Modular assembly technology of cell-penetrating peptide-mediated polypeptide or microprotein targeting chimeras and applications thereof

Abstract

The application provides a modular assembly technology of a cell penetrating peptide-mediated polypeptide or microprotein targeting chimeric compound and application thereof, and the targeting chimeric compound comprises at least one penetrating peptide module, at least one targeting polypeptide module and at least one small molecule ligand module which are connected with each other, and the targeting polypeptide module is a polypeptide sequence capable of being combined with a target protein. The application has the characteristics and advantages that the cell penetrating peptide-mediated polypeptide or microprotein modular assembly targeting chimeric compound provided by the application adopts modular design, and each sequence or small molecule compound module with different functions can be replaced and superimposed according to needs, and all polypeptide module parts can be circularized or modified with a secondary microprotein structure. The design idea greatly enhances the use effect and application range of the targeting drug.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, specifically to modular assembly technology and its application of peptide or microprotein targeted chimeras mediated by cell-penetrating peptides. Background Technology

[0002] Protein degradation agent technology has become increasingly popular worldwide in recent years, with the earliest and most prevalent being the protein degradation targeting chimera (PROTAC) technology. Structurally, PROTAC consists of three parts: a small molecule E3 ubiquitin ligase ligand and a small molecule target protein ligand. The two active ligands are linked together by a specially designed "linker" structure, ultimately forming the active form of the small molecule triplet "PROTAC," with the structure: small molecule ligand (target protein) + linker + small molecule ligand (binding to E3 ligase). The target protein binds to the small molecule target protein ligand, while the E3 ligase ligand also binds to it. The E3 ligase adds a ubiquitin tag to the target protein, and after multiple rounds of ubiquitination, multiple ubiquitin tags are formed. The polyubiquitinated target protein is then recognized and degraded by the proteasome.

[0003] PROTAC technology has been successfully applied to the induced degradation of various pathological proteins. Under natural conditions, E3 ubiquitin ligases require a specific recognition signal to recruit and ubiquitinate their target proteins. The advent of PROTAC technology makes it possible for E3 to ubiquitinate any protein. This technology designs a dual-function molecule, with one end binding to the target protein and the other end binding to the E3 ligase, forming a polymer. E3 then ubiquitinates the target protein and guides it into the degradation pathway. The most attractive aspect of targeted protein degradation is its ability to target proteins traditionally considered undrugable, which may constitute more than 80% of the human proteome. Because targeted protein degradation strategies can selectively degrade proteins by binding to almost any site on the protein, rather than the active site, theoretically this strategy can be used for any protein.

[0004] However, in actual research or operation, the small molecule ligands of the above-mentioned small molecule "triads" PROTACs that bind to target proteins are extremely difficult to screen. Many targets cannot be targeted with drugs because suitable ligands cannot be found. Moreover, in nature, many protein targets are not naturally suitable for small molecule binding, which may lead to the failure of drug development for small molecule PROTACs.

[0005] Existing therapeutic drugs mainly fall into two categories: small molecule drugs and biologics. However, due to the limitations of their inherent biophysical properties, these two types of therapeutic drugs cannot effectively cover all the confirmed important molecular targets. Peptide drugs, on the other hand, are another class of targeted molecules that have attracted widespread attention and interest. Similar to biological macromolecules, peptide molecules also have high binding affinity and selectivity to their targets, exhibiting less off-target effects compared to small molecule drugs. Furthermore, the metabolic products of peptides in vivo are amino acids, minimizing toxicity. Compared to small molecule drugs, peptide drugs have unparalleled advantages, mainly manifested in their ease of modification, target recognition specificity, and broad targeting range. Summary of the Invention

[0006] The purpose of this invention is to provide a modular assembly technology and its application of a cell-penetrating-peptide-induced-targeting chimera mediated by cell-penetrating peptides that can target and effectively degrade target proteins, namely, the cell-penetrating-peptide-induced-targeting chimera (CePPiTAC) technology for targeted protein degradation.

[0007] The above-mentioned objectives of the present invention can be achieved by the following technical solutions:

[0008] The first objective of this invention is to provide a modular assembly of peptides or microproteins mediated by cell-penetrating peptides, comprising at least one interlinked membrane-penetrating peptide module, at least one targeting peptide module, one (or none) small molecule linker module and at least one small molecule ligand module, wherein the targeting peptide module is a peptide sequence capable of binding to a target protein.

[0009] Optionally, the above-mentioned target chimera mediated by cell-penetrating peptides and modular assembly of polypeptides or microproteins further includes at least one linker module, wherein the target polypeptide module and the small molecule ligand module are intercalated through the linker module.

[0010] Optionally, in the above-mentioned target chimera assembled by a modular assembly of a polypeptide or microprotein mediated by a cell-penetrating peptide, the cell-penetrating peptide module is connected to the free end of the target polypeptide module and is used to guide the target chimera to penetrate the cell membrane.

[0011] Optionally, in the above-mentioned targeted chimeric assembly of polypeptides or microproteins mediated by cell-penetrating peptides, the small molecule ligand module is a small molecule E3 ligand that can bind to E3 ligase; preferably, the protease degrading agent adapted to the small molecule E3 ligand is one or more of CRBN (Cereblon protein), VHL (von Hippel-Lindau), and IAP (inhibitor of apoptosis proteins).

[0012] Optionally, in the above-mentioned targeted chimeric assembly of polypeptides or microproteins mediated by cell-penetrating peptides, the amino acid sequence of the cell-penetrating peptide module is any one of SEQ ID No. 1-SEQ ID No. 3.

[0013] Optionally, in the above-mentioned targeted chimeric assembly of polypeptide or microprotein modular assembly mediated by cell-penetrating peptides, the amino acid sequence of the targeted polypeptide module is any one or more of SEQ ID No. 4-SEQ ID No. 17.

[0014] Optionally, in the above-mentioned modular assembly of polypeptides or microproteins mediated by cell-penetrating peptides, the linker module is a small molecule compound with the structural formula shown in Formula I. Formula I.

[0015] Optionally, when the above-mentioned target chimeric assembly of polypeptide or microprotein modular assembly mediated by cell-penetrating peptides is adapted to CRBN as a protease degrading agent, the structural formula of the small molecule ligand module is shown in Formula II. Formula II; When the suitable protease degrading agent is VHL, the structural formula of the small molecule ligand module is shown in Formula III. Formula III; When the suitable protease degrading agent is IAP, the structural formula of the small molecule ligand module is shown in Formula IV. Formula IV.

[0016] Optionally, the above-mentioned modular assembly and targeting chimeric complexes of polypeptides or microproteins mediated by cell-penetrating peptides have any one or more of the following structures:

[0017] 1) A membrane-penetrating peptide SEQ ID No. 1 + a targeting peptide SEQ ID No. 4 + a linker of formula I + a small molecule ligand of formula II;

[0018] 2) Membrane-penetrating peptide SEQ ID No. 2 + targeting peptide SEQ ID No. 5 + linker of formula I + small molecule ligand of formula III;

[0019] 3) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 5 + linker of formula I + small molecule ligand of formula IV;

[0020] 4) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 6 + linker of formula I + small molecule ligand of formula II;

[0021] 5) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 7 + linker of formula I + small molecule ligand of formula III;

[0022] 6) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 8 + linker of formula I + small molecule ligand of formula II;

[0023] 7) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 9 + linker of formula I + small molecule ligand of formula III;

[0024] 8) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 10 + linker of formula I + small molecule ligand of formula II;

[0025] 9) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 11 + linker of formula I + small molecule ligand of formula IV;

[0026] 10) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 12 + linker of formula I + small molecule ligand of formula III;

[0027] 11) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 13 + linker of formula I + small molecule ligand of formula III;

[0028] 12) Membrane-penetrating peptide SEQ ID No. 3 + targeting peptide SEQ ID No. 14 + linker of formula I + small molecule ligand of formula IV;

[0029] 13) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 15 + linker of formula I + small molecule ligand of formula II;

[0030] 14) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 16 + linker of formula I + small molecule ligand of formula II;

[0031] 15) Membrane-penetrating peptide SEQ ID No. 2 + targeting peptide SEQ ID No. 17 + linker of formula I + small molecule ligand of formula IV;

[0032] 16) Membrane-penetrating peptide SEQ ID No. 3 + targeting peptide SEQ ID No. 14 + linker of formula I structure + (double E3 ligand: small molecule ligand of formula II structure + small molecule ligand of formula III structure);

[0033] 17) Membrane-penetrating peptide SEQ ID No.1 + (dual-target: targeting peptide SEQ ID No.4 + targeting peptide SEQ ID No.5) + linker of Formula I + small molecule ligand of Formula II.

[0034] Optionally, the target polypeptide module of the above-mentioned target chimera mediated by cell-penetrating peptides (PPPs) further includes a modified stapled peptide sequence or a cyclic peptide sequence, which has membrane penetration function. In this case, the target chimera mediated by PPPs may not have a cell-penetrating peptide.

[0035] Optionally, in the above-mentioned modular assembly of targeted chimeras using cell-penetrating peptides mediated by cell-penetrating peptides, the structural formula of the stapled peptide is shown in Formula V. Formula V; the structural formula of the cyclic peptide is shown in Formula VI. Formula VI.

[0036] Optionally, the above-mentioned target chimera of polypeptide or microprotein modular assembly mediated by cell-penetrating peptide, which contains a staple peptide, has the following structure: staple peptide of formula V + linker of formula I + small molecule ligand of formula II.

[0037] Optionally, the above-mentioned modular assembly and targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides, containing cyclic peptides, has the following structure: a cyclic peptide of Formula VI + a linker of Formula I + a small molecule ligand of Formula II.

[0038] A second objective of this invention is to provide the application of the above-described modularly assembled targeted chimeric polypeptides or microproteins mediated by cell-penetrating peptides in the preparation of products for degrading targeted proteins or for degrading targeted proteins with variable amino acid sites.

[0039] Optionally, in the above applications, the targeted degradation proteins include one or more of the following: SARS-CoV-2 S protein HR2, SARS-CoV-2 N protein, SARS-CoV-2 M protein, SARS-CoV-2 E protein, SARS-CoV-2 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 KRAS protein G12V variant.

[0040] Based on the above technical description, the core idea of ​​this invention is to connect multiple freely interchangeable "module" sequences or small molecule compounds into "modular" targeted chimeras that have strong targeting, good membrane penetration, and high degradation efficiency.

[0041] In the technical solution of this invention, the most basic structure consists of a membrane-penetrating peptide module, a targeting polypeptide module, and a small molecule ligand module, which are interconnected. Further configuration can be a membrane-penetrating peptide module, a targeting polypeptide module, a linker module, and a small molecule ligand module, forming a basic structure of membrane-penetrating peptide-targeting polypeptide-linker-small molecule ligand.

[0042] The basic structure of transmembrane peptide-targeting peptide-small molecule ligand can directly guide the small molecule ligand to the target protein. Although the chimera under this basic structure can exert targeted therapeutic properties, it has certain defects. The connection between the three is not stable and is easy to detach.

[0043] The membrane-penetrating peptide-targeting peptide-linker-small molecule ligand is an upgraded structure of the above basic structure. It overcomes the defects of poor membrane penetration performance of the targeting peptide and the instability of direct connection between the targeting peptide and the small molecule ligand. The membrane-penetrating peptide penetrates the membrane, and the targeting peptide guides the target protein, thereby achieving a directional penetration effect. The use of a linker to connect the two can effectively reduce the chance of detachment. At the same time, it overcomes the two major defects of poor membrane penetration and unstable connection, and almost perfectly solves the technical effect of targeted therapy, improving the targeting efficiency. The most important technical point is that in this optimal structure, the membrane-penetrating peptide + targeting peptide "replaces" the target protein in the existing PROTAC technology. The targeting peptide can greatly expand the selectivity of the target protein, and through the linker (E3), it can target and degrade almost all known target proteins.

[0044] Penetrating the cell membrane to enter the cell is a prerequisite for many biomolecules to exert their effects within the cell. However, the biological barrier function of the biomembrane prevents many macromolecules from entering the cell, thus greatly limiting their application in the therapeutic field. Therefore, how to guide these substances to penetrate the cell membrane is an urgent problem to be solved. Peptides, as intermediate products of protein hydrolysis, have poor membrane penetration. In recent years, with the development of technology, it has been discovered that the transactivator of transcription (TAT) in human immunodeficiency virus (HIV) can effectively cross the cell membrane and enter the cell. Subsequently, a number of proteins with the ability to cross the cell membrane were discovered and named cell-penetrating peptides (CPPs). Generally, cell-penetrating peptides are usually polypeptide molecules of no more than 30 amino acids, capable of crossing the cell membrane independently without relying on specific membrane receptors. These cell-penetrating peptides, as intracellular transport tools for bioactive molecules, have the characteristics of low toxicity, convenience, and effectiveness compared with other iontophoresis and nanocarriers. They are playing an increasingly important role in drug development, and some drugs containing CPP have even passed FDA clinical trials.

[0045] The permeation peptide-coupled chimeric technology (CePPiTAC) of this invention uses a polypeptide that can bind to the target protein to "replace" the small molecule target protein ligand part in the ordinary "triad" PROTAC structure. This polypeptide connects the linker and the small molecule E3 ligand, and a permeation peptide sequence is added to form a structure of: permeation peptide (permeation peptide) + polypeptide (target) + linker + E3 ligand. If necessary, the linker can be removed so that the polypeptide (target) containing the permeation peptide is directly linked to the E3 ligand.

[0046] The drug synthesized in this invention is a complex of a polypeptide and a small molecule, which can be linked by a small molecule linker or removed. A membrane-entering polypeptide sequence can be added to the non-small molecule linker segment of the polypeptide. This sequence can carry the polypeptide-small molecule complex (CePPiTAC complex) into the cell. At this time, the polypeptide portion targeting the target protein can bind to the target protein. Meanwhile, the small molecule E3 ligand at the other end of the linker can bind to the E3 ligase and trigger the E3 ubiquitinase reaction to ubiquitinate the target protein, thereby enabling the intracellular 26S protease to recognize the target protein and degrade it.

[0047] The features and advantages of this invention are as follows: The target chimera provided by this invention, mediated by cell-penetrating peptides, is a modular assembly of peptides or microproteins. Through the interconnection of membrane-penetrating peptides, targeting peptides, and small molecule ligands, it can penetrate the cell membrane and target all target proteins. The connected small molecule ligands can bind to immobilized ligases and initiate ubiquitinase reactions, thereby ubiquitinizing the target protein. This allows intracellular proteases to target and degrade the target protein, thus enabling a broader screening of targeted drugs. Because it uses peptide modules to bind to target proteins, theoretically, it can target all target proteins, which is not possible with other degradation agent technologies. In addition, because this technology uses highly efficient 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, the degradation of target proteins can be achieved at the nmol level in cell experiments.

[0048] In this chimeric structure, a linker can also be added, which can further solidify the connection between the targeting peptide and the small molecule ligand.

[0049] Meanwhile, the targeted chimeric compound provided by this invention adopts a modular design, and the different functional sequences or small molecule compound modules can be replaced and stacked as needed. This design concept greatly enhances the effectiveness and applicability of targeted drugs.

[0050] Small molecule triplet PROTACs have limited target targeting capabilities, while peptide-based PROTAC / degraders have low degradation efficiency, often requiring micromole levels to degrade targets on cells. This technology, however, can target all targets while achieving highly efficient nmol-level degradation (cellular degradation), truly realizing the goal of "a cure for all diseases." Furthermore, this invention is revolutionary compared to previous technologies. For example, targeting viral-related proteins has traditionally focused on proteins involved in viral infection of human cells (such as the S protein of SARS-CoV-2) and enzymes required for viral synthesis, resulting in limited target selection. This technology, however, uses peptide sequences to bind to the inactive sites of targets, enabling it to target and degrade all viral proteins. It is also effective in overcoming drug resistance caused by viral mutations, significantly increasing the likelihood and convenience of successful viral drug development. Additionally, this technology can degrade a single mutation of a target while leaving wild-type homologous proteins without the mutation largely unaffected—a capability unattainable by other degradation agent technologies such as PROTACs. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0052] Figure 1 This is shown as the conventional design method of modular assembly of targeted chimeric compounds (target degrading agents) mediated by cell-penetrating peptides in Embodiment 2 of the present invention, namely the general membrane-penetrating peptide-targeting peptide-linker-small molecule ligand mode.

[0053] Figure 2 The target chimera shown in Example 3 of this invention is a modular assembly of polypeptides or microproteins mediated by cell-penetrating peptides. It is specifically designed for a certain type or class of difficult-to-degrade pathogenic proteins and can bind the target polypeptide to two or more different E3 ligase conjugates to effectively degrade the target pathogenic protein.

[0054] Figure 3 The target chimera of polypeptide or microprotein modular assembly mediated by cell-penetrating peptides shown in Example 4 of the present invention can effectively target multiple targets related to the formation of protein-protein complexes of pathogenic proteins, thereby achieving the effect of targeting pathogenic proteins, effectively inhibiting the entire pathogenic pathway, and completely inhibiting a specific disease by degrading multiple targets.

[0055] Figure 4 The diagram shows the solid-phase synthesis process flow of the polypeptide in Example 5, where the synthesized polypeptide is labeled as 1.

[0056] Figure 5 The diagram shows the synthesis reaction of lenalidomide and succinic anhydride in Example 5, where lenalidomide is labeled as 2, succinic anhydride as 3, and the synthesized product as 4.

[0057] Figure 6 The diagram shown is a flow chart of the solid-phase synthesis process of the LEN-binding peptide in Example 5, wherein the synthesized product (diastereomeric mixture) is labeled as 5.

[0058] Figure 7 This invention illustrates an embodiment of a targeted chimera mediated by cell-penetrating peptides, used to degrade the SARS-CoV-2 S protein HR2, and its efficacy verification. Figure 7 A is a structural diagram of a targeted chimera assembled from modular peptides or microproteins mediated by cell-penetrating peptides. Figure 7 B represents the verification of the protein degradation effect. Figure 7C represents the effect of protease degrading agents, verified by using the protease inhibitor MG132.

[0059] Figure 8 This invention illustrates a modular assembly and targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides for degrading the SARS-CoV-2 N protein, and its efficacy verification, as shown in one embodiment of the invention. Figure 8 A is a structural diagram of a targeted chimera assembled from modular peptides or microproteins mediated by cell-penetrating peptides. Figure 8 B represents the verification of the protein degradation effect. Figure 8 C represents the effect of protease degrading agents, verified by using the protease inhibitor MG132.

[0060] Figure 9 This invention illustrates a modular assembly of peptides or microproteins mediated by cell-penetrating peptides for degrading the SARS-CoV-2 M protein, and its efficacy verification. Figure 9 A is a structural diagram of a targeted chimera assembled from modular peptides or microproteins mediated by cell-penetrating peptides. Figure 9 B represents the verification of the protein degradation effect. Figure 9 C represents the effect of protease degrading agents, verified by using the protease inhibitor MG132.

[0061] Figure 10 This invention illustrates a modular assembly and targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides for degrading SARS-CoV-2 E protein, and its efficacy verification, as shown in one embodiment of the invention. Figure 10 A is a structural diagram of a targeted chimera assembled from modular peptides or microproteins mediated by cell-penetrating peptides. Figure 10 B represents the verification of the protein degradation effect. Figure 10 C represents the effect of protease degrading agents, verified by using the protease inhibitor MG132.

[0062] Figure 11 This invention illustrates a modular assembly of peptides or microproteins mediated by cell-penetrating peptides for degrading the Orf6 protein of COVID-19, and its efficacy verification. Figure 11 A is a structural diagram of a targeted chimera assembled from modular peptides or microproteins mediated by cell-penetrating peptides. Figure 11 B represents the verification of the protein degradation effect. Figure 11 C represents the effect of protease degrading agents, verified by using the protease inhibitor MG132.

[0063] Figure 12 This invention illustrates an embodiment of a targeted chimera of polypeptides or microproteins mediated by cell-penetrating peptides for degrading Lag-3 protein, and its effectiveness verification. Figure 12A is a structural diagram of a targeted chimera assembled from modular peptides or microproteins mediated by cell-penetrating peptides. Figure 12 B represents the verification of the protein degradation effect. Figure 12 C represents the effect of protease degrading agents, verified by using the protease inhibitor MG132.

[0064] Figure 13 This invention illustrates an embodiment of the modular assembly and targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides for degrading Her2 protein, and its effectiveness verification. Figure 13 A is a structural diagram of a targeted chimera assembled from modular peptides or microproteins mediated by cell-penetrating peptides. Figure 13 B represents the verification of the protein degradation effect. Figure 13 C represents the effect of protease degrading agents, verified by using the protease inhibitor MG132.

[0065] Figure 14 This invention illustrates an embodiment of the modular assembly and targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides for degrading SHP-2 protein, and its effectiveness verification. Figure 14 A is a structural diagram of a targeted chimera assembled from modular peptides or microproteins mediated by cell-penetrating peptides. Figure 14 B represents the verification of the protein degradation effect. Figure 14 C represents the effect of protease degrading agents, verified by using the protease inhibitor MG132.

[0066] Figure 15 This invention illustrates an embodiment of the modular assembly and targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides for degrading STAT5B protein, and its effectiveness verification. Figure 15 A is a structural diagram of a targeted chimera assembled from modular peptides or microproteins mediated by cell-penetrating peptides. Figure 15 B represents the verification of the protein degradation effect. Figure 15 C represents the effect of protease degrading agents, verified by using the protease inhibitor MG132.

[0067] Figure 16 This invention illustrates an embodiment of the modular assembly and targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides for degrading MUC16 protein, and its effectiveness verification. Figure 16 A is a structural diagram of a targeted chimera assembled from modular peptides or microproteins mediated by cell-penetrating peptides. Figure 16 B represents the verification of the protein degradation effect. Figure 16 C represents the effect of protease degrading agents, verified by using the protease inhibitor MG132.

[0068] Figure 17 This invention illustrates an embodiment of a targeted chimera mediated by cell-penetrating peptides for degrading CTLA-4 protein, using modular assembly of polypeptides or microproteins, and its effectiveness verification. Figure 17 A is a structural diagram of a targeted chimera assembled from modular peptides or microproteins mediated by cell-penetrating peptides. Figure 17 B represents the verification of the protein degradation effect.

[0069] Figure 18 This invention illustrates a modular assembly of peptides or microproteins mediated by cell-penetrating peptides for degrading PCSK9 protein, and its effectiveness verification, as shown in one embodiment of the invention. Figure 18 A is a structural diagram of a targeted chimera assembled from modular peptides or microproteins mediated by cell-penetrating peptides. Figure 18 B is to verify that the effect is indeed produced by the protease degrader by using the protease inhibitor MG132.

[0070] Figure 19 This invention illustrates an embodiment of the modular assembly and targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides for degrading PD-1 protein, and its efficacy verification. Figure 19 A is a structural diagram of a targeted chimera assembled from modular peptides or microproteins mediated by cell-penetrating peptides. Figure 19 B is to verify that the effect is indeed produced by the protease degrader by using the protease inhibitor MG132.

[0071] Figure 20 This invention illustrates an embodiment of a modularly assembled targeted chimera of polypeptides or microproteins mediated by cell-penetrating peptides for degrading PD-L1 protein, and its effectiveness verification. Figure 20 A is a structural diagram of a targeted chimera assembled from modular peptides or microproteins mediated by cell-penetrating peptides. Figure 20 B is to verify that the effect is indeed produced by the protease degrader by using the protease inhibitor MG132.

[0072] Figure 21 This invention illustrates an embodiment of a modularly assembled targeted chimera of polypeptides or microproteins mediated by cell-penetrating peptides for precisely targeting and degrading KRAS proteins carrying the G12V mutation, and its effectiveness verification. Figure 21 A is a structural diagram of a targeted chimera assembled from modular peptides or microproteins mediated by cell-penetrating peptides. Figure 21 B is the validation of the degradation effect on the G12V variant KRAS protein. Figure 21 C represents the validation of the degradation effect on unmutated KRAS protein (wild type).

[0073] Figure 22This invention illustrates a modular transmembrane peptide-mediated dual E3 ligand targeting chimera for PCSK9 protein degradation and its effectiveness verification in one embodiment. Figure 22 A is a structural diagram of a modular transmembrane peptide-mediated dual E3 ligand (CRBN+VHL) targeting chimera. Figure 22 B was developed using the protease inhibitor MG132 to verify that the effect was indeed produced by the protease degrader and that the overall drug dosage was lower.

[0074] Figure 23 This invention illustrates a modular transmembrane peptide-mediated dual-target chimera for the simultaneous degradation of HR2 and N proteins, and its efficacy verification, as shown in one embodiment of the invention. Figure 23 A is a structural diagram of a modular transmembrane peptide-mediated dual-target (COVID-19 HR2+ COVID-19 N protein target) targeting chimera. Figure 23 B is to verify that the effect is indeed produced by the protease degrader by using the protease inhibitor MG132.

[0075] Figure 24 This invention illustrates a modular transmembrane peptide-mediated stapled peptide-modified targeted chimera for degrading PD-L1 protein and its efficacy verification, as shown in one embodiment of the invention. Figure 24 A is a structural diagram of a modular transmembrane peptide-mediated stapled peptide-modified targeted chimera. Figure 24 B represents the verification of the protein degradation effect.

[0076] Figure 25 This invention illustrates a modular transmembrane peptide-mediated cyclic peptide-modified targeted chimera for degrading PD-L1 protein and its efficacy verification, as shown in one embodiment of the invention. Figure 25 A is a structural diagram of a modular transmembrane peptide-mediated cyclic peptide-modified targeted chimeric compound. Figure 25 B represents the verification of the protein degradation effect.

[0077] Figure 26 Displayed as Figures 12-14 Transmembrane staining verification diagram of targeted chimeric polypeptide or microprotein modular assembly mediated by cell transmembrane peptides.

[0078] Figure 27 Displayed as Figure 15 , Figure 16 Transmembrane staining verification diagram of targeted chimeric polypeptide or microprotein modular assembly mediated by cell transmembrane peptides.

[0079] Figure 28 Displayed as Figure 17 , Figure 18 Transmembrane staining verification diagram of targeted chimeric polypeptide or microprotein modular assembly mediated by cell transmembrane peptides.

[0080] Figure 29 Displayed as Figure 19 , Figure 20 Transmembrane staining verification diagram of targeted chimeric polypeptide or microprotein modular assembly mediated by cell transmembrane peptides.

[0081] Figures 30-32 The diagram shows the structure of a staple peptide + small molecule ligand chimeric compound. Figure 30 Part A and Figure 31 Part A is connected. Figure 31 Part B and Figure 32 The B part is connected, and the whole structure is a chimeric compound containing staple peptide.

[0082] Figures 33-35 The diagram shows the structure of a cyclic peptide + small molecule ligand chimeric compound. Figure 33 Part A and Figure 34 Part A is connected. Figure 33 Part B and Figure 34 Part B is connected. Figure 34 Part D and Figure 35 The D portion is connected, and the whole structure is a chimeric compound containing a cyclic peptide. Detailed Implementation

[0083] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0084] Example 1:

[0085] A targeted chimera mediated by cell-penetrating peptides and modularly assembled from polypeptides or microproteins includes at least one interlinked membrane-penetrating peptide module, at least one targeting polypeptide module, and at least one small molecule ligand module, wherein the targeting polypeptide module is a polypeptide sequence capable of binding to a target protein.

[0086] Chimeric molecules formed by membrane-penetrating peptides, targeting peptides, and small molecule ligands can effectively target the small molecule ligands to the pathogenic target proteins through the targeting peptides, thereby achieving targeted and specific drug therapy. In the treatment of various diseases (especially tumors), they can exert more stable and broader effects.

[0087] There is one membrane-penetrating peptide module, and there can be one, two, three or even more targeting peptide modules, as well as one, two, three or even more small molecule ligand modules.

[0088] The modular assembly of targeted chimeras using cell-penetrating peptides as a mediated method for polypeptides or microproteins also includes at least one linker module for chimerizing the targeted polypeptide module with the small molecule ligand module. There can be one, two, three, or even more linker modules.

[0089] The membrane-penetrating peptide module is attached to the free end of the targeting peptide module and is used to guide the targeting chimeric compound to penetrate the cell membrane.

[0090] The small molecule ligand module is a small molecule E3 ligand that can bind to E3 ligase;

[0091] Preferably, the protease degrading agent adapted to the small molecule E3 ligand is one or more of CRBN, VHL, and IAP.

[0092] The amino acid sequence of the transmembrane peptide module is any one of SEQ ID No. 1-SEQ ID No. 3.

[0093] SEQ ID No. 1: YGRKKRRQRRR;

[0094] SEQ ID No. 2: RRRRRRRR;

[0095] SEQ ID No. 3: RQIKIWFQNRRMKWK.

[0096] The amino acid sequence of the targeted peptide module is any one or more of SEQ ID No. 4-SEQ ID No. 17.

[0097] SEQ ID No.4: SAIGKIQDSLSSTAS;

[0098] SEQ ID No. 5: PQEESEEEVEEP;

[0099] SEQ ID No. 6: GGKGLGKacGGA;

[0100] SEQ ID No.7: DTMVGWDKDARTK;

[0101] SEQ ID No. 8: FNGARSFIDI;

[0102] SEQ ID No. 9: WARLWNYLYR;

[0103] SEQ ID No. 10: RSFIDIGSGT;

[0104] SEQ ID No.11: KAVDG(p)YVKPQI;

[0105] SEQ ID No. 12: WIDPVNGDTE;

[0106] SEQ ID No.13: ARHPSWYRPFEGCG;

[0107] SEQ ID No. 14: MESFPGWNLV(homoR)IGLLR.

[0108] SEQ ID No.15: FNWDYSLEELREKAKYK;

[0109] SEQ ID No.16:MPIFLDHILNKFWILHYA;

[0110] SEQ ID No. 17: LYDVAGSDKY.

[0111] The linker module is a small molecule compound, and its structural formula is shown in Formula I. Formula I.

[0112] When a targeted chimeric compound is assembled from a modular polypeptide or microprotein mediated by cell-penetrating peptides, and the appropriate protease degrading agent is CRBN, the structural formula of the small molecule ligand module is shown in Formula II. Formula II; When the suitable protease degrading agent is VHL, the structural formula of the small molecule ligand module is shown in Formula III. Formula III; When the suitable protease degrading agent is IAP, the structural formula of the small molecule ligand module is shown in Formula IV. Formula IV.

[0113] Targeted chimeric compounds assembled from modular polypeptide or microprotein modules mediated by cell-penetrating peptides, having any one or more of the following structures:

[0114] 1) A membrane-penetrating peptide SEQ ID No. 1 + a targeting peptide SEQ ID No. 4 + a linker of formula I + a small molecule ligand of formula II;

[0115] 2) Membrane-penetrating peptide SEQ ID No. 2 + targeting peptide SEQ ID No. 5 + linker of formula I + small molecule ligand of formula III;

[0116] 3) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 5 + linker of formula I + small molecule ligand of formula IV;

[0117] 4) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 6 + linker of formula I + small molecule ligand of formula II;

[0118] 5) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 7 + linker of formula I + small molecule ligand of formula III;

[0119] 6) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 8 + linker of formula I + small molecule ligand of formula II;

[0120] 7) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 9 + linker of formula I + small molecule ligand of formula III;

[0121] 8) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 10 + linker of formula I + small molecule ligand of formula II;

[0122] 9) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 11 + linker of formula I + small molecule ligand of formula IV;

[0123] 10) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 12 + linker of formula I + small molecule ligand of formula III;

[0124] 11) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 13 + linker of formula I + small molecule ligand of formula III;

[0125] 12) Membrane-penetrating peptide SEQ ID No. 3 + targeting peptide SEQ ID No. 14 + linker of formula I + small molecule ligand of formula IV;

[0126] 13) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 15 + linker of formula I + small molecule ligand of formula II;

[0127] 14) Membrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 16 + linker of formula I + small molecule ligand of formula II;

[0128] 15) Membrane-penetrating peptide SEQ ID No. 2 + targeting peptide SEQ ID No. 17 + linker of formula I + small molecule ligand of formula IV;

[0129] 16) Membrane-penetrating peptide SEQ ID No. 3 + targeting peptide SEQ ID No. 14 + linker of formula I structure + (double E3 ligand: small molecule ligand of formula II structure + small molecule ligand of formula III structure);

[0130] 17) Membrane-penetrating peptide SEQ ID No.1 + (dual-target: targeting peptide SEQ ID No.4 + targeting peptide SEQ ID No.5) + linker of Formula I + small molecule ligand of Formula II.

[0131] The targeted peptide modules in the modular assembly of peptides or microproteins mediated by cell-penetrating peptides also include modified stapled peptide sequences or cyclic peptide sequences.

[0132] The structural formula of the staple peptide is shown in Formula V. Formula V; the structural formula of the cyclic peptide is shown in Formula VI. Formula VI.

[0133] The structure of a modularly assembled targeted chimera of polypeptides or microproteins containing a staple peptide mediated by a cell-penetrating peptide is as follows: a staple peptide of formula V + a linker of formula I + a small molecule ligand of formula II.

[0134] Modular assembly of targeted chimeric polypeptides or microproteins containing cyclic peptides mediated by cell-penetrating peptides, with the following structure: cyclic peptide of Formula VI + linker of Formula I + small molecule ligand of Formula II.

[0135] Example 2:

[0136] The modular assembly of peptides or microproteins mediated by cell-penetrating peptides in Example 1 can be used to prepare products that degrade target proteins or products that degrade target proteins with variable amino acid sites.

[0137] The targeted degradation proteins include one or more of the following: SARS-CoV-2 S protein HR2, SARS-CoV-2 N protein, SARS-CoV-2 M protein, SARS-CoV-2 E protein, SARS-CoV-2 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 KRAS protein G12V variant.

[0138] To date, targeting, inhibiting, and drug-treating proteins involved in protein-protein interactions has been virtually impossible with the help of inhibitory molecules. Targeting harmful / pathogenic proteins using proteosome degradation mechanisms is a promising therapeutic approach. With the aid of the ubiquitin-proteasome system (UPS), target protein-protein interactions (PPIs) selectively degrade “drug-unmanageable” target proteins by interacting with the target protein via a key peptide sequence. The inventors have designed a method for such a bifunctional peptide-based degrader that targets and degrades target proteins involved in PPIs. The inventors achieved the degradation of the desired target protein by binding a peptide with high affinity and selective interaction to an E3 ligase via a linker. To achieve cell permeability of the peptide degrader, the inventors further bound the peptide degrader sequence to a cell-permeable peptide (cell-penetrating peptide). The typical design of a target degrader is as follows: Figure 1 As shown.

[0139] The inventors discovered that the peptide degrader is designed to degrade >15,000 targets involved in protein-protein interactions, using corresponding targeting ligands / peptides. These ligands can couple to approximately 1,100 linkers (including approximately 300 PEG-type linkers) and approximately 65 E3 ligase-binding ligands. To improve cell permeability, the inventors further combined the peptide PROTAC technology with approximately 800 cell-penetrating peptides.

[0140] SMILES (Simplified Molecular Input Line Entry Specification) is a specification that explicitly describes molecular structures using ASCII strings. InChI Key (International Chemical Identifier) ​​is a unique identifier for the chemical structure of each compound, given by the International Union of Pure and Applied Chemistry (IUPAC). Using the InChI key, the corresponding compound can be easily found in the PubMed ChemCompound database (https: / / www.ncbi.nlm.nih.gov / pccompound).

[0141] Table 1 shows the selection of linker modules, including but not limited to the molecular structures represented by SMILES and the compounds corresponding to the InChI key, as detailed in Table 1 below.

[0142]

[0143] Table 2 shows another part of the Linkers module, namely the selection of PEG type linkers, including but not limited to the compounds represented by "name" and corresponding to the EnamineStore ID, as detailed in Table 2 below. EnamineStore is a compound database (website: https: / / www.enaminestore.com / search).

[0144]

[0145] Table 3 shows the selection of E3 ligase binding ligand modules, including but not limited to the molecular structures represented by SMILES and the compounds corresponding to the InChI key, as detailed in Table 3 below.

[0146]

[0147] Table 4 shows the selection of cell-penetrating peptide sequence modules, including but not limited to the sequences indicated as "cell-penetrating peptide sequences", see Table 4 below for details.

[0148]

[0149] Table 5 shows examples of the selection of target peptides, including but not limited to the target proteins indicated by "target protein name" and the corresponding peptide sequences; there are approximately 19,813 target proteins, including all known target proteins and all targeting peptides targeting these target proteins. However, due to their large size, the inventors have only selected a few dozen representative target proteins as examples. But the scope of protection claimed by this invention includes all target proteins and targeting peptides known in the art, and is not limited to these few dozen target proteins, as shown in Table 5 below.

[0150]

[0151] In Tables 1-5, Table 3 "e3 ligand" represents all currently applicable small molecule ligands for E3 ligases. There are two types of linkers: one is "PEG linkers" as shown in Table 2, and the other linkers are collected in Table 1 "linkers". Table 4 "CPP list" contains all currently applicable transmembrane peptides, and Table 5 "Target interacting peptide" contains examples of target peptides for all currently applicable targets.

[0152] Currently, due to technological limitations, only about 10-20% of targets can be developed. However, the CePPiTAC technology provided by this invention changes the target protein from a small molecule to a polypeptide and attaches a transmembrane peptide sequence to ensure that the complex can enter the cell membrane. Since any target protein can be screened to bind to resistant polypeptides that have a binding affinity, theoretically, it can degrade any target protein and cause proteases to degrade it. Therefore, the application market is extremely broad. Previously "untargetable" target proteins can be developed into drugs for degradation. Moreover, since the relationships between many proteins are already clear, screening ligand polypeptides is very convenient. Several highly efficient small molecule E3 ligase ligands have emerged, and their binding to polypeptides is very simple. Using this invention to design drugs will be very convenient, saving time and effort, and enabling the rapid and economical development of various new drugs.

[0153] Example 3:

[0154] The inventors also effectively degraded the target by binding the target-binding peptide to two or more different E3 ligase conjugates, as shown below. Specifically, as follows... Figure 2 As shown.

[0155] Example 4:

[0156] The inventors also considered degrading multiple targets involved in the formation of disease-causing protein-protein complexes to inhibit the entire pathogenic pathway, and to completely inhibit a specific disease by degrading multiple targets. To achieve this peptide degrader, the inventors designed the peptide degrader as follows: Figure 3 As shown.

[0157] Example 5:

[0158] Synthesis of a representative PROTAC peptide conjugate, PEN-FFW-LINK-LEN. Here, PEN, FFW, and LEN represent the membrane-penetrating peptide, the targeting peptide, and the small molecule ligand, respectively.

[0159] a. Solid-phase synthesis of peptide 1:

[0160] Option 1:

[0161] like Figure 4As shown, peptide 1 was synthesized using 0.15 mmol as the standard.

[0162] The SYRO automated peptide synthesizer was used to extend the full-length sequence. 0.5 g of Fmoc-Ile Wang resin (0.3 mmol / g) was swollen in DMF and deprotected with 20% piperidine / DMF (twice, for 5 min and 20 min). After each deprotection, the resin was washed with DMF (3 x 10 mL). On the instrument, each fmoc-amino acid residue (4 eq, 0.6 mmol) was dual-coupled with two different activators in DMF: DIC / Oxyma (4 eq, 0.6 mmol, 30 min) and HATU / DIPEA (4 eq, 0.6 mmol, 45 min). Final deprotection of the Fmoc-part was performed using 20% ​​piperidine / DMF (twice, for 5 min and 20 min), followed by washing with DMF (3 x 10 mL) to provide resin-bound linear peptide 1. The desired quality was determined by micro-cleavage.

[0163] b. Synthesis of lenalidomide coupled with succinic anhydride:

[0164] Option 1:

[0165] like Figure 5 As shown, nalidomide 2 (200 mg, 0.77 mmol) was added to a round-bottom flask containing succinic anhydride 3 (90 mg, 0.93 mmol) and toluene (8 mL), and a reflux condenser was used. The mixture was refluxed for 3 h, and the precipitate was separated by vacuum filtration. The filter cake was washed with ethyl acetate (20 mL x 2) and dried under vacuum to give 4-(2-(2,6-dioxoperidin-3-yl)-1-oxoisoindol-4-yl)amino)-4-oxobutyric acid product 4. Yield: 120 mg, 43.4%.

[0166] c. Solid-phase synthesis and resin cleavage of LEN-coupled peptides:

[0167] Option 3:

[0168] 1. Solid-phase synthesis:

[0169] like Figure 6 As shown. Lenalidomide coupled with succinic anhydride (4) was activated with DIC / Oxyma (4 eq) in DMF, added to pre-dissolved resin-bound amine (200 mg, 0.06 mmol), and shaken or shaken for 2 hours. The resin was filtered and washed with DMF (3 x 10 mL) and DCM (3 x 10 mL), and finally washed with ether (2 x 10 mL). After vacuum drying, the resin was pyrolyzed.

[0170] 2. Resin pyrolysis:

[0171] The product was separated from the resin within 10 ml, containing trifluoroacetic acid, triisopropylsilane, and water (95:2.5:2.5), providing 120 mg of crude peptide. Purification was performed using reversed-phase high-performance liquid chromatography (RP-HPLC) to yield 10 mg of diastereomeric mixture 5 with a maximum purity of 97.15% and a 214 nm purity of 93.19%, yielding 10 mg at 6.68%.

[0172] Table 6 shows the preparation conditions for high performance liquid chromatography.

[0173]

[0174] Table 7 shows the gradient table.

[0175]

[0176] Example 6:

[0177] 1. Many existing diseases target membrane proteins, such as PD-1 and PD-L1. Although their inhibitors are commonly used, their efficacy is low and they are prone to drug resistance. This is because PD-1 and PD-L1 target proteins are located on the cell membrane, and it is difficult to develop small molecule PD-1 / PD-L1 degrading agents. The technology of this invention can target the intracellular portion of these two proteins and degrade them.

[0178] 2. Some disease targets are difficult to bind to small molecules due to their structure, so it is difficult to design PROTACs for ordinary small molecules, such as the G12V mutation of the Kras protein. However, the technology of this invention can utilize peptides to bind to and degrade them.

[0179] 3. Some virus-related proteins, such as those of SARS-CoV-2 or HIV, are difficult to target with traditional antiviral drugs, which focus on neutralizing antibodies or proteases that inhibit the virus. This approach has limited targets, and the drugs become ineffective if the virus mutates. The CePPiTAC technology provided by this invention can target viral structural proteins or proteases by binding to them with peptides, then degrading them. This impairs protein synthesis or prevents the formation of viral packaging. Firstly, it expands the number of targets for antiviral drug development, allowing targeting of many previously untargetable viral proteins. Secondly, because it can bind to structural protein portions that are less prone to mutation and degrade the entire target protein, the resulting drug is immune to any viral mutations.

[0180] Example 7:

[0181] To illustrate the free combination and multiple selectivity of the four "modules" in the basic structure of the modular assembly and targeting chimera of polypeptides or microproteins mediated by cell-penetrating peptides presented in this application, the inventors designed a modular targeting chimera for degrading the SARS-CoV-2 S protein HR2. Figure 7 A); where the transmembrane peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequence is SAIGKIQDSLSSTAS; and the linker module is a small molecule composed of (PEG)4, with the following structural formula: The E3 small molecule ligand module is the E3 ligand for the target CRBN, with the following structural formula: .

[0182] Western blotting was used to verify the protein degradation effects of different dosages (nmol) of the targeting chimeric compound. Figure 7 (B) It can be seen that as the dosage increases, the protein has been degraded (no expression).

[0183] To verify that the targeted chimera provided in this application functions as degradation rather than inhibition, the inventors also designed and added the protease inhibitor MG132. The protease inhibitor can inhibit the effect of the targeted chimera in this application, as demonstrated by experimental results (…). Figure 7 C) It can be seen that the protein with only the target chimera added still cannot be expressed, but the protein with the target chimera + MG132 added can be expressed normally, and its expression level is comparable to that of the protein without the target chimera. This result shows that the target chimera degrades rather than inhibits the protein.

[0184] Studies have shown that the S protein on the surface of coronaviruses mediates the viral infection process of target cells. It consists of two subunits, S1 and S2. The S1 subunit is responsible for binding to receptors on the cell surface, while the S2 subunit functions in viral fusion with the cell membrane. The S2 subunit contains important functional regions such as heptapeptide repeat domain 1 (HR1) and heptapeptide repeat domain 2 (HR2). During viral membrane fusion, HR1 and HR2 fold to form a six-helix bundle structure (6HB) to bring the viral membrane and cell membrane closer together, allowing viral genetic material to enter the target cell through the fusion pore. The targeting chimera can bind to and degrade the HR2 subunit of the SARS-CoV-2 S protein, inhibiting the formation of the six-helix bundle structure and thus interfering with viral fusion with the cell membrane, preventing viral invasion of cells and fundamentally achieving the goal of preventing and controlling COVID-19.

[0185] Example 8:

[0186] Similar to Example 7, to illustrate the "modular" design of this application, the inventors have also provided modular targeting chimeras for degrading the SARS-CoV-2 N protein, SARS-CoV-2 M protein, SARS-CoV-2 E protein, SARS-CoV-2 Orf6 protein, Lag-3 protein, Her2 protein, SHP-2 protein, STAT5B protein, MUC16 protein, CTLA-4 protein, PCSK9 protein, PD-1 protein, and PD-L1 protein. Figure 8 A- Figure 20 A).

[0187] A modular targeting chimeric compound for degrading the SARS-CoV-2 N protein has the following sequence: RRRRRRRR; targeting peptide module sequence: PQEESEEEVEEP; and linker module, a small molecule composed of (PEG)4, with the following structural formula: The E3 small molecule ligand module is the E3 ligand for the target VHL, with the following structural formula: .

[0188] A modular targeting chimeric compound for degrading the SARS-CoV-2 M protein has the following sequence: transmembrane peptide module sequence YGRKKRRQRRR; targeting peptide module sequence PQEESEEEVEEP; and linker module consisting of a small molecule composed of (PEG)4, with the following structural formula: The E3 small molecule ligand module is the E3 ligand for the target IAP, with the following structural formula: .

[0189] A modular targeting chimeric compound for degrading the SARS-CoV-2 E protein has the following sequence: transmembrane peptide module sequence YGRKKRRQRRR; targeting peptide module sequence GGKGLGKacGGA; and linker module consisting of a small molecule composed of (PEG)4, with the following structural formula: The E3 small molecule ligand module is the E3 ligand for the target CRBN, with the following structural formula: .

[0190] A modular targeting chimeric compound for degrading the Orf6 protein of SARS-CoV-2 has the following sequence: transmembrane peptide module sequence YGRKKRRQRRR; targeting peptide module sequence DTMVGWDKDARTK; and linker module consisting of a small molecule composed of (PEG)4, with the following structural formula: The E3 small molecule ligand module is the E3 ligand for the target VHL, with the following structural formula: .

[0191] A modular targeting chimeric compound for degrading Lag-3 protein, comprising a transmembrane peptide module sequence of YGRKKRRQRRR; a targeting peptide module sequence of FNGARSFIDI; and a linker module consisting of a small molecule composed of (PEG)4, with the following structural formula: The E3 small molecule ligand module is the E3 ligand for the target CRBN, with the following structural formula: .

[0192] A modular targeting chimera for degrading Her2 protein has the following sequence: transmembrane peptide module sequence YGRKKRRQRRR; targeting peptide module sequence WARLWNYLYR; and linker module consisting of a small molecule composed of (PEG)4, with the following structural formula: The E3 small molecule ligand module is the E3 ligand for the target VHL, with the following structural formula: .

[0193] A modular targeting chimera for degrading SHP-2 protein has the following sequence: transmembrane peptide module sequence YGRKKRRQRRR; targeting peptide module sequence RSFIDIGSGT; and linker module consisting of a small molecule composed of (PEG)4, with the following structural formula: The E3 small molecule ligand module is the E3 ligand for the target CRBN, with the following structural formula: .

[0194] A modular targeting chimeric compound for degrading STAT5B protein has the following sequence: transmembrane peptide module sequence YGRKKRRQRRR; targeting peptide module sequence KAVDG(p)YVKPQI; and linker module consisting of a small molecule composed of (PEG)4, with the following structural formula: The E3 small molecule ligand module is the E3 ligand for the target IAP, with the following structural formula: .

[0195] A modular targeting chimera for degrading MUC16 protein has the following sequence: transmembrane peptide module sequence YGRKKRRQRRR; targeting peptide module sequence WIDPVNGDTE; and linker module consisting of a small molecule composed of (PEG)4, with the following structural formula: The E3 small molecule ligand module is the E3 ligand for the target VHL, with the following structural formula: .

[0196] A modular targeting chimeric compound for degrading CTLA-4 protein, comprising a transmembrane peptide module sequence of YGRKKRRQRRR; a targeting peptide module sequence of ARHPSWYRPFEGCG; and a linker module consisting of a small molecule composed of (PEG)4, with the following structural formula: The E3 small molecule ligand module is the E3 ligand for the target VHL, with the following structural formula: .

[0197] A modular targeting chimeric compound for degrading PCSK9 protein has the following sequence: transmembrane peptide module sequence RQIKIWFQNRRMKWK; targeting peptide module sequence MESFPGWNLV(homoR)IGLLR; and linker module consisting of a small molecule composed of (PEG)4, with the following structural formula: The E3 small molecule ligand module is the E3 ligand for the target IAP, with the following structural formula: .

[0198] A modular targeting chimeric compound for degrading PD-1 protein, comprising a transmembrane peptide module sequence of YGRKKRRQRRR; a targeting peptide module sequence of FNWDYSLEELREKAKYK; and a linker module consisting of a small molecule composed of (PEG)4, with the following structural formula: The E3 small molecule ligand module is the E3 ligand for the target CRBN, with the following structural formula: .

[0199] A modular targeting chimeric compound for degrading PD-L1 protein, comprising a transmembrane peptide module sequence of YGRKKRRQRRR; a targeting peptide module sequence of MPIFLDHILNKFWILHYA; and a linker module consisting of a small molecule composed of (PEG)4, with the following structural formula: The E3 small molecule ligand module is the E3 ligand for the target CRBN, with the following structural formula: .

[0200] The degradation effects of different dosages (nmol) of the targeted chimera on SARS-CoV-2 N protein, SARS-CoV-2 M protein, SARS-CoV-2 E protein, SARS-CoV-2 Orf6 protein, Lag-3 protein, Her2 protein, SHP-2 protein, STAT5B protein, MUC16 protein, and CTLA-4 protein were also verified using Western blotting. Figure 8 B- Figure 17 (B) It can be seen that as the dosage increases, the protein has been degraded (no expression).

[0201] Similarly, to verify that the targeted chimera provided in this application functions as degradation rather than inhibition, the inventors also designed and added the protease inhibitor MG132. The protease inhibitor can inhibit the effect of the targeted chimera in this application, as demonstrated by experimental results (…). Figure 8 C- Figure 16 C and Figure 18 As can be seen from B-20B, the protein with only the target chimera added still could not be expressed, but the protein with the target chimera + MG132 added could be expressed normally, and its expression level was comparable to that of the protein without the target chimera. This result indicates that the target chimera degrades rather than inhibits the protein.

[0202] The SARS-CoV-2 N protein, abundant in coronaviruses, is a highly immunogenic protein involved in genome replication and cell signaling pathway regulation. By targeting and degrading this protein with the chimeric compound described in this application, effective inhibition and treatment of SARS-CoV-2 can be achieved.

[0203] The SARS-CoV-2 M protein, a membrane glycoprotein (M), is a component of the viral particle envelope. The M protein participates in the assembly and release of the next generation of viral particles and plays an important role in the structural stability and functional expression of other structural proteins (S, E, and N proteins). By degrading this protein through targeted chimeras, the stability of the viral structure can be effectively disrupted, and viral function can be inhibited.

[0204] The COVID-19 E protein (E, Envelope Protein) is a component of the viral particle envelope and is a small envelope glycoprotein. The main function of the E protein is to protect the RNA gene chain inside the virus. By degrading this protein through targeted chimeras, the protective mechanism of viral RNA can be reduced or even eliminated, making the RNA chain easier to break, thereby effectively inhibiting viral function.

[0205] The Orf6 protein of the novel coronavirus is the most cytotoxic protein to human cells among the novel coronavirus proteins. Current research has found that introducing it into human cells can kill about half of the human cells. It can effectively inhibit the innate immune activity of host cells. By degrading this protein through targeted chimeras, the toxicity of the novel coronavirus to the human immune system can be greatly reduced.

[0206] Lag-3 protein, also known as CD233 or lymphocyte activation gene 3, is a type I transmembrane protein belonging to the immunoglobulin (Ig) superfamily. It is mainly expressed on the surface of activated T cells and NK cells. LAG-3 is a very promising immunotherapy target. By targeting and degrading this protein with chimeric compounds, it is possible to effectively block the inhibitory signals in the interaction between tumor cells and TILs in the tumor microenvironment, restore the immune surveillance function of TILs against tumor cells, and achieve an anti-tumor effect.

[0207] Her2 protein is a transmembrane protein with tyrosine protein kinase activity and belongs to the EGFR family. HER2 gene amplification is one of the most important factors affecting the growth and metastasis of breast cancer. By degrading this protein through targeted chimeras, apoptosis of breast tumors can be promoted and their proliferation inhibited.

[0208] SHP-2 protein, encoded by protein tyrosine phosphatase nonreceptor 11 (PTPN11), is a well-established PTP oncoprotein in humans and is becoming an important target for cancer treatment. Overactivation of SHP2 plays a crucial pathogenic role. By degrading this protein through targeted chimeras, the activation of the SHP2 pathway can be effectively blocked or inhibited, thereby significantly improving tumor treatment.

[0209] STAT5B protein is a signal transduction and transcription activator-5b. STAT signaling is a regulatory signal in various tumors. By degrading this protein through targeted chimeras, STAT signaling can be effectively dysregulated, thereby inhibiting the proliferation and colony formation of tumor cells (such as osteosarcoma cells) and inducing G0 / G1 phase cell arrest and apoptosis.

[0210] MUC16 protein is the largest transmembrane mucoprotein. Because it is known to be overexpressed on the surface of ovarian cancer cells and to cleave / shed into the bloodstream, it is a recognized serum biomarker for ovarian cancer. At the same time, MUC16 is also believed to play an anti-apoptotic role in cancer cells. Ectopic expression of its C-terminal domain induces cisplatin resistance in ovarian cancer cells, and this effect is mediated by p53 inhibition. By targeting and degrading this protein, apoptosis of cancer cells can be effectively regulated and their proliferation inhibited.

[0211] CTLA-4 protein, cytotoxic T-lymphocyte-associated protein 4, also known as CD152 (differentiation cluster 152), is a protein receptor that acts as an immune checkpoint and downregulates the immune response. Mutations in the CTLA-4 gene are not only associated with cancer, but also with type 1 diabetes, Graves' disease, Hashimoto's thyroiditis, celiac disease, systemic lupus erythematosus, thyroid-associated orbital disease, primary biliary cirrhosis, and other autoimmune diseases. Degrading this protein through targeted chimeras can effectively increase the body's immune activity.

[0212] Example 9:

[0213] In particular, to illustrate that the "modular" design presented in this application can also precisely target proteins with mutated amino acids, the inventors designed a modular targeting chimera for degrading KRAS proteins with G12V mutations. Figure 21 A), in which the transmembrane peptide module sequence is RRRRRRRR; the targeting peptide module sequence is LYDVAGSDKY; the linker module is a small molecule composed of (PEG)4, with the structural formula as follows: The E3 small molecule ligand module is the E3 ligand for the target IAP, with the following structural formula: .

[0214] Western blotting was also used to verify the degradation effect of different amounts (nmol) of the targeting chimeric compound on KRAS protein with G12V mutation, and the expression level was statistically analyzed. Figure 21 (B) It can be seen that as the dosage increases, the protein has been degraded (the expression level gradually decreases).

[0215] To verify the precise targeting of this targeting chimeric compound, the inventors also used Western blotting to verify the degradation effect of different dosages (nmol) of the targeting chimeric compound on unmutated KRAS protein (wild type) and statistically analyzed the expression levels. Figure 21 C) It can be seen that with the increase of dosage, the protein change is not obvious (the expression level decreases slightly, but not significantly), which fully demonstrates that the targeted chimera designed in this way can accurately target KRAS protein with G12V mutation, while basically not degrading wild-type KRAS protein, and the targeting and degradation accuracy is extremely high.

[0216] The KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene is a GDP / GTP binding protein. KRAS is activated when it binds to GTP and deactivated when it binds to GDP. KRAS can be transiently activated by growth factors or tyrosine kinases (such as EGFR). Activated KRAS can activate downstream pathways such as the PI3K-AKT-mTOR signaling pathway, which controls cell production, and the RAS-RAF-MEK-ERK signaling pathway, which controls cell proliferation. Mutated KRAS can remain continuously activated even without EGFR or other kinase activation, leading to continuous cell proliferation and ultimately carcinogenesis. KRAS mutations have been found in various tumors, most commonly lung cancer and pancreatic cancer. Targeted chimeras can precisely degrade KRAS proteins with the G12V mutation while having no effect on wild-type KRAS proteins, significantly improving the efficiency of targeted therapy for mutant proteins.

[0217] Example 10:

[0218] In particular, to illustrate that the "modular" design presented in this application can also connect dual E3 ligand structures and increase protein degradation efficiency, the inventors designed a dual E3 ligand modular targeting chimera for degrading PCSK9. Figure 22A), where the transmembrane peptide module sequence is RQIKIWFQNRRMKWK; the target peptide module sequence is MESFPGWNLV(homoR)IGLLR; and two linker modules are connected to two different E3 small molecule ligand modules, which are the E3 ligands for the targets CRBN and IAP, respectively. The overall structural formula of the linker modules and the E3 small molecule ligand modules is as follows: .

[0219] Similarly, to verify that the targeted chimera provided in this application functions as degradation rather than inhibition, the inventors also designed and added the protease inhibitor MG132. The protease inhibitor can inhibit the effect of the targeted chimera in this application, as demonstrated by experimental results (…). Figure 22 B) It can be seen that the protein with only the target chimera added still cannot be expressed, but the protein with the target chimera + MG132 added can be expressed normally, and its expression level is comparable to that of the protein without the target chimera. This result shows that the target chimera degrades rather than inhibits the protein.

[0220] At the same time, with Figure 18 A and Figure 18 Compared to B (also used for PCSK9 protein degradation), the targeted chimera with dual E3 ligands requires a lower dosage (from 25 nmol to 15 nmol) to achieve the same degradation effect. Furthermore, because this targeted chimera uses dual E3 ligands, even if one of the E3 ubiquitinases mutates and develops resistance, the other E3 ubiquitinase can still function, increasing the reliability of the targeted chimera.

[0221] Example 11:

[0222] In particular, to illustrate that the "modular" design presented in this application can simultaneously degrade two or more proteins targeting different protein sites, the inventors designed a dual-target modular targeting chimera for simultaneously degrading the SARS-CoV-2 HR2 protein and the SARS-CoV-2 N protein. Figure 23 A), where the transmembrane peptide module sequence is YGRKKRRQRRR; the targeting peptide module sequences are SAIGKIQDSLSSTAS and PQEESEEEVEEP; the linker module is a small molecule composed of (PEG)4, with the structural formula […]. The E3 small molecule ligand module is the E3 ligand for the target CRBN, with the following structural formula: .

[0223] Similarly, to verify that the dual-target chimera provided in this application functions as degradation rather than inhibition, the inventors also designed and added the protease inhibitor MG132. The protease inhibitor can inhibit the effect of the target chimera in this application, as demonstrated by experimental results ( Figure 23 B) It can be seen that the protein with only the target chimera added still cannot be expressed, but the protein with the target chimera + MG132 added can be expressed normally, and its expression level is comparable to that of the protein without the target chimera. This result shows that the target chimera degrades rather than inhibits the protein.

[0224] Meanwhile, this dual-target chimera can simultaneously degrade HR2 and N proteins. Based on this, the inventors can also design chimeras targeting three, four, or even more targets to simultaneously degrade multiple proteins, thereby achieving better applicability and a wider range of application conditions. Considering the space limitations of this application, it will not be elaborated here, but the dual-target description in this embodiment should not limit the technical protection of more target chimeras.

[0225] Example 12:

[0226] In particular, to illustrate that the "modular" design presented in this application can also modify the targeting peptide module to achieve the purpose of eliminating transmembrane peptide linkage or increasing structural stability, the inventors designed a modular targeting chimera using stapled peptide modification and cyclic peptide modification. Figure 24 A and Figure 25 A).

[0227] The degradation effect of different dosages (nmol) of the targeting chimeric compound on PD-L1 protein was also verified using Western blotting. Figure 24 B and Figure 25 (B) It can be seen that as the dosage increases, the protein has been degraded (the expression level gradually decreases).

[0228] The principle behind modifying stapled peptides and cyclic peptides is to make the target peptide module present a state similar to that of a protein secondary structure, forming a "miniature protein". Even without connecting to the membrane-penetrating peptide module, the breakdown of peptide segments during membrane entry can still be avoided, effectively increasing the stability of the target peptide module.

[0229] The modification process of the staple peptide (targeted peptide module) is as follows: CGIQDTNSKKQSDTHLEETC is modified and linked using two compounds, R8 and S5 (structures shown below), to make the peptide become: CGIQDT(R8)NSKKQS(S5)DTHLEET-;

[0230] R8 is Fmoc-R8-OH, with the following structural formula:

[0231] ;

[0232] S5 is Fmoc-S5-OH, with the following structural formula:

[0233] .

[0234] Structure of the staple peptide (or microprotein) + E3 small molecule ligand chimeric compound:

[0235] .

[0236] The structural formula of the targeting chimeric compound containing the staple peptide is as follows:

[0237] .

[0238] Overall structural formula as follows Figure 30-32 As shown.

[0239] Cyclic peptide + small molecule E3 ligand intercalation structure:

[0240] .

[0241] Structural formula of the cyclic peptide (targeting peptide module): Linker-3PEG, Binder (ligand): CRBN (full name Cereblon):

[0242] ;

[0243] The linker module is a small molecule composed of (PEG)4, with the following structural formula: ;

[0244] The E3 small molecule ligand module is the E3 ligand for the target CRBN, with the following structural formula: Cyclation method: Cyclate the two cysteine ​​disulfide bonds in the diagram above.

[0245] Overall structural formula as follows Figure 33-35 As shown.

[0246] Example 13:

[0247] The applicant retrieved three prior art documents related to this technology and compared their technologies with the technical solution of this invention.

[0248] Comparative Reference 1:

[0249] "Specific Knockdown of a-Synuclein by Peptide-Directed ProteasomeDegradation Rescued ItsAssociated Neurotoxicity" (Jing Qu, Xiaoxi Ren, FenqinXue, ..., Haixia Huang, Wei Wang, Jianliang Zhang, "Cell Chemical Biology", 2020).

[0250] Comparative Reference 2:

[0251] "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).

[0252] Comparative Reference 3:

[0253] "A PROTAC peptide induces durable β-catenin degradation and suppresses Wnt-dependent intestinal cancer" (Hongwei Liao1, Xiang Li2, Lianzheng Zhao1, Yalong Wang1, Xiaodan Wang1, Ye Wu2, Xin Zhou3, Wei Fu3, Lei Liu4, Hong-GangHu2,5 and Ye-Guang Chen1, Cell Discovery, 2020).

[0254] Among them, comparative literature 1 and comparative literature 2 disclose the following composition: peptide transmembrane peptide + target peptide + peptide linker + peptide binder. Comparative literature 1 shows a significant effect on the degradation of the target protein down to 50 μm (Figure 3); comparative literature 2 shows a significant effect on the target protein down to 100 μm (Figure 2).

[0255] In contrast, reference 3 discloses a composition of staple peptide + peptide linker + peptide binder, while reference 3 requires a depth of 70 μm to show significant degradation of the target site (Figure 1).

[0256] The targeted chimera provided by the technical solution of this invention can achieve the degradation of target proteins at the nm level, as shown in Table 8.

[0257] Table 8

[0258]

[0259] As can be seen from Table 8, the targeting chimeric compound provided in this application can degrade the target protein at the nmol level (up to 100 nmol, i.e. 0.1 μmol), while in comparison, the minimum amount required in references 1-3 is at least 50 μmol to produce a significant degradation effect on the target protein. The two differ by at least 500 times in the amount of degrading agent used, showing a very significant difference in effect.

[0260] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A targeted chimera assembled from polypeptides or microproteins mediated by cell-penetrating peptides, characterized in that, It includes at least one transmembrane peptide module, at least one targeting peptide module, at least one small molecule ligand module, and at least one linker module that are interconnected. The targeted polypeptide module is a polypeptide sequence that can bind to the target protein. The targeted peptide module and the small molecule ligand module are integrated through a linker module; The membrane-penetrating peptide module is connected to the free end of the targeting peptide module and is used to guide the targeting chimeric compound to penetrate the cell membrane. The small molecule ligand module is a small molecule E3 ligand that can bind to E3 ligase, and the protease degrading agent adapted to the small molecule E3 ligand is CRBN (Cereblon protein). The amino acid sequence of the transmembrane peptide module is the sequence shown in SEQ ID No. 1; The amino acid sequence of the targeted polypeptide module is the sequence shown in SEQ ID No. 4; The Linker module is a small molecule compound, and its structural formula is shown in Formula I. Formula I; The structural formula of the small molecule ligand module is shown in Formula II. Formula II; The structure of the intercalation is as follows: Transmembrane-penetrating peptide SEQ ID No. 1 + targeting peptide SEQ ID No. 4 + linker of formula I + small molecule ligand of formula II.

2. The application of the target chimera of polypeptide or microprotein modular assembly mediated by cell-penetrating peptides as described in claim 1 in the preparation of products for degrading targeted proteins, characterized in that, The target protein is the SARS-CoV-2 S protein HR2.

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