CN117959457A - Bifunctional compound serving as target protein degradation agent and application of bifunctional compound in degradation of target protein lysosome - Google Patents

Bifunctional compound serving as target protein degradation agent and application of bifunctional compound in degradation of target protein lysosome Download PDF

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CN117959457A
CN117959457A CN202211318398.4A CN202211318398A CN117959457A CN 117959457 A CN117959457 A CN 117959457A CN 202211318398 A CN202211318398 A CN 202211318398A CN 117959457 A CN117959457 A CN 117959457A
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group
receptor
protein
activity
molecule
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房丽晶
陈亮
李红昌
郑纪维
何婉怡
李晶
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Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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Priority to PCT/CN2022/137378 priority patent/WO2024087332A1/en
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Abstract

The invention belongs to the technical field of medicines, and provides a difunctional compound serving as a target protein degradation agent and application thereof in target protein lysosome degradation. The bifunctional compound comprises a protein-binding small molecule of interest, an integrin recognition ligand and a linking group for linking the protein-binding small molecule of interest and the integrin recognition ligand. The invention provides a difunctional compound, on the basis of which a novel integrin-promoted target protein lysosome degradation (IFLD) strategy is established and verified, and a novel technical choice is provided for regulating extracellular proteins and membrane-associated proteins. The invention uses the bifunctional compound with targeting effect, easy synthesis and modification to replace the expensive and difficult-to-synthesize antibody, nano-antibody, nucleic acid aptamer and glycopeptide polymer or longer polypeptide, and establishes a bridge between the target protein on the cell surface and a specific receptor, thereby promoting the endocytosis and lysosome degradation of the target protein.

Description

Bifunctional compound serving as target protein degradation agent and application of bifunctional compound in degradation of target protein lysosome
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a difunctional compound serving as a target protein degradation agent and application thereof in target protein lysosome degradation.
Background
The Target Protein Degradation (TPD) technology developed in recent years is a novel technology that specifically recognizes a target protein (target protein) and directly degrades the target protein using an intracellular intrinsic protein degradation pathway. At present, the TPD field develops PROTAC, molecular gel, degradation label, lysosome targeting chimera, autophagy small body binding compound and other technologies, and greatly expands the range of degradable target proteins. Targeting Protein Degradation (TPD) is currently mainly carried out by ubiquitin-proteasome and lysosomal pathways, which can be subdivided into approximately 9 different technical routes according to specific principles of action, wherein the technologies by ubiquitin proteasome degradation include PROTAC technology and molecular gel technology; techniques for degrading a target protein by lysosomes include LYTAC techniques, bispecific aptamer techniques, glueTAC techniques, AUTAC techniques, ATTEC techniques, AUTOTAC techniques, and CMA techniques.
The PROTAC technology is the fastest growing and most widely used TPD technology, PROTACs is capable of forming a ternary complex with the target protein and E3 ligase, inducing ubiquitination of the target protein, followed by proteasome degradation. However, the mechanism of ubiquitin-proteinase system (UPS) degradation of proteins determines PROTACs that is characterized by degradation of target Proteins (POIs) in cells, but not on extracellular and cell membranes. Molecular gel technology cannot be obtained by large-scale screening of the components like PROTAC, and design is difficult.
The antibody or oligosaccharide in LYTAC technical molecules has stronger immunogenicity, the dose ratio and the connecting site are uncertain when the oligosaccharide is coupled with the antibody, the glycopeptide chain is a high molecular mixture, and the in vivo clearance rate is higher; bispecific aptamer technologies are inefficient in delivery and unstable in vivo; the GlueTAC technology has the advantages that the covalent binding safety of the nano antibody with unnatural amino acid and the target protein is to be evaluated, and the in vivo half-life is short; AUTAC technology has slower degradation speed; ATTEC technology is difficult to reasonably design, the cost is high, and whether the whole autophagy of the cell is influenced is unknown; AUTOTAC technology has slow degradation speed; the stability and the conveying efficiency of the CMA technology are required to be improved.
Based on the above problems of the prior art, the present invention provides a bifunctional compound for use as a target protein degrading agent and its use in the degradation of a target protein lysosome.
Disclosure of Invention
In order to solve the problems that the Targeting Protein Degradation (TPD) technology in the prior art is difficult to design, has poor in vivo stability, short in vivo half-life and slow degradation rate, and can not effectively degrade target proteins outside cells or on cell membranes, the invention provides a bifunctional compound which can be used as a target protein degradation agent.
The bifunctional compound comprises a target protein binding unit, an integrin recognition unit and a linking unit for linking the target protein binding unit and the integrin recognition unit.
Further, the difunctional compound is synthesized by the reaction of A molecule, B molecule and L,
The A molecule comprises an A1 unit and a reactive group A2 connected with the A1 unit, wherein the A1 unit is a target protein binding unit and comprises a ligand bound with the target protein;
the B molecule comprises a B1 unit and a reactive group B2 connected with the B1 unit, wherein the B1 unit is an integrin recognition unit and comprises a ligand combined with integrin;
The L molecule comprises an active group L1 which reacts with the A2 active group, an active group L2 which reacts with the B2 active group and an L3 unit which connects the active group L1 and the active group L2, wherein the L3 unit is a connecting unit which generates a covalent bond with the A1 unit and the B1 unit;
The structural general formula of the difunctional compound is A1-L3-B1.
Further, the active group A2 is a group or a functional group that undergoes a substitution reaction, elimination reaction, addition reaction, or rearrangement reaction with the active group L1, and is selected from one or more of an alkane group, an arene group, a heterocyclic arene group, an alkenyl group, an alkynyl group, a halogeno group, an alcoholic hydroxyl group, a mercapto group, an aldehyde group, a ketone group, a carboxyl group, an amino group, an enol group, an azide group, a maleimide, a tetrazine group, and variants thereof, and an alcoholic hydroxyl group containing an α hydrogen;
The active group B2 is a group or a functional group which is subjected to substitution reaction, elimination reaction, addition reaction or rearrangement reaction with the active group L2 and is selected from one or more of an alkane group, an arene group, a heterocyclic arene group, an alkenyl group, an alkynyl group, a halogeno group, an alcoholic hydroxyl group, a sulfhydryl group, an aldehyde group, a ketone group, a carboxyl group, an aldehyde group, an amino group, an enol group, an azide group, maleimide, a tetrazine group and variants thereof and an alcoholic hydroxyl group containing alpha hydrogen;
The L3 unit includes one or more of an alkyl chain, an aromatic ring, a heterocyclic ring, a heteroatom, and a functional group.
Further, the target protein is selected from structural proteins; a receptor; cell surface proteins such as enzymes; proteins associated with cellular integration functions, including proteins involved in catalytic activity, aromatase activity, locomotor activity, helicase activity, metabolic processes, antioxidant activity, proteolysis, biosynthesis; proteins having kinase activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, ligase activity, enzyme modulating activity, signal transduction activity, structural molecule activity, binding activity, receptor activity, cell motility, membrane fusion, cell communication, biological process regulation, development, cell differentiation, stimulating reactions; behavioural proteins, cell adhesion proteins; proteins involved in cell necrosis; and one or more of proteins involved in transport.
Further, the protein of interest is selected from the group consisting of apoptosis-ligand 1 (i.e., PD-L1), apoptosis receptor 1 (i.e., PD-1), epidermal growth factor receptor (i.e., EGFR), human epidermal growth factor receptor-2 (i.e., HER 2), G-protein coupled receptor (i.e., GPCR), fibroblast growth factor receptor (i.e., FGFRs), vascular endothelial growth factor receptor family (i.e., VEGFR, VEGF stands for vascular endothelial growth factor), cytotoxic T lymphocyte-associated protein 4 (i.e., CTLA4 or CTLA-4), human interleukin 5 receptor alpha (IL-5Ralpha), apolipoprotein, apolipoprotein E4 (i.e., apoE 4), beta-amyloid, angiotensin converting enzyme 2 (ACE 2), sodium-taurocholate cotransporter (NTCP), B7.1 and B7, TI FR1m, TNFR2, NADPH oxidase, bc1IBax and other ligands in the apoptotic pathway, C5a receptor, HMG-CoA reductase, PDE V phosphodiesterase type, PDE IV phosphodiesterase type 4, PDE I, PDE II, PDE III, squalene cyclase inhibitor, CXCR1, CXCR2, nitric oxide synthase, cyclooxygenase 1, cyclooxygenase 2, 5HT receptor, dopamine receptor, G protein, histamine receptor, 5-lipoxygenase, protease-like serine protease, thymidylate synthase, purine nucleoside phosphorylase, glyceraldehyde-3-phosphate dehydrogenase (i.e., GAPDH), glycogen phosphorylase, carbonic anhydrase, chemokine receptor, JAW, RXR and the like, HIV1 protease, HIV1 integrase, influenza neuraminidase, hepatitis B reverse transcriptase, sodium channel, protein P-glycoprotein, P glycoprotein and MRP complex amino acid kinase, CD23, CD73, CD124, tyrosine kinase P561ck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-alpha R, ICAM1, ca2+ channel, VCAM, VLA-4 integrin, selectin, CD40/CD40L, newokinins and receptor, inosine monophosphate dehydrogenase, P38 MAP kinase, ras/Raf/MEW/ERK pathway, interleukin-1 converting enzyme, caspase, HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotidyl transferase, rhinovirus, 3C protease, herpes simplex virus-1, protease, cytomegalovirus protease, poly (ADP-ribose) polymerase, cyclin dependent kinase, vascular endothelial growth factor, oxytocin receptor, microsomal transfer protein inhibitors, bile acid transport inhibitors, 5α reductase inhibitors, angiotensin 11, glycine receptors, norepinephrine reuptake receptors, endothelin receptors, neuropeptide Y and receptors, adenosine kinase and AMP dehydrogenases, purinergic receptors, farnesyl transferase, geranyl transferase, trkA receptor of NCF, tyrosine kinase Flk-IIKDR, vitronectin receptor, integrin receptors, her-21 sphingomyelin, telomerase inhibition, cytosolic phosphate A2 and EGF receptor tyrosine kinases, ecdysone 20-monooxygenase, GABA-gated chloride ion channels, acetylcholinesterase, voltage-sensitive sodium channel proteins, calcium release channels and chloride ion channels, acetyl coa carboxylase, adenylate succinate synthase, one or more of protoporphyrinogen oxidase and enolpyruvylshikimate phosphate synthase, and/or one or more of all variants, mutants, splice variants, insertion deletions, and fusions of the above proteins.
Further, the A molecule is BMS-8, biotin-NHS or PH-002, and the integrin recognition ligand is cRGD.
Further, when the A molecule is BMS-8, the active group A2 is carboxyl, the active group B2 connected with the integrin recognition unit comprises alkynyl, the active group L1 in the L molecule is amino, the amino and the carboxyl form an amide bond, the active group L2 is an azide group, and the azide group and the alkynyl form a five-membered heterocycle of 1,2, 3-triazole;
when the A molecule is Biotin-NHS, the active group A2 is-NHS, the active group B2 connected with the integrin recognition unit comprises alkynyl, the active group L1 in the L molecule is amino, the amino replaces the active group NHS and a Biotin group to form an amide bond, the active group L2 in the L molecule is an azide group, and the azide group and the alkynyl form a five-membered heterocycle of 1,2, 3-triazole;
When the A molecule is PH-002, the A2 group contains amino protected by tert-butoxycarbonyl, the active group B2 connected with the integrin recognition unit comprises alkynyl, the active group L1 in the L molecule is carboxyl, the carboxyl and the exposed amino after the tert-butoxycarbonyl is removed on the PH-002 form an amide bond, the active group L2 in the L molecule is azido, and the azido and the alkynyl form five-membered heterocycle of 1,2, 3-triazole.
It is an object of the present invention to provide a pharmaceutical composition of a bifunctional compound as defined in any one of the preceding claims, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, benign proliferative disorders, infectious or non-infectious inflammatory events, autoimmune diseases, inflammatory diseases, systemic inflammatory response syndrome, viral infections and viral diseases, and eye diseases.
It is an object of the present invention to provide a use of a bifunctional compound or a pharmaceutical composition as defined in any one of the preceding claims for modulating the protein activity of a protein of interest in a patient in need thereof.
It is an object of the present invention to provide the use of a bifunctional compound or a pharmaceutical composition as defined in any one of the preceding claims for the degradation of a target protein lysosome.
The invention provides a difunctional compound, on the basis of which a novel integrin-promoted target protein lysosome degradation (IFLD) strategy is established and verified, and a novel technical choice is provided for regulating extracellular proteins and membrane-associated proteins. The invention uses the bifunctional compound with targeting effect, easy synthesis and modification to replace the expensive and difficult-to-synthesize antibody, nano-antibody, nucleic acid aptamer and glycopeptide polymer or longer polypeptide, and establishes a bridge between the target protein on the cell surface and a specific receptor, thereby promoting the endocytosis and lysosome degradation of the target protein. In addition, by utilizing the strategy, a BMS-L1-RGD bifunctional molecule degradation agent with high activity is designed, and the capability of regulating the in-vivo and in-vitro PD-L1 protein level and the anti-tumor activity are confirmed, so that a novel tool is provided for future tumor targeted treatment.
Drawings
FIG. 1 is a schematic diagram of an integrin-promoted target protein lysosomal degradation (IFLD) strategy provided by the present invention;
FIG. 2 shows the structural formulas of BMS-L3 1-RGD、BMS-L32 -RGD and BMS-L3 3 -RGD provided by the invention;
FIG. 3 is an HRMS spectrum of BMS-L3 1 -Azide provided in example 1 of the present invention;
FIG. 4 is a 1H NMR spectrum of BMS-L3 1 -RGD provided in example 1 of the present invention;
FIG. 5 is an HRMS spectrum of BMS-L3 1 -RGD provided in example 1 of the present invention;
FIG. 6 is an HPLC analysis of BMS-L3 1 -RGD provided in example 1 of the present invention;
FIG. 7 is a fluorescent chart of the degradation test of the membrane protein PD-L1 provided in example 3 of the present invention;
FIG. 8 is a diagram showing that BMS-L3 1 -RGD provided in example 2 of the present invention promotes degradation of membrane-associated protein PD-L1 by the integrin-lysosomal pathway;
FIG. 9 is an in vivo antitumor activity evaluation of BMS-L3 1 -RGD provided in example 3 of the present invention;
FIG. 10 is a graph showing the degradation of extracellular protein PD-L1 by BMS-L3 1 -RGD through an integrin-promoted lysosomal degradation strategy provided in example 4 of the present invention;
FIG. 11 is a HRMS spectrum of Biotin-L3 1 -Azide provided in example 5 of the present invention;
FIG. 12 is a 1H NMR spectrum of Biotin-L3 1 -RGD provided in example 5 of the present invention;
FIG. 13 is a HRMS spectrum of Biotin-L3 1 -RGD provided in example 5 of the present invention;
FIG. 14 shows the HPLC analysis of Biotin-L3 1 -RGD provided in example 5 of the present invention;
FIG. 15 shows that Biotin-L3 1 -RGD provided in example 6 of the present invention promotes degradation of extracellular proteins via the integrin-lysosomal pathway;
FIG. 16 is a HRMS spectrum of pH002-L3 4 -Azide provided in example 7 of the present invention;
FIG. 17 is a HRMS spectrum of PH002-L3 4 -RGD provided in example 7 of the present invention;
FIG. 18 shows the lysosomal degradation of the extracellular protein, apolipoprotein E4 (APOE 4-AF 488) promoted by integrin with PH002-L3 4 -RGD provided in example 8 of the present invention.
Detailed Description
In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments that are illustrated in the appended drawings, but are not to be construed as limiting the scope of the invention.
Lysosomes mediate the degradation of proteins and organelles by endocytosis, phagocytosis, and autophagy. With intensive research into endosome-lysosomal and autophagosome-lysosomal degradation pathways, TPD technologies such as LYTAC, abTAC, ATTEC, AUTAC, AUTOTAC and the like have been developed in recent years through the lysosomal pathway. Compared with TPD based on ubiquitin-proteasome mechanism, TPD based on lysosome can degrade not only intracellular proteins, but also protein aggregates, damaged organelles and extracellular proteins, and has wider potential in application fields. Extracellular and membrane proteins account for 40% of the encoded proteins and are closely related to neurodegenerative diseases, autoimmune diseases and cancers. To degrade such proteins, researchers have developed lysosomal targeting chimeras (Lysosome-TARGETING CHIMAERAS, LYTAC) that induce extracellular and membrane proteins to degrade via the endosomal-lysosomal pathway by creating a bridge between the target protein and the Lysosomal Transporter (LTR). In addition, bispecific aptamer chimeras (Bispecific APTAMER CHIMERA) and LYTAC, like that, mediate target protein degradation via the endosomal-lysosomal pathway, are coupled from two aptamers targeting the target protein and LTR, respectively, and promote target protein endocytosis and lysosomal degradation by forming ternary complexes with LTR and target protein. Antibody-based PROTAC (anti-based PROTAC, abTAC) also induces degradation of extracellular and membrane proteins by the endosome-lysosomal pathway. AbTAC is essentially a recombinant bispecific antibody, one end of which targets the target protein on the cell surface, and the other end of which targets the transmembrane E3 ligase, and compared with LYTAC, abTAC can reduce the immunogenicity of the chimeric molecule, but specific mechanisms such as recycling and reutilization are yet to be studied. PROTAC (Covalent Nanobody-BasedPROTAC, glueTAC) based on covalent antibodies consists of nanobodies capable of covalently binding to target proteins and a transmembrane peptide-lysosome sorting peptide, glueTAC covalently binding to target proteins and then transporting the target proteins to lysosomes and degrading by clathrin-mediated endocytosis under the action of the transmembrane peptide-lysosome sorting peptide. GlueTAC molecules, while exhibiting strong degradability, incorporate unnatural amino acids into nanobodies and form covalent bonds with target proteins, and require careful assessment of the toxicity of this modification to target and non-target cells. In the above techniques, the portion for recognizing the target protein on the cell membrane is an antibody, nanobody or aptamer chimera, and there may be problems in immunogenicity and stability. At present, strategies for mediating degradation of cell membrane proteins using structurally single bifunctional compounds have not been reported.
As lysosomal transporters, the mannose 6-phosphate/IGF-II receptor (M6P/IGFIIR) and the salivary glycoprotein receptor (ASGPR) have been shown to be effective in inducing degradation of secreted or membrane proteins into the lysosome. In addition to M6P/IGFIIR and ASGPR, receptor-ligand mediated delivery systems involve other cell surface receptors, such as transferrin receptor, folate receptor, integrins, and the like, which also have the ability to deliver fluorophores, drugs, or nanomaterials into cells via receptor-mediated endocytosis. Here we focus on integrins, which are cell adhesion receptors expressed on the cell surface, playing an important role in cell-matrix interactions. Since integrin alpha vβ3 is overexpressed in solid tumor vessels, proliferating tumor endothelial cells and various tumor cells, there is a great interest in tumor-targeted therapies. However, while the α vβ3 integrin recognition motif Arg-Gly-Asp (RGD) sequence has been widely used to deliver various therapeutic agents into tumors, the possibility of RGD-integrin mediated TPD has not been investigated prior to our work.
Referring to FIG. 1 of the drawings, the present invention establishes a novel integrin-promoted target protein lysosome degradation (IFLD) strategy for degrading extracellular and cell membrane proteins using bifunctional compounds as molecular degrading agents. The bifunctional compound referred to in the present invention is a compound in which a target protein-binding ligand and an integrin recognition ligand-RGD-containing polypeptide sequence are bound together by a Linker (Linker, which includes a linking unit in the present invention and a residue after reaction with the reactive group A1, B1). As a molecular degradant, such bifunctional compounds have been shown to be capable of efficiently inducing internalization and subsequent degradation of extracellular or cell membrane proteins in an integrin-and lysosomal-dependent manner.
The bifunctional compound provided by the invention comprises a target protein binding unit, an integrin recognition unit and a connection unit for connecting the target protein binding unit and the integrin recognition unit.
In one embodiment, the bifunctional compound is synthesized by reacting an A molecule, a B molecule, and an L molecule,
The A molecule comprises an A1 unit and a reactive group A2 connected with the A1 unit, wherein the A1 unit is a target protein binding unit and comprises a ligand bound with the target protein;
The B molecule comprises a B1 unit and a reactive group B2 connected with the B1 unit, wherein the B1 unit is an integrin recognition unit and comprises a ligand combined with integrin;
The L molecule comprises an active group L1 which reacts with the active group A2, an active group L2 which reacts with the active group B2 and an L3 unit which connects the active group L1 and the active group L2, wherein the L3 unit is a connecting unit which generates a covalent bond with the unit A1 and the unit B1;
The structural general formula of the difunctional compound is A1-L3-B1.
In one embodiment, reactive group A2 is a group or functional group that undergoes a substitution, elimination, addition, or rearrangement reaction with reactive group L1 and is selected from one or more of an alkane group, an arene group, a heterocyclic arene group, an alkenyl group, an alkynyl group, a halo group, an alcoholic hydroxyl group, a sulfhydryl group, an aldehyde group, a ketone group, a carboxyl group, an amino group, an enol group, an azide group, a maleimide, a tetrazine group, and variants thereof, and an alcoholic hydroxyl group containing an alpha hydrogen.
The active group B2 is a group or a functional group which undergoes a substitution reaction, elimination reaction, addition reaction or rearrangement reaction with the active group L2, and is selected from one or more of an alkane group, an arene group, a heterocyclic arene group, an alkenyl group, an alkynyl group, a halogen group, an alcoholic hydroxyl group, a mercapto group, an aldehyde group, a ketone group, a carboxyl group, an amino group, an enol group, an azide group, a maleimide group, a tetrazine group, a variant thereof, and an alcoholic hydroxyl group containing an alpha hydrogen.
The L3 unit includes one or more of an alkyl chain, an aromatic ring, a heterocyclic ring, a heteroatom, and a functional group.
The active group A2, the active group B2, the active group L1 and the active group L2 have the functions of forming a connecting arm among A1, B1 and L3 to obtain a bifunctional compound A1-L3-B1, binding a target protein through an A1 unit, binding an integrin receptor by a B1 unit, and forming a ternary complex among the target protein, the integrin receptor and the bifunctional compound A1-L3-B1, so that the target protein bound with the A1 unit can be transferred from outside cells to inside cells for degradation. Based on the above principle, the reactive group A2 and the reactive group L1 may be any two groups capable of reacting and forming a covalent bond between the A1 unit and the L3 unit, and similarly, the reactive group B2 and the reactive group L2 may also be any two groups capable of reacting and forming a covalent bond between the B1 unit and the L3 unit. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention; meanwhile, as those skilled in the art will vary in the specific embodiments and application scope according to the idea of the present invention, the contents of the present specification should not be construed as limiting the present invention in summary.
In one embodiment, the protein of interest is selected from structural proteins; a receptor; an enzymatic cell surface protein; proteins associated with cellular integration functions, including proteins involved in catalytic activity, aromatase activity, locomotor activity, helicase activity, metabolic processes, antioxidant activity, proteolysis, biosynthesis; proteins having kinase activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, ligase activity, enzyme modulating activity, signal transduction activity, structural molecule activity, binding activity, receptor activity, cell motility, membrane fusion, cell communication, biological process regulation, development, cell differentiation, stimulating reactions; behavioural proteins, cell adhesion proteins; proteins involved in cell necrosis; and one or more of proteins involved in transport.
In one embodiment, the protein of interest is selected from the group consisting of: apoptosis-ligand 1 (i.e., PD-L1), apoptosis receptor 1 (i.e., PD-1), epidermal growth factor receptor (i.e., EGFR), human epidermal growth factor receptor-2 (i.e., HER 2), G-protein coupled receptor (i.e., GPCR), fibroblast growth factor receptor (i.e., FGFRs), vascular endothelial growth factor receptor family (i.e., VEGFR, VEGF representing vascular endothelial growth factor), cytotoxic T lymphocyte-associated protein 4 (i.e., CTLA4 or CTLA-4), human interleukin 5 receptor alpha (IL-5Ralpha), apolipoprotein, apolipoprotein E4 (i.e., apoE 4), beta-amyloid, angiotensin converting enzyme 2 (ACE 2), sodium-taurocholate cotransporter (NTCP), B7.1 and B7, TI FR1m, TNFR2, NADPH oxidase, bc1IBax and other ligands in the apoptotic pathway, C5a receptor, HMG-CoA reductase, PDE V phosphodiesterase type, PDE IV phosphodiesterase type 4, PDE I, PDE II, PDE III, squalene cyclase inhibitor, CXCR1, CXCR2, nitric oxide synthase, cyclooxygenase 1, cyclooxygenase 2, 5HT receptor, dopamine receptor, G protein, histamine receptor, 5-lipoxygenase, protease-like serine protease, thymidylate synthase, purine nucleoside phosphorylase, glyceraldehyde-3-phosphate dehydrogenase (i.e., GAPDH), glycogen phosphorylase, carbonic anhydrase, chemokine receptor, JAW, RXR and the like, HIV1 protease, HIV1 integrase, influenza ammonia-phase, STAT reverse transcriptase, sodium channel, protein P-glycoprotein, P-glycoprotein and MRP-tyrosine kinase, CD23, CD73, CD124, tyrosine kinase P561ck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-alpha R, ICAM1, ca2+ channel, VCAM, VLA-4 integrin, selectin, CD40/CD40L, newokinins and receptor, inosine monophosphate dehydrogenase, P38 MAP kinase, ras/Raf/MEW/ERK pathway, interleukin-1 converting enzyme, caspase, HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotidyl transferase, rhinovirus, 3C protease, herpes simplex virus-1, protease, cytomegalovirus protease, poly (ADP-ribose) polymerase, cyclin-dependent kinase, vascular endothelial growth factor, oxytocin receptor, microsomal transfer protein inhibitor, bile acid transport inhibitor 5 alpha reductase inhibitor, angiotensin 11, glycine receptor, noradrenaline reuptake receptor, endothelin receptor, neuropeptide Y and receptor, adenosine kinase and AMP dehydrogenase, purinergic receptor, farnesyl transferase, geranyltransferase, trkA receptor for NCF, tyrosine kinase Flk-IIKDR, vitronectin receptor, integrin receptor, her-21 sphingosine, telomerase inhibition, cytosolic phosphate A2 and EGF receptor tyrosine kinase, ecdysone 20-monooxygenase, GABA-gated chloride ion channel, acetylcholinesterase, voltage-sensitive sodium channel protein, calcium release channel and chloride ion channel, acetyl-CoA carboxylase, adenylyl succinate synthase, protoporphyrinogen oxidase and enolpyruvylshikimate phosphate synthase, and/or one or more of all variants, mutants, splice variants, insertion deletions and fusions of the above proteins.
In one embodiment, the A molecule is BMS-8, biotin-NHS or PH-002 and the integrin recognition ligand is cRGD.
In one embodiment, when the A molecule is BMS-8, the active group A2 is carboxyl, the active group B2 connected with the integrin recognition unit comprises alkynyl, the active group L1 in the L molecule is amino, the amino and the carboxyl form an amide bond, the active group L2 is azide group, and the azide group and the alkynyl form five-membered heterocycle of 1,2, 3-triazole;
when the A molecule is Biotin-NHS, the active group A2 is-NHS, the active group B2 connected with the integrin recognition unit comprises alkynyl, the active group L1 in the L molecule is amino, the amino replaces NHS and the Biotin group to form an amide bond, the active group L2 in the L molecule is an azide group, and the azide group and the alkynyl form five-membered heterocycle of 1,2, 3-triazole;
When the A molecule is PH-002, the A2 group contains amino protected by tert-butoxycarbonyl, the active group B2 connected with the integrin recognition unit comprises alkynyl, the active group L1 in the L molecule is carboxyl, the carboxyl and the amino exposed after the tert-butoxycarbonyl is removed on PH-002 form an amide bond, the active group L2 in the L molecule is azide group, and the azide group and the alkynyl form five-membered heterocycle of 1,2, 3-triazole.
The pharmaceutical composition of the bifunctional compound of any one of the above claims or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, benign proliferative disorders, infectious or non-infectious inflammatory events, autoimmune diseases, inflammatory diseases, systemic inflammatory response syndrome, viral infections and viral diseases, and eye diseases.
The use of a bifunctional compound or pharmaceutical composition of any one of the above claims to modulate the protein activity of a protein of interest in a patient in need thereof.
Use of a bifunctional compound or pharmaceutical composition of any one of the preceding claims in the degradation of a target protein lysosome.
Example 1 BMS-Synthesis of L3 1 -RGD
The synthetic route for BMS-L3 1 -RGD is shown in the following figure:
Step 1 Synthesis of Compound BMS-L3 1 -Azide
BMS-8 small molecule (10.0 mg, 20.2. Mu. Mol), HATU (11.5 mg, 30.3. Mu. Mol) and DIEA (10.0. Mu.L, 60.6. Mu. Mol) were added sequentially to the reaction flask, dissolved with 1.0mL of anhydrous N, N-Dimethylformamide (DMF) with stirring, and 3-azidopropylamine (3.0. Mu.L, 30.3. Mu. Mol) was added. After stirring the reaction mixture at room temperature for 3 hours, it was purified by HPLC and lyophilized to give white powder BMS-L31-Azide(11.0mg,95％).HRMS(ESI)m/z:calcd.for C30H35BrN5O2[M+H]+576.1969,found 576.1973.( as shown in FIG. 3)
Step 2 Synthesis of Compound BMS-L3 1 -RGD
To the flask was added Alkyne-cRGD(9.4mg,13.1μmol)﹑BMS-L31-Azide(5.0mg,8.7μmol)﹑CuSO4·5H2O(1.3mg,5.2μmol) and NaVc (6.9 mg, 34.7. Mu. Mol) in sequence and dissolved in a DMF/H 2 O (1.5 mL, 2:1) mixture. After stirring the reaction mixture at room temperature for 6 hours, it was separated and purified by HPLC and lyophilized to give a white powder BMS-L1-RGD(9.5mg,85％).1H NMR(400MHz,MeOD)δ:8.78–8.68(m,1H),8.14(s,1H),7.79(s,1H),7.67(dd,J＝12.6,7.3Hz,1H),7.51(d,J＝8.2Hz,2H),7.45(t,J＝7.3Hz,2H),7.41–7.34(m,2H),7.34–7.28(m,3H),7.28–7.21(m,1H),7.02(d,J＝8.4Hz,2H),6.71(d,J＝8.3Hz,2H),5.30(s,1H),4.82–4.74(m,1H),4.58(t,J＝6.8Hz,2H),4.53–4.36(m,3H),4.37–4.20(m,2H),4.07(d,J＝13.0Hz,1H),4.00–3.79(m,3H),3.55–3.42(m,3H),3.28–3.00(m,7H),2.99–2.74(m,5H),2.65–2.53(m,2H),2.44–2.14(m,8H),2.03(dd,J＝12.3,5.1Hz,3H),1.97–1.79(m,5H),1.80–1.50(m,9H),1.47–1.34(m,4H),1.05(d,J＝7.0Hz,3H),0.92(t,J＝6.8Hz,1H).(, 4).HRMS(ESI)m/z:calcd.for C63H82O11N14Br[M+H]+1289.5465,found 1289.5471.(, FIG. 5
Replacement of 3-azidopropylamine in step 1 with N- (2- (2-aminoethoxy) ethyl) -6-azidohexanamide or N- (2- (2- (2-aminoethoxy) ethoxy) ethoxy) ethyl) -6-azidohexanamide can produce BMS-L3 2-RGD、BMS-L33 -RGD with the structure shown in FIG. 2. (figure) 2)BMS-L32-RGD,HRMS(ESI)m/z:calcd.for C70H95O13N15Br[M+H]+1432.6412,found 1432.6403;BMS-L33-RGD,HRMS(ESI)m/z:calcd.for C74H103BrN15O15[M+H]+1520.6963,found 1520.6948
Example 2 is an application example of the BMS-L3 1 -Azide compound prepared in example 1, and the properties of BMS-L3 1 -RGD compound in degrading the membrane protein PD-L1 in vitro were screened and evaluated
Immunofluorescence method: cells on coverslips were washed 2 times in PBS, fixed with 4% PFA for 15 min, rinsed 3 times with PBS (10 mM, pH 7.4), 5min each. If it is necessary to observe changes in intracellular PD-L1, an additional 0.10% Triton X-100 is required to incubate the cells for 15 minutes at room temperature. Cells were then blocked with 3% bsa for 30min and then incubated with the indicated primary and secondary antibodies for 2 and 1 hour, respectively, at room temperature. Following this step, nuclei were stained with DAPI C1005 for 15 minutes in the dark. The cells were then washed three times with PBS (10 mM, pH 7.4) for 5 minutes each. The fluorescence image was imaged by LEICA STELLARIS confocal fluorescence microscope.
Immunofluorescence analysis of membrane protein PD-L1 degradation assay. Hela cells stably expressing PD-L1 were incubated in 24-well coverslips to a confluency of about 40% to 50%. cRGD, BMS-8, BMS-8+cRGD and BMS-L3 1 -RGD were diluted to 25nM with medium, respectively, and incubated in 24-well plates at 37℃for 8 hours. Cells were treated by immunofluorescence and imaged by laser confocal imaging.
By blocking the alpha vβ3 integrin with cRGD, BMS-L3 1 -RGD mediated PD-L1 degradation is competitively inhibited. Hela cells stably expressing PD-L1 were incubated in 24-well coverslips to a confluency of about 40% to 50%. A24-well plate was incubated with cRGD at a high concentration (5. Mu.M) at 4℃for 1h, and BMS-L3 1 -RGD was added to the wells and incubated at 37℃for 8h. The remaining steps were performed according to the immunofluorescence method described above.
Immunoblot experiments analyzed PD-L1 levels. MDA-MB-231 cells were cultured in 6-well cell plates at a density of 70-80%. To determine the optimal concentration of BMS-L3 1 -RGD, dilution to 5, 25, 50, 100nM was done with DMEM and incubated with cells for 8h. BMS-L3 1 -RGD was diluted to 25nM for different time gradients and added to cells at different times (0, 4, 8, 12, 24 hours). To screen for BMS-L3 1-RGD、BMS-L32-RGD、BMS-L33 -RGD for different linkers, we diluted to 25nM and 50nM with DMEM and incubated with cells for 8h.
In verifying the degradation pathway, cells were pretreated with bafilomycin A1 (100 nM) for 2h in one well, then BMS-L3 1 -RGD was added and cells were cultured at 37℃for an additional 8h. In another well, MG132 (5. Mu.M) and BMS-L3 1 -RGD were added to the cells, and the cells were cultured at 37℃for 8 hours. After the incubation, the cells were washed 2-3 times with cooled PBS and scraped with a cell scraper. After centrifugation at 450g for 5min, 120. Mu.L of CSK buffer (20 mM HEPES-NaOH, pH=7.5, 40mM sodium chloride, 300mM sucrose, 1mM protease inhibitor) was added to the cell pellet and the mixture was ice-coated for 20min. Repeatedly blowing with insulin syringe for 45 times, centrifuging at 15000rpm for 40min, collecting supernatant as cytoplasmic component, and collecting the precipitate as cell membrane component. The two components were added to 120. Mu.L SDS lysis buffer containing protease inhibitor SDS (1 mM), ice-coated for 30min, then added to loading buffer (5X), and boiled for 20min. Finally, the samples were subjected to immunoblot analysis. The results of the assay are shown in the middle of the lower panel of FIG. 8, where the addition of the lysosomal inhibitor bafilomycin A1 was able to attenuate the degradation effect of BMS-L3 1 -RGD on PD-L1 protein, while the addition of the proteasome inhibitor MG132 did not affect the degradation of PD-L1 protein by BMS-L3 1 -RGD, which experiment indicated that BMS-L3 1 -RGD degraded PD-L1 protein by the lysosomal pathway rather than the proteasome pathway.
In competition experiments, cRGD (5. Mu.M) was incubated with cells for 1 hour at 4℃and then BMS-L3 1 -RGD was added, cells were incubated for another 8 hours at 37℃and then washed with twice cold PBS, 150-200. Mu.L SDS lysis buffer (containing protease inhibitor 1. Mu.M) was added and centrifuged at 14000rpm for 5min, protein samples were boiled for 20min and then SDS-PAGE sample loading buffer (5X) for 10min. Protein samples were electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to 0.45 μm polyvinylidene fluoride (PVDF) membranes (microwells). The membrane was then blocked with 5% nonfat dry milk in PBST buffer (PBS+0.1% Tween-20) with gentle shaking at room temperature for 2 hours. The membranes were incubated with primary antibodies (PD-L1 antibody, cell Signaling Technology (CST), rabbit source, 1:1000; GAPDH antibody, protein, murine source, 1:10000) at 4℃with gentle shaking overnight. Then, the membrane was washed 3 times (5 min each) with PBST buffer. Membranes were incubated with horseradish peroxidase (HRP) -conjugated anti-rabbit IgG antibody (1:5000 dilution) and anti-mouse IgG antibody (1:5000 dilution) for 1 hour at room temperature. Finally, the membrane was washed 3 times (5 min each) with PBST buffer and western blot strips were detected using Electrochemiluminescent (ECL) western blot substrates. The detection result is shown on the right side of the lower diagram in fig. 8. In the case of preincubation with cRGD, the binding of Integrin on the cell surface to its ligand cRGD competitively inhibited BMS-L3 1 -RGD from binding to Integrin, resulting in reduced degradation of PD-L1 protein by BMS-L3 1 -RGD. This experiment shows that BMS-L3 1 -RGD promotes the degradation of PD-L1 protein by binding to Integrin on the cell surface.
Example 3 evaluation of the ability of BMS-L3 1 -RGD to degrade PD-L1 levels and anti-tumor Activity in vivo
Sterile female C57BL/6J mice of 5-6 weeks of age were used to establish tumor xenograft models. B16F10 cells (2X 10 5) were suspended in 100. Mu.L PBS and injected subcutaneously on the right side of 5-6 week old sterile female C57BL/6J mice. After the tumor size reached 30mm 3-50 mm3 (L.times.W.times.1/2W), animals were randomly divided into three groups of 5 animals each. BMS-8 (2 mg/kg), BMS-L3 1 -RGD (5 mg/kg) and 10% DMSO/PBS (100. Mu.L) were each applied to a group of animals, once every two days, by tail vein intravenous injection, 5 times in total. Animals injected with 10% DMSO/PBS were control groups. Tumor size and mouse body weight were measured prior to each injection. Mice were sacrificed 18 days after injection of B16F10 cells, tumor specimens were collected, weighed and further analyzed, including slice fluorescent staining. Data represent mean ± SEM (n=5), and statistical significance was assessed using two methods of P <0.05, P <0.01, P < 0.001. All animal experiments were conducted in accordance with the relevant guidelines and regulations and were approved by the institutional animal care and use committee of SIAT. The test results are shown in fig. 9, in which graph a shows the flow of the operation of the experiment, graph B shows the change in the body weight of the animal in the injected B16F10 cells 18, graph C shows the change in the volume of the tumor tissue in the injected B16F10 cells 18, graph D shows the change in the size of the tumor tissue in the injected B16F10 cells 18, and graph E shows the average value of the tumor tissue weight in the injected B16F10 cells 18. The results of immunohistochemical imaging of the tumor sections in panel F show that the level of PD-L1 in the BMS-L3 1 -RGD drug group is significantly reduced compared to the control group and the BMS-8 drug group; the lower tumor section immunofluorescence imaging results in panel F showed a significant increase in apoptosis levels in the BMS-L3 1 -RGD drug group compared to the control group and BMS-8 drug group. This experiment shows that BMS-L3 1 -RGD can effectively degrade PD-L1 level in mice and cause apoptosis of tumor cells, thereby producing remarkable anti-tumor effect.
Example 4 is an application example of BMS-L3 1 -RGD prepared in example 1, verifying that integrins promote degradation of extracellular protein PD-L1 by lysosomal degradation strategy (IFLD strategy)
Fluorescent staining method: cells on coverslips were washed 2 times with PBS, fixed with 4% paraformaldehyde solution for 15min, and rinsed 3 times with PBS (10 mM, pH 7.4) for 5min each. Following this step, nuclei were stained with DAPI C1005 for 15 minutes in the dark. The cells were then washed three times with PBS (10 mM, pH 7.4) for 5 minutes each. The fluorescence image was imaged by LEICA STELLARIS confocal fluorescence microscope.
Extracellular protein uptake assay. Huh7 cells were incubated in 24-well coverslips to achieve about 40% to 50% confluency. BMS-L3 1 -RGD was diluted to 400nM,Alexa Fluor 488 labeled protein PD-L1 (PD-L1-AF 488) in medium and diluted to 400nM in medium, and incubated for 30 min after mixing to give RGD labeled PD-L1-AF488. The resulting RGD-labeled PD-L1-AF488 protein solution was added to a 24-well plate and co-cultured with the fused cells at 37℃for 20 hours. To verify the effect of lysosomal inhibitors (chloroquine, CQ) on protein uptake, the fused cells were incubated with the RGD-labeled PD-L1-AF488 protein solution and CQ solution (50 μm in medium) described above for 12 hours. As a control, BMS-Azide was pre-incubated with APOE4-AF488 using the same procedure as described above to give azide-labeled PD-L1-AF488 protein solution. The resulting azide-labeled PD-L1-AF488 protein solution was added to a 24-well plate and co-cultured with the fused cells at 37℃for 20h. Cells were fluorescent stained and imaged by laser confocal microscopy.
Example 5 Biotin Synthesis of L3 1 -RGD
The synthesis route of Biotin-L3 1 -RGD is shown in the following figure:
Step 1 Synthesis of Biotin-L3 1 -Azide
To the reaction flask was added 3-azidopropylamine (12.9. Mu.L, 132. Mu. Mol), dissolved in 1.0mL of anhydrous DMF, followed by triethylamine (24.5. Mu.L, 176. Mu. Mol), and after stirring well, a solution of Biotin-NHS (30.0 mg, 88. Mu. Mol) in DMF (1.0 mL) was added dropwise. After stirring the reaction mixture at room temperature for 6 hours, it was purified by HPLC separation and lyophilized to give white powder Biotin-L31-Azide(26.1mg,91％).HRMS(ESI)m/z:calcd.for C13H23O2N6S[M+H]+327.1598,found 327.1597;calcd.for C13H22O2N6NaS[M+Na]+349.1417,found 349.1416.( FIG. 6)
Step 2 Synthesis of Biotin-L3 1 -RGD
To the flask was added Alkyne-cRGD(8.9mg,12.5μmol)﹑Biotin-L31-Azide(5.3mg,16.2μmol)﹑CuSO4·5H2O(1.9mg,7.5μmol) and NaVc (9.9 mg, 50.0. Mu. Mol) in sequence and dissolved in a DMF/H 2 O (1.8 mL, 1:3) mixture. After stirring the reaction mixture at room temperature for 6 hours, it was purified by HPLC and lyophilized to give a white powder Biotin-L31-RGD(10.8mg,83％).1H NMR(400MHz,D2O)δ：8.16(s,1H),6.95(d,J＝8.4Hz,2H),6.66(d,J＝8.3Hz,2H),4.63(d,J＝7.0Hz,3H),4.49–4.41(m,6H),4.27–4.19(m,3H),4.07(s,1H),4.03(s,1H),3.71(dd,J＝10.6,4.0Hz,2H),3.41(s,1H),3.37(s,1H),3.16–3.09(m,4H),3.07–3.02(m,2H),2.99–2.91(m,3H),2.86–2.79(m,3H),2.78–2.72(m,4H),2.64–2.55(m,3H),2.21(t,J＝7.4Hz,3H),1.91–1.85(m,2H),1.77–1.68(m,2H),1.58–1.45(m,5H),1.39(dd,J＝15.6,6.9Hz,6H),1.23(d,J＝9.4Hz,6H),0.89–0.78(m,3H).( FIG. 7)HRMS(ESI)m/z:calcd.for C46H70N15O11S[M+H]+1040.5095,found 1040.5090.( FIG. 8
Example 6 is an application example of Biotin-L3 1 -RGD prepared in example 5, and it was verified that integrin promotes degradation of extracellular proteins by lysosomal degradation strategy (IFLD strategy).
Fluorescent staining method: cells on coverslips were washed 2 times with PBS, fixed with 4% paraformaldehyde solution for 15min, and rinsed 3 times with PBS (10 mM, pH 7.4) for 5min each. Following this step, nuclei were stained with DAPI C1005 for 15 minutes in the dark. The cells were then washed three times with PBS (10 mM, pH 7.4) for 5 minutes each. The fluorescence image was imaged by LEICA STELLARIS confocal fluorescence microscope.
Extracellular protein uptake assay. A549 cells and Huh7 cells were incubated in 24-well coverslips to achieve about 40% to 50% confluence. Biotin-L3 1 -RGD was diluted to 5. Mu.M in medium, NAP-650 protein was diluted to 400nM in medium, and mixed and incubated for 30 minutes to form an RGD-labeled NAP-650 solution. The resulting RGD-labeled NAP-650 solution was added to a 24-well plate and co-cultured with the fused cells at 37℃for 20 hours. Using the same procedure as described above, azide-labeled NAP-650 solutions were formed using Biotin-L-Azide and NAP-650. The resulting azide-labeled NAP-650 solution was added to a 24-well plate and co-cultured with the confluent cells at 37℃for 20h. When verifying the effect of lysosomal inhibitors (chloroquine, CQ) on protein uptake, the fused cells were incubated with RGD-labeled NAP-650 solution and CQ solution (50 μm in medium) for 12 hours. Cells were fluorescent stained and imaged by laser confocal microscopy.
Co-localization of extracellular protein NAP-650 with lysosomal markers. A549 cells and Huh7 cells were incubated in 24-well coverslips to about 40% to 50% confluency. RGD-labeled NAP-650 solution was added to a 24-well plate and incubated at 37℃for 20h. The sample-containing medium was then replaced with an equal volume of medium containing Lyso-TRACKER GREEEN (lysosome-localized green fluorescent dye) and the cells were further incubated for 1h. The remaining steps were performed according to the above-described fluorescent staining method.
Co-localization of extracellular proteins with early endosomal markers (Rab 5). A549 cells and Huh7 cells were incubated in 24-well coverslips to a confluency of about 60% to 70%. The Rab5-RFP plasmid was transfected into A549 cells or Huh7 cells using PEI transfection reagent. The cells were cultured for 24h to allow Rab5-RFP expression in early endosomes. Biotin-L3 1 -RGD was diluted to 5. Mu.M in medium, NAP-FITC proteins were diluted to 400nM in medium, and mixed incubated for 30min to form RGD-labeled NAP-FITC solutions. The resulting NAP-FITC solution was added to 24 wells and incubated at 37℃for 20h. The remaining steps were performed according to the above-described fluorescent staining method.
EXAMPLE 7 Synthesis of PH002-L3 4 -RGD
The synthetic route for PH002-L3 4 -RGD is shown in the following figure:
Step 1 Synthesis of the Compound PH002-L3 4 -Azide
The reaction flask was charged with compound PH-002 (5.0 mg, 10.2. Mu. Mol,1.0 eq) and 20% TFA/DCM (2.0 mL) was added under ice-bath. After 2 hours of reaction, the solvent was distilled off under reduced pressure, the residue was dissolved in anhydrous DMF (1.0 mL), N3-C5-PEG2-COOH(4.61mg,15.2μmol,1.5eq),HATU(5.03mg,13.2μmol,1.3eq),DIEA(13.5μl,81.6μmol,8.0eq), was added to the above solution and the reaction mixture was stirred at room temperature for 3 hours, and then purified by HPLC separation, and freeze-dried to give PH002-L34-Azide(5.4mg,78％).HRMS(ESI)m/z:calcd.for C34H45O6N9Na[M+H]+698.3385,found 698.3377.( FIG. 16)
Step 2 Synthesis of the Compound PH002-L3 4 -RGD
To the reaction flask was added Alkyne-cRGD(3.0mg,4.2μmol,1.0eq)﹑PH002-Azide(3.4mg,5.0μmol,1.2eq)﹑CuSO4·5H2O(0.63mg,2.52μmol,0.6eq) and NaVc (3.33 mg, 16.8. Mu. Mol,4.0 eq) in sequence, and dissolved in a DMF/H 2 O (0.7 mL, 1:1) mixture. After stirring the reaction mixture at room temperature for 3 hours, it was purified by HPLC and lyophilized to give white powder PH002-L34-RGD(5.3mg,91％).HRMS(ESI)m/z:calcd.for C67H93O15N18[M+H]+1389.7062,found 1389.7098.( as shown in FIG. 17)
Example 8 is an application example of PH002-L3 4 -RGD prepared in example 7, verifying that integrin promotes degradation of extracellular protein apolipoprotein E4 (ApoE 4) by lysosomal degradation strategy (IFLD strategy)
Fluorescent staining method: cells on coverslips were washed 2 times with PBS, fixed with 4% paraformaldehyde solution for 15min, and rinsed 3 times with PBS (10 mM, pH 7.4) for 5min each. Following this step, nuclei were stained with DAPI C1005 for 15 minutes in the dark. The cells were then washed three times with PBS (10 mM, pH 7.4) for 5 minutes each. The fluorescence image was imaged by LEICA STELLARIS confocal fluorescence microscope.
Extracellular protein uptake assay. Huh7 cells were incubated in 24-well coverslips to achieve about 40% to 50% confluency. Dilution of PH002-L3 4 -RGD to 400nM,Alexa Fluor 488 labeled apolipoprotein E4 (APOE 4-AF 488) in medium was performed to 400nM, and incubation was performed for 30 minutes after mixing to give RGD labeled APOE4-AF488. The resulting RGD-labeled APOE4-AF488 protein solution was added to a 24-well plate and co-cultured with the fused cells at 37℃for 20h. To verify the effect of lysosomal inhibitors (chloroquine, CQ) on protein uptake, the fused cells were incubated with the RGD-labeled APOE4-AF488 protein solution and CQ solution (50 μm in medium) described above for 12 hours. As a control, ph002-L3 4 -Azide was preincubated with APOE4-AF488 using the same procedure as described above to give an azide-labeled APOE4-AF488 protein solution. The resulting azide-labeled APOE4-AF488 protein solution was added to a 24-well plate and incubated with the fused cells for 20h at 37 ℃. Cells were fluorescent stained and imaged by laser confocal microscopy. (FIG. 18, wherein a panel labeled pH002 is shown as pH002-L3 4 -Azide pre-incubated with APOE4-AF488 to provide fluorescence detection of azide-labeled APOE4-AF488 protein solution)
Experimental results:
Referring to the description of fig. 8, immunoblotting experiments indicate that: three BMS-L3-RGD compounds containing connecting arms with different lengths can induce the degradation of PD-L1, wherein the degradation rate of BMS-L3 1 -RGD on PD-L1 is higher at a lower concentration (25 nM). We studied the PD-L1 levels in the cell membrane-cytoplasmic and total fractions after treatment with different drug concentrations and found that BMS-L3 1 -RGD showed better degradation at 25 nM. Screening incubation time experiments showed that 8 hours was sufficient to maximize the degradation of PD-L1 protein. We found that lysosomal inhibitor Bafilomycin A1 inhibited the degradation of PD-L1, whereas proteasome inhibitor MG132 had no inhibitory effect on protein degradation, confirming that its degradation was dependent on the lysosomal pathway. We also used the cyclic peptide cRGD to pretreat MDA-MB-231 cells to block integrins on the cell surface, and then incubated the cells with BMS-L1-RGD. Under these conditions, BMS-L3 1 -RGD did not induce degradation of PD-L1, indicating that PD-L1 degradation is mediated by integrins.
Referring to fig. 9 of the specification, in vivo animal experiments indicate that: the tail vein is injected with BMS-L3 1 -RGD (injected once every two days for 5 times), and the body weight of mice in the administration group and the control group is not changed obviously; the tumor volume of the administration group is slowly increased, and the tumor volume of the control group is in a steady-state increasing trend; after the experiment is finished, mice are killed, the tumor tissues peeled off by weighing are obviously smaller than that of a control group, wherein the average diameter of the tumor of the administration group injected with BMS-L3 1 -RGD is minimum, which indicates that the BMS-L3 1 -RGD can effectively inhibit the growth of the tumor, and has smaller toxic and side effects.
Referring to fig. 10 of the specification, bms-L3 1 -RGD degrades extracellular protein PD-L1: incubation with RGD-labeled PD-L1-AF488 significantly increased cellular uptake compared to azide-labeled PD-L1-AF 488-treated cells, while inhibition of lysosomal proteolytic activity with CQ resulted in the highest accumulation of the protein. These can demonstrate that binding of cRGD cyclic peptide to small molecule ligands of extracellular POI can promote integrin-mediated internalization and degradation of extracellular protein-PD-L1 (APOE 4-AF 488) via the endosomal-lysosomal pathway.
Referring to figure 14 of the drawings, integrin-promoted lysosomal degradation of extracellular proteins: incubation with RGD-labeled NAP-650 significantly increased cellular uptake compared to azide-labeled NAP-650 treated cells, while inhibition of lysosomal proteolytic activity with CQ resulted in the highest accumulation of the protein. In addition, the fluorescent signal of FITC-labeled neutralizing viral protein (NAP-FITC) co-localizes with early endosomal labeling (Rab 5), NAP-650 co-localizes with lysosomal labeling (LysoTracker), indicating that internalized protein is transported to lysosomes through the endosome. These may demonstrate that binding of cRGD cyclic peptides to small molecule ligands of extracellular POI can promote integrin-mediated internalization and degradation of extracellular proteins via the endosomal-lysosomal pathway.
Referring to figure 17 of the drawings, integrin-promoted lysosomal degradation of extracellular protein-apolipoprotein E4 (APOE 4-AF 488): incubation with RGD-labeled APOE4-AF488 significantly increased cellular uptake compared to azide-labeled APOE4-AF 488-treated cells, while inhibition of lysosomal proteolytic activity with CQ resulted in the highest accumulation of the protein. These can demonstrate that binding of cRGD cyclic peptide to small molecule ligands of extracellular POI can promote integrin-mediated internalization and degradation of extracellular protein-apolipoprotein E4 (APOE 4-AF 488) via the endosomal-lysosomal pathway.
By adopting the strategy, the synthesized BMS-L3-RGD series compound is proved to be a high-efficiency apoptosis ligand 1 (PD-L1) degradation agent in-vivo and in-vitro experiments; PH002-L34-RGD is a highly potent extracellular protein apolipoprotein E4 degrading agent. From this, we propose IFLD strategies that extend the toolbox for modulating secreted and membrane-associated protein levels, thus having great application potential in the fields of chemical biology and drug discovery.

Claims (10)

1. A bifunctional compound comprising a target protein binding unit, an integrin recognition unit, and a linking unit for linking the target protein binding unit and the integrin recognition unit.
2. The bifunctional compound of claim 1,
The difunctional compound is synthesized by the reaction of A molecule, B molecule and L,
The A molecule comprises an A1 unit and a reactive group A2 connected with the A1 unit, wherein the A1 unit is a target protein binding unit and comprises a ligand bound with the target protein;
the B molecule comprises a B1 unit and a reactive group B2 connected with the B1 unit, wherein the B1 unit is an integrin recognition unit and comprises a ligand combined with integrin;
The L molecule comprises an active group L1 which reacts with the A2 active group, an active group L2 which reacts with the B2 active group and an L3 unit which connects the active group L1 and the active group L2, wherein the L3 unit is a connecting unit which generates a covalent bond with the A1 unit and the B1 unit;
The structural general formula of the difunctional compound is A1-L3-B1.
3. The bifunctional compound of claim 2,
The active group A2 is a group or a functional group which is subjected to substitution reaction, elimination reaction, addition reaction or rearrangement reaction with the active group L1 and is selected from one or more of an alkane group, an arene group, a heterocyclic arene group, an alkenyl group, an alkynyl group, a halogeno group, an alcoholic hydroxyl group, a sulfhydryl group, an aldehyde group, a ketone group, a carboxyl group, an amino group, an enol group, an azide group, maleimide, a tetrazine group and variants thereof and an alcoholic hydroxyl group containing alpha hydrogen;
The active group B2 is a group or a functional group which is subjected to substitution reaction, elimination reaction, addition reaction or rearrangement reaction with the active group L2 and is selected from one or more of an alkane group, an arene group, a heterocyclic arene group, an alkenyl group, an alkynyl group, a halogeno group, an alcoholic hydroxyl group, a sulfhydryl group, an aldehyde group, a ketone group, a carboxyl group, an amino group, an enol group, an azide group, maleimide, a tetrazine group and variants thereof and an alcoholic hydroxyl group containing alpha hydrogen;
The L3 unit includes one or more of an alkyl chain, an aromatic ring, a heterocyclic ring, a heteroatom, and a functional group.
4. The bifunctional compound of claim 1, wherein the protein of interest is selected from the group consisting of structural proteins; a receptor; an enzymatic cell surface protein; proteins associated with cellular integration functions, including proteins involved in catalytic activity, aromatase activity, locomotor activity, helicase activity, metabolic processes, antioxidant activity, proteolysis, biosynthesis; proteins having kinase activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, ligase activity, enzyme modulating activity, signal transduction activity, structural molecule activity, binding activity, receptor activity, cell motility, membrane fusion, cell communication, biological process regulation, development, cell differentiation, stimulating reactions; behavioural proteins, cell adhesion proteins; proteins involved in cell necrosis; and one or more of proteins involved in transport.
5. The bifunctional compound of claim 1, wherein the protein of interest is selected from the group consisting of: apoptosis-ligand 1 (i.e., PD-L1), apoptosis receptor 1 (i.e., PD-1), epidermal growth factor receptor (i.e., EGFR), human epidermal growth factor receptor-2 (i.e., HER 2), G-protein coupled receptor (i.e., GPCR), fibroblast growth factor receptor (i.e., FGFRs), vascular endothelial growth factor receptor family (i.e., VEGFR, VEGF representing vascular endothelial growth factor), cytotoxic T lymphocyte-associated protein 4 (i.e., CTLA4 or CTLA-4), human interleukin 5 receptor alpha (IL-5Ralpha), apolipoprotein, apolipoprotein E4 (i.e., apoE 4), beta-amyloid, angiotensin converting enzyme 2 (ACE 2), sodium-taurocholate cotransporter (NTCP), B7.1 and B7, TI FR1m, TNFR2, NADPH oxidase, bc1IBax and other ligands in the apoptotic pathway, C5a receptor, HMG-CoA reductase, PDE V phosphodiesterase type, PDE IV phosphodiesterase type 4, PDE I, PDE II, PDE III, squalene cyclase inhibitor, CXCR1, CXCR2, nitric oxide synthase, cyclooxygenase 1, cyclooxygenase 2, 5HT receptor, dopamine receptor, G protein, histamine receptor, 5-lipoxygenase, protease-like serine protease, thymidylate synthase, purine nucleoside phosphorylase, glyceraldehyde-3-phosphate dehydrogenase (i.e., GAPDH), glycogen phosphorylase, carbonic anhydrase, chemokine receptor, JAW, RXR and the like, HIV1 protease, HIV1 integrase, influenza ammonia-phase, STAT reverse transcriptase, sodium channel, protein P-glycoprotein, P-glycoprotein and MRP-tyrosine kinase, CD23, CD73, CD124, tyrosine kinase P561ck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-alpha R, ICAM1, ca2+ channel, VCAM, VLA-4 integrin, selectin, CD40/CD40L, newokinins and receptor, inosine monophosphate dehydrogenase, P38 MAP kinase, ras/Raf/MEW/ERK pathway, interleukin-1 converting enzyme, caspase, HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotidyl transferase, rhinovirus, 3C protease, herpes simplex virus-1, protease, cytomegalovirus protease, poly (ADP-ribose) polymerase, cyclin-dependent kinase, vascular endothelial growth factor, oxytocin receptor, microsomal transfer protein inhibitor, bile acid transport inhibitor 5 alpha reductase inhibitor, angiotensin 11, glycine receptor, noradrenaline reuptake receptor, endothelin receptor, neuropeptide Y and receptor, adenosine kinase and AMP dehydrogenase, purinergic receptor, farnesyl transferase, geranyltransferase, trkA receptor for NCF, tyrosine kinase Flk-IIKDR, vitronectin receptor, integrin receptor, her-21 sphingosine, telomerase inhibition, cytosolic phosphate A2 and EGF receptor tyrosine kinase, ecdysone 20-monooxygenase, GABA-gated chloride ion channel, acetylcholinesterase, voltage-sensitive sodium channel protein, calcium release channel and chloride ion channel, acetyl-CoA carboxylase, adenylyl succinate synthase, protoporphyrinogen oxidase and enolpyruvylshikimate phosphate synthase, and/or one or more of all variants, mutants, splice variants, insertion deletions and fusions of the above proteins.
6. The bifunctional compound of claim 3, wherein the A molecule is BMS-8, biotin-NHS or PH-002 and the integrin recognition ligand is cRGD.
7. The bifunctional compound of claim 6,
When the A molecule is BMS-8, the active group A2 is carboxyl, the active group B2 connected with the integrin recognition unit comprises alkynyl, the active group L1 in the L molecule is amino, the amino and the carboxyl form an amide bond, the active group L2 is azide group, and the azide group and the alkynyl form five-membered heterocycle of 1,2, 3-triazole;
when the A molecule is Biotin-NHS, the active group A2 is-NHS, the active group B2 connected with the integrin recognition unit comprises alkynyl, the active group L1 in the L molecule is amino, the amino replaces the active group NHS and a Biotin group to form an amide bond, the active group L2 in the L molecule is an azide group, and the azide group and the alkynyl form a five-membered heterocycle of 1,2, 3-triazole;
When the A molecule is PH-002, the A2 group contains amino protected by tert-butoxycarbonyl, the active group B2 connected with the integrin recognition unit comprises alkynyl, the active group L1 in the L molecule is carboxyl, the carboxyl and the exposed amino after the tert-butoxycarbonyl is removed on the PH-002 form an amide bond, the active group L2 in the L molecule is azido, and the azido and the alkynyl form five-membered heterocycle of 1,2, 3-triazole.
8. A pharmaceutical composition of the bifunctional compound of any one of claims 1-7, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, benign proliferative disorders, infectious or non-infectious inflammatory events, autoimmune diseases, inflammatory diseases, systemic inflammatory response syndrome, viral infections and viral diseases, and eye diseases.
9. Use of the bifunctional compound of any one of claims 1-7 or the pharmaceutical composition of claim 8 to modulate the protein activity of a protein of interest in a patient in need thereof.
10. Use of a bifunctional compound of any one of claims 1-7 or a pharmaceutical composition of claim 8 for degradation of a target protein lysosome.
CN202211318398.4A 2022-10-26 2022-10-26 Bifunctional compound serving as target protein degradation agent and application of bifunctional compound in degradation of target protein lysosome Pending CN117959457A (en)

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