WO2024087332A1

WO2024087332A1 - Bifunctional compound used as target protein degradation agent and use thereof in target protein lysosomal degradation

Info

Publication number: WO2024087332A1
Application number: PCT/CN2022/137378
Authority: WO
Inventors: 房丽晶; 陈亮; 郑纪维; 何婉怡; 李晶; 李红昌
Original assignee: 深圳先进技术研究院
Priority date: 2022-10-26
Filing date: 2022-12-07
Publication date: 2024-05-02
Also published as: CN117959457A

Abstract

The present invention relates to the technical field of medicines, and provides a bifunctional compound used as a target protein degradation agent and a use thereof in target protein lysosomal degradation. The bifunctional compound comprises a target protein-binding small molecule, an integrin-recognition ligand, and a linking group for linking the target protein-binding small molecule and the integrin-recognition ligand. The present invention provides a bifunctional compound. On this basis, a novel integrin-facilitated lysosomal degradation (IFLD) strategy of the target protein is established and verified, and a new technical option for the regulation of extracellular proteins and membrane-associated proteins is provided. According to the present invention, the bifunctional compound that has a targeting function and is easy to synthesize and modify is used for replacing an antibody that is expensive and not easy to synthesize, a nano-antibody, a nucleic acid aptamer and a glycopeptide polymer or a long polypeptide, and then bridges are established between target proteins and specific receptors on the surface of cells, so that endocytosis and lysosomal degradation of the target protein are promoted.

Description

一种用作靶蛋白降解剂的双功能化合物及其在靶蛋白溶酶体降解中的应用A bifunctional compound used as a target protein degrader and its application in lysosomal degradation of target protein

技术领域Technical Field

本发明属于医药技术领域，具体涉及一种用作靶蛋白降解剂的双功能化合物及其在靶蛋白溶酶体降解中的应用。The invention belongs to the field of medical technology, and specifically relates to a bifunctional compound used as a target protein degradation agent and application of the compound in lysosomal degradation of the target protein.

背景技术Background technique

近年来开发的靶向蛋白降解(TPD)技术是一项特异性地识别靶蛋白(目标蛋白)，并利用细胞内固有的蛋白质降解途径直接降解靶蛋白的新技术。目前，TPD领域发展出了PROTAC、分子胶、降解标签、溶酶体靶向嵌合体、自噬小体绑定化合物等技术，大大拓展了可降解的靶蛋白的范围。靶向蛋白质降解(TPD)目前主要通过泛素-蛋白酶体和溶酶体途径来进行，根据具体作用原理又可细分为近9个不同技术路线，其中，通过泛素蛋白酶体降解的技术包括PROTAC技术和分子胶技术；通过溶酶体降解靶蛋白的技术包括LYTAC技术、双特异性核酸适体技术、GlueTAC技术、AUTAC技术、ATTEC技术、AUTOTAC技术和CMA技术。The targeted protein degradation (TPD) technology developed in recent years is a new technology that specifically identifies target proteins (target proteins) and directly degrades target proteins by utilizing the intrinsic protein degradation pathways in cells. At present, the TPD field has developed technologies such as PROTAC, molecular glue, degradation tags, lysosomal targeting chimeras, and autophagosome binding compounds, which have greatly expanded the range of degradable target proteins. Targeted protein degradation (TPD) is currently mainly carried out through the ubiquitin-proteasome and lysosomal pathways. According to the specific principles of action, it can be subdivided into nearly 9 different technical routes. Among them, the technologies for degradation through ubiquitin proteasomes include PROTAC technology and molecular glue technology; the technologies for degradation of target proteins through lysosomes include LYTAC technology, bispecific nucleic acid aptamer technology, GlueTAC technology, AUTAC technology, ATTEC technology, AUTOTAC technology, and CMA technology.

PROTAC技术在TPD技术中发展最快、应用最广，PROTACs能够与靶蛋白和E3连接酶之间形成三元复合物，诱导靶蛋白泛素化，随后被蛋白酶体降解。但是泛素-蛋白酶体***(UPS)降解蛋白质的机制决定PROTACs的特点是降解细胞内的目标蛋白(POI)，而难以降解细胞外与细胞膜上的靶蛋白。分子胶技术无法像PROTAC那样通过对各组分的大规模筛选来获得，设计困难。PROTAC technology is the fastest growing and most widely used technology in TPD. PROTACs can form a ternary complex between the target protein and the E3 ligase, induce ubiquitination of the target protein, and then be degraded by the proteasome. However, the mechanism of protein degradation by the ubiquitin-proteasome system (UPS) determines that the characteristic of PROTACs is to degrade the target protein (POI) inside the cell, but it is difficult to degrade the target protein outside the cell and on the cell membrane. Molecular glue technology cannot be obtained through large-scale screening of each component like PROTAC, and it is difficult to design.

LYTAC技术分子中的抗体或寡糖存在较强免疫原性、寡糖与抗体偶联时剂量比与连接位点不确定、糖肽链为高分子混合物、体内清除率较高；双特异性核酸适体技术递送效率差，体内不稳定；GlueTAC技术存在非天然氨基酸的纳米抗体与靶蛋白的共价结合安全性有待评估，体内半衰期短；AUTAC技术降解速度较慢；ATTEC技术难以合理设计，发现成本高，对细胞整体自噬是否有影响未知；AUTOTAC技术降解速度慢；CMA技术稳定性与输送效率有待提高。The antibodies or oligosaccharides in the LYTAC technology molecules have strong immunogenicity, the dose ratio and connection site are uncertain when the oligosaccharides are coupled to the antibodies, the glycopeptide chains are high molecular weight mixtures, and the in vivo clearance rate is high; the bispecific nucleic acid aptamer technology has poor delivery efficiency and is unstable in vivo; the GlueTAC technology has non-natural amino acids, the safety of the covalent binding of nano-antibodies to target proteins needs to be evaluated, and the in vivo half-life is short; the AUTAC technology degrades slowly; the ATTEC technology is difficult to design rationally, the discovery cost is high, and it is unknown whether it has an impact on the overall autophagy of cells; the AUTOTAC technology degrades slowly; the stability and delivery efficiency of the CMA technology need to be improved.

基于以上现有技术中存在的问题，本发明提供一种用作靶蛋白降解剂的双功能化合物及其在靶蛋白溶酶体降解中的应用。Based on the above problems existing in the prior art, the present invention provides a bifunctional compound used as a target protein degrading agent and its application in lysosomal degradation of the target protein.

发明内容Summary of the invention

为了解决现有技术中靶向蛋白降解(TPD)技术设计困难、体内稳定性差、体内半衰期短、降解速率慢、不能有效降解细胞外或细胞膜上的靶蛋白的问题，本发明提供了一种可作为靶蛋白降解剂的双功能化合物。In order to solve the problems in the prior art of targeted protein degradation (TPD) technology, such as difficult design, poor in vivo stability, short in vivo half-life, slow degradation rate, and inability to effectively degrade target proteins outside cells or on cell membranes, the present invention provides a bifunctional compound that can be used as a target protein degrader.

该双功能化合物包括目标蛋白结合单元、整合素识别单元和用于连接所述目标蛋白结合单元和所述整合素识别单元的连接单元。The bifunctional compound comprises a target protein binding unit, an integrin recognition unit and a connecting unit for connecting the target protein binding unit and the integrin recognition unit.

进一步地，所述双功能化合物经A分子、B分子和L反应合成，Furthermore, the bifunctional compound is synthesized by reacting molecule A, molecule B and L.

所述A分子包括A1单元和与所述A1单元连接的活性基团A2，所述A1单元为目标蛋白结合单元，包括与目标蛋白结合的配体；The A molecule includes an A1 unit and an active group A2 connected to the A1 unit, wherein the A1 unit is a target protein binding unit and includes a ligand that binds to the target protein;

所述B分子包括B1单元和与所述B1单元连接的活性基团B2，所述B1单元为整合素识别单元，包括与整合素结合的配体；The B molecule includes a B1 unit and an active group B2 connected to the B1 unit, wherein the B1 unit is an integrin recognition unit and includes a ligand that binds to the integrin;

所述L分子包括与所述A2活性基团反应的活性基团L1、与所述B2活性基团反应的活性基团L2以及所述连接活性L1和所述活性基团L2的L3单元，所述L3单元为与所述A1单元和所述B1单元生成共价键的连接单元；The L molecule includes an active group L1 that reacts with the A2 active group, an active group L2 that reacts with the B2 active group, and an L3 unit that connects the active group L1 and the active group L2, wherein the L3 unit is a connecting unit that forms a covalent bond with the A1 unit and the B1 unit;

所述双功能化合物的结构通式为A1-L3-B1。The general structural formula of the bifunctional compound is A1-L3-B1.

进一步地，所述活性基团A2是与所述活性基团L1发生取代反应、消去反应、加成反应或重排反应的基团或官能团，选自烷烃基团、芳烃基团、杂环芳烃基团、烯基、炔基、卤代基、醇羟基、巯基、醛基、酮基、羧基、氨基、烯醇基团、叠氮基团、马来酰亚胺、四嗪基团及其变体和含有α氢的醇羟基中的一种或多种；Further, the active group A2 is a group or functional group that undergoes 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 aromatic group, a heterocyclic aromatic group, an alkenyl group, an alkynyl group, a halide group, an alcoholic hydroxyl group, a thiol 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 and variants thereof, and an alcoholic hydroxyl group containing α hydrogen;

所述活性基团B2是与所述活性基团L2发生取代反应、消去反应、加成反应或重排反应的基团或官能团，选自烷烃基团、芳烃基团、杂环芳烃基团、烯基、炔基、卤代基、醇羟基、巯基、醛基、酮基、羧基、醛基、氨基、烯醇基团、叠氮基团、马来酰亚胺、四嗪基团及其变体和含有α氢的醇羟基中的一种或多种；The active group B2 is a group or functional group that undergoes 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 aromatic group, a heterocyclic aromatic group, an alkenyl group, an alkynyl group, a halide group, an alcoholic hydroxyl group, a thiol group, an aldehyde group, a keto group, a carboxyl group, an aldehyde group, an amino group, an enol group, an azide group, a maleimide group, a tetrazine group and variants thereof, and an alcoholic hydroxyl group containing α hydrogen;

L3单元中包括烷基链、芳香环、杂环、杂原子和官能团中的一种或多种。The L3 unit includes one or more of an alkyl chain, an aromatic ring, a heterocycle, a heteroatom and a functional group.

进一步地，所述目标蛋白选自结构蛋白质；受体；酶等细胞表面蛋白质；与细胞整合功能相关的蛋白质，包括涉及催化活性、芳香酶活性、运动活性、解旋酶活性、代谢过程、抗氧化活性、蛋白水解、生物合成的蛋白质；具有激酶活性、氧化还原酶活性、转移酶活性、水解酶活性、裂解酶活性、异构酶活性、连接酶活性、酶调节活性、信号转导活性、结构分子活性、结合活性、受体活性、细胞运动性、膜融合、细胞通信、生物过程调节、发育、细胞分化、刺激反应的蛋白质；行为蛋白质、细胞黏附蛋白质；涉及细胞坏死的蛋白质；和涉及转运的蛋白质中的一种或多种。Furthermore, the target protein is selected from structural proteins; receptors; cell surface proteins such as enzymes; proteins related to cell integration functions, including proteins involved in catalytic activity, aromatase activity, motor activity, helicase activity, metabolic processes, antioxidant activity, proteolysis, and biosynthesis; proteins with kinase activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, ligase activity, enzyme regulatory activity, signal transduction activity, structural molecule activity, binding activity, receptor activity, cell motility, membrane fusion, cell communication, biological process regulation, development, cell differentiation, and stimulus response; behavioral proteins, cell adhesion proteins; proteins involved in cell necrosis; and one or more of proteins involved in transport.

进一步地，所述目标蛋白选自细胞程序性死亡-配体1(即PD-L1)、程序性死亡受体1(即PD-1)、表皮生长因子受体(即EGFR)、人表皮生长因子受体-2(即HER2)、G蛋白偶联受体(即GPCR)、成纤维细胞生长因子受体(即FGFRs)、血管内皮生长因子受体家族(即VEGFR，VEGF表示血管内皮生长因子)、细胞毒性T淋巴细胞相关蛋白4(即CTLA4或CTLA-4)、人白介素5受体α(IL-5Rα)、载脂蛋白，载脂蛋白E4(即ApoE4)、β-淀粉样蛋白、血管紧张素转化酶2(ACE2)、钠离子-牛磺胆酸共转运蛋白(NTCP)、B7.1和B7、TI FR1m、TNFR2、NADPH氧化酶、Bc1IBax和在细胞凋亡通路中的其他配体、C5a受体、HMG-CoA还原酶、PDEⅤ磷酸二酯酶型、PDEⅣ磷酸二酯酶4型、PDEⅠ、PDEⅡ、PDEⅢ、鲨烯环化酶抑制剂、CXCR1、CXCR2、一氧化氮合成酶、环氧化酶1、环氧化酶2、5HT受体、多巴胺受体、G蛋白、组胺受体、5-脂肪氧化酶、类蛋白酶丝氨酸蛋白酶、胸苷酸合成酶、嘌呤核苷磷酸化酶、甘油醛-3-磷酸脱氢酶(即GAPDH)、糖原磷酸化酶、碳酸酐酶、趋化因子受体、JAW STAT、RXR和类似物、HIV1蛋白酶、HIV1整合酶、流感神经氨酸酶、乙型肝炎逆转录酶、钠通道、蛋白质P-糖蛋白、P糖蛋白和MRP络氨酸激酶、CD23、CD73、CD124、酪氨酸激酶p561ck、CD4、CD5、IL-2受体、IL-1受体、TNF-αR、ICAM1、Ca2+通道、VCAM、VLA-4整合素、选择素、CD40/CD40L、newokinins和受体、肌苷一磷酸脱氢酶、p38MAP激酶、Ras/Raf/MEW/ERK通路、白介素-1转化酶、半胱天冬酶、HCV、NS3蛋白酶、HCV NS3 RNA解旋酶、甘氨酰胺核糖核苷酸甲酰转移酶、鼻病毒、3C蛋白酶、单纯性疱疹病毒-1、蛋白酶、巨细胞病毒蛋白酶、聚(ADP-核糖)聚合酶、细胞周期蛋白依赖性激酶、血管内皮生长因子、催产素受体、微粒体转移蛋白质抑制子、胆汁酸转运抑制子、5α还原酶抑制子、血管紧张素11、甘氨酸受体、去甲肾上腺激素再摄取受体、内皮素受体、神经肽Y和受体、腺苷受体、腺苷激酶和AMP脱氢酶、嘌呤能受体、法尼基转移酶、香叶基转移酶、NCF的TrkA受体、酪氨酸激酶Flk-IIKDR、玻连蛋白受体、整合素受体、Her-21神经鞘、端粒酶抑制、细胞溶质磷酸酯A2和EGF受体酪氨酸激酶、蜕皮激素20-单氧酶、GABA门控的氯离子通道、乙酰胆碱酯酶、电压敏感的钠通道蛋白、钙释放通道和氯离子通道、乙酰辅酶A羧化酶、腺苷酸琥珀酸合成酶、原卟啉原氧化酶和烯醇丙酮酰莽草酸磷酸合成酶中的一种或多种，和/或上述蛋白质所有变体、突变体、剪接变体、***缺失体和融合体中的一种或多种。Furthermore, the target protein is selected from programmed cell death-ligand 1 (i.e. PD-L1), programmed death receptor 1 (i.e. PD-1), epidermal growth factor receptor (i.e. EGFR), human epidermal growth factor receptor-2 (i.e. HER2), G protein-coupled receptor (i.e. GPCR), fibroblast growth factor receptor (i.e. FGFRs), vascular endothelial growth factor receptor family (i.e. VEGFR, VEGF represents vascular endothelial growth factor), cytotoxic T lymphocyte-associated protein 4 (i.e. CTLA4 or CTLA-4), human interleukin 5 receptor alpha (IL-5Rα), apolipoprotein, apolipoprotein E4 (i.e. ApoE4), β-amyloid protein, angiotensin converting enzyme 2 (ACE2), sodium ion-taurocholic acid co-transporter (NTCP), B7.1 and B7, TI FR1m, TNFR2, NADPH oxidase, Bc1IBax and other ligands in the apoptosis pathway, C5a receptor, HMG-CoA reductase, PDEⅤ phosphodiesterase type, PDEⅣ phosphodiesterase type 4, PDEⅠ, PDEⅡ, PDEⅢ, squalene cyclase inhibitor, CXCR1, CXCR2, nitric oxide synthase, cyclooxygenase 1, cyclooxygenase 2, 5HT receptor, dopamine receptor, G protein, histamine receptor, 5-lipoxygenase, protease-like serine protease, thymidylate synthase, purine nucleoside phosphorylase, glyceraldehyde-3-phosphate dehydrogenase (i.e. GAPDH), glycogen phosphorylase, carbonic anhydrase, chemokine receptor, JAW STAT, RXR and analogs, HIV1 protease, HIV1 integrase, influenza neuraminidase, hepatitis B reverse transcriptase, sodium channel, protein P-glycoprotein, P-glycoprotein and MRP tyrosine kinase, CD23, CD7 3. CD124, tyrosine kinase p561ck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-αR, ICAM1, Ca2+ channel, VCAM, VLA-4 integrin, selectin, CD40/CD40L, newokinins and receptors, inosine monophosphate dehydrogenase, p38MAP kinase, Ras/Raf/MEW/ERK pathway, interleukin-1 converting enzyme, caspase, HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotide formyltransferase, rhinovirus, 3C protease, herpes simplex virus-1, protease, cytomegalovirus protease, poly (ADP-ribose) polymerase, cyclin-dependent kinase, vascular endothelial growth factor, oxytocin receptor, microsomal transfer protein inhibitor, bile acid transport inhibitor, 5α-reductase inhibitor One or more of the following: adenosine receptors, angiotensin 11, glycine receptors, norepinephrine reuptake receptors, endothelin receptors, neuropeptide Y and receptors, adenosine receptors, adenosine kinases and AMP dehydrogenases, purinergic receptors, farnesyltransferases, geranyltransferases, TrkA receptors of NCF, tyrosine kinase Flk-IIKDR, vitronectin receptors, integrin receptors, Her-21 nerve sheaths, telomerase inhibition, cytosolic phosphate A2 and EGF receptor tyrosine kinases, ecdysone 20-monooxygenase, GABA-gated chloride channels, acetylcholinesterase, voltage-sensitive sodium channel proteins, calcium release channels and chloride channels, acetyl-CoA carboxylase, adenylate succinate synthetase, protoporphyrinogen oxidase and enolpyruvylshikimate phosphate synthetase, and/or one or more of all variants, mutants, splice variants, insertions, deletions and fusions of the above proteins.

进一步地，所述A分子为BMS-8、Biotin-NHS或PH-002，所述整合素识别配体为cRGD。Furthermore, the A molecule is BMS-8, Biotin-NHS or PH-002, and the integrin recognition ligand is cRGD.

进一步地，所述A分子为BMS-8时，所述活性基团A2为羧基，连接所述整合素识别单元的所述活性基团B2包括炔基，所述L分子中的所述活性基团L1为氨基，所述氨基和所述羧基形成酰胺键，所述活性基团L2为叠氮基团，所述叠氮基团和所述炔基形成1,2,3-三氮唑的五元杂环；Further, when the A molecule is BMS-8, the active group A2 is a carboxyl group, the active group B2 connected to the integrin recognition unit includes an alkynyl group, the active group L1 in the L molecule is an amino group, the amino group and the carboxyl group form an amide bond, the active group L2 is an azide group, and the azide group and the alkynyl group form a five-membered heterocyclic ring of 1,2,3-triazole;

所述A分子为Biotin-NHS时，所述活性基团A2为-NHS，连接所述整合素识别单元的活性基团B2包括炔基，所述L分子中的所述活性基团L1为氨基，所述氨基取代所述活性基团NHS和Biotin基团形成酰胺键，所述L分子中的所述活性基团L2为叠氮基团，所述叠氮基团和所述炔基形成1,2,3-三氮唑的五元杂环；When the A molecule is Biotin-NHS, the active group A2 is -NHS, the active group B2 connected to the integrin recognition unit includes an alkynyl group, the active group L1 in the L molecule is an amino group, the amino group replaces the active group 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 group form a five-membered heterocyclic ring of 1,2,3-triazole;

所述A分子为PH-002时，所述A2基团包含叔丁氧羰基保护的氨基，连接所述整合素识别单元的所述活性基团B2包括炔基，所述L分子中的所述活性基团L1为羧基，所述羧基与所述PH-002上脱除所述叔丁氧羰基后裸露的氨基形成酰胺键，所述L分子中的所述活性基团L2为叠氮基团，所述叠氮基团和所述炔基形成1,2,3-三氮唑的五元杂环。When the A molecule is PH-002, the A2 group includes an amino group protected by a tert-butyloxycarbonyl group, the active group B2 connected to the integrin recognition unit includes an alkynyl group, the active group L1 in the L molecule is a carboxyl group, and the carboxyl group forms an amide bond with the amino group exposed after the tert-butyloxycarbonyl group is removed from the PH-002, and the active group L2 in the L molecule is an azide group, and the azide group and the alkynyl group form a five-membered heterocyclic ring of 1,2,3-triazole.

本发明的一个目的是提供一种如上任一项所述的双功能化合物或其药物上可接受的盐的药物组合物，所述药物组合物用于治疗癌症、良性增生性失常、感染性或非感染性炎症事件、自身免疫性疾病、炎性疾病、全身性炎症反应综合征、病毒性感染和病毒性疾病以及眼疾。An object of the present invention is to provide a pharmaceutical composition of a bifunctional compound as described above or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is used to treat 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.

本发明的一个目的是提供一种如上任一项所述的双功能化合物或药物组合物在有需要的患者中调节目标蛋白的蛋白质活性的应用。One object of the present invention is to provide a use of a bifunctional compound or pharmaceutical composition as described above for regulating the protein activity of a target protein in a patient in need thereof.

本发明的一个目的是提供一种如上任一项所述的双功能化合物或药物组合物在靶蛋白溶酶体降解中的应用。One object of the present invention is to provide a use of a bifunctional compound or pharmaceutical composition as described above in lysosomal degradation of a target protein.

本发明提供了一种双功能化合物，在此基础上建立并验证了一种新颖的整合素促进的靶蛋白溶酶体降解(IFLD)策略，为胞外蛋白和膜相关蛋白的调控提供了新的技术选择。本发明使用具有靶向作用、易合成和修饰的双功能化合物代替价格昂贵、不易合成的抗体、纳米抗体、核酸适配子以及糖肽聚合物或较长的多肽，在细胞表面的靶蛋白以及特定的受体之间建立桥梁，从而促进了靶蛋白的內吞和溶酶体降解。此外，利用本策略，我们设计了具有高活性的BMS-L1-RGD双功能分子降解剂，并且证实了其调节体内、体外PD-L1蛋白水平的能力及抗肿瘤活性，这为未来的肿瘤靶向治疗提供了新的工具。The present invention provides a bifunctional compound, on the basis of which a novel integrin-promoted target protein lysosomal degradation (IFLD) strategy is established and verified, providing a new technical option for the regulation of extracellular proteins and membrane-associated proteins. The present invention uses bifunctional compounds with targeted effects, easy synthesis and modification to replace expensive and difficult-to-synthesize antibodies, nanobodies, nucleic acid aptamers, and glycopeptide polymers or longer polypeptides to establish a bridge between the target protein on the cell surface and a specific receptor, thereby promoting the endocytosis and lysosomal degradation of the target protein. In addition, using this strategy, we designed a highly active BMS-L1-RGD bifunctional molecular degrader, and confirmed its ability to regulate PD-L1 protein levels in vivo and in vitro and its anti-tumor activity, which provides a new tool for future targeted tumor therapy.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1为本发明提供的整合素促进的靶蛋白溶酶体降解(IFLD)策略；FIG1 is an integrin-promoted target protein lysosomal degradation (IFLD) strategy provided by the present invention;

图2为本发明提供的BMS-L3 ¹-RGD、BMS-L3 ²-RGD和BMS-L3 ³-RGD结构式； FIG2 is the structural formula of BMS-L3 ¹ -RGD, BMS-L3 ² -RGD and BMS-L3 ³ -RGD provided by the present invention;

图3为本发明实施例1提供的BMS-L3 ¹-Azide的HRMS图谱； FIG3 is the HRMS spectrum of BMS-L3 ¹ -Azide provided in Example 1 of the present invention;

图4为本发明实施例1提供的BMS-L3 ¹-RGD的1H NMR图谱； FIG4 is a 1H NMR spectrum of BMS-L3 ¹ -RGD provided in Example 1 of the present invention;

图5为本发明实施例1提供的BMS-L3 ¹-RGD的HRMS图谱； FIG5 is the HRMS spectrum of BMS-L3 ¹ -RGD provided in Example 1 of the present invention;

图6为本发明实施例1提供的BMS-L3 ¹-RGD的HPLC分析； FIG6 is an HPLC analysis of BMS-L3 ¹ -RGD provided in Example 1 of the present invention;

图7为本发明实施例3提供的膜蛋白PD-L1降解试验的荧光图；FIG7 is a fluorescence image of the membrane protein PD-L1 degradation test provided in Example 3 of the present invention;

图8为本发明实施例2提供的BMS-L3 ¹-RGD通过整合素-溶酶体途径促进膜相关蛋白PD-L1的降解； FIG8 shows that BMS-L3 ¹ -RGD provided in Example 2 of the present invention promotes the degradation of membrane-associated protein PD-L1 through the integrin-lysosome pathway;

图9为本发明实施例3提供的BMS-L3 ¹-RGD的体内抗肿瘤活性评估； FIG9 is an in vivo anti-tumor activity evaluation of BMS-L3 ¹ -RGD provided in Example 3 of the present invention;

图10为本发明实施例4提供的验证BMS-L3 ¹-RGD通过整合素促进溶酶体降解策略降解胞外蛋白PD-L1； FIG. 10 is a diagram for verifying that BMS-L3 ¹ -RGD degrades the extracellular protein PD-L1 by promoting lysosomal degradation through integrin provided in Example 4 of the present invention;

图11为本发明实施例5提供的Biotin-L3 ¹-Azide的HRMS图谱； FIG11 is the HRMS spectrum of Biotin-L3 ¹ -Azide provided in Example 5 of the present invention;

图12为本发明实施例5提供的Biotin-L3 ¹-RGD的1H NMR图谱； FIG12 is the 1H NMR spectrum of Biotin-L3 ¹ -RGD provided in Example 5 of the present invention;

图13为本发明实施例5提供的Biotin-L3 ¹-RGD的HRMS图谱； FIG13 is the HRMS spectrum of Biotin-L3 ¹ -RGD provided in Example 5 of the present invention;

图14为本发明实施例5提供的Biotin-L3 ¹-RGD的HPLC分析； FIG14 is an HPLC analysis of Biotin-L3 ¹ -RGD provided in Example 5 of the present invention;

图15为本发明实施例6提供的Biotin-L3 ¹-RGD通过整合素-溶酶体途径促进细胞外蛋白的降解； FIG15 shows that Biotin-L3 ¹ -RGD provided in Example 6 of the present invention promotes the degradation of extracellular proteins through the integrin-lysosome pathway;

图16为本发明实施例7提供的PH002-L3 ⁴-Azide的HRMS图谱； FIG16 is the HRMS spectrum of PH002-L3 ⁴ -Azide provided in Example 7 of the present invention;

图17为本发明实施例7提供的PH002-L3 ⁴-RGD的HRMS图谱； FIG17 is the HRMS spectrum of PH002-L3 ⁴ -RGD provided in Example 7 of the present invention;

图18为本发明实施例8提供的PH002-L3 ⁴-RGD通过整合素促进的胞外蛋白—载脂蛋白E4(APOE4-AF488)的溶酶体降解。 FIG. 18 shows the lysosomal degradation of the extracellular protein apolipoprotein E4 (APOE4-AF488) promoted by PH002-L3 ⁴ -RGD provided in Example 8 of the present invention through integrin.

具体实施方式Detailed ways

为了使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention are described in detail below with reference to the accompanying drawings, but they should not be construed as limiting the applicable scope of the present invention.

溶酶体通过内吞、吞噬和自噬作用介导蛋白和细胞器的降解。随着对内体-溶酶体和自噬体-溶酶体降解途径的深入研究，近年来不断有通过溶酶体途径的TPD技术被开发出来，如LYTAC、AbTAC、ATTEC、AUTAC、AUTOTAC等。相比基于泛素-蛋白酶体机制的TPD，基于溶酶体的TPD不仅可以降解胞内蛋白，还可以降解蛋白聚集体、损伤的细胞器以及细胞外蛋白，在应用场景上有更广泛的潜力。细胞外蛋白和膜蛋白占编码蛋白的40％，并且与神经退行性疾病、自身免疫性疾病和癌症密切相关。为了降解此类蛋白，研究者开发了溶酶体靶向嵌合体(Lysosome-targeting chimaeras,LYTAC)，通过在靶蛋白与溶酶体转运蛋白(LTR)之间建立桥梁，从而诱导细胞外和膜蛋白通过内体-溶酶体途径降解。此外，双特异性核酸适体嵌合体(Bispecific aptamer chimera)和LYTAC类似，也通过内体-溶酶体途径介导目标蛋白降解，其由两个分别靶向目标蛋白和LTR的核酸适体偶联得到，也是通过与LTR及目标蛋白形成三元复合物，促使目标蛋白的内吞与溶酶体降解。基于抗体的PROTAC(Antibody-based PROTAC,AbTAC)也是通过内体-溶酶体途径诱导胞外蛋白和膜蛋白的降解。AbTAC本质上是一个重组的双特异性抗体，一端靶向细胞表面的目标蛋白，另一端靶向跨膜的E3连接酶，相对LYTAC，AbTAC能降低嵌合体分子的免疫原性，但其是否可以回收再利用等具体机制还有待研究。基于共价抗体的PROTAC(Covalent Nanobody-BasedPROTAC,GlueTAC)由可共价结合目标蛋白的纳米抗体与穿膜肽-溶酶体分选肽组成，GlueTAC共价结合目标蛋白后在穿膜肽-溶酶体分选肽的作用下，通过网格蛋白介导的内吞将目标蛋白运送到溶酶体并降解。GlueTAC分子虽然表现出较强的降解能力，但纳米抗体中引入了非天然氨基酸并与目标蛋白形成共价键，还需要仔细评估这一改造对靶细胞和非靶细胞的毒性。在以上技术中，用于识别细胞膜上目标蛋白的部分为抗体、纳米抗体或核酸适体嵌合体，可能存在免疫原性以及对稳定性的问题。目前，使用结构单一的双功能化合物介导细胞膜蛋白降解的策略还未见报道。Lysosomes mediate the degradation of proteins and organelles through endocytosis, phagocytosis, and autophagy. With the in-depth study of the endosomal-lysosomal and autophagosome-lysosomal degradation pathways, TPD technologies through the lysosomal pathway have been continuously developed in recent years, such as LYTAC, AbTAC, ATTEC, AUTAC, AUTOTAC, etc. Compared with TPD based on the ubiquitin-proteasome mechanism, lysosome-based TPD can not only degrade intracellular proteins, but also protein aggregates, damaged organelles, and extracellular proteins, and has a wider potential in application scenarios. Extracellular proteins and membrane proteins account for 40% of the encoded proteins and are closely related to neurodegenerative diseases, autoimmune diseases, and cancer. In order to degrade such proteins, researchers have developed lysosome-targeting chimaeras (LYTAC), which induces the degradation of extracellular and membrane proteins through the endosomal-lysosomal pathway by establishing a bridge between the target protein and the lysosomal transporter (LTR). In addition, bispecific aptamer chimera is similar to LYTAC and also mediates the degradation of target proteins through the endosomal-lysosomal pathway. It is obtained by coupling two aptamers that target the target protein and LTR respectively. It also promotes the endocytosis and lysosomal degradation of the target protein by forming a ternary complex with LTR and the target protein. Antibody-based PROTAC (AbTAC) also induces the degradation of extracellular and membrane proteins through the endosomal-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 targets the transmembrane E3 ligase. Compared with LYTAC, AbTAC can reduce the immunogenicity of the chimeric molecule, but the specific mechanism such as whether it can be recycled and reused remains to be studied. Covalent antibody-based PROTAC (Covalent Nanobody-Based PROTAC, GlueTAC) consists of a nanobody that can covalently bind to the target protein and a membrane-penetrating peptide-lysosomal sorting peptide. After GlueTAC covalently binds to the target protein, under the action of the membrane-penetrating peptide-lysosomal sorting peptide, the target protein is transported to the lysosome and degraded through clathrin-mediated endocytosis. Although the GlueTAC molecule exhibits strong degradation ability, non-natural amino acids are introduced into the nanobody and form covalent bonds with the target protein. The toxicity of this modification to target cells and non-target cells needs to be carefully evaluated. In the above technologies, the part used to recognize the target protein on the cell membrane is an antibody, a nanobody or a nucleic acid aptamer chimera, which may have problems with immunogenicity and stability. At present, the strategy of using a single structural bifunctional compound to mediate the degradation of cell membrane proteins has not been reported.

作为溶酶体转运蛋白，6-磷酸甘露糖/IGF-II受体(M6P/IGFIIR)和唾液糖蛋白受体(ASGPR)被证明可以有效地诱导分泌蛋白或膜蛋白到溶酶体进行降解。除M6P/IGFIIR和ASGPR外，受体-配体介导的递送***涉及其它细胞表面受体，如转铁蛋白受体、叶酸受体和整合素等，它们也有能力通过受体介导的内吞作用将荧光团、药物或纳米材料递送到细胞中。在此，我们重点关注整合素，它是表达在细胞表面的细胞粘附受体，在细胞-基质相互作用中发挥重要作用。由于整合素α _vβ ₃在实体肿瘤血管、增殖肿瘤内皮细胞和各种肿瘤细胞中过表达，在肿瘤靶向治疗中得到了广泛的关注。然而，虽然α _vβ ₃整合素识别基序Arg-Gly-Asp(RGD)序列已经被广泛用于将各种治疗药物传递到肿瘤中，但在我们的工作之前，RGD-整合素介导的TPD的可能性尚未被研究。 As lysosomal transporters, 6-phosphate mannose/IGF-II receptor (M6P/IGFIIR) and asialoglycoprotein receptor (ASGPR) have been shown to effectively induce secretory or membrane proteins to lysosomes for degradation. In addition to M6P/IGFIIR and ASGPR, receptor-ligand mediated delivery systems involve other cell surface receptors, such as transferrin receptor, folate receptor, and integrin, 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 and play an important role in cell-matrix interactions. Integrin α _v β ₃ has received extensive attention in tumor targeted therapy due to its overexpression in solid tumor blood vessels, proliferating tumor endothelial cells, and various tumor cells. However, although the α _v β ₃ integrin recognition motif Arg-Gly-Asp (RGD) sequence has been widely used to deliver various therapeutic drugs into tumors, the possibility of RGD-integrin-mediated TPD has not been investigated before our work.

参阅说明书图1，本发明建立了一种新颖的整合素促进的靶蛋白溶酶体降解(IFLD)策略，即利用双功能化合物作为分子降解剂来降解细胞外蛋白和细胞膜蛋白。本发明中所指的双功能化合物是通过一个链接臂(Linker，在本发明中包括连接单元和与活性基团A1、活性基团B1反应后的残基)将目标蛋白结合配体与整合素识别配体-含有RGD的多肽序列结合在一起。作为分子降解剂，这种双功能化合物被证实能够高效地以整合素和溶酶体依赖的方式诱导细胞外或细胞膜蛋白的内化和随后的降解。Referring to Figure 1 of the specification, the present invention establishes a novel integrin-promoted target protein lysosomal degradation (IFLD) strategy, that is, using bifunctional compounds as molecular degradation agents to degrade extracellular proteins and cell membrane proteins. The bifunctional compound referred to in the present invention is a linker arm (Linker, in the present invention, includes a connecting unit and the residue after the reaction with the active group A1 and the active group B1) that combines the target protein binding ligand with the integrin recognition ligand-RGD-containing polypeptide sequence. As a molecular degradation agent, this bifunctional compound has been proven to be able to efficiently induce the internalization and subsequent degradation of extracellular or cell membrane proteins in an integrin- and lysosome-dependent manner.

本发明提供的双功能化合物包括目标蛋白结合单元、整合素识别单元和用于连接所述目标蛋白结合单元和整合素识别单元的连接单元。The bifunctional compound provided by the present invention comprises a target protein binding unit, an integrin recognition unit and a connecting unit for connecting the target protein binding unit and the integrin recognition unit.

在一个实施例中，双功能化合物经A分子、B分子和L反应合成，In one embodiment, the bifunctional compound is synthesized by reacting molecule A, molecule B and molecule L.

A分子包括A1单元和与A1单元连接的活性基团A2，A1单元为目标蛋白结合单元，包括与目标蛋白结合的配体；The A molecule includes an A1 unit and an active group A2 connected to the A1 unit, wherein the A1 unit is a target protein binding unit and includes a ligand that binds to the target protein;

B分子包括B1单元和与B1单元连接的活性基团B2，B1单元为整合素识别单元，包括与整合素结合的配体；The B molecule includes a B1 unit and an active group B2 connected to the B1 unit. The B1 unit is an integrin recognition unit and includes a ligand that binds to the integrin.

L分子包括与A2活性基团反应的活性基团L1、与B2活性基团反应的活性基团L2以及连接活性L1和活性基团L2的L3单元，L3单元为与A1单元和B1单元生成共价键的连接单元；The L molecule includes an active group L1 that reacts with the active group A2, an active group L2 that reacts with the active group B2, and an L3 unit that connects the active group L1 and the active group L2, wherein the L3 unit is a connecting unit that forms a covalent bond with the A1 unit and the B1 unit;

双功能化合物的结构通式为A1-L3-B1。The general structural formula of the bifunctional compound is A1-L3-B1.

在一个实施例中，活性基团A2是与活性基团L1发生取代反应、消去反应、加成反应或重排反应的基团或官能团，选自烷烃基团、芳烃基团、杂环芳烃基团、烯基、炔基、卤代基、醇羟基、巯基、醛基、酮基、羧基、氨基、烯醇基团、叠氮基团、马来酰亚胺、四嗪基团及其变体和含有α氢的醇羟基中的一种或多种。In one embodiment, the active group A2 is a group or functional group that undergoes 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 aromatic group, a heterocyclic aromatic group, an alkenyl group, an alkynyl group, a halide group, an alcoholic hydroxyl group, a thiol group, an aldehyde group, a keto group, a carboxyl group, an amino group, an enol group, an azide group, a maleimide, a tetrazine group and its variants and an alcoholic hydroxyl group containing alpha hydrogen.

活性基团B2是与活性基团L2发生取代反应、消去反应、加成反应或重排反应的基团或官能团，选自烷烃基团、芳烃基团、杂环芳烃基团、烯基、炔基、卤代基、醇羟基、巯基、醛基、酮基、羧基、氨基、烯醇基团、叠氮基团、马来酰亚胺、四嗪基团及其变体和含有α氢的醇羟基中的一种或多种。The active group B2 is a group or functional group that undergoes 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 aromatic group, a heterocyclic aromatic group, an alkenyl group, an alkynyl group, a halide group, an alcoholic hydroxyl group, a thiol 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 and its variants and an alcoholic hydroxyl group containing α hydrogen.

活性基团A2、活性基团B2、活性基团L1和活性基团L2的作用是在A1、B1和L3之间形成连接臂，得到双功能化合物A1-L3-B1，通过A1单元结合目标蛋白，B1单元结合整合素受体，在目标蛋白、整合素受体与双功能化合物A1-L3-B1之间形成三元复合物，从而促使与A1单元结合的目标蛋白可从细胞外转移到细胞内进行降解。基于以上原理，活性基团A2和活性基团L1可以是任意两种能够发生反应且使A1单元和L3单元之间形成共价键的基团，同理，活性基团B2和活性基团L2也可以是任意两种能够发生反应且使B1单元和L3单元之间形成共价键的基团。只要在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内；同时，对于本领域的一般技术人员，依据本发明的思想，在具体实施方式及应用范围上均会有改变之处，综上，本说明书的内容不应理解为对本发明的限制。The role of the active group A2, the active group B2, the active group L1 and the active group L2 is to form a connecting arm between A1, B1 and L3 to obtain a bifunctional compound A1-L3-B1, through the A1 unit to bind to the target protein, the B1 unit to bind to the integrin receptor, a ternary complex is formed between the target protein, the integrin receptor and the bifunctional compound A1-L3-B1, thereby promoting the target protein bound to the A1 unit to be transferred from the extracellular to the intracellular for degradation. Based on the above principles, the active group A2 and the active group L1 can be any two groups that can react and form a covalent bond between the A1 unit and the L3 unit. Similarly, the active group B2 and the active group L2 can also be any two groups that can react and form a covalent bond between the B1 unit and the L3 unit. As long as it is within the spirit and principle of the present invention, any modifications, equivalent substitutions, improvements, etc. made should be included in the scope of protection of the present invention; at the same time, for those of ordinary skill in the art, according to the idea of the present invention, there will be changes in the specific implementation and scope of application. In summary, the content of this specification should not be understood as limiting the present invention.

在一个实施例中，目标蛋白选自结构蛋白质；受体；酶细胞表面蛋白质；与细胞整合功能相关的蛋白质，包括涉及催化活性、芳香酶活性、运动活性、解旋酶活性、代谢过程、抗氧化活性、蛋白水解、生物合成的蛋白质；具有激酶活性、氧化还原酶活性、转移酶活性、水解酶活性、裂解酶活性、异构酶活性、连接酶活性、酶调节活性、信号转导活性、结构分子活性、结合活性、受体活性、细胞运动性、膜融合、细胞通信、生物过程调节、发育、细胞分化、刺激反应的蛋白质；行为蛋白质、细胞黏附蛋白质；涉及细胞坏死的蛋白质；和涉及转运的蛋白质中的一种或多种。In one embodiment, the target protein is selected from structural proteins; receptors; enzyme cell surface proteins; proteins related to cell integration functions, including proteins involved in catalytic activity, aromatase activity, motor activity, helicase activity, metabolic processes, antioxidant activity, proteolysis, biosynthesis; proteins with kinase activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, ligase activity, enzyme regulatory activity, signal transduction activity, structural molecule activity, binding activity, receptor activity, cell motility, membrane fusion, cell communication, biological process regulation, development, cell differentiation, stimulus response; behavioral proteins, cell adhesion proteins; proteins involved in cell necrosis; and one or more of proteins involved in transport.

在一个实施例中，所述目标蛋白选自：细胞程序性死亡-配体1(即PD-L1)、程序性死亡受体1(即PD-1)、表皮生长因子受体(即EGFR)、人表皮生长因子受体-2(即HER2)、G蛋白偶联受体(即GPCR)、成纤维细胞生长因子受体(即FGFRs)、血管内皮生长因子受体家族(即VEGFR，VEGF表示血管内皮生长因子)、细胞毒性T淋巴细胞相关蛋白4(即CTLA4或CTLA-4)、人白介素5受体α(IL-5Rα)、载脂蛋白，载脂蛋白E4(即ApoE4)、β-淀粉样蛋白、血管紧张素转化酶2(ACE2)、钠离子-牛磺胆酸共转运蛋白(NTCP)、B7.1和B7、TI FR1m、TNFR2、NADPH氧化酶、Bc1IBax和在细胞凋亡通路中的其他配体、C5a受体、HMG-CoA还原酶、PDEⅤ磷酸二酯酶型、PDEⅣ磷酸二酯酶4型、PDEⅠ、PDEⅡ、PDEⅢ、鲨烯环化酶抑制剂、CXCR1、CXCR2、一氧化氮合成酶、环氧化酶1、环氧化酶2、5HT受体、多巴胺受体、G蛋白、组胺受体、5-脂肪氧化酶、类蛋白酶丝氨酸蛋白酶、胸苷酸合成酶、嘌呤核苷磷酸化酶、甘油醛-3-磷酸脱氢酶(即GAPDH)、糖原磷酸化酶、碳酸酐酶、趋化因子受体、JAW STAT、RXR和类似物、HIV1蛋白酶、HIV1整合酶、流感神经氨酸酶、乙型肝炎逆转录酶、钠通道、蛋白质P-糖蛋白、P糖蛋白和MRP络氨酸激酶、CD23、CD73、CD124、酪氨酸激酶p561ck、CD4、CD5、IL-2受体、IL-1受体、TNF-αR、ICAM1、Ca2+通道、VCAM、VLA-4整合素、选择素、CD40/CD40L、newokinins和受体、肌苷一磷酸脱氢酶、p38MAP激酶、Ras/Raf/MEW/ERK通路、白介素-1转化酶、半胱天冬酶、HCV、NS3蛋白酶、HCV NS3 RNA解旋酶、甘氨酰胺核糖核苷酸甲酰转移酶、鼻病毒、3C蛋白酶、单纯性疱疹病毒-1、蛋白酶、巨细胞病毒蛋白酶、聚(ADP-核糖)聚合酶、细胞周期蛋白依赖性激酶、血管内皮生长因子、催产素受体、微粒体转移蛋白质抑制子、胆汁酸转运抑制子、5α还原酶抑制子、血管紧张素11、甘氨酸受体、去甲肾上腺激素再摄取受体、内皮素受体、神经肽Y和受体、腺苷受体、腺苷激酶和AMP脱氢酶、嘌呤能受体、法尼基转移酶、香叶基转移酶、NCF的TrkA受体、酪氨酸激酶Flk-IIKDR、玻连蛋白受体、整合素受体、Her-21神经鞘、端粒酶抑制、细胞溶质磷酸酯A2和EGF受体酪氨酸激酶、蜕皮激素20-单氧酶、GABA门控的氯离子通道、乙酰胆碱酯酶、电压敏感的钠通道蛋白、钙释放通道和氯离子通道、乙酰辅酶A羧化酶、腺苷酸琥珀酸合成酶、原卟啉原氧化酶和烯醇丙酮酰莽草酸磷酸合成酶中的一种或多种，和/或上述蛋白质所有变体、突变体、剪接变体、***缺失体和融合体中的一种或多种。In one embodiment, the target protein is selected from: programmed cell death-ligand 1 (i.e., PD-L1), programmed death receptor 1 (i.e., PD-1), epidermal growth factor receptor (i.e., EGFR), human epidermal growth factor receptor-2 (i.e., HER2), G protein-coupled receptor (i.e., GPCR), fibroblast growth factor receptor (i.e., FGFRs), vascular endothelial growth factor receptor family (i.e., VEGFR, VEGF represents vascular endothelial growth factor), cytotoxic T lymphocyte-associated protein 4 (i.e., CTLA4 or CTLA-4), human interleukin 5 receptor α (IL-5Rα), apolipoprotein, apolipoprotein E4 (i.e., ApoE4), β-amyloid protein, angiotensin converting enzyme 2 (ACE2), sodium ion-taurocholic acid cotransporter (NTCP), B7.1 and B7, TI FR1m, TNFR2, NADPH oxygen phosphodiesterase, BclIBax and other ligands in the apoptosis pathway, C5a receptor, HMG-CoA reductase, PDEⅤ phosphodiesterase type, PDEⅣ phosphodiesterase type 4, PDEⅠ, PDEⅡ, PDEⅢ, squalene cyclase inhibitor, CXCR1, CXCR2, nitric oxide synthase, cyclooxygenase 1, cyclooxygenase 2, 5HT receptor, dopamine receptor, G protein, histamine receptor, 5-lipoxygenase, protease-like serine protease, thymidylate synthase, purine nucleoside phosphorylase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycogen phosphorylase, carbonic anhydrase, chemokine receptor, JAW STAT, RXR and analogs, HIV1 protease, HIV1 integrase, influenza neuraminidase, hepatitis B reverse transcriptase, sodium channel, protein P-glycoprotein, P-glycoprotein and MRP tyrosine kinase, CD23, C D73, CD124, tyrosine kinase p561ck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-αR, ICAM1, Ca2+ channel, VCAM, VLA-4 integrin, selectin, CD40/CD40L, newokinins and receptors, inosine monophosphate dehydrogenase, p38MAP kinase, Ras/Raf/MEW/ERK pathway, interleukin-1 converting enzyme, caspase, HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotide formyltransferase, rhinovirus, 3C protease, herpes simplex virus-1, protease, cytomegalovirus protease, poly (ADP-ribose) polymerase, cyclin-dependent kinase, vascular endothelial growth factor, oxytocin receptor, microsomal transfer protein inhibitor, bile acid transport inhibitor, 5α-reductase Inhibitor, angiotensin 11, glycine receptor, norepinephrine reuptake receptor, endothelin receptor, neuropeptide Y and receptor, adenosine receptor, adenosine kinase and AMP dehydrogenase, purinergic receptor, farnesyl transferase, geranyl transferase, TrkA receptor of NCF, tyrosine kinase Flk-IIKDR, vitronectin receptor, integrin receptor, Her-21 nerve sheath, telomerase inhibition, cytosolic phosphate A2 and EGF receptor tyrosine kinase, ecdysone 20-monooxygenase, GABA-gated chloride channel, acetylcholinesterase, voltage-sensitive sodium channel protein, calcium release channel and chloride channel, acetyl-CoA carboxylase, adenylate succinate synthetase, protoporphyrinogen oxidase and enolpyruvylshikimate phosphate synthetase, and/or one or more of all variants, mutants, splice variants, insertions, deletions and fusions of the above proteins.

在一个实施例中，A分子为BMS-8、Biotin-NHS或PH-002，整合素识别配体为cRGD。In one embodiment, the A molecule is BMS-8, Biotin-NHS or PH-002, and the integrin recognition ligand is cRGD.

在一个实施例中，A分子为BMS-8时，活性基团A2为羧基，连接整合素识别单元的活性基团B2包括炔基，L分子中的活性基团L1为氨基，氨基和羧基形成酰胺键，活性基团L2为叠氮基团，叠氮基团和炔基形成1,2,3-三氮唑的五元杂环；In one embodiment, when the A molecule is BMS-8, the active group A2 is a carboxyl group, the active group B2 connected to the integrin recognition unit includes an alkynyl group, the active group L1 in the L molecule is an amino group, the amino group and the carboxyl group form an amide bond, the active group L2 is an azide group, and the azide group and the alkynyl group form a five-membered heterocyclic ring of 1,2,3-triazole;

A分子为Biotin-NHS时，活性基团A2为-NHS，连接整合素识别单元的活性基团B2包括炔基，L分子中的活性基团L1为氨基，氨基取代NHS和Biotin基团形成酰胺键，L分子中的活性基团L2为叠氮基团，叠氮基团和炔基形成1,2,3-三氮唑的五元杂环；When the A molecule is Biotin-NHS, the active group A2 is -NHS, the active group B2 connected to the integrin recognition unit includes an alkynyl group, the active group L1 in the L molecule is an amino group, the amino group replaces the NHS and the Biotin group to form an amide bond, and the active group L2 in the L molecule is an azide group, and the azide group and the alkynyl group form a five-membered heterocyclic ring of 1,2,3-triazole;

A分子为PH-002时，A2基团包含叔丁氧羰基保护的氨基，连接整合素识别单元的活性基团B2包括炔基，L分子中的活性基团L1为羧基，羧基与PH-002上脱除叔丁氧羰基后裸露的氨基形成酰胺键，L分子中的活性基团L2为叠氮基团，叠氮基团和炔基形成1,2,3-三氮唑的五元杂环。When the A molecule is PH-002, the A2 group includes an amino group protected by a tert-butyloxycarbonyl group, the active group B2 connected to the integrin recognition unit includes an alkynyl group, the active group L1 in the L molecule is a carboxyl group, and the carboxyl group forms an amide bond with the exposed amino group after the tert-butyloxycarbonyl group is removed from PH-002, and the active group L2 in the L molecule is an azide group, and the azide group and the alkynyl group form a five-membered heterocyclic ring of 1,2,3-triazole.

本发明如上任一项所述的双功能化合物或其药物上可接受的盐的药物组合物，该药物组合物用于治疗癌症、良性增生性失常、感染性或非感染性炎症事件、自身免疫性疾病、炎性疾病、全身性炎症反应综合征、病毒性感染和病毒性疾病以及眼疾。The present invention provides a pharmaceutical composition of a bifunctional compound or a pharmaceutically acceptable salt thereof as described in any one of the above items, which is used to treat 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.

本发明如上任一项所述的双功能化合物或药物组合物在有需要的患者中调节目标蛋白的蛋白质活性的应用。The invention provides a use of the bifunctional compound or pharmaceutical composition as described above for regulating the protein activity of a target protein in a patient in need thereof.

本发明如上任一项所述的双功能化合物或药物组合物在靶蛋白溶酶体降解中的应用。The invention relates to use of the bifunctional compound or pharmaceutical composition as described in any one of the above items in lysosomal degradation of target protein.

实施例1 BMS-L3 ¹-RGD的合成 Example 1 Synthesis of BMS-L3 ¹ -RGD

BMS-L3 ¹-RGD的合成路线如下图所示： The synthetic route of BMS-L3 ¹ -RGD is shown in the figure below:

步骤1.化合物BMS-L3 ¹-Azide的合成 Step 1. Synthesis of compound BMS-L3 ¹ -Azide

将BMS-8小分子(10.0mg，20.2μmol)﹑HATU(11.5mg，30.3μmol)以及DIEA(10.0μL,60.6μmol)依次加入反应瓶，用1.0mL的无水N,N-二甲基甲酰胺(DMF)搅拌溶解，再加入3-叠氮基丙胺(3.0μL,30.3μmol)。反应混合物室温搅拌3小时后，采用HPLC分离纯化，冷冻干燥后得到白色粉末BMS-L3 ¹-Azide(11.0mg,95％)。HRMS(ESI)m/z:calcd.for C ₃₀H ₃₅BrN ₅O ₂[M+H] ⁺576.1969,found 576.1973.(图3) BMS-8 small molecule (10.0 mg, 20.2 μmol), HATU (11.5 mg, 30.3 μmol) and DIEA (10.0 μL, 60.6 μmol) were added to the reaction bottle in sequence, stirred and dissolved with 1.0 mL of anhydrous N, N-dimethylformamide (DMF), and then 3-azidopropylamine (3.0 μL, 30.3 μmol) was added. After the reaction mixture was stirred at room temperature for 3 hours, it was separated and purified by HPLC, and freeze-dried to obtain white powder BMS-L3 ¹ -Azide (11.0 mg, 95%). HRMS (ESI) m/z: calcd. for C ₃₀ H ₃₅ BrN ₅ O ₂ [M+H] ⁺ 576.1969, found 576.1973. (Figure 3)

步骤2.化合物BMS-L3 ¹-RGD的合成 Step 2. Synthesis of compound BMS-L3 ¹ -RGD

向反应瓶中依次加入Alkyne-cRGD(9.4mg,13.1μmol)﹑BMS-L3 ¹-Azide(5.0mg,8.7μmol)﹑CuSO ₄·5H ₂O(1.3mg,5.2μmol)和NaVc(6.9mg,34.7μmol)，用DMF/H ₂O(1.5mL,2:1)混合溶液溶解。反应混合物在室温条件下搅拌6小时后，采用HPLC分离纯化，冷冻干燥后得到白色粉末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).(图4)。HRMS(ESI)m/z:calcd.for C ₆₃H ₈₂O ₁₁N ₁₄Br[M+H] ⁺1289.5465,found 1289.5471.(图5) Alkyne-cRGD (9.4 mg, 13.1 μmol), BMS-L3 ¹ -Azide (5.0 mg, 8.7 μmol), CuSO ₄ ·5H ₂ O (1.3 mg, 5.2 μmol) and NaVc (6.9 mg, 34.7 μmol) were added to the reaction flask in sequence and dissolved in a DMF/H ₂ O (1.5 mL, 2:1) mixed solution. The reaction mixture was stirred at room temperature for 6 hours, separated and purified by HPLC, and freeze-dried to obtain white powder ^BMS -L1-RGD (9.5 mg, 85%). NMR (400 MHz, MeOD) δ: 8.78–8.68 (m, 1H), 8.14 (s, 1H), 7.79 (s, 1H), 7.67 (dd, J = 12.6, 7.3 Hz, 1H), 7.51 (d, J = 8.2 Hz, 2H), 7.45 (t, J = 7.3 Hz, 2H), 7.41–7.34 (m, 2H), 7.34–7.28 (m, 3H), 7.28–7.21 (m, 1H), 7.02 (d, J = 8.4 Hz, 2H), 6.71 (d, J = 8.3 Hz, 2H), 5.30 (s, 1H), 4.82–4.74 (m, 1H), 4.58 (t, J = 6.8 Hz, 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).(Figure 4). HRMS (ESI) m/z: calcd. for C ₆₃ H ₈₂ O ₁₁ N ₁₄ Br [M+H] ⁺ 1289.5465, found 1289.5471. (Figure 5)

步骤1中的3-叠氮基丙胺替换为N-(2-(2-aminoethoxy)ethyl)-6-azidohexanamide或N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-6-azidohexanamide可制备结构如图2所示的BMS-L3 ²-RGD、BMS-L3 ³-RGD。(图2)BMS-L3 ²-RGD，HRMS(ESI)m/z:calcd.for C ₇₀H ₉₅O ₁₃N ₁₅Br[M+H] ⁺1432.6412,found 1432.6403；BMS-L3 ³-RGD，HRMS(ESI)m/z:calcd.for C ₇₄H ₁₀₃BrN ₁₅O ₁₅[M+H] ⁺1520.6963,found 1520.6948 Replacing 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 prepare BMS-L3 ² -RGD and BMS-L3 ³ -RGD with the structures shown in Figure 2. (Figure 2) BMS-L3 ² -RGD, HRMS (ESI) m/z: calcd. for C ₇₀ H ₉₅ O ₁₃ N ₁₅ Br [M+H] ⁺ 1432.6412, found 1432.6403; BMS-L3 ³ -RGD, HRMS (ESI) m/z: calcd. for C ₇₄ H ₁₀₃ BrN ₁₅ O ₁₅ [M+H] ⁺ 1520.6963, found 1520.6948

实施例2 为实施例1制备的BMS-L3 ¹-Azide化合物的应用例，筛选、评价BMS-L3 ¹-RGD化合物在体外降解膜蛋白PD-L1的性能 Example 2 is an application example of the BMS-L3 ¹ -Azide compound prepared in Example 1, screening and evaluating the performance of the BMS-L3 ¹ -RGD compound in degrading the membrane protein PD-L1 in vitro

免疫荧光法：将盖玻片上的细胞在PBS中洗涤2次，用4％PFA固定15分钟，用PBS(10mM，pH 7.4)漂洗3次，每次5分钟。如果有必要观察细胞内PD-L1的变化，则需要额外的0.10％Triton X-100在室温下与细胞共孵育15分钟。然后，用3％BSA封闭细胞30分钟，然后在室温下分别与指定的一抗和二抗孵育2小时和1小时。在此步骤之后，在黑暗中用DAPI C1005对细胞核染色15分钟。然后用PBS(10mM，pH 7.4)洗涤细胞三次，每次5分钟。荧光图像由Leica STELLARIS 5共焦荧光显微镜成像。Immunofluorescence: The cells on the coverslips were washed twice in PBS, fixed with 4% PFA for 15 minutes, and rinsed three times with PBS (10mM, pH 7.4) for 5 minutes each time. If it is necessary to observe the changes of intracellular PD-L1, additional 0.10% Triton X-100 is required to incubate with the cells for 15 minutes at room temperature. Then, the cells were blocked with 3% BSA for 30 minutes and then incubated with the designated primary and secondary antibodies for 2 hours and 1 hour at room temperature, respectively. After this step, the nuclei were stained with DAPI C1005 for 15 minutes in the dark. The cells were then washed three times with PBS (10mM, pH 7.4) for 5 minutes each time. Fluorescence images were imaged by a Leica STELLARIS 5 confocal fluorescence microscope.

膜蛋白PD-L1降解试验的免疫荧光分析。稳定表达PD-L1的Hela细胞在24孔室的盖玻片中孵育至约40％至50％的融合度。用培养基将cRGD、BMS-8、BMS-8+cRGD和BMS-L3 ¹-RGD分别稀释至25nM，加入24孔板，在37℃下培养8h。以免疫荧光法对细胞进行处理，采用激光共聚焦成像。 Immunofluorescence analysis of membrane protein PD-L1 degradation assay. Hela cells stably expressing PD-L1 were incubated in coverslips in 24-well chambers to a confluency of approximately 40% to 50%. cRGD, BMS-8, BMS-8+cRGD, and BMS-L3 ¹ -RGD were diluted to 25 nM in culture medium, added to 24-well plates, and incubated at 37°C for 8 h. Cells were processed by immunofluorescence and imaged using confocal laser scanning.

通过与cRGD阻断α _vβ ₃整合素，竞争性抑制BMS-L3 ¹-RGD介导的PD-L1降解。稳定表达PD-L1的Hela细胞在24孔室的盖玻片中孵育至约40％至50％的融合度。24孔板中加入高浓度(5μM)的cRGD，4℃培养1h，孔中加入BMS-L3 ¹-RGD，37℃培养8h。其余步骤按照上述免疫荧光法进行。 Competitive inhibition of BMS-L3 ¹ -RGD-mediated PD-L1 degradation by blocking α _v β ₃ integrin with cRGD. Hela cells stably expressing PD-L1 were incubated in coverslips in 24-well chambers to a confluence of about 40% to 50%. High concentration (5 μM) of cRGD was added to the 24-well plate and cultured at 4°C for 1 h, and BMS-L3 ¹ -RGD was added to the wells and cultured at 37°C for 8 h. The remaining steps were performed according to the above-mentioned immunofluorescence method.

免疫印迹实验分析PD-L1水平。MDA-MB-231细胞在6孔细胞板中培养，密度为70-80％。为确定BMS-L3 ¹-RGD的最佳浓度，用DMEM稀释至5、25、50、100nM，并与细胞共孵育8h。对于不同的时间梯度，将BMS-L3 ¹-RGD稀释至25nM，并在不同的时间(0、4、8、12、24小时)加入到细胞中。为了筛选不同连接子的BMS-L3 ¹-RGD、BMS-L3 ²-RGD、BMS-L3 ³-RGD，我们用DMEM将其稀释至25nM和50nM，并与细胞共孵育8h。 Immunoblotting assay to analyze 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, it was diluted to 5, 25, 50, 100 nM with DMEM and incubated with cells for 8 h. For different time gradients, BMS-L3 ¹ -RGD was diluted to 25 nM and added to the cells at different times (0, 4, 8, 12, 24 hours). In order to screen BMS-L3 ¹ -RGD, BMS-L3 ² -RGD, and BMS-L3 ³ -RGD of different linkers, we diluted them to 25 nM and 50 nM with DMEM and incubated them with cells for 8 h.

在验证降解途径时，在一个孔中，先用bafilomycin A1(100nM)预处理细胞2h，然后加入BMS-L3 ¹-RGD，并在37℃下再培养细胞8h。在另一个孔中，细胞中加入MG132(5μM)和BMS-L3 ¹-RGD，细胞在37℃下培养8h。培养结束后，用冷却的PBS洗涤细胞2-3次，用细胞刮刀刮取。450g离心5min后，将120μL CSK缓冲液(20mM HEPES-NaOH,pH＝7.5,40mM氯化钠，300mM蔗糖，1mM蛋白酶抑制剂)加入细胞沉淀中，混合物冰敷20min。用胰岛素注射器重复吹气45次，15000rpm离心40min后，上清液为细胞质成分，沉淀部分为细胞膜成分。将这两种成分加入含有蛋白酶抑制剂SDS(1mM)的120μL SDS裂解缓冲液，冰敷30min，然后加入上样缓冲液(5×)，煮沸20min。最后，对样品进行免疫印迹分析。检测结果显示在图8下方的中间，其中加入溶酶体抑制剂bafilomycin A1能够减弱BMS-L3 ¹-RGD对PD-L1蛋白的降解效果，而加入蛋白酶体抑制剂MG132并不影响BMS-L3 ¹-RGD对PD-L1蛋白的降解，这一实验表明BMS-L3 ¹-RGD是通过溶酶体途径而非蛋白酶体途径来降解PD-L1蛋白的。 When verifying the degradation pathway, in one well, cells were pretreated with bafilomycin A1 (100 nM) for 2 h, then BMS-L3 ¹ -RGD was added, and the cells were cultured at 37 °C for another 8 h. In another well, MG132 (5 μM) and BMS-L3 ¹ -RGD were added to the cells, and the cells were cultured at 37 °C for 8 h. After the culture, the cells were washed 2-3 times with cooled PBS and scraped with a cell scraper. After centrifugation at 450 g for 5 min, 120 μL CSK buffer (20 mM HEPES-NaOH, pH = 7.5, 40 mM sodium chloride, 300 mM sucrose, 1 mM protease inhibitor) was added to the cell pellet, and the mixture was iced for 20 min. After repeated blowing with an insulin syringe for 45 times and centrifugation at 15000 rpm for 40 min, the supernatant was the cytoplasmic component and the precipitate was the cell membrane component. The two components were added to 120 μL SDS lysis buffer containing the protease inhibitor SDS (1 mM), placed on ice for 30 min, and then the loading buffer (5×) was added and boiled for 20 min. Finally, the samples were analyzed by immunoblotting. The test results are shown in the middle of the lower part of Figure 8, where the addition of the lysosomal inhibitor bafilomycin A1 can weaken the degradation effect of BMS-L3 ¹ -RGD on PD-L1 protein, while the addition of the proteasome inhibitor MG132 does not affect the degradation of PD-L1 protein by BMS-L3 ¹ -RGD. This experiment shows that BMS-L3 ¹ -RGD degrades PD-L1 protein through the lysosomal pathway rather than the proteasome pathway.

在竞争实验中，将cRGD(5μM)与细胞在4℃下共孵育1小时，然后加入BMS-L3 ¹-RGD，细胞在37℃下再培养8小时，然后细胞经两次冷PBS洗涤，加入后150-200μL SDS裂解缓冲液(含有蛋白酶抑制剂1μM)用14000rpm转速离心5min，蛋白质样品煮20min，然后煮SDS-PAGE样品加载缓冲液(5x)10min。蛋白质样品用10％十二烷基硫酸钠聚丙烯酰胺凝胶(SDS-PAGE)进行电泳，转移到0.45μm聚偏氟乙烯(PVDF)膜(微孔)。然后，用5％脱脂奶粉在PBST缓冲液(PBS+0.1％吐温-20)中，在室温下轻微摇晃，封闭膜2小时。膜分别与一抗(PD-L1抗体、细胞信号技术(CST)、兔源，1：1000；GAPDH抗体、蛋白、鼠源，1：10000)在4℃下轻微摇晃孵育过夜。然后，用PBST缓冲液洗涤膜3次(每次5min)。将膜与辣根过氧化物酶(HRP)偶联的抗兔IgG抗体(1：5000稀释)和抗小鼠IgG抗体(1：5000稀释)在室温下孵育1小时。最后，用PBST缓冲液洗涤膜3次(每次5min)，采用电化学发光(ECL)蛋白印迹底物检测蛋白印迹条带。检测结果显示在图8中下图的右侧。在采用cRGD与细胞进行预孵育的情况下，细胞表面的Integrin与其配体cRGD的结合，竞争性抑制了BMS-L3 ¹-RGD与Integrin的结合，从而导致BMS-L3 ¹-RGD对PD-L1蛋白的降解效果减弱。这一实验表明BMS-L3 ¹-RGD通过与细胞表面的Integrin结合来促进PD-L1蛋白的降解。 In the competition experiment, cRGD (5 μM) was incubated with cells at 4°C for 1 hour, then BMS-L3 ¹ -RGD was added, and the cells were cultured at 37°C for another 8 hours. The cells were then washed twice with cold PBS, and 150-200 μL of SDS lysis buffer (containing 1 μM protease inhibitor) was added and centrifuged at 14000 rpm for 5 minutes. The protein samples were boiled for 20 minutes, and then boiled in SDS-PAGE sample loading buffer (5x) for 10 minutes. The protein samples were electrophoresed with 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to a 0.45 μm polyvinylidene fluoride (PVDF) membrane (Millipore). Then, the membrane was blocked with 5% skim milk powder in PBST buffer (PBS + 0.1% Tween-20) at room temperature for 2 hours with gentle shaking. The membranes were incubated with primary antibodies (PD-L1 antibody, cell signaling technology (CST), rabbit source, 1:1000; GAPDH antibody, protein, mouse source, 1:10000) at 4°C with gentle shaking overnight. Then, the membranes were washed three times with PBST buffer (5 min each time). The membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (1:5000 dilution) and anti-mouse IgG antibody (1:5000 dilution) at room temperature for 1 hour. Finally, the membranes were washed three times with PBST buffer (5 min each time), and the protein blot bands were detected using electrochemiluminescence (ECL) protein blot substrate. The detection results are shown on the right side of the lower figure in Figure 8. When cRGD was used to pre-incubate cells, the binding of Integrin on the cell surface to its ligand cRGD competitively inhibited the binding of BMS-L3 ¹ -RGD to Integrin, resulting in a weakened degradation effect of BMS-L3 ¹ -RGD on PD-L1 protein. This experiment showed that BMS-L3 ¹ -RGD promoted the degradation of PD-L1 protein by binding to Integrin on the cell surface.

实施例3 评价BMS-L3 ¹-RGD在体内降解PD-L1水平的能力与抗肿瘤活性 Example 3 Evaluation of the ability of BMS-L3 ¹ -RGD to degrade PD-L1 levels in vivo and its anti-tumor activity

5-6周龄的无菌雌性C57BL/6J小鼠用于建立肿瘤异种移植模型。将B16F10细胞(2×10 ⁵)悬浮于100μL PBS中，并注射于5-6周龄无菌雌性C57BL/6J小鼠右侧皮下。在肿瘤大小达到30mm ³-50mm ³(L×W×1/2W)后，将动物随机分为三组，每组5只。BMS-8(2mg/kg)、BMS-L3 ¹-RGD(5mg/kg)和10％DMSO/PBS(100μL)分别应用于一组动物，每两天通过尾静脉静脉注射一次，共5次。注射10％DMSO/PBS的动物为对照组。每次注射前测量肿瘤大小和小鼠体重。注射B16F10细胞后18天处死小鼠，采集肿瘤标本，称重并进一步分析，包括进行切片荧光染色。数据代表平均±SEM(n＝5)，采用*P<0.05、**P<0.01、***P<0.001两种方法评估其统计学意义。所有的动物实验均按照相关的指导方针和规定进行，并得到了SIAT的机构动物护理和使用委员会的批准。测试结果显示在图9中，A图表示本实验的操作流程，B图注射B16F10细胞18内动物体重的变化，C图表示注射B16F10细胞18内肿瘤组织体积的变化，D图表示表示注射B16F10细胞18内肿瘤组织大小的变化，E图表示表示注射B16F10细胞18内肿瘤组织重量平均值。F图中上面的肿瘤切片免疫组化成像结果显示，与对照组和BMS-8药物组相比，BMS-L3 ¹-RGD药物组中PD-L1的水平显著降低；F图中下面的肿瘤切片免疫荧光成像结果显示与对照组和BMS-8药物组相比，BMS-L3 ¹-RGD药物组中细胞凋亡水平显著上升。这一实验表明，BMS-L3 ¹-RGD能够有效降解小鼠体内的PD-L1水平，并导致肿瘤细胞凋亡，从而产生显著的抗肿瘤效果。 5-6 week old germ-free female C57BL/6J mice were used to establish tumor xenograft models. B16F10 cells (2×10 ⁵ ) were suspended in 100 μL PBS and injected subcutaneously into the right side of 5-6 week old germ-free female C57BL/6J mice. After the tumor size reached 30 mm ³ -50 mm ³ (L×W×1/2W), the animals were randomly divided into three groups, 5 in each group. BMS-8 (2 mg/kg), BMS-L3 ¹ -RGD (5 mg/kg) and 10% DMSO/PBS (100 μL) were applied to one group of animals, respectively, and injected intravenously through the tail vein once every two days for a total of 5 times. Animals injected with 10% DMSO/PBS served as the control group. Tumor size and mouse weight were measured before each injection. Mice were killed 18 days after injection of B16F10 cells, and tumor specimens were collected, weighed and further analyzed, including sectioning for fluorescent staining. Data represent mean ± SEM (n = 5), and statistical significance was evaluated by *P < 0.05, **P < 0.01, and ***P < 0.001. All animal experiments were performed in accordance with relevant guidelines and regulations and were approved by the Institutional Animal Care and Use Committee of SIAT. The test results are shown in Figure 9, where Figure A shows the operation flow of this experiment, Figure B shows the change in animal body weight within 18 days after injection of B16F10 cells, Figure C shows the change in tumor tissue volume within 18 days after injection of B16F10 cells, Figure D shows the change in tumor tissue size within 18 days after injection of B16F10 cells, and Figure E shows the average weight of tumor tissue within 18 days after injection of B16F10 cells. The results of immunohistochemistry imaging of the upper tumor slices in Figure F show that the level of PD-L1 in the BMS-L3 ¹ -RGD drug group was significantly reduced compared with the control group and the BMS-8 drug group; the results of immunofluorescence imaging of the lower tumor slices in Figure F show that the level of cell apoptosis in the BMS-L3 ¹ -RGD drug group was significantly increased compared with the control group and the BMS-8 drug group. This experiment showed that BMS-L3 ¹ -RGD can effectively degrade the PD-L1 level in mice and cause tumor cell apoptosis, thereby producing a significant anti-tumor effect.

实施例4 为实施例1制备的BMS-L3 ¹-RGD的应用例，验证整合素促进溶酶体降解策略(IFLD策略)降解胞外蛋白PD-L1 Example 4 is an application example of BMS-L3 ¹ -RGD prepared in Example 1, verifying the integrin-promoted lysosomal degradation strategy (IFLD strategy) for degradation of extracellular protein PD-L1

荧光染色法：将盖玻片上的细胞用PBS洗涤2次，用4％多聚甲醛溶液固定15分钟，并用PBS(10mM，pH 7.4)漂洗3次，每次5分钟。在此步骤之后，在黑暗中用DAPI C1005 对细胞核染色15分钟。然后用PBS(10mM，pH 7.4)洗涤细胞三次，每次5分钟。荧光图像由Leica STELLARIS 5共焦荧光显微镜成像。Fluorescence staining: The cells on the coverslips were washed twice with PBS, fixed with 4% paraformaldehyde solution for 15 minutes, and rinsed three times with PBS (10mM, pH 7.4) for 5 minutes each. After this step, the nuclei were stained with DAPI C1005 for 15 minutes in the dark. The cells were then washed three times with PBS (10mM, pH 7.4) for 5 minutes each. Fluorescence images were taken by a Leica STELLARIS 5 confocal fluorescence microscope.

细胞外蛋白摄取试验。Huh7细胞在24孔的盖玻片中孵育，达到约40％至50％的融合。将BMS-L3 ¹-RGD在培养基中稀释至400nM，Alexa Fluor 488标记的蛋白PD-L1(PD-L1-AF488)在培养基中稀释至400nM，混合后孵育30分钟，得到RGD标记的PD-L1-AF488。将所得RGD标记的PD-L1-AF488蛋白溶液加入到24孔板中，在37℃下与融合细胞共培养20h。为了验证溶酶体抑制剂(氯喹，CQ)对蛋白质摄取的影响时，将融合细胞与上述RGD标记的PD-L1-AF488蛋白溶液和CQ溶液(培养基中50μM)共孵育12小时。作为对照，采用上述同样的程序将BMS-Azide与APOE4-AF488预孵育，得到叠氮化物标记的PD-L1-AF488蛋白溶液。将所得叠氮化物标记的PD-L1-AF488蛋白溶液加入到24孔板中，在37℃下与融合细胞共培养20h。对细胞进行荧光染色，激光共聚焦显微镜成像。 Extracellular protein uptake assay. Huh7 cells were incubated in 24-well coverslips to achieve about 40% to 50% confluence. BMS-L3 ¹ -RGD was diluted to 400nM in culture medium, and Alexa Fluor 488-labeled protein PD-L1 (PD-L1-AF488) was diluted to 400nM in culture medium, mixed and incubated for 30 minutes to obtain 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 fused cells at 37°C for 20h. In order to verify the effect of lysosomal inhibitors (chloroquine, CQ) on protein uptake, the fused cells were incubated with the above-mentioned RGD-labeled PD-L1-AF488 protein solution and CQ solution (50μM in culture medium) for 12 hours. As a control, BMS-Azide was pre-incubated with APOE4-AF488 using the same procedure as above to obtain an azide-labeled PD-L1-AF488 protein solution. The obtained azide-labeled PD-L1-AF488 protein solution was added to a 24-well plate and co-cultured with fusion cells at 37°C for 20 hours. The cells were fluorescently stained and imaged using a laser confocal microscope.

实施例5 Biotin-L3 ¹-RGD的合成 Example 5 Synthesis of Biotin-L3 ¹ -RGD

Biotin-L3 ¹-RGD的合成路线如下图所示： The synthesis route of Biotin-L3 ¹ -RGD is shown in the figure below:

步骤1.化合物Biotin-L3 ¹-Azide的合成 Step 1. Synthesis of Biotin-L3 ¹ -Azide

在反应瓶中先加入3-叠氮基丙胺(12.9μL,132μmol)，并用1.0mL无水DMF溶解，随后加入三乙胺(24.5μL,176μmol)，搅拌均匀后滴加Biotin-NHS(30.0mg,88μmol)的DMF溶液(1.0mL)。反应混合物室温搅拌6小时后，采用HPLC分样纯化，冷冻干燥后得到白色粉末Biotin-L3 ¹-Azide(26.1mg,91％)。HRMS(ESI)m/z:calcd.for C ₁₃H ₂₃O ₂N ₆S[M+H] ⁺327.1598,found 327.1597；calcd.for C ₁₃H ₂₂O ₂N ₆NaS[M+Na] ⁺349.1417,found 349.1416.(图6) First, 3-azidopropylamine (12.9 μL, 132 μmol) was added to the reaction flask and dissolved in 1.0 mL of anhydrous DMF. Then, triethylamine (24.5 μL, 176 μmol) was added. After stirring evenly, a DMF solution (1.0 mL) of Biotin-NHS (30.0 mg, 88 μmol) was added dropwise. After the reaction mixture was stirred at room temperature for 6 hours, it was purified by HPLC and freeze-dried to obtain a white powder Biotin-L3 ¹ -Azide (26.1 mg, 91%). HRMS (ESI) m/z: calcd. for C ₁₃ H ₂₃ O ₂ N ₆ S [M + H] ⁺ 327.1598, found 327.1597; calcd. for C ₁₃ H ₂₂ O ₂ N ₆ NaS [M + Na] ⁺ 349.1417, found 349.1416. (Figure 6)

步骤2.化合物Biotin-L3 ¹-RGD的合成 Step 2. Synthesis of the compound Biotin-L3 ¹ -RGD

往反应瓶依次加入Alkyne-cRGD(8.9mg,12.5μmol)﹑Biotin-L3 ¹-Azide(5.3mg,16.2μmol)﹑CuSO ₄·5H ₂O(1.9mg,7.5μmol)和NaVc(9.9mg,50.0μmol)，用DMF/H ₂O(1.8mL,1：3)混合溶液溶解。反应混合物室温搅拌6小时后，采用HPLC分离纯化，冷冻干燥后得到白色粉末Biotin-L3 ¹-RGD(10.8mg,83％)。 ¹H NMR(400MHz,D ₂O)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).(图7)HRMS(ESI)m/z:calcd.for C ₄₆H ₇₀N ₁₅O ₁₁S[M+H] ⁺1040.5095,found 1040.5090.(图8) Alkyne-cRGD (8.9 mg, 12.5 μmol), Biotin-L3 ¹ -Azide (5.3 mg, 16.2 μmol), CuSO ₄ ·5H ₂ O (1.9 mg, 7.5 μmol) and NaVc (9.9 mg, 50.0 μmol) were added to the reaction flask in sequence and dissolved in a DMF/H ₂ O (1.8 mL, 1:3) mixed solution. The reaction mixture was stirred at room temperature for 6 hours, separated and purified by HPLC, and freeze-dried to obtain a white powder Biotin-L3 ¹ -RGD (10.8 mg, 83%). ¹ H NMR (400 MHz, D ₂ O) δ: 8.16 (s, 1H), 6.95 (d, J = 8.4 Hz, 2H), 6.66 (d, J = 8.3 Hz, 2H), 4.63 (d, J = 7.0 Hz, 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, 4.0 Hz, 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).(Figure 7) HRMS(ESI)m/z:calcd.for C ₄₆ H ₇₀ N ₁₅ O ₁₁ S[M+H] ⁺ 1040.5095,found 1040.5090.(Figure 8)

实施例6 为实施例5制备的Biotin-L3 ¹-RGD的应用例，验证整合素促进溶酶体降解策略(IFLD策略)降解胞外蛋白。 Example 6 is an application example of Biotin-L3 ¹ -RGD prepared in Example 5, and verifies the integrin-promoted lysosomal degradation strategy (IFLD strategy) for degrading extracellular proteins.

荧光染色法：将盖玻片上的细胞用PBS洗涤2次，用4％多聚甲醛溶液固定15分钟，并用PBS(10mM，pH 7.4)漂洗3次，每次5分钟。在此步骤之后，在黑暗中用DAPI C1005对细胞核染色15分钟。然后用PBS(10mM，pH 7.4)洗涤细胞三次，每次5分钟。荧光图像由Leica STELLARIS 5共焦荧光显微镜成像。Fluorescence staining: The cells on the coverslips were washed twice with PBS, fixed with 4% paraformaldehyde solution for 15 minutes, and rinsed three times with PBS (10mM, pH 7.4) for 5 minutes each time. After this step, the nuclei were stained with DAPI C1005 for 15 minutes in the dark. The cells were then washed three times with PBS (10mM, pH 7.4) for 5 minutes each time. Fluorescence images were taken by a Leica STELLARIS 5 confocal fluorescence microscope.

细胞外蛋白摄取试验。A549细胞和Huh7细胞在24孔的盖玻片中孵育，达到约40％至50％的融合。将Biotin-L3 ¹-RGD在培养基中稀释至5μM，NAP-650蛋白在培养基中稀释至400nM，混合后孵育30分钟形成RGD标记的NAP-650溶液。将所得RGD标记的NAP-650溶液加入到24孔板中，在37℃下与融合细胞共培养20h。采用上述同样的程序，采用Biotin-L-Azide和NAP-650形成叠氮化物标记的NAP-650溶液。将所得叠氮化物标记的NAP-650溶液加入到24孔板中，在37℃下与融合细胞共培养20h。当验证溶酶体抑制剂(氯喹，CQ)对蛋白质摄取的影响时，将融合细胞与RGD标记的NAP-650溶液和CQ溶液(培养基中50μM)共孵育12小时。对细胞进行荧光染色，激光共聚焦显微镜成像。 Extracellular protein uptake assay. A549 cells and Huh7 cells were incubated in 24-well coverslips to achieve approximately 40% to 50% confluence. Biotin-L3 ¹ -RGD was diluted to 5 μM in culture medium, and NAP-650 protein was diluted to 400 nM in culture medium, 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 confluent cells at 37°C for 20 h. Using the same procedure as above, Biotin-L-Azide and NAP-650 were used to form an azide-labeled NAP-650 solution. The resulting azide-labeled NAP-650 solution was added to a 24-well plate and co-cultured with the confluent cells at 37°C for 20 h. When verifying the effect of lysosomal inhibitor (chloroquine, CQ) on protein uptake, confluent cells were incubated with RGD-labeled NAP-650 solution and CQ solution (50 μM in culture medium) for 12 hours. Cells were fluorescently stained and imaged with laser confocal microscopy.

细胞外蛋白NAP-650与溶酶体标记物的共定位。A549细胞和Huh7细胞在24孔的盖玻片中孵育至约40％至50％的融合。将RGD标记的NAP-650溶液加入24孔板中，在37℃下培养20h。然后用等体积的含Lyso-tracker Greeen(定位于溶酶体的绿色荧光染料)的培养基代替含样品的培养基，并将细胞进一步孵育1h。其余步骤按照上述荧光染色法进行。Colocalization of the extracellular protein NAP-650 with lysosomal markers. A549 cells and Huh7 cells were incubated in 24-well coverslips to approximately 40% to 50% confluence. RGD-labeled NAP-650 solution was added to the 24-well plate and incubated at 37°C for 20 h. The medium containing the sample was then replaced with an equal volume of medium containing Lyso-tracker Greeen (a green fluorescent dye localized to lysosomes), and the cells were further incubated for 1 h. The remaining steps were performed according to the fluorescent staining method described above.

细胞外蛋白与早期核内体标记物(Rab5)的共定位。A549细胞和Huh7细胞在24孔的盖玻片中孵育，达到约60％至70％的融合度。用PEI转染试剂将Rab5-RFP质粒转染A549细胞或Huh7细胞。该细胞培养24h，使Rab5-RFP在早期核内体中表达。将Biotin-L3 ¹-RGD在培养基中分别稀释至5μM，NAP-FITC蛋白在培养基中分别稀释至400nM，混合孵育30min，形成RGD标记的NAP-FITC溶液。将所得NAP-FITC溶液溶液加入到24孔中，在37℃下培养20h。其余步骤按照上述荧光染色法进行。 Colocalization of extracellular proteins with early endosomal markers (Rab5). A549 cells and Huh7 cells were incubated in 24-well coverslips to achieve a confluency of about 60% to 70%. A549 cells or Huh7 cells were transfected with Rab5-RFP plasmid using PEI transfection reagent. The cells were cultured for 24 hours to allow Rab5-RFP to be expressed in early endosomes. Biotin-L3 ¹ -RGD was diluted to 5 μM in the culture medium, and NAP-FITC protein was diluted to 400 nM in the culture medium, mixed and incubated for 30 minutes to form an RGD-labeled NAP-FITC solution. The resulting NAP-FITC solution was added to the 24 wells and cultured at 37°C for 20 hours. The remaining steps were performed according to the above-mentioned fluorescent staining method.

实施例7 PH002-L3 ⁴-RGD的合成 Example 7 Synthesis of PH002-L3 ⁴ -RGD

PH002-L3 ⁴-RGD的合成路线如下图所示： The synthetic route of PH002-L3 ⁴ -RGD is shown in the figure below:

步骤1.化合物PH002-L3 ⁴-Azide的合成 Step 1. Synthesis of compound PH002-L3 ⁴ -Azide

在反应瓶中加入化合物PH-002(5.0mg,10.2μmol,1.0eq)，在冰浴下加入20％TFA/DCM(2.0mL)。反应2小时后，减压蒸除溶剂，残余物用无水DMF混合物(1.0mL)溶解，向上述溶液中加入N ₃-C5-PEG2-COOH(4.61mg,15.2μmol,1.5eq)，HATU(5.03mg,13.2μmol,1.3eq)，DIEA(13.5μl,81.6μmol,8.0eq)，反应混合物室温搅拌3小时后，采用HPLC分样纯化，冷冻干燥后得到PH002-L3 ⁴-Azide(5.4mg,78％)。HRMS(ESI)m/z:calcd.for C ₃₄H ₄₅O ₆N ₉Na[M+H] ⁺698.3385,found 698.3377.(图16) Compound PH-002 (5.0 mg, 10.2 μmol, 1.0 eq) was added to the reaction flask, and 20% TFA/DCM (2.0 mL) was added under ice bath. After reacting for 2 hours, the solvent was evaporated under reduced pressure, and the residue was dissolved with anhydrous DMF mixture (1.0 mL). N ₃ -C5-PEG2-COOH (4.61 mg, 15.2 μmol, 1.5 eq), HATU (5.03 mg, 13.2 μmol, 1.3 eq), DIEA (13.5 μl, 81.6 μmol, 8.0 eq) were added to the above solution. After the reaction mixture was stirred at room temperature for 3 hours, it was purified by HPLC and freeze-dried to obtain PH002-L3 ⁴ -Azide (5.4 mg, 78%). HRMS (ESI) m/z: calcd. for C ₃₄ H ₄₅ O ₆ N ₉ Na [M+H] ⁺ 698.3385, found 698.3377. (Figure 16)

步骤2.化合物PH002-L3 ⁴-RGD的合成 Step 2. Synthesis of compound PH002-L3 ⁴ -RGD

往反应瓶依次加入Alkyne-cRGD(3.0mg,4.2μmol,1.0eq)﹑PH002-Azide(3.4mg,5.0μmol,1.2eq)﹑CuSO ₄·5H ₂O(0.63mg,2.52μmol,0.6eq)和NaVc(3.33mg,16.8μmol,4.0eq)，用DMF/H ₂O(0.7mL,1:1)混合溶液溶解。反应混合物室温搅拌3小时后，采用HPLC分离纯化，冷冻干燥后得到白色粉末PH002-L3 ⁴-RGD(5.3mg,91％)。HRMS(ESI)m/z:calcd.for C ₆₇H ₉₃O ₁₅N ₁₈[M+H] ⁺1389.7062,found 1389.7098.(图17) Alkyne-cRGD (3.0 mg, 4.2 μmol, 1.0 eq), PH002-Azide (3.4 mg, 5.0 μmol, 1.2 eq), CuSO ₄ ·5H ₂ O (0.63 mg, 2.52 μmol, 0.6 eq) and NaVc (3.33 mg, 16.8 μmol, 4.0 eq) were added to the reaction flask in sequence and dissolved in a DMF/H ₂ O (0.7 mL, 1:1) mixed solution. After the reaction mixture was stirred at room temperature for 3 hours, it was separated and purified by HPLC and freeze-dried to obtain white powder PH002-L3 ⁴ -RGD (5.3 mg, 91%). HRMS (ESI) m/z: calcd. for C ₆₇ H ₉₃ O ₁₅ N ₁₈ [M+H] ⁺ 1389.7062, found 1389.7098. (Figure 17)

实施例8 为实施例7制备的PH002-L3 ⁴-RGD的应用例，验证整合素促进溶酶体降解策略(IFLD策略)降解胞外蛋白载脂蛋白E4(ApoE4) Example 8 is an application example of PH002-L3 ⁴ -RGD prepared in Example 7, verifying the integrin-promoted lysosomal degradation strategy (IFLD strategy) for degrading the extracellular protein apolipoprotein E4 (ApoE4)

细胞外蛋白摄取试验。Huh7细胞在24孔的盖玻片中孵育，达到约40％至50％的融合。将PH002-L3 ⁴-RGD在培养基中稀释至400nM，Alexa Fluor 488标记的载脂蛋白E4(APOE4-AF488)在培养基中稀释至400nM，混合后孵育30分钟，得到RGD标记的APOE4-AF488。将所得RGD标记的APOE4-AF488蛋白溶液加入到24孔板中，在37℃下与融合细胞共培养20h。为了验证溶酶体抑制剂(氯喹，CQ)对蛋白质摄取的影响时，将融合细胞与上述RGD标记的APOE4-AF488蛋白溶液和CQ溶液(培养基中50μM)共孵育12小时。作为对照，采用上述同样的程序将Ph002-L3 ⁴-Azide与APOE4-AF488预孵育，得到叠氮化物标记的APOE4-AF488蛋白溶液。将所得叠氮化物标记的APOE4-AF488蛋白溶液加入到24孔板中，在37℃下与融合细胞共培养20h。对细胞进行荧光染色，激光共聚焦显微镜成像。(图18，其中标记为PH002的一组图片为Ph002-L3 ⁴-Azide与APOE4-AF488预孵育得到叠氮化物标记的APOE4-AF488蛋白溶液的荧光检测结果) Extracellular protein uptake assay. Huh7 cells were incubated in 24-well coverslips to achieve approximately 40% to 50% confluence. PH002-L3 ⁴ -RGD was diluted to 400 nM in culture medium, and Alexa Fluor 488-labeled apolipoprotein E4 (APOE4-AF488) was diluted to 400 nM in culture medium, mixed and incubated for 30 minutes to obtain 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°C for 20 hours. In order to verify the effect of lysosomal inhibitors (chloroquine, CQ) on protein uptake, the fused cells were incubated with the above-mentioned RGD-labeled APOE4-AF488 protein solution and CQ solution (50 μM in culture medium) for 12 hours. As a control, Ph002-L3 ⁴ -Azide was pre-incubated with APOE4-AF488 using the same procedure as above to obtain an azide-labeled APOE4-AF488 protein solution. The obtained azide-labeled APOE4-AF488 protein solution was added to a 24-well plate and co-cultured with the fusion cells at 37°C for 20 hours. The cells were fluorescently stained and imaged using a laser confocal microscope. (Figure 18, in which the group of pictures marked as PH002 are the fluorescence detection results of the azide-labeled APOE4-AF488 protein solution obtained by pre-incubating Ph002-L3 ⁴ -Azide with APOE4-AF488)

实验结果：Experimental results:

参阅说明书图8，免疫印记实验表明表明：三种包含不同长度的连接臂的BMS-L3-RGD化合物均能诱导PD-L1的降解，其中的BMS-L3 ¹-RGD在较低浓度(25nM)下对PD-L1的降解率更高。我们研究了不同药物浓度处理后的细胞膜-胞质组分和总组分中的PD-L1水平，发现BMS-L3 ¹-RGD在25nM时表现出更好的降解效果。筛选孵育时间实验显示，8小时足以最大限度地降解PD-L1蛋白。我们发现溶酶体抑制剂Bafilomycin A1抑制了PD-L1的降解，而蛋白酶体抑制剂MG132对蛋白降解没有抑制效果，这证实了其降解依赖于溶酶体途径。我们也使用环肽cRGD对MDA-MB-231细胞进行预处理，以阻断细胞表面的整合素，然后再将细胞与BMS-L1-RGD孵育。在这种条件下，BMS-L3 ¹-RGD并没有诱导PD-L1的降解，这就说明PD-L1的降解是由整合素介导的。 Referring to Figure 8 of the specification, immunoblotting experiments show that three BMS-L3-RGD compounds containing different lengths of connecting arms can induce the degradation of PD-L1, among which BMS-L3 ¹ -RGD has a higher degradation rate of PD-L1 at a lower concentration (25nM). We studied the PD-L1 levels in the cell membrane-cytoplasmic fraction and the total fraction after treatment with different drug concentrations, and found that BMS-L3 ¹ -RGD showed better degradation effect at 25nM. The screening incubation time experiment showed that 8 hours was sufficient to maximize the degradation of PD-L1 protein. We found that the lysosomal inhibitor Bafilomycin A1 inhibited the degradation of PD-L1, while the proteasome inhibitor MG132 had no inhibitory effect on protein degradation, which confirmed that its degradation depends on the lysosomal pathway. We also pretreated MDA-MB-231 cells with cyclic peptide cRGD to block integrins on the cell surface, and then incubated the cells with BMS-L1-RGD. Under this condition, BMS-L3 ¹ -RGD did not induce the degradation of PD-L1, indicating that the degradation of PD-L1 is mediated by integrin.

参阅说明书图9，体内动物实验表明：尾静脉注射5mg/kg的BMS-L3 ¹-RGD(每两天注射一次，共5次)，用药组与对照组老鼠的体重没有明显变化；用药组肿瘤的体积增长缓慢，而对照组肿瘤的体积呈稳态增长趋势；实验结束后，处死老鼠，称重剥离出的肿瘤组织，用药组肿瘤的平均质量明显小于对照组，其中，注射BMS-L3 ¹-RGD的用药组肿瘤的平均直径最小，这说明BMS-L3 ¹-RGD能够有效抑制肿瘤的生长，并且毒副作用较小。 Referring to Figure 9 of the specification, in vivo animal experiments showed that: after tail vein injection of 5 mg/kg BMS-L3 ¹ -RGD (injected once every two days, for a total of 5 times), there was no significant change in the body weight of mice in the treatment group and the control group; the volume of the tumor in the treatment group grew slowly, while the volume of the tumor in the control group showed a steady growth trend; after the experiment, the mice were killed and the excised tumor tissues were weighed. The average mass of the tumors in the treatment group was significantly smaller than that in the control group. Among them, the average diameter of the tumors in the treatment group injected with BMS-L3 ¹ -RGD was the smallest, which shows that BMS-L3 ¹ -RGD can effectively inhibit tumor growth and has fewer toxic side effects.

参阅说明书图10，BMS-L3 ¹-RGD降解胞外蛋白PD-L1：与叠氮化物标记的PD-L1-AF488处理的细胞相比，与RGD标记的PD-L1-AF488共孵育显著增加了细胞摄取，而用CQ抑制溶酶体蛋白水解活性导致了该蛋白的最高积累。这些可以证明将cRGD环肽与细胞外POI的小分子配体结合，可以通过内体-溶酶体途径促进整合素介导的细胞外蛋白—PD-L1(APOE4-AF488)的内化和降解。 See Figure 10 of the specification, BMS-L3 ¹ -RGD degrades extracellular protein PD-L1: Compared with cells treated with azide-labeled PD-L1-AF488, co-incubation with RGD-labeled PD-L1-AF488 significantly increased cellular uptake, while inhibition of lysosomal proteolytic activity with CQ led to the highest accumulation of the protein. These can prove that the combination of cRGD cyclic peptide with small molecule ligands of extracellular POI can promote integrin-mediated internalization and degradation of extracellular protein-PD-L1 (APOE4-AF488) through the endosomal-lysosomal pathway.

参阅说明书图14，整合素促进的胞外蛋白的溶酶体降解：与叠氮化物标记的NAP-650处理的细胞相比，与RGD标记的NAP-650共孵育显著增加了细胞摄取，而用CQ抑制溶酶体蛋白水解活性导致了该蛋白的最高积累。此外，FITC标记的中和病毒蛋白(NAP-FITC)的荧光信号与早期核内体标记(Rab5)共定位，NAP-650与溶酶体标记(LysoTracker)共定位，表明内化的蛋白通过内体运输到溶酶体。这些可以证明将cRGD环肽与细胞外POI的小分子配体选结合，可以通过内体-溶酶体途径促进整合素介导的细胞外蛋白的内化和降解。See Figure 14 of the specification for lysosomal degradation of extracellular proteins promoted by integrins: compared with cells treated with azide-labeled NAP-650, co-incubation with RGD-labeled NAP-650 significantly increased cellular uptake, while inhibition of lysosomal proteolytic activity with CQ resulted in the highest accumulation of the protein. In addition, the fluorescence signal of the FITC-labeled neutralizing virus protein (NAP-FITC) co-localized with the early endosome marker (Rab5), and NAP-650 co-localized with the lysosomal marker (LysoTracker), indicating that the internalized protein was transported to the lysosome through the endosome. These can prove that the selective combination of cRGD cyclic peptide with small molecule ligands of extracellular POI can promote the internalization and degradation of extracellular proteins mediated by integrin through the endosomal-lysosomal pathway.

参阅说明书图17，整合素促进的胞外蛋白—载脂蛋白E4(APOE4-AF488)的溶酶体降解：与叠氮化物标记的APOE4-AF488处理的细胞相比，与RGD标记的APOE4-AF488共孵育显著增加了细胞摄取，而用CQ抑制溶酶体蛋白水解活性导致了该蛋白的最高积累。这些可以证明将cRGD环肽与细胞外POI的小分子配体结合，可以通过内体-溶酶体途径促进整合素介导的细胞外蛋白—载脂蛋白E4(APOE4-AF488)的内化和降解。See Figure 17 of the specification, integrin-promoted lysosomal degradation of extracellular protein-apolipoprotein E4 (APOE4-AF488): compared with cells treated with azide-labeled APOE4-AF488, co-incubation with RGD-labeled APOE4-AF488 significantly increased cellular uptake, while inhibition of lysosomal proteolytic activity with CQ led to the highest accumulation of the protein. These can prove that the combination of cRGD cyclic peptide with small molecule ligands of extracellular POI can promote integrin-mediated internalization and degradation of extracellular protein-apolipoprotein E4 (APOE4-AF488) through the endosomal-lysosomal pathway.

采用上述策略，我们设计合成的BMS-L3-RGD系列化合物在体内与体外实验中均被证明是一个高效的程序死亡配体1(PD-L1)降解剂；PH002-L34-RGD是一个高效的胞外蛋白载脂蛋白E4降解剂。由此可见，我们提出的IFLD策略扩展了调节分泌蛋白和膜相关蛋白水平的工具箱，因此在化学生物学和药物发现领域有巨大的应用潜力。Using the above strategy, we designed and synthesized a series of BMS-L3-RGD compounds, which were proved to be highly efficient programmed death ligand 1 (PD-L1) degraders in both in vivo and in vitro experiments; PH002-L34-RGD is a highly efficient degrader of extracellular protein apolipoprotein E4. It can be seen that the IFLD strategy we proposed has expanded the toolbox for regulating the levels of secretory proteins and membrane-associated proteins, and therefore has great application potential in the fields of chemical biology and drug discovery.

Claims

一种双功能化合物，其特征在于，包括目标蛋白结合单元、整合素识别单元和用于连接所述目标蛋白结合单元和所述整合素识别单元的连接单元。A bifunctional compound, characterized in that it comprises a target protein binding unit, an integrin recognition unit and a connecting unit for connecting the target protein binding unit and the integrin recognition unit.
如权利要求1所述的双功能化合物，其特征在于，The bifunctional compound according to claim 1, characterized in that

所述双功能化合物经A分子、B分子和L反应合成，The bifunctional compound is synthesized by reacting A molecule, B molecule and L.

所述A分子包括A1单元和与所述A1单元连接的活性基团A2，所述A1单元为目标蛋白结合单元，包括与目标蛋白结合的配体；The A molecule includes an A1 unit and an active group A2 connected to the A1 unit, wherein the A1 unit is a target protein binding unit and includes a ligand that binds to the target protein;

所述B分子包括B1单元和与所述B1单元连接的活性基团B2，所述B1单元为整合素识别单元，包括与整合素结合的配体；The B molecule includes a B1 unit and an active group B2 connected to the B1 unit, wherein the B1 unit is an integrin recognition unit and includes a ligand that binds to the integrin;

所述L分子包括与所述A2活性基团反应的活性基团L1、与所述B2活性基团反应的活性基团L2以及所述连接活性L1和所述活性基团L2的L3单元，所述L3单元为与所述A1单元和所述B1单元生成共价键的连接单元；The L molecule includes an active group L1 that reacts with the A2 active group, an active group L2 that reacts with the B2 active group, and an L3 unit that connects the active group L1 and the active group L2, wherein the L3 unit is a connecting unit that forms a covalent bond with the A1 unit and the B1 unit;

所述双功能化合物的结构通式为A1-L3-B1。The general structural formula of the bifunctional compound is A1-L3-B1.
如权利要求2所述的双功能化合物，其特征在于，The bifunctional compound according to claim 2, characterized in that

所述活性基团A2是与所述活性基团L1发生取代反应、消去反应、加成反应或重排反应的基团或官能团，选自烷烃基团、芳烃基团、杂环芳烃基团、烯基、炔基、卤代基、醇羟基、巯基、醛基、酮基、羧基、氨基、烯醇基团、叠氮基团、马来酰亚胺、四嗪基团及其变体和含有α氢的醇羟基中的一种或多种；The active group A2 is a group or functional group that undergoes 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 aromatic group, a heterocyclic aromatic group, an alkenyl group, an alkynyl group, a halide group, an alcoholic hydroxyl group, a thiol 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 and variants thereof, and an alcoholic hydroxyl group containing α hydrogen;

所述活性基团B2是与所述活性基团L2发生取代反应、消去反应、加成反应或重排反应的基团或官能团，选自烷烃基团、芳烃基团、杂环芳烃基团、烯基、炔基、卤代基、醇羟基、巯基、醛基、酮基、羧基、氨基、烯醇基团、叠氮基团、马来酰亚胺、四嗪基团及其变体和含有α氢的醇羟基中的一种或多种；The active group B2 is a group or functional group that undergoes 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 aromatic group, a heterocyclic aromatic group, an alkenyl group, an alkynyl group, a halide group, an alcoholic hydroxyl group, a thiol 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 and variants thereof, and an alcoholic hydroxyl group containing α hydrogen;

L3单元中包括烷基链、芳香环、杂环、杂原子和官能团中的一种或多种。The L3 unit includes one or more of an alkyl chain, an aromatic ring, a heterocycle, a heteroatom and a functional group.
如权利要求1所述的双功能化合物，其特征在于，所述目标蛋白选自结构蛋白质；受体；酶细胞表面蛋白质；与细胞整合功能相关的蛋白质，包括涉及催化活性、芳香酶活性、运动活性、解旋酶活性、代谢过程、抗氧化活性、蛋白水解、生物合成的蛋白质；具有激酶活性、氧化还原酶活性、转移酶活性、水解酶活性、裂解酶活性、异构酶活性、连接酶活性、酶调节活性、信号转导活性、结构分子活性、结合活性、受体活性、细胞运动性、膜融合、细胞通信、生物过程调节、发育、细胞分化、刺激反应的蛋白质；行为蛋白质、细胞黏附蛋白质；涉及细胞坏死的蛋白质；和涉及转运的蛋白质中的一种或多种。The bifunctional compound as claimed in claim 1, characterized in that the target protein is selected from structural proteins; receptors; enzyme cell surface proteins; proteins related to cell integration function, including proteins involved in catalytic activity, aromatase activity, motor activity, helicase activity, metabolic process, antioxidant activity, proteolysis, biosynthesis; proteins with kinase activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, ligase activity, enzyme regulatory activity, signal transduction activity, structural molecule activity, binding activity, receptor activity, cell motility, membrane fusion, cell communication, biological process regulation, development, cell differentiation, stimulus response; behavioral proteins, cell adhesion proteins; proteins involved in cell necrosis; and proteins involved in transport.
如权利要求1所述的双功能化合物，其特征在于，所述目标蛋白选自：细胞程序性死亡-配体1(即PD-L1)、程序性死亡受体1(即PD-1)、表皮生长因子受体(即EGFR)、人表皮生长因子受体-2(即HER2)、G蛋白偶联受体(即GPCR)、成纤维细胞生长因子受体(即FGFRs)、血管内皮生长因子受体家族(即VEGFR，VEGF表示血管内皮生长因子)、细胞毒性T淋巴细胞相关蛋白4(即CTLA4或CTLA-4)、人白介素5受体α(IL-5Rα)、载脂蛋白，载脂蛋白E4(即ApoE4)、β-淀粉样蛋白、血管紧张素转化酶2(ACE2)、钠离子-牛磺胆酸共转运蛋白(NTCP)、B7.1和B7、TI FR1m、TNFR2、NADPH氧化酶、Bc1IBax和在细胞凋亡通路中的其他配体、C5a受体、HMG-CoA还原酶、PDEV磷酸二酯酶型、PDE IV磷酸二酯酶4型、PDE I、PDE II、PDE III、鲨烯环化酶抑制剂、CXCR1、CXCR2、一氧化氮合成酶、环氧化酶1、环氧化酶2、5HT受体、多巴胺受体、G蛋白、组胺受体、5-脂肪氧化酶、类蛋白酶丝氨酸蛋白酶、胸苷酸合成酶、嘌呤核苷磷酸化酶、甘油醛-3-磷酸脱氢酶(即GAPDH)、糖原磷酸化酶、碳酸酐酶、趋化因子受体、JAW STAT、RXR和类似物、HIV1蛋白酶、HIV1整合酶、流感神经氨酸酶、乙型肝炎逆转录酶、钠通道、蛋白质P-糖蛋白、P糖蛋白和MRP络氨酸激酶、CD23、CD73、CD124、酪氨酸激酶p561ck、CD4、CD5、IL-2受体、IL-1受体、TNF-αR、ICAM1、Ca2+通道、VCAM、VLA-4整合素、选择素、CD40/CD40L、newokinins和受体、肌苷一磷酸脱氢酶、p38 MAP激酶、Ras/Raf/MEW/ERK通路、白介素-1转化酶、半胱天冬酶、HCV、NS3蛋白酶、HCV NS3 RNA解旋酶、甘氨酰胺核糖核苷酸甲酰转移酶、鼻病毒、3C蛋白酶、单纯性疱疹病毒-1、蛋白酶、巨细胞病毒蛋白酶、聚(ADP-核糖)聚合酶、细胞周期蛋白依赖性激酶、血管内皮生长因子、催产素受体、微粒体转移蛋白质抑制子、胆汁酸转运抑制子、5α还原酶抑制子、血管紧张素11、甘氨酸受体、去甲肾上腺激素再摄取受体、内皮素受体、神经肽Y和受体、腺苷受体、腺苷激酶和AMP脱氢酶、嘌呤能受体、法尼基转移酶、香叶基转移酶、NCF的TrkA受体、酪氨酸激酶Flk-IIKDR、玻连蛋白受体、整合素受体、Her-21神经鞘、端粒酶抑制、细胞溶质磷酸酯A2和EGF受体酪氨酸激酶、蜕皮激素20-单氧酶、GABA门控的氯离子通道、乙酰胆碱酯酶、电压敏感的钠通道蛋白、钙释放通道和氯离子通道、乙酰辅酶A羧化酶、腺苷酸琥珀酸合成酶、原卟啉原氧化酶和烯醇丙酮酰莽草酸磷酸合成酶中的一种或多种，和/或上述蛋白质所有变体、突变体、剪接变体、***缺失体和融合体中的一种或多种。The bifunctional compound according to claim 1, characterized in that the target protein is selected from: programmed cell death-ligand 1 (i.e., PD-L1), programmed death receptor 1 (i.e., PD-1), epidermal growth factor receptor (i.e., EGFR), human epidermal growth factor receptor-2 (i.e., HER2), G protein-coupled receptor (i.e., GPCR), fibroblast growth factor receptor (i.e., FGFRs), vascular endothelial growth factor receptor family (i.e., VEGFR, VEGF represents vascular endothelial growth factor), cytotoxic T lymphocyte-associated protein 4 (i.e., CTLA4 or CTLA-4), human interleukin 5 receptor α (IL-5Rα), apolipoprotein, apolipoprotein E4 (i.e., ApoE4), β-amyloid protein, angiotensin converting enzyme 2 (ACE2), sodium ion-taurocholate cotransporter (NTCP), B7.1 and B7, TI FR1m, TNFR2, NADPH oxidase, Bc1IBax and other ligands in the apoptosis pathway, C5a receptor, HMG-CoA reductase, PDEV phosphodiesterase type, P DE IV phosphodiesterase type 4, PDE I, PDE II, PDE III, squalene cyclase inhibitors, 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 STAT, RXR and analogs, HIV1 protease, HIV1 integrase, influenza neuraminidase, hepatitis B reverse transcriptase, sodium channel, protein P-glycoprotein, P-glycoprotein and MRP tyrosine kinase, CD23, CD73, CD124, tyrosine kinase p561ck, CD4, CD5, I L-2 receptor, IL-1 receptor, TNF-αR, ICAM1, Ca2+ channel, VCAM, VLA-4 integrin, selectin, CD40/CD40L, newokinins and receptors, inosine monophosphate dehydrogenase, p38 MAP kinase, Ras/Raf/MEW/ERK pathway, interleukin-1 converting enzyme, caspase, HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotide formyltransferase, rhinovirus, 3C protease, herpes simplex virus-1, protease, cytomegalovirus protease, poly (ADP-ribose) polymerase, cyclin-dependent kinase, vascular endothelial growth factor, oxytocin receptor, microsomal transfer protein inhibitor, bile acid transport inhibitor, 5α-reductase inhibitor, angiotensin 11, glycine receptors, norepinephrine reuptake receptors, endothelin receptors, neuropeptide Y and receptors, adenosine receptors, adenosine kinases and AMP dehydrogenases, purinergic receptors, farnesyltransferases, geranyltransferases, TrkA receptors of NCF, tyrosine kinase Flk-IIKDR, vitronectin receptors, integrin receptors, Her-21 nerve sheaths, telomerase inhibition, cytosolic phosphate A2 and EGF receptor tyrosine kinases, ecdysone 20-monooxygenase, GABA-gated chloride channels, acetylcholinesterase, voltage-sensitive sodium channel proteins, calcium release channels and chloride channels, acetyl-CoA carboxylase, adenylate succinate synthetase, protoporphyrinogen oxidase and enolpyruvylshikimate phosphate synthetase, and/or one or more of all variants, mutants, splice variants, indels and fusions of the above proteins.
如权利要求3所述的双功能化合物，其特征在于，所述A分子为BMS-8、Biotin-NHS或PH-002，所述整合素识别配体为cRGD。The bifunctional compound according to claim 3, characterized in that the A molecule is BMS-8, Biotin-NHS or PH-002, and the integrin recognition ligand is cRGD.
如权利要求6所述的双功能化合物，其特征在于，The bifunctional compound according to claim 6, characterized in that

所述A分子为BMS-8时，所述活性基团A2为羧基，连接所述整合素识别单元的所述活性基团B2包括炔基，所述L分子中的所述活性基团L1为氨基，所述氨基和所述羧基形成酰胺键，所述活性基团L2为叠氮基团，所述叠氮基团和所述炔基形成1，2，3-三氮唑的五元杂环；When the A molecule is BMS-8, the active group A2 is a carboxyl group, the active group B2 connected to the integrin recognition unit includes an alkynyl group, the active group L1 in the L molecule is an amino group, the amino group and the carboxyl group form an amide bond, the active group L2 is an azide group, and the azide group and the alkynyl group form a five-membered heterocyclic ring of 1,2,3-triazole;

所述A分子为Biotin-NHS时，所述活性基团A2为-NHS，连接所述整合素识别单元的活性基团B2包括炔基，所述L分子中的所述活性基团L1为氨基，所述氨基取代所述活性基团NHS和Biotin基团形成酰胺键，所述L分子中的所述活性基团L2为叠氮基团，所述叠氮基团和所述炔基形成1，2，3-三氮唑的五元杂环；When the A molecule is Biotin-NHS, the active group A2 is -NHS, the active group B2 connected to the integrin recognition unit includes an alkynyl group, the active group L1 in the L molecule is an amino group, the amino group replaces the active group 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 group form a five-membered heterocyclic ring of 1,2,3-triazole;

所述A分子为PH-002时，所述A2基团包含叔丁氧羰基保护的氨基，连接所述整合素识别单元的所述活性基团B2包括炔基，所述L分子中的所述活性基团L1为羧基，所述羧基与所述PH-002上脱除所述叔丁氧羰基后裸露的氨基形成酰胺键，所述L分子中的所述活性基团L2为叠氮基团，所述叠氮基团和所述炔基形成1，2，3-三氮唑的五元杂环。When the A molecule is PH-002, the A2 group includes an amino group protected by a tert-butyloxycarbonyl group, the active group B2 connected to the integrin recognition unit includes an alkynyl group, the active group L1 in the L molecule is a carboxyl group, and the carboxyl group forms an amide bond with the amino group exposed after the tert-butyloxycarbonyl group is removed from the PH-002, and the active group L2 in the L molecule is an azide group, and the azide group and the alkynyl group form a five-membered heterocyclic ring of 1,2,3-triazole.
如权利要求1-7任一项所述的双功能化合物或其药物上可接受的盐的药物组合物，所述药物组合物用于治疗癌症、良性增生性失常、感染性或非感染性炎症事件、自身免疫性疾病、炎性疾病、全身性炎症反应综合征、病毒性感染和病毒性疾病以及眼疾。A pharmaceutical composition of a bifunctional compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 7, wherein the pharmaceutical composition is used to treat 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.
如权利要求1-7中任一项所述的双功能化合物或权利要求8所述的药物组合物在有需要的患者中调节目标蛋白的蛋白质活性的应用。Use of the bifunctional compound according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 8 for regulating the protein activity of a target protein in a patient in need thereof.
如权利要求1-7中任一项所述的双功能化合物或权利要求8所述的药物组合物在靶蛋白溶酶体降解中的应用。Use of the bifunctional compound according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 8 in lysosomal degradation of a target protein.