 [[ID=⑦]]H 174 [[ID=⑨]]N 50 O 20 S[M + 4H] 4+ 597.8464, found 597.8459.( Figure 9 )

[0126] Example 2 Synthesis of BMS-L(1 - 3)-CPP1 and BMS-L1-CPP2

[0127] Step 1. Synthesis of compound BMS-L(1 - 3)-Azide

[0128]

[0129] BMS-L1-Azide. BMS-8 small molecule (10.0 mg, 20.2 μmol), HATU (11.5 mg, 30.3 μmol), and triethylamine (11.3 μL, 60.6 μmol) were successively added to a reaction flask. After stirring and dissolving in 1.0 mL of anhydrous DMF, 3-azidopropylamine (3.0 μL, 30.3 μmol) was added. After the reaction mixture was stirred at room temperature for 3 hours, a sample was taken and the reaction progress was monitored by analytical HPLC. The retention time of the target substance BMS-L1-Azide was t R = 17.2 min. The reaction mixture was directly fractionated and purified by preparative HPLC, and white powder BMS-L1-Azide (11.0 mg, 95%) was obtained after lyophilization. HRMS (ESI) m / z: calcd. for C 30 H 35 BrN5O2[M + H] + 576.1969, found 576.1973.( Figure 10 )

[0130]

[0131] BMS-L2-Azide. The BMS-8 small molecule (20 mg, 40.45 μmol), HATU (18.5 mg, 48.65 μmol), and DIEA (36 μL, 202.25 μmol) were successively added to the reaction flask. After stirring and dissolving with 2.0 mL of anhydrous DMF, Azide-PEG3-amine (10.6 mg, 48.54 μmol) was added. After the reaction mixture was stirred at room temperature for 2 hours, a sample was taken and the reaction progress was monitored by analytical HPLC. The retention time of the target substance BMS-L2-Azide was t R = 17.18 min. The reaction mixture was directly fractionated and purified by preparative HPLC, and white powder BMS-L2-Azide (21.1 mg, 75%) was obtained after lyophilization. HRMS (ESI) m / z: calcd. for C 35 H 45 BrN5O5[M+H] + 694.2599, found 694.2606.( Figure 12 )

[0132]

[0133] BMS-L3-Azide. The BMS-8 small molecule (6.0 mg, 12.1 μmol), HATU (6.9 mg, 18.1 μmol), and DIEA (6.0 μL, 36.3 μmol) were successively added to the reaction flask. After stirring and dissolving with 1.0 mL of anhydrous DMF, NH2-PEG3-NH2 (9.3 mg, 48.4 μmol) was added. The reaction mixture was stirred overnight at room temperature. A sample was taken and the reaction progress was monitored by analytical HPLC. The retention time of the target substance BMS-L3-NH2 was t R = 13.5 min. The reaction mixture was directly fractionated and purified by preparative HPLC, and white powder BMS-L3-NH2 (5.3 mg, 64%) was obtained after lyophilization. In the reaction flask, BMS-L3-NH2 (5.3 mg, 7.7 μmol), N3-C5-NHS (2.9 mg, 11.4 μmol), and triethylamine (10.7 μL, 77.0 μmol) were dissolved in 2 mL of anhydrous DMF and stirred at room temperature. A sample was taken and the reaction progress was monitored by analytical HPLC. The retention time of the target substance BMS-L3-Azide was t R= 16.9 min. The reaction mixture was directly fractionated and purified by preparative HPLC, and after freeze-drying, a white powder BMS-L3-Azide (5.4 mg, 87%) was obtained. HRMS (ESI) m / z: calcd. for C 41 H 56 O6N6Br[M+H] + 807.3439, found 807.3441.( Figure 13 )

[0134] Step 2. Synthesis of compound BMS-L(1-3)-CPP1

[0135]

[0136] BMS-L1-CPP1. CPP1-Alkyne (5.0 mg, 2.14 μmol), BMS-L1-Azide (1.85 mg, 3.21 μmol), CuSO4·5H2O (0.32 mg, 1.28 μmol) and NaVc (1.7 mg, 8.58 μmol) were successively added to a reaction flask and dissolved with a DMF / H2O (600 μL) mixed solution. After the reaction mixture was stirred at room temperature for 6 hours, the reaction progress was monitored by analytical HPLC. The retention time of the target substance BMS-CPP1 was t R = 11.98 min( Figure 15 ). The reaction mixture was directly fractionated and purified by preparative HPLC, and after freeze-drying, a white powder BMS-L1-CPP1 (5.1 mg, 82%) was obtained. HRMS (ESI) m / z: calcd. for C 140 H 212 BrN 40 O 22 S[M+4H] 4+ 729.3901, found 729.3905.( Figure 14 )

[0137]

[0138] BMS-L2-CPP1. CPP1-Alkyne (5.0 mg, 2.14 μmol), BMS-L2-Azide (2.96 mg, 4.28 μmol), CuSO4·5H2O (0.32 mg, 1.28 μmol) and NaVc (1.7 mg, 8.58 μmol) were successively added to a reaction flask and dissolved with a DMF / H2O (500 μL) mixed solution. After the reaction mixture was stirred at room temperature for 6 hours, the reaction progress was monitored by analytical HPLC. The retention time of the target substance BMS-L2-CPP1 was t R= 11.34 min( Figure 17 ). The reaction mixture was directly fractionated and purified by preparative HPLC, and after lyophilization, a white powder BMS-L2-CPP1 (4.3 mg, 73%) was obtained. HRMS (ESI) m / z: calcd. for C 145 H 222 BrN 40 O 25 S[M + 3H] 3+ 1011.5411, found 1011.5408.( Figure 16 )

[0139]

[0140] BMS-L3-CPP1. CPP1-Alkyne (5.0 mg, 2.14 μmol), BMS-L3-Azide (3.45 mg, 4.28 μmol), CuSO4·5H2O (0.32 mg, 1.28 μmol) and NaVc (1.7 mg, 8.58 μmol) were successively added to a reaction flask and dissolved with a mixed solution of DMF / H2O (2.5:1, 500 μL). After stirring the reaction mixture at room temperature for 6 hours, the reaction progress was monitored by analytical HPLC. The retention time of the target substance BMS-L3-CPP1 was t R = 11.51 min( Figure 19 ). The reaction mixture was directly fractionated and purified by preparative HPLC, and after lyophilization, a white powder BMS-L3-CPP1 (4.3 mg, 73%) was obtained. HRMS (ESI) m / z: calcd. for C 151 H 235 BrN 41 O 26 S[M + 5H] 5+ 629.9446, found 629.9457.( Figure 18 )

[0141]

[0142] BMS-L1-CPP2. CPP2-Alkyne (5.0 mg, 3.02 μmol), BMS-L1-Azide (2.62 mg, 4.53 μmol), CuSO4·5H2O (0.68 mg, 2.72 μmol) and NaVc (2.4 mg, 12.08 μmol) were successively added to a reaction flask and dissolved with a mixed solution of DMF / H2O (600 μL). After stirring the reaction mixture at room temperature for 6 hours, the reaction progress was monitored by analytical HPLC. The retention time of the target substance BMS-CPP2 was t R= 11.49 min( Figure 21 ). The reaction mixture was directly fractionated and purified by preparative HPLC, and after freeze-drying, a white powder BMS-L1-CPP2 (5.0 mg, 74%) was obtained. HRMS (ESI) m / z: calcd. for C 100 H 162 BrN 38 O 16 [M + 3H] 3+ 743.4071, found 743.4100.( Figure 20 )

[0143] Example 3 Synthesis of Sul-L4-CPP1 and Sul-L4-CPP2

[0144] Step 1. Synthesis of compound Sul-L4-Azide

[0145]

[0146] The small molecule Sul-COOH (10.0 mg, 35.68 μmol), HATU (16.3 mg, 42.90 μmol), and DIEA (18.7 μL, 107 μmol) were successively added to a reaction flask. After stirring and dissolving in 1.0 mL of anhydrous DMF, NH2-C6-NH-Boc (9.3 mg, 48.4 μmol) was added. The reaction mixture was reacted at room temperature for 3 hours. The reaction progress was monitored by analytical HPLC. The retention time of the target substance Sul-C6-NH-Boc was t R = 12.6 min. The reaction mixture was directly fractionated and purified by preparative HPLC, and after freeze-drying, a white powder Sul-C6-NH-Boc (15.4 mg) was obtained. The white powder was dissolved in 20% TFA / DCM under an ice bath, then the reaction solution was brought to room temperature and stirred for 1 hour, and the solvent was evaporated under reduced pressure to obtain the crude amine after removal of the Boc protecting group, without further purification. The crude deprotected amine, N3-C5-COOH (7.6 mg, 48.4 μmol), DIEA (53.2 μL, 357 μmol), and HATU (16.28 mg, 42.82 μmol) were dissolved in 2 mL of anhydrous DMF. The reaction solution was stirred at room temperature for 3 hours. The reaction progress was monitored by analytical HPLC. The retention time of the target substance Sul-L4-Azide was t R = 11.6 min. The reaction mixture was directly fractionated and purified by preparative HPLC, and after freeze-drying, a white powder Sul-L4-Azide (13.3 mg, 80%) was obtained. HRMS (ESI) m / z: calcd. for C 18 H 32N9O5S2[M+H] + 518.1962, found 518.1965.( Figure 22 )

[0147] Step 2. Synthesis of Compound Sul-L4-CPP1 and Compound Sul-L4-CPP2

[0148]

[0149] Sul-L4-CPP1. Add CPP1-Alkyne (5.0 mg, 2.14 μmol), Sul-L4-Azide (0.72 mg, 2.57 μmol), CuSO4·5H2O (0.267 mg, 1.07 μmol) and NaVc (1.412 mg, 7.13 μmol) into the reaction flask in sequence, and dissolve with the mixed solution of DMF / H2O (700 μL). After stirring the reaction mixture at room temperature for 6 hours, monitor the reaction progress by analytical HPLC. The retention time of the target substance Sul-CPP1 is t R = 9.48 min( Figure 24 ). The reaction mixture is directly fractionated and purified by preparative HPLC, and white powder Sul-L4-CPP1 (4.3 mg, 77%) is obtained after freeze-drying. HRMS (ESI) m / z: calcd. for C 128 H 210 N 44 O 25 S3[M+4H] 4+ 714.8919, found 714.8935.( Figure 23 )

[0150]

[0151] Sul-L4-CPP2. Add CPP2-Alkyne (5.0 mg, 3.02 μmol), Sul-L4-Azide (2.35 mg, 4.54 μmol), CuSO4·5H2O (0.453 mg, 1.81 μmol) and NaVc (2.4 mg, 12.1 μmol) into the reaction flask in sequence, and dissolve with the mixed solution of DMF / H2O (700 μL). After stirring the reaction mixture at room temperature for 6 hours, monitor the reaction progress by analytical HPLC. The retention time of the target substance Sul-CPP2 is t R = 7.44 min( Figure 26 ). The reaction mixture is directly fractionated and purified by preparative HPLC, and white powder Sul-L4-CPP2 (4.6 mg, 70%) is obtained after freeze-drying. HRMS (ESI) m / z: calcd. for C88 H 159 N 42 O 19 S2[M+3H] 3+ 724.5448, found 724.4089.( Figure 25 )

[0152] Synthesis of Example 4 HU-CPP1

[0153] Step 1. Synthesis of HU-308-derived primary amine

[0154]

[0155] Compound HU-308-derived primary amine was synthesized according to the method reported in the literature (M.V. Westphal.; R.C. Sarott.; E.A. Zirwes.; A. Osterwald.; W. Guba.; C. Ullmer.; U. Grether.; E.M. Carreira. Highly selective, amine-derived cannabinoid receptor2 probes. Chem. Eur. J. 2020, 26, 13807). HRMS(ESI) m / z: calcd. for C 27 H 43 N4O2[M+H] + 455.3381, found 455.3381.( Figure 27 )

[0156] Step 2. Synthesis of compound HU-CPP1

[0157]

[0158] HU-CPP1. CPP1-Alkyne (5.0 mg, 2.14 μmol), HU-308-derived primary amine (1.9 mg, 4.18 μmol μmol), CuSO4·5H2O (0.53 mg, 2.12 μmol) and NaVc (1.69 mg, 8.53 μmol) were successively added to the reaction flask and dissolved in a mixed solution of DMF / H2O (700 μL). After the reaction mixture was stirred at room temperature for 6 hours, the reaction process was monitored by analytical HPLC. The retention time of the target substance HU-CPP1 was t R = 11.26 min( Figure 29)。The reaction mixture was directly fractionated and purified by preparative HPLC, and a white powder HU-CPP1 (4.2 mg, 71%) was obtained after lyophilization. HRMS (ESI) m / z: calcd. for C 137 H 220 N 39 O 22 S[M+3H] 3+ 931.9005, found 931.8970.( Figure 28 )

[0159] Example 5 Synthesis of Compound PH002-L5-CPP1 and Compound PH002-L5-CPP2

[0160] Step 1. Synthesis of Compound PH002-L5-Azide

[0161]

[0162] Under an ice bath, 20% TFA / DCM solution (2.0 mL) was added to a round-bottom flask containing the small molecule PH-002 (5.0 mg, 10.2 μmol, 1.0 eq). The resulting reaction mixture was allowed to warm to room temperature naturally and stirred for 2 h. The solvent was evaporated under reduced pressure, and the resulting substance was dissolved in DMF (1.0 mL). Then, N3-C5-PEG2-COOH (4.61 mg, 15.2 μmol, 1.5 eq), HATU (5.03 mg, 13.2 μmol, 1.3 eq), and Et3N (14.2 μl, 81.4 μmol, 8.0 eq) were added. The resulting reaction mixture was stirred at room temperature for 3 h, and the product was detected by HPLC (PH002-Azide, t R =10.35 min). The reaction mixture was directly purified by HPLC and lyophilized to obtain a white solid product PH002-Azide (5.3 mg, 8.0 μmol, 78%). HRMS (ESI) m / z: calcd. for C 34 H 45 O6N9Na[M+H] + 698.3385, found 698.3377.

[0163] Step 2. Synthesis of Compound PH002-L5-CPP1 and Compound PH002-L5-CPP2

[0164]

[0165] PH002-L5-CPP1. Add CPP1-Alkyne (5.0 mg, 2.14 μmol), PH002-Azide (1.96 mg, 2.5 μmol), CuSO4·5H2O (0.267 mg, 1.07 μmol), and NaVc (1.412 mg, 7.13 μmol) to the reaction flask in sequence, and dissolve them with the mixed solution of DMF / H2O (700 μL). Monitor the reaction progress by analytical HPLC after stirring the reaction mixture at room temperature for 6 hours. Purify the reaction mixture by preparative HPLC fractionation directly, and obtain white powder PH002-L5-CPP1 (4.5 mg, 71%) after freeze-drying. HRMS (ESI) m / z: calcd. for C 144 H 223 O 44 N 26 S[M+3H] 3+ 1005.5728 found 1005.5752.

[0166]

[0167] PH002-L5-CPP2. Add CPP2-Alkyne (5.0 mg, 3.02 μmol), PH002-Azide (2.74 mg, 3.5 μmol), CuSO4·5H2O (0.453 mg, 1.81 μmol), and NaVc (2.4 mg, 12.1 μmol) to the reaction flask in sequence, and dissolve them with the mixed solution of DMF / H2O (700 μL). Monitor the reaction progress by analytical HPLC after stirring the reaction mixture at room temperature for 6 hours. Purify the reaction mixture by preparative HPLC fractionation directly, and obtain white powder PH002-L5-CPP2 (4.2 mg, 65%) after freeze-drying. HRMS (ESI) m / z: calcd. for C 104 H 173 O 42 N 20 S[M+3H] 3+ 776.7932, found 776.7964.

[0168] Example 6 Screening and verification of the efficiency and mechanism of action of CPPs in inducing extracellular protein internalization

[0169] Extracellular protein uptake experiment. A549 cells were grown in 24-well glass coverslips to a density of approximately 40% to 50%. Biotin-CPPs (Biotin-L1-CPP1, Biotin-L1-CPP2, Biotin-L1-CPP3, Biotin-L1-CPP4) and NAP-650 were diluted to 1 μM and 400 nM respectively and co-incubated for 30 min to form CPPs-labeled NAP-650. Similarly, Biotin-Azide and NAP-650 were co-incubated to form azide-labeled NAP-650. At the same time, the solvent without Biotin-CPPs was used as a blank, and the resulting solution was co-incubated with the cells for 20 h; when verifying the effect of lysosome inhibitor (chloroquine, CQ) on protein uptake, the cells were co-incubated with CPPs-labeled NAP-650 and CQ solution (50 μM) for 12 h, and then fluorescence analysis was performed respectively.

[0170] The test results are shown in Figure 30 a. The group with Biotin-CPPs added and without CQ added was the NC group of Biotin-CPPs. From the test results, it can be seen that only adding Biotin-Azide cannot make Biotin-Azide enter the cells, while in all groups with Biotin-CPPs added, whether lysosome inhibitor was added or not, it showed that Biotin-CPPs could enter the cells, and the addition of lysosome inhibitor inhibited the proteolytic activity of lysosomes, resulting in an increase in the accumulation of NAP-650, indicating that extracellular proteins are degraded in lysosomes after entering the cells through the CPPTACs of the present invention. In the comparison of the four compounds, biotin-L1-CPP1 and biotin-L1-CPP2 containing PEN and TAT sequences showed stronger efficiency than other sequences in promoting the internalization of NAP-650.

[0171] In addition, in order to study the endocytosis pathway of A549 cells, A549 cells were pretreated with 50 μM Nystatin, 10 μg / mL chlorpromazine (CPZ) or 10 μg / mL Ethylisopropylamiloride (EIPA) for 1 h, then CPPs-labeled NAP-650 was added and incubated for 8 h, and then fluorescence staining or flow cytometry was performed.

[0172] The test results are shown in Figure 30bc, Ethylisopropylamiloride (EIPA) is an inhibitor of the macropinocytosis pathway. From the experimental results, the concentration of NAP-650 labeled with CPPs in the cells of the group added with EIPA remained basically the same and did not decrease. Chlorpromazine (CPZ) is an inhibitor of the clathrin-mediated endocytosis pathway. From the experimental results, the concentration of NAP-650 labeled with CPPs in the cells of the group added with CPZ decreased. Nystatin is an inhibitor of caveolin-mediated endocytosis. From the experimental results, the concentration of NAP-650 labeled with CPPs in the cells of the group added with nystatin decreased. This indicates that Biotin-CPPs enter cells through endocytosis. In endocytosis, whether it is through the caveolin pathway or the clathrin pathway specifically depends on the sequence of CPPs.

[0173] Co-localization of extracellular proteins and lysosome markers (lysotracker). A549 cells were grown in 24-well glass coverslips until the density reached about 40% to 50%. The solution of NAP-650 labeled with CPPs was co-incubated with the cells for 20 h. Then, the medium containing the sample was replaced with an equal volume of medium containing lysotracker-green, and incubation in the cells was continued for 1 h. Subsequently, fluorescence staining was performed.

[0174] First, co-incubate NAP-FITC (0.4 μM) with Biotin-L1-CPP1 at specified concentrations (0.1 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM) for 30 minutes, and then co-incubate with A549 cells for 20 hours. Fluorescence and flow cytometry were used to analyze the cellular uptake of NAP-FITC (green). Quantify the mean fluorescence intensity of NAP-FITC relative to the untreated group (n = 3 biological replicates, means ± SD).

[0175] The experimental results are shown in Figure 30 , and the experimental results show that the endocytosis of Biotin-L1-CPP1 for NAP-FITC is dose-dependent. As the concentration of Biotin-L1-CPP1 increases, the concentration of NAP-FITC entering the cells also increases.

[0176] The experimental results are shown in Figure 30 h, the fluorescence signal in A549 cells shows the fluorescence signal (red) of the co-localization of NAP-650 with the lysosome marker (LysoTracker, green) mediated by Biotin-CPP1 or Biotin-CPP2. The experimental results show that the fluorescence imaging of the lysosome marker coincides with that of NAP-650 labeled with CPPs, indicating that after NAP-650 labeled with CPPs is endocytosed into cells, it is transported to lysosomes through endosomes and then degraded.

[0177] Co-localization of extracellular proteins with early endosome marker (Rab5). HeLa cells were grown in a 24-well glass coverslip at a density of approximately 60% to 70%. The Rab5-RFP plasmid was transfected into the cells using PEI transfection reagent and cultured for 24 h to express Rab5-RFP in early endosomes.

[0178] The test results are shown in Figure 30 g, Fluorescence signals of co-localization of NAP-FITC (green) with early endosome marker (Rab5, red) in HeLa cells mediated by Biotin-CPP1 or Biotin-CPP2. The test results indicate that NAP-650 labeled with CPPs is endocytosed into the cells.

[0179] Example 7 Application of the CPPTAC strategy to degrade membrane protein PD-L1, and evaluate the degradation efficiency and mechanism of action of the BMS-CPPs series of compounds in vitro and in vivo

[0180] Membrane protein PD-L1 degradation experiment.

[0181] MDA-MB-231 cells or PD-L1 overexpressing HeLa cell line were cultured in a 12-well cell plate at a density of 70 - 80%. To determine the optimal concentration of BMS-CPPs, it was diluted to 5, 25, 50, 100, 250 nM with medium and co-incubated with the cells for 8 h.

[0182] To determine the optimal degradation time, BMS-CPPs was diluted to the optimal concentration and co-incubated with the cells for 4 h, 8 h, 12 h, 24 h respectively.

[0183] The test results are shown in Figure 31 a - e in, Western blot analysis of PD-L1 levels after treating MDA-MB-231 or Hela-HA-PD-L1 cells with BMS-L1-CPP1 (a, c) or BMS-L1-CPP2 (b, d) at the specified concentration or specified time. The test results indicate that PD-L1 degradation can be achieved at very low concentrations of BMS-L1-CPP1 or BMS-L1-CPP2 (5 - 25 nM), and the degradation rate is relatively fast, with significant degradation occurring after 4 - 8 hours.

[0184] To investigate the effect of BMS-CPP1 on PD-L1 degradation in different cell types, BMS-8, CPP1, and BMS-L1-CPP1 were diluted to 25 nM and incubated with U87-MG, NCI-H292, and NCI-H1975 cells for 8 hours. PD-L1 levels in the different cell lines were analyzed by Western blot after treatment with 25 nM BMS-CPP1, BMS-8, or CPP1 for 8 hours.

[0185] The test results are shown in Figure 31 The results showed that the blank, BMS-8, and CPP1 groups showed almost no changes in U87-MG, NCI-H292, and NCI-H1975 cells, while the addition of BMS-L1-CPP1 significantly reduced PD-L1 levels. This indicates that the CPPTACs approach of the present invention is not affected by cell type and that PD-L1 degradation cannot be achieved by using BMS-8 or CPP1 alone.

[0186] To evaluate the effect of BMS-linker-CPP1 with different linker lengths on PD-L1 degradation, BMS-L2-CPP1 or BMS-L3-CPP1 was diluted to 5, 25, 50, 100, and 250 nM, incubated with cells for 8 h, and analyzed by Western blot.

[0187] The test results are shown in Figure 31 The experimental results showed that PD-L1 degradation can be achieved at an extremely low BMS-L2-CPP1 or BMS-L3-CPP1 concentration (5nM) and within a wide concentration range, indicating that the length of the linker has little effect on the CPPTACs pathway.

[0188] When verifying the degradation pathway, bafilomycin A1 (BAF, 100 nM) was incubated with cells for 2 hours in advance, and then BMS-CPPs were added and incubated for another 8 hours; or MG132 (5 μM) and BMS-CPPs were added to the cells at the same time and incubated for 8 hours, and the PD-L1 level in MDA-MB-231 cells was analyzed by Western blot.

[0189] The test results are shown in Figure 31 The results of j and k show that after the addition of the lysosomal inhibitor BAF, PD-L1 was almost not degraded. MG132 is a proteasome inhibitor, and the addition of MG132 did not affect CAIX degradation, indicating that PD-L1 is degraded through the lysosomal pathway after entering the cell, rather than the proteasome pathway.

[0190] When studying the endocytic pathway, MDA-MB-231 cells were pretreated with 50 μM nystatin, 10 μg / mL chlorpromazine (CPZ), or 10 μg / mL Ethylisopropylamiloride (EIPA) for 1 h, and then BMS-CPPs were added and incubated for 8 h. Protein samples were collected using SDS lysis buffer for western blot experiments to detect the expression of PD-L1 protein.

[0191] The test results are shown in Figure 31 l and m. The test results showed that BMS-L-CPPs entered the cells through endocytosis.

[0192] Immunofluorescence analysis of PD-L1 degradation level. The HeLa cell line overexpressing PD-L1 was cultured in the coverslips of 24-well plates at a density of approximately 40% to 50%. BMS-8, CPP1, the mixture of BMS-8 and CPP1 (BMS-8 + CPP1), and BMS-L1-CPP1 were diluted to 25 nM respectively and added to the cells for incubation for 8 h. Subsequently, immunofluorescence analysis of PD-L1 (red) degradation was performed on the cell membrane or whole cells.

[0193] The test results are shown in Figure 31 g-h. The test results showed that when using BMS-8, CPP1, and the mixture of BMS-8 and CPP1, the fluorescence intensity and range of PD-L1 on the surface were almost the same as those of the blank group, while after using BMS-L1-CPP1, the fluorescence intensity of PD-L1 decreased significantly, indicating that PD-L1 on the cell surface was degraded after using BMS-L1-CPP1. Among them, g shows only the target protein on the cell membrane, and h shows the target protein on the cell membrane and inside the cell. It can be seen that the degraded part is on the membrane.

[0194] In summary, the test results excluded the influence of BMS-8 and CPPs themselves on PD-L1. BMS-CPPs can induce the rapid degradation of endogenous and exogenous PD-L1 through the lysosomal pathway within a wide concentration range, degrade 65 - 80% of PD-L1 within 8 h, the degradation lasts for 24 h, and there is no "hook effect", and it is widely applicable to different cell types. In addition, it has a high tolerance to the length and working concentration of the "linker".

[0195] In vivo experiment. Sterile female C57BL / 6J mice at 5 - 6 weeks of age were used to establish a tumor xenograft model. B16F10 cells (2×10 5 ) were resuspended in 100 μL of PBS and injected subcutaneously into the right side of the mice. When the tumor size reached 30 mm 3 -50 mm 3After (L×W×1 / 2W), the animals were randomly divided into three groups of 5 animals each. BMS-8 (2 mg / kg) and BMS-L1-CPP1 (5 mg / kg) were dissolved in 1% DMSO / PBS (100 μL) and injected peritumorally once a day for two consecutive days. The control group was injected with an equal volume of 1% DMSO / PBS. Tumor specimens were collected and subjected to tissue immunoblotting and immunohistochemistry experiments. All animal experiments were conducted in accordance with relevant guidelines and regulations and were approved by the Institutional Animal Care and Use Committee of SIAT.

[0196] The test results are shown in Figure 32 d - g, where (d) Schematic diagram of in vivo experiment in B16 F10 tumor xenograft C57BL / 6J mouse model. (e, f) Results of Western blot analysis. (g) Representative immunohistochemical staining of PD-L1 in tumor tissues. The test results showed that BMS-CPP1 treatment led to a significant decrease in the PD-L1 level in tumor tissues by approximately 75%, while the effect of BMS-8 itself on PD-L1 expression in vivo was negligible. The above results showed that after using BMS-L1-CPP1 in animals, the level of PD-L1 in tumor tissues decreased significantly, indicating that this type of compound can effectively degrade the target protein in vivo.

[0197] Example 8 Apply the CPPTACs strategy to degrade membrane proteins CAIX and CB2R and evaluate their degradation efficiency and mechanism of action in vitro.

[0198] Membrane protein CAIX and CB2R degradation experiments. MDA-MB-231 cells or HT-29 cells were seeded in 12-well plates at a cell density of 70 - 80%. To determine the optimal concentration of Sul-CPPs / HU-CPP1, they were diluted to 5, 25, 50, 100, 250, 500, 1000 nM respectively and co-incubated with cells for 8 h. To determine the optimal degradation time, Sul-CPPs were diluted to the optimal concentration and co-incubated with cells for 4 h, 8 h, 12 h, 24 h respectively.

[0199] The test results are shown in Figure 33 b - c, m - n of, Sul-L4-CPPs / HU-CPP1 can show high degradation efficiency at low concentration conditions and can induce the rapid degradation of CAIX and CB2R via the lysosomal pathway respectively within a wide concentration range, with degradation efficiencies of 75 - 80% and 70 - 75% respectively.

[0200] To study whether small molecule ligands can degrade CAIX and CB2R, Sul-acid / HU, CPP1 and Sul-CPP1 / HU-CPP1 were diluted to 25 nM respectively and incubated with cells for 8 h.

[0201] The test results are shown in Figure 33 d and m of Figure 33 . The test results show that the degradation is brought about by the compounds of the present invention, excluding the influence of the protein-binding molecule and the cell-penetrating peptide itself.

[0202] When verifying the degradation pathway, bafilomycin A1 (BAF, 100 nM) was pre-incubated with cells for 2 h in advance and then Sul-L4-CPP1 was added and incubated for another 8 h; MG132 (5 μM) and Sul-L4-CPP1 were added to cells simultaneously and incubated for 8 h.

[0203] The test results are shown in Figure 33 e of Figure 33 . The results show that after adding the lysosomal inhibitor BAF, CAIX was hardly degraded. MG132 is a proteasome inhibitor. After adding MG132, it did not affect the degradation of CAIX, indicating that after CAIX enters the cell, it is degraded through the lysosomal pathway rather than the proteasome pathway.

[0204] When studying the endocytic pathway, cells were pretreated with 50 μM nystatin or 10 μg / mL chlorpromazine (CPZ) for 1 h, and then Sul-L4-CPPs were added and incubated for 8 h. SDS lysis buffer was used to collect protein samples for western blot experiments to detect the expression levels of CAIX and CB2R proteins. To study the degradation of CAIX in a hypoxic environment, cells were pretreated with CoCl2 solution (100 μM) for 48 h in advance, and then Sul-L4-CPP1 was added and incubated with cells for another 8 h, and flow cytometry was performed to detect the degradation of CAIX.

[0205] The test results are shown in Figure 33 f and g of Figure 33 . The test results show that Sul-L-CPPs enter cells through endocytosis.

[0206] CCK8 analysis of cell survival experiment. MDA-MB-231 cells were seeded in 96-well plates at a cell density of approximately 40% to 50%. Sul-acid, CPP1, Sul-L4-CPP1, SLC-0111, and CoCl2 solution were diluted to 25 nM, 25 nM, 25 nM, 25 nM, and 100 μM, respectively. The solutions were incubated with cells for 24, 48, and 72 h, respectively. Flow cytometry analysis was performed to evaluate the degradation of CAIX mediated by Sul-CPP1 in MDA-MB-231 cells under CoCl2-induced hypoxic conditions. The mean fluorescence intensity of CAIX-FITC relative to the isotype group was quantitatively measured (n = 3 biological replicates, means ± SD). Subsequently, a CCK8 experiment was performed to detect cell survival.

[0207] Example 9 Evaluation of the Antitumor Activity of BMS-L1-CPP1

[0208] Female germ-free C57BL / 6J mice at 5 - 6 weeks of age were used to establish a tumor xenograft model. B16F10 cells (5×10 5 ) were suspended in 100 μL of PBS and injected subcutaneously into the right side of female germ-free C57BL / 6J mice at 5 - 6 weeks of age. When the tumor size reached 30 mm 3 - 50 mm 3 (L×W×1 / 2W), the animals were randomly divided into four groups of 5 each. BMS-8 (2 mg / kg), CPP1 (4 mg / kg), BMS-CPP1 (10 mg / kg), and 1% DMSO / PBS (100 μL) were applied to one group of animals respectively, and injected intravenously via the tail vein once every two days for a total of 6 times. The animals injected with 1% DMSO / PBS served as the control group. The tumor size and mouse body weight were measured before each injection. The mice were sacrificed 18 days after injection of B16F10 cells, tumor specimens were collected, weighed and further analyzed. The data represent mean±SEM (n = 5), and two-tailed Student’s t-tests and two way ANOVA were used to evaluate the statistical significance. All animal experiments were conducted in accordance with relevant guidelines and regulations and were approved by the Institutional Animal Care and Use Committee of SIAT.

[0209] The test results are shown in Figure 34 (in the figure, BMS-CPP1 corresponds to the result of BMS-L1-CPP1). Compared with BMS-8, CPP1 and the control group, the tumor growth of mice treated with BMS-L1-CPP1 was significantly inhibited, and the body weight of the mice was not significantly affected. At the end of the treatment, the tumors were excised and weighed. The average tumor volume of the BMS-L1-CPP1 treatment group was significantly smaller than that of the other two groups. In addition, there were fewer spleen metastases in the BMS-L1-CPP1 treatment group, indicating that BMS-L1-CPP1 may have the ability to inhibit tumor metastasis. Subsequently, tumor lymphocytes were extracted for flow cytometry analysis. The results showed that the levels of T lymphocyte markers CD3, CD4, CD8 and cytokine IFN-γ in the BMS-L1-CPP1 drug group were significantly higher than those in the control group, CPP1 and BMS-8 drug groups.

[0210] The above results indicate that BMS-L1-CPP1 can exhibit obvious tumor inhibitory effects by enhancing the function of effector T cells and has no obvious toxic side effects.

[0211] Example 10 Experiment on the uptake of extracellular protein PD-L1-FITC and APOE4.

[0212] A549 cells were grown in 24-well glass coverslips to a density of approximately 40% to 50%. BMS-L1-CPP1 and FITC-labeled PD-L1 protein (PD-L1-FITC) were diluted to 1 μM and 400 nM, respectively, and co-incubated for 30 min to form CPP1-labeled PD-L1-FITC. Similarly, BMS-8 and PD-L1-FITC were co-incubated for 30 min. At the same time, the solvent without BMS-L1-CPP1 was used as a blank, and the resulting solution was co-incubated with the cells for 20 h; when verifying the effect of the lysosome inhibitor (chloroquine, CQ) on protein uptake, the cells were co-incubated with CPP1-labeled PD-L1-FITC and CQ solution (50 μM) for 12 h, and then fluorescence analysis was performed separately.

[0213] The test results are shown in Figure 35 as shown in a of (in the figure, BMS-CPP1 is the result of BMS-L1-CPP1). The result of the group with BMS-L1-CPP1 added and CQ not added is the NC group of BMS-L1-CPP1. From the test results, it can be seen that only adding BMS-8 cannot make PD-L1-FITC enter the cells, while in all groups with BMS-L1-CPP1 added, whether the lysosome inhibitor is added or not, it shows that BMS-CPP1 can mediate the entry of PD-L1-FITC protein into the cells, and the addition of the lysosome inhibitor inhibits the proteolytic activity of lysosomes, resulting in an increase in the accumulation of PD-L1-FITC, indicating that extracellular proteins are degraded in lysosomes after entering the cells through the CPPTACs of the present invention.

[0214] A549 cells were grown in 24-well glass coverslips to a density of approximately 40% to 50%. PH002-L5-CPP1 and FITC-labeled APOE4 protein (APOE4-FITC) were diluted to 1 μM and 400 nM, respectively, and co-incubated for 30 min to form CPP1-labeled APOE4-FITC. Similarly, PH002 and APOE4-FITC were co-incubated for 30 min. At the same time, the solvent without PH002-CPP1 was used as a blank, and the resulting solution was co-incubated with the cells for 20 h; when verifying the effect of the lysosome inhibitor (chloroquine, CQ) on protein uptake, the cells were co-incubated with CPP1-labeled APOE4-FITC and CQ solution (50 μM) for 12 h, and then fluorescence analysis was performed separately.

[0215] The test results are as follows Figure 35, among which, as shown in b (in the figure, PH002-CPP1 is the result of PH002-L5-CPP1), the result of the group that added PH002-L5-CPP1 and did not add CQ is the NC group of PH002-L5-CPP1. It can be seen from the experimental results that only adding PH002 cannot make APOE4-FITC enter the cells, while in all groups that added PH002-L5-CPP1, whether lysosome inhibitors were added or not, it was shown that PH002-L5-CPP1 could mediate the entry of APOE4-FITC protein into the cells, and the addition of lysosome inhibitors inhibited the proteolytic activity of lysosomes, resulting in an increase in the accumulation of APOE4-FITC, indicating that extracellular proteins are degraded in lysosomes after entering the cells through the CPPTACs of the present invention.

[0216] In summary, the preparation methods of Biotin-CPPs, BMS-CPPs, Sul-CPPs, HU-CPPs and PH002-CPPs series compounds in this invention have been proven to be feasible through experiments, and the experimental conditions are reproducible with stable yields. Four different CPPs were synthesized respectively in the research. Among them, CPP1, CPP2 and CPP3 are composed of PEN, TAT and R9 sequences respectively, while CPP4 is a fusion peptide containing both a cell-penetrating sequence and a lysosomal sorting sequence. First, the cellular uptake and lysosomal delivery capabilities of these four CPPs for extracellular protein NAP-650 were evaluated. The results showed that biotin-CPP1 and biotin-CPP2 containing PEN and TAT sequences respectively showed stronger efficiency in promoting NAP-650 internalization compared with other sequences. In addition, inhibiting the proteolytic activity of lysosomes with chloroquine led to an increase in the accumulation of NAP-650, and NAP-650 co-localized with early endosome and lysosome markers, thus proving that the internalized protein was degraded after being transported to lysosomes through endosomes. Then, CPZ, nystatin and EIPA were used to block clathrin- and caveolin-mediated endocytosis and macropinocytosis respectively. The results showed that the efficiency of internalization and the pathway of endocytosis depended on the specific sequence of CPPs in CPPTACs. Subsequently, the CPPTACs strategy was used to degrade plasma membrane proteins PD-L1, CAIX, APOE4 and CB2R. The results showed that, excluding the influence of BMS-8 and CPPs themselves on PD-L1, BMS-CPPs could induce the rapid degradation of endogenous and exogenous PD-L1 through the lysosomal pathway within a wide concentration range, degrade 65-80% of PD-L1 within 8 h, the degradation lasted for 24 h, and there was no "hook effect", and it was widely applicable in different cell types. In addition, it had high tolerance to the length and working concentration of the "linker". Next, a B16 F10 tumor xenograft C57BL / 6J mouse model was used to examine the effect of CPPTACs on degrading PD-L1 in vivo. The results showed that BMS-CPP1 treatment led to a significant decrease in the PD-L1 level in tumor tissues by about 75%, while the effect of BMS-8 itself on PD-L1 expression in vivo was negligible. Finally, the CPPTACs strategy was used to degrade plasma membrane proteins CAIX and CB2R to expand the application of this strategy in degrading other plasma membrane proteins. The results showed that both Sul-CPPs and HU-CPP1 could induce the rapid degradation of CAIX and CB2R through the lysosomal pathway within a wide concentration range, and the degradation efficiencies were 75-80% and 70-75% respectively, and there was no "hook effect", and the influence of the protein-binding molecule and the cell-penetrating peptide itself was excluded at the same time. In addition, the endocytic pathway of different CPPTACs was determined by the specific peptide sequence adopted, rather than being affected by the POI target.Meanwhile, the effects of Sul-CPP1 on tumor cells were investigated under a hypoxia microenvironment induced by cobalt chloride. The results showed that Sul-CPP1 significantly reduced the cell surface CAIX in the hypoxia microenvironment and had the ability to significantly inhibit the viability of tumor cells, comparable to the CAIX inhibitor SLC-0111 currently being evaluated in clinical trials. In summary, the findings of this invention provide convincing evidence for the wide application of the CPPTACs platform in effectively degrading various plasma membrane proteins.

[0217] The linker used in this invention can be replaced by other types and lengths of linkers; the small molecules BMS-8 that bind to the PD-L1 target protein, Sul-acid that binds to the CAIX target protein, HU-308-derived primary amine that binds to the CB2R target protein, and PH002 that binds to APOE4 can all be replaced by other small molecule or polypeptide compounds that can bind to PD-L1, CAIX, CB2R, and APOE4; meanwhile, the CPP peptide used can also be a polypeptide sequence containing PEN, TAT, R9, or other transmembrane-active polypeptide sequences and their sequence combinations; in terms of protein targets, this invention is not limited to the target proteins PD-L1, CAIX, CB2R, and APOE4 in the examples, and bifunctional molecules obtained by combining binding molecules of other target proteins with the above polypeptides have the ability to degrade the corresponding target proteins.

Claims

1. A bifunctional compound for degrading cell membrane proteins or extracellular proteins by utilizing cell lysosomes, characterized in that: The bifunctional compound includes a linked target protein binding compound and a cell-penetrating peptide, The target protein binding compound is a compound or a derivative thereof that can bind to a cell membrane protein or an extracellular protein; The target protein binding compound and the cell penetrating peptide are connected via a linker, or the target protein binding compound and the cell penetrating peptide are directly connected.

2. The bifunctional compound according to claim 1, characterized in that The linker is coupled to the target protein binding compound and the cell-penetrating peptide respectively through chemical bonds; Preferably, the chemical bond is selected from an amide bond, an ester bond, a triazole, an alkynyl group, and a C—C, C═C, or a heteroatom-containing CN, CO, CS, C═S, and a CP bond; Preferably, the linker comprises a chemical structure formed by coupling a compound that binds to a cell membrane protein or an extracellular protein or a derivative thereof with a cell-penetrating peptide through a click chemistry reaction, and the groups that undergo click chemistry reaction include any pair of sulfhydryl SH and maleimide MA, azide N3 and alkynyl, azide N3 and DBCO or BCN, and tetrazine and trans-cyclooctene or their derivatives; Preferably, the linker is coupled to the C-terminus and N-terminus of the cell-penetrating peptide or to the active group on the amino acid side chain of the cell-penetrating peptide through a chemical bond; More preferably, the linker is selected from Wherein, L1 and L2 are independently selected from none, -(CH2)n-, -(COC-(CH2)n)m-, -((CH2)n-COC)m-, -((CH2)n-COC-(CH2)n)m-, -(C=C-(CH2)n)m-, -((CH2)nC=C)m-, -((CH2)nC=C-(CH2)n)m, -(CO-(CH2)n)m-, -((CH2)n-CO)m-, -((CH2)n-CO-(CH2)n)m-, -(CONH-(CH2)n)m-, -((CH2)n-CONH)m-, -((CH2)n-CONH-(CH2)n)m-, -(NHCO-(CH2)n)m-, -((CH2)n-NHCO)m-, -((CH2)n-NHCO-(CH2)n)m-, -(OCH2CH2)n-; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; More preferably, the linker is selected from L1 and L2, and the sum of all n values and m values is 4-20; More preferably, the linker is selected from 3. The bifunctional compound according to claim 1, characterized in that The extracellular protein or cell membrane protein is selected from APOE4, PD-L1, β-amyloid protein, programmed cell death-ligand 1 (i.e., PD-L1), programmed death receptor 1 (i.e., PD-1), epidermal growth factor receptor (i.e., EGFR), human epidermal growth factor receptor-2 (i.e., HER2), G protein-coupled receptor (i.e., GPCR), fibroblast growth factor receptor (i.e., FGFRs), vascular endothelial growth factor receptor family (i.e., VEGFR, VEGF represents vascular endothelial growth factor), cytotoxic T lymphocyte-associated protein 4 (i.e., CTLA4 or CTLA-4), human interleukin 5 receptor alpha (IL-5Rα), apolipoprotein, apolipoprotein E4 (i.e., ApoE4), β-amyloid protein, angiotensin-converting enzyme 2 (ACE2), sodium-taurocholate cotransporter (NTCP), B7.1 and B7, TI FR1m, TNFR2, NADPH oxidase, Bc1IBax and other ligands in the apoptosis pathway, C5a receptor, HMG-CoA reductase, PDEⅤ phosphodiesterase type, PDEⅣ phosphodiesterase type 4, PDEⅠ, PDEⅡ, PDEⅢ, squalene cyclase inhibitor, CXCR1, CXCR2, nitric oxide synthase, cyclooxygenase 1, cyclooxygenase 2, 5HT receptor, dopamine receptor, G protein, histamine receptor, 5-lipoxygenase, protease-like serine protease, thymidylate synthase, purine nucleoside phosphorylase, glyceraldehyde-3-phosphate dehydrogenase (ie, GAPDH), glycogen phosphorylase, carbonic anhydrase, chemokine receptor, JAW STAT, RXR and analogs, HIV1 protease, HIV1 integrase, influenza neuraminidase, hepatitis B reverse transcriptase, sodium channels, protein P-glycoprotein, P-glycoprotein and MRP tyrosine kinase, CD23, CD73, CD124, tyrosine kinase p561ck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-αR, ICAM1, Ca2+ channels, VCAM, VLA-4 integrin, selectins, CD40 / CD40L, newokinins and receptors, inosine monophosphate dehydrogenase, p38 MAP kinase, Ras / Raf / MEW / ERK pathway, interleukin-1 converting enzyme, caspases, HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotide formyltransferase, rhinovirus, 3C protease, herpes simplex virus-1, protease, cytomegalovirus protease, poly (ADP-ribose) polymerase, cyclin-dependent kinase, vascular endothelial growth factor, oxytocin receptor, inhibitor of microsomal transfer protein, bile acid transport inhibitor, 5-alpha reductase inhibitor, angiotensin 11, glycine receptor, norepinephrine reuptake receptor, endothelin receptor, neuropeptide Y and receptor, adenosine receptor, adenosine kinase and AMP dehydrogenase, purinergic receptor, farnesyltransferase, geranyltransferase, Tr of NCF One or more of: α-κB receptor, tyrosine kinase Flk-IIKDR, vitronectin receptor, integrin receptor, Her-21 neuropilin, telomerase inhibitor, cytosolic phospho-A2 and EGF receptor tyrosine kinase, ecdysone 20-monooxygenase, GABA-gated chloride channel, acetylcholinesterase, voltage-sensitive sodium channel protein, calcium release channel and chloride channel, acetyl-CoA carboxylase, adenylate succinate synthetase, protoporphyrinogen oxidase and enolpyruvylshikimate phosphate synthetase, and / or one or more of all variants, mutants, splice variants, indels and fusions of the aforementioned proteins; Preferably, the compound capable of binding to cell membrane proteins or extracellular proteins or a derivative thereof is selected from any one of Biotin, 8HU-308, Sul-COOH, PH002 or a derivative thereof.

4. The bifunctional compound according to claim 1, characterized in that The cell-penetrating peptide is selected from cationic cell-penetrating peptides, amphipathic cell-penetrating peptides, hydrophobic cell-penetrating peptides, synthetic cell-penetrating peptides, or a fusion peptide of a cell-penetrating sequence and a lysosomal sorting sequence; Preferably, the cell-penetrating peptide is selected from any one of the sequences shown in Table 1 or has any one of the sequences shown in Table 1 and has a modification and / or protecting group; Preferably, the C-terminal α-carboxyl group of the cell-penetrating peptide is protected by an amide bond.

5. A method for degrading cell membrane proteins or extracellular proteins, the method comprising the step of contacting the bifunctional compound with cells containing cell membrane proteins, or the method comprising the step of incubating the bifunctional compound with cells and extracellular proteins; The target protein binding compound in the bifunctional compound can specifically bind to the cell membrane protein to be degraded or the degraded extracellular protein; Preferably, the method of degrading cell membrane proteins or extracellular proteins is carried out in vitro or in vivo; Preferably, the cell membrane protein or the extracellular protein is degraded via the intracellular lysosomal pathway; Preferably, the cell membrane protein or the extracellular protein enters the cell via endocytosis; Preferably, the cells are normal cells or tumor cells.

6. A method for designing a bifunctional compound capable of utilizing lysosomes to degrade cell membrane proteins or extracellular proteins, the method comprising the following steps: S1) determining the cell membrane protein or extracellular protein target to be degraded; S2) obtaining a small molecule compound that can specifically bind to a cell membrane protein or extracellular protein target to be degraded; S3) connecting the small molecule compound obtained in S2) with a cell-penetrating peptide to obtain a bifunctional compound capable of utilizing cell lysosomes to degrade cell membrane proteins or extracellular proteins.

7. The design method according to claim 6, wherein the connection in S3) is to directly connect the small molecule compound and the cell-penetrating peptide through a chemical bond; or By modifying small molecule compounds and / or cell-penetrating peptides and then chemically linking them; or The small molecule compound and the cell-penetrating peptide are chemically linked via a linker; Preferably, the chemical bond is selected from an amide bond, an ester bond, a triazole, an alkynyl group, and a C—C, C═C, or a heteroatom-containing CN, CO, CS, C═S, and a CP bond; More preferably, the linker is selected from in, L1 and L2 are independently selected from none, -(CH2)n-, -(COC-(CH2)n)m-, -((CH2)n-COC)m-, -((CH2)n-COC-(CH2)n)m-, -(C=C-(CH2)n)m-, -((CH2)nC=C)m-, -((CH2)nC=C-(CH2)n)m, -(CO-(CH2)n)m-, -((CH2)n-CO)m-, - A combination of one or more of ((CH2)n-CO-(CH2)n)m-, -(CONH-(CH2)n)m-, -((CH2)n-CONH)m-, -((CH2)n-CONH-(CH2)n)m-, -(NHCO-(CH2)n)m-, -((CH2)n-NHCO)m-, -((CH2)n-NHCO-(CH2)n)m-, and -(OCH2CH2)n-; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; More preferably, the linker is selected from L1 and L2, and the sum of all n values and m values is 4-20; More preferably, the linker is selected from Preferably, the cell-penetrating peptide is selected from cationic cell-penetrating peptides, amphiphilic cell-penetrating peptides, hydrophobic cell-penetrating peptides, synthetic cell-penetrating peptides, or a fusion peptide of a cell-penetrating sequence and a lysosomal sorting sequence; More preferably, the cell-penetrating peptide is selected from any one of the sequences shown in Table 1 or has any one of the sequences shown in Table 1 and has a modification and / or protecting group; Preferably, the C-terminal α-carboxyl group of the cell-penetrating peptide is protected by an amide bond.

8. Use of the bifunctional compound according to claim 1 in the preparation of a reagent for degrading cell membrane proteins or extracellular proteins, wherein the target protein binding compound in the bifunctional compound can specifically bind to the cell membrane protein to be degraded or the degraded extracellular protein.

9. Use of the bifunctional compound according to claim 1 in the preparation of drugs for anti-tumor, hypolipidemic and neurological disease treatment, The extracellular protein in the bifunctional compound is selected from APOE4, PD-L1, and β-amyloid protein, and the cell membrane protein is selected from programmed cell death-ligand 1 (i.e., PD-L1), programmed death receptor 1 (i.e., PD-1), epidermal growth factor receptor (i.e., EGFR), human epidermal growth factor receptor-2 (i.e., HER2), G protein-coupled receptor (i.e., GPCR), fibroblast growth factor receptor (i.e., FGFRs), vascular endothelial growth factor receptor family (i.e., VEGFR, VEGF represents vascular endothelial growth factor), cytotoxic T lymphocyte-associated protein 4 (i.e., CTLA4 or CTLA-4), human interleukin 5 receptor alpha (IL-5Rα), apolipoprotein, apolipoprotein E4 (i.e., ApoE4), β-amyloid protein, angiotensin-converting enzyme 2 (ACE2), sodium-taurocholate cotransporter (NTCP), B7.1 and B7, TI FR1m, TNFR2, NADPH oxidase, Bc1IBax and other ligands in the apoptosis pathway, C5a receptor, HMG-CoA reductase, PDEⅤ phosphodiesterase type, PDEⅣ phosphodiesterase type 4, PDEⅠ, PDEⅡ, PDEⅢ, squalene cyclase inhibitor, CXCR1, CXCR2, nitric oxide synthase, cyclooxygenase 1, cyclooxygenase 2, 5HT receptor, dopamine receptor, G protein, histamine receptor, 5-lipoxygenase, protease-like serine protease, thymidylate synthase, purine nucleoside phosphorylase, glyceraldehyde-3-phosphate dehydrogenase (ie, GAPDH), glycogen phosphorylase, carbonic anhydrase, chemokine receptor, JAW STAT, RXR and analogs, HIV1 protease, HIV1 integrase, influenza neuraminidase, hepatitis B reverse transcriptase, sodium channels, protein P-glycoprotein, P-glycoprotein and MRP tyrosine kinase, CD23, CD73, CD124, tyrosine kinase p561ck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-αR, ICAM1, Ca2+ channels, VCAM, VLA-4 integrin, selectins, CD40 / CD40L, newokinins and receptors, inosine monophosphate dehydrogenase, p38 MAP kinase, Ras / Raf / MEW / ERK pathway, interleukin-1 converting enzyme, caspases, HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotide formyltransferase, rhinovirus, 3C protease, herpes simplex virus-1, protease, cytomegalovirus protease, poly (ADP-ribose) polymerase, cyclin-dependent kinase, vascular endothelial growth factor, oxytocin receptor, inhibitor of microsomal transfer protein, bile acid transport inhibitor, 5-alpha reductase inhibitor, angiotensin 11, glycine receptor, norepinephrine reuptake receptor, endothelin receptor, neuropeptide Y and receptor, adenosine receptor, adenosine kinase and AMP dehydrogenase, purinergic receptor, farnesyltransferase, geranyltransferase, Trk of NCF One or more of A receptor, tyrosine kinase Flk-IIKDR, vitronectin receptor, integrin receptor, Her-21 nerve sheath, telomerase inhibition, cytosolic phospho-A2 and EGF receptor tyrosine kinase, ecdysone 20-monooxygenase, GABA-gated chloride channel, acetylcholinesterase, voltage-sensitive sodium channel protein, calcium release channel and chloride channel, acetyl-CoA carboxylase, adenylate succinate synthetase, protoporphyrinogen oxidase and enolpyruvylshikimate phosphate synthetase, and / or one or more of all variants, mutants, splice variants, indels and fusions of the above proteins.

10. The method for preparing the bifunctional compound according to claim 1, comprising the following steps: S01) obtaining target protein binding compounds and cell-penetrating peptides; S02) directly coupling the target protein binding compound and the cell-penetrating peptide or coupling them through a linker; Preferably, the step S01) further comprises the step of modifying the obtained target protein binding compound and cell-penetrating peptide; Preferably, the modification is to allow the target protein binding compound and the cell-penetrating peptide to contain an active reactive group having a chemical bond capable of forming a coupling; Preferably, the active reactive group capable of forming a coupled chemical bond is selected from any pair of a combination of a carboxyl group and an amino group, a combination of a carboxyl group and a hydroxyl group, a combination of an azide group and an alkynyl group, a group of a thiol group SH and a maleimide group MA, a combination of an azide N3 and an alkynyl group, a combination of an azide N3 and DBCO or BCN, and a combination of a tetrazine and trans-cyclooctene, or their derivatives.

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