WO2023274347A1 - Technology for modular assembly of cell-penetrating peptide-mediated polypeptide or microprotein targeting chimera, and use thereof - Google Patents

Technology for modular assembly of cell-penetrating peptide-mediated polypeptide or microprotein targeting chimera, and use thereof Download PDF

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WO2023274347A1
WO2023274347A1 PCT/CN2022/102648 CN2022102648W WO2023274347A1 WO 2023274347 A1 WO2023274347 A1 WO 2023274347A1 CN 2022102648 W CN2022102648 W CN 2022102648W WO 2023274347 A1 WO2023274347 A1 WO 2023274347A1
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targeting
formula
linker
protein
small molecule
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PCT/CN2022/102648
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French (fr)
Chinese (zh)
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刘淼
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刘淼
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • the invention relates to the field of bioengineering, in particular to the modular assembly technology of polypeptide or microprotein targeting chimera mediated by cell-penetrating peptides and its application.
  • PROTAC Protein degrader technology has been popular in the world in recent years, among which the earliest and most popular is the protein degradation targeting chimera (Proteolysis targeting Chimera, PROTAC) technology.
  • PROTAC includes three parts: a small molecule E3 ubiquitin linker
  • its structure is: small molecule Ligand (targeting target protein) + linker + small molecule ligand (binding to E3 ligase).
  • the target protein binds to the small molecule target protein ligand, and at the same time the E3 ligase ligand is also bound by its ligand.
  • the E3 ligase adds a ubiquitin tag to the target protein, and then after multiple rounds of ubiquitination, there is With multiple ubiquitin tags, the target protein after polyubiquitination will be recognized and degraded by the proteasome.
  • PROTAC technology has been successfully applied to the induced degradation of various diseased proteins.
  • E3 ubiquitin ligase requires a specific recognition signal to recruit and ubiquitinate its target protein.
  • the emergence of PROTAC technology makes it possible for E3 to ubiquitinate any protein.
  • This technique designs a dual-functional molecule that binds a target protein at one end and an E3 ligase at the other end, and forms a polymer of the two.
  • E3 can ubiquitinate the target protein and guide it into the degradation pathway.
  • the most attractive aspect of targeting protein degradation is that it can target protein targets that have traditionally been considered undruggable, and these proteins may account for more than 80% of the human proteome. Since the targeted protein degradation strategy can achieve the purpose of selectively degrading proteins by binding to almost any site on the protein instead of the active site, this strategy can theoretically be used for any protein.
  • Peptide drugs are another class of targeting molecules that have attracted widespread attention and interest. Similar to biomacromolecules, polypeptide molecules also have higher binding force and selectivity for targets, and have smaller off-target effects than small molecule drugs. The metabolites of polypeptides in the body are amino acids, which minimizes toxicity. Compared with small molecule drugs, peptide drugs have incomparable advantages, mainly in the easy modification of peptide molecules, target recognition specificity, and wide targeting range.
  • the purpose of the present invention is to provide a modular assembly technology and its application of a polypeptide or microprotein targeting chimera mediated by cell-penetrating peptides that can target the target protein, thereby effectively degrading the targeted protein.
  • the first object of the present invention is to provide a targeting chimera mediated by cell-penetrating peptide modular assembly of polypeptides or microproteins, including at least one penetrating peptide module, at least one targeting polypeptide module, One (or no) small molecule Linker module and at least one small molecule ligand module, the targeting polypeptide module is a polypeptide sequence that can bind to the target protein.
  • the above-mentioned cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera also includes at least one linker Linker module, and the targeting polypeptide module and the small molecule ligand module pass through Connector Linker module for chimerism.
  • the penetrating peptide module is connected to the free end of the targeting polypeptide module and used to guide the targeting chimera Penetrate the cell membrane.
  • the small molecule ligand module is a small molecule E3 ligand that can bind to E3 ligase; preferably
  • the protease degradation agent adapted to the small molecule E3 ligand is CRBN (Cereblon protein, Cereblon protein), VHL (Hippel-Lindau, von Hippel-Lindau), IAP (apoptosis inhibitory protein, Inhibitor of apoptosis proteins) one or more.
  • the amino acid sequence of the penetrating peptide module is SEQ ID No.1-SEQ ID No.3 any one.
  • the amino acid sequence of the targeting polypeptide module is SEQ ID No.4-SEQ ID No.17 Any one or more.
  • the Linker module is a small molecule compound, and its structural formula is shown in formula I;
  • the structural formula of the small molecule ligand module is as shown in Formula II Show, when the suitable protease degradation agent is VHL, the structural formula of the small molecule ligand module is shown in formula III, When the suitable protease degradation agent is IAP, the structural formula of the small molecule ligand module is shown in formula IV,
  • the above-mentioned cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera has any one or more of the following structures:
  • the targeting polypeptide module also includes a modified staple peptide sequence or cyclic peptide sequence, staple peptide Sequences or cyclic peptide sequences have the function of entering the membrane.
  • the targeting chimera for modular assembly of polypeptides or microproteins mediated by cell-penetrating peptides may not have a membrane-penetrating peptide.
  • the above cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera the cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera containing staple peptide
  • Its structure is as follows: Staple peptide with the structure of formula V + linker Linker with the structure of formula I + small molecule ligand with the structure of formula II.
  • the above-mentioned cell-penetrating peptide-mediated modular assembly of polypeptides or microproteins targeting chimera and the cell-penetrating peptide-mediated modular assembly of polypeptides or microproteins targeting chimeras containing cyclic peptides Its structure is as follows: cyclic peptide of formula VI + linker Linker of formula I + small molecule ligand of formula II.
  • the second object of the present invention is to provide the above-mentioned cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera in the preparation of products for degrading the target protein or for degrading the presence of mutated amino acid positions. Point of view for the application of products targeting target proteins.
  • the targeted degradation protein includes new crown S protein HR2, new crown N protein, new crown M protein, new crown E protein, new crown Orf6 protein, Lag-3 protein, Her2 protein, SHP-2 protein, One or more of STAT5B protein, MUC16 protein, CTLA-4 protein, PCSK9 protein, PD-1 protein, PD-L1 protein, KRAS protein G12V variation.
  • the core idea of the present invention is to link multiple freely transformable "module” sequences or small molecule compounds into a “modular” targeting chimera with strong targeting, good membrane penetration and high degradation efficiency. thing.
  • the most basic composition is a membrane-penetrating peptide module, a targeting polypeptide module and a small molecule ligand module, the three are connected to each other, and further constitute a membrane-penetrating peptide module and a targeting polypeptide module , a linker Linker module and a small molecule ligand module, forming a basic structure of penetrating peptide-targeting polypeptide-linker Linker-small molecule ligand.
  • the basic structure of penetrating peptide-targeting polypeptide-small molecule ligand can directly direct the small molecule ligand to the target protein. Although some chimeras under this basic structure can exert targeted therapeutic properties, they have Certain defects, the connection between the three is not stable, easy to fall off.
  • Membrane-penetrating peptide-targeting polypeptide-linker Linker-small molecule ligand is an upgraded structure of the above basic structure, which overcomes the defect of poor membrane-penetrating performance of targeting polypeptide, and also overcomes the direct connection between targeting polypeptide and small molecule ligand
  • the membrane-penetrating peptide is used to penetrate the membrane, and the targeting polypeptide is directed to the target protein, thereby achieving the effect of directional penetration.
  • the linker Linker is used to connect the two, which can effectively reduce the probability of falling off; at the same time, it overcomes the poor membrane penetration and connection
  • the two major defects of instability have almost perfectly solved the technical effect of targeted therapy and improved the targeting efficiency.
  • this optimal structure uses membrane-penetrating peptide + targeting polypeptide to "replace"
  • the targeting protein in the existing PROTAC technology the targeting polypeptide can greatly expand the selectivity of targeting the target protein, and through the attached small molecule ligand (E3), it can target and degrade almost all known target proteins.
  • TAT Transactivator
  • HIV human immunodeficiency virus
  • cell-penetrating peptides are polypeptide molecules of no more than 30 amino acids, which can independently pass through the cell membrane without relying on specific membrane receptors. These cell-penetrating peptides are used as intracellular transport tools for bioactive molecules. Compared with other iontophoresis and nanocarriers, they have the characteristics of low toxicity, convenience and effectiveness, and play an increasingly important role in drug development. There are even related drugs containing CPP that have passed FDA clinical trials.
  • the penetrating peptide coupling chimera technology (CePPiTAC) technology of the present invention is to "replace" the small-molecule target protein ligand part in the ordinary "triple" PROTAC structure with a polypeptide that can bind to the target protein.
  • Peptides are used to connect the Linker and the small molecule E3 ligand, and the penetrating peptide sequence is added to form a structure: penetrating peptide (penetrating peptide) + polypeptide (targeting target) + Linker + E3 ligand, and the Linker can be removed if necessary , so that the polypeptide (targeting target) containing the penetrating peptide is directly linked to the E3 ligand.
  • the medicine synthesized by the present invention is a complex of polypeptide and small molecule, which can be connected by small molecule linker or removed.
  • a membrane-entrying polypeptide sequence can be added, and this sequence can connect polypeptide and small molecule.
  • the complex (CePPiTAC complex) is brought into the cell, at this time, the polypeptide part targeting the target protein can bind to the target protein, and the small molecule E3 ligand at the other end of the linker can bind the E3 ligase and trigger E3 ubiquitination The ubiquitination of the target protein by the enzyme reaction, so that the 26S protease in the cell can recognize the target protein and degrade it.
  • the polypeptide or microprotein modular assembly targeting chimera provided by the present invention is mediated by cell-penetrating peptides, through the interconnected penetrating peptides, targeting polypeptides and small molecule ligands , can penetrate the cell membrane and target all targeted proteins, the connected small molecule ligand can bind to the immobilized ligase and trigger the ubiquitinase reaction, thereby ubiquitinating the target protein and making the protease in the cell Targeted recognition of target protein and degradation of target protein, so that targeted drugs can be screened out more broadly.
  • the peptide module is used to bind the target protein, in theory, all target proteins can be targeted, which was not possible with other degrader technologies in the past.
  • this technology combines high-efficiency small molecule E3 ligands to achieve ubiquitination, it is much more efficient than other Peptide-based PROTAC/degraders that use peptide ligands. In most cases, it can be used in cell experiments. The degradation of the target protein is achieved at the nmol level.
  • a linker Linker can also be added, and the addition of the linker Linker can further solidify the connection between the targeting polypeptide and the small molecule ligand.
  • the targeting chimera provided by the present invention adopts a modular design, and each sequence or small molecule compound module with different functions can be replaced and superimposed according to needs. This design idea greatly enhances the use of targeted drugs. effect and scope of application.
  • Small-molecule triplet PROTACs can be developed for limited targets, while Peptide-Based PROTAC/degrader has low degradation efficiency and often needs to be at the umol level to degrade targets on cells, while this technology can target all At the same time as the target, it can also achieve efficient degradation (cellular degradation) at the nmol level, and truly realize the goal of "there are medicines for all diseases".
  • the present invention also has subversive significance to the previous technical concepts. Taking virus-related proteins as an example, the related targets for viruses in the past are mainly related proteins of viruses that infect human cells (such as the S protein of the new crown), and virus synthesis. The required enzymes, etc., the target selection is relatively small.
  • this technology uses the inactive site of the polypeptide sequence to bind the target, which can target and degrade all proteins of all viruses, and is also effective in overcoming drug resistance caused by virus mutation, which greatly improves the possibility and convenience of the successful development of viral drugs sex.
  • this technology can degrade a single variation of a certain target, while the homologous protein of the wild type (Wild Type) without some variation will not be affected or will be less affected, which is also impossible for other degrader technologies such as PROTAC. Achieved.
  • Figure 1 shows the conventional design method of the cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera (target degradation agent) described in Example 2 of the present invention, that is, the general-purpose membrane-penetrating peptide-target To the peptide-connector Linker-small molecule ligand mode.
  • Figure 2 shows the targeted chimera for modular assembly of polypeptides or microproteins mediated by cell-penetrating peptides described in Example 3 of the present invention, which is specially designed for a certain type or types of pathogenic proteins that are difficult to degrade, and can Combining the targeting polypeptide with two or more different E3 ligase conjugates to effectively degrade the target disease-causing protein.
  • Figure 3 shows the cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera described in Example 4 of the present invention, which can effectively target the protein-protein complex formation related to pathogenic proteins It can effectively inhibit the entire pathogenic pathway and completely inhibit a specific disease by degrading multiple targets.
  • FIG. 4 is a flow chart of the solid-phase synthesis process of the polypeptide in Example 5, wherein the synthetic product polypeptide is marked as 1.
  • Example 5 shows the synthesis reaction of lenalidomide and succinic anhydride synthesis compound in Example 5, wherein lenalidomide is marked as 2, succinic anhydride is marked as 3, and the synthetic product is marked as 4.
  • FIG. 6 shows a flowchart of the solid-phase synthesis process of the LEN-binding peptide in Example 5, wherein the synthesized product (diastereomeric mixture) is marked as 5 .
  • Figure 7 shows a cell-penetrating peptide-mediated peptide or microprotein modular assembly targeting chimera and its effect verification for degrading the new crown S protein HR2 in an embodiment of the present invention
  • Figure 7A is a cell-based The structural diagram of the targeted chimera for the modular assembly of peptides or microproteins mediated by penetrating peptides
  • Figure 7B is the verification of the degradation effect of the protein
  • Figure 7C is the protease inhibitor MG132 to verify that it is indeed produced by the protease degradation agent Effect.
  • Figure 8 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading the new crown N protein in an embodiment of the present invention, in which Figure 8A is a cell-penetrating peptide Structural diagram of membrane peptide-mediated modular assembly of polypeptides or microproteins targeting chimera, Figure 8B is the verification of the degradation effect of the protein, and Figure 8C is the protease inhibitor MG132 to verify the effect produced by the protease degradation agent .
  • Figure 9 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading the new crown M protein in an embodiment of the present invention.
  • Structural diagram of membrane peptide-mediated modular assembly of polypeptides or microproteins targeting chimera is the verification of the degradation effect of the protein
  • Figure 9C is the protease inhibitor MG132 to verify the effect produced by the protease degradation agent .
  • Figure 10 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading the new crown E protein in an embodiment of the present invention.
  • Structural diagram of membrane peptide-mediated modular assembly of polypeptides or microproteins targeting chimera is the verification of the degradation effect of the protein
  • Figure 10C is the protease inhibitor MG132 to verify the effect produced by the protease degradation agent .
  • Figure 11 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading the new crown Orf6 protein in an embodiment of the present invention, in which Figure 11A is a cell-penetrating peptide Structural diagram of membrane peptide-mediated modular assembly of polypeptides or microproteins targeting chimeras, Figure 11B is the verification of the degradation effect of the protein, and Figure 11C is the protease inhibitor MG132 to verify the effect produced by the protease degradation agent .
  • Figure 12 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading Lag-3 protein in an embodiment of the present invention.
  • Figure 12B is the verification of the degradation effect of the protein
  • Figure 12C is the protease inhibitor MG132 to verify that it is indeed produced by the protease degradation agent Effect.
  • Figure 13 shows the targeting chimera for degrading Her2 protein mediated by cell-penetrating peptides or microprotein modular assembly and its effect verification, in which Figure 13A is a cell-penetrating peptide The peptide-mediated peptide or microprotein modular assembly targeting chimera structure diagram, Figure 13B is the verification of the degradation effect of the protein, and Figure 13C is the protease inhibitor MG132 to verify the effect produced by the protease degradation agent.
  • Figure 14 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading SHP-2 protein in an embodiment of the present invention.
  • the structural diagram of the targeting chimera for the modular assembly of peptides or microproteins mediated by penetrating peptides is the verification of the degradation effect of the protein
  • Figure 14C is the protease inhibitor MG132 to verify that it is indeed produced by the protease degradation agent Effect.
  • Figure 15 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading STAT5B protein in an embodiment of the present invention.
  • the peptide-mediated peptide or microprotein modular assembly targeting chimera structure diagram is the verification of the degradation effect of the protein, and
  • Figure 15C is the protease inhibitor MG132 to verify the effect produced by the protease degradation agent.
  • Figure 16 shows the targeting chimera for degrading MUC16 protein mediated by cell-penetrating peptides or microprotein modular assembly and its effect verification in one embodiment of the present invention, wherein Figure 16A is a cell-penetrating peptide The structural diagram of peptide-mediated modular assembly of polypeptides or microproteins targeting chimeras, Figure 16B is the verification of the degradation effect on proteins, and Figure 16C is the protease inhibitor MG132 to verify the effect produced by the protease degradation agent.
  • Figure 17 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading CTLA-4 protein in an embodiment of the present invention.
  • Figure 18 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading PCSK9 protein in an embodiment of the present invention.
  • Peptide-mediated peptide or microprotein modular assembly targeting chimera structure diagram, Figure 18B is a protease inhibitor MG132 to verify the effect of the protease degradation agent.
  • FIG. 19 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading PD-1 protein in an embodiment of the present invention.
  • the structural diagram of the targeting chimera for the modular assembly of polypeptides or microproteins mediated by penetrating peptides, FIG. 19B is a protease inhibitor MG132 to verify the effect of the protease degradation agent.
  • FIG. 20 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading PD-L1 protein in an embodiment of the present invention.
  • FIG. 20B is a protease inhibitor MG132 to verify the effect of the protease degradation agent.
  • Figure 21 shows a cell-penetrating peptide-mediated peptide or microprotein modular assembly targeting chimera and its effect verification for precise targeting and degradation of KRAS protein with G12V mutation in an embodiment of the present invention
  • Figure 21A is a structural diagram of a peptide or microprotein modular assembly targeting chimera mediated by a cell-penetrating peptide
  • Figure 21B is a verification of the degradation effect of a G12V mutant KRAS protein
  • Figure 21C is a diagram of a non-mutant KRAS protein (wild type) degradation effect verification.
  • Figure 22 shows a modular penetrating peptide-mediated dual E3 ligand targeting chimera and its effect verification for degrading PCSK9 protein in an embodiment of the present invention, wherein Figure 22A is a modular penetrating peptide-mediated The structural diagram of the double E3 ligand (CRBN+VHL) targeting chimera, Figure 22B is a protease inhibitor MG132 to verify the effect of the protease degradation agent and the overall dosage of the drug is less.
  • Figure 23 shows a modular membrane-penetrating peptide-mediated dual-target targeting chimera and its effect verification for simultaneously degrading HR2 protein and N protein in an embodiment of the present invention, wherein Figure 23A is a modular membrane-penetrating peptide The structural diagram of the peptide-mediated dual-target (new crown HR2 + new crown N protein target) targeting chimera, Figure 23B is the protease inhibitor MG132 to verify the effect of the protease degradation agent.
  • Figure 24 shows a modular penetrating peptide-mediated staple peptide modification targeting chimera and its effect verification for degrading PD-L1 protein in an embodiment of the present invention, wherein Figure 24A is a modular penetrating peptide
  • Figure 24A is a modular penetrating peptide
  • the structural diagram of the mediated staple peptide modification targeting chimera, Figure 20B is the verification of the degradation effect on the protein.
  • Figure 25 shows a modular penetrating peptide-mediated circular peptide modification targeting chimera and its effect verification for degrading PD-L1 protein in an embodiment of the present invention, wherein Figure 25A is a modular penetrating peptide-mediated The structural diagram of the guided cyclic peptide modification targeting chimera, and Fig. 25B is the verification of the degradation effect on the protein.
  • Fig. 26 shows the transmembrane staining verification diagram of the modular assembly of polypeptides or microproteins mediated by cell-penetrating peptides in Figs. 12-14.
  • Fig. 27 shows the transmembrane staining verification diagram of the peptide or microprotein modular assembly targeting chimera mediated by cell-penetrating peptides in Fig. 15 and Fig. 16 .
  • Fig. 28 shows the transmembrane staining verification diagram of the peptide or microprotein modular assembly targeting chimera mediated by cell-penetrating peptides in Fig. 17 and Fig. 18.
  • Fig. 29 shows the transmembrane staining verification diagram of the modular assembly of polypeptides or microproteins mediated by cell-penetrating peptides in Fig. 19 and Fig. 20 .
  • Figures 30-32 show schematic representations of the structure of the staple peptide + small molecule ligand chimera. Wherein, part A of Fig. 30 is connected with part A of Fig. 31, part B of Fig. 31 is connected with part B of Fig. 32, and the whole is a chimeric compound structure containing staple peptide.
  • Figures 33-35 show schematic representations of the structures of cyclic peptide + small molecule ligand chimeras. Among them, part A of Figure 33 is connected to part A of Figure 34, part B of Figure 33 is connected to part B of Figure 34, part D of Figure 34 is connected to part D of Figure 35, and the whole is a chimera containing a cyclic peptide Compound structure.
  • Cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera including at least one penetrating peptide module, at least one targeting polypeptide module and at least one small molecule ligand module connected to each other, said The targeting polypeptide module is a polypeptide sequence that can bind to the targeting target protein.
  • the chimeric molecule formed by penetrating peptide + targeting polypeptide + small molecule ligand can effectively target the small molecule ligand to the pathogenic target protein through the targeting polypeptide, so as to achieve the pertinence and specificity of drug treatment.
  • various diseases especially tumor diseases
  • it can exert more stable and wider effects.
  • penetrating peptide module There is one penetrating peptide module, one, two, three or even multiple targeting polypeptide modules, and one, two, three or even multiple small molecule ligand modules.
  • the modular assembly of polypeptides or microproteins mediated by cell-penetrating peptides also includes at least one linker module in the targeting chimera, which is used for chimeric targeting polypeptide modules and small molecule ligand modules. There can be one, two, three or even multiple linker modules.
  • the membrane-penetrating peptide module is linked to the free end of the targeting polypeptide module and used to guide the targeting chimera to penetrate the cell membrane.
  • the small molecule ligand module is a small molecule E3 ligand that can bind to E3 ligase
  • the protease degradation agent adapted to the small molecule E3 ligand is one or more of CRBN, VHL, and IAP.
  • the amino acid sequence of the penetrating peptide module is any one of SEQ ID No.1-SEQ ID No.3.
  • SEQ ID No.1 YGRKKRRQRRR;
  • SEQ ID No. 3 RQIKIWFQNRRMKWK.
  • the amino acid sequence of the targeting polypeptide module is any one or more of SEQ ID No.4-SEQ ID No.17.
  • SEQ ID No.4 SAIGKIQDSLSSTAS;
  • SEQ ID No. 14 MESFPGWNLV(homoR)IGLLR.
  • SEQ ID No. 17 LYDVAGSDKY.
  • the linker module is a small molecule compound, and its structural formula is shown in formula I;
  • Cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera its structure is any one or more of the following structures:
  • the targeting polypeptide module in the targeting chimera also includes modified stapled peptide sequences or circular peptide sequences.
  • Cell-penetrating peptide-mediated peptide or microprotein modular assembly targeting chimera containing cyclic peptides its structure is as follows: cyclic peptide of formula VI + linker Linker of formula I + small structure of formula II molecular ligand.
  • Example 1 The cell-penetrating peptide-mediated modular assembly of polypeptides or microproteins targeting chimeras in Example 1 can be used to prepare products that degrade target proteins or degrade products that target target proteins with mutated amino acid positions .
  • Targeted degradation proteins include new crown S protein HR2, new crown N protein, new crown M protein, new crown E protein, new crown Orf6 protein, Lag-3 protein, Her2 protein, SHP-2 protein, STAT5B protein, MUC16 protein, CTLA-4 protein , PCSK9 protein, PD-1 protein, PD-L1 protein, and one or more of KRAS protein G12V mutations.
  • Targeting, inhibiting and drug-treating proteins involved in protein-protein interactions has so far been almost impossible with the help of inhibitor molecules.
  • Targeting harmful/pathogenic proteins using the proteosome degradation machinery is a promising therapeutic approach.
  • the target protein-protein interaction PPI
  • PPI target protein-protein interaction
  • the inventors devised this method of bifunctional peptidyl degraders, which target and degrade target proteins involved in PPIs.
  • the inventors combined the peptide with high affinity and selective interaction with the target protein to the E3 ligase, and realized the degradation of the expected target protein by means of the linker.
  • the inventors further combined the peptide degrader sequence with a cell-permeable peptide (cell penetrating peptide).
  • a cell-permeable peptide cell penetrating peptide
  • peptide degraders can degrade >15,000 targets involved in protein-protein interactions, and these ligands can be coupled to about 1,100 linkers with the help of corresponding targeting ligands/peptides, (of which about 300 PEG-type linkers), about 65 E3 ligase-binding ligands.
  • targeting ligands/peptides of which about 300 PEG-type linkers
  • 65 E3 ligase-binding ligands about 65 E3 ligase-binding ligands.
  • the inventors further combined the peptide PROTAC technology with about 800 cell penetrating peptides.
  • SMILES Simple molecular input line entry specification
  • simplified molecular linear input specification is a specification that clearly describes the molecular structure with ASCII strings
  • InChI Key International Compound Identification
  • InCHI code is the international pure
  • the unique identification code of the chemical structure of each compound given by the International Union of Pure and Applied Chemistry (IUPAC) can be found in the PubMed ChemCompound database through the InChI key (https://www.ncbi.nlm.nih.gov /pccompound) is easy to find its unique corresponding compound.
  • Table 1 shows the selection of the linker Linkers module, including but not limited to the molecular structure represented by SMILES and the compound corresponding to the InChI key, as shown in Table 1 below.
  • Table 2 shows another part of the linker Linkers module, that is, the selection of PEG type linkers, including but not limited to the compounds represented by "name” and corresponding to EnamineStore ID, see Table 2 below for details.
  • EnamineStore is a compound database (URL is https://www.enaminestore.com/search).
  • Table 3 shows the selection of the E3 ligase binding ligand module, including but not limited to the molecular structure represented by SMILES and the compound corresponding to the InChI key, see Table 3 below for details.
  • Table 4 shows the selection of cell-penetrating peptide sequence modules, including but not limited to the sequence indicated by "cell-penetrating peptide sequence", see Table 4 below for details.
  • Table 5 shows an example of the selection of target peptides, including but not limited to the target protein indicated by "target protein name” and the peptide sequence corresponding to the target protein; there are about 19813 target proteins in total, including the existing known All target proteins and all target polypeptides targeting these target proteins, but due to their large size, the inventors only screened a few dozen representative target proteins as an example, but the requirements of the present invention
  • the content of protection is all target proteins and targeting polypeptides known in the art, not limited to these dozens of target proteins, as shown in Table 5 below.
  • e3ligand in Table 3 represents the small molecule ligands of all E3 ligases that can be applied at present.
  • linkers There are two types of linkers, one is the “PEG linkers” shown in Table 2, and the other linkers are It is collected in Table 1 "linkers”, “CPP list” Table 4 is all the currently available penetrating peptides, and Table 5 “Target interacting peptide” is an example of targeted peptides for all currently applicable targets.
  • the CePPiTAC technology changes the part of the target protein from a small molecule to a polypeptide, and connects the penetrating peptide sequence to ensure its The complex can enter the cell membrane, because any target protein can be screened out and combined with a resistant polypeptide that is connected to it, so it can theoretically degrade any target protein and allow proteases to degrade it, so the application market is extremely broad.
  • the "non-targetable" target protein of the drug may be degraded by the drug developed by it.
  • it is very convenient to screen ligand peptides.
  • enzyme ligands which are very simple to connect with polypeptides, will be very convenient to use the invention to design drugs, which can save time and effort, and develop various new drugs quickly and economically.
  • the inventors also efficiently degrade the target by combining the target binding peptide with two or more different E3 ligase conjugates as shown below. Specifically shown in Figure 2.
  • the inventors also contemplated degrading multiple targets involved in the formation of disease-causing protein-protein complexes to inhibit the entire pathogenic pathway and completely inhibit a specific disease by degrading multiple targets.
  • the inventors designed the peptide degrading agent as shown in FIG. 3 .
  • lenalidomide 2 200 mg, 0.77 mmol was added to a round bottom flask containing succinic anhydride 3 (90 mg, 0.93 mmol) in toluene (8 mL) and equipped with a reflux condenser. The mixture was refluxed for 3 hours, the precipitate was separated by vacuum filtration, the filter cake was washed with ethyl acetate (20mLx2), and dried in vacuum to obtain 4-(2-(2,,6-dioxopiperidin-3-yl)-1-oxoiso Indol-4-yl)amino)-4-oxobutanoic acid product 4. Yield: 120 mg, 43.4%.
  • the product was isolated in 10 ml from the resin containing trifluoroacetic acid, triisopropylsilane and water (95:2.5:2.5) to provide 120 mg of crude peptide. Purification by reverse-phase high-performance liquid chromatography afforded 10 mg of diastereomeric mixture 5 with a maximum purity of 97.15% and a purity of 93.19% at 214 nm. Yield: 10 mg, 6.68%.
  • Table 6 shows the preparatory conditions for HPLC.
  • Table 7 is shown as a gradient table.
  • Embodiment 6 is a diagrammatic representation of Embodiment 6
  • the target proteins of many existing diseases are membrane proteins, such as PD-1 and PD-L1. Although their inhibitors are very commonly used, their effectiveness is not high and they are prone to drug resistance, because PD-1 and PD-L1 target The point protein is on the cell membrane, and small molecule PD-1/PD-L1 degradation agents are difficult to develop, but the technology of the present invention can target the intracellular part of these two proteins to degrade them.
  • Some virus-related proteins such as new coronavirus or HIV virus-related proteins, because traditional virus drugs focus on neutralizing antibodies or virus-inhibiting proteases to inhibit viruses, there are few targets, and once the virus mutates, so The drugs developed will be for naught.
  • the CePPiTAC technology provided by the present invention can select viral structural proteins or proteases, use polypeptides to bind and target them, and then degrade them, so that protein synthesis is damaged or virus packaging cannot be formed, which will first expand the target of viral drug research and development. According to the number of points, many viral proteins that could not be targeted in the past can be targeted. Secondly, because it can bind to the structural protein part that is not easy to mutate and then degrade the entire target protein, it can be used without fear of any virus mutation.
  • Embodiment 7 is a diagrammatic representation of Embodiment 7:
  • the inventor In order to verify that the targeting chimera provided by the application is degraded rather than inhibited, the inventor also designed and added protease inhibitor MG132, which can inhibit the effect of the targeting chimera of the application, through the experimental results (Fig. 7C) It can be seen that the protein that only added the targeting chimera still cannot be expressed, but the protein after adding the targeting chimera+MG132 can be expressed normally, and its expression level is the same as that of the protein without the targeting chimera The expression levels were comparable, suggesting that the targeting chimera was degrading rather than inhibiting the protein.
  • the S protein on the surface of the coronavirus mediates the infection process of the virus to the target cell, which is composed of two subunits, S1 and S2.
  • the S1 subunit is responsible for binding to the receptor on the cell surface
  • the S2 subunit is responsible for The function of the virus to fuse with the cell membrane.
  • the S2 subunit contains important functional regions such as the heptad repeat domain 1 (HR1) and the heptad repeat domain 2 (HR2).
  • HR1 and HR2 fold to form a six-helix bundle structure (6HB) to draw the viral membrane closer
  • 6HB six-helix bundle structure
  • the targeting chimera can bind to the HR2 subunit of the new coronavirus S protein and degrade it, inhibiting the formation of the six-helix bundle structure, thereby interfering with the fusion of the virus and the cell membrane, preventing the virus from invading cells, and fundamentally achieving the prevention and treatment of the new coronavirus. the goal of.
  • Embodiment 8 is a diagrammatic representation of Embodiment 8
  • the inventors also provided methods for degrading the new crown N protein, the new crown M protein, the new crown E protein, the new crown Orf6 protein, the Lag-3 protein, and the Her2 protein.
  • the modular targeting chimera used to degrade the new crown N protein, its membrane-penetrating peptide module sequence is RRRRRRRRR; the targeting peptide module sequence is PQEESEEEVEEP; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
  • the E3 small molecule ligand module is the E3 ligand of the target VHL, and the structural formula is
  • the modular targeting chimera used to degrade the M protein of the new crown, its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is PQEESEEEVEEP; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
  • the E3 small molecule ligand module is the E3 ligand of the target IAP, and the structural formula is
  • the modular targeting chimera used to degrade the new crown E protein, its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is GGKGLGKacGGA; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
  • the E3 small molecule ligand module is the E3 ligand of the target CRBN, and the structural formula is
  • the modular targeting chimera used to degrade the new crown Orf6 protein, its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is DTMVGWDKDARTK; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
  • the E3 small molecule ligand module is the E3 ligand of the target VHL, and the structural formula is
  • Modular targeting chimera for degrading Lag-3 protein its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is FNGARSFIDI; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is The E3 small molecule ligand module is the E3 ligand of the target CRBN, and the structural formula is
  • the modular targeting chimera used to degrade Her2 protein, its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is WARLWNYLYR; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
  • the E3 small molecule ligand module is the E3 ligand of the target VHL, and the structural formula is
  • the modular targeting chimera used to degrade SHP-2 protein, its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is RSFIDIGSGT; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
  • the E3 small molecule ligand module is the E3 ligand of the target CRBN, and the structural formula is
  • Modular targeting chimera for degrading STAT5B protein its membrane-penetrating peptide module sequence is YGRKKRRQRRR; targeting peptide module sequence is KAVDG(p)YVKPQI; linker Linker module is a small molecule composed of (PEG)4, structural formula for The E3 small molecule ligand module is the E3 ligand of the target IAP, and the structural formula is
  • the modular targeting chimera used to degrade MUC16 protein, its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is WIDPVNGDTE; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
  • the E3 small molecule ligand module is the E3 ligand of the target VHL, and the structural formula is
  • Modular targeting chimera for degrading CTLA-4 protein its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is ARHPSWYRPFEGCG; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is The E3 small molecule ligand module is the E3 ligand of the target VHL, and the structural formula is
  • Modular targeting chimera for degrading PCSK9 protein its membrane-penetrating peptide module sequence is RQIKIWFQNRRMKWK; targeting peptide module sequence is MESFPGWNLV(homoR)IGLLR; linker Linker module is a small molecule composed of (PEG)4, structural formula for The E3 small molecule ligand module is the E3 ligand of the target IAP, and the structural formula is
  • Modular targeting chimera for degrading PD-1 protein its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is FNWDYSLEELREKAKYK; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
  • the E3 small molecule ligand module is the E3 ligand of the target CRBN, and the structural formula is
  • the modular targeting chimera used to degrade PD-L1 protein, its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is MPIFLDHILNKFWILHYA; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
  • the E3 small molecule ligand module is the E3 ligand of the target CRBN, and the structural formula is
  • the Western Blot method was also used to verify the effect of different dosages (nmol) of targeted chimeras on the new crown N protein, new crown M protein, new crown E protein, new crown Orf6 protein, Lag-3 protein, Her2 protein, SHP-2 protein, STAT5B protein , MUC16 protein, and CTLA-4 protein degradation effects (Figure 8B- Figure 17B), it can be seen that with the increase of dosage, the protein has been degraded (no expression).
  • the inventor also designed and added a protease inhibitor MG132, which can inhibit the effect of the targeting chimera of the application, through the experimental results ( (Fig. 8C-Fig. 16C and Fig. 18B-20B) It can be seen that the protein only adding targeting chimera still cannot be expressed, but the protein after adding targeting chimera + MG132 can be expressed normally, and its expression level is the same as that of The expression level of protein without targeting chimera was comparable, which indicated that the function of targeting chimera was to degrade rather than inhibit protein.
  • MG132 protease inhibitor
  • the new coronavirus N protein which is abundant in the coronavirus, is a highly immunogenic protein that participates in genome replication and regulation of cell signaling pathways. Through this application, targeting chimeras to degrade this protein can effectively inhibit the new coronavirus and treatment.
  • the M protein of the new crown is a membrane glycoprotein (M, Membrane Protein), which is an integral part of the viral particle envelope.
  • M membrane glycoprotein
  • S, E, N proteins structural proteins
  • New crown E protein (E, Envelope Protein) is a component of the virus particle envelope, a small envelope glycoprotein, the main function of E protein is to protect the RNA gene chain inside the virus, and degrade this protein by targeting chimeras, It can reduce or even remove the protective mechanism of viral RNA, making the RNA chain easier to break, thereby effectively inhibiting the function of the virus.
  • the new coronavirus Orf6 protein is the most toxic protein to human cells among the new coronavirus proteins.
  • Existing studies have found that it can kill about half of human cells after being introduced into human cells, and it can effectively inhibit the innate immunity of host cells Activity, by targeting the chimera to degrade this protein, can greatly reduce the toxicity of the new coronavirus to the human immune system.
  • Lag-3 protein, lymphocyte activation gene 3, also known as CD233, is a type I transmembrane protein belonging to the immunoglobulin (Ig) superfamily, mainly expressed on the surface of activated T cells and NK cells, LAG-3 is a It is a very promising immunotherapy target. By targeting chimera to degrade this protein, it can effectively block the inhibitory signal in the interaction between tumor cells and TIL in the tumor microenvironment, and restore the immune surveillance function of TIL on tumor cells. , to achieve anti-tumor effect.
  • Ig immunoglobulin
  • Her2 protein is a transmembrane protein with tyrosine protein kinase activity and belongs to one of the EGFR family members. HER2 gene amplification is one of the most important factors affecting the growth and metastasis of breast cancer. It can be degraded by targeting chimeras This protein can promote the apoptosis and inhibit the proliferation of breast tumors.
  • SHP-2 protein encoded by protein tyrosine phosphatase nonreceptor 11 (PTP nonreceptor 11, PTPN11), SHP2 is a well-validated PTP oncoprotein in humans and is becoming an important target for cancer therapy, hyperactivation of SHP2 It plays a crucial role in pathogenicity. By targeting chimera to degrade this protein, it can effectively block or inhibit the activation of SHP2 pathway, thereby significantly improving tumor treatment.
  • PTP nonreceptor 11 protein tyrosine phosphatase nonreceptor 11
  • STAT5B protein is signal transducer and transcription activator-5b.
  • STAT signal is a regulatory signal of various tumors. By targeting chimera to degrade this protein, it can effectively cause the disorder of STAT signal, thereby inhibiting tumor cells (such as Osteosarcoma cells) proliferation and colony formation, and induce G0/G1 phase cell arrest and apoptosis.
  • tumor cells such as Osteosarcoma cells
  • MUC16 protein the largest transmembrane mucin, is a well-recognized serum biomarker for ovarian cancer because it is known to be overexpressed on the surface of ovarian cancer cells and split/shedding into the blood.
  • MUC16 is also It is believed to play an anti-apoptotic role in cancer cells, and the ectopic expression of its C-terminal domain induces resistance to cisplatin in ovarian cancer cells, which is mediated by inhibiting p53, by targeting chimeric Degradation of this protein can effectively regulate the apoptosis of cancer cells and inhibit their proliferation.
  • CTLA-4 protein cytotoxic T lymphocyte-associated protein 4, also known as CD152 (cluster of differentiation 152), is a protein receptor that functions as an immune checkpoint and downregulates immune responses, mutations in the CTLA-4 gene, Linked not only to cancer but also to type 1 diabetes, Graves' disease, Hashimoto's thyroiditis, celiac disease, systemic lupus erythematosus, thyroid-associated orbitopathy, primary biliary cirrhosis, and other autoimmune diseases , after degrading this protein by targeting the chimera, it can effectively increase the immune activity of the body.
  • CD152 cluster of differentiation 152
  • Embodiment 9 is a diagrammatic representation of Embodiment 9:
  • the inventors designed a modular targeting chimera for degrading KRAS proteins with G12V mutations (Fig. 21A), wherein the membrane-penetrating peptide module sequence is RRRRRRRRR; the targeting peptide module sequence is LYDVAGSDKY; the linker Linker module is a small molecule composed of (PEG)4, and the structural formula is The E3 small molecule ligand module is the E3 ligand of the target IAP, and the structural formula is
  • the Western Blot method was also used to verify the degradation effect of different dosages (nmol) of targeting chimeras on the KRAS protein with G12V mutation, and the expression level was counted (Figure 21B). It can be seen that as the dosage increases, The protein has been degraded (expression gradually decreased).
  • the inventors also used Western Blot method to verify the degradation effect of different dosages (nmol) of targeting chimera on unmutated KRAS protein (wild type) and the effect on The expression level was counted ( Figure 21C), and it can be seen that with the increase of the dosage, the protein change is not obvious (the expression level is slightly reduced, but not obvious), which fully demonstrates that the targeting chimera of this design can accurately target To the KRAS protein with G12V mutation, it will basically not degrade the wild-type KRAS protein, and the targeted degradation accuracy is extremely high.
  • KRAS Kersten Rat Sarcoma Viral Oncogene Homolog gene is a GDP/GTP-binding protein. KRAS is activated when combined with GTP, and closed when combined with GDP. KRAS can be temporarily activated by growth factors or tyrosine kinases (such as EGFR). The latter KRAS can activate downstream such as the PI3K-AKT-mTOR signaling pathway that controls cell production, and the RAS-RAF-MEK-ERK signaling pathway that controls cell proliferation. Mutant KRAS will continue to be activated even in the absence of EGFR and other kinase activation , resulting in continued proliferation of cells and eventually canceration.
  • EGFR tyrosine kinases
  • KRAS mutations are found in a variety of tumors, the most common ones include lung cancer and pancreatic cancer. KRAS protein with G12V mutation can be accurately degraded by targeting chimera, but has no effect on wild-type KRAS protein, which greatly improves the efficiency of targeted treatment of mutated protein.
  • the inventors designed a dual E3 ligand modular targeting chimera for degradation of PCSK9 (Figure 22A), wherein the membrane-penetrating peptide module sequence is RQIKIWFQNRRMKWK; the targeting peptide module sequence is MESFPGWNLV(homoR)IGLLR; the Linker module is connected to two different E3 small molecule ligand modules through two linkers, and the two different The E3 small molecule ligand modules are the E3 ligands of the target CRBN and IAP respectively, and the overall structural formula of the linker module and the E3 small molecule ligand module is
  • the inventor also designed and added a protease inhibitor MG132, which can inhibit the effect of the targeting chimera of the application, through the experimental results ( Figure 22B) It can be seen that the protein that only added the targeting chimera still cannot be expressed, but the protein after adding the targeting chimera + MG132 can be expressed normally, and its expression level is the same as that of the protein without targeting chimera Protein expression was comparable, suggesting that the targeting chimera was degrading rather than inhibiting the protein.
  • MG132 protease inhibitor
  • the targeting chimera using double E3 ligands has a lower dosage under the same degradation effect (from 25nmol to 15nmol); and this kind of targeting chimera adopts double E3 ligands, after one E3 ubiquitinase connected therein mutates and produces drug resistance, another E3 ubiquitinase can continue to play a role, increasing The reliability of the targeting chimera was improved.
  • Target modular targeting chimera ( Figure 23A)), wherein the membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequences are respectively SAIGKIQDSLSSTAS and PQEESEEEVEEP; the linker Linker module is a small molecule composed of (PEG) 4, the structural formula for The E3 small molecule ligand module is the E3 ligand of the target CRBN, and the structural formula is
  • HR2 protein and N protein can be degraded at the same time.
  • the inventors can also design targets for three targets, four targets or even more targets. It can degrade multiple proteins at the same time to achieve better applicability and wider use conditions. Considering the space limit of the application, it will not be repeated here, but it should not be because of the duality described in this example. The technical protection of more target-targeted chimeras is limited.
  • the Western Blot method was also used to verify the degradation effect of different dosages (nmol) of targeting chimeras on PD-L1 protein ( Figure 24B and Figure 25B). It can be seen that with the increase of dosage, the protein has been degraded (expression level Gradually decreases).
  • the principle of the modification of the staple peptide and the cyclic peptide is that the targeting peptide module can be presented in a state similar to that of the secondary structure of the imitated protein, forming a "miniature protein", which can still be avoided without connecting the membrane-penetrating peptide module
  • the decomposition of the peptide segment during the membrane entry process effectively increases the stability of the targeting peptide module.
  • the modification process of the staple peptide is: use the two compounds R 8 and S 5 (structure as follows) to modify and link CGIQDTNSKKQSDTHLEETC, so that the polypeptide becomes: CGIQDT(R8)NSKKQS(S5)DTHLEET- ;
  • R8 is Fmoc-R8-OH, the structural formula is as follows:
  • S5 is Fmoc-S5-OH, the structural formula is as follows:
  • the targeting chimera containing staple peptide has a simplified structural formula:
  • the linker module is a small molecule composed of (PEG)4, and its structural formula is
  • the E3 small molecule ligand module is the E3 ligand of the target CRBN, and the structural formula is Cyclization method: The two cysteine disulfide bonds in the above figure form a ring.
  • a PROTAC peptide induces durable ⁇ -catenindegradation and suppresses Wnt-dependentintestinal cancer" (Hongwei Liao1, Xiang Li2, Lianzheng Zhao1, Yalong Wang1, Xiaodan Wang1, Ye Wu2, Xin Zhou3, Wei Fu3, Lei Liu 4, 2 Hong-Gang Hu Ye-Guang Chen1, “Cell Discovery", 2020).
  • Comparative Document 1 and Comparative Document 2 disclosed: polypeptide membrane-penetrating peptide + target-targeting polypeptide + polypeptide Linker + polypeptide Binder, and Comparative Document 1 had no obvious effect on target protein degradation until 50 ⁇ m (Figure 3); Document 2 has no obvious effect on the target protein until the size is 100 ⁇ m ( Figure 2).
  • Comparative Document 3 discloses: staple peptide + polypeptide Linker + polypeptide Binder composition, Comparative Document 3 does not have significant degradation effect on the target until it reaches 70 ⁇ m ( Figure 1).
  • the targeting chimera provided by the technical solution of the present invention can achieve the degradation of the target protein at the nm level, as shown in Table 8 for the specific comparison.
  • Example 7 new crown S protein HR2, Figure 7) 50-100nmol Example 8 (new crown N protein, Figure 8) 50-100nmol Example 8 (new crown M protein, Figure 9) 50-100nmol Example 8 (new crown E protein, Figure 10) 10-100nmol Example 8 (new crown Orf6 protein, Figure 11) 50-100nmol Embodiment 8 (Lag-3 protein, Fig. 12) 75-100nmol Embodiment 8 (Her2 protein, Fig. 13) 10-100nmol Embodiment 8 (SHP-2 protein, Fig. 14) 10-100nmol Embodiment 8 (STAT5B protein, Fig. 15) 75-100nmol Embodiment 8 (MUC16 protein, Fig.
  • the targeting chimera provided by the present application can degrade the target protein in the order of nmol (the highest is 100 nmol, that is, 0.1 ⁇ mol), while in Comparative Literature 1-3, the lowest is also in Only on the order of 50 ⁇ mol can it produce obvious degradation effect on the target protein, and the difference in the amount of degradation agent used between the two is at least 500 times, which has a very obvious difference in effect.

Abstract

Provided are a technology for modular assembly of a cell-penetrating peptide-mediated polypeptide or microprotein targeting chimera, and the use thereof. The targeting chimera comprises at least one cell-penetrating peptide module, at least one targeting polypeptide module and at least one small molecule ligand module which are mutually connected, wherein the targeting polypeptide module is a polypeptide sequence capable of binding to a targeting target protein. The technology has the following characteristics and advantages of the provided cell-penetrating peptide-mediated polypeptide or microprotein modularly-assembled targeting chimera being designed in a modular manner, virous sequences or small molecule compound modules having different functions being able to be replaced and overlapped according to requirements, and all the polypeptide module parts being able to be subjected to cyclization or secondary microprotein structure modification. The design idea greatly enhances the use effect and application range of a targeting drug.

Description

以细胞穿膜肽介导的多肽或微蛋白靶向嵌合物的模块化组装技术及其应用Modular assembly technology of peptide or microprotein targeting chimera mediated by cell-penetrating peptides and its application 技术领域technical field
本发明涉及生物工程领域，具体涉及以细胞穿膜肽介导的多肽或微蛋白靶向嵌合物的模块化组装技术及其应用。The invention relates to the field of bioengineering, in particular to the modular assembly technology of polypeptide or microprotein targeting chimera mediated by cell-penetrating peptides and its application.
背景技术Background technique
蛋白降解剂技术近几年来风行世界，其中最早也是最流行的是蛋白降解靶向嵌合体(Proteolysis targeting Chimera，，PROTAC)技术，在结构上，PROTAC包括三个部分：一个小分子E3泛素连接酶配体和一个小分子靶蛋白配体，两个活性配体通过特殊设计的“Linker”结构连接在一起，最终形成了小分子三联体的“PROTAC”的活性形式，其结构为：小分子配体(靶向目标蛋白)+linker+小分子配体(与E3连接酶结合)。靶点蛋白与小分子靶蛋白配体结合，同时E3连接酶配体也由其配体结合，由E3连接酶给靶点蛋白加上泛素标签，然后经过多轮泛素化后就有了多个泛素标签，多聚泛素化之后的靶点蛋白会被蛋白酶体识别并被降解。Protein degrader technology has been popular in the world in recent years, among which the earliest and most popular is the protein degradation targeting chimera (Proteolysis targeting Chimera, PROTAC) technology. Structurally, PROTAC includes three parts: a small molecule E3 ubiquitin linker The enzyme ligand and a small molecule target protein ligand, the two active ligands are connected together through a specially designed "Linker" structure, and finally form the active form of the small molecule triplet "PROTAC", its structure is: small molecule Ligand (targeting target protein) + linker + small molecule ligand (binding to E3 ligase). The target protein binds to the small molecule target protein ligand, and at the same time the E3 ligase ligand is also bound by its ligand. The E3 ligase adds a ubiquitin tag to the target protein, and then after multiple rounds of ubiquitination, there is With multiple ubiquitin tags, the target protein after polyubiquitination will be recognized and degraded by the proteasome.
PROTAC技术被成功的应用到多种病变蛋白的诱导降解中。在自然情况下，E3泛素连接酶需要一个特殊的识别信号来招募并泛素化它的目标蛋白。而PROTAC技术的出现使E3泛素化任何一个蛋白成为可能。这项技术设计一个双重功能的分子，其一端可以结合目标蛋白，另一端结合E3连接酶，并将两者形成一个聚合物。此时E3就能够泛素化目标蛋白并引导其进入降解通路。靶向蛋白降解最有吸引力的地方在于它可以针对那些传统上认为不可成药的蛋白靶点，这些蛋白可能占了人类蛋 白质组的80％以上。由于靶向蛋白降解策略可以通过结合蛋白上的几乎任何一个位点，而不是活性位点，来达到选择性的降解蛋白的目的，因此理论上这个策略可以用于任何一个蛋白质。PROTAC technology has been successfully applied to the induced degradation of various diseased proteins. In nature, E3 ubiquitin ligase requires a specific recognition signal to recruit and ubiquitinate its target protein. The emergence of PROTAC technology makes it possible for E3 to ubiquitinate any protein. This technique designs a dual-functional molecule that binds a target protein at one end and an E3 ligase at the other end, and forms a polymer of the two. At this time, E3 can ubiquitinate the target protein and guide it into the degradation pathway. The most attractive aspect of targeting protein degradation is that it can target protein targets that have traditionally been considered undruggable, and these proteins may account for more than 80% of the human proteome. Since the targeted protein degradation strategy can achieve the purpose of selectively degrading proteins by binding to almost any site on the protein instead of the active site, this strategy can theoretically be used for any protein.
但在实际研究或操作中，以上的小分子“三联体”PROTAC，与靶点蛋白相结合的小分子配体部分，筛查难度极大，很多靶点由于无法找到合适的配体而无法开发出针对性的药物，而且，自然界中，有很多蛋白靶点天然不适合小分子结合，因此可能导致小分子PROTAC的药物研发失败。However, in actual research or operation, the above small-molecule "triple" PROTAC, the small-molecule ligand part that binds to the target protein, is extremely difficult to screen, and many targets cannot be developed due to the inability to find suitable ligands. Moreover, there are many protein targets in nature that are not suitable for small molecule binding, which may lead to the failure of small molecule PROTAC drug development.
现有的治疗性药物主要集中在两类：小分子药物(small molecules)、蛋白类药物(biologics)。而这两类治疗性药物由于其自身生物物理性质的局限性，并不能有效覆盖这所有已确证的重要分子靶点。多肽类药物则是另一类引起人们广泛关注和兴趣的靶向分子。与生物大分子类似，多肽类分子对于靶点也有较高的结合力与选择性，相对于小分子类药物具有更小的脱靶效应。而多肽在体内的代谢产物为氨基酸，最大限度地降低了毒性。相较于小分子药物，多肽类药物具有不可比拟的优势，主要表现在多肽类分子的易修饰、靶点识别特异性、靶向范围广等方面。Existing therapeutic drugs are mainly concentrated in two categories: small molecules and biologicals. However, due to the limitations of their own biophysical properties, these two types of therapeutic drugs cannot effectively cover all the confirmed important molecular targets. Peptide drugs are another class of targeting molecules that have attracted widespread attention and interest. Similar to biomacromolecules, polypeptide molecules also have higher binding force and selectivity for targets, and have smaller off-target effects than small molecule drugs. The metabolites of polypeptides in the body are amino acids, which minimizes toxicity. Compared with small molecule drugs, peptide drugs have incomparable advantages, mainly in the easy modification of peptide molecules, target recognition specificity, and wide targeting range.
发明内容Contents of the invention
本发明的目的是提供一种可以靶向目标蛋白，从而可以有效降解靶向蛋白的以细胞穿膜肽介导的多肽或微蛋白靶向嵌合物的模块化组装技术及其应用，即用于靶向蛋白降解的穿膜肽偶联嵌合体技术(Cell-penetrating-peptide Induced Targeting Chimera，CePPiTAC)技术。The purpose of the present invention is to provide a modular assembly technology and its application of a polypeptide or microprotein targeting chimera mediated by cell-penetrating peptides that can target the target protein, thereby effectively degrading the targeted protein. Cell-penetrating-peptide Induced Targeting Chimera (CePPiTAC) technology for targeting protein degradation.
本发明的上述目的可采用下列技术方案来实现：Above-mentioned purpose of the present invention can adopt following technical scheme to realize:
本发明的第一个目的是提供了以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，包括有相互连接的至少一个穿膜肽模块、至少一个靶向多肽模块、一个(或没有)小分子Linker模块和至少一个小分子 配体模块，所述靶向多肽模块为可与靶向目标蛋白结合的多肽序列。The first object of the present invention is to provide a targeting chimera mediated by cell-penetrating peptide modular assembly of polypeptides or microproteins, including at least one penetrating peptide module, at least one targeting polypeptide module, One (or no) small molecule Linker module and at least one small molecule ligand module, the targeting polypeptide module is a polypeptide sequence that can bind to the target protein.
可选地，上述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，还包括有至少一个连接体Linker模块，所述靶向多肽模块与小分子配体模块通过连接体Linker模块进行嵌合。Optionally, the above-mentioned cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera also includes at least one linker Linker module, and the targeting polypeptide module and the small molecule ligand module pass through Connector Linker module for chimerism.
可选地，上述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，所述穿膜肽模块连接于靶向多肽模块的游离端并用于引导靶向嵌合物穿透细胞膜。Optionally, the above-mentioned cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera, the penetrating peptide module is connected to the free end of the targeting polypeptide module and used to guide the targeting chimera Penetrate the cell membrane.
可选地，上述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，所述小分子配体模块为可与E3连接酶结合的小分子E3配体；优选地，小分子E3配体适配的蛋白酶降解剂为CRBN(Cereblon蛋白，Cereblon protein)、VHL(希佩尔-林道，von Hippel-Lindau)、IAP(凋亡抑制蛋白，Inhibitor of apoptosis proteins)中的一种或多种。Optionally, the above-mentioned cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera, the small molecule ligand module is a small molecule E3 ligand that can bind to E3 ligase; preferably The protease degradation agent adapted to the small molecule E3 ligand is CRBN (Cereblon protein, Cereblon protein), VHL (Hippel-Lindau, von Hippel-Lindau), IAP (apoptosis inhibitory protein, Inhibitor of apoptosis proteins) one or more.
可选地，上述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，所述穿膜肽模块的氨基酸序列为SEQ ID No.1-SEQ ID No.3中的任意一条。Optionally, the above-mentioned cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera, the amino acid sequence of the penetrating peptide module is SEQ ID No.1-SEQ ID No.3 any one.
可选地，上述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，所述靶向多肽模块的氨基酸序列为SEQ ID No.4-SEQ ID No.17中的任意一条或多条。Optionally, the above-mentioned cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera, the amino acid sequence of the targeting polypeptide module is SEQ ID No.4-SEQ ID No.17 Any one or more.
可选地，上述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，所述连接体Linker模块为小分子化合物，其结构式如式I所示；Optionally, the above-mentioned cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera, the Linker module is a small molecule compound, and its structural formula is shown in formula I;
Figure PCTCN2022102648-appb-000001
Figure PCTCN2022102648-appb-000001
可选地，上述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，适配的蛋白酶降解剂为CRBN时，所述小分子配体模块的结构 式如式Ⅱ所示，
Figure PCTCN2022102648-appb-000002
适配的蛋白酶降解剂为VHL时，所述小分子配体模块的结构式如式Ⅲ所示，
Figure PCTCN2022102648-appb-000003
Figure PCTCN2022102648-appb-000004
适配的蛋白酶降解剂为IAP时，所述小分子配体模块的结构式如式Ⅳ所示，
Figure PCTCN2022102648-appb-000005
Optionally, when the above-mentioned cell-penetrating peptide-mediated modular assembly of polypeptides or microproteins targets chimeras, and the suitable protease degradation agent is CRBN, the structural formula of the small molecule ligand module is as shown in Formula II Show,
Figure PCTCN2022102648-appb-000002
When the suitable protease degradation agent is VHL, the structural formula of the small molecule ligand module is shown in formula III,
Figure PCTCN2022102648-appb-000003
Figure PCTCN2022102648-appb-000004
When the suitable protease degradation agent is IAP, the structural formula of the small molecule ligand module is shown in formula IV,
Figure PCTCN2022102648-appb-000005
可选地，上述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其结构为以下各结构中的任意一种或多种：Optionally, the above-mentioned cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera has any one or more of the following structures:
1)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.4+式I结构的连接体Linker+式Ⅱ结构的小分子配体；1) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.4+linker Linker with formula I structure+small molecule ligand with formula II structure;
2)穿膜肽SEQ ID No.2+靶向多肽SEQ ID No.5+式I结构的连接体Linker+式Ⅲ结构的小分子配体；2) Membrane-penetrating peptide SEQ ID No.2+targeting peptide SEQ ID No.5+linker Linker with formula I structure+small molecule ligand with formula III structure;
3)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.5+式I结构的连接体Linker+式Ⅳ结构的小分子配体；3) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.5+linker Linker with formula I structure+small molecule ligand with formula IV structure;
4)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.6+式I结构的连接体Linker+式Ⅱ结构的小分子配体；4) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.6+linker Linker with formula I structure+small molecule ligand with formula II structure;
5)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.7+式I结构的连接体Linker+ 式Ⅲ结构的小分子配体；5) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.7+linker Linker with formula I structure+small molecule ligand with formula III structure;
6)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.8+式I结构的连接体Linker+式Ⅱ结构的小分子配体；6) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.8+linker Linker with formula I structure+small molecule ligand with formula II structure;
7)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.9+式I结构的连接体Linker+式Ⅲ结构的小分子配体；7) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.9+linker Linker with formula I structure+small molecule ligand with formula III structure;
8)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.10+式I结构的连接体Linker+式Ⅱ结构的小分子配体；8) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.10+linker Linker with formula I structure+small molecule ligand with formula II structure;
9)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.11+式I结构的连接体Linker+式Ⅳ结构的小分子配体；9) Membrane-penetrating peptide SEQ ID No.1+targeting polypeptide SEQ ID No.11+linker Linker with formula I structure+small molecule ligand with formula IV structure;
10)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.12+式I结构的连接体Linker+式Ⅲ结构的小分子配体；10) Membrane-penetrating peptide SEQ ID No.1+targeting polypeptide SEQ ID No.12+linker Linker with formula I structure+small molecule ligand with formula III structure;
11)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.13+式I结构的连接体Linker+式Ⅲ结构的小分子配体；11) Membrane-penetrating peptide SEQ ID No.1+targeting polypeptide SEQ ID No.13+linker Linker with formula I structure+small molecule ligand with formula III structure;
12)穿膜肽SEQ ID No.3+靶向多肽SEQ ID No.14+式I结构的连接体Linker+式Ⅳ结构的小分子配体；12) Membrane-penetrating peptide SEQ ID No.3 + targeting polypeptide SEQ ID No.14 + linker Linker with formula I structure + small molecule ligand with formula IV structure;
13)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.15+式I结构的连接体Linker+式Ⅱ结构的小分子配体；13) Membrane-penetrating peptide SEQ ID No.1+targeting polypeptide SEQ ID No.15+linker Linker with formula I structure+small molecule ligand with formula II structure;
14)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.16+式I结构的连接体Linker+式Ⅱ结构的小分子配体；14) Membrane-penetrating peptide SEQ ID No.1+targeting polypeptide SEQ ID No.16+linker Linker with formula I structure+small molecule ligand with formula II structure;
15)穿膜肽SEQ ID No.2+靶向多肽SEQ ID No.17+式I结构的连接体Linker+式Ⅳ结构的小分子配体；15) Membrane-penetrating peptide SEQ ID No.2+targeting polypeptide SEQ ID No.17+linker Linker with formula I structure+small molecule ligand with formula IV structure;
16)穿膜肽SEQ ID No.3+靶向多肽SEQ ID No.14+式I结构的连接体Linker+(双E3配体：式Ⅱ结构的小分子配体+式Ⅲ结构的小分子配体)；16) Membrane-penetrating peptide SEQ ID No.3+ targeting polypeptide SEQ ID No.14+ linker Linker with formula I structure + (double E3 ligand: small molecule ligand with formula II structure + small molecule ligand with formula III structure );
17)穿膜肽SEQ ID No.1+(双靶点：靶向多肽SEQ ID No.4+靶向多肽SEQ ID No.5)+式I结构的连接体Linker+式Ⅱ结构的小分子配体。17) Membrane-penetrating peptide SEQ ID No.1+ (dual targets: targeting peptide SEQ ID No.4+targeting peptide SEQ ID No.5)+linker Linker with formula I structure+small molecule ligand with formula II structure .
可选地，上述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，所述靶向多肽模块还包括经过修饰的订书肽序列或环形肽序列，订书肽序列或环形肽序列具有入膜功能，此种情况下的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物可以没有穿膜肽。Optionally, the above-mentioned cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera, the targeting polypeptide module also includes a modified staple peptide sequence or cyclic peptide sequence, staple peptide Sequences or cyclic peptide sequences have the function of entering the membrane. In this case, the targeting chimera for modular assembly of polypeptides or microproteins mediated by cell-penetrating peptides may not have a membrane-penetrating peptide.
可选地，上述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，所述订书肽的结构式如式Ⅴ所示，Optionally, the above-mentioned cell-penetrating peptide-mediated modular assembly of polypeptides or microproteins targeting chimeras, the structural formula of the staple peptide is shown in formula V,
Figure PCTCN2022102648-appb-000006
Figure PCTCN2022102648-appb-000006
所述环形肽的结构式如式Ⅵ所示，The structural formula of the cyclic peptide is shown in formula VI,
Figure PCTCN2022102648-appb-000007
Figure PCTCN2022102648-appb-000007
可选地，上述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，含有订书肽的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其结构如下所示：式Ⅴ结构的订书肽+式I结构的连接体Linker+式Ⅱ结构的小分子配体。Optionally, the above cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera, the cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera containing staple peptide Its structure is as follows: Staple peptide with the structure of formula V + linker Linker with the structure of formula I + small molecule ligand with the structure of formula II.
可选地，上述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，含有环形肽的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其结构如下所示：式Ⅵ结构的环形肽+式I结构的连接体 Linker+式Ⅱ结构的小分子配体。Optionally, the above-mentioned cell-penetrating peptide-mediated modular assembly of polypeptides or microproteins targeting chimera, and the cell-penetrating peptide-mediated modular assembly of polypeptides or microproteins targeting chimeras containing cyclic peptides Its structure is as follows: cyclic peptide of formula VI + linker Linker of formula I + small molecule ligand of formula II.
本发明的第二个目的是提供了上述以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物在制备用于降解靶向目标蛋白的产品或用于降解存在变异氨基酸位点的靶向目标蛋白的产品中的应用。The second object of the present invention is to provide the above-mentioned cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera in the preparation of products for degrading the target protein or for degrading the presence of mutated amino acid positions. Point of view for the application of products targeting target proteins.
可选地，上述的应用，所述靶向的降解蛋白包括新冠S蛋白HR2、新冠N蛋白、新冠M蛋白、新冠E蛋白、新冠Orf6蛋白、Lag-3蛋白、Her2蛋白、SHP-2蛋白、STAT5B蛋白、MUC16蛋白、CTLA-4蛋白、PCSK9蛋白、PD-1蛋白、PD-L1蛋白、KRAS蛋白G12V变异中的一种或多种。Optionally, in the above-mentioned application, the targeted degradation protein includes new crown S protein HR2, new crown N protein, new crown M protein, new crown E protein, new crown Orf6 protein, Lag-3 protein, Her2 protein, SHP-2 protein, One or more of STAT5B protein, MUC16 protein, CTLA-4 protein, PCSK9 protein, PD-1 protein, PD-L1 protein, KRAS protein G12V variation.
基于上述技术描述，本发明的核心思路是以多个可自由变换的“模块”序列或小分子化合物连接成靶向性强、穿膜效果好、降解效率高的“模块化”靶向嵌合物。Based on the above technical description, the core idea of the present invention is to link multiple freely transformable "module" sequences or small molecule compounds into a "modular" targeting chimera with strong targeting, good membrane penetration and high degradation efficiency. thing.
本发明技术方案中，最基础的构成为一个穿膜肽模块、一个靶向多肽模块和一个小分子配体模块，三者相互连接，进一步构成可以为一个穿膜肽模块、一个靶向多肽模块、一个连接体Linker模块和一个小分子配体模块，形成穿膜肽-靶向多肽-连接体Linker-小分子配体的基本结构。In the technical solution of the present invention, the most basic composition is a membrane-penetrating peptide module, a targeting polypeptide module and a small molecule ligand module, the three are connected to each other, and further constitute a membrane-penetrating peptide module and a targeting polypeptide module , a linker Linker module and a small molecule ligand module, forming a basic structure of penetrating peptide-targeting polypeptide-linker Linker-small molecule ligand.
穿膜肽-靶向多肽-小分子配体的基础结构可将小分子配体直接导向至目标靶点蛋白，虽然此种基础结构下部分嵌合体是可以发挥靶向治疗特性的，但其具有一定缺陷，三者的连接并不稳定，容易脱落。The basic structure of penetrating peptide-targeting polypeptide-small molecule ligand can directly direct the small molecule ligand to the target protein. Although some chimeras under this basic structure can exert targeted therapeutic properties, they have Certain defects, the connection between the three is not stable, easy to fall off.
穿膜肽-靶向多肽-连接体Linker-小分子配体是上述基本结构的升级结构，其克服了靶向多肽穿膜性能差的缺陷，也克服了靶向多肽和小分子配体直接连接不稳定的缺陷，以穿膜肽穿膜，靶向多肽导向目标蛋白，从而实现了定向穿透的效果，采用连接体Linker连接二者，能够有效降低脱落几率；同时克服了穿膜性差和连接不稳定的两大缺陷，近乎完美的解决了靶向治疗的技术效果，提升了靶向效率，最重要的技术点在于， 此种最优结构中采用穿膜肽+靶向多肽“替换了”现有PROTAC技术中的靶向蛋白，靶向多肽可以大大扩展靶向目标蛋白的选择性，通过连接的小分子配体(E3)，几乎可以靶向降解已知的所有目标蛋白。Membrane-penetrating peptide-targeting polypeptide-linker Linker-small molecule ligand is an upgraded structure of the above basic structure, which overcomes the defect of poor membrane-penetrating performance of targeting polypeptide, and also overcomes the direct connection between targeting polypeptide and small molecule ligand For unstable defects, the membrane-penetrating peptide is used to penetrate the membrane, and the targeting polypeptide is directed to the target protein, thereby achieving the effect of directional penetration. The linker Linker is used to connect the two, which can effectively reduce the probability of falling off; at the same time, it overcomes the poor membrane penetration and connection The two major defects of instability have almost perfectly solved the technical effect of targeted therapy and improved the targeting efficiency. The most important technical point is that this optimal structure uses membrane-penetrating peptide + targeting polypeptide to "replace" The targeting protein in the existing PROTAC technology, the targeting polypeptide can greatly expand the selectivity of targeting the target protein, and through the attached small molecule ligand (E3), it can target and degrade almost all known target proteins.
穿透细胞膜进入细胞内是许多作用靶点在细胞内的生物大分子发挥作用的先决条件，然而生物膜的生物屏障作用阻止了许多高分子物质进入细胞内，从而很大程度地限制了这些物质在治疗领域的应用。因此，如何引导这些物质穿透细胞膜是一个迫切需要解决的问题。多肽做为一种蛋白质水解的中间产物，其穿膜性较差。近些年来随着技术的发展，逐渐发现人类免疫缺陷病毒(Human immunodeficiency virus，HIV)中的反式转录激活因子(Transactivator，，TAT)，能够有效穿过细胞膜进入细胞内，紧接着多种具有穿越细胞膜能力的蛋白被发现，并被命名为细胞穿膜肽(Cell-penetrating peptides，CPPs)。一般来说细胞穿膜肽通常是不超过30个氨基酸的多肽分子，能够不依赖特异膜受体独立穿过细胞膜。这些细胞穿透肽作为生物活性分子细胞内转运工具，与其它的离子导入、纳米载体等相比，，具有低毒、便捷、有效的特性，在药物开发中起到了越来越重要的作用，甚至有相关含有CPP的药物通过了FDA的临床实验。Penetrating the cell membrane and entering the cell is a prerequisite for many biological macromolecules with targets in the cell to function. applications in therapeutic areas. Therefore, how to guide these substances to penetrate the cell membrane is an urgent problem to be solved. As an intermediate product of protein hydrolysis, polypeptide has poor membrane penetration. In recent years, with the development of technology, it has been gradually discovered that the transactivator (Transactivator, TAT) in human immunodeficiency virus (Human immunodeficiency virus, HIV) can effectively pass through the cell membrane and enter the cell, followed by a variety of Proteins with the ability to cross cell membranes were discovered and named as cell-penetrating peptides (CPPs). In general, cell-penetrating peptides are polypeptide molecules of no more than 30 amino acids, which can independently pass through the cell membrane without relying on specific membrane receptors. These cell-penetrating peptides are used as intracellular transport tools for bioactive molecules. Compared with other iontophoresis and nanocarriers, they have the characteristics of low toxicity, convenience and effectiveness, and play an increasingly important role in drug development. There are even related drugs containing CPP that have passed FDA clinical trials.
本发明的穿膜肽偶联嵌合体技术(CePPiTAC)技术，是由可与靶点蛋白结合的多肽来“代替”普通“三联体”PROTAC结构中的小分子靶点蛋白配体部分，以此多肽来连接Linker与小分子E3配体，并加入穿膜肽序列，形成结构为：穿膜肽(穿膜肽)+多肽(靶向靶点)+Linker+E3配体，必要时可以去除Linker，使包含穿膜肽的多肽(靶向靶点)直接连接E3配体。The penetrating peptide coupling chimera technology (CePPiTAC) technology of the present invention is to "replace" the small-molecule target protein ligand part in the ordinary "triple" PROTAC structure with a polypeptide that can bind to the target protein. Peptides are used to connect the Linker and the small molecule E3 ligand, and the penetrating peptide sequence is added to form a structure: penetrating peptide (penetrating peptide) + polypeptide (targeting target) + Linker + E3 ligand, and the Linker can be removed if necessary , so that the polypeptide (targeting target) containing the penetrating peptide is directly linked to the E3 ligand.
本发明所合成的药物为多肽与小分子的复合物，其间可由小分子linker连接，也可以去除，在多肽的非小分子连接段，可以加入入膜多肽 序列，此序列可以将多肽与小分子的复合物(CePPiTAC复合物)带入至细胞内部，此时靶向目标蛋白的多肽部分即可与目标蛋白结合，而linker另一端的小分子E3配体，可结合E3连接酶并引发E3泛素酶反应，将目标蛋白泛素化，从而使细胞内的26S蛋白酶可以识别目标蛋白，将其降解。The medicine synthesized by the present invention is a complex of polypeptide and small molecule, which can be connected by small molecule linker or removed. In the non-small molecule linking section of polypeptide, a membrane-entrying polypeptide sequence can be added, and this sequence can connect polypeptide and small molecule. The complex (CePPiTAC complex) is brought into the cell, at this time, the polypeptide part targeting the target protein can bind to the target protein, and the small molecule E3 ligand at the other end of the linker can bind the E3 ligase and trigger E3 ubiquitination The ubiquitination of the target protein by the enzyme reaction, so that the 26S protease in the cell can recognize the target protein and degrade it.
本发明的的特点及优点是：本发明提供的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，通过相互连接的穿膜肽、靶向多肽和小分子配体，能够穿透细胞膜并靶向定位所有靶向目标蛋白，所连接的小分子配体，可与固定的连接酶结合并引发泛素酶反应，从而将目标蛋白泛素化，使细胞内的蛋白酶可靶向识别目标蛋白，并降解目标蛋白，从而可更加广阔的筛选出靶向药物。由于是使用多肽模块结合靶点蛋白，因此在理论上，可以靶向所有靶点蛋白，这是以往其它降解剂技术所不能实现的。另外，由于本技术是结合高效小分子E3配体从而实现泛素化，比其它使用多肽配体的Peptide-based的PROTAC/degrader要高效的多，大部分情况下，可以在细胞实验中可以在nmol级水平实现对靶点蛋白的降解。The characteristics and advantages of the present invention are: the polypeptide or microprotein modular assembly targeting chimera provided by the present invention is mediated by cell-penetrating peptides, through the interconnected penetrating peptides, targeting polypeptides and small molecule ligands , can penetrate the cell membrane and target all targeted proteins, the connected small molecule ligand can bind to the immobilized ligase and trigger the ubiquitinase reaction, thereby ubiquitinating the target protein and making the protease in the cell Targeted recognition of target protein and degradation of target protein, so that targeted drugs can be screened out more broadly. Since the peptide module is used to bind the target protein, in theory, all target proteins can be targeted, which was not possible with other degrader technologies in the past. In addition, since this technology combines high-efficiency small molecule E3 ligands to achieve ubiquitination, it is much more efficient than other Peptide-based PROTAC/degraders that use peptide ligands. In most cases, it can be used in cell experiments. The degradation of the target protein is achieved at the nmol level.
此嵌合物结构中，还可加入连接体Linker，连接体Linker的加入可进一步固化靶向多肽和小分子配体的连接。In this chimeric structure, a linker Linker can also be added, and the addition of the linker Linker can further solidify the connection between the targeting polypeptide and the small molecule ligand.
同时，本发明所提供的靶向嵌合物，采用模块化设计，各个不同功能的序列或小分子化合物模块，能够根据需要进行替换和叠加，此种设计思路，大大增强了靶向药物的使用效果和适用范围。At the same time, the targeting chimera provided by the present invention adopts a modular design, and each sequence or small molecule compound module with different functions can be replaced and superimposed according to needs. This design idea greatly enhances the use of targeted drugs. effect and scope of application.
小分子三联体的PROTAC，所能开发针对的靶点有限，而Peptide-Based PROTAC/degrader，降解效率低，往往要在umol水平才能在细胞上对靶点进行降解，而本技术可以靶向所有靶点的同时，还可以实现nmol水平的高效降解(细胞降解)，真正实现“诸病皆有药”的目 的。同时，本发明还对以往技术理念有着颠覆性的意义，以病毒相关蛋白为例，以往针对病毒的相关靶点主要为病毒的感染人体细胞的相关蛋白(比如新冠的S蛋白)，以及病毒合成所需要的酶类等等，靶点选择比较少。但本技术使用多肽序列结合靶点的非活性位点，可以靶向所有病毒所有蛋白并降解，对克服病毒变异引起的耐药也有效果，大0大提高了病毒药物研发成功的可能性与便捷性。另外，本技术可以针对某靶点的单个变异进行降解，而没有些变异的野生型(Wild Type)同源蛋白则不会受到影响或影响较小，这也是其它如PROTAC等降解剂技术所不能实现的。Small-molecule triplet PROTACs can be developed for limited targets, while Peptide-Based PROTAC/degrader has low degradation efficiency and often needs to be at the umol level to degrade targets on cells, while this technology can target all At the same time as the target, it can also achieve efficient degradation (cellular degradation) at the nmol level, and truly realize the goal of "there are medicines for all diseases". At the same time, the present invention also has subversive significance to the previous technical concepts. Taking virus-related proteins as an example, the related targets for viruses in the past are mainly related proteins of viruses that infect human cells (such as the S protein of the new crown), and virus synthesis. The required enzymes, etc., the target selection is relatively small. However, this technology uses the inactive site of the polypeptide sequence to bind the target, which can target and degrade all proteins of all viruses, and is also effective in overcoming drug resistance caused by virus mutation, which greatly improves the possibility and convenience of the successful development of viral drugs sex. In addition, this technology can degrade a single variation of a certain target, while the homologous protein of the wild type (Wild Type) without some variation will not be affected or will be less affected, which is also impossible for other degrader technologies such as PROTAC. Achieved.
附图说明Description of drawings
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings that need to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those skilled in the art, other drawings can also be obtained based on these drawings without creative effort.
图1显示为本发明实施例2所述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物(目标降解剂)的常规设计方式，即通用的穿膜肽-靶向多肽-连接体Linker-小分子配体模式。Figure 1 shows the conventional design method of the cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera (target degradation agent) described in Example 2 of the present invention, that is, the general-purpose membrane-penetrating peptide-target To the peptide-connector Linker-small molecule ligand mode.
图2显示为本发明实施例3所述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其针对某种或某类难以降解的致病蛋白专门设计，可将靶向多肽与两种或多种不同的E3连接酶结合物结合来有效地降解目标致病蛋白。Figure 2 shows the targeted chimera for modular assembly of polypeptides or microproteins mediated by cell-penetrating peptides described in Example 3 of the present invention, which is specially designed for a certain type or types of pathogenic proteins that are difficult to degrade, and can Combining the targeting polypeptide with two or more different E3 ligase conjugates to effectively degrade the target disease-causing protein.
图3显示的本发明实施例4所述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其可有效靶向与致病蛋白的蛋白-蛋白复合物形成有关的多个靶点，从而实现靶向致病蛋白的功效，能够有效抑制整个致病途径，并通过降解多个靶点来完全抑制某种特定疾病。Figure 3 shows the cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera described in Example 4 of the present invention, which can effectively target the protein-protein complex formation related to pathogenic proteins It can effectively inhibit the entire pathogenic pathway and completely inhibit a specific disease by degrading multiple targets.
图4显示为实施例5中多肽的固相合成工艺流程图，其中，合成产物多肽标记为1。FIG. 4 is a flow chart of the solid-phase synthesis process of the polypeptide in Example 5, wherein the synthetic product polypeptide is marked as 1.
图5显示为实施例5中来那度胺与丁二酸酐合成化合物的合成反应，其中，来那度胺标记为2，丁二酸酐标记为3，合成产物标记为4。5 shows the synthesis reaction of lenalidomide and succinic anhydride synthesis compound in Example 5, wherein lenalidomide is marked as 2, succinic anhydride is marked as 3, and the synthetic product is marked as 4.
图6显示为实施例5中LEN结合肽的固相合成工艺流程图，其中，合成产物(非对映体混合物)标记为5。FIG. 6 shows a flowchart of the solid-phase synthesis process of the LEN-binding peptide in Example 5, wherein the synthesized product (diastereomeric mixture) is marked as 5 .
图7显示为本发明一实施例中用于降解新冠S蛋白HR2的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物及其效果验证，其中，图7A为以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的结构图，图7B为对蛋白的降解效果验证，图7C为通过蛋白酶抑制剂MG132以验证确实是蛋白酶降解剂所产生的效果。Figure 7 shows a cell-penetrating peptide-mediated peptide or microprotein modular assembly targeting chimera and its effect verification for degrading the new crown S protein HR2 in an embodiment of the present invention, wherein Figure 7A is a cell-based The structural diagram of the targeted chimera for the modular assembly of peptides or microproteins mediated by penetrating peptides, Figure 7B is the verification of the degradation effect of the protein, and Figure 7C is the protease inhibitor MG132 to verify that it is indeed produced by the protease degradation agent Effect.
图8显示为本发明一实施例中用于降解新冠N蛋白的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物及其效果验证，其中，图8A为以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的结构图，图8B为对蛋白的降解效果验证，图8C为通过蛋白酶抑制剂MG132以验证确实是蛋白酶降解剂所产生的效果。Figure 8 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading the new crown N protein in an embodiment of the present invention, in which Figure 8A is a cell-penetrating peptide Structural diagram of membrane peptide-mediated modular assembly of polypeptides or microproteins targeting chimera, Figure 8B is the verification of the degradation effect of the protein, and Figure 8C is the protease inhibitor MG132 to verify the effect produced by the protease degradation agent .
图9显示为本发明一实施例中用于降解新冠M蛋白的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物及其效果验证，其中，图9A为以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的结构图，图9B为对蛋白的降解效果验证，图9C为通过蛋白酶抑制剂MG132以验证确实是蛋白酶降解剂所产生的效果。Figure 9 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading the new crown M protein in an embodiment of the present invention. Structural diagram of membrane peptide-mediated modular assembly of polypeptides or microproteins targeting chimera, Figure 9B is the verification of the degradation effect of the protein, and Figure 9C is the protease inhibitor MG132 to verify the effect produced by the protease degradation agent .
图10显示为本发明一实施例中用于降解新冠E蛋白的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物及其效果验证，其中，图10A为以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的结构图，图10B为对蛋白的降解效果验证，图10C为通过蛋白酶抑制剂MG132 以验证确实是蛋白酶降解剂所产生的效果。Figure 10 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading the new crown E protein in an embodiment of the present invention. Structural diagram of membrane peptide-mediated modular assembly of polypeptides or microproteins targeting chimera, Figure 10B is the verification of the degradation effect of the protein, and Figure 10C is the protease inhibitor MG132 to verify the effect produced by the protease degradation agent .
图11显示为本发明一实施例中用于降解新冠Orf6蛋白的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物及其效果验证，其中，图11A为以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的结构图，图11B为对蛋白的降解效果验证，图11C为通过蛋白酶抑制剂MG132以验证确实是蛋白酶降解剂所产生的效果。Figure 11 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading the new crown Orf6 protein in an embodiment of the present invention, in which Figure 11A is a cell-penetrating peptide Structural diagram of membrane peptide-mediated modular assembly of polypeptides or microproteins targeting chimeras, Figure 11B is the verification of the degradation effect of the protein, and Figure 11C is the protease inhibitor MG132 to verify the effect produced by the protease degradation agent .
图12显示为本发明一实施例中用于降解Lag-3蛋白的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物及其效果验证，其中，图12A为以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的结构图，图12B为对蛋白的降解效果验证，图12C为通过蛋白酶抑制剂MG132以验证确实是蛋白酶降解剂所产生的效果。Figure 12 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading Lag-3 protein in an embodiment of the present invention. The structural diagram of the targeting chimera for the modular assembly of peptides or microproteins mediated by penetrating peptides. Figure 12B is the verification of the degradation effect of the protein, and Figure 12C is the protease inhibitor MG132 to verify that it is indeed produced by the protease degradation agent Effect.
图13显示为本发明一实施例中用于降解Her2蛋白的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物及其效果验证，其中，图13A为以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的结构图，图13B为对蛋白的降解效果验证，图13C为通过蛋白酶抑制剂MG132以验证确实是蛋白酶降解剂所产生的效果。Figure 13 shows the targeting chimera for degrading Her2 protein mediated by cell-penetrating peptides or microprotein modular assembly and its effect verification, in which Figure 13A is a cell-penetrating peptide The peptide-mediated peptide or microprotein modular assembly targeting chimera structure diagram, Figure 13B is the verification of the degradation effect of the protein, and Figure 13C is the protease inhibitor MG132 to verify the effect produced by the protease degradation agent.
图14显示为本发明一实施例中用于降解SHP-2蛋白的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物及其效果验证，其中，图14A为以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的结构图，图14B为对蛋白的降解效果验证，图14C为通过蛋白酶抑制剂MG132以验证确实是蛋白酶降解剂所产生的效果。Figure 14 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading SHP-2 protein in an embodiment of the present invention. The structural diagram of the targeting chimera for the modular assembly of peptides or microproteins mediated by penetrating peptides, Figure 14B is the verification of the degradation effect of the protein, and Figure 14C is the protease inhibitor MG132 to verify that it is indeed produced by the protease degradation agent Effect.
图15显示为本发明一实施例中用于降解STAT5B蛋白的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物及其效果验证，其中，图15A为以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的结构图，图15B为对蛋白的降解效果验证，图15C为通过蛋白酶抑制剂MG132 以验证确实是蛋白酶降解剂所产生的效果。Figure 15 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading STAT5B protein in an embodiment of the present invention. The peptide-mediated peptide or microprotein modular assembly targeting chimera structure diagram, Figure 15B is the verification of the degradation effect of the protein, and Figure 15C is the protease inhibitor MG132 to verify the effect produced by the protease degradation agent.
图16显示为本发明一实施例中用于降解MUC16蛋白的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物及其效果验证，其中，图16A为以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的结构图，图16B为对蛋白的降解效果验证，图16C为通过蛋白酶抑制剂MG132以验证确实是蛋白酶降解剂所产生的效果。Figure 16 shows the targeting chimera for degrading MUC16 protein mediated by cell-penetrating peptides or microprotein modular assembly and its effect verification in one embodiment of the present invention, wherein Figure 16A is a cell-penetrating peptide The structural diagram of peptide-mediated modular assembly of polypeptides or microproteins targeting chimeras, Figure 16B is the verification of the degradation effect on proteins, and Figure 16C is the protease inhibitor MG132 to verify the effect produced by the protease degradation agent.
图17显示为本发明一实施例中用于降解CTLA-4蛋白的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物及其效果验证，其中，图17A为以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的结构图，图17B为对蛋白的降解效果验证。Figure 17 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading CTLA-4 protein in an embodiment of the present invention. The structural diagram of the targeting chimera for the modular assembly of polypeptides or microproteins mediated by membrane-penetrating peptides, and Figure 17B is a verification of the degradation effect on proteins.
图18显示为本发明一实施例中用于降解PCSK9蛋白的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物及其效果验证，其中，图18A为以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的结构图，图18B为通过蛋白酶抑制剂MG132以验证确实是蛋白酶降解剂所产生的效果。Figure 18 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading PCSK9 protein in an embodiment of the present invention. Peptide-mediated peptide or microprotein modular assembly targeting chimera structure diagram, Figure 18B is a protease inhibitor MG132 to verify the effect of the protease degradation agent.
图19显示为本发明一实施例中用于降解PD-1蛋白的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物及其效果验证，其中，图19A为以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的结构图，图19B为通过蛋白酶抑制剂MG132以验证确实是蛋白酶降解剂所产生的效果。Figure 19 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading PD-1 protein in an embodiment of the present invention. The structural diagram of the targeting chimera for the modular assembly of polypeptides or microproteins mediated by penetrating peptides, FIG. 19B is a protease inhibitor MG132 to verify the effect of the protease degradation agent.
图20显示为本发明一实施例中用于降解PD-L1蛋白的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物及其效果验证，其中，图20A为以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的结构图，图20B为通过蛋白酶抑制剂MG132以验证确实是蛋白酶降解剂所产生的效果。Figure 20 shows the modular assembly targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides and its effect verification for degrading PD-L1 protein in an embodiment of the present invention. The structural diagram of the targeted chimera for the modular assembly of polypeptides or microproteins mediated by penetrating peptides. FIG. 20B is a protease inhibitor MG132 to verify the effect of the protease degradation agent.
图21显示为本发明一实施例中用于精准靶向并降解带有G12V变异的KRAS蛋白的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物及其效果验证，其中，图21A为以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的结构图，图21B为对G12V变异KRAS蛋白的降解效果验证，图21C为对无变异KRAS蛋白(野生型)的降解效果验证。Figure 21 shows a cell-penetrating peptide-mediated peptide or microprotein modular assembly targeting chimera and its effect verification for precise targeting and degradation of KRAS protein with G12V mutation in an embodiment of the present invention, Among them, Figure 21A is a structural diagram of a peptide or microprotein modular assembly targeting chimera mediated by a cell-penetrating peptide, Figure 21B is a verification of the degradation effect of a G12V mutant KRAS protein, and Figure 21C is a diagram of a non-mutant KRAS protein (wild type) degradation effect verification.
图22显示为本发明一实施例中用于降解PCSK9蛋白的模块化穿膜肽介导的双E3配体靶向嵌合物及其效果验证，其中，图22A为模块化穿膜肽介导的双E3配体(CRBN+VHL)靶向嵌合物的结构图，图22B为通过蛋白酶抑制剂MG132以验证确实是蛋白酶降解剂所产生的效果且药物整体用量更少。Figure 22 shows a modular penetrating peptide-mediated dual E3 ligand targeting chimera and its effect verification for degrading PCSK9 protein in an embodiment of the present invention, wherein Figure 22A is a modular penetrating peptide-mediated The structural diagram of the double E3 ligand (CRBN+VHL) targeting chimera, Figure 22B is a protease inhibitor MG132 to verify the effect of the protease degradation agent and the overall dosage of the drug is less.
图23显示为本发明一实施例中用于同时降解HR2蛋白和N蛋白的模块化穿膜肽介导的双靶点靶向嵌合物及其效果验证，其中，图23A为模块化穿膜肽介导的双靶点(新冠HR2+新冠N蛋白靶点)靶向嵌合物的结构图，图23B为通过蛋白酶抑制剂MG132以验证确实是蛋白酶降解剂所产生的效果。Figure 23 shows a modular membrane-penetrating peptide-mediated dual-target targeting chimera and its effect verification for simultaneously degrading HR2 protein and N protein in an embodiment of the present invention, wherein Figure 23A is a modular membrane-penetrating peptide The structural diagram of the peptide-mediated dual-target (new crown HR2 + new crown N protein target) targeting chimera, Figure 23B is the protease inhibitor MG132 to verify the effect of the protease degradation agent.
图24显示为本发明一实施例中用于降解PD-L1蛋白的模块化穿膜肽介导的订书肽修饰靶向嵌合物及其效果验证，其中，图24A为模块化穿膜肽介导的订书肽修饰靶向嵌合物的结构图，图20B为对蛋白的降解效果验证。Figure 24 shows a modular penetrating peptide-mediated staple peptide modification targeting chimera and its effect verification for degrading PD-L1 protein in an embodiment of the present invention, wherein Figure 24A is a modular penetrating peptide The structural diagram of the mediated staple peptide modification targeting chimera, Figure 20B is the verification of the degradation effect on the protein.
图25显示为本发明一实施例中用于降解PD-L1蛋白的模块化穿膜肽介导的环形肽修饰靶向嵌合物及其效果验证，其中，图25A为模块化穿膜肽介导的环形肽修饰靶向嵌合物的结构图，图25B为对蛋白的降解效果验证。Figure 25 shows a modular penetrating peptide-mediated circular peptide modification targeting chimera and its effect verification for degrading PD-L1 protein in an embodiment of the present invention, wherein Figure 25A is a modular penetrating peptide-mediated The structural diagram of the guided cyclic peptide modification targeting chimera, and Fig. 25B is the verification of the degradation effect on the protein.
图26显示为图12-图14中以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的穿膜染色验证图。Fig. 26 shows the transmembrane staining verification diagram of the modular assembly of polypeptides or microproteins mediated by cell-penetrating peptides in Figs. 12-14.
图27显示为图15、图16中以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的穿膜染色验证图。Fig. 27 shows the transmembrane staining verification diagram of the peptide or microprotein modular assembly targeting chimera mediated by cell-penetrating peptides in Fig. 15 and Fig. 16 .
图28显示为图17、图18中以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的穿膜染色验证图。Fig. 28 shows the transmembrane staining verification diagram of the peptide or microprotein modular assembly targeting chimera mediated by cell-penetrating peptides in Fig. 17 and Fig. 18.
图29显示为图19、图20中以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物的穿膜染色验证图。Fig. 29 shows the transmembrane staining verification diagram of the modular assembly of polypeptides or microproteins mediated by cell-penetrating peptides in Fig. 19 and Fig. 20 .
图30-图32显示为订书肽+小分子配体嵌合物的结构图示。其中，图30的A部分与图31的A部分相连，图31的B部分与图32的B部分相连，整体为含有订书肽的嵌合物化合物结构。Figures 30-32 show schematic representations of the structure of the staple peptide + small molecule ligand chimera. Wherein, part A of Fig. 30 is connected with part A of Fig. 31, part B of Fig. 31 is connected with part B of Fig. 32, and the whole is a chimeric compound structure containing staple peptide.
图33-图35显示为环形肽+小分子配体嵌合物的结构图示。其中，图33的A部分与图34的A部分相连，图33的B部分与图34的B部分相连，图34的D部分与图35的D部分相连，整体为含有环形肽的嵌合物化合物结构。Figures 33-35 show schematic representations of the structures of cyclic peptide + small molecule ligand chimeras. Among them, part A of Figure 33 is connected to part A of Figure 34, part B of Figure 33 is connected to part B of Figure 34, part D of Figure 34 is connected to part D of Figure 35, and the whole is a chimera containing a cyclic peptide Compound structure.
具体实施方式detailed description
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
实施例1：Example 1:
以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，包括有相互连接的至少一个穿膜肽模块、至少一个靶向多肽模块和至少一个小分子配体模块，所述靶向多肽模块为可与靶向目标蛋白结合的多肽序列。Cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera, including at least one penetrating peptide module, at least one targeting polypeptide module and at least one small molecule ligand module connected to each other, said The targeting polypeptide module is a polypeptide sequence that can bind to the targeting target protein.
穿膜肽+靶向多肽+小分子配体形成的嵌合分子，可有效将小分子配 体通过靶向多肽靶向至致病的目标蛋白，从而可以实现药物治疗的针对性和特异性，在多种疾病(尤其是肿瘤病症)的治疗领域，能够发挥更加稳定和更加广泛的效果。The chimeric molecule formed by penetrating peptide + targeting polypeptide + small molecule ligand can effectively target the small molecule ligand to the pathogenic target protein through the targeting polypeptide, so as to achieve the pertinence and specificity of drug treatment. In the field of treatment of various diseases (especially tumor diseases), it can exert more stable and wider effects.
穿膜肽模块为一个，靶向多肽模块可以为一个、两个、三个甚至多个，小分子配体模块可以为一个、两个、三个甚至多个。There is one penetrating peptide module, one, two, three or even multiple targeting polypeptide modules, and one, two, three or even multiple small molecule ligand modules.
以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物中还包括有至少一个连接体Linker模块，用于嵌合靶向多肽模块与小分子配体模块。连接体Linker模块可以为一个、两个、三个甚至多个。The modular assembly of polypeptides or microproteins mediated by cell-penetrating peptides also includes at least one linker module in the targeting chimera, which is used for chimeric targeting polypeptide modules and small molecule ligand modules. There can be one, two, three or even multiple linker modules.
穿膜肽模块连接于靶向多肽模块的游离端并用于引导靶向嵌合物穿透细胞膜。The membrane-penetrating peptide module is linked to the free end of the targeting polypeptide module and used to guide the targeting chimera to penetrate the cell membrane.
小分子配体模块为可与E3连接酶结合的小分子E3配体；The small molecule ligand module is a small molecule E3 ligand that can bind to E3 ligase;
优选地，小分子E3配体适配的蛋白酶降解剂为CRBN、VHL、IAP中的一种或多种。Preferably, the protease degradation agent adapted to the small molecule E3 ligand is one or more of CRBN, VHL, and IAP.
穿膜肽模块的氨基酸序列为SEQ ID No.1-SEQ ID No.3中的任意一条。The amino acid sequence of the penetrating peptide module is any one of SEQ ID No.1-SEQ ID No.3.
SEQ ID No.1：YGRKKRRQRRR；SEQ ID No.1: YGRKKRRQRRR;
SEQ ID No.2：RRRRRRRR；SEQ ID No.2: RRRRRRRR;
SEQ ID No.3：RQIKIWFQNRRMKWK。SEQ ID No. 3: RQIKIWFQNRRMKWK.
靶向多肽模块的氨基酸序列为SEQ ID No.4-SEQ ID No.17中的任意一条或多条。The amino acid sequence of the targeting polypeptide module is any one or more of SEQ ID No.4-SEQ ID No.17.
SEQ ID No.4：SAIGKIQDSLSSTAS；SEQ ID No.4: SAIGKIQDSLSSTAS;
SEQ ID No.5：PQEESEEEVEEP；SEQ ID No.5: PQEESEEEVEEP;
SEQ ID No.6：GGKGLGKacGGA；SEQ ID No.6: GGKGLGKacGGA;
SEQ ID No.7：DTMVGWDKDARTK；SEQ ID No.7: DTMVGWDKDARTK;
SEQ ID No.8：FNGARSFIDI；SEQ ID No.8: FNGARSFIDI;
SEQ ID No.9：WARLWNYLYR；SEQ ID No.9: WARLWNYLYR;
SEQ ID No.10：RSFIDIGSGT；SEQ ID No. 10: RSFIDIGSGT;
SEQ ID No.11：KAVDG(p)YVKPQI；SEQ ID No.11: KAVDG(p)YVKPQI;
SEQ ID No.12：WIDPVNGDTE；SEQ ID No.12: WIDPVNGDTE;
SEQ ID No.13：ARHPSWYRPFEGCG；SEQ ID No.13: ARHPSWYRPFEGCG;
SEQ ID No.14：MESFPGWNLV(homoR)IGLLR。SEQ ID No. 14: MESFPGWNLV(homoR)IGLLR.
SEQ ID No.15：FNWDYSLEELREKAKYK；SEQ ID No.15: FNWDYSLEELREKAKYK;
SEQ ID No.16：MPIFLDHILNKFWILHYA；SEQ ID No.16: MPIFLDHILNKFWILHYA;
SEQ ID No.17：LYDVAGSDKY。SEQ ID No. 17: LYDVAGSDKY.
连接体Linker模块为小分子化合物，其结构式如式I所示；The linker module is a small molecule compound, and its structural formula is shown in formula I;
Figure PCTCN2022102648-appb-000008
Figure PCTCN2022102648-appb-000008
以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，适配的蛋白酶降解剂为CRBN时，所述小分子配体模块的结构式如式Ⅱ所示，
Figure PCTCN2022102648-appb-000009
适配的蛋白酶降解剂为VHL时，所述小分子配体模块的结构式如式Ⅲ所示，
Figure PCTCN2022102648-appb-000010
适配的蛋白酶降解剂为IAP时，所述小分子配体模块的结构式如式Ⅳ所示， Cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera, when the suitable protease degradation agent is CRBN, the structural formula of the small molecule ligand module is shown in formula II,
Figure PCTCN2022102648-appb-000009
When the suitable protease degradation agent is VHL, the structural formula of the small molecule ligand module is shown in formula III,
Figure PCTCN2022102648-appb-000010
When the suitable protease degradation agent is IAP, the structural formula of the small molecule ligand module is shown in formula IV,
Figure PCTCN2022102648-appb-000011
Figure PCTCN2022102648-appb-000011
以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其结构为以下各结构中的任意一种或多种：Cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera, its structure is any one or more of the following structures:
1)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.4+式I结构的连接体Linker+式Ⅱ结构的小分子配体；1) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.4+linker Linker with formula I structure+small molecule ligand with formula II structure;
2)穿膜肽SEQ ID No.2+靶向多肽SEQ ID No.5+式I结构的连接体Linker+式Ⅲ结构的小分子配体；2) Membrane-penetrating peptide SEQ ID No.2+targeting peptide SEQ ID No.5+linker Linker with formula I structure+small molecule ligand with formula III structure;
3)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.5+式I结构的连接体Linker+式Ⅳ结构的小分子配体；3) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.5+linker Linker with formula I structure+small molecule ligand with formula IV structure;
4)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.6+式I结构的连接体Linker+式Ⅱ结构的小分子配体；4) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.6+linker Linker with formula I structure+small molecule ligand with formula II structure;
5)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.7+式I结构的连接体Linker+式Ⅲ结构的小分子配体；5) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.7+linker Linker with formula I structure+small molecule ligand with formula III structure;
6)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.8+式I结构的连接体Linker+式Ⅱ结构的小分子配体；6) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.8+linker Linker with formula I structure+small molecule ligand with formula II structure;
7)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.9+式I结构的连接体Linker+式Ⅲ结构的小分子配体；7) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.9+linker Linker with formula I structure+small molecule ligand with formula III structure;
8)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.10+式I结构的连接体Linker+式Ⅱ结构的小分子配体；8) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.10+linker Linker with formula I structure+small molecule ligand with formula II structure;
9)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.11+式I结构的连接体Linker+式Ⅳ结构的小分子配体；9) Membrane-penetrating peptide SEQ ID No.1+targeting polypeptide SEQ ID No.11+linker Linker with formula I structure+small molecule ligand with formula IV structure;
10)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.12+式I结构的连接体Linker+式Ⅲ结构的小分子配体；10) Membrane-penetrating peptide SEQ ID No.1+targeting polypeptide SEQ ID No.12+linker Linker with formula I structure+small molecule ligand with formula III structure;
11)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.13+式I结构的连接体Linker+式Ⅲ结构的小分子配体；11) Membrane-penetrating peptide SEQ ID No.1+targeting polypeptide SEQ ID No.13+linker Linker with formula I structure+small molecule ligand with formula III structure;
12)穿膜肽SEQ ID No.3+靶向多肽SEQ ID No.14+式I结构的连接体Linker+式Ⅳ结构的小分子配体；12) Membrane-penetrating peptide SEQ ID No.3 + targeting polypeptide SEQ ID No.14 + linker Linker with formula I structure + small molecule ligand with formula IV structure;
13)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.15+式I结构的连接体Linker+式Ⅱ结构的小分子配体；13) Membrane-penetrating peptide SEQ ID No.1+targeting polypeptide SEQ ID No.15+linker Linker with formula I structure+small molecule ligand with formula II structure;
14)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.16+式I结构的连接体Linker+式Ⅱ结构的小分子配体；14) Membrane-penetrating peptide SEQ ID No.1+targeting polypeptide SEQ ID No.16+linker Linker with formula I structure+small molecule ligand with formula II structure;
15)穿膜肽SEQ ID No.2+靶向多肽SEQ ID No.17+式I结构的连接体Linker+式Ⅳ结构的小分子配体；15) Membrane-penetrating peptide SEQ ID No.2+targeting polypeptide SEQ ID No.17+linker Linker with formula I structure+small molecule ligand with formula IV structure;
16)穿膜肽SEQ ID No.3+靶向多肽SEQ ID No.14+式I结构的连接体Linker+(双E3配体：式Ⅱ结构的小分子配体+式Ⅲ结构的小分子配体)；16) Membrane-penetrating peptide SEQ ID No.3+ targeting polypeptide SEQ ID No.14+ linker Linker with formula I structure + (double E3 ligand: small molecule ligand with formula II structure + small molecule ligand with formula III structure );
17)穿膜肽SEQ ID No.1+(双靶点：靶向多肽SEQ ID No.4+靶向多肽SEQ ID No.5)+式I结构的连接体Linker+式Ⅱ结构的小分子配体。17) Membrane-penetrating peptide SEQ ID No.1+ (dual targets: targeting peptide SEQ ID No.4+targeting peptide SEQ ID No.5)+linker Linker with formula I structure+small molecule ligand with formula II structure .
以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物中的靶向多肽模块还包括经过修饰的订书肽序列或环形肽序列。Cell-penetrating peptide-mediated polypeptide or microprotein modular assembly The targeting polypeptide module in the targeting chimera also includes modified stapled peptide sequences or circular peptide sequences.
订书肽的结构式如式Ⅴ所示，The structural formula of the staple peptide is shown in formula V,
Figure PCTCN2022102648-appb-000012
Figure PCTCN2022102648-appb-000013
环形肽的结构式如式Ⅵ所示，
Figure PCTCN2022102648-appb-000014
Figure PCTCN2022102648-appb-000012
Figure PCTCN2022102648-appb-000013
The structural formula of the cyclic peptide is shown in formula VI,
Figure PCTCN2022102648-appb-000014
含有订书肽的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其结构如下所示：式Ⅴ结构的订书肽+式I结构的连接体Linker+式Ⅱ结构的小分子配体。Cell-penetrating peptide-mediated peptide or microprotein modular assembly targeting chimera containing staple peptide, its structure is as follows: staple peptide of formula V structure + linker Linker of formula I structure + formula II structure small molecule ligands.
含有环形肽的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其结构如下所示：式Ⅵ结构的环形肽+式I结构的连接体Linker+式Ⅱ结构的小分子配体。Cell-penetrating peptide-mediated peptide or microprotein modular assembly targeting chimera containing cyclic peptides, its structure is as follows: cyclic peptide of formula VI + linker Linker of formula I + small structure of formula II molecular ligand.
实施例2：Example 2:
实施例1的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，可以用于制备降解靶向目标蛋白的产品或降解存在变异氨基酸位点的靶向目标蛋白的产品。The cell-penetrating peptide-mediated modular assembly of polypeptides or microproteins targeting chimeras in Example 1 can be used to prepare products that degrade target proteins or degrade products that target target proteins with mutated amino acid positions .
靶向的降解蛋白包括新冠S蛋白HR2、新冠N蛋白、新冠M蛋白、新冠E蛋白、新冠Orf6蛋白、Lag-3蛋白、Her2蛋白、SHP-2蛋白、STAT5B 蛋白、MUC16蛋白、CTLA-4蛋白、PCSK9蛋白、PD-1蛋白、PD-L1蛋白、KRAS蛋白G12V变异中的一种或多种。Targeted degradation proteins include new crown S protein HR2, new crown N protein, new crown M protein, new crown E protein, new crown Orf6 protein, Lag-3 protein, Her2 protein, SHP-2 protein, STAT5B protein, MUC16 protein, CTLA-4 protein , PCSK9 protein, PD-1 protein, PD-L1 protein, and one or more of KRAS protein G12V mutations.
到目前为止，在抑制剂分子的帮助下，靶向、抑制和药物治疗参与蛋白质-蛋白质相互作用的蛋白质几乎是不可能的。利用蛋白质体降解机制靶向有害/致病蛋白是一种很有前途的治疗方法。借助泛素蛋白酶体***(UPS)的帮助，靶标蛋白-蛋白相互作用(PPI)借助关键肽序列与目标蛋白相互作用，选择性降解“不可药物化”的靶标蛋白。发明人设计了这种双功能肽基降解剂的方法，它针对并降解涉及PPI的目标蛋白。发明人通过将与目标蛋白具有高亲和力、选择性相互作用的肽结合到E3连接酶上，借助连接体实现了预期目标蛋白的降解。为了实现肽降解剂的细胞渗透性，发明人进一步将肽降解剂序列与细胞渗透性肽(细胞穿透肽)结合。一般情况下的目标降解剂的设计如下图1所示。Targeting, inhibiting and drug-treating proteins involved in protein-protein interactions has so far been almost impossible with the help of inhibitor molecules. Targeting harmful/pathogenic proteins using the proteosome degradation machinery is a promising therapeutic approach. With the help of the ubiquitin-proteasome system (UPS), the target protein-protein interaction (PPI) interacts with the target protein through key peptide sequences to selectively degrade the "undruggable" target protein. The inventors devised this method of bifunctional peptidyl degraders, which target and degrade target proteins involved in PPIs. The inventors combined the peptide with high affinity and selective interaction with the target protein to the E3 ligase, and realized the degradation of the expected target protein by means of the linker. In order to achieve the cell permeability of the peptide degrader, the inventors further combined the peptide degrader sequence with a cell-permeable peptide (cell penetrating peptide). In general, the design of the target degradation agent is shown in Figure 1 below.
发明人发现肽降解剂的设计可以降解蛋白-蛋白相互作用中涉及的>15000个靶点，借助于对应的靶向配体/肽，这些配体可以偶联到约1100个linkers，(其中约300个PEG型linkers)，约65个E3连接酶结合配体。为了提高细胞的通透性，发明人将肽PROTAC技术进一步与约800个细胞穿透肽结合。The inventors found that the design of peptide degraders can degrade >15,000 targets involved in protein-protein interactions, and these ligands can be coupled to about 1,100 linkers with the help of corresponding targeting ligands/peptides, (of which about 300 PEG-type linkers), about 65 E3 ligase-binding ligands. In order to increase the permeability of cells, the inventors further combined the peptide PROTAC technology with about 800 cell penetrating peptides.
SMILES(Simplified molecular input line entry specification)，简化分子线性输入规范，是一种用ASCII字符串明确描述分子结构的规范；InChI Key(国际化合物标识)是International Chemical Identifier的缩写，InCHI编码是国际纯粹与应用化学联合会International Union of Pure and Applied Chemistry(IUPAC)给出的每种化合物化学结构的唯一识别码，通过InChI key可在PubMed ChemCompound数据库(网址https://www.ncbi.nlm.nih.gov/pccompound)中很容易找到其唯一对应的化合物。SMILES (Simplified molecular input line entry specification), simplified molecular linear input specification, is a specification that clearly describes the molecular structure with ASCII strings; InChI Key (International Compound Identification) is the abbreviation of International Chemical Identifier, InCHI code is the international pure and The unique identification code of the chemical structure of each compound given by the International Union of Pure and Applied Chemistry (IUPAC) can be found in the PubMed ChemCompound database through the InChI key (https://www.ncbi.nlm.nih.gov /pccompound) is easy to find its unique corresponding compound.
表1显示为连接体Linkers模块的选择，包括但不限于其中以SMILES表示的分子结构和与InChI key所对应的化合物，具体如下表1所示。Table 1 shows the selection of the linker Linkers module, including but not limited to the molecular structure represented by SMILES and the compound corresponding to the InChI key, as shown in Table 1 below.
Figure PCTCN2022102648-appb-000015
Figure PCTCN2022102648-appb-000015
Figure PCTCN2022102648-appb-000016
Figure PCTCN2022102648-appb-000016
Figure PCTCN2022102648-appb-000017
Figure PCTCN2022102648-appb-000017
Figure PCTCN2022102648-appb-000018
Figure PCTCN2022102648-appb-000018
Figure PCTCN2022102648-appb-000019
Figure PCTCN2022102648-appb-000019
Figure PCTCN2022102648-appb-000020
Figure PCTCN2022102648-appb-000020
Figure PCTCN2022102648-appb-000021
Figure PCTCN2022102648-appb-000021
Figure PCTCN2022102648-appb-000022
Figure PCTCN2022102648-appb-000022
Figure PCTCN2022102648-appb-000023
Figure PCTCN2022102648-appb-000023
Figure PCTCN2022102648-appb-000024
Figure PCTCN2022102648-appb-000024
Figure PCTCN2022102648-appb-000025
Figure PCTCN2022102648-appb-000025
Figure PCTCN2022102648-appb-000026
Figure PCTCN2022102648-appb-000026
Figure PCTCN2022102648-appb-000027
Figure PCTCN2022102648-appb-000027
Figure PCTCN2022102648-appb-000028
Figure PCTCN2022102648-appb-000028
Figure PCTCN2022102648-appb-000029
Figure PCTCN2022102648-appb-000029
Figure PCTCN2022102648-appb-000030
Figure PCTCN2022102648-appb-000030
Figure PCTCN2022102648-appb-000031
Figure PCTCN2022102648-appb-000031
Figure PCTCN2022102648-appb-000032
Figure PCTCN2022102648-appb-000032
Figure PCTCN2022102648-appb-000033
Figure PCTCN2022102648-appb-000033
Figure PCTCN2022102648-appb-000034
Figure PCTCN2022102648-appb-000034
Figure PCTCN2022102648-appb-000035
Figure PCTCN2022102648-appb-000035
Figure PCTCN2022102648-appb-000036
Figure PCTCN2022102648-appb-000036
表2显示为另一部分连接体Linkers模块，即PEG type linkers的选择，包括但不限于其中以“名称”表示的和与EnamineStore ID所对应的化合物，具体参见下表2。其中，EnamineStore为化合物数据库(网址为https://www.enaminestore.com/search)。Table 2 shows another part of the linker Linkers module, that is, the selection of PEG type linkers, including but not limited to the compounds represented by "name" and corresponding to EnamineStore ID, see Table 2 below for details. Wherein, EnamineStore is a compound database (URL is https://www.enaminestore.com/search).
Figure PCTCN2022102648-appb-000037
Figure PCTCN2022102648-appb-000037
Figure PCTCN2022102648-appb-000038
Figure PCTCN2022102648-appb-000038
Figure PCTCN2022102648-appb-000039
Figure PCTCN2022102648-appb-000039
Figure PCTCN2022102648-appb-000040
Figure PCTCN2022102648-appb-000040
Figure PCTCN2022102648-appb-000041
Figure PCTCN2022102648-appb-000041
Figure PCTCN2022102648-appb-000042
Figure PCTCN2022102648-appb-000042
Figure PCTCN2022102648-appb-000043
Figure PCTCN2022102648-appb-000043
Figure PCTCN2022102648-appb-000044
Figure PCTCN2022102648-appb-000044
表3显示为E3连接酶结合配体模块的选择，包括但不限于其中以SMILES表示的分子结构和与InChI key所对应的化合物，具体见下表3。Table 3 shows the selection of the E3 ligase binding ligand module, including but not limited to the molecular structure represented by SMILES and the compound corresponding to the InChI key, see Table 3 below for details.
Figure PCTCN2022102648-appb-000045
Figure PCTCN2022102648-appb-000045
Figure PCTCN2022102648-appb-000046
Figure PCTCN2022102648-appb-000046
Figure PCTCN2022102648-appb-000047
Figure PCTCN2022102648-appb-000047
Figure PCTCN2022102648-appb-000048
Figure PCTCN2022102648-appb-000048
表4显示为细胞穿膜肽序列模块的选择，包括但不限于其中以“细胞穿透肽序列”所示的序列，具体参见下表4。Table 4 shows the selection of cell-penetrating peptide sequence modules, including but not limited to the sequence indicated by "cell-penetrating peptide sequence", see Table 4 below for details.
Figure PCTCN2022102648-appb-000049
Figure PCTCN2022102648-appb-000049
Figure PCTCN2022102648-appb-000050
Figure PCTCN2022102648-appb-000050
Figure PCTCN2022102648-appb-000051
Figure PCTCN2022102648-appb-000051
Figure PCTCN2022102648-appb-000052
Figure PCTCN2022102648-appb-000052
Figure PCTCN2022102648-appb-000053
Figure PCTCN2022102648-appb-000053
Figure PCTCN2022102648-appb-000054
Figure PCTCN2022102648-appb-000054
Figure PCTCN2022102648-appb-000055
Figure PCTCN2022102648-appb-000055
Figure PCTCN2022102648-appb-000056
Figure PCTCN2022102648-appb-000056
Figure PCTCN2022102648-appb-000057
Figure PCTCN2022102648-appb-000057
Figure PCTCN2022102648-appb-000058
Figure PCTCN2022102648-appb-000058
表5显示为目标作用肽的选择示例，包括但不限于其中以“目标蛋白名称”所示的目标蛋白以及目标蛋白所对应的肽序列；共约有19813种目标蛋白，包含了现有已知的所有目标蛋白以及靶向这些目标蛋白的所有靶向多肽，但由于其体量太大，发明人只筛选了其中比较具有代表性的几十种目标蛋白做为示例，但本发明所要求的保护的内容为本领域内已知的所有目标蛋白及靶向多肽，并不限于此几十种目标蛋白，见下表5所示。Table 5 shows an example of the selection of target peptides, including but not limited to the target protein indicated by "target protein name" and the peptide sequence corresponding to the target protein; there are about 19813 target proteins in total, including the existing known All target proteins and all target polypeptides targeting these target proteins, but due to their large size, the inventors only screened a few dozen representative target proteins as an example, but the requirements of the present invention The content of protection is all target proteins and targeting polypeptides known in the art, not limited to these dozens of target proteins, as shown in Table 5 below.
Figure PCTCN2022102648-appb-000059
Figure PCTCN2022102648-appb-000059
Figure PCTCN2022102648-appb-000060
Figure PCTCN2022102648-appb-000060
Figure PCTCN2022102648-appb-000061
Figure PCTCN2022102648-appb-000061
Figure PCTCN2022102648-appb-000062
Figure PCTCN2022102648-appb-000062
表1-表5中，表3“e3ligand”代表目前可以应用的所有E3连接酶的小分子配体，linker分为两种，一种是表2所示的“PEG linkers”，其它的linker则被收集到表1“linkers”中，“CPP list”表4中是目前可以应用的所有穿膜肽，表5“Target interacting peptide”是目前可以应用的所有靶点的靶向多肽示例。In Table 1-Table 5, "e3ligand" in Table 3 represents the small molecule ligands of all E3 ligases that can be applied at present. There are two types of linkers, one is the "PEG linkers" shown in Table 2, and the other linkers are It is collected in Table 1 "linkers", "CPP list" Table 4 is all the currently available penetrating peptides, and Table 5 "Target interacting peptide" is an example of targeted peptides for all currently applicable targets.
现如今由于技术限制，大概只有10-20％的靶点可以被开发，而本发明提供的CePPiTAC技术，在靶向目标蛋白的部分由小分子改为多肽，并连接上穿膜肽序列确保其复合物可以进入细胞膜，因为任何目标蛋白都可以筛选出对其有连接性的抗性多肽相结合，所以理论上可以降解靶向任何目标蛋白并使蛋白酶将其降解，因此应用市场极为广阔，以往的“不可靶向性”靶点蛋白都可能被其开发出药物降解，而且，由于很多蛋白与蛋白之间的关系早已明确，筛选配体多肽非常方便，现已有多个高效小分子E3连接酶配体出现，其与多肽连接非常简单，使用本发明设计药物将会非常便利，可以省时省力，快速经济的研发出各种新药。Nowadays, due to technical limitations, only about 10-20% of the targets can be developed, and the CePPiTAC technology provided by the present invention changes the part of the target protein from a small molecule to a polypeptide, and connects the penetrating peptide sequence to ensure its The complex can enter the cell membrane, because any target protein can be screened out and combined with a resistant polypeptide that is connected to it, so it can theoretically degrade any target protein and allow proteases to degrade it, so the application market is extremely broad. The "non-targetable" target protein of the drug may be degraded by the drug developed by it. Moreover, since the relationship between many proteins has been clarified, it is very convenient to screen ligand peptides. There are now several high-efficiency small molecule E3 link The emergence of enzyme ligands, which are very simple to connect with polypeptides, will be very convenient to use the invention to design drugs, which can save time and effort, and develop various new drugs quickly and economically.
实施例3：Example 3:
发明人还通过将目标结合肽与如下所示的两个或多个不同的E3连接酶结合物结合，有效地降解目标物。具体如图2所示。The inventors also efficiently degrade the target by combining the target binding peptide with two or more different E3 ligase conjugates as shown below. Specifically shown in Figure 2.
实施例4：Example 4:
发明人还考虑降解涉及导致疾病的蛋白质-蛋白质复合物的形成的多个靶点，以抑制整个致病途径，并通过降解多个靶点来完全抑制特定疾病。为了实现这种肽降解剂，发明人将肽降解剂设计如图3所示。The inventors also contemplated degrading multiple targets involved in the formation of disease-causing protein-protein complexes to inhibit the entire pathogenic pathway and completely inhibit a specific disease by degrading multiple targets. In order to realize this peptide degrading agent, the inventors designed the peptide degrading agent as shown in FIG. 3 .
实施例5：Example 5:
具有代表性的PROTAC肽结合物PEN-FFW-LINK-LEN的合成。其 中，PEN、FFW和LEN分别代表穿膜肽、靶向多肽和小分子配体。Synthesis of a representative PROTAC peptide conjugate PEN-FFW-LINK-LEN. Among them, PEN, FFW and LEN represent penetrating peptide, targeting peptide and small molecule ligand, respectively.
a.肽1的固相合成：a. Solid phase synthesis of peptide 1:
方案1：plan 1:
如图4所示，肽1的合成以0.15mmol为标准。As shown in Figure 4, the synthesis of peptide 1 was based on 0.15 mmol.
SYRO自动肽合成器辅助用于延长全序列。0.5g Fmoc-Ile Wang树脂(0.3mmol/g)在DMF中溶胀，20％哌啶/DMF去保护Fmoc-部分(2次，持续5分钟和20分钟)。每次去保护后，用DMF(3x10mL)清洗树脂。在仪器上，每个fmoc-氨基酸残基(4eq，，0.6mmol)分别用DMF中的DIC/Oxyma(4eq，，0.6mmol，，30min)和HATU/DIPEA(4eq，，0.6mmol，，45min)两种不同的激活剂双偶联树脂。使用20％哌啶/DMF(2次，持续5和20分钟)最终Fmoc-部分去保护，用DMF(3x10mL)洗涤，以提供树脂结合线性肽1。所期望的质量是由微裂解确定的。The SYRO automated peptide synthesizer aid was used to extend the full sequence. 0.5g Fmoc-Ile Wang resin (0.3mmol/g) was swollen in DMF, and 20% piperidine/DMF deprotected the Fmoc-moiety (2 times for 5 min and 20 min). After each deprotection, the resin was washed with DMF (3x10 mL). On the instrument, each fmoc-amino acid residue (4eq, 0.6mmol) was treated separately with DIC/Oxyma (4eq, 0.6mmol, 30min) and HATU/DIPEA (4eq, 0.6mmol, 45min) in DMF Two different activator double coupling resins. Final Fmoc-partial deprotection using 20% piperidine/DMF (2x for 5 and 20 min), washing with DMF (3x10 mL) afforded resin-bound linear peptide 1 . The expected mass was determined by microlysis.
b.来那度胺偶联丁二酸酐的合成：b. Synthesis of lenalidomide-coupled succinic anhydride:
方案1：plan 1:
如图5所示，将来那度胺2(200mg，，0.77mmol)加入含有丁二酸酐3(90mg，，0.93mmol)甲苯(8mL)溶液的圆底烧瓶中，并配备回流冷凝器。混合液回流3h，真空过滤分离沉淀，用乙酸乙酯(20mLx2)洗涤滤饼，真空干燥，得到4-(2-(2，，6-二氧哌啶-3-基)-1-氧异吲哚-4-基)氨基)-4-氧丁酸产物4。收率:120mg，，43.4％。As shown in Figure 5, lenalidomide 2 (200 mg, 0.77 mmol) was added to a round bottom flask containing succinic anhydride 3 (90 mg, 0.93 mmol) in toluene (8 mL) and equipped with a reflux condenser. The mixture was refluxed for 3 hours, the precipitate was separated by vacuum filtration, the filter cake was washed with ethyl acetate (20mLx2), and dried in vacuum to obtain 4-(2-(2,,6-dioxopiperidin-3-yl)-1-oxoiso Indol-4-yl)amino)-4-oxobutanoic acid product 4. Yield: 120 mg, 43.4%.
c.LEN偶联肽的固相合成和树脂裂解：c. Solid-phase synthesis and resin cleavage of LEN-coupled peptides:
方案3：Option 3:
1.固相合成：1. Solid phase synthesis:
如图6所示。用DMF中的DIC/Oxyma(4eq)活化来那度胺偶联丁二酸酐(4)，加入预溶化树脂结合胺(200mg，，0.06mmol)中，摇匀或摇匀2小时，用DMF(3x10mL)和DCM(3x10mL)过滤和洗涤树脂，最后用乙 醚(2x10mL)清洗树脂，真空干燥后进行树脂裂解。As shown in Figure 6. Activate lenalidomide-coupled succinic anhydride (4) with DIC/Oxyma (4eq) in DMF, add to pre-dissolved resin-bound amine (200mg,, 0.06mmol), shake or shake for 2 hours, and use DMF ( 3x10mL) and DCM (3x10mL) to filter and wash the resin, and finally wash the resin with ether (2x10mL) and dry the resin in vacuo for resin cleavage.
2.树脂裂解：2. Resin cracking:
该产品在10ml内从树脂中分离出来，其中包含三氟乙酸、三异丙基硅烷和水(95:2.5:2.5)，提供120毫克粗肽。采用反相高效液相色谱法进行纯化，得到10mg的非对映体混合物5，最大纯度为97.15％，214nm纯度为93.19％，收率：10mg，6.68％。The product was isolated in 10 ml from the resin containing trifluoroacetic acid, triisopropylsilane and water (95:2.5:2.5) to provide 120 mg of crude peptide. Purification by reverse-phase high-performance liquid chromatography afforded 10 mg of diastereomeric mixture 5 with a maximum purity of 97.15% and a purity of 93.19% at 214 nm. Yield: 10 mg, 6.68%.
表6显示为高效液相色谱的准备条件。Table 6 shows the preparatory conditions for HPLC.
仪器instrument Agilent Technologies 1260 infinityAgilent Technologies 1260 infinity
色谱柱Chromatographic column X-Select CSH C18(250*19)mm 5微米X-Select CSH C18(250*19)mm 5 microns
流动相Amobile phase A 0.1％TFA水0.1% TFA water
流动相Bmobile phase B 乙腈Acetonitrile
流量flow 15mL/min15mL/min
反应时间Reaction time 22mins22mins
负载能力load capacity 10mg/每次注入10mg/per injection
表7显示为梯度表。Table 7 is shown as a gradient table.
时间(min)time (min) 流动相A的百分比％% of mobile phase A 流动相B的百分比％% of mobile phase B
0.00.0 9090 1010
1515 5050 5050
15.115.1 00 100100
1919 00 100100
19.119.1 9090 1010
22twenty two 9090 1010
实施例6：Embodiment 6:
1、现有很多疾病的靶点蛋白为膜蛋白，如PD-1与PD-L1，虽然其抑制剂药物非常常用，但有效率不高且容易耐药，因为PD-1与PD-L1靶点蛋白在细胞膜上，小分子的PD-1/PD-L1降解剂很难开发，而本发明的技术可以定点靶向这两个蛋白的胞内部分，将其降解。1. The target proteins of many existing diseases are membrane proteins, such as PD-1 and PD-L1. Although their inhibitors are very commonly used, their effectiveness is not high and they are prone to drug resistance, because PD-1 and PD-L1 target The point protein is on the cell membrane, and small molecule PD-1/PD-L1 degradation agents are difficult to develop, but the technology of the present invention can target the intracellular part of these two proteins to degrade them.
2、有些疾病靶点因为结构的原因，难以与小分子结合，所以普通小分子的PROTAC很难设计，比如Kras蛋白的G12V变异，但是本发明的技术可以利用多肽与其结合，将其降解。2. Some disease targets are difficult to combine with small molecules due to structural reasons, so it is difficult to design ordinary small molecule PROTACs, such as the G12V mutation of Kras protein, but the technology of the present invention can use polypeptides to bind to it and degrade it.
3、有些病毒相关蛋白，比如新冠病毒或者HIV病毒相关蛋白，由于传统的病毒药物是着重于以中和性抗体或抑制病毒的蛋白酶类来抑制病毒，靶点少，而且一旦病毒发生变异，所开发的药物将前功尽弃。本发明提供的CePPiTAC技术，可以选择病毒的结构蛋白或者蛋白酶类，使用多肽与其结合靶向，然后将其降解，使其蛋白合成受损或者无法形成病毒包装，这样首先会扩大病毒药物研发的靶点数量，以往很多无法靶向的病毒蛋白都可以靶向，其次由于可以结合不容易变异的结构蛋白部分进而降解整个目标蛋白，因此成药后可以无惧任何病毒变异。3. Some virus-related proteins, such as new coronavirus or HIV virus-related proteins, because traditional virus drugs focus on neutralizing antibodies or virus-inhibiting proteases to inhibit viruses, there are few targets, and once the virus mutates, so The drugs developed will be for naught. The CePPiTAC technology provided by the present invention can select viral structural proteins or proteases, use polypeptides to bind and target them, and then degrade them, so that protein synthesis is damaged or virus packaging cannot be formed, which will first expand the target of viral drug research and development. According to the number of points, many viral proteins that could not be targeted in the past can be targeted. Secondly, because it can bind to the structural protein part that is not easy to mutate and then degrade the entire target protein, it can be used without fear of any virus mutation.
实施例7：Embodiment 7:
为了说明本申请给出的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其基本结构中的四种“模块”的自由组合以及多种选择性，发明人设计了用于降解新冠S蛋白HR2的模块化靶向嵌合物(图7A)；其中穿膜肽模块序列为YGRKKRRQRRR；靶向肽模块序列为SAIGKIQDSLSSTAS；连接体Linker模块为(PEG)4组成的小分子，结构式为
Figure PCTCN2022102648-appb-000063
E3小分子配体模块为靶点CRBN的E3配体，结构式为
Figure PCTCN2022102648-appb-000064
In order to illustrate the targeted chimeric assembly of polypeptides or microproteins mediated by cell-penetrating peptides in this application, the free combination of four "modules" in its basic structure and various selectivities, the inventors designed A modular targeting chimera for degrading the new crown S protein HR2 (Figure 7A); wherein the membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is SAIGKIQDSLSSTAS; the linker Linker module is a small (PEG)4 Molecule, the structural formula is
Figure PCTCN2022102648-appb-000063
The E3 small molecule ligand module is the E3 ligand of the target CRBN, and the structural formula is
Figure PCTCN2022102648-appb-000064
采用Western Blot方法验证了不同用量(nmol)的靶向嵌合物对蛋白的降解效果(图7B)，可以看出随着用量的增加，蛋白已被降解(无表达)。The Western Blot method was used to verify the degradation effect of different dosages (nmol) of targeting chimeras on the protein (Figure 7B). It can be seen that the protein has been degraded (no expression) with the increase of dosage.
为了验证本申请提供的靶向嵌合物，其作用是降解而非抑制，发明人还设计加入了蛋白酶抑制剂MG132，蛋白酶抑制剂可以抑制本申请靶 向嵌合体的作用，通过实验结果(图7C)可以看出，只加入靶向嵌合物的蛋白依旧无法表达，但加入靶向嵌合物+MG132后的蛋白却可以正常表达，而且其表达量与未加靶向嵌合物的蛋白表达量相当，此结果说明了靶向嵌合物的作用是降解而非抑制了蛋白。In order to verify that the targeting chimera provided by the application is degraded rather than inhibited, the inventor also designed and added protease inhibitor MG132, which can inhibit the effect of the targeting chimera of the application, through the experimental results (Fig. 7C) It can be seen that the protein that only added the targeting chimera still cannot be expressed, but the protein after adding the targeting chimera+MG132 can be expressed normally, and its expression level is the same as that of the protein without the targeting chimera The expression levels were comparable, suggesting that the targeting chimera was degrading rather than inhibiting the protein.
有研究表明，冠状病毒表面的S蛋白介导了病毒对靶细胞的侵染过程，其由S1和S2两个亚基组成，S1亚基负责与细胞表面的受体结合，S2亚基则发挥病毒与细胞膜融合的功能。S2亚基含有七肽重复域1(HR1)、七肽重复域2(HR2)等重要功能区，在病毒膜融合过程中，HR1与HR2折叠形成六螺旋束结构(6HB)以拉近病毒膜和细胞膜发生融合反应，从而使病毒的基因物质通过融合孔进入到靶细胞内。而靶向嵌合物，可与新冠病毒S蛋白HR2亚基结合并使其降解，抑制六螺旋束结构的形成，从而干扰病毒与细胞膜的融合，防止病毒入侵细胞，从根本上达到了防治新冠的目的。Studies have shown that the S protein on the surface of the coronavirus mediates the infection process of the virus to the target cell, which is composed of two subunits, S1 and S2. The S1 subunit is responsible for binding to the receptor on the cell surface, and the S2 subunit is responsible for The function of the virus to fuse with the cell membrane. The S2 subunit contains important functional regions such as the heptad repeat domain 1 (HR1) and the heptad repeat domain 2 (HR2). During the fusion process of the viral membrane, HR1 and HR2 fold to form a six-helix bundle structure (6HB) to draw the viral membrane closer A fusion reaction occurs with the cell membrane, so that the genetic material of the virus enters the target cell through the fusion hole. The targeting chimera can bind to the HR2 subunit of the new coronavirus S protein and degrade it, inhibiting the formation of the six-helix bundle structure, thereby interfering with the fusion of the virus and the cell membrane, preventing the virus from invading cells, and fundamentally achieving the prevention and treatment of the new coronavirus. the goal of.
实施例8：Embodiment 8:
与实施例7相似，为了说明本申请的“模块化”设计，发明人还分别给出了用于降解新冠N蛋白、新冠M蛋白、新冠E蛋白、新冠Orf6蛋白、Lag-3蛋白、Her2蛋白、SHP-2蛋白、STAT5B蛋白、MUC16蛋白、CTLA-4蛋白、PCSK9蛋白、PD-1蛋白、PD-L1蛋白的模块化靶向嵌合物(图8A-图20A)。Similar to Example 7, in order to illustrate the "modular" design of the present application, the inventors also provided methods for degrading the new crown N protein, the new crown M protein, the new crown E protein, the new crown Orf6 protein, the Lag-3 protein, and the Her2 protein. , SHP-2 protein, STAT5B protein, MUC16 protein, CTLA-4 protein, PCSK9 protein, PD-1 protein, PD-L1 protein modular targeting chimera (Figure 8A-Figure 20A).
用于降解新冠N蛋白的模块化靶向嵌合物，其穿膜肽模块序列为RRRRRRRR；靶向肽模块序列为PQEESEEEVEEP；连接体Linker模块为(PEG)4组成的小分子，结构式为
Figure PCTCN2022102648-appb-000065
E3小分子 配体模块为靶点VHL的E3配体，结构式为
Figure PCTCN2022102648-appb-000066
The modular targeting chimera used to degrade the new crown N protein, its membrane-penetrating peptide module sequence is RRRRRRRRR; the targeting peptide module sequence is PQEESEEEVEEP; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
Figure PCTCN2022102648-appb-000065
The E3 small molecule ligand module is the E3 ligand of the target VHL, and the structural formula is
Figure PCTCN2022102648-appb-000066
用于降解新冠M蛋白的模块化靶向嵌合物，其穿膜肽模块序列为YGRKKRRQRRR；靶向肽模块序列为PQEESEEEVEEP；连接体Linker模块为(PEG)4组成的小分子，结构式为
Figure PCTCN2022102648-appb-000067
E3小分子配体模块为靶点IAP的E3配体，结构式为
Figure PCTCN2022102648-appb-000068
The modular targeting chimera used to degrade the M protein of the new crown, its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is PQEESEEEVEEP; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
Figure PCTCN2022102648-appb-000067
The E3 small molecule ligand module is the E3 ligand of the target IAP, and the structural formula is
Figure PCTCN2022102648-appb-000068
用于降解新冠E蛋白的模块化靶向嵌合物，其穿膜肽模块序列为YGRKKRRQRRR；靶向肽模块序列为GGKGLGKacGGA；连接体Linker模块为(PEG)4组成的小分子，结构式为
Figure PCTCN2022102648-appb-000069
E3小分子配体模块为靶点CRBN的E3配体，结构式为
Figure PCTCN2022102648-appb-000070
The modular targeting chimera used to degrade the new crown E protein, its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is GGKGLGKacGGA; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
Figure PCTCN2022102648-appb-000069
The E3 small molecule ligand module is the E3 ligand of the target CRBN, and the structural formula is
Figure PCTCN2022102648-appb-000070
用于降解新冠Orf6蛋白的模块化靶向嵌合物，其穿膜肽模块序列为YGRKKRRQRRR；靶向肽模块序列为DTMVGWDKDARTK；连接体Linker模块为(PEG)4组成的小分子，结构式为
Figure PCTCN2022102648-appb-000071
E3小分子配体模块为靶点VHL的E3配体，结构式为
Figure PCTCN2022102648-appb-000072
The modular targeting chimera used to degrade the new crown Orf6 protein, its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is DTMVGWDKDARTK; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
Figure PCTCN2022102648-appb-000071
The E3 small molecule ligand module is the E3 ligand of the target VHL, and the structural formula is
Figure PCTCN2022102648-appb-000072
用于降解Lag-3蛋白的模块化靶向嵌合物，其穿膜肽模块序列为YGRKKRRQRRR；靶向肽模块序列为FNGARSFIDI；连接体Linker模块为(PEG)4组成的小分子，结构式为
Figure PCTCN2022102648-appb-000073
E3小分子配体模块为靶点CRBN的E3配体，结构式为
Figure PCTCN2022102648-appb-000074
Modular targeting chimera for degrading Lag-3 protein, its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is FNGARSFIDI; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
Figure PCTCN2022102648-appb-000073
The E3 small molecule ligand module is the E3 ligand of the target CRBN, and the structural formula is
Figure PCTCN2022102648-appb-000074
用于降解Her2蛋白的模块化靶向嵌合物，其穿膜肽模块序列为YGRKKRRQRRR；靶向肽模块序列为WARLWNYLYR；连接体Linker模块为(PEG)4组成的小分子，结构式为
Figure PCTCN2022102648-appb-000075
E3小分子配体模块为靶点VHL的E3配体，结构式为
Figure PCTCN2022102648-appb-000076
The modular targeting chimera used to degrade Her2 protein, its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is WARLWNYLYR; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
Figure PCTCN2022102648-appb-000075
The E3 small molecule ligand module is the E3 ligand of the target VHL, and the structural formula is
Figure PCTCN2022102648-appb-000076
用于降解SHP-2蛋白的模块化靶向嵌合物，其穿膜肽模块序列为YGRKKRRQRRR；靶向肽模块序列为RSFIDIGSGT；连接体Linker模块为(PEG)4组成的小分子，结构式为
Figure PCTCN2022102648-appb-000077
E3小分子 配体模块为靶点CRBN的E3配体，结构式为
Figure PCTCN2022102648-appb-000078
The modular targeting chimera used to degrade SHP-2 protein, its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is RSFIDIGSGT; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
Figure PCTCN2022102648-appb-000077
The E3 small molecule ligand module is the E3 ligand of the target CRBN, and the structural formula is
Figure PCTCN2022102648-appb-000078
用于降解STAT5B蛋白的模块化靶向嵌合物，其穿膜肽模块序列为YGRKKRRQRRR；靶向肽模块序列为KAVDG(p)YVKPQI；连接体Linker模块为(PEG)4组成的小分子，结构式为
Figure PCTCN2022102648-appb-000079
E3小分子配体模块为靶点IAP的E3配体，结构式为
Figure PCTCN2022102648-appb-000080
Modular targeting chimera for degrading STAT5B protein, its membrane-penetrating peptide module sequence is YGRKKRRQRRR; targeting peptide module sequence is KAVDG(p)YVKPQI; linker Linker module is a small molecule composed of (PEG)4, structural formula for
Figure PCTCN2022102648-appb-000079
The E3 small molecule ligand module is the E3 ligand of the target IAP, and the structural formula is
Figure PCTCN2022102648-appb-000080
用于降解MUC16蛋白的模块化靶向嵌合物，其穿膜肽模块序列为YGRKKRRQRRR；靶向肽模块序列为WIDPVNGDTE；连接体Linker模块为(PEG)4组成的小分子，结构式为
Figure PCTCN2022102648-appb-000081
E3小分子配体模块为靶点VHL的E3配体，结构式为
Figure PCTCN2022102648-appb-000082
The modular targeting chimera used to degrade MUC16 protein, its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is WIDPVNGDTE; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
Figure PCTCN2022102648-appb-000081
The E3 small molecule ligand module is the E3 ligand of the target VHL, and the structural formula is
Figure PCTCN2022102648-appb-000082
用于降解CTLA-4蛋白的模块化靶向嵌合物，其穿膜肽模块序列为YGRKKRRQRRR；靶向肽模块序列为ARHPSWYRPFEGCG；连接体Linker模块为(PEG)4组成的小分子，结构式为
Figure PCTCN2022102648-appb-000083
E3小分子配体模块为靶点VHL的E3配体，结构式为
Figure PCTCN2022102648-appb-000084
Modular targeting chimera for degrading CTLA-4 protein, its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is ARHPSWYRPFEGCG; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
Figure PCTCN2022102648-appb-000083
The E3 small molecule ligand module is the E3 ligand of the target VHL, and the structural formula is
Figure PCTCN2022102648-appb-000084
用于降解PCSK9蛋白的模块化靶向嵌合物，其穿膜肽模块序列为RQIKIWFQNRRMKWK；靶向肽模块序列为MESFPGWNLV(homoR)IGLLR；连接体Linker模块为(PEG)4组成的小分子，结构式为
Figure PCTCN2022102648-appb-000085
E3小分子配体模块为靶点IAP的E3配体，结构式为
Figure PCTCN2022102648-appb-000086
Modular targeting chimera for degrading PCSK9 protein, its membrane-penetrating peptide module sequence is RQIKIWFQNRRMKWK; targeting peptide module sequence is MESFPGWNLV(homoR)IGLLR; linker Linker module is a small molecule composed of (PEG)4, structural formula for
Figure PCTCN2022102648-appb-000085
The E3 small molecule ligand module is the E3 ligand of the target IAP, and the structural formula is
Figure PCTCN2022102648-appb-000086
用于降解PD-1蛋白的模块化靶向嵌合物，其穿膜肽模块序列为YGRKKRRQRRR；靶向肽模块序列为FNWDYSLEELREKAKYK；连接体Linker模块为(PEG)4组成的小分子，结构式为
Figure PCTCN2022102648-appb-000087
E3小分子配体模块为靶点CRBN的E3配体，结构式为
Figure PCTCN2022102648-appb-000088
Modular targeting chimera for degrading PD-1 protein, its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is FNWDYSLEELREKAKYK; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
Figure PCTCN2022102648-appb-000087
The E3 small molecule ligand module is the E3 ligand of the target CRBN, and the structural formula is
Figure PCTCN2022102648-appb-000088
用于降解PD-L1蛋白的模块化靶向嵌合物，其穿膜肽模块序列为YGRKKRRQRRR；靶向肽模块序列为MPIFLDHILNKFWILHYA；连接体Linker模块为(PEG)4组成的小分子，结构式为
Figure PCTCN2022102648-appb-000089
E3小分子配体模块为靶点CRBN的E3配体，结构式为
Figure PCTCN2022102648-appb-000090
The modular targeting chimera used to degrade PD-L1 protein, its membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is MPIFLDHILNKFWILHYA; the linker Linker module is a small molecule composed of (PEG)4, and its structural formula is
Figure PCTCN2022102648-appb-000089
The E3 small molecule ligand module is the E3 ligand of the target CRBN, and the structural formula is
Figure PCTCN2022102648-appb-000090
同样采用Western Blot方法验证了不同用量(nmol)的靶向嵌合物对新冠N蛋白、新冠M蛋白、新冠E蛋白、新冠Orf6蛋白、Lag-3蛋白、Her2蛋白、SHP-2蛋白、STAT5B蛋白、MUC16蛋白、CTLA-4蛋白的降解效果(图8B-图17B)，可以看出随着用量的增加，蛋白已被降解(无表达)。The Western Blot method was also used to verify the effect of different dosages (nmol) of targeted chimeras on the new crown N protein, new crown M protein, new crown E protein, new crown Orf6 protein, Lag-3 protein, Her2 protein, SHP-2 protein, STAT5B protein , MUC16 protein, and CTLA-4 protein degradation effects (Figure 8B-Figure 17B), it can be seen that with the increase of dosage, the protein has been degraded (no expression).
同样为了验证本申请提供的靶向嵌合物，其作用是降解而非抑制，发明人还设计加入了蛋白酶抑制剂MG132，蛋白酶抑制剂可以抑制本申请靶向嵌合体的作用，通过实验结果(图8C-图16C及图18B-20B)可以看出，只加入靶向嵌合物的蛋白依旧无法表达，但加入靶向嵌合物+MG132后的蛋白却可以正常表达，而且其表达量与未加靶向嵌合物的蛋白表达量相当，此结果说明了靶向嵌合物的作用是降解而非抑制了蛋白。Also in order to verify that the targeting chimera provided by the application is degraded rather than inhibited, the inventor also designed and added a protease inhibitor MG132, which can inhibit the effect of the targeting chimera of the application, through the experimental results ( (Fig. 8C-Fig. 16C and Fig. 18B-20B) It can be seen that the protein only adding targeting chimera still cannot be expressed, but the protein after adding targeting chimera + MG132 can be expressed normally, and its expression level is the same as that of The expression level of protein without targeting chimera was comparable, which indicated that the function of targeting chimera was to degrade rather than inhibit protein.
新冠N蛋白，在冠状病毒中含量丰富，是一种高度免疫原性蛋白，参与基因组复制和细胞信号通路调节，通过本申请靶向嵌合物降解此种蛋白，能够实现对新冠病毒的有效抑制和治疗。The new coronavirus N protein, which is abundant in the coronavirus, is a highly immunogenic protein that participates in genome replication and regulation of cell signaling pathways. Through this application, targeting chimeras to degrade this protein can effectively inhibit the new coronavirus and treatment.
新冠M蛋白，为膜糖蛋白(M，Membrane Protein)，是病毒颗粒包膜的组成部分，M蛋白参与下一代病毒颗粒的组装和释放，对其他结构蛋白(S、E、N蛋白)的结构稳定和功能表达有重要作用，通过靶向嵌合物降解此种蛋白，能够有效破坏病毒结构的稳定性，抑制病毒功能。The M protein of the new crown is a membrane glycoprotein (M, Membrane Protein), which is an integral part of the viral particle envelope. The M protein participates in the assembly and release of the next generation of viral particles, and the structure of other structural proteins (S, E, N proteins) Stability and functional expression play an important role. By targeting chimeras to degrade this protein, it can effectively destabilize the virus structure and inhibit virus function.
新冠E蛋白(E，Envelope Protein)是病毒颗粒包膜的组成部分，为小包膜糖蛋白，E蛋白的主要功能是保护病毒内部的RNA基因链条，通过靶向嵌合物降解此种蛋白，能够减少甚至去除病毒RNA的保护机制，使RNA链更易断裂，从而有效抑制病毒功能。New crown E protein (E, Envelope Protein) is a component of the virus particle envelope, a small envelope glycoprotein, the main function of E protein is to protect the RNA gene chain inside the virus, and degrade this protein by targeting chimeras, It can reduce or even remove the protective mechanism of viral RNA, making the RNA chain easier to break, thereby effectively inhibiting the function of the virus.
新冠Orf6蛋白，是新冠病毒蛋白中对人体细胞毒性最强的一种蛋白，现有研究发现，将其导入人体细胞后能够杀死大约一半的人类细胞，其能够有效地抑制宿主细胞的先天免疫活性，通过靶向嵌合物降解此种蛋白，能够大大降低新冠病毒对人类免疫***的毒害。The new coronavirus Orf6 protein is the most toxic protein to human cells among the new coronavirus proteins. Existing studies have found that it can kill about half of human cells after being introduced into human cells, and it can effectively inhibit the innate immunity of host cells Activity, by targeting the chimera to degrade this protein, can greatly reduce the toxicity of the new coronavirus to the human immune system.
Lag-3蛋白，淋巴细胞活化基因3，又称为CD233，是一个Ⅰ型跨膜蛋白，属于免疫球蛋白(Ig)超家族，主要表达在活化T细胞和NK细胞表面，LAG-3是一个非常有应用前景的免疫治疗靶点，通过靶向嵌合物降解此种蛋白，能够有效阻断肿瘤微环境中肿瘤细胞与TIL相互作用中的抑制性信号，恢复TIL对肿瘤细胞的免疫监视功能，达到抗肿瘤的作用。Lag-3 protein, lymphocyte activation gene 3, also known as CD233, is a type I transmembrane protein belonging to the immunoglobulin (Ig) superfamily, mainly expressed on the surface of activated T cells and NK cells, LAG-3 is a It is a very promising immunotherapy target. By targeting chimera to degrade this protein, it can effectively block the inhibitory signal in the interaction between tumor cells and TIL in the tumor microenvironment, and restore the immune surveillance function of TIL on tumor cells. , to achieve anti-tumor effect.
Her2蛋白，是具有酪氨酸蛋白激酶活性的跨膜蛋白，属于EGFR家族成员之一，HER2基因扩增是影响乳腺癌生长与转移的最重要的因素之一，通过靶向嵌合物降解此种蛋白，能够促进乳腺肿瘤的凋亡、抑制其增殖。Her2 protein is a transmembrane protein with tyrosine protein kinase activity and belongs to one of the EGFR family members. HER2 gene amplification is one of the most important factors affecting the growth and metastasis of breast cancer. It can be degraded by targeting chimeras This protein can promote the apoptosis and inhibit the proliferation of breast tumors.
SHP-2蛋白，由蛋白酪氨酸磷酸酶非受体11(PTP nonreceptor 11，PTPN11)编码，SHP2是人类中经过充分验证的PTP癌蛋白，并且正在成为治疗癌症的重要靶标，SHP2的过度激活起着至关重要的致病作用，通过靶向嵌合物降解此种蛋白，能够有效阻断或抑制SHP2的通路激活，从而明显改善肿瘤治疗。SHP-2 protein, encoded by protein tyrosine phosphatase nonreceptor 11 (PTP nonreceptor 11, PTPN11), SHP2 is a well-validated PTP oncoprotein in humans and is becoming an important target for cancer therapy, hyperactivation of SHP2 It plays a crucial role in pathogenicity. By targeting chimera to degrade this protein, it can effectively block or inhibit the activation of SHP2 pathway, thereby significantly improving tumor treatment.
STAT5B蛋白，是信号转导和转录激活因子-5b，STAT信号是多种肿瘤的调节信号，通过靶向嵌合物降解此种蛋白，能够有效引起STAT信号的失调，从而抑制了肿瘤细胞(如骨肉瘤细胞)的增殖和克隆形成，并诱导了G0/G1期细胞阻滞和凋亡。STAT5B protein is signal transducer and transcription activator-5b. STAT signal is a regulatory signal of various tumors. By targeting chimera to degrade this protein, it can effectively cause the disorder of STAT signal, thereby inhibiting tumor cells (such as Osteosarcoma cells) proliferation and colony formation, and induce G0/G1 phase cell arrest and apoptosis.
MUC16蛋白，是最大的跨膜粘蛋白，由于已知MUC16在卵巢癌细胞表面过表达并***/脱落到血液中，因此它是卵巢癌的一种公认的血清 生物标记物，同时，MUC16也被认为在癌细胞中起着抗凋亡的作用，其c端结构域的异位表达诱导卵巢癌细胞对顺铂产生耐药性，该作用是通过抑制p53介导的，通过靶向嵌合物降解此种蛋白，能够有效调节癌细胞的凋亡，抑制其增殖。MUC16 protein, the largest transmembrane mucin, is a well-recognized serum biomarker for ovarian cancer because it is known to be overexpressed on the surface of ovarian cancer cells and split/shedding into the blood. At the same time, MUC16 is also It is believed to play an anti-apoptotic role in cancer cells, and the ectopic expression of its C-terminal domain induces resistance to cisplatin in ovarian cancer cells, which is mediated by inhibiting p53, by targeting chimeric Degradation of this protein can effectively regulate the apoptosis of cancer cells and inhibit their proliferation.
CTLA-4蛋白，细胞毒性T淋巴细胞相关蛋白4，也称为CD152(分化簇152)，是一种蛋白受体，其作为免疫检查点起作用并下调免疫应答，CTLA-4基因的变异，不仅与癌症相关，同时与1型糖尿病，格雷夫斯病，桥本氏甲状腺炎，乳糜泻，***性红斑狼疮，甲状腺相关性眼眶病，原发性胆汁性肝硬化和其他自身免疫性疾病有关，通过靶向嵌合物降解此种蛋白后，能够有效增加机体的免疫活性。CTLA-4 protein, cytotoxic T lymphocyte-associated protein 4, also known as CD152 (cluster of differentiation 152), is a protein receptor that functions as an immune checkpoint and downregulates immune responses, mutations in the CTLA-4 gene, Linked not only to cancer but also to type 1 diabetes, Graves' disease, Hashimoto's thyroiditis, celiac disease, systemic lupus erythematosus, thyroid-associated orbitopathy, primary biliary cirrhosis, and other autoimmune diseases , after degrading this protein by targeting the chimera, it can effectively increase the immune activity of the body.
实施例9：Embodiment 9:
特别地，为了说明本申请给出的“模块化”设计还能够精准靶向具有变异氨基酸的蛋白，发明人设计了用于降解带有G12V变异的KRAS蛋白的模块化靶向嵌合物(图21A)，其中穿膜肽模块序列为RRRRRRRR；靶向肽模块序列为LYDVAGSDKY；连接体Linker模块为(PEG)4组成的小分子，结构式为
Figure PCTCN2022102648-appb-000091
E3小分子配体模块为靶点IAP的E3配体，结构式为
Figure PCTCN2022102648-appb-000092
In particular, in order to illustrate that the "modular" design proposed in this application can also accurately target proteins with mutated amino acids, the inventors designed a modular targeting chimera for degrading KRAS proteins with G12V mutations (Fig. 21A), wherein the membrane-penetrating peptide module sequence is RRRRRRRRR; the targeting peptide module sequence is LYDVAGSDKY; the linker Linker module is a small molecule composed of (PEG)4, and the structural formula is
Figure PCTCN2022102648-appb-000091
The E3 small molecule ligand module is the E3 ligand of the target IAP, and the structural formula is
Figure PCTCN2022102648-appb-000092
同样采用Western Blot方法验证了不同用量(nmol)的靶向嵌合物对带有G12V变异的KRAS蛋白的降解效果并对表达量进行了统计(图21B)，可以看出随着用量的增加，蛋白已被降解(表达量逐渐降低)。The Western Blot method was also used to verify the degradation effect of different dosages (nmol) of targeting chimeras on the KRAS protein with G12V mutation, and the expression level was counted (Figure 21B). It can be seen that as the dosage increases, The protein has been degraded (expression gradually decreased).
为了验证此种靶向嵌合物的精准靶向性，发明人还采用Western Blot 方法验证了不同用量(nmol)的靶向嵌合物对未变异的KRAS蛋白(野生型)的降解效果并对表达量进行了统计(图21C)，可以看出随着用量的增加，蛋白变化不明显(表达量略有降低，但不明显)，充分说明了此种设计的靶向嵌合物能够精准靶向带有G12V变异的KRAS蛋白，而基本不会降解野生型KRAS蛋白，靶向降解精准度极高。In order to verify the precise targeting of this targeting chimera, the inventors also used Western Blot method to verify the degradation effect of different dosages (nmol) of targeting chimera on unmutated KRAS protein (wild type) and the effect on The expression level was counted (Figure 21C), and it can be seen that with the increase of the dosage, the protein change is not obvious (the expression level is slightly reduced, but not obvious), which fully demonstrates that the targeting chimera of this design can accurately target To the KRAS protein with G12V mutation, it will basically not degrade the wild-type KRAS protein, and the targeted degradation accuracy is extremely high.
KRAS(Kirsten Rat Sarcoma Viral Oncogene Homolog)基因是GDP/GTP结合蛋白，KRAS与GTP结合呈激活状态，与GDP结合呈关闭状态，KRAS可被生长因子或酪氨酸激酶(如EGFR)短暂活化，活化后的KRAS可以激活下游如控制细胞生成的PI3K-AKT-mTOR信号通路，以及控制细胞增殖的RAS-RAF-MEK-ERK信号通路，突变的KRAS即使无EGFR等激酶激活的情况下都会发生持续活化，导致细胞持续增殖最终发生癌变。KRAS突变类在多种肿瘤中被发现，最为常见的包括肺癌、胰腺癌等。通过靶向嵌合物能够精准降解带有G12V变异的KRAS蛋白，而对野生型KRAS蛋白则无影响，大大提高了变异蛋白质的针对性治疗效率。KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene is a GDP/GTP-binding protein. KRAS is activated when combined with GTP, and closed when combined with GDP. KRAS can be temporarily activated by growth factors or tyrosine kinases (such as EGFR). The latter KRAS can activate downstream such as the PI3K-AKT-mTOR signaling pathway that controls cell production, and the RAS-RAF-MEK-ERK signaling pathway that controls cell proliferation. Mutant KRAS will continue to be activated even in the absence of EGFR and other kinase activation , resulting in continued proliferation of cells and eventually canceration. KRAS mutations are found in a variety of tumors, the most common ones include lung cancer and pancreatic cancer. KRAS protein with G12V mutation can be accurately degraded by targeting chimera, but has no effect on wild-type KRAS protein, which greatly improves the efficiency of targeted treatment of mutated protein.
实施例10：Example 10:
特别地，为了说明本申请给出的“模块化”设计还能够连接双E3配体结构，增加蛋白的降解效率，发明人设计了用于降解PCSK9的双E3配体模块化靶向嵌合物(图22A)，其中穿膜肽模块序列为RQIKIWFQNRRMKWK；靶向肽模块序列为MESFPGWNLV(homoR)IGLLR；通过两条连接体Linker模块分别与两个不同的E3小分子配体模块连接，两个不同的E3小分子配体模块分别为靶点CRBN和IAP的E3配体，连接体Linker模块和E3小分子配体模块 的整体结构式为
Figure PCTCN2022102648-appb-000093
In particular, in order to illustrate that the "modular" design given in this application can also connect double E3 ligand structures and increase protein degradation efficiency, the inventors designed a dual E3 ligand modular targeting chimera for degradation of PCSK9 (Figure 22A), wherein the membrane-penetrating peptide module sequence is RQIKIWFQNRRMKWK; the targeting peptide module sequence is MESFPGWNLV(homoR)IGLLR; the Linker module is connected to two different E3 small molecule ligand modules through two linkers, and the two different The E3 small molecule ligand modules are the E3 ligands of the target CRBN and IAP respectively, and the overall structural formula of the linker module and the E3 small molecule ligand module is
Figure PCTCN2022102648-appb-000093
同样为了验证本申请提供的靶向嵌合物，其作用是降解而非抑制，发明人还设计加入了蛋白酶抑制剂MG132，蛋白酶抑制剂可以抑制本申请靶向嵌合体的作用，通过实验结果(图22B)可以看出，只加入靶向嵌合物的蛋白依旧无法表达，但加入靶向嵌合物+MG132后的蛋白却可以正常表达，而且其表达量与未加靶向嵌合物的蛋白表达量相当，此结果说明了靶向嵌合物的作用是降解而非抑制了蛋白。Also in order to verify that the targeting chimera provided by the application is degraded rather than inhibited, the inventor also designed and added a protease inhibitor MG132, which can inhibit the effect of the targeting chimera of the application, through the experimental results ( Figure 22B) It can be seen that the protein that only added the targeting chimera still cannot be expressed, but the protein after adding the targeting chimera + MG132 can be expressed normally, and its expression level is the same as that of the protein without targeting chimera Protein expression was comparable, suggesting that the targeting chimera was degrading rather than inhibiting the protein.
同时，与图18A和图18B(同样用于降解PCSK9蛋白的靶向嵌合物)相比，采用了双E3配体的靶向嵌合物在相同的降解效果下用量更低(从25nmol降低至15nmol)；并且此种靶向嵌合物由于采用了双E3配体，在其中连接的一个E3泛素酶变异并产生耐药性后，另一个E3泛素酶还可以继续发挥作用，增加了靶向嵌合物的可靠性。At the same time, compared with Figure 18A and Figure 18B (the targeting chimera also used to degrade PCSK9 protein), the targeting chimera using double E3 ligands has a lower dosage under the same degradation effect (from 25nmol to 15nmol); and this kind of targeting chimera adopts double E3 ligands, after one E3 ubiquitinase connected therein mutates and produces drug resistance, another E3 ubiquitinase can continue to play a role, increasing The reliability of the targeting chimera was improved.
实施例11：Example 11:
特别地，为了说明本申请给出的“模块化”设计还能够针对不同的蛋白靶点，同时降解两种甚至多种蛋白，发明人设计了用于同时降解新冠HR2蛋白和新冠N蛋白的双靶点模块化靶向嵌合物(图23A))，其中穿膜肽模块序列为YGRKKRRQRRR；靶向肽模块序列分别为SAIGKIQDSLSSTAS和PQEESEEEVEEP；连接体Linker模块为(PEG) 4组成的小分子，结构式为
Figure PCTCN2022102648-appb-000094
E3小分子配体模块为靶点CRBN的E3配体，结构式为
Figure PCTCN2022102648-appb-000095
In particular, in order to illustrate that the "modular" design given in this application can also degrade two or more proteins at the same time for different protein targets, the inventors designed a dual-purpose protein for simultaneous degradation of the new crown HR2 protein and the new crown N protein. Target modular targeting chimera (Figure 23A)), wherein the membrane-penetrating peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequences are respectively SAIGKIQDSLSSTAS and PQEESEEEVEEP; the linker Linker module is a small molecule composed of (PEG) 4, the structural formula for
Figure PCTCN2022102648-appb-000094
The E3 small molecule ligand module is the E3 ligand of the target CRBN, and the structural formula is
Figure PCTCN2022102648-appb-000095
同样为了验证本申请提供的双靶点靶向嵌合物，其作用是降解而非抑制，发明人还设计加入了蛋白酶抑制剂MG132，蛋白酶抑制剂可以抑制本申请靶向嵌合体的作用，通过实验结果(图23B)可以看出，只加入靶向嵌合物的蛋白依旧无法表达，但加入靶向嵌合物+MG132后的蛋白却可以正常表达，而且其表达量与未加靶向嵌合物的蛋白表达量相当，此结果说明了靶向嵌合物的作用是降解而非抑制了蛋白。Also in order to verify that the dual-target targeting chimera provided by this application is degraded rather than inhibited, the inventor also designed and added a protease inhibitor MG132, which can inhibit the effect of the targeting chimera of this application, through From the experimental results (Figure 23B), it can be seen that the protein that only added the targeting chimera could not be expressed, but the protein after adding the targeting chimera + MG132 could be expressed normally, and its expression level was the same as that without the targeting chimera. The protein expression of the chimera was comparable to that of the chimera, indicating that the targeting chimera was degrading rather than inhibiting the protein.
同时，此种双靶点靶向嵌合物使用后，可同时降解HR2蛋白和N蛋白，基于此种方式考虑，发明人还可以设计针对三靶点、四靶点甚至更多靶点的靶向嵌合物，可以同时降解多种蛋白，以实现更好的适用性以及更广泛的使用条件，考虑到申请的篇幅限制，在此不再赘述，但并不应当因为本实施例记载的双靶点而限制更多靶点靶向嵌合物的技术保护。At the same time, after the use of this dual-target targeting chimera, HR2 protein and N protein can be degraded at the same time. Based on this method, the inventors can also design targets for three targets, four targets or even more targets. It can degrade multiple proteins at the same time to achieve better applicability and wider use conditions. Considering the space limit of the application, it will not be repeated here, but it should not be because of the duality described in this example. The technical protection of more target-targeted chimeras is limited.
实施例12：Example 12:
特别地，为了说明本申请给出的“模块化”设计还能够对靶向肽模块进行修饰以实现免除穿膜肽连接或增加结构稳定性的目的，发明人设计了采用订书肽修饰和环形肽修饰的模块化靶向嵌合物(图24A和图25A)。In particular, in order to illustrate that the "modular" design given in this application can also modify the targeting peptide module to achieve the purpose of exempting the connection of the penetrating peptide or increasing the structural stability, the inventors designed a modification using a stapled peptide and a circular Peptide-modified modular targeting chimeras (Figure 24A and Figure 25A).
同样采用Western Blot方法验证了不同用量(nmol)的靶向嵌合物对PD-L1蛋白的降解效果(图24B和图25B)，可以看出随着用量的增加，蛋白已被降解(表达量逐渐降低)。The Western Blot method was also used to verify the degradation effect of different dosages (nmol) of targeting chimeras on PD-L1 protein (Figure 24B and Figure 25B). It can be seen that with the increase of dosage, the protein has been degraded (expression level Gradually decreases).
订书肽和环形肽的修饰，其原理是可以使靶向肽模块呈现类似于模仿蛋白质二级结构的状态，形成一种“微型蛋白质”，在不连接穿膜肽模块的情形下仍可避免入膜过程中对肽段的分解，有效增加了靶向肽模块的稳定性。The principle of the modification of the staple peptide and the cyclic peptide is that the targeting peptide module can be presented in a state similar to that of the secondary structure of the imitated protein, forming a "miniature protein", which can still be avoided without connecting the membrane-penetrating peptide module The decomposition of the peptide segment during the membrane entry process effectively increases the stability of the targeting peptide module.
订书肽(靶向肽模块)的修饰过程为：使用R 8和S 5这两个化合物(结构如下)对CGIQDTNSKKQSDTHLEETC进行修饰和链接，使多肽变为：CGIQDT(R8)NSKKQS(S5)DTHLEET-；The modification process of the staple peptide (targeting peptide module) is: use the two compounds R 8 and S 5 (structure as follows) to modify and link CGIQDTNSKKQSDTHLEETC, so that the polypeptide becomes: CGIQDT(R8)NSKKQS(S5)DTHLEET- ;
R8为Fmoc-R8-OH，结构式如下：R8 is Fmoc-R8-OH, the structural formula is as follows:
Figure PCTCN2022102648-appb-000096
Figure PCTCN2022102648-appb-000096
S5为Fmoc-S5-OH，结构式如下：S5 is Fmoc-S5-OH, the structural formula is as follows:
Figure PCTCN2022102648-appb-000097
Figure PCTCN2022102648-appb-000097
订书肽(或称为微蛋白)+E3小分子配体嵌合物结构：Staple peptide (or microprotein) + E3 small molecule ligand chimeric structure:
Figure PCTCN2022102648-appb-000098
Figure PCTCN2022102648-appb-000098
含有订书肽的靶向嵌合物，其结构简式为：The targeting chimera containing staple peptide has a simplified structural formula:
Figure PCTCN2022102648-appb-000099
Figure PCTCN2022102648-appb-000099
整体结构式如图30-32所示。The overall structural formula is shown in Figure 30-32.
环形肽+小分子E3配体嵌合物结构：Cyclic peptide + small molecule E3 ligand chimeric structure:
Figure PCTCN2022102648-appb-000100
Figure PCTCN2022102648-appb-000100
环形肽(靶向肽模块)的结构简式：Linker-3PEG，Binder(配体)：CRBN(全名Cereblon)：The structural formula of the cyclic peptide (targeting peptide module): Linker-3PEG, Binder (ligand): CRBN (full name Cereblon):
Figure PCTCN2022102648-appb-000101
Figure PCTCN2022102648-appb-000101
连接体Linker模块为(PEG)4组成的小分子，结构式为
Figure PCTCN2022102648-appb-000102
The linker module is a small molecule composed of (PEG)4, and its structural formula is
Figure PCTCN2022102648-appb-000102
E3小分子配体模块为靶点CRBN的E3配体，结构式为
Figure PCTCN2022102648-appb-000103
环化方法：在上图的两个半胱氨酸二硫键成环。 The E3 small molecule ligand module is the E3 ligand of the target CRBN, and the structural formula is
Figure PCTCN2022102648-appb-000103
Cyclization method: The two cysteine disulfide bonds in the above figure form a ring.
整体结构式如图33-35所示。The overall structural formula is shown in Figure 33-35.
实施例13：Example 13:
申请人检索到了与本技术相关的三篇现有技术文献，并将其技术做为对比，与本发明技术方案进行了效果对比。The applicant retrieved three prior art documents related to this technology, and compared their technologies with the technical solutions of the present invention.
对比文献1：Comparative literature 1:
《Specific Knockdown of a-Synuclein by Peptide-Directed Proteasome Degradation Rescued ItsAssociated Neurotoxicity》(Jing Qu，Xiaoxi Ren，Fenqin Xue，...，Haixia Huang，Wei Wang，Jianliang Zhang，《Cell Chemical Biology》，2020)。"Specific Knockdown of a-Synuclein by Peptide-Directed Proteasome Degradation Rescued Its Associated Neurotoxicity" (Jing Qu, Xiaoxi Ren, Fenqin Xue,..., Haixia Huang, Wei Wang, Jianliang Zhang, "Cell Chemical Biology", 2020).
对比文献2：Comparative Document 2:
《Specific Knockdown of Endogenous Tau Protein byPeptide-Directed Ubiquitin-ProteasomeDegradation》(Ting-Ting Chu，Na Gao，Qian-Qian Li，...，Yong-Xiang Chen，Yu-Fen Zhao，Yan-Mei Li《Cell Chemical Biology》，2016)。"Specific Knockdown of Endogenous Tau Protein by Peptide-Directed Ubiquitin-Proteasome Degradation" (Ting-Ting Chu, Na Gao, Qian-Qian Li,..., Yong-Xiang Chen, Yu-Fen Zhao, Yan-Mei Li "Cell Chemical Biology ", 2016).
对比文献3：Comparative Document 3:
《A PROTAC peptide induces durableβ-catenindegradation and suppresses Wnt-dependentintestinal cancer》(Hongwei Liao1，Xiang Li2，Lianzheng Zhao1，Yalong Wang1，Xiaodan Wang1，Ye Wu2，Xin Zhou3，Wei Fu3，Lei Liu 4，Hong-Gang Hu2，5and Ye-Guang Chen1，《Cell Discovery》，2020)。"A PROTAC peptide induces durableβ-catenindegradation and suppresses Wnt-dependentintestinal cancer" (Hongwei Liao1, Xiang Li2, Lianzheng Zhao1, Yalong Wang1, Xiaodan Wang1, Ye Wu2, Xin Zhou3, Wei Fu3, Lei Liu 4, 2 Hong-Gang Hu Ye-Guang Chen1, "Cell Discovery", 2020).
其中，对比文献1与对比文献2公开了：多肽穿膜肽+靶向靶点的多肽+多肽Linker+多肽Binder组成，对比文献1对靶点蛋白降解要到50μm才有明显效果(Figure3)；对比文献2对靶点蛋白要到100μm才有明显效果(Figure 2)。Among them, Comparative Document 1 and Comparative Document 2 disclosed: polypeptide membrane-penetrating peptide + target-targeting polypeptide + polypeptide Linker + polypeptide Binder, and Comparative Document 1 had no obvious effect on target protein degradation until 50 μm (Figure 3); Document 2 has no obvious effect on the target protein until the size is 100 μm (Figure 2).
对比文献3公开了：订书肽+多肽Linker+多肽Binder组成，对比文献3要到70μm才对靶点有明显降解作用(Figure 1)。 Comparative Document 3 discloses: staple peptide + polypeptide Linker + polypeptide Binder composition, Comparative Document 3 does not have significant degradation effect on the target until it reaches 70 μm (Figure 1).
而本发明技术方案所给出的靶向嵌合物，其在nm一级即可实现靶点蛋白的降解，具体对比如表8所示。However, the targeting chimera provided by the technical solution of the present invention can achieve the degradation of the target protein at the nm level, as shown in Table 8 for the specific comparison.
表8Table 8
 the 靶点蛋白降解时药物用量Drug dosage when target protein is degraded
实施例7(新冠S蛋白HR2，图7)Example 7 (new crown S protein HR2, Figure 7) 50-100nmol50-100nmol
实施例8(新冠N蛋白，图8)Example 8 (new crown N protein, Figure 8) 50-100nmol50-100nmol
实施例8(新冠M蛋白，图9)Example 8 (new crown M protein, Figure 9) 50-100nmol50-100nmol
实施例8(新冠E蛋白，图10)Example 8 (new crown E protein, Figure 10) 10-100nmol10-100nmol
实施例8(新冠Orf6蛋白，图11)Example 8 (new crown Orf6 protein, Figure 11) 50-100nmol50-100nmol
实施例8(Lag-3蛋白，图12)Embodiment 8 (Lag-3 protein, Fig. 12) 75-100nmol75-100nmol
实施例8(Her2蛋白，图13)Embodiment 8 (Her2 protein, Fig. 13) 10-100nmol10-100nmol
实施例8(SHP-2蛋白，图14)Embodiment 8 (SHP-2 protein, Fig. 14) 10-100nmol10-100nmol
实施例8(STAT5B蛋白，图15)Embodiment 8 (STAT5B protein, Fig. 15) 75-100nmol75-100nmol
实施例8(MUC16蛋白，图16)Embodiment 8 (MUC16 protein, Fig. 16) 50-100nmol50-100nmol
实施例8(CTLA-4蛋白，图17)Embodiment 8 (CTLA-4 protein, Fig. 17) 10-75nmol10-75nmol
实施例8(PCSK9蛋白，图18)Embodiment 8 (PCSK9 protein, Fig. 18) 25nmol25nmol
实施例8(PD-1蛋白，图19)Example 8 (PD-1 protein, Figure 19) 25nmol25nmol
实施例8(PD-L1蛋白，图20)Example 8 (PD-L1 protein, Figure 20) 30nmol30nmol
实施例9(KRAS蛋白G12V变异，图21)Example 9 (KRAS protein G12V variation, Figure 21) 30nmol30nmol
实施例10(双E3，PCSK9蛋白，图22)Example 10 (double E3, PCSK9 protein, Figure 22) 15nmol15nmol
实施例11(双靶点，新冠HR2蛋白+新冠N蛋白，图23)Example 11 (dual targets, new crown HR2 protein + new crown N protein, Figure 23) 15nmol15nmol
实施例12(订书肽修饰，PD-L1蛋白，图24)Example 12 (Stapled peptide modification, PD-L1 protein, Figure 24) 50-75nmol50-75nmol
实施例12(环形肽修饰，PD-L1蛋白，图25)Example 12 (cyclic peptide modification, PD-L1 protein, Figure 25) 30nmol30nmol
对比文献1Reference 1 50μmol50μmol
对比文献2 Reference 2 100μmol 100μmol
对比文献3Reference 3 70μmol70μmol
从表8可以看出，本申请所提供的靶向嵌合物，在nmol的数量级(最高为100nmol，即0.1μmol)即可降解靶点蛋白，而对比文献1-3中，最低也要在50μmol数量级才能够对靶点蛋白产生明显的降解作用，二者在降解剂的使用量上相差了至少500倍，具有极为明显的效果差异。It can be seen from Table 8 that the targeting chimera provided by the present application can degrade the target protein in the order of nmol (the highest is 100 nmol, that is, 0.1 μmol), while in Comparative Literature 1-3, the lowest is also in Only on the order of 50 μmol can it produce obvious degradation effect on the target protein, and the difference in the amount of degradation agent used between the two is at least 500 times, which has a very obvious difference in effect.
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. scope.

Claims (15)

  1. 以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其特征在于，包括有相互连接的至少一个穿膜肽模块、至少一个靶向多肽模块和至少一个小分子配体模块，所述靶向多肽模块为可与靶向目标蛋白结合的多肽序列。Cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera, characterized in that it includes at least one penetrating peptide module, at least one targeting polypeptide module and at least one small molecule ligand connected to each other module, the targeting polypeptide module is a polypeptide sequence that can bind to the targeting target protein.
  2. 根据权利要求1所述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其特征在于，还包括有至少一个连接体Linker模块，所述靶向多肽模块与小分子配体模块通过连接体Linker模块进行嵌合。According to the cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera according to claim 1, it is characterized in that it also includes at least one linker Linker module, and the targeting polypeptide module and the small The molecular ligand module is chimerized through the linker module.
  3. 根据权利要求2所述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其特征在于，所述穿膜肽模块连接于靶向多肽模块的游离端并用于引导靶向嵌合物穿透细胞膜。The polypeptide or microprotein modular assembly targeting chimera mediated by cell-penetrating peptides according to claim 2, characterized in that the membrane-penetrating peptide module is connected to the free end of the targeting polypeptide module and used to guide The targeting chimera penetrates the cell membrane.
  4. 根据权利要求3所述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其特征在于，所述小分子配体模块为可与E3连接酶结合的小分子E3配体；优选地，小分子E3配体适配的蛋白酶降解剂为CRBN(Cereblon蛋白，Cereblon protein)、VHL(希佩尔-林道，von Hippel-Lindau)、IAP(凋亡抑制蛋白，Inhibitor of apoptosis proteins)中的一种或多种。According to the cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera according to claim 3, it is characterized in that the small molecule ligand module is a small molecule E3 that can be combined with E3 ligase Ligand; preferably, the protease degradation agent that the small molecule E3 ligand fits is CRBN (Cereblon protein, Cereblon protein), VHL (Hippel-Lindau, von Hippel-Lindau), IAP (apoptosis inhibitory protein, Inhibitor of One or more of apoptosis proteins).
  5. 根据权利要求1-4任一所述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其特征在于，所述穿膜肽模块的氨基酸序列为SEQ ID No.1-SEQ ID No.3中的任意一条。According to any one of claims 1-4, the cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera is characterized in that the amino acid sequence of the penetrating peptide module is SEQ ID No. Any one of 1-SEQ ID No.3.
  6. 根据权利要求1-4任一所述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其特征在于，所述靶向多肽模块的氨基酸序列为SEQ ID No.4-SEQ ID No.17中的任意一条或多条。According to any one of claims 1-4, the polypeptide or microprotein modular assembly targeting chimera mediated by cell-penetrating peptides is characterized in that the amino acid sequence of the targeting polypeptide module is SEQ ID No. Any one or more of 4-SEQ ID No.17.
  7. 根据权利要求1-4任一所述的以细胞穿膜肽介导的多肽或微蛋白模块 化组装靶向嵌合物，其特征在于，所述连接体Linker模块为小分子化合物，其结构式如式I所示；
    Figure PCTCN2022102648-appb-100001
    According to the cell-penetrating peptide-mediated modular assembly targeting chimera of polypeptides or microproteins according to any one of claims 1-4, it is characterized in that the linker module is a small molecule compound, and its structural formula is as follows Shown in formula I;
    Figure PCTCN2022102648-appb-100001
  8. 根据权利要求1-4任一所述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其特征在于，适配的蛋白酶降解剂为CRBN时，所述小分子配体模块的结构式如式Ⅱ所示，
    Figure PCTCN2022102648-appb-100002
    适配的蛋白酶降解剂为VHL时，所述小分子配体模块的结构式如式Ⅲ所示，
    Figure PCTCN2022102648-appb-100003
    适配的蛋白酶降解剂为IAP时，所述小分子配体模块的结构式如式Ⅳ所示，
    Figure PCTCN2022102648-appb-100004
    According to the cell-penetrating peptide-mediated modular assembly targeting chimera of polypeptides or microproteins according to any one of claims 1-4, it is characterized in that when the adapted protease degradation agent is CRBN, the small molecule The structural formula of the ligand module is shown in formula II,
    Figure PCTCN2022102648-appb-100002
    When the suitable protease degradation agent is VHL, the structural formula of the small molecule ligand module is shown in formula III,
    Figure PCTCN2022102648-appb-100003
    When the suitable protease degradation agent is IAP, the structural formula of the small molecule ligand module is shown in formula IV,
    Figure PCTCN2022102648-appb-100004
  9. 根据权利要求5或6或7或8所述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其特征在于，其结构为以下各结构中的任意一种或多种：According to claim 5 or 6 or 7 or 8, the polypeptide or microprotein modular assembly targeting chimera mediated by cell-penetrating peptides is characterized in that its structure is any one of the following structures or Various:
    1)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.4+式I结构的连接体Linker+ 式Ⅱ结构的小分子配体；1) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.4+linker Linker with formula I structure+small molecule ligand with formula II structure;
    2)穿膜肽SEQ ID No.2+靶向多肽SEQ ID No.5+式I结构的连接体Linker+式Ⅲ结构的小分子配体；2) Membrane-penetrating peptide SEQ ID No.2+targeting peptide SEQ ID No.5+linker Linker with formula I structure+small molecule ligand with formula III structure;
    3)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.5+式I结构的连接体Linker+式Ⅳ结构的小分子配体；3) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.5+linker Linker with formula I structure+small molecule ligand with formula IV structure;
    4)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.6+式I结构的连接体Linker+式Ⅱ结构的小分子配体；4) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.6+linker Linker with formula I structure+small molecule ligand with formula II structure;
    5)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.7+式I结构的连接体Linker+式Ⅲ结构的小分子配体；5) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.7+linker Linker with formula I structure+small molecule ligand with formula III structure;
    6)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.8+式I结构的连接体Linker+式Ⅱ结构的小分子配体；6) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.8+linker Linker with formula I structure+small molecule ligand with formula II structure;
    7)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.9+式I结构的连接体Linker+式Ⅲ结构的小分子配体；7) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.9+linker Linker with formula I structure+small molecule ligand with formula III structure;
    8)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.10+式I结构的连接体Linker+式Ⅱ结构的小分子配体；8) Membrane-penetrating peptide SEQ ID No.1+targeting peptide SEQ ID No.10+linker Linker with formula I structure+small molecule ligand with formula II structure;
    9)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.11+式I结构的连接体Linker+式Ⅳ结构的小分子配体；9) Membrane-penetrating peptide SEQ ID No.1+targeting polypeptide SEQ ID No.11+linker Linker with formula I structure+small molecule ligand with formula IV structure;
    10)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.12+式I结构的连接体Linker+式Ⅲ结构的小分子配体；10) Membrane-penetrating peptide SEQ ID No.1+targeting polypeptide SEQ ID No.12+linker Linker with formula I structure+small molecule ligand with formula III structure;
    11)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.13+式I结构的连接体Linker+式Ⅲ结构的小分子配体；11) Membrane-penetrating peptide SEQ ID No.1+targeting polypeptide SEQ ID No.13+linker Linker with formula I structure+small molecule ligand with formula III structure;
    12)穿膜肽SEQ ID No.3+靶向多肽SEQ ID No.14+式I结构的连接体Linker+式Ⅳ结构的小分子配体；12) Membrane-penetrating peptide SEQ ID No.3 + targeting polypeptide SEQ ID No.14 + linker Linker with formula I structure + small molecule ligand with formula IV structure;
    13)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.15+式I结构的连接体Linker+式Ⅱ结构的小分子配体；13) Membrane-penetrating peptide SEQ ID No.1+targeting polypeptide SEQ ID No.15+linker Linker with formula I structure+small molecule ligand with formula II structure;
    14)穿膜肽SEQ ID No.1+靶向多肽SEQ ID No.16+式I结构的连接体Linker+式Ⅱ结构的小分子配体；14) Membrane-penetrating peptide SEQ ID No.1+targeting polypeptide SEQ ID No.16+linker Linker with formula I structure+small molecule ligand with formula II structure;
    15)穿膜肽SEQ ID No.2+靶向多肽SEQ ID No.17+式I结构的连接体Linker+式Ⅳ结构的小分子配体；15) Membrane-penetrating peptide SEQ ID No.2+targeting polypeptide SEQ ID No.17+linker Linker with formula I structure+small molecule ligand with formula IV structure;
    16)穿膜肽SEQ ID No.3+靶向多肽SEQ ID No.14+式I结构的连接体Linker+(双E3配体：式Ⅱ结构的小分子配体+式Ⅲ结构的小分子配体)；16) Membrane-penetrating peptide SEQ ID No.3+ targeting peptide SEQ ID No.14+ linker Linker with formula I structure + (double E3 ligand: small molecule ligand with formula II structure + small molecule ligand with formula III structure );
    17)穿膜肽SEQ ID No.1+(双靶点：靶向多肽SEQ ID No.4+靶向多肽SEQ ID No.5)+式I结构的连接体Linker+式Ⅱ结构的小分子配体。17) Membrane-penetrating peptide SEQ ID No.1+ (dual target: targeting peptide SEQ ID No.4+targeting peptide SEQ ID No.5)+linker Linker with formula I structure+small molecule ligand with formula II structure .
  10. 根据权利要求5或6或7或8所述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其特征在于，所述靶向多肽模块还包括有经过修饰的订书肽序列或环形肽序列，订书肽序列或环形肽序列具有入膜功能。According to the cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera according to claim 5 or 6 or 7 or 8, it is characterized in that the targeting polypeptide module also includes a modified The staple peptide sequence or the cyclic peptide sequence, the staple peptide sequence or the cyclic peptide sequence has a membrane entry function.
  11. 根据权利要求10所述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其特征在于，所述订书肽的结构式如式Ⅴ所示，According to the cell-penetrating peptide-mediated modular assembly targeting chimera of polypeptides or microproteins according to claim 10, it is characterized in that the structural formula of the staple peptide is shown in formula V,
    Figure PCTCN2022102648-appb-100005
    Figure PCTCN2022102648-appb-100005
    所述环形肽的结构式如式Ⅵ所示，The structural formula of the cyclic peptide is shown in formula VI,
    Figure PCTCN2022102648-appb-100006
    Figure PCTCN2022102648-appb-100006
  12. 根据权利要求11所述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其特征在于，含有订书肽的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其结构如下所示：式Ⅴ结构的订书肽+式I结构的连接体Linker+式Ⅱ结构的小分子配体。The cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera according to claim 11, characterized in that the cell-penetrating peptide-mediated polypeptide or microprotein module containing staple peptide The chemically assembled targeting chimera, its structure is as follows: staple peptide of formula V structure + linker Linker of formula I structure + small molecule ligand of formula II structure.
  13. 根据权利要求11所述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其特征在于，含有环形肽的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物，其结构如下所示：式Ⅵ结构的环形肽+式I结构的连接体Linker+式Ⅱ结构的小分子配体。According to the cell-penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimera according to claim 11, it is characterized in that the cell-penetrating peptide-mediated polypeptide or microprotein modularization containing a cyclic peptide The targeting chimera is assembled, and its structure is as follows: cyclic peptide of formula VI + linker Linker of formula I + small molecule ligand of formula II.
  14. 权利要求1-13任一所述的以细胞穿膜肽介导的多肽或微蛋白模块化组装靶向嵌合物在制备用于降解靶向目标蛋白的产品或用于降解存在变异氨基酸位点的靶向目标蛋白的产品中的应用。According to any one of claims 1-13, the targeted chimeric assembly of polypeptides or microproteins mediated by cell-penetrating peptides has variable amino acid positions in the preparation of products for degradation targeting target proteins or for degradation The application of products targeting target proteins.
  15. 根据权利要求14所述的应用，其特征在于，所述靶向的降解蛋白包括新冠S蛋白HR2、新冠N蛋白、新冠M蛋白、新冠E蛋白、新冠Orf6蛋白、Lag-3蛋白、Her2蛋白、SHP-2蛋白、STAT5B蛋白、MUC16蛋白、CTLA-4蛋白、PCSK9蛋白、PD-1蛋白、PD-L1蛋白、KRAS蛋白G12V变异中的一种或多种。The application according to claim 14, characterized in that, the targeted degradation protein comprises new crown S protein HR2, new crown N protein, new crown M protein, new crown E protein, new crown Orf6 protein, Lag-3 protein, Her2 protein, One or more of SHP-2 protein, STAT5B protein, MUC16 protein, CTLA-4 protein, PCSK9 protein, PD-1 protein, PD-L1 protein, KRAS protein G12V variation.
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