CN117357511A - Application of Ginkgetin in preparation of STING inhibitor - Google Patents
Application of Ginkgetin in preparation of STING inhibitor Download PDFInfo
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- CN117357511A CN117357511A CN202210773943.2A CN202210773943A CN117357511A CN 117357511 A CN117357511 A CN 117357511A CN 202210773943 A CN202210773943 A CN 202210773943A CN 117357511 A CN117357511 A CN 117357511A
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Abstract
Description
技术领域Technical field
本发明属于医药技术领域，具体而言，涉及Ginkgetin在制备STING抑制剂中的应用。The invention belongs to the field of medical technology, and specifically relates to the application of Ginkgetin in the preparation of STING inhibitors.
背景技术Background technique
先天免疫反应被称为人体的第一道防线，它对于宿主有效抵抗外来病原微生物的入侵是至关重要的。人体内含有特殊模式识别受体(pattern recognition receptors,PRRs)，入侵的病原体会被宿主细胞中的模式识别受体识别，PRRs可启动一系列信号传导的活动，如诱导产生I型干扰素(IFN)、分泌促炎性细胞因子以及激活其他下游区域有关抗病原微生物的蛋白的表达，进而启动免疫应答[1]。在过去几年中，越来越多的证据表明，介导DNA的免疫应答最主要的途径是：cGAS-STING信号传导通路。The innate immune response is known as the body's first line of defense and is crucial for the host to effectively resist the invasion of foreign pathogenic microorganisms. The human body contains special pattern recognition receptors (PRRs). Invading pathogens will be recognized by the pattern recognition receptors in host cells. PRRs can initiate a series of signaling activities, such as inducing the production of type I interferon (IFN). ), secrete pro-inflammatory cytokines and activate the expression of proteins related to anti-pathogenic microorganisms in other downstream regions, thereby initiating immune responses [1] . In the past few years, increasing evidence has shown that the most important pathway mediating immune responses to DNA is the cGAS-STING signaling pathway.
干扰素基因刺激因子(stimulator of interferon gene,STING)是一种胞内模式识别受体，是内质网(endoplasmic reticulum,ER)上的跨膜蛋白，在固有免疫信号通路中发挥着重要的作用[2]。胞质中的DNA感受器环状GMP-AMP合成酶(cyclic GMP-AMPsynthase,cGAS)可以识别来自病毒、细菌和自身胞质的DNA，当cGAS识别胞质中积累的DNA时，cGAS与其结合，活性位点构象会发生改变，催化ATP和GTP合成第二信使cGAMP[3]，cGAMP与ER膜上的STING蛋白结合，诱导其构象发生变化，形成多聚体，活化的STING从ER转移到高尔基体，在高尔基体中招募TANK结合激酶1 (TANK-binding kinase 1,TBK1)等激酶，磷酸化干扰素调节因子3(Interferon regulatory factor,IRF3)，磷酸化的IRF3会发生二聚化并移位至细胞核，促进干扰素的分泌，进而发挥免疫调节作用，影响机体病毒防御、炎症和肿瘤治疗[4]。Stimulator of interferon gene (STING) is an intracellular pattern recognition receptor and a transmembrane protein on the endoplasmic reticulum (ER). It plays an important role in the innate immune signaling pathway. [2] . The DNA sensor cyclic GMP-AMP synthase (cGAS) in the cytoplasm can recognize DNA from viruses, bacteria and its own cytoplasm. When cGAS recognizes DNA accumulated in the cytoplasm, cGAS binds to it and becomes active. The conformation of the site will change, catalyzing ATP and GTP to synthesize the second messenger cGAMP [3] . cGAMP binds to the STING protein on the ER membrane, inducing its conformational change to form a multimer, and the activated STING is transferred from the ER to the Golgi apparatus. , recruits kinases such as TANK-binding kinase 1 (TBK1) in the Golgi apparatus to phosphorylate interferon regulatory factor 3 (IRF3), and the phosphorylated IRF3 will dimerize and translocate to The cell nucleus promotes the secretion of interferon, thereby exerting an immune regulatory effect and affecting the body's virus defense, inflammation and tumor treatment [4] .
基于STING信号通路在肿瘤治疗和先天免疫中发挥的重要作用以及重要意义，目前已经有很多文献报道了诸多STING激动剂，其中包括ADU-S100[5]、diABZI(GSK3) [6]、SR-717[7]以及MSA-2[8]等等，它们在抗病毒免疫和抗肿瘤免疫中发挥着重要的作用。但是STING的点突变或DNA酶功能受损时，STING的慢性激活会导致自身免疫性疾病的发生，包括系统性红斑狼疮(systemic lupu erythematosu,SLE)、家族性冻疮(familial chilblainlupus,FCL)、STING相关性血管病变(STING-associated vasculopathy,SAVI) 以及血管和肺综合征(vascular and pulmonary syndrome,VAPS)[9,10]等，因此抑制STING 信号通路自激活对于治疗很多自身免疫紊乱疾病是非常有帮助的。目前，STING抑制剂主要包括共价和非共价两大类，共价抑制剂主要有硝基脂肪酸(NO2-FA)[11]和硝基呋喃[12]两大类，两者都可以共价结合半胱氨酸88或91从而抑制氨基酸的棕榈酰化并阻断通路，其中硝基呋喃具有比硝基脂肪酸(NO2-FA)有更强的选择性。此外，非共价抑制剂Astin C[13]和四氢异喹啉衍生物[14]能够竞争性抑制内源性CDNs与STING结合。STING抑制剂已被设计用于治疗自身免疫性疾病，并且正在陆续开发针对其他炎症性疾病的新型免疫疗法。然而，现在市场上用于治疗自身免疫性疾病的药物非常稀少，现有文献中靶向人源STING的抑制剂亦屈指可数。Based on the important role and significance of the STING signaling pathway in tumor treatment and innate immunity, many STING agonists have been reported in many literatures, including ADU-S100 [5] , diABZI(GSK3) [6] , SR- 717 [7] and MSA-2 [8] , etc., which play an important role in anti-viral immunity and anti-tumor immunity. However, when STING point mutations or DNA enzyme function is impaired, chronic activation of STING can lead to the occurrence of autoimmune diseases, including systemic lupus erythematosus (SLE), familial chilblainlupus (FCL), STING STING-associated vasculopathy (SAVI) and vascular and pulmonary syndrome (VAPS) [9,10] , etc. Therefore, inhibiting the self-activation of the STING signaling pathway is very effective in treating many autoimmune disorders. helpful. At present, STING inhibitors mainly include two categories: covalent and non-covalent. Covalent inhibitors mainly include nitro fatty acids (NO 2 -FA) [11] and nitrofurans [12] , both of which can be used. Covalently binds to cysteine 88 or 91 to inhibit palmitoylation of amino acids and block the pathway, in which nitrofurans have stronger selectivity than nitro fatty acids (NO 2 -FA). In addition, non-covalent inhibitors Astin C [13] and tetrahydroisoquinoline derivatives [14] can competitively inhibit the binding of endogenous CDNs to STING. STING inhibitors have been designed to treat autoimmune diseases, and novel immunotherapies are being developed for other inflammatory diseases. However, there are very few drugs on the market for the treatment of autoimmune diseases, and there are only a handful of inhibitors targeting human STING in the existing literature.
发明内容Contents of the invention
本发明致力于发现针对人源和鼠源STING的具有高选择性的STING小分子抑制剂，可作用于人源STING的抑制剂的发现，可为治疗自身免疫性疾病提供新的思路与方法。The present invention is dedicated to discovering highly selective small molecule inhibitors of STING against human and mouse STING. The discovery of inhibitors that can act on human STING can provide new ideas and methods for the treatment of autoimmune diseases.
Ginkgetin是一种从银杏叶中分离得到的双黄酮，也称为银杏素、银杏黄酮甙、银杏黄酮苷、白果双黄酮、银杏双黄酮、银杏黄素等，结构如下：Ginkgetin is a biflavonoid isolated from Ginkgo leaves, also known as ginkgo biloba, ginkgo flavonoid glycoside, ginkgo flavonoid glycoside, ginkgo biflavonoid, ginkgo biflavonoid, ginkgo flavonoid, etc. Its structure is as follows:
本发明通过虚拟筛选发现Ginkgetin可以靶向STING，后通过分子水平实验(蛋白热迁移，表面等离子体共振)发现Ginkgetin可以直接与STING蛋白结合；又在细胞水平发现Ginkgetin通过特异性结合STING阻断STING与TBK1的相互作用，使得STING 通路相关蛋白磷酸化减弱，Ⅰ型干扰素和其他促炎细胞因子等表达下降，即可特异性地阻断双链DNA以及多种STING小分子激动剂刺激引起的STING信号通路激活；另通过动物实验发现小分子化合物缓解了自身免疫性疾病和急性结肠炎等病症的发生发展。The present invention found that Ginkgetin can target STING through virtual screening, and later found through molecular level experiments (protein thermal migration, surface plasmon resonance) that Ginkgetin can directly bind to STING protein; and at the cellular level, it was found that Ginkgetin blocks STING by specifically binding to STING. The interaction with TBK1 weakens the phosphorylation of STING pathway-related proteins and reduces the expression of type I interferons and other pro-inflammatory cytokines, which can specifically block the stimulation of double-stranded DNA and various STING small molecule agonists. The STING signaling pathway is activated; in addition, animal experiments have found that small molecule compounds can alleviate the occurrence and development of autoimmune diseases, acute colitis and other diseases.
因此，一方面，本发明提供了Ginkgetin在制备STING抑制剂中的应用。Therefore, in one aspect, the present invention provides the use of Ginkgetin in the preparation of STING inhibitors.
另一方面，本发明提供了Ginkgetin在制备用于预防或治疗与STING信号通路激活相关的疾病的药物中的应用。On the other hand, the present invention provides the use of Ginkgetin in the preparation of medicaments for preventing or treating diseases related to STING signaling pathway activation.
再一方面，本发明提供了Ginkgetin，其用于制备STING抑制剂，或者用于预防或治疗与STING信号通路激活相关的疾病。In another aspect, the present invention provides Ginkgetin, which is used for preparing STING inhibitors, or for preventing or treating diseases related to STING signaling pathway activation.
再一方面，本发明提供了一种药物组合物，其用于预防或治疗与STING信号通路激活相关的疾病，包含预防或治疗有效量的Ginkgetin，和任选的药学上可接受的载体。In another aspect, the present invention provides a pharmaceutical composition for preventing or treating diseases related to STING signaling pathway activation, comprising a preventive or therapeutically effective amount of Ginkgetin, and optionally a pharmaceutically acceptable carrier.
所述药物组合物可以被配制用于任何适宜的施用途径，例如静脉内施用、肌内施用、口服施用、皮下施用、直肠施用、吸入施用、鼻施用、局部施用、眼睛施用、耳施用，但不限于此。所述药物组合物可以是固体制剂、液体制剂、气溶胶等，例如可以是片剂、丸剂、胶囊、液体剂、吸入剂、鼻喷雾溶液剂、栓剂、糖浆剂、锭剂、栓剂、溶液剂、乳剂、软膏剂、滴眼剂或滴耳剂，但不限于此。The pharmaceutical composition may be formulated for any suitable route of administration, such as intravenous administration, intramuscular administration, oral administration, subcutaneous administration, rectal administration, inhalation administration, nasal administration, topical administration, ocular administration, otic administration, but Not limited to this. The pharmaceutical composition can be a solid preparation, a liquid preparation, an aerosol, etc., for example, it can be a tablet, pill, capsule, liquid, inhalant, nasal spray solution, suppository, syrup, lozenge, suppository, solution , emulsions, ointments, eye drops or ear drops, but are not limited to these.
Ginkgetin的典型的剂量范围是0.001-1000mg活性成分/公斤体重/天。所述剂量可以每日一次或分多次给予。适宜剂量的确定由主治医师根据要治疗的疾病的种类及其严重性、个体的健康状况和既往病史、共用药物、和施用途径等酌情确定。根据需要，Ginkgetin的用量可以超出该剂量范围。Typical dosage ranges for Ginkgetin are 0.001-1000mg active ingredient/kg body weight/day. The dosage may be administered once daily or in divided doses. The appropriate dosage is determined at the discretion of the attending physician based on the type and severity of the disease to be treated, the individual's health status and past medical history, shared medications, and route of administration. Ginkgetin can be used outside this dosage range as needed.
本发明中，所述与STING信号通路激活相关的疾病是指炎症性疾病，包括但不限于自身免疫性疾病(例如，Trex1缺陷型炎症)、急性结肠炎等。In the present invention, the diseases related to STING signaling pathway activation refer to inflammatory diseases, including but not limited to autoimmune diseases (for example, Trex1-deficient inflammation), acute colitis, etc.
本发明中，Trex1缺陷型炎症指的是由Trex1基因缺陷导致的系统性炎症反应。In the present invention, Trex1-deficient inflammation refers to the systemic inflammatory reaction caused by Trex1 gene deficiency.
在一个实施方式中，所述自身免疫性疾病，包括但不限于，系统性红斑狼疮(SLE)、家族性冻疮(FCL)、STING相关性血管病变(SAVI)以及血管和肺综合征(VAPS)等。In one embodiment, the autoimmune diseases include, but are not limited to, systemic lupus erythematosus (SLE), familial chilblains (FCL), STING-associated vasculopathy (SAVI), and vascular and pulmonary syndrome (VAPS). wait.
在一个实施方式中，所述与STING信号通路激活相关的疾病可以是急性结肠炎。In one embodiment, the disease associated with STING signaling pathway activation may be acute colitis.
再一方面，本发明提供了一种预防或治疗与STING信号通路激活相关的疾病的方法，包括向有此需要的受试者施用预防或治疗有效量的Ginkgetin，或者上述药物组合物。In another aspect, the present invention provides a method for preventing or treating diseases related to STING signaling pathway activation, which includes administering a preventive or therapeutically effective amount of Ginkgetin, or the above-mentioned pharmaceutical composition, to a subject in need.
本发明中，所述受试者可以是哺乳动物，例如，包括但不限于，人，鼠，马，猪，狗，牛，羊等。In the present invention, the subject may be a mammal, such as, but not limited to, humans, mice, horses, pigs, dogs, cattle, sheep, etc.
如本文中所使用的，术语“治疗有效量”是指该数量具有治疗性的效果而可用于预防或治疗本文所述的特定疾病、病症或病状。例如，“治疗有效量”可指在接受治疗的个体上提供治疗性或所欲功效所需的数量。如本领域技术人员所知，治疗有效量会因为施用途径、赋形剂的使用以及与其他疗法共用时的可能性而有所变化。As used herein, the term "therapeutically effective amount" refers to an amount that has a therapeutic effect and is useful in preventing or treating a particular disease, disorder, or condition described herein. For example, a "therapeutically effective amount" may refer to an amount necessary to provide a therapeutic or desired effect in a subject being treated. As is known to those skilled in the art, the therapeutically effective amount will vary depending on the route of administration, use of excipients, and the possibility of co-administration with other therapies.
如本文所用，“药学上可接受的载体”包括任何和所有的溶剂、分散介质、包衣、表面活性剂、抗氧化剂、防腐剂(例如抗菌剂、抗真菌剂)、等渗剂、吸收延迟剂、盐、防腐剂、药物、药物稳定剂、粘合剂、赋形剂、崩解剂、润滑剂、甜味剂、矫味剂、染料，所述类似的物质和其组合，其是本领域普通技术人员所公知的。除非任何常规载体是与活性成分不能共存的，可以考虑在治疗或药物组合物中使用它。As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, Agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, similar substances and combinations thereof, which are It is well known to those of ordinary skill in the art. Unless any conventional carrier is incompatible with the active ingredient, it is contemplated for use in the therapeutic or pharmaceutical compositions.
本发明通过研究证实，Ginkgetin可以作为靶向STING的小分子抑制剂，在抑制STING通路激活过程中发挥作用。同时，Ginkgetin可以特异性地结合STING，阻断了 STING和TBK1的相互作用，IRF3的磷酸化以及随后的IFN免疫应答和促炎细胞因子的释放，以此达到抑制cGAS-STING通路激活的作用，但是不影响其他模式识别受体通路，为后续STING相关的自身免疫性疾病和急性结肠炎的治疗提供参考性的策略。The present invention has confirmed through research that Ginkgetin can be used as a small molecule inhibitor targeting STING and plays a role in inhibiting the activation of the STING pathway. At the same time, Ginkgetin can specifically bind to STING, blocking the interaction between STING and TBK1, the phosphorylation of IRF3, and the subsequent IFN immune response and release of pro-inflammatory cytokines, thereby inhibiting the activation of the cGAS-STING pathway. However, it does not affect other pattern recognition receptor pathways and provides a reference strategy for the subsequent treatment of STING-related autoimmune diseases and acute colitis.
本发明所述小分子化合物Ginkgetin可有效地抑制抑制自身免疫性疾病和急性结肠炎的炎症表型，将很有可能为今后这些疾病的临床治疗提供新的药物研发指导。The small molecule compound Ginkgetin of the present invention can effectively suppress the inflammatory phenotype of autoimmune diseases and acute colitis, and will very likely provide new drug research and development guidance for the clinical treatment of these diseases in the future.
在上文中已经详细地描述了本发明，但是上述实施方式本质上仅是例示性，且并不欲限制本发明。此外，本文并不受前述现有技术或发明内容或以下实施例中所描述的任何理论的限制。The present invention has been described in detail above, but the above-mentioned embodiments are only illustrative in nature and are not intended to limit the present invention. Furthermore, there is no intention to be bound by any theory presented in the preceding prior art or summary of the invention or described in the following examples.
除非另有明确说明，在整个申请文件中的数值范围包括其中的任何子范围和以其中给定值的最小子单位递增的任何数值。除非另有明确说明，在整个申请文件中的数值表示对包括与给定值的微小偏差以及具有大约所提及的值以及具有所提及的精确值的实施方案的范围的近似度量或限制。除了在详细描述最后提供的工作实施例之外，本申请文件(包括所附权利要求)中的参数(例如，数量或条件)的所有数值在所有情况下都应被理解为被术语“大约”修饰，不管“大约”是否实际出现在该数值之前。“大约”表示所述的数值允许稍微不精确(在该值上有一些接近精确；大约或合理地接近该值；近似)。如果“大约”提供的不精确性在本领域中没有以这个普通含义来理解，则本文所用的“大约”至少表示可以通过测量和使用这些参数的普通方法产生的变化。例如，“大约”可以包括小于或等于10％，小于或等于5％，小于或等于4％，小于或等于3％，小于或等于 2％，小于或等于1％或者小于或等于0.5％的变化。Unless expressly stated otherwise, numerical ranges throughout this application include any subrange therein and any numerical value incrementing the smallest subunit of the value given therein. Unless expressly stated otherwise, numerical values throughout this application represent approximate measures or limitations of the scope of embodiments that include minor deviations from the given values and have about the recited value as well as having the precise value recited. With the exception of the working examples provided at the end of the detailed description, all numerical values for parameters (eg quantities or conditions) in this application document (including the appended claims) are to be understood in all cases as being expressed by the term "about" Modification, regardless of whether "about" actually appears before the value. "Approximately" means that the stated value is allowed to be slightly imprecise (somewhat close to exact on the value; about or reasonably close to the value; approximation). If the imprecision provided by "approximately" is not understood in this ordinary sense in the art, "approximately" as used herein at least means the variation that can be produced by ordinary methods of measuring and using these parameters. For example, "about" may include a variation of less than or equal to 10%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, or less than or equal to 0.5% .
附图说明Description of the drawings
图1为显示实施例1中小分子化合物Ginkgetin直接与STING蛋白的结合情况的图。其中：A：蛋白热迁移实验(PTS)分析Ginkgetin与STINGH232蛋白的结合情况；B：蛋白热迁移实验(PTS)分析Ginkgetin与STINGR232蛋白的结合情况；C：蛋白热迁移实验(PTS)分析Ginkgetin与mSTING蛋白的结合情况；D：表面等离子体共振实验(SPR) 分析Ginkgetin与STINGH232蛋白的结合情况。Figure 1 is a diagram showing the direct binding of the small molecule compound Ginkgetin to the STING protein in Example 1. Among them: A: Protein Thermal Shift Test (PTS) analyzes the binding of Ginkgetin and STING H232 protein; B: Protein Thermal Shift Test (PTS) analyzes the binding of Ginkgetin and STING R232 protein; C: Protein Thermal Shift Test (PTS) analyzes The binding situation of Ginkgetin and mSTING protein; D: Surface plasmon resonance experiment (SPR) to analyze the binding situation of Ginkgetin and STING H232 protein.
图2为显示实施例2中小分子化合物Ginkgetin直接作用于STING的图。其中：A：在2’,3’-cGAMP存在下，用Ginkgetin或其结构类似物Amentoflavone处理不同人源细胞后上清中的SEAP活性；B：在2’,3’-cGAMP存在下，用Ginkgetin或其结构类似物 Amentoflavone处理不同鼠源细胞后上清中的Lucia荧光素酶活性；C：Ginkgetin结构类似物Amentoflavone的结构；D：THP-1细胞经Ginkgetin处理后的细胞存活率；E：Raw 264.7细胞经Ginkgetin处理后的细胞存活率；F：用Ginkgetin处理分别过表达 Flag-STINGR232、Flag-TBK1、IRF3-5D的HEK293T细胞，测定细胞中IFNB1 mRNA的表达水平。Figure 2 is a diagram showing the direct effect of the small molecule compound Ginkgetin on STING in Example 2. Among them: A: SEAP activity in the supernatant of different human cells treated with Ginkgetin or its structural analog Amentoflavone in the presence of 2',3'-cGAMP; B: In the presence of 2',3'-cGAMP, SEAP activity in the supernatant of different human cells Lucia luciferase activity in the supernatant of different mouse cells treated with Ginkgetin or its structural analog Amentoflavone; C: Structure of Ginkgetin structural analog Amentoflavone; D: Cell survival rate of THP-1 cells after Ginkgetin treatment; E: Cell survival rate of Raw 264.7 cells after Ginkgetin treatment; F: HEK293T cells overexpressing Flag-STING R232 , Flag-TBK1, and IRF3-5D were treated with Ginkgetin, and the expression level of IFNB1 mRNA in the cells was measured.
图3为显示实施例3中小分子化合物Ginkgetin抑制了2’,3’-cGAMP激活的STING通路的图。其中：A-C：对2’,3’-cGAMP或者2’,3’-cGAMP和Ginkgetin处理后的THP-1 来源的巨噬细胞进行RNA测序分析。A：用火山图表示两组的差异基因；B：热图显示 Ginkgetin与2’,3’-cGAMP联用组中相较于2’,3’-cGAMP组降低最显著的50个基因；C：两组之间差异基因的富集分析；D：Western Blot实验检测Ginkgetin与2’,3’-cGAMP联用后对STING通路激活后关键蛋白磷酸化的影响；E-F：IF实验检测Ginkgetin与 2’,3’-cGAMP联用后对STING通路激活后IRF3(E)和p65(F)核易位的影响。Figure 3 is a diagram showing that the small molecule compound Ginkgetin in Example 3 inhibits the STING pathway activated by 2',3'-cGAMP. Among them: A-C: RNA sequencing analysis of THP-1-derived macrophages treated with 2’,3’-cGAMP or 2’,3’-cGAMP and Ginkgetin. A: A volcano plot is used to represent the differential genes between the two groups; B: A heat map shows the 50 genes that were most significantly reduced in the Ginkgetin and 2',3'-cGAMP combined group compared to the 2',3'-cGAMP group; C : Enrichment analysis of differential genes between the two groups; D: Western Blot experiment to detect the effect of Ginkgetin combined with 2',3'-cGAMP on the phosphorylation of key proteins after STING pathway activation; E-F: IF experiment to detect the association between Ginkgetin and 2 Effects of combined use of ',3'-cGAMP on the nuclear translocation of IRF3(E) and p65(F) after STING pathway activation.
图4为显示实施例3中小分子化合物Ginkgetin对不同刺激物触发的基因表达的影响的图。其中：A-B：THP-1来源的巨噬细胞中，Ginkgetin对各种刺激物引起的相关基因表达水平的影响；C-E：Raw 264.7细胞中，Ginkgetin对各种刺激物引起的相关基因表达水平的影响；＊＊＊表示P<0.001，**表示P<0.01，*表示P<0.05。Figure 4 is a graph showing the effect of the small molecule compound Ginkgetin on gene expression triggered by different stimuli in Example 3. Among them: A-B: In macrophages derived from THP-1, the effect of Ginkgetin on the expression levels of related genes caused by various stimuli; C-E: In Raw 264.7 cells, the effect of Ginkgetin on the expression levels of related genes caused by various stimuli. ; ＊＊＊ means P<0.001, ** means P<0.01, * means P<0.05.
图5为显示实施例3中小分子化合物Ginkgetin抑制细胞内双链DNA刺激引起的基因表达的图。其中：A-C：Ginkgetin对THP-1细胞内G3-YSD触发的IFNB1(A)、CXCL10 (B)和IL6(C)mRNA表达的影响。Figure 5 is a graph showing that the small molecule compound Ginkgetin in Example 3 inhibits gene expression induced by intracellular double-stranded DNA stimulation. Among them: A-C: Effect of Ginkgetin on G3-YSD-triggered IFNB1 (A), CXCL10 (B) and IL6 (C) mRNA expression in THP-1 cells.
图6为显示实施例3中小分子化合物Ginkgetin抑制细胞内双链DNA刺激引起的基因表达的图。其中：A-C：Ginkgetin对Raw 264.7细胞内ISD触发的Ifnb1(A)、Cxcl10 (B)和Il6(C)mRNA表达的影响。Figure 6 is a graph showing that the small molecule compound Ginkgetin in Example 3 inhibits gene expression induced by intracellular double-stranded DNA stimulation. Among them: A-C: Effect of Ginkgetin on ISD-triggered Ifnb1 (A), Cxcl10 (B) and Il6 (C) mRNA expression in Raw 264.7 cells.
图7为显示实施例3中小分子化合物Ginkgetin对STING通路激活的关键蛋白表达水平的抑制的图。其中：A-C：Western Blot检测Ginkgetin与GSK3(A)或ADU-S100 (B)或SR-717(C)联用后对THP-1细胞内STING通路关键蛋白磷酸化水平的影响； D-H：Western Blot检测Ginkgetin与2’,3’-cGAMP(D)或GSK3(E)或ADU-S100(F) 或SR-717(G)或MSA2(H)联用后对Raw 264.7细胞内STING通路关键蛋白磷酸化水平的影响。Figure 7 is a graph showing the inhibition of the expression levels of key proteins activated by the STING pathway by the small molecule compound Ginkgetin in Example 3. Among them: A-C: Western Blot detects the effect of Ginkgetin combined with GSK3 (A) or ADU-S100 (B) or SR-717 (C) on the phosphorylation level of key proteins of the STING pathway in THP-1 cells; D-H: Western Blot Detection of the phosphorylation of key proteins of the STING pathway in Raw 264.7 cells after Ginkgetin combined with 2',3'-cGAMP (D) or GSK3 (E) or ADU-S100 (F) or SR-717 (G) or MSA2 (H) The impact of the level of
图8为显示实施例4中小分子化合物Ginkgetin阻断了STING与TBK1的相互作用的图。其中：A-B：HEK293T细胞转染Flag-STINGH232和Flag-TBK1两种质粒后，用 Ginkgetin和2’,3’-cGAMP共处理，通过Co-Immunoprecipitation检测Ginkgetin对STING 与TBK1相互作用的影响；C：同时用Ginkgetin和2’,3’-cGAMP处理转染STINGH232质粒24h的HeLa细胞，通过IF实验检测STING与TBK1的共定位。Figure 8 is a graph showing that the small molecule compound Ginkgetin blocks the interaction between STING and TBK1 in Example 4. Among them: AB: After HEK293T cells were transfected with two plasmids, Flag-STING H232 and Flag-TBK1, they were co-treated with Ginkgetin and 2',3'-cGAMP, and the effect of Ginkgetin on the interaction between STING and TBK1 was detected by Co-Immunoprecipitation; C : HeLa cells transfected with STING H232 plasmid were treated with Ginkgetin and 2',3'-cGAMP at the same time for 24 hours, and the co-localization of STING and TBK1 was detected by IF experiment.
图9为显示实施例5中小分子化合物Ginkgetin抑制Trex1-/-小鼠BMDMs细胞中免疫相关因子的表达的图。其中：A-F:Trex1-/-小鼠的BMDMs细胞用Ginkgetin处理后检测Ifnb1(A)、Cxcl10(B)、Isg15(C)、Il6(D)、Il1b(E)和Isg56(F)mRNA的表达水平；＊＊＊表示P<0.00，**表示P<0.01。Figure 9 is a graph showing that the small molecule compound Ginkgetin in Example 5 inhibits the expression of immune-related factors in Trex1 -/- mouse BMDMs cells. Among them: BMDMs cells of AF:Trex1 -/- mice were treated with Ginkgetin to detect the expression of Ifnb1 (A), Cxcl10 (B), Isg15 (C), Il6 (D), Il1b (E) and Isg56 (F) mRNA. Level; ＊＊＊ means P<0.00, ** means P<0.01.
图10为显示实施例5中小分子化合物Ginkgetin抑制Trex1-/-小鼠组织中免疫相关因子的表达的图。其中：A-D：收集WT和Trex1-/-小鼠的心脏(A)、胃(B)、舌头(C) 和肌肉(D)组织，分别提取RNA，以使用qPCR实验测量指定基因的mRNA表达水平；＊＊＊表示P<0.001，**表示P<0.01，*表示P<0.05。Figure 10 is a graph showing that the small molecule compound Ginkgetin in Example 5 inhibits the expression of immune-related factors in Trex1 -/- mouse tissues. Among them: AD: Heart (A), stomach (B), tongue (C) and muscle (D) tissues of WT and Trex1 -/- mice were collected, and RNA was extracted respectively to measure the mRNA expression levels of the specified genes using qPCR experiments. ; ＊＊＊ means P<0.001, ** means P<0.01, * means P<0.05.
图11为显示实施例5中小分子化合物Ginkgetin缓解Trex1-/-小鼠组织炎症表型的图。Figure 11 is a graph showing that the small molecule compound Ginkgetin in Example 5 alleviates the tissue inflammation phenotype of Trex1 -/- mice.
图12为显示实施例6中小分子化合物Ginkgetin改善DSS诱导的小鼠急性结肠炎的图。其中：A：三组小鼠每日的体重变化情况；B：有代表性的结肠影像；C：三组小鼠的结肠长度；D：小鼠结肠组织的代表性H&E图像；E：用qPCR方法检测小鼠结肠组织中Il6、Il1b、TnfmRNA的转录水平；F：用Elisa法测定小鼠血清中IL-6、IL-1β和TNF的浓度；＊＊＊表示P<0.001，**表示P<0.01，*表示P<0.05。Figure 12 is a graph showing that the small molecule compound Ginkgetin in Example 6 improves DSS-induced acute colitis in mice. Among them: A: Daily weight changes of mice in three groups; B: Representative colon images; C: Colon length of mice in three groups; D: Representative H&E images of mouse colon tissue; E: qPCR Methods to detect the transcription levels of Il6, Il1b and TnfmRNA in mouse colon tissue; F: Use Elisa method to determine the concentrations of IL-6, IL-1β and TNF in mouse serum; ** indicates P<0.001, ** indicates P <0.01, * indicates P<0.05.
具体实施方式Detailed ways
下述实施例所使用的实验方法如无特殊说明，均为常规方法。The experimental methods used in the following examples are all conventional methods unless otherwise specified.
下述实施例所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.
实施例1小分子化合物Ginkgetin与STING蛋白的相互作用Example 1 Interaction between the small molecule compound Ginkgetin and STING protein
1.1.蛋白热迁移实验(Protein Thermal Shift，PTS)分析Ginkgetin与STING蛋白的结合情况1.1. Protein Thermal Shift (PTS) analysis of the binding of Ginkgetin and STING protein
实验方法：experimental method:
用实时荧光定量PCR仪检测STING蛋白的热稳定性。先用分子筛缓冲液配制终浓度为2μM STING蛋白和5×SYPRO orange(Invitrogene,S6651)的混合液，混匀后加入96孔无裙边PCR板(DN Biotech)中，每孔19μL，再加入1μL化合物Ginkgetin，封板后1000rpm离心1min，放入PCR仪启动程序，在25～90℃内监测和采集荧光信号，用蛋白质热迁移软件(Bio-Rad)测定STING的Tm值。Real-time fluorescence quantitative PCR instrument was used to detect the thermal stability of STING protein. First use molecular sieve buffer to prepare a mixture of STING protein and 5×SYPRO orange (Invitrogene, S6651) with a final concentration of 2 μM, mix well, and add it to a 96-well skirtless PCR plate (DN Biotech), adding 19 μL to each well, and then add 1 μL For the compound Ginkgetin, centrifuge at 1000 rpm for 1 min after sealing the plate, put it into the PCR machine to start the program, monitor and collect fluorescence signals at 25-90°C, and use protein thermal migration software (Bio-Rad) to measure the T m value of STING.
实验结果：Experimental results:
如图1中A-C所示，PTS实验表明小分子化合物使得STINGH232、STINGR232和 mSTING这三种蛋白的Tm值均增大，并且随着浓度的增大，蛋白稳定性逐渐增加，说明Ginkgetin可以直接与STING蛋白结合。As shown in AC in Figure 1, PTS experiments show that small molecule compounds increase the T m values of the three proteins STING H232 , STING R232 and mSTING, and as the concentration increases, the protein stability gradually increases, indicating that Ginkgetin Can directly bind to STING protein.
1.2.表面等离子体共振实验(Surface Plasmon Resonance，SPR)分析Ginkgetin与 STING蛋白的结合情况1.2. Surface Plasmon Resonance (SPR) experiment to analyze the binding of Ginkgetin and STING protein
实验方法：experimental method:
使用Biacore T200仪器(GE Healthcare)进行SPR结合分析。在10mM醋酸钠 (pH＝4.0)中，运行缓冲液HBS-EP(50mM Hepes pH 7.4,150mM NaCl，0.05％v/v P20)，用标准的氨基偶联法将STINGH232蛋白共价偶联到CM5传感器芯片上。将化合物 Ginkgetin用缓冲液梯度稀释，以30μL/min的流速注入到传感器芯片上进行动力学实验，结合120s，解离180s。使用Biacore T200评估软件(GE Healthcare)分析得出小分子化合物Ginkgetin和STINGH232蛋白的平衡解离常数(KD)值。SPR binding analysis was performed using a Biacore T200 instrument (GE Healthcare). STING H232 protein was covalently coupled using standard amino coupling methods in 10mM sodium acetate (pH=4.0), running buffer HBS-EP (50mM Hepes pH 7.4, 150mM NaCl, 0.05% v/v P20). On the CM5 sensor chip. The compound Ginkgetin was gradient diluted with buffer and injected onto the sensor chip at a flow rate of 30 μL/min for kinetic experiments, binding for 120 s and dissociating for 180 s. The equilibrium dissociation constant (K D ) values of the small molecule compound Ginkgetin and STING H232 protein were analyzed using Biacore T200 evaluation software (GE Healthcare).
实验结果：Experimental results:
如图1中D所示，SPR实验表明小分子化合物Ginkgetin与STINGH232蛋白有直接结合并且其解离常数(KD)为4.5μM。As shown in D in Figure 1, SPR experiments show that the small molecule compound Ginkgetin directly binds to the STING H232 protein and its dissociation constant (K D ) is 4.5 μM.
实施例2小分子化合物Ginkgetin直接作用于STINGExample 2 The small molecule compound Ginkgetin directly acts on STING
2.1.报告基因实验分析Ginkgetin对STING通路的抑制不涉及上游cGAS2.1. Reporter gene experimental analysis: Ginkgetin’s inhibition of the STING pathway does not involve upstream cGAS
实验方法：experimental method:
THP1-Blue ISG细胞(简称THP-1)、THP1-Dual KO-cGAS细胞(简称cGAS KO THP-1)、Raw-Lucia细胞(简称Raw-Lucia)、Raw-Lucia ISG-KO-cGAS细胞(简称cGAS KO Raw-Lucia)(密度5×105个/mL)分别种于96孔板中过夜培养后，用不同浓度的 Ginkgetin或Amentoflavone处理1h，然后加入2’,3’-cGAMP，同时用DMSO做阴性对照。24h后THP1-BlueISG细胞和THP1-Dual KO-cGAS细胞分别按说明书用Quanti-Blue 试剂盒(Invivogen)测定SEAP活性。具体地，将Quanti-Blue溶液按照每孔180μL分配到96孔板中，加入20μL的样品(表达SEAP的细胞培养上清)或阴性对照(细胞培养液)，37℃孵育30分钟后，使用Spark酶标仪在650nm处测量光密度(OD)。 Raw-Lucia细胞和Raw-Lucia ISG-KO-cGAS细胞分别按说明书用Quanti-Luc试剂盒 (Invivogen)测定Lucia荧光素酶活性。具体地，从96孔板中取出20μL细胞上清，然后每孔加入50μL的Quanti-Luc分析溶液。轻轻拍打几次使其混合，立即用Spark多功能酶标仪(Tecan)检测。THP1-Blue ISG cells (referred to as THP-1), THP1-Dual KO-cGAS cells (referred to as cGAS KO THP-1), Raw-Lucia cells (referred to as Raw-Lucia), Raw-Lucia ISG-KO-cGAS cells (referred to as cGAS KO Raw-Lucia) (density 5 × 10 5 cells/mL) were seeded in 96-well plates overnight and cultured overnight, then treated with different concentrations of Ginkgetin or Amentoflavone for 1 hour, then 2',3'-cGAMP was added, and DMSO was added at the same time. Make a negative control. After 24 hours, the SEAP activity of THP1-BlueISG cells and THP1-Dual KO-cGAS cells was measured using Quanti-Blue kit (Invivogen) according to the instructions. Specifically, the Quanti-Blue solution was distributed into a 96-well plate at 180 μL per well, and 20 μL of sample (SEAP-expressing cell culture supernatant) or negative control (cell culture medium) was added, and after incubation at 37°C for 30 minutes, Spark was used. A microplate reader measures optical density (OD) at 650 nm. Raw-Lucia cells and Raw-Lucia ISG-KO-cGAS cells were used to measure Lucia luciferase activity using the Quanti-Luc kit (Invivogen) according to the instructions. Specifically, 20 μL of cell supernatant was taken from the 96-well plate, and then 50 μL of Quanti-Luc assay solution was added to each well. Gently tap several times to mix, and immediately detect with Spark multifunctional microplate reader (Tecan).
实验结果：Experimental results:
如图2中A-B所示，通过碱性磷酸酶和荧光素酶报告基因实验数据可知，小分子化合物Ginkgetin抑制了THP-1细胞和Raw-Lucia细胞中SEAP和Lucia荧光素酶的活性，对STING通路产生了抑制作用，并且该化合物仍对敲除cGAS的THP-1细胞 (THP1-Dual KO-cGAS细胞)和Raw-Lucia细胞(Raw-Lucia ISG-KO-cGAS细胞)中酶的活性具有抑制作用，所以考虑此化合物并不是作用于上游cGAS，而是作用于STING 或者其下游；其结构类似物Amentoflavone对STING通路的激活并没有抑制作用。如图 2中D-E，还评价了化合物Ginkgetin的细胞毒性。化合物处理THP-1细胞24h后，检测其细胞活力发现Ginkgetin在25μM以内均无明显毒性；化合物处理Raw 264.7细胞 24h后，检测其细胞活力发现Ginkgetin在50μM以内均无明显毒性。因此，排除了化合物毒性造成抑制作用的可能性。As shown in A-B in Figure 2, the alkaline phosphatase and luciferase reporter gene experimental data show that the small molecule compound Ginkgetin inhibits the activities of SEAP and Lucia luciferase in THP-1 cells and Raw-Lucia cells, and has a negative impact on STING The pathway produced an inhibitory effect, and the compound still inhibited the enzyme activity in THP-1 cells with cGAS knockout (THP1-Dual KO-cGAS cells) and Raw-Lucia cells (Raw-Lucia ISG-KO-cGAS cells). Therefore, it is considered that this compound does not act on the upstream cGAS, but on STING or its downstream; its structural analog Amentoflavone does not inhibit the activation of the STING pathway. As shown in Figure 2D-E, the cytotoxicity of compound Ginkgetin was also evaluated. After the compound treated THP-1 cells for 24 hours, the cell viability was tested and it was found that Ginkgetin had no obvious toxicity within 25 μM. After the compound treated Raw 264.7 cells for 24 hours, the cell viability was tested and it was found that Ginkgetin had no obvious toxicity within 50 μM. Therefore, the possibility of inhibitory effects due to toxicity of the compounds was ruled out.
2.2.小分子化合物Ginkgetin明显抑制STING诱导的IFNB1 mRNA的表达2.2. The small molecule compound Ginkgetin significantly inhibits STING-induced IFNB1 mRNA expression.
实验方法：experimental method:
HEK293T提前种于六孔板中分别通过转染过表达Flag-STINGR232(Gene ID:340061)、Flag-TBK1(Gene ID:29110)、IRF3-5D(Gene ID:3661)，三种质粒均使用 PolyJet试剂(SignaGen)转染，转染24h后，用不同浓度的Ginkgetin(0.098-25μM) 处理6h，通过qPCR检测细胞中IFNB1 mRNA的转录水平。HEK293T was seeded in six-well plates in advance and overexpressed Flag-STING R232 (Gene ID: 340061), Flag-TBK1 (Gene ID: 29110), and IRF3-5D (Gene ID: 3661) by transfection. All three plasmids were used The cells were transfected with PolyJet reagent (SignaGen). After 24 hours of transfection, they were treated with different concentrations of Ginkgetin (0.098-25 μM) for 6 hours, and the transcription level of IFNB1 mRNA in the cells was detected by qPCR.
实验结果：Experimental results:
如图2中F所示，Ginkgetin明显减弱了STINGR232诱导的IFNB1 mRNA的表达，而对TBK1或IRF3-5D诱导的IFNB1 mRNA的表达并没有影响。考虑到这些信号分子之间的上下游关系，再结合上面提到的报告基因实验，更加确定Ginkgetin是直接作用于STING而不是cGAS、TBK1或者IRF3。As shown in F in Figure 2 , Ginkgetin significantly attenuated the expression of IFNB1 mRNA induced by STING R232 , but had no effect on the expression of IFNB1 mRNA induced by TBK1 or IRF3-5D. Considering the upstream and downstream relationships between these signaling molecules, combined with the reporter gene experiments mentioned above, it is more certain that Ginkgetin directly acts on STING rather than cGAS, TBK1 or IRF3.
实施例3小分子化合物Ginkgetin在细胞内抑制了STING通路的激活和信号传导Example 3 The small molecule compound Ginkgetin inhibits the activation and signaling of the STING pathway in cells
3.1.Ginkgetin抑制了2’,3’-cGAMP刺激引起的STING通路基因的表达3.1. Ginkgetin inhibits the expression of STING pathway genes induced by 2’,3’-cGAMP stimulation
实验方法：experimental method:
DMSO或2’,3’-cGAMP处理后的THP-1细胞(存在或不存在Ginkgetin)的RNA测序分析。用DMSO或Ginkgetin(10μM)预处理THP-1细胞，然后用或不用2’,3’-cGAMP (5μM)刺激6小时。2’,3’-cGAMP刺激后，用PBS洗涤收集细胞，细胞沉淀加入RNA isolater Total RNAExtraction Reagent(Vazyme,R401-01)，吹打均匀后，装入预冷的冻存管中，液氮速冻半小时，先放入-80℃暂存。样本后续的质检、RNA测序和差异基因表达分析由美吉生物完成。归一化值的热图和火山图使用RStudio中的pheatmap函数和 ggplot函数进行作图，差异基因功能富集分析使用GESA软件运行分析得到结果。RNA sequencing analysis of THP-1 cells treated with DMSO or 2’,3’-cGAMP (in the presence or absence of Ginkgetin). THP-1 cells were pretreated with DMSO or Ginkgetin (10 μM) and then stimulated with or without 2’,3’-cGAMP (5 μM) for 6 hours. After stimulation with 2',3'-cGAMP, collect cells by washing with PBS. Add RNA isolator Total RNAExtraction Reagent (Vazyme, R401-01) to the cell pellet. After pipetting evenly, put it into a pre-cooled cryovial and freeze half in liquid nitrogen. hours, first put it into -80℃ for temporary storage. The subsequent quality inspection, RNA sequencing and differential gene expression analysis of the samples were completed by Meiji Biotech. The heat map and volcano plot of normalized values were plotted using the pheatmap function and ggplot function in RStudio, and the differential gene functional enrichment analysis was run using GESA software to obtain the results.
实验结果：Experimental results:
如图3中A-C所示，分析Ginkgetin对THP-1细胞中由2’,3’-cGAMP启动的转录程序的影响表明，上调最多的基因中关于STING通路比较有代表性的基因大幅减少，比如IFNB1、IL6、CXCL10等，另外，在差异基因的功能富集分析的比对结果显示3个最明显下调的基因集为：‘interferon gamma response’,‘interferon alpha response’, ‘inflammatoryresponse’，这更加证明了Ginkgetin明显抑制了STING下游信号通路的传导。As shown in Figure 3A-C, analysis of the effect of Ginkgetin on the transcription program initiated by 2',3'-cGAMP in THP-1 cells showed that among the most up-regulated genes, the genes that are more representative of the STING pathway were significantly reduced, such as IFNB1, IL6, CXCL10, etc. In addition, the comparison results of functional enrichment analysis of differential genes showed that the three most significantly down-regulated gene sets are: 'interferon gamma response', 'interferon alpha response', 'inflammatory response', which is more It was proved that Ginkgetin significantly inhibited the conduction of STING downstream signaling pathway.
3.2.Ginkgetin对2’,3’-cGAMP激活STING通路的抑制3.2. Ginkgetin inhibits the STING pathway activated by 2’,3’-cGAMP
实验方法：experimental method:
Ginkgetin与内源性STING激动剂2’,3’-cGAMP共同处理THP-1细胞2h，然后通过Western Blot检测化合物对STING通路中关键蛋白磷酸化的作用，或者利用免疫荧光实验检测对IRF3和p65核易位的影响。THP-1 cells were treated with Ginkgetin and the endogenous STING agonist 2',3'-cGAMP for 2 hours, and then the effect of the compound on the phosphorylation of key proteins in the STING pathway was detected through Western Blot, or the effects on IRF3 and p65 were detected using immunofluorescence experiments. Effects of nuclear translocation.
实验结果：Experimental results:
如图3中D所示，2’,3’-cGAMP等激动剂能刺激诱导STING、TBK1、IRF3、p65 和STAT3的磷酸化，而Ginkgetin显著降低了它们的磷酸化水平，且呈浓度依赖性，同样地，免疫荧光实验结果(图3中E-F)显示单用Ginkgetin处理对IRF3和p65并无明显影响，但是在Ginkgetin的存在下，显著减弱了2’,3’-cGAMP刺激引起的IRF3和p65 的核易位。以上结果均证明Ginkgetin对2’,3’-cGAMP引起的STING通路的激活具有抑制作用。As shown in D in Figure 3, 2',3'-cGAMP and other agonists can stimulate and induce the phosphorylation of STING, TBK1, IRF3, p65 and STAT3, while Ginkgetin significantly reduces their phosphorylation levels in a concentration-dependent manner. , Similarly, the results of immunofluorescence experiments (E-F in Figure 3) show that Ginkgetin treatment alone has no obvious effect on IRF3 and p65, but in the presence of Ginkgetin, it significantly weakens the IRF3 and p65 induced by 2',3'-cGAMP stimulation. Nuclear translocation of p65. The above results all prove that Ginkgetin has an inhibitory effect on the activation of the STING pathway caused by 2’,3’-cGAMP.
3.3.Ginkgetin特异性抑制细胞内STING通路的激活3.3. Ginkgetin specifically inhibits the activation of the STING pathway in cells
实验方法：experimental method:
用Ginkgetin(10μM)处理THP-1细胞或Raw 264.7细胞1h后，再用不同的STING 激动剂(包括2’,3’-cGAMP、GSK3、ADU-S100、SR-717、MSA-2)刺激5h，利用实时荧光定量PCR(qPCR)方法检测THP-1细胞的IFNB1、TNF以及Raw 264.7细胞Ifnb1、 Cxcl10、Il6基因的表达水平。THP-1 cells or Raw 264.7 cells were treated with Ginkgetin (10μM) for 1 hour, and then stimulated with different STING agonists (including 2',3'-cGAMP, GSK3, ADU-S100, SR-717, MSA-2) for 5 hours. , real-time fluorescence quantitative PCR (qPCR) method was used to detect the expression levels of IFNB1 and TNF in THP-1 cells and Ifnb1, Cxcl10 and Il6 genes in Raw 264.7 cells.
双链RNA类似物poly(I:C)用Lipofectamine 2000试剂(Thermo Fisher)转染进THP-1 细胞或Raw 264.7细胞；在脂多糖(LPS,5μg/mL)前2h加入化合物；干扰素刺激DNA(ISD)和G3-YSD(一段dsDNA)均用PolyJet试剂转染进THP-1细胞与Raw 264.7 细胞。在指定时间提取细胞内RNA，逆转录后通过qPCR方法检测相关mRNA的表达。The double-stranded RNA analog poly(I:C) was transfected into THP-1 cells or Raw 264.7 cells using Lipofectamine 2000 reagent (Thermo Fisher); the compound was added 2 hours before lipopolysaccharide (LPS, 5 μg/mL); interferon stimulated DNA (ISD) and G3-YSD (a segment of dsDNA) were transfected into THP-1 cells and Raw 264.7 cells using PolyJet reagent. Intracellular RNA was extracted at the specified time, and the expression of relevant mRNA was detected by qPCR method after reverse transcription.
Western Blot检测Ginkgetin与GSK3、ADU-S100、SR-717等STING激动剂联用后对细胞内STING通路关键蛋白磷酸化的影响。Western Blot detects the effect of Ginkgetin combined with STING agonists such as GSK3, ADU-S100, SR-717, etc. on the phosphorylation of key proteins in the STING pathway in cells.
实验结果：Experimental results:
如图4-7所示，Ginkgetin在不同STING激动剂的作用下抑制了THP-1细胞内IFNB1、TNF以及Raw 264.7细胞中Ifnb1、Cxcl10、Il6基因的转录，而且Ginkgetin对细胞内STING通路激活引起的关键蛋白的磷酸化水平也存在抑制作用。更重要的是，它可以特异性抑制双链DNA引起的基因表达，并且呈剂量依赖性，但并不影响poly(I:C) 触发的RIG-I介导的信号和LPS刺激的TLR4通路的激活，这些数据表明Ginkgetin可以选择性地抑制STING介导的下游信号通路的激活，且而不影响其他模式识别受体通路。As shown in Figure 4-7, Ginkgetin inhibited the transcription of IFNB1, TNF in THP-1 cells and Ifnb1, Cxcl10, and Il6 genes in Raw 264.7 cells under the action of different STING agonists, and Ginkgetin caused activation of the STING pathway in cells. There is also an inhibitory effect on the phosphorylation levels of key proteins. More importantly, it can specifically inhibit gene expression caused by double-stranded DNA in a dose-dependent manner, but does not affect poly(I:C)-triggered RIG-I-mediated signaling and LPS-stimulated TLR4 pathway. Activation, these data indicate that Ginkgetin can selectively inhibit the activation of STING-mediated downstream signaling pathways without affecting other pattern recognition receptor pathways.
实施例4小分子化合物Ginkgetin阻断了STING与TBK1的相互作用Example 4 The small molecule compound Ginkgetin blocks the interaction between STING and TBK1
实验方法：experimental method:
HEK293T细胞转染Flag-STINGH232(Gene ID:340061)和Flag-TBK1(Gene ID:29110)两个质粒24h后加入Ginkgetin和2’,3’-cGAMP，处理6h后提取HEK293T细胞的裂解物，通过免疫共沉淀方法检测STING和TBK1之间的相互作用。用Ginkgetin 和2’,3’-cGAMP刺激表达STINGH232的HeLa细胞2h后，用免疫荧光实验检测STING 与TBK1的位置情况。HEK293T cells were transfected with two plasmids, Flag-STING H232 (Gene ID: 340061) and Flag-TBK1 (Gene ID: 29110). After 24 hours, Ginkgetin and 2',3'-cGAMP were added. After 6 hours of treatment, the lysates of HEK293T cells were extracted. The interaction between STING and TBK1 was detected by co-immunoprecipitation method. After stimulating HeLa cells expressing STING H232 with Ginkgetin and 2',3'-cGAMP for 2 hours, immunofluorescence experiments were used to detect the positions of STING and TBK1.
实验结果：Experimental results:
如图8所示，通过抗Flag和抗Myc的免疫共沉淀结果以及免疫荧光实验中STING 和TBK1的位置可知，Ginkgetin阻断了STING和TBK1的相互作用。As shown in Figure 8, it can be seen from the co-immunoprecipitation results of anti-Flag and anti-Myc and the positions of STING and TBK1 in immunofluorescence experiments that Ginkgetin blocks the interaction between STING and TBK1.
实施例5小分子化合物Ginkgetin缓解Trex1-/-小鼠的自身炎症反应Example 5 The small molecule compound Ginkgetin alleviates the autoinflammatory response in Trex1 -/- mice
实验方法：experimental method:
为了评价Ginkgetin在Trex1-/-小鼠的体内抑制作用，对实验小鼠(由中科院上海营养与健康研究所赠送，在中科院上海药物研究所海科路园区动物房饲养繁殖，许可证号：SYKX(沪)2020-0042，SPF级别。)分为四组：①WT小鼠，注射溶剂组；②WT小鼠，注射Ginkgetin组；③Trex1-/-小鼠，注射溶剂组；④Trex1-/-小鼠，注射Ginkgetin组。选用6周龄小鼠，每隔一天腹腔注射Ginkgetin(5mg/kg)，共3周，收集血清和组织作进一步分析。In order to evaluate the inhibitory effect of Ginkgetin in Trex1 -/- mice, experimental mice (gifted from Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, raised and bred in the animal room of Haike Road Park, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, license number: SYKX (Shanghai) 2020-0042, SPF level.) were divided into four groups: ①WT mice, solvent injection group; ②WT mice, Ginkgetin injection group; ③Trex1 -/- mice, solvent injection group; ④Trex1 -/- mice, Ginkgetin injection group. Six-week-old mice were selected and intraperitoneally injected with Ginkgetin (5mg/kg) every other day for a total of 3 weeks. Serum and tissues were collected for further analysis.
实验结果：Experimental results:
如图9-11所示，Ginkgetin抑制Trex1-/-小鼠BMDMs细胞和组织中的I型干扰素和促炎细胞因子(Ifnb1、Cxcl10、Il6、Tnf、Isg15和Isg56)的表达。组织学结果表明，给予Ginkgetin可以缓解Trex1-/-小鼠的心脏、胃、舌头、肌肉组织的自身炎症症状。因此，这些数据共同说明Ginkgetin有力地减轻了Trex1-/-小鼠的炎症表型。As shown in Figures 9-11, Ginkgetin inhibits the expression of type I interferons and pro-inflammatory cytokines (Ifnb1, Cxcl10, Il6, Tnf, Isg15 and Isg56) in Trex1 -/- mouse BMDMs cells and tissues. Histological results showed that administration of Ginkgetin could alleviate autoinflammatory symptoms in the heart, stomach, tongue, and muscle tissue of Trex1 -/- mice. Therefore, these data collectively demonstrate that Ginkgetin potently reduces the inflammatory phenotype in Trex1 −/− mice.
实施例6小分子化合物Ginkgetin改善了DSS诱导的小鼠急性结肠炎Example 6 The small molecule compound Ginkgetin improves DSS-induced acute colitis in mice
实验方法：experimental method:
选用6-8周龄的C57BL/6J小鼠(由中科院上海药物研究所祖冲之路园区动物房统一订购，IACUC号：2021-10-JHL-24，SPF级别饲养。)，用无菌水配制3％(w/v)葡聚糖硫酸钠(DSS)饮用水，连续饮用7天诱导小鼠急性结肠炎。对实验小鼠分为三组：①正常水，注射溶剂(5％DMSO+生理盐水)组；②3％DSS水，注射溶剂组；③3％DSS 水，注射Ginkgetin组。在DSS诱导的结肠炎期间，化合物以20mg/kg的剂量腹腔注射，每日一次并记录其体重变化。9天后采集血样，收集血清，利用酶联免疫吸附实验 (ELISA)试剂盒检测血清中特定抗体的含量。同时，分离取出小鼠的结肠，进行肠长的测量、组织病理学和细胞因子测定。C57BL/6J mice aged 6-8 weeks were selected (ordered from the animal room of Zuchongzhizhi Park, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, IACUC number: 2021-10-JHL-24, raised at SPF level.), and prepared with sterile water 3 % (w/v) dextran sodium sulfate (DSS) in drinking water for 7 days to induce acute colitis in mice. The experimental mice were divided into three groups: ① normal water, solvent injection group (5% DMSO + physiological saline) group; ② 3% DSS water, solvent injection group; ③ 3% DSS water, Ginkgetin injection group. During DSS-induced colitis, the compound was injected intraperitoneally at a dose of 20 mg/kg once daily and body weight changes were recorded. Blood samples were collected 9 days later, and serum was collected, and an enzyme-linked immunosorbent assay (ELISA) kit was used to detect the content of specific antibodies in the serum. At the same time, the colon of the mice was isolated and the intestinal length was measured, histopathologically and cytokines determined.
实验结果：Experimental results:
如图12所示，在上述三组急性结肠炎模型中，小鼠饮用3％DSS水，注射溶剂组显示出体重的急速下降，而在Ginkgetin处理的小鼠中，服用DSS后第5天到第8天体重下降显著减轻；DSS处理可显著缩短小鼠的结肠长度，而化合物处理均可显著改善小鼠的结肠长度。组织学检查显示，第9天模型组小鼠结肠切片中炎性细胞浸润、上皮严重破坏、隐窝丢失，而化合物治疗组小鼠上述特征得到改善。相应地，在模型小鼠中，Il6、 Il1b和Tnf在结肠切片中的表达显著增加，但在化合物处理的模型小鼠中的表达明显降低。同样，模型组小鼠血清中IL-6、IL-1β和TNF的浓度升高，而化合物治疗的模型组小鼠血清中的浓度也明显降低。综上所述，这些结果表明，Ginkgetin对DSS诱导的体内急性结肠炎有显著的治疗作用。As shown in Figure 12, in the above three groups of acute colitis models, the mice drank 3% DSS water, and the solvent injection group showed a rapid decrease in body weight, while in the Ginkgetin-treated mice, on the 5th day after taking DSS The weight loss was significantly reduced on the 8th day; DSS treatment could significantly shorten the colon length of mice, while compound treatment could significantly improve the colon length of mice. Histological examination showed that on day 9, inflammatory cell infiltration, severe epithelial destruction, and crypt loss were found in the colon sections of the mice in the model group, while the above characteristics of the mice in the compound treatment group were improved. Correspondingly, in model mice, the expression of Il6, Il1b and Tnf in colon slices was significantly increased, but the expression was significantly decreased in compound-treated model mice. Similarly, the concentrations of IL-6, IL-1β and TNF in the serum of mice in the model group increased, while the concentrations in the serum of mice in the compound-treated model group also decreased significantly. Taken together, these results indicate that Ginkgetin has a significant therapeutic effect on DSS-induced acute colitis in vivo.
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Claims (7)
- Application of ginkgetin in preparation of STING inhibitor.
- Use of ginkgetin for the manufacture of a medicament for the prevention or treatment of a disease associated with the activation of STING signaling pathway.
- 3. Use according to claim 2, wherein the disease associated with the activation of STING signalling pathway is selected from inflammatory diseases.
- 4. The use according to claim 3, wherein the inflammatory disease is selected from autoimmune diseases and acute colitis.
- 5. The use according to claim 4, wherein the autoimmune disease is selected from systemic lupus erythematosus, familial chilblain, STING-related vascular lesions, and vascular and pulmonary syndrome.
- 6. The use of claim 4, wherein the autoimmune disease is selected from Trex 1-deficient inflammation.
- 7. The use according to claim 2, wherein the disease associated with the activation of STING signaling pathway is acute colitis.
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| CN118161479A (en) * | 2024-03-12 | 2024-06-11 | 中国动物卫生与流行病学中心 | Application of Ginkgo biloba flavonoids in the preparation of preparations against porcine Seneca virus |
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| CN118161479B (en) * | 2024-03-12 | 2025-01-28 | 中国动物卫生与流行病学中心 | Application of Ginkgo biloba flavonoids in the preparation of preparations against porcine Seneca virus |
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