技术领域Technical field
本发明属于生物技术和生物医药技术领域，尤其涉及一种FTO蛋白降解靶向嵌合体及其制备方法和应用。The invention belongs to the fields of biotechnology and biomedicine technology, and particularly relates to an FTO protein degradation targeting chimera and its preparation method and application.
背景技术Background technique
FTO是通过全基因组关联分析发现的与肥胖相关，并在脂肪形成中发挥调节作用的关键基因。其编码的蛋白属于AlkB家族，是一种依赖Fe2+和α-酮戊二酸的加氧酶，能够催化RNA去甲基化。近期的研究发现肝脏FTO的异常活性与肥胖、2型糖尿病和非酒精性脂肪肝病等代谢损伤有关。其能通过改变参与脂肪酸氧化、脂肪分解和脂肪生成的基因的甲基化状态以及转录因子的活性来调节肝糖脂代谢。这些调节机制的损坏与肥胖和代谢性疾病(如2型糖尿病和非酒精性脂肪肝)的发病密切相关，而这些损伤恢复可能有益于逆转代谢异常。因此，通过抑制FTO的表达或活性可能是治疗肥胖、糖尿病和非酒精性脂肪肝的治疗方法。然而，目前抑制FTO的小分子由于缺乏特异性，无法应对其他信号通路的代偿，并且作用于DNA或RNA水平的CRISPR-CAS9和siRNA/shRNA技术面临不可逆编辑、存在脱靶效应等弊端。FTO is a key gene found to be associated with obesity through genome-wide association analysis and plays a regulatory role in adipogenesis. The protein it encodes belongs to the AlkB family and is an oxygenase that relies on Fe2+ and α-ketoglutarate and can catalyze RNA demethylation. Recent studies have found that abnormal activity of hepatic FTO is related to metabolic damage such as obesity, type 2 diabetes, and non-alcoholic fatty liver disease. It can regulate hepatic glucose and lipid metabolism by changing the methylation status of genes involved in fatty acid oxidation, lipolysis and lipogenesis and the activity of transcription factors. Impairment of these regulatory mechanisms is closely associated with the development of obesity and metabolic diseases (such as type 2 diabetes and non-alcoholic fatty liver disease), and restoration of these impairments may be beneficial in reversing metabolic abnormalities. Therefore, inhibiting the expression or activity of FTO may be a therapeutic approach to treat obesity, diabetes, and non-alcoholic fatty liver disease. However, current small molecules that inhibit FTO lack specificity and cannot cope with the compensation of other signaling pathways, and CRISPR-CAS9 and siRNA/shRNA technologies that act on the DNA or RNA level face drawbacks such as irreversible editing and off-target effects.
靶向降解嵌合体PROTAC作为一种新兴的蛋白降解技术在近年来脱颖而出，受到了广大学者和制药公司的关注。蛋白降解靶向嵌合体作为一种双功能小分子化合物，共由三部分组成，一端为结合靶蛋白的配体，另一端为结合E3泛素连接酶的配体，两个配体之间通过连接链进行连接。在细胞中，PROTAC分子能够识别并选择性的结合靶蛋白并招募特定的E3泛素连接酶，拉近E3泛素连接酶与靶蛋白之间的空间距离从而形成“靶蛋白-PROTAC-E3泛素连接酶”三元复合物，而后通过与E2泛素结合酶共同作用，促进靶蛋白的泛素化，使其进入泛素-蛋白酶体途径，从而达到降解靶蛋白的目的。与传统小分子通过直接与靶蛋白的活性位点结合阻断蛋白的功能不同，PROTAC属于“事件驱动”型，不需要直接抑制靶蛋白的功能活性且对靶蛋白配体的亲和力要求不高，仅需较低浓度就能发挥蛋白降解功能，因此能获得更持久的效果并且能够很好的克服现有药物的耐药性问题。Targeted degradation chimeric PROTAC has emerged as an emerging protein degradation technology in recent years and has attracted the attention of scholars and pharmaceutical companies. As a bifunctional small molecule compound, the protein degradation targeting chimera consists of three parts. One end is a ligand that binds to the target protein, and the other end is a ligand that binds to E3 ubiquitin ligase. The two ligands pass through Connect the chain to connect. In cells, PROTAC molecules can recognize and selectively bind target proteins and recruit specific E3 ubiquitin ligases, shortening the spatial distance between E3 ubiquitin ligases and target proteins to form a "target protein-PROTAC-E3 ubiquitin ligase". The protein ligase" ternary complex then works with E2 ubiquitin-conjugating enzyme to promote the ubiquitination of the target protein and make it enter the ubiquitin-proteasome pathway, thereby achieving the purpose of degrading the target protein. Unlike traditional small molecules that block protein function by directly binding to the active site of the target protein, PROTAC is an "event-driven" type that does not need to directly inhibit the functional activity of the target protein and does not require high affinity for the target protein ligand. It only needs a lower concentration to exert its protein degradation function, so it can achieve a longer-lasting effect and can well overcome the resistance problem of existing drugs.
发明内容Contents of the invention
有鉴于此，本发明目的在于提供一种FTO蛋白降解靶向嵌合体及其制备方法和应用，本发明提供的FTO蛋白降解靶向嵌合体，作为FTO蛋白降解剂，能够有效降解FTO蛋白，从而改善肥胖和脂肪肝，可以作为新的预防和/或治疗肥胖、脂肪肝和糖尿病药物进行开发应用。In view of this, the purpose of the present invention is to provide an FTO protein degradation targeting chimera and its preparation method and application. The FTO protein degradation targeting chimera provided by the invention, as an FTO protein degradation agent, can effectively degrade FTO protein, thereby Improve obesity and fatty liver and can be developed and applied as new drugs to prevent and/or treat obesity, fatty liver and diabetes.
本发明的目的由以下的技术方案实现：The object of the present invention is achieved by the following technical solutions:
一种FTO蛋白降解靶向嵌合体，为具有FTO降解活性的蛋白降解靶向嵌合体，其结构通式如式I或式II所示：An FTO protein degradation-targeted chimera is a protein degradation-targeted chimera with FTO degradation activity, and its general structural formula is as shown in Formula I or Formula II:
其中，E为具有泛素化功能的E3连接酶配体；Among them, E is an E3 ligase ligand with ubiquitination function;
L为连接基团，所述连接基团为亚烷基或者烷氧基中的一种。L is a connecting group, and the connecting group is one of alkylene or alkoxy.
作为优选的实施方式，所述的E3连接酶配体，为VHL(von Hippel-Lindau,希佩尔-林道)类蛋白配体或CRBN(Cereblon)蛋白配体(即CRBN基因编码的蛋白酶小分子配体)。As a preferred embodiment, the E3 ligase ligand is a VHL (von Hippel-Lindau) protein ligand or a CRBN (Cereblon) protein ligand (that is, a protease small molecule encoded by the CRBN gene Ligand).
进一步的，所述CRBN蛋白配体，为泊马度胺(结构式为)及其衍生物、来那度胺(结构式为/>)及其衍生物、沙利度胺(结构式为/>)及其衍生物、酰胺类化合物或者邻苯二酰亚胺类化合物中的一种或多种；Further, the CRBN protein ligand is pomalidomide (structural formula is ) and its derivatives, lenalidomide (structural formula is/> ) and its derivatives, thalidomide (structural formula is/> ) and one or more of its derivatives, amide compounds or phthalimide compounds;
所述VHL类蛋白配体为VHL蛋白配体(结构式为)或带甲基VHL蛋白配体(结构式为/>)中的一种或多种。The VHL protein ligand is a VHL protein ligand (structural formula is ) or methylated VHL protein ligand (structural formula is/> ) one or more.
作为优选的实施方式，所述的亚烷基或者烷氧基为-(CH2)n-、-(CH2)nCO-、-NR1(CH2)nCO-、-NR1(CH2)n-、-NR1CO-、-(CH2CH2O)n-、-(OCH2CH2O)n-、-(OCH2CH2OCH2)n-、-(CH2CH2OCH2)n-、-(CH2CH2OCH2CH2)n-、亚烯基、亚炔基、亚环烷基或亚杂芳羟基中的一种或多种的组合(组合包括直链或带支链的组合)，其中n为1至20的自然数，R1为H或者为C1-10烷基。As a preferred embodiment, the alkylene or alkoxy group is -(CH2 )n -, -(CH2 )n CO-, -NR1 (CH2 )n CO-, -NR1 (CH2 )n-, -NR1 CO-, -(CH2 CH2 O)n -, -(OCH2 CH2 O)n -, -(OCH2 CH2 OCH2 )n -, -(CH2 CH2 OCH2 )n -, -(CH2 CH2 OCH2 CH2 )n -, one or more combinations of alkenylene, alkynylene, cycloalkylene or heteroarylene hydroxyl (combinations include Linear or branched chain combination), where n is a natural number from 1 to 20, and R1 is H or C1-10 alkyl.
作为优选的实施方式，所述的FTO蛋白降解靶向嵌合体，具体包括以下表1中化合物的一种或多种：As a preferred embodiment, the FTO protein degradation targeting chimera specifically includes one or more of the compounds in the following Table 1:
表1Table 1
更进一步地，所述的FTO蛋白降解靶向嵌合体优选为式Ⅳ所示的化合物。Furthermore, the FTO protein degradation targeting chimera is preferably a compound represented by Formula IV.
作为优选的实施方式，一种FTO蛋白降解靶向嵌合体，包含嵌合体化合物药理或生理上可接受的盐。As a preferred embodiment, an FTO protein degradation targeting chimera includes a pharmacologically or physiologically acceptable salt of the chimera compound.
作为优选的实施方式，上述的FTO蛋白降解靶向嵌合体在制备FTO蛋白降解剂中应用；该FTO蛋白降解剂在肥胖、脂肪肝和糖尿病相关的研究和治疗中应用。As a preferred embodiment, the above-mentioned FTO protein degradation targeting chimera is used in the preparation of FTO protein degradation agent; the FTO protein degradation agent is used in research and treatment related to obesity, fatty liver and diabetes.
作为优选的实施方式，上述的FTO蛋白降解靶向嵌合体或其药理或生理上可接受的盐在制备预防和/或治疗肥胖、脂肪肝或糖尿病药物中的应用，所述预防和/或治疗肥胖、脂肪肝或糖尿病，包括减少体内脂肪沉积、抗肥胖、缓解脂肪肝和糖尿病等方面的预防和治疗。As a preferred embodiment, the above-mentioned FTO protein degradation-targeting chimera or its pharmacologically or physiologically acceptable salt is used in the preparation of drugs for preventing and/or treating obesity, fatty liver or diabetes, and the prevention and/or treatment Obesity, fatty liver or diabetes, including prevention and treatment of reducing fat deposition in the body, anti-obesity, alleviating fatty liver and diabetes.
作为优选的实施方式，所述预防和/或治疗肥胖、脂肪肝和糖尿病药物，除包含FTO蛋白降解靶向嵌合体或其药理或生理上可接受的盐外，还包含药学上可接受的载体或赋形剂。As a preferred embodiment, the medicine for preventing and/or treating obesity, fatty liver and diabetes, in addition to containing the FTO protein degradation targeting chimera or a pharmacologically or physiologically acceptable salt thereof, also contains a pharmaceutically acceptable carrier. or excipients.
上述FTO蛋白降解靶向嵌合体的制备方法，具体包括以下步骤：The preparation method of the above-mentioned FTO protein degradation targeting chimera specifically includes the following steps:
1)连接基团供体与E3连接酶配体发生取代反应，获得连接基团取代的E3连接酶配体；1) The linking group donor undergoes a substitution reaction with the E3 ligase ligand to obtain the linking group-substituted E3 ligase ligand;
2)连接基团取代的E3连接酶配体与FTO配体经过缩合反应，得到FTO蛋白降解靶向嵌合体；2) The E3 ligase ligand substituted by the linking group undergoes a condensation reaction with the FTO ligand to obtain the FTO protein degradation targeting chimera;
其中，所述连接基团供体为连接基团(亚烷基或者烷氧基)在未连接前的供体化合物；Wherein, the connecting group donor is a donor compound in which the connecting group (alkylene or alkoxy group) is not connected;
所述FTO配体为式ⅩⅢ或式ⅩⅣ中的一种；The FTO ligand is one of formula XIII or formula XIV;
更进一步地，所述FTO蛋白降解靶向嵌合体为式I所示化合物，L为n＝1-20的烷氧基链，E为泊马度胺；本发明提供一种FTO蛋白降解靶向嵌合体(如式ⅩⅨ所示)的制备方法，包括以下步骤：Furthermore, the FTO protein degradation targeting chimera is a compound shown in Formula I, L is an alkoxy chain with n=1-20, and E is pomalidomide; the invention provides an FTO protein degradation targeting chimera. The preparation method of chimera (shown as formula XIX) includes the following steps:
丙酸叔丁酯聚乙二醇链取代的E3配体化合物与FTO配体发生缩合反应，得到FTO蛋白降解剂。The E3 ligand compound substituted by tert-butyl propionate polyethylene glycol chain undergoes a condensation reaction with the FTO ligand to obtain an FTO protein degradation agent.
所述丙酸叔丁酯聚乙二醇链取代的E3配体化合物如式ⅩⅤ所示：The E3 ligand compound substituted by the polyethylene glycol chain of tert-butyl propionate is shown in formula XV:
所示FTO配体如式ⅩⅢ所示。The FTO ligand shown is represented by formula XIII.
更进一步地，所述丙酸叔丁酯聚乙二醇链取代的E3配体化合物(如式ⅩⅤ所示)的制备方法，包括以下步骤：Furthermore, the preparation method of the tert-butyl propionate polyethylene glycol chain-substituted E3 ligand compound (shown as formula XV) includes the following steps:
(1)由式ⅩⅥ所示结构的聚乙二醇化合物与式ⅩⅦ所示结构的化合物进行取代反应，得到具有式ⅩⅧ所示结构的丙酸叔丁酯聚乙二醇链杂环化合物；(1) A polyethylene glycol compound having a structure represented by formula XVI and a compound having a structure represented by formula XVII are subjected to a substitution reaction to obtain a tert-butyl propionate polyethylene glycol chain heterocyclic compound having a structure represented by formula XVIII;
(2)具有式ⅩⅧ所示结构的化合物通过取代反应得到具有式ⅩⅤ所示结构的丙酸叔丁酯聚乙二醇链取代的E3配体化合物。(2) The compound having the structure represented by formula
(3)具有式ⅩⅤ所示结构的化合物通过与式ⅩⅢ所示结构的FTO配体进行缩合反应，得到具有式ⅩⅨ所示的FTO蛋白降解靶向嵌合体化合物；(3) The compound having the structure shown in Formula XV is subjected to a condensation reaction with the FTO ligand having the structure shown in Formula XIII to obtain the FTO protein degradation-targeting chimera compound shown in Formula XIX;
本发明成功制备得到了靶向FTO的PROTAC分子，能够有效靶向目的蛋白，并降低细胞中FTO蛋白的含量，减少细胞内去甲基化，从而抑制细胞内脂肪沉积。细胞水平和动物体内的活性评价表明，本发明提供的FTO蛋白降解靶向嵌合体具有良好的FTO蛋白降解剂生物活性，在纳摩尔水平下，此降解剂能够快速高效的诱导HepG2细胞中FTO蛋白的泛素化降解；基于FTO蛋白降解靶向嵌合体的药效验证，本发明提供的FTO蛋白降解靶向嵌合体，作为FTO蛋白降解剂，能够有效降解FTO蛋白，从而改善肥胖和脂肪肝，可以作为新的抗肥胖、缓解脂肪肝和治疗糖尿病药物进行开发，具有广泛的应用前景。The present invention successfully prepares FTO-targeting PROTAC molecules, which can effectively target the target protein, reduce the content of FTO protein in cells, reduce intracellular demethylation, thereby inhibiting intracellular fat deposition. Activity evaluation at the cell level and in animals shows that the FTO protein degradation targeting chimera provided by the present invention has good FTO protein degradation agent biological activity. At the nanomolar level, this degradation agent can quickly and efficiently induce FTO protein in HepG2 cells. Ubiquitination degradation; based on the efficacy verification of the FTO protein degradation targeting chimera, the FTO protein degradation targeting chimera provided by the present invention, as an FTO protein degradation agent, can effectively degrade FTO protein, thereby improving obesity and fatty liver. It can be developed as a new anti-obesity, fatty liver alleviation and diabetes treatment drug, and has broad application prospects.
附图说明Description of drawings
图1为本发明所实施例6的蛋白降解嵌合体进行验证的LC-MS、HP-LC和HNMR图谱结果图；其中：A为LC-MS图谱结果图，B为HP-LC图谱结果图，C为HNMR图谱结果图。Figure 1 shows the LC-MS, HP-LC and HNMR spectrum results for verification of the protein degradation chimera in Example 6 of the present invention; wherein: A is the LC-MS spectrum result chart, B is the HP-LC spectrum result chart, C is the HNMR spectrum result picture.
图2为本发明实施例6的FTO蛋白降解嵌合体的降解检测结果图；其中：A为不同浓度降解剂处理HepG2细胞24h后细胞内FTO蛋白含量检测结果图；B为加入0.33nM降解剂不同时间后FTO蛋白含量检测结果图；C为加入降解剂清洗后培养不同时间后细胞内FTO蛋白含量检测结果图。Figure 2 is a graph of the degradation detection results of the FTO protein degradation chimera in Example 6 of the present invention; wherein: A is a graph of the detection results of intracellular FTO protein content after HepG2 cells were treated with different concentrations of degradation agents for 24 hours; B is a graph of the detection results of FTO protein content in HepG2 cells after adding 0.33nM degradation agent. Picture of the detection results of FTO protein content after time; C is the picture of the detection results of FTO protein content in cells after adding degradation agent and cleaning and culturing for different times.
图3为根据本发明一个实施例的FTO蛋白降解嵌合体降解FTO蛋白的Western Blot检测结果图；其中，A为FTO抑制剂甲氯芬那酸和CRBN抑制剂泊马度胺对降解剂降解FTO的竞争试验检测结果图；B为蛋白酶体抑制剂硼佐替米对降解机制验证试验检测结果图。Figure 3 is a diagram showing the Western Blot detection results of the FTO protein degradation chimera degrading FTO protein according to one embodiment of the present invention; wherein, A is the degradation of FTO by the FTO inhibitor meclofenamic acid and the CRBN inhibitor pomalidomide. The picture of the competition test results; B is the picture of the verification test results of the degradation mechanism of the proteasome inhibitor borzotimi.
图4为根据本发明一个实施例的蛋白降解嵌合体对HepG2细胞脂肪沉积抑制和甲基化水平研究试验结果图；其中，A为不同浓度降解剂对HepG2细胞甘油三酯含量的影响检测结果图，B为HepG2细胞油红O染色检测结果图，C为油红O染色ImageJ软件分析结果图，D为加入降解剂后mRNA的N6-甲基腺苷RNA修饰水平检测结果图。Figure 4 is a graph showing the results of a research test on the inhibition of fat deposition and methylation levels in HepG2 cells by protein degradation chimeras according to one embodiment of the present invention; wherein, A is a graph showing the effects of different concentrations of degradation agents on the triglyceride content of HepG2 cells. , B is the result of Oil Red O staining of HepG2 cells, C is the result of ImageJ software analysis of Oil Red O staining, and D is the result of detection of N6-methyladenosine RNA modification level of mRNA after adding degradation agent.
图5为根据本发明一个实施例的蛋白降解嵌合体对高脂饮食小鼠肝脏FTO蛋白降解、小鼠体重和脂肪率的影响检测结果图；其中：A为肝脏中FTO蛋白降解情况检测结果图，B为小鼠体重变化趋势检测结果图，C为小鼠脂肪率的变化趋势检测结果图，D为小鼠电子计算机断层扫描图。Figure 5 is a graph showing the detection results of the effect of the protein degradation chimera on FTO protein degradation, mouse body weight and fat rate in the liver of mice on a high-fat diet according to one embodiment of the present invention; wherein: A is a graph showing the detection results of FTO protein degradation in the liver , B is the result of detecting the change trend of mouse body weight, C is the result of detecting the change trend of fat percentage of mice, and D is the computerized tomography scan of mouse.
图6为根据本发明一个实施例的蛋白降解嵌合体对小鼠糖代谢的影响结果图；其中，A为小鼠葡萄糖耐量检测结果图；B为A图的药时曲线下面积统计结果图；C为胰岛素耐量检测结果；D为C图的药时曲线下面积统计结果图。Figure 6 is a graph showing the effect of the protein degradation chimera on mouse glucose metabolism according to one embodiment of the present invention; wherein, A is the mouse glucose tolerance test result graph; B is the statistical result graph of the area under the drug-time curve of graph A; C is the insulin tolerance test result; D is the statistical result of the area under the drug-time curve in Figure C.
图7为根据本发明一个实施例的蛋白降解嵌合体对小鼠肝脏脂质沉积的影响结果图；其中，A为肝脏照片结果图，B为肝脏H&E染色结果图，C为肝脏油红O染色结果图，D为肝脏甘油三酯含量检测结果图，E为肝脏胆固醇含量检测结果图。Figure 7 is a graph showing the effect of protein degradation chimera on lipid deposition in mouse liver according to one embodiment of the present invention; A is a photograph of the liver, B is a H&E staining of the liver, and C is Oil Red O staining of the liver. The result chart, D is the result chart of liver triglyceride content detection, and E is the result chart of liver cholesterol content detection.
图8为根据本发明一个实施例的蛋白降解嵌合体对小鼠血清生化指标的影响检测结果图；其中，A为血清甘油三酯含量检测结果图，B为血清胆固醇含量检测结果图，C为血清谷草转氨酶含量检测结果图，D为血清谷丙转氨酶含量检测结果图。Figure 8 is a graph showing the detection results of the effect of protein degradation chimeras on mouse serum biochemical indicators according to one embodiment of the present invention; wherein, A is the detection result of serum triglyceride content, B is the detection result of serum cholesterol content, and C is The result chart of serum aspartate aminotransferase content, D is the result chart of serum alanine aminotransferase content.
具体实施方式Detailed ways
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.
另外，各个实施例之间的技术方案可以相互结合，但是必须是以本领域普通技术人员能够实现为基础，当技术方案的结合出现相互矛盾或无法实现时应当认为这种技术方案的结合不存在，也不在本发明要求的保护范围之内。In addition, the technical solutions in various embodiments can be combined with each other, but it must be based on the realization by those of ordinary skill in the art. When the combination of technical solutions is contradictory or cannot be realized, it should be considered that such a combination of technical solutions does not exist. , nor within the protection scope required by the present invention.
目前，缺少有效的抗肥胖和缓解脂肪肝Currently, there is a lack of effective anti-obesity and fatty liver disease treatments
药物，为了解决上述技术问题，本发明提出了一种FTO蛋白降解靶向嵌合体及其制备方法和应用。Drug, in order to solve the above technical problems, the present invention proposes an FTO protein degradation targeting chimera and its preparation method and application.
实施例1Example 1
称取化合物b(900mg,3.46mmol,1.00eq),化合物a(731mg，4.15mmol，1.20eq)、Pd2(dba)3(316mg，346μmol，0.10eq)、BINAP(215mg，346μmol，0.10eq)和Cs2CO3(2.26g，6.92mmol，2.00eq)的混合物在甲苯(20mL)中并用氮气脱气清洗3次，然后搅拌混合物12小时；用50mL乙酸乙酯稀释反应混合物，并通过硅胶垫(20.0g)过滤，在减压下浓缩滤液以得到残渣；通过反相高效液相色谱(0.1％FA条件)纯化粗产物并冷冻干燥，得到化合物c(750mg，2.11mmol，61.01％产率)，其为黄色油，经HNMR确认。Weigh compound b (900mg, 3.46mmol, 1.00eq), compound a (731mg, 4.15mmol, 1.20eq), Pd2 (dba)3 (316mg, 346μmol, 0.10eq), BINAP (215mg, 346μmol, 0.10eq) and Cs2 CO3 (2.26 g, 6.92 mmol, 2.00 eq) in toluene (20 mL) and degassed with nitrogen 3 times, then the mixture was stirred for 12 h; the reaction mixture was diluted with 50 mL of ethyl acetate and passed through a silica gel pad (20.0g) was filtered, and the filtrate was concentrated under reduced pressure to obtain a residue; the crude product was purified by reversed-phase high-performance liquid chromatography (0.1% FA conditions) and freeze-dried to obtain compound c (750 mg, 2.11 mmol, 61.01% yield) , which is a yellow oil, confirmed by HNMR.
1H NMR:化合物c(400MHz,DMSO-d6)1 H NMR: Compound c (400MHz, DMSO-d6 )
δ8.11(s,1H),7.52(d,J＝8.3Hz,1H),7.47-7.34(m,3H),6.51(dd,J＝0.9,8.3Hz,1H),3.83(s,3H),2.38(s,3H).δ8.11(s,1H),7.52(d,J＝8.3Hz,1H),7.47-7.34(m,3H),6.51(dd,J＝0.9,8.3Hz,1H),3.83(s,3H) ,2.38(s,3H).
实施例2Example 2
向溶解在THF(7mL)中的化合物c(700mg，1.97mmol，1.00eq)中添加LiOH·H2O(3M，3.64mL)；将混合物在55℃下搅拌8小时；分离反应混合物，在Na2SO4上干燥有机层，过滤并减压浓缩，得到黄色油状化合物d(600mg，1.76mmol，89.2％产率)。To compound c (700 mg, 1.97 mmol, 1.00 eq) dissolved in THF (7 mL) was added LiOH·H2 O (3 M, 3.64 mL); the mixture was stirred at 55 °C for 8 h; the reaction mixture was separated and dissolved in Na The organic layer was dried over2 SO4 , filtered and concentrated under reduced pressure to obtain compound d as a yellow oil (600 mg, 1.76 mmol, 89.2% yield).
实施例3Example 3
在氮气环境下，将Pt/V/C(956mg，73.2μmol，2％纯度，0.05eq)添加到化合物d(500mg，1.47mmol，1.00eq)在THF(10mL)中的溶液中；在25℃的氢气(15Psi)下搅拌混合物6小时；反应混合物利用硅藻土垫过滤，滤液在真空下浓缩，得到化合物e(450mg，1.45mmol，98.6％产率)，为淡黄色固体，经HNMR确认。Pt/V/C (956 mg, 73.2 μmol, 2% purity, 0.05 eq) was added to a solution of compound d (500 mg, 1.47 mmol, 1.00 eq) in THF (10 mL) under nitrogen atmosphere; at 25 °C The mixture was stirred under hydrogen gas (15 Psi) for 6 hours; the reaction mixture was filtered through a diatomaceous earth pad, and the filtrate was concentrated under vacuum to obtain compound e (450 mg, 1.45 mmol, 98.6% yield) as a light yellow solid, which was confirmed by HNMR.
1H NMR:化合物e(400MHz,DMSO-d6)1 H NMR: Compound e (400MHz, DMSO-d6 )
δ12.29-11.65(m,1H),7.37(d,J＝8.2Hz,1H),7.15(d,J＝8.2Hz,1H),6.57(t,J＝8.0Hz,1H),5.89(d,J＝7.9Hz,1H),5.18(d,J＝7.9Hz,1H),2.33(s,3H).δ12.29-11.65(m,1H),7.37(d,J＝8.2Hz,1H),7.15(d,J＝8.2Hz,1H),6.57(t,J＝8.0Hz,1H),5.89(d ,J＝7.9Hz,1H),5.18(d,J＝7.9Hz,1H),2.33(s,3H).
实施例4Example 4
向化合物f(3.50g，12.66mmol，1.00eq)和化合物g(3.51g，12.6mmol，1.00eq)在二氧六环(70.0mL)中的溶液中添加DIEA(2.46g，19.0mmol，3.31mL，1.50eq)进行反应；将反应混合物倒入水中(250mL)，用乙酸乙酯(300mL×3)萃取；用盐水(200mL)洗涤合并的有机层，在Na2SO4上干燥，过滤并减压浓缩，得到残渣；粗产物通过反相高效液相色谱(0.1％FA条件)纯化并冻干，得到化合物h(2.70g，5.06mmol，39.9％产率)，其为黄色油，经HNMR证实。To a solution of compound f (3.50g, 12.66mmol, 1.00eq) and compound g (3.51g, 12.6mmol, 1.00eq) in dioxane (70.0mL) was added DIEA (2.46g, 19.0mmol, 3.31mL , 1.50eq) for reaction; pour the reaction mixture into water (250mL), extract with ethyl acetate (300mL×3); wash the combined organic layer with brine (200mL), dry over Na2 SO4 , filter and reduce Concentrate under pressure to obtain a residue; the crude product was purified by reversed-phase high-performance liquid chromatography (0.1% FA conditions) and lyophilized to obtain compound h (2.70g, 5.06mmol, 39.9% yield), which was a yellow oil, confirmed by HNMR .
1H NMR:化合物h(400MHz,DMSO-d6)1 H NMR: Compound h (400MHz, DMSO-d6 )
δ11.33-10.85(m,1H),7.58(dd,J＝7.3,8.4Hz,1H),7.15(d,J＝8.6Hz,1H),7.04(d,J＝7.0Hz,1H),6.60(t,J＝5.7Hz,1H),5.05(dd,J＝5.4,12.8Hz,1H),3.64-3.44(m,14H),2.95-2.82(m,1H),2.69-2.55(m,2H),2.39(t,J＝6.2Hz,2H),2.06-1.97(m,1H),1.38(s,9H)。δ11.33-10.85(m,1H),7.58(dd,J＝7.3,8.4Hz,1H),7.15(d,J＝8.6Hz,1H),7.04(d,J＝7.0Hz,1H),6.60 (t,J＝5.7Hz,1H),5.05(dd,J＝5.4,12.8Hz,1H),3.64-3.44(m,14H),2.95-2.82(m,1H),2.69-2.55(m,2H ), 2.39 (t, J = 6.2Hz, 2H), 2.06-1.97 (m, 1H), 1.38 (s, 9H).
实施例5Example 5
向化合物h(2.30g，4.31mmol，1.00eq)的溶液中添加HCl/二氧六环(2M，46.0mL，21.3eq)在减压下浓缩反应，得到化合物i(1.70g，3.56mmol，82.6％产率)，其为黑棕色油，经HNMR证实。To the solution of compound h (2.30g, 4.31mmol, 1.00eq), HCl/dioxane (2M, 46.0mL, 21.3eq) was added and the reaction was concentrated under reduced pressure to obtain compound i (1.70g, 3.56mmol, 82.6 % yield), which was a dark brown oil, confirmed by HNMR.
1H NMR:化合物i(400MHz,DMSO-d6)1 H NMR: Compound i (400MHz, DMSO-d6 )
δ11.09(s,1H),7.59(dd,J＝7.2,8.5Hz,1H),7.16(d,J＝8.6Hz,1H),7.05(d,J＝7.0Hz,1H),6.61(br s,1H),5.06(dd,J＝5.4,12.8Hz,1H),3.64-3.59(m,4H),3.56-3.47(m,10H),2.94-2.83(m,1H),2.63-2.53(m,2H),2.43(t,J＝6.3Hz,2H),2.08-2.00(m,1H),1.78-1.75(m,1H).δ11.09(s,1H),7.59(dd,J＝7.2,8.5Hz,1H),7.16(d,J＝8.6Hz,1H),7.05(d,J＝7.0Hz,1H),6.61(br s,1H),5.06(dd,J＝5.4,12.8Hz,1H),3.64-3.59(m,4H),3.56-3.47(m,10H),2.94-2.83(m,1H),2.63-2.53( m,2H),2.43(t,J＝6.3Hz,2H),2.08-2.00(m,1H),1.78-1.75(m,1H).
实施例6Example 6
向溶解化合物i(300mg，628μmol，1.00eq)的DMF(4mL)中添加DIEA(243mg，1.88mmol，328uL，3.00eq)和HATU(238.75mg，627.5μmol，1eq)反应0.5小时；然后添加化合物e(195mg，628μmol，1.00eq)，将所得反应混合物搅拌1小时；反应混合物通过反相高效液相色谱(0.1％FA条件)纯化并冻干，得到化合物f(120.04mg，149μmol，23.8％产率，96.2％纯度)为黄色固体，其经LCMS(图1A)、HPLC(图1B)、HNMR(图1C)确认。DIEA (243mg, 1.88mmol, 328uL, 3.00eq) and HATU (238.75mg, 627.5μmol, 1eq) were added to DMF (4mL) in which compound i (300mg, 628μmol, 1.00eq) was dissolved and reacted for 0.5 hours; then compound e was added (195 mg, 628 μmol, 1.00 eq), and the resulting reaction mixture was stirred for 1 hour; the reaction mixture was purified by reversed-phase high-performance liquid chromatography (0.1% FA conditions) and lyophilized to obtain compound f (120.04 mg, 149 μmol, 23.8% yield , 96.2% purity) as a yellow solid, which was confirmed by LCMS (Figure 1A), HPLC (Figure 1B), and HNMR (Figure 1C).
1H NMR(400MHz,DMSO-d6)1 H NMR (400MHz, DMSO-d6 )
δ11.10(s,1H),10.58(br d,J＝2.1Hz,1H),9.11-8.68(m,1H),7.56(t,J＝7.8Hz,1H),7.48(d,J＝8.3Hz,1H),7.43(d,J＝8.0Hz,1H),7.31(d,J＝8.4Hz,1H),7.21-7.09(m,2H),7.03(d,J＝7.0Hz,1H),6.59(br s,1H),5.91(d,J＝8.3Hz,1H),5.05(dd,J＝5.3,12.8Hz,1H),3.68(t,J＝6.1Hz,2H),3.61-3.57(m,2H),3.52(d,J＝2.4Hz,8H),3.44(br d,J＝4.8Hz,3H),2.94-2.81(m,1H),2.60(br s,1H),2.54(br t,J＝6.3Hz,3H),2.36(s,3H),2.06-1.97(m,1H)。δ11.10(s,1H),10.58(br d,J＝2.1Hz,1H),9.11-8.68(m,1H),7.56(t,J＝7.8Hz,1H),7.48(d,J＝8.3 Hz,1H),7.43(d,J＝8.0Hz,1H),7.31(d,J＝8.4Hz,1H),7.21-7.09(m,2H),7.03(d,J＝7.0Hz,1H), 6.59(br s,1H),5.91(d,J＝8.3Hz,1H),5.05(dd,J＝5.3,12.8Hz,1H),3.68(t,J＝6.1Hz,2H),3.61-3.57( m,2H),3.52(d,J＝2.4Hz,8H),3.44(br d,J＝4.8Hz,3H),2.94-2.81(m,1H),2.60(br s,1H),2.54(br t,J＝6.3Hz,3H),2.36(s,3H),2.06-1.97(m,1H).
以下对上述实施例6制备获得的FTO蛋白降解靶向嵌合体(化合物f)进行生物学测试：The following biological tests were performed on the FTO protein degradation targeting chimera (compound f) prepared in the above Example 6:
效果实施例1：降解剂诱导HepG2细胞FTO蛋白降解Effect Example 1: Degrading agent induces degradation of FTO protein in HepG2 cells
将细胞接种于24孔板中，24小时后加入不同浓度梯度(0nM，0.1nM，0.33nM，1nM，3.33nM、10nM)的FTO降解剂(化合物f)培养24小时，收集细胞，加入含有蛋白酶抑制剂的细胞裂解液收集蛋白进行Western Blot试验。或者加入相同浓度(0.33nM)的FTO降解剂后培养不同时间(0h，2h，4h，8h，12h，24h)，收集细胞，加入裂解液收集蛋白进行Western Blot试验。以及加入FTO降解剂相同时间后更换培养基培养不同时间后收集细胞进行WesternBlot试验。通过Western Blot试验，本发明对涉及合成的降解剂分子的HepG2人肝癌细胞FTO蛋白降解能力进行了检测。检测了以聚乙二醇类(PEG)结构作为连接基的降解剂。FTO蛋白降解嵌合体的降解结果及其具有浓度依赖性和时间依赖性结果如图2所示，图2A为不同浓度降解剂处理HepG2细胞24h后细胞内FTO蛋白含量；图2B为加入0.33nM降解剂不同时间后FTO蛋白含量；图2C为加入降解剂清洗后培养不同时间后细胞内FTO蛋白含量。上述结果表明，降加入降解剂0.33到1nM出现了明显降解，加入0.33nM降解剂后在2h出现了明显降解，且降解效果持续到清洗后36h。The cells were seeded in a 24-well plate. After 24 hours, FTO degradation agent (compound f) with different concentration gradients (0 nM, 0.1 nM, 0.33 nM, 1 nM, 3.33 nM, 10 nM) was added and cultured for 24 hours. The cells were collected and protease was added. Proteins were collected from cell lysates of inhibitors for Western Blot testing. Or add the same concentration (0.33nM) of FTO degradation agent and incubate for different times (0h, 2h, 4h, 8h, 12h, 24h), collect cells, add lysate to collect proteins for Western Blot testing. And after adding FTO degrading agent for the same time, the culture medium was replaced and cultured for different times, and then the cells were collected for Western Blot test. Through Western Blot test, the present invention detects the degradation ability of HepG2 human liver cancer cell FTO protein involving synthetic degradation agent molecules. Degradants using polyethylene glycol (PEG) structures as linkers were tested. The degradation results of the FTO protein degradation chimera and its concentration-dependent and time-dependent results are shown in Figure 2. Figure 2A shows the intracellular FTO protein content in HepG2 cells treated with different concentrations of degradation agents for 24 hours; Figure 2B shows the degradation by adding 0.33nM FTO protein content after adding degradation agent for different times; Figure 2C shows the FTO protein content in cells after adding degradation agent for cleaning and culturing for different times. The above results show that obvious degradation occurred when the degradation agent was added from 0.33 to 1 nM. After adding 0.33 nM degradation agent, obvious degradation occurred within 2 hours, and the degradation effect continued until 36 hours after cleaning.
效果实施例2：降解剂通过泛素-蛋白酶体途径降解FTO蛋白Effect Example 2: Degrading agent degrades FTO protein through ubiquitin-proteasome pathway
本发明使用FTO抑制剂甲氯芬那酸、CRBN抑制剂泊马度胺、蛋白酶体抑制剂硼佐替米分别先孵育细胞2h后加入FTO降解剂处理，最后通过Western Blot检测FTO蛋白条带变化，结果如图3所示，图3A为FTO抑制剂甲氯芬那酸和CRBN抑制剂泊马度胺对降解剂降解FTO的竞争试验；图3B为蛋白酶体抑制剂硼佐替米对降解机制验证试验。从图3A可以看出，加入甲氯芬那酸和泊马度胺后，降解剂对FTO蛋白的降解被竞争抑制。从图3B中可以看出，降解剂对FTO蛋白的降解作用可以被硼佐替米明显阻断，说明降解剂诱导FTO蛋白得降解是通过泛素-蛋白酶体途径实现。The present invention uses the FTO inhibitor meclofenamic acid, the CRBN inhibitor pomalidomide, and the proteasome inhibitor borzotimi to incubate the cells for 2 hours respectively, then add an FTO degradation agent for treatment, and finally detect the changes in the FTO protein band through Western Blot. , the results are shown in Figure 3. Figure 3A shows the competition test of the FTO inhibitor meclofenamic acid and the CRBN inhibitor pomalidomide on the degradation of FTO by the degradation agent; Figure 3B shows the degradation mechanism of the proteasome inhibitor borzotimid. Verification test. As can be seen from Figure 3A, after adding meclofenamic acid and pomalidomide, the degradation of FTO protein by the degradation agent was competitively inhibited. As can be seen from Figure 3B, the degradation of FTO protein by the degradation agent can be significantly blocked by borzotimi, indicating that the degradation of FTO protein induced by the degradation agent is achieved through the ubiquitin-proteasome pathway.
效果实施例3：降解剂减少OA/PA诱导的HepG2细胞脂肪沉积和m6A水平Effect Example 3: Degradant reduces OA/PA-induced fat deposition and m6A levels in HepG2 cells
(1)降解剂减少OA/PA诱导的HepG2细胞脂肪沉积(1) Degradants reduce OA/PA-induced fat deposition in HepG2 cells
将HepG2细胞接种于24孔板中，用生长培养基培养24h后加入0.33nM降解剂和新鲜OA/PA培养基，以未加药组作为对照，培养24h后将孔板的培养基吸完后，每孔加入一定量的PBS进行清洗后吸出，随后每孔加入200μL裂解液，于4℃摇床上裂解30min，之后利用枪头刮下贴壁细胞并将裂解液移入1.5mL离心管中，室温12000rpm离心10min，取部分上清用于蛋白含量检测。剩余部分用70℃干预锅加热10min后12000rpm离心10min，在96孔酶标板中每孔加入64μL样品，200μL工作液，放于37℃培养箱静置10min。用酶标仪检测510nm处的吸光度数值。按照以下公式测定甘油三酯含量。此外，将上述方案培养后的细胞进行油红O染色，并用ImageJ软件分析脂滴数量和大小。结果如图4所示，图4A为不同浓度降解剂对HepG2细胞甘油三酯含量的影响，图4B为HepG2细胞油红O染色结果，图4C为油红O染色ImageJ软件分析结果，加入降解剂后，细胞内甘油三酯含量减少，表明降解剂能够减少HepG2细胞脂肪积累，增加小脂滴比例，减少大脂滴比例。HepG2 cells were seeded in a 24-well plate, cultured in growth medium for 24 hours, and then 0.33nM degradation agent and fresh OA/PA medium were added. The unmedicated group was used as a control. After 24 hours of culture, the medium in the well plate was drained. , add a certain amount of PBS to each well, wash and aspirate, then add 200 μL of lysis solution to each well, lyse on a shaking table at 4°C for 30 minutes, then use the pipette tip to scrape off the adherent cells and move the lysate into a 1.5mL centrifuge tube at room temperature. Centrifuge at 12,000 rpm for 10 min, and take part of the supernatant for protein content detection. The remaining part was heated in an intervention pot at 70°C for 10 minutes and centrifuged at 12,000 rpm for 10 minutes. Add 64 μL of sample and 200 μL of working solution to each well of a 96-well enzyme plate, and place it in a 37°C incubator for 10 minutes. Use a microplate reader to detect the absorbance value at 510 nm. Determine triglyceride content according to the following formula. In addition, the cells cultured according to the above protocol were stained with Oil Red O, and the number and size of lipid droplets were analyzed using ImageJ software. The results are shown in Figure 4. Figure 4A shows the effect of different concentrations of degradants on the triglyceride content of HepG2 cells. Figure 4B shows the results of Oil Red O staining of HepG2 cells. Figure 4C shows the results of Oil Red O staining ImageJ software analysis. Adding degradants After treatment, the intracellular triglyceride content decreased, indicating that the degradation agent could reduce fat accumulation in HepG2 cells, increase the proportion of small lipid droplets, and reduce the proportion of large lipid droplets.
公式：formula:
(2)降解剂减少HepG2细胞m6A水平(2) Degradants reduce m6A levels in HepG2 cells
将HepG2细胞接种于24孔板种，用用生长培养基培养24h后加入0.33nM降解剂和新鲜OA/PA培养基，以未加药组作为对照，培养24h后将孔板的培养基吸完后，每孔加入一定量的PBS进行清洗后吸出，随后利用Trizol法提取RNA，利用无RNAaes的水将RNA稀释至20ng/ul。在95℃下加热3分钟以破坏二级结构，随后立即在冰上冷却以防止二级结构恢复。将2ul的mRNA直接滴加到硝酸纤维素膜上，利用紫外灯进行交联，随后在清洁的洗涤盘中加入10mL PBST在室温中清洗硝酸纤维素膜5分钟，清洗掉没有结合的mRNA。随后按照斑点印迹的实验方法进行后续实验。结果如图4D所示，过表达FTO后mRNA的N6-甲基腺苷RNA修饰水平降低，而加入降解剂后mRNA的N6-甲基腺苷RNA修饰水平增加。表明降解剂能够通过降解FTO从而增加HepG2细胞中N6-甲基腺苷RNA修饰水平。HepG2 cells were seeded in a 24-well plate. After culturing for 24 hours, 0.33nM degradation agent and fresh OA/PA culture medium were added. The unmedicated group was used as a control. After culturing for 24 hours, the culture medium from the well plate was drained. Finally, a certain amount of PBS was added to each well for washing and aspiration, and then the RNA was extracted using the Trizol method, and the RNA was diluted to 20ng/ul with RNAaes-free water. Heat at 95°C for 3 minutes to destroy secondary structure, followed by immediate cooling on ice to prevent recovery of secondary structure. Drop 2 ul of mRNA directly onto the nitrocellulose membrane and use UV light for cross-linking. Then add 10 mL PBST to a clean washing dish and wash the nitrocellulose membrane at room temperature for 5 minutes to remove unbound mRNA. Follow-up experiments were then carried out according to the experimental method of dot blotting. The results are shown in Figure 4D. The N6-methyladenosine RNA modification level of mRNA decreased after overexpression of FTO, while the N6-methyladenosine RNA modification level of mRNA increased after adding a degradation agent. It shows that the degrader can increase the level of N6-methyladenosine RNA modification in HepG2 cells by degrading FTO.
效果实施例4：降解剂对高脂饮食诱导的脂肪肝小鼠的影响Effect Example 4: Effect of degradation agent on mice with fatty liver induced by high-fat diet
本发明通过高脂饮食构建了肝脏脂肪变性的小鼠模型，用来评价FTO降解剂的体内药效学作用。The present invention constructs a mouse model of liver steatosis through a high-fat diet to evaluate the in vivo pharmacodynamic effect of FTO degrading agents.
(1)使用降解剂(0.04mg/kg和0.2mg/kg)以及空白溶剂分别对小鼠进行腹腔注射，每四天给药一次，在饲喂14周后麻醉小鼠处死后，收集肝脏组织，并提取蛋白进行WesternBlot试验，所得结果如图5A所示，肝脏中FTO蛋白出现降解现象。此外，我们在饲养期间每周称量一次体重，每两周测量一次脂肪率并在处死前进行电子计算机断层扫描，从而比较多次给药后对小鼠体内脂肪沉积的影响。结果如图5所示，图5B为小鼠体重变化趋势，凸5C为小鼠脂肪率的变化趋势，图5D为小鼠电子计算机断层扫描图像。从图5中可以看出，高脂模型组小鼠体重和脂肪率明显上升，给药后这一比率均有所下降，同时脂肪率也呈现下降趋势。上述结果表明，降解剂在体内试验中对肝脏仍具有明显的FTO蛋白降解效果，并且能够有效抑制高脂饮食导致的体重增加和脂肪率升高。(1) Use degradation agents (0.04mg/kg and 0.2mg/kg) and blank solvent to intraperitoneally inject mice respectively, once every four days. After feeding for 14 weeks, the mice were anesthetized and sacrificed, and the liver tissues were collected. , and extracted the protein for Western Blot test. The results are shown in Figure 5A. FTO protein in the liver is degraded. In addition, we weighed the body weight once a week during the feeding period, measured the fat percentage every two weeks, and performed electronic computed tomography scans before sacrifice to compare the effects of multiple doses on fat deposition in the mice. The results are shown in Figure 5. Figure 5B shows the changing trend of mouse body weight. Figure 5C shows the changing trend of mouse fat percentage. Figure 5D shows the computerized tomography image of mice. As can be seen from Figure 5, the body weight and fat rate of mice in the high-fat model group increased significantly. After administration, this ratio decreased, and the fat rate also showed a downward trend. The above results show that the degradation agent still has a significant FTO protein degradation effect on the liver in in vivo tests, and can effectively inhibit the increase in weight gain and fat rate caused by high-fat diet.
(2)降解剂可以改善小鼠葡萄糖耐量、胰岛素耐量(2) Degradants can improve glucose tolerance and insulin tolerance in mice
本发明在高脂饮食处理后第12.13周分别进行了葡萄糖耐量和胰岛素耐量试验，按照2g/Kg的剂量给小鼠腹腔注射葡萄糖溶液，以及0.5U/kg的剂量给小鼠腹腔注射胰岛素并于0，15，30，60，90，120min时通过尾部取血测量小鼠血糖含量。结果如图6所示，图6中A-B为小鼠葡萄糖耐量，C-D为胰岛素耐量。从图6中可以看出，高脂会导致小鼠葡萄糖耐量和胰岛素耐量降低，而降解剂给药组能够改善高脂饮食所导致的葡萄糖和胰岛素耐量降低。The present invention conducted glucose tolerance and insulin tolerance tests respectively at 12.13 weeks after high-fat diet treatment. The mice were intraperitoneally injected with glucose solution at a dose of 2g/Kg, and mice were intraperitoneally injected with insulin at a dose of 0.5U/kg. The blood glucose content of the mice was measured by taking blood from the tail at 0, 15, 30, 60, 90, and 120 minutes. The results are shown in Figure 6. In Figure 6, A-B are the glucose tolerance of mice, and C-D are insulin tolerance. As can be seen from Figure 6, high fat can lead to reduced glucose tolerance and insulin tolerance in mice, and the degradation agent administration group can improve the reduced glucose and insulin tolerance caused by high-fat diet.
(3)降解剂对小鼠肝脏脂质沉积的影响(3) Effect of degradation agents on liver lipid deposition in mice
本发明将小鼠高脂饮食处理后第14周进行麻醉处死，将肝脏分离拍照后取部分放于组织固定液中，用于H&E和油红O染色。其余取适量放入1.5ml离心管中，加入1mL裂解液和钢珠并于组织破碎仪中破碎组织以提取甘油三酯和蛋白质。提取完成后12000rpm多次离心取上清，在96孔酶标板中分别取2.5μL和5μL用于检测甘油三酯和胆固醇，并从剩余部分中取10μL用于蛋白含量检测。结果如图7所示，图7中A为肝脏照片和肝脏重量，B为肝脏H&E染色，C为肝脏油红O染色，D为肝脏甘油三酯含量，E为肝脏胆固醇含量。从图7中可以看出，高脂模型组小鼠肝脏更大，更苍白，H&E染色显示高脂模型组的肝脏有未染色的脂质，而给药组减少了未染色的脂质数量，油红O染色进一步证实，与对照组相比，模型组肝脏中的中性脂质积聚更广泛，给药组可以在一定程度上减少肝脏中脂质积累。此外，模型组肝脏中的甘油三酯和胆固醇水平显著升高，而注射降解剂以剂量依赖的方式逆转了这一现象，将甘油三酯水平降至对照水平。上述结果表明，降解剂能够有效缓解高脂饮食导致的肝脏脂质积累，缓解脂肪肝的发生。In the present invention, mice were anesthetized and killed at 14 weeks after being treated with a high-fat diet. The livers were separated and photographed, and the parts were placed in tissue fixatives for H&E and Oil Red O staining. Put an appropriate amount of the rest into a 1.5 ml centrifuge tube, add 1 mL of lysis solution and steel beads, and crush the tissue in a tissue disruptor to extract triglycerides and proteins. After the extraction is completed, centrifuge at 12,000 rpm multiple times to take the supernatant, take 2.5 μL and 5 μL respectively in a 96-well microplate for detecting triglycerides and cholesterol, and take 10 μL from the remaining part for protein content detection. The results are shown in Figure 7. In Figure 7, A is the liver photo and liver weight, B is the H&E staining of the liver, C is the Oil Red O staining of the liver, D is the triglyceride content of the liver, and E is the cholesterol content of the liver. As can be seen from Figure 7, the livers of mice in the high-fat model group are larger and paler. H&E staining shows that the livers of the high-fat model group have unstained lipids, while the drug administration group has reduced the amount of unstained lipids. Oil Red O staining further confirmed that compared with the control group, neutral lipid accumulation in the liver was more extensive in the model group, and the drug administration group could reduce lipid accumulation in the liver to a certain extent. Furthermore, triglyceride and cholesterol levels were significantly elevated in the liver of the model group, while injection of the degrader reversed this phenomenon in a dose-dependent manner, reducing triglyceride levels to control levels. The above results show that the degradation agent can effectively alleviate the accumulation of liver lipids caused by high-fat diet and alleviate the occurrence of fatty liver.
(4)降解剂对血液生化指标的影响(4) Effect of degradation agents on blood biochemical indicators
本发明将小鼠高脂饮食处理后第14周进行麻醉后，通过眼眶取血收集血液于3000rpm离心收集血清用于生理生化指标检测。结果如图8所示，图8中A为血清甘油三酯含量，B为血清胆固醇含量，C为血清谷草转氨酶含量，D为血清谷丙转氨酶含量。从图中可以看出，高脂模型组的血清中甘油三酯含量、胆固醇含量、谷草转氨酶含量和谷丙转氨酶含量均明显上升，而注射降解剂以剂量依赖性的方式减少了这些指标的升高。上述结果表明，降解剂能够有效减少高脂饮食导致的血清脂质转运增多，减少肝脏损伤。In the present invention, mice are anesthetized on the 14th week after being treated with a high-fat diet, and then blood is collected from the orbit and centrifuged at 3000 rpm to collect serum for physiological and biochemical index detection. The results are shown in Figure 8. In Figure 8, A is the serum triglyceride level, B is the serum cholesterol level, C is the serum aspartate aminotransferase level, and D is the serum alanine aminotransferase level. It can be seen from the figure that the serum triglyceride content, cholesterol content, aspartate aminotransferase content and alanine aminotransferase content in the high-fat model group all increased significantly, and the injection of degradation agents reduced the increase in these indicators in a dose-dependent manner. high. The above results indicate that the degradation agent can effectively reduce the increase in serum lipid transport caused by high-fat diet and reduce liver damage.
本发明成功制备得到了靶向FTO的PROTAC分子，能够有效靶向目的蛋白，并降低细胞中FTO蛋白的含量，减少细胞内去甲基化，从而抑制细胞内脂肪沉积。细胞水平和动物体内的活性评价表明，本发明提供的FTO蛋白降解靶向嵌合体具有良好的FTO蛋白降解剂生物活性，在纳摩尔水平下，此降解剂能够快速高效的诱导HepG2细胞中FTO蛋白的泛素化降解；基于FTO蛋白降解靶向嵌合体的药效验证，本发明提供的FTO蛋白降解靶向嵌合体，作为FTO蛋白降解剂，能够有效降解FTO蛋白，从而改善肥胖和脂肪肝，可以作为新的抗肥胖和缓解脂肪肝药物进行开发，具有广泛的应用前景。The present invention successfully prepares FTO-targeting PROTAC molecules, which can effectively target the target protein, reduce the content of FTO protein in cells, reduce intracellular demethylation, thereby inhibiting intracellular fat deposition. Activity evaluation at the cell level and in animals shows that the FTO protein degradation targeting chimera provided by the present invention has good FTO protein degradation agent biological activity. At the nanomolar level, this degradation agent can quickly and efficiently induce FTO protein in HepG2 cells. Ubiquitination degradation; based on the efficacy verification of the FTO protein degradation targeting chimera, the FTO protein degradation targeting chimera provided by the present invention, as an FTO protein degradation agent, can effectively degrade FTO protein, thereby improving obesity and fatty liver. It can be developed as a new anti-obesity and fatty liver alleviation drug and has broad application prospects.
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.
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| CN108524482A (en)* | 2017-03-02 | 2018-09-14 | 中国科学院上海药物研究所 | The purposes of 2- (substitution phenylamino) benzoic acids FTO inhibitor for treating leukaemia |
| US20210395244A1 (en)* | 2020-06-01 | 2021-12-23 | Icahn School Of Medicine At Mount Sinai | Mitogen-activated protein kinase kinase (mek) degradation compounds and methods of use |
| US20220348556A1 (en)* | 2018-07-06 | 2022-11-03 | Kymera Therapeutics, Inc. | Protein degraders and uses thereof |
| WO2022261220A1 (en)* | 2021-06-08 | 2022-12-15 | University Of Maryland, Baltimore | Proteolysis targeting chimeras and methods of use thereof |
| CN115504963A (en)* | 2021-06-22 | 2022-12-23 | 苏州开拓药业股份有限公司 | A c-Myc protein degradation agent |
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108524482A (en)* | 2017-03-02 | 2018-09-14 | 中国科学院上海药物研究所 | The purposes of 2- (substitution phenylamino) benzoic acids FTO inhibitor for treating leukaemia |
| US20200079727A1 (en)* | 2017-03-02 | 2020-03-12 | Shanghai Institute Of Materia Medica, Chinese Academy Of Sciences | 2-(substituted benzene matrix) aromatic formate fto inhibitor, preparation method therefor, and applications thereof |
| US20220348556A1 (en)* | 2018-07-06 | 2022-11-03 | Kymera Therapeutics, Inc. | Protein degraders and uses thereof |
| US20210395244A1 (en)* | 2020-06-01 | 2021-12-23 | Icahn School Of Medicine At Mount Sinai | Mitogen-activated protein kinase kinase (mek) degradation compounds and methods of use |
| WO2022261220A1 (en)* | 2021-06-08 | 2022-12-15 | University Of Maryland, Baltimore | Proteolysis targeting chimeras and methods of use thereof |
| CN115504963A (en)* | 2021-06-22 | 2022-12-23 | 苏州开拓药业股份有限公司 | A c-Myc protein degradation agent |
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