WO2023274347A1 - Technologie pour l'assemblage modulaire d'un polypeptide à médiation par un peptide de pénétration cellulaire ou d'une chimère ciblant une microprotéine, et son utilisation - Google Patents

Technologie pour l'assemblage modulaire d'un polypeptide à médiation par un peptide de pénétration cellulaire ou d'une chimère ciblant une microprotéine, et son utilisation 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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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.

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Abstract

L'invention concerne une technologie pour l'assemblage modulaire d'un polypeptide à médiation par un peptide de pénétration cellulaire ou d'une chimère ciblant une microprotéine, et son utilisation. La chimère de ciblage comprend au moins un module peptidique de pénétration cellulaire, au moins un module polypeptidique de ciblage et au moins un module ligand à petites molécules qui sont mutuellement connectés, le module polypeptidique de ciblage étant une séquence polypeptidique capable de se lier à une protéine cible de ciblage. La technologie présente les caractéristiques et les avantages suivants : le polypeptide à médiation par un peptide de pénétration cellulaire ou la chimère de ciblage à assemblage modulaire de microprotéines est conçu de manière modulaire, des séquences vireuses ou des modules de composés à petites molécules ayant différentes fonctions pouvant être remplacées et chevauchées selon les exigences, et toutes les parties du module polypeptidique peuvent être soumises à une cyclisation ou une modification de structure de microprotéine secondaire. L'idée de conception améliore considérablement l'effet d'utilisation et la plage d'application d'un médicament de ciblage.
PCT/CN2022/102648 2021-06-30 2022-06-30 Technologie pour l'assemblage modulaire d'un polypeptide à médiation par un peptide de pénétration cellulaire ou d'une chimère ciblant une microprotéine, et son utilisation WO2023274347A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160264627A1 (en) * 2015-03-11 2016-09-15 California Institute Of Technology Cyclic peptide binder against oncogenic k-ras
CN107794280A (zh) * 2017-09-26 2018-03-13 天津大学 靶向穿膜肽基因载体及其应用
CN108948178A (zh) * 2018-07-20 2018-12-07 安他杰（杭州）生物医药科技有限公司 一种改良的人il-37蛋白及其应用
CN111225976A (zh) * 2017-08-18 2020-06-02 剑桥企业有限公司 模块化结合蛋白

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160264627A1 (en) * 2015-03-11 2016-09-15 California Institute Of Technology Cyclic peptide binder against oncogenic k-ras
CN111225976A (zh) * 2017-08-18 2020-06-02 剑桥企业有限公司 模块化结合蛋白
CN107794280A (zh) * 2017-09-26 2018-03-13 天津大学 靶向穿膜肽基因载体及其应用
CN108948178A (zh) * 2018-07-20 2018-12-07 安他杰（杭州）生物医药科技有限公司 一种改良的人il-37蛋白及其应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHEN SHUPING, YANG HAN, JIANG JINLU, YU SIYUAN, LI TINGDONG, GE SHENGXIANG: "Advances of targeted protein degradation technology and its applications in diseases therapy", CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 37, no. 11, 25 November 2021 (2021-11-25), pages 3915 - 3932, XP093017945, DOI: 10.13345/j.cjb.200816 *
CHU TING-TING; GAO NA; LI QIAN-QIAN; CHEN PU-GUANG; YANG XI-FEI; CHEN YONG-XIANG; ZHAO YU-FEN; LI YAN-MEI: "Specific Knockdown of Endogenous Tau Protein by Peptide-Directed Ubiquitin-Proteasome Degradation", CELL CHEMICAL BIOLOGY , ELSEVIER, AMSTERDAM, NL, vol. 23, no. 4, 21 April 2016 (2016-04-21), AMSTERDAM, NL , pages 453 - 461, XP029518406, ISSN: 2451-9456, DOI: 10.1016/j.chembiol.2016.02.016 *
LIANG QIWEN, RAO BEN-QIANG, XU XI, XIAO GANG, WANG JIANPING: "Screening of tissue factor targeting peptides alternately from phage dis- play peptide library", CHINESE JOURNAL OF PATHOPHYSIOLOGY, ZHONGGUO BINGLI SHENGLI XUEHUI, BEIJING, CN, vol. 27, no. 12, 31 December 2011 (2011-12-31), CN , pages 2429 - 2432, XP093017947, ISSN: 1000-4718, DOI: 10.3969/j.issn.1000-4718.201.12.038 *
QU JING; REN XIAOXI; XUE FENQIN; HE YI; ZHANG RUIHUA; ZHENG YAN; HUANG HAIXIA; WANG WEI; ZHANG JIANLIANG: "Specific Knockdown of alpha-Synuclein by Peptide-Directed Proteasome Degradation Rescued Its Associated Neurotoxicity", CELL CHEMICAL BIOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 27, no. 6, 30 April 2020 (2020-04-30), AMSTERDAM, NL , pages 751, XP086196119, ISSN: 2451-9456, DOI: 10.1016/j.chembiol.2020.03.010 *

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