WO2023097610A1 - 马来酸茚达特罗在作为cGAS-STING通路靶向激动剂中的应用 - Google Patents

马来酸茚达特罗在作为cGAS-STING通路靶向激动剂中的应用 Download PDF

Info

Publication number
WO2023097610A1
WO2023097610A1 PCT/CN2021/135088 CN2021135088W WO2023097610A1 WO 2023097610 A1 WO2023097610 A1 WO 2023097610A1 CN 2021135088 W CN2021135088 W CN 2021135088W WO 2023097610 A1 WO2023097610 A1 WO 2023097610A1
Authority
WO
WIPO (PCT)
Prior art keywords
cgas
indacaterol maleate
protein
sting pathway
purchased
Prior art date
Application number
PCT/CN2021/135088
Other languages
English (en)
French (fr)
Inventor
何庆瑜
李杨葭
Original Assignee
暨南大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 暨南大学 filed Critical 暨南大学
Priority to PCT/CN2021/135088 priority Critical patent/WO2023097610A1/zh
Publication of WO2023097610A1 publication Critical patent/WO2023097610A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4704—2-Quinolinones, e.g. carbostyril
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the technical field of medicine, and specifically relates to the application of indacaterol maleate as a cGAS-STING pathway targeting agonist.
  • indacaterol maleate is as follows:
  • COPD chronic obstructive pulmonary disease
  • cyclic GMP-AMP synthase (cGAS) and interferon gene stimulator (stimulator of interferon genes, STING) pathway is a pattern recognition pathway that senses cytoplasmic double-stranded DNA through cGAS, thereby activating STING lead to the activation of inflammatory pathways [4].
  • cGAS can catalyze the generation of cyclic guanine nucleotide-adenine nucleotide (cyclic GMP-AMP, cGAMP), and cGAMP further activates STING and its downstream pathways[5].
  • Targeted activators of the cGAS-STING pathway have strong application value [6].
  • the present invention aims to demonstrate the application of indacaterol maleate as a cGAS-STING pathway targeting agonist, and provide ideas for the treatment of diseases such as tumors related to colorectal cancer.
  • the present invention shows that indacaterol maleate can target and activate the cGAS-STING pathway.
  • the invention provides a new application of indacaterol maleate, and provides a new drug source for adjuvant therapy of cancer.
  • indacaterol maleate is a drug that has been approved by the FDA for the treatment of chronic obstructive pulmonary disease. This study found that indacaterol maleate can target the cGAS-STING pathway as its activator in addition to its existing effects. Compared with the development of new drugs, it has lower cost, higher safety and better development prospects.
  • Figure 1 is the identification of indacaterol maleate targeting cGAS-STING pathway by biotin photoaffinity labeling combined with proteomics.
  • Figure 2 is the verification of indacaterol maleate targeting cGAS in vitro.
  • the cells were lysed with RIPA lysate (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.), 100 ⁇ L per well, centrifuged at 4°C, 14000 ⁇ g for 20 minutes, and the protein supernatant was collected, and BCA reagent (purchased from Thermo Fisher Scientific ) to measure the protein concentration.
  • RIPA lysate purchased from Shanghai Biyuntian Biotechnology Co., Ltd.
  • BCA reagent purchased from Thermo Fisher Scientific
  • the reaction was terminated with 500 ⁇ M pre-cooled acetone, and the protein was precipitated by incubation at -20°C for 1 hour.
  • the precipitated protein was separated by centrifugation at 4°C/14000 ⁇ g, and redissolved with 30 ⁇ L of SDS lysate containing 1 ⁇ loading buffer, and bathed in 95°C water for 10 minutes. Equal samples from each group were electrophoresed on a 10% SDS-PAGE gel. After the electrophoresis, the gel was scanned for fluorescence imaging with a Typhoon 9500 fluorescent gel scanner (Amersham Biosciences), stained with Coomassie brilliant blue, and photographed.
  • Spread colorectal cancer cells HT29 (purchased from ATCC) into a 10cm cell dish, wait until the cell density grows to 80%-90%, remove the culture medium, add indacaterol maleate-probe concentration is 0, Serum-free 1640 medium (purchased from GIBCO) at 30 ⁇ M was incubated in a 37° C./5% CO 2 incubator for 4.5 hours. After the incubation, the culture medium was removed, washed twice with pre-cooled PBS, the cell dish was placed on ice, and irradiated with 365nm UV light for 10 minutes.
  • the reaction was terminated with 500 ⁇ M pre-cooled acetone, and the protein was precipitated by incubation at -20°C for 1 hour. Redissolve the protein with PBS containing 1% SDS and incubate overnight at 4 °C with 80 ⁇ L streptavidin sepharose beads. The agarose beads were collected by centrifugation at 4°C/2500 rpm for 10 minutes, and the protein was lysed with 50 ⁇ L of SDS lysis buffer.
  • Each group was added with an equal volume of 8M urea solution and DTT solution with a final concentration of 50mM, and reacted in a water bath at 37°C for 1 hour.
  • the reacted solution was added to a 30kD ultrafiltration tube previously rinsed with 50 mM TEAB (purchased from Sigma-Aldrich), rinsed twice with 200 ⁇ L urea and five times with 200 ⁇ L TEAB.
  • Add 20 ⁇ g of mass spectrometry-grade trypsin purchasedd from Beijing Hualishi Technology Co., Ltd.
  • the peptide solution after enzymatic hydrolysis was desalted by MonoTIPTM C 18 desalting column (purchased from GL Sciences), and mass spectrometry was performed with Orbitrap Fusion Lumos mass spectrometer mass spectrometer (purchased from Thermo Fisher Scientific), and the original data was analyzed by Spectronaut software (Omicsolution Co. ., Ltd.) to search the library.
  • Spread colorectal cancer cells HT29 (purchased from ATCC) into a 10cm cell dish, wait until the cell density grows to 80%-90%, remove the culture medium, add indacaterol maleate-probe concentration is 0, Serum-free 1640 medium (purchased from GIBCO) at 30 ⁇ M was incubated in a 37° C./5% CO 2 incubator for 4.5 hours. After the incubation, the culture medium was removed, washed twice with pre-cooled PBS, the cell dish was placed on ice, and irradiated with 365nm UV light for 10 minutes.
  • the reaction was terminated with 500 ⁇ M pre-cooled acetone, and the protein was precipitated by incubation at -20°C for 1 hour. Redissolve the protein with PBS containing 1% SDS and incubate overnight at 4 °C with 80 ⁇ L streptavidin sepharose beads. Collect the agarose beads by centrifugation at 4°C/2500rpm for 10 minutes, and lyse the protein with 50 ⁇ L of SDS lysate containing 1 ⁇ loading buffer. An equal amount of samples from each group was run by 10% SDS-PAGE gel electrophoresis for Western blot experiments, and the bands were incubated with the target protein cGAS antibody and developed.
  • HT29 cell line knocked out of endogenous cGAS protein was constructed with sgcGAS plasmid.
  • the sgcGAS plasmid was purchased from Guangzhou Aiji Biotechnology Co., Ltd., the sgRNA fragment was CGCATCCCTCCGTACGAGAA, and the plasmid vector was lentiCRISPRv2.
  • 293T cells in logarithmic phase (purchased from ATCC) were inoculated into 6-well plates at a density of 50%, and cultivated for 12 hours; sgcGAS plasmid and packaging plasmid PSPAX2 (purchased from Thermo Fisher Scientific) were mixed in opti-MEM (purchased from Addgene) and PMD2G (purchased from Addgene) were mixed separately, and p3000 (purchased from Life Science) was added, and another tube of opti-MEM was added to Lipo3000 (Life Science), and the two tubes were mixed separately.
  • the HT29 cells knocked out of cGAS and the cells in the control group were lysed to get the protein, and the concentration was measured by the BCA method, and an equal amount was added to 5 ⁇ loading buffer to prepare samples, and a 10% SDS-PAGE gel was used for Western blot experiments.
  • cGAS knockout was detected by incubating with cGAS antibody (purchased from ABclonal).
  • the HT29 cells in the control group and the cGAS knockout group were plated into 96-well plates, with 3000 cells per well.
  • cGAMP enzyme-linked immunosorbent assay was used to identify the level of cGAMP in colorectal cancer cells treated with indacaterol maleate.
  • the culture medium was removed, washed twice with PBS, the cells were lysed with M-PER MAMMALIAN PROTEIN EXTRACTION REAGENT lysate (purchased from Thermo Fisher Scientific), and cGAMP in the cells was detected with cGAMP ELISA kit (purchased from Cayman Chemical) level.
  • the cells in each group were lysed to take the protein, and after the concentration was measured by the BCA method, an equal amount was added to 5 ⁇ loadingbuffer for sample preparation, and a 10% SDS-PAGE gel was used for Western blotting experiments, and the strips after transfer were incubated with pSTING antibodies , pTBK1 antibody, pIRF3 antibody (all purchased from ABcloncal) and ⁇ -Actin antibody (purchased from Bioworld) to detect the downstream activation of the cGAS-STING pathway.
  • IFN ⁇ enzyme-linked immunosorbent assay was used to identify the level of IFN ⁇ in the supernatant of colorectal cancer cells treated with indacaterol maleate.
  • the cell supernatant was collected, centrifuged at 4°C/1000 ⁇ g to remove cell debris, and the Human IFN- ⁇ ELISA kit (purchased from Hangzhou Lianke Biotechnology Co., Ltd.) was used to detect the IFN ⁇ level in the cell culture supernatant.
  • cGAS is a potential target of indacaterol maleate that can be detected by the pull-down of the indacaterol maleate probe, but not detected in the control group protein.
  • the cGAS protein expression of the HT29 cell line knocked out of the endogenous cGAS protein and the control cell line were detected by Western blotting, and the cGAS endogenous protein was successfully knocked out; as shown in Figure 2C, the cGAS knockout
  • the sensitivity of HT29 cells to indacaterol maleate was weaker than that of the control group. Under the treatment of indacaterol maleate at the same concentration and time, the viability of HT29 cells knocked out of cGAS was stronger, indicating that Malayan Indacaterol inhibited the growth of HT29 cells through cGAS.
  • Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science. 2013; 339:786–791.5. Civril, F. et al.

Landscapes

  • Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

提供了马来酸茚达特罗在作为cGAS-STING通路靶向激动剂中的应用。通过体外水平验证、生物素光亲和标记，结合蛋白质组学、生物信息学、分子生物学结果表明马来酸茚达特罗可以靶向激活cGAS-STING通路，抑制癌细胞增殖，对于辅助治疗结直肠癌相关疾病具有良好的治疗前景。

Description

马来酸茚达特罗在作为cGAS-STING通路靶向激动剂中的应用 技术领域
本发明属于医药技术领域，具体涉及为马来酸茚达特罗在作为cGAS-STING通路靶向激动剂中的应用。
背景技术
马来酸茚达特罗结构式如图：
Figure PCTCN2021135088-appb-000001
慢性阻塞性肺病(chronic obstructive pulmoriary disease,COPD)是一种持续性、进展性气流受限的疾病[1]。由于外周肌肉功能受损、通气受阻及心肺功能受损等因素，COPD患者常常出现呼吸困难、肌肉无力等症状，进一步引起活动耐量受损，造成生活质量下降[2]。马来酸茚达特罗作为一种支气管扩张剂，是一种长效β2受体激动剂，能够持续舒张支气管平滑肌，缓解肺过度膨胀，从而改善气流受限，减轻COPD患者的症状[3]。
环状GMP-AMP合成酶(cyclic GMP-AMP synthase,cGAS)和干扰素基因刺激因子(stimulator of interferon genes,STING)通路是一种模式识别通路，通过cGAS感知胞质双链DNA，进而激活STING导致炎症通路的激活[4]。cGAS能够催化环状鸟嘌呤核苷酸-腺嘌呤核苷酸(cyclic GMP-AMP,cGAMP)的生成，cGAMP进一步激活STING及其下游通路[5]。cGAS-STING通路的靶向激活剂具有很强的应用价值[6]。
目前尚未有文章表明马来酸茚达特罗和cGAS-STING通路的关系，尚无专利申请。
发明内容
本发明旨在展示马来酸茚达特罗在作为cGAS-STING通路靶向激动剂中的应用，为治疗与结直肠癌相关肿瘤等疾病提供思路。
本发明通过体外实验，结合蛋白质组学、生物信息学、分子生物学结果表明马来酸茚达特罗可以靶向激活cGAS-STING通路。
利用生物素光亲和标记结合蛋白质组学鉴定马来酸茚达特罗靶向cGAS-STING通路：将马来酸茚达特罗与光交联探针结合，活细胞原位标记鉴定马来酸茚达特罗-探针与潜在靶点蛋白的结合，下拉-质谱联用鉴定马来酸茚达特罗潜在靶点蛋白。
利用体外水平验证马来酸茚达特罗靶向cGAS：下拉-蛋白质印迹法联用验证马来酸茚达特罗靶向cGAS，细胞水平敲除cGAS验证马来酸茚达特罗靶向cGAS。
利用体外水平验证马来酸茚达特罗激活cGAS-STING通路：cGAMP酶联免疫吸附法鉴定马来酸茚达特罗处理后结直肠癌细胞内cGAMP水平，蛋白质印迹法鉴定马来酸茚达特罗处理后cGAS-STING下游蛋白水平，IFNβ酶联免疫吸附法鉴定马来酸茚达特罗处理后结直肠癌细胞上清中IFNβ水平。
本发明提供马来酸茚达特罗的新应用，为癌症的辅助治疗提供一种新的药物来源。
与现有技术相比，本发明具有以下有益效果：马来酸茚达特罗是一种FDA已批准上市用于治疗慢性阻塞性肺病的药物。本研究发现马来酸茚达特罗除了现有作用之外，还可以靶向cGAS-STING通路，作为其激活剂，较开发新药物而言成本低，安全性高，开发前景较好。
附图说明
图1为生物素光亲和标记结合蛋白质组学鉴定马来酸茚达特罗靶向cGAS-STING通路。
图2为体外水平验证马来酸茚达特罗靶向cGAS。
图3体外水平验证马来酸茚达特罗激活cGAS-STING通路。
具体实施方式
以下通过实施例形式的具体实施方式，对本发明的上述内容作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下实施例。
实施例1
为了达到上述目的，本发明采用以下技术方案：
1、生物素光亲和标记结合蛋白质组学鉴定马来酸茚达特罗靶向cGAS-STING通路。具体步骤为：
(1)将马来酸茚达特罗与光交联探针结合。将50mg马来酸茚达特罗(购自上海陶素生化科技有限公司)溶于5mL二甲基甲酰胺(N,N-Dimethylformamide,DMF)中，并加入25.8mg探针二丫吡啶(3-(but-3-yn-1-yl)-3-(2-iodoethyl)-3H-diazirine)(购自上海毕得医药科技股份有限公司)及26mg碳酸钾，混匀溶液后在60℃搅拌12小时。反应结束后，加入10mL 超纯水降温，并通过乙酸乙酯萃取(2次，每次10mL)，用10mL盐水结合并洗涤有机层两次，用无水硫酸钠干燥。用闪蒸柱(二氯甲烷:甲醇＝30:1)层析纯化溶剂蒸发后剩余的残渣，得到带上光交联探针的马来酸茚达特罗(7mg)。
(2)活细胞原位标记鉴定马来酸茚达特罗-探针与潜在靶点蛋白的结合。将结直肠癌细胞HT29(购自ATCC)铺入6孔板中，待细胞密度长至80％-90％，去培养基，分别加入含有马来酸茚达特罗-探针浓度为0,7.5,15,30μM的无血清1640培养基(购自GIBCO)，在37℃/5％CO 2培养箱中孵育4.5小时。孵育结束后，去培养基，用预冷PBS洗两次，将6孔板置于冰上，用365nm UV光照10分钟。光照结束后，用RIPA裂解液(购自上海碧云天生物技术有限公司)裂解细胞，每孔100μL，4℃，14000×g离心20分钟后收集蛋白上清，并用BCA试剂(购自Thermo Fisher Scientific)测蛋白浓度。各组取等量蛋白，加入现配的点击化学反应试剂(50μM TAMEA-N3，0.1mM TBTA，1mM TCEP，1mM CuSO 4，购自Sigma-Aldrich及Click Chemistry Tools)，在混匀仪上室温反应两小时。反应结束后，用500μM预冷丙酮结束反应，在-20℃孵育1小时沉淀蛋白。沉淀的蛋白通过4℃/14000×g离心分离，并用30μL含有1×loading buffer的SDS裂解液重溶，95℃水浴10分钟。各组取等量样品用10％SDS-PAGE胶跑电泳，电泳结束后的凝胶用Typhoon 9500荧光凝胶扫描仪(Amersham Biosciences)扫描荧光成像，并用考马斯亮蓝染色、拍照。
(3)下拉-质谱联用鉴定马来酸茚达特罗潜在靶点蛋白。
将结直肠癌细胞HT29(购自ATCC)铺入10cm细胞皿中，待细胞密度长至80％-90％，去培养基，分别加入含有马来酸茚达特罗-探针浓度为0,30μM的无血清1640培养基(购自GIBCO)，在37℃/5％CO 2培养箱中孵育4.5小时。孵育结束后，去培养基，用预冷PBS洗两次，将细胞皿置于冰上，用365nm UV光照10分钟。光照结束后，用RIPA裂解液(购自上海碧云天生物技术有限公司)裂解细胞，每皿1mL，4℃，14000×g离心20分钟后收集蛋白上清，并用BCA试剂(购自Thermo Fisher Scientific)测蛋白浓度。各组取等量蛋白，加入现配的点击化学反应试剂(50μM Biotin-N3，0.1mM TBTA，1mM TCEP，1mM CuSO 4，购自Sigma-Aldrich及Click Chemistry Tools)，在混匀仪上室温反应两小时。反应结束后，用500μM预冷丙酮结束反应，在-20℃孵育1小时沉淀蛋白。用含1％SDS的PBS重溶蛋白，并与80μL链霉亲和素琼脂糖珠4℃孵育过夜。通过4℃/2500rpm离心10分钟收集琼脂糖珠，并用50μL SDS裂解液裂解蛋白。
各组加入等体积的8M尿素溶液，加入终浓度为50mM的DTT溶液，37℃水浴反应1小时。加入终浓度为150mM的IAA溶液，室温避光反应30分钟。将反应后的溶液加入预先用50mM TEAB(购自Sigma-Aldrich)润洗的30kD超滤管中，并分别用200μL尿素润 洗两次，用200μLTEAB润洗五次。在超滤管中加入20μg质谱级胰酶(购自北京华利世科技有限公司)，37℃孵育过夜。酶解后的肽段溶液通过MonoTIPTM C 18除盐柱(购自GL Sciences)除盐，并用Orbitrap Fusion Lumos mass spectrometer质谱仪(购自Thermo Fisher Scientific)进行质谱分析，原始数据用Spectronaut软件(Omicsolution Co.,Ltd.)进行搜库。
2、体外水平验证马来酸茚达特罗靶向cGAS。具体步骤为：
(1)下拉-蛋白质印迹法联用验证马来酸茚达特罗靶向cGAS。
将结直肠癌细胞HT29(购自ATCC)铺入10cm细胞皿中，待细胞密度长至80％-90％，去培养基，分别加入含有马来酸茚达特罗-探针浓度为0,30μM的无血清1640培养基(购自GIBCO)，在37℃/5％CO 2培养箱中孵育4.5小时。孵育结束后，去培养基，用预冷PBS洗两次，将细胞皿置于冰上，用365nm UV光照10分钟。光照结束后，用RIPA裂解液(购自上海碧云天生物技术有限公司)裂解细胞，每皿1mL，4℃，14000×g离心20分钟后收集蛋白上清，并用BCA试剂(购自Thermo Fisher Scientific)测蛋白浓度。各组取等量蛋白，加入现配的点击化学反应试剂(50μM Biotin-N3，0.1mM TBTA，1mM TCEP，1mM CuSO 4，购自Sigma-Aldrich及Click Chemistry Tools)，在混匀仪上室温反应两小时。反应结束后，用500μM预冷丙酮结束反应，在-20℃孵育1小时沉淀蛋白。用含1％SDS的PBS重溶蛋白，并与80μL链霉亲和素琼脂糖珠4℃孵育过夜。通过4℃/2500rpm离心10分钟收集琼脂糖珠，并用50μL含1×loading buffer的SDS裂解液裂解蛋白。各组取等量样品用10％SDS-PAGE胶跑电泳进行蛋白印迹实验，用目标蛋白cGAS抗体孵育条带并显影。
(2)细胞水平敲除cGAS验证马来酸茚达特罗靶向cGAS。
用sgcGAS质粒构建敲除内源cGAS蛋白的HT29细胞株。sgcGAS质粒购自广州艾基生物技术有限公司，sgRNA片段为CGCATCCCTCCGTACGAGAA，质粒载体为lentiCRISPRv2。取对数期的293T细胞(购自ATCC)以50％的密度接种到6孔板中，培养12小时；在opti-MEM(购自Thermo Fisher Scientific)中将sgcGAS质粒与包装质粒PSPAX2(购自Addgene)及PMD2G(购自Addgene)分别混合，并加入p3000(购自Life Science)，同时另取一管opti-MEM加入Lipo3000(Life science)，两管分别混匀静置5分钟后，混匀静置20分钟；将293T细胞换液，并加入上述混合好的转染试剂，置于培养箱中培养6小时后更换新鲜的完全培养基培养48小时，收集细胞上清离心去除细胞碎片，并用0.45μm滤器过滤彻底去除碎片。将过滤后的上清加入到提前1天以30％的密度接种好HT29细胞的6孔板中感染24小时，去除含病毒培养基换成新鲜培养基。48小时后加入嘌呤霉素(1μg/mL)进行筛选，每两天换一次液，筛选一周后更换成正常培养基培养，检测cGAS蛋白敲除情况，保种备用。
敲除cGAS的HT29细胞及对照组细胞分别裂解取蛋白，用BCA法测浓度后取等量加入 5×loading buffer制样，用10％SDS-PAGE凝胶进行蛋白印迹实验，转膜后的条带孵育cGAS抗体(购自ABclonal)检测cGAS敲除情况。将对照组及敲除cGAS组的HT29细胞分别铺入96孔板中，每孔3000个细胞。培养12小时后，去培养基，两组各分两个马来酸茚达特罗处理浓度(0,15μM)处理24小时，用CCK8试剂(购自上海陶素生化科技有限公司)检测细胞增殖。
3、体外水平验证马来酸茚达特罗激活cGAS-STING通路。具体步骤为：
(1)cGAMP酶联免疫吸附法鉴定马来酸茚达特罗处理后结直肠癌细胞内cGAMP水平。将HT29细胞铺入6孔板中，每孔1.2×10 6个细胞，培养12小时。更换含有马来酸茚达特罗浓度分别为0,15,30μM的1640完全培养基培养48小时。处理结束后，去培养基，用PBS洗两次，用M-PER MAMMALIAN PROTEIN EXTRACTION REAGENT裂解液(购自Thermo Fisher Scientific)裂解细胞，用cGAMP ELISA试剂盒(购自Cayman Chemical)检测胞内的cGAMP水平。
(2)蛋白质印迹法鉴定马来酸茚达特罗处理后cGAS-STING下游蛋白水平。将HT29细胞铺入6孔板中，待细胞密度达到60％后，分别加入含有马来酸茚达特罗浓度分别为0,15,30μM的1640完全培养基培养48小时。处理结束后，各组细胞分别裂解取蛋白，用BCA法测浓度后取等量加入5×loadingbuffer制样，用10％SDS-PAGE凝胶进行蛋白印迹实验，转膜后的条带孵育pSTING抗体、pTBK1抗体、pIRF3抗体(均购自ABcloncal)及β-Actin抗体(购自Bioworld)检测cGAS-STING通路下游激活情况。
(3)IFNβ酶联免疫吸附法鉴定马来酸茚达特罗处理后结直肠癌细胞上清中IFNβ水平。将HT29细胞铺入6孔板中，每孔1.2×10 6个细胞，培养12小时。更换含有马来酸茚达特罗浓度分别为0,15,30μM的无血清1640培养基培养48小时。处理结束后，收集细胞上清，4℃/1000×g离心去除细胞碎片，用Human IFN-βELISA试剂盒(购自杭州联科生物技术股份有限公司)检测细胞培养上清中的IFNβ水平。
生物素光亲和标记结合蛋白质组学鉴定马来酸茚达特罗靶向cGAS-STING通路。
马来酸茚达特罗与光交联探针结合后的核磁共振氢谱如图1A所示，结果显示二者成功结合。活细胞原位标记实验结果如图1B所示，在荧光成像(左)中，随着马来酸茚达特罗-探针浓度的增加，其下拉的蛋白量也呈现递增趋势，考马斯亮蓝染色的全蛋白(右)作为内参，表明马来酸茚达特罗-探针能够下拉与其结合的潜在靶点蛋白。如图1C所示，通过下拉-质谱联用分析，cGAS是在马来酸茚达特罗-探针能够下拉检测到，而对照组中检测不到的马来酸茚达特罗潜在靶点蛋白。
体外水平验证马来酸茚达特罗靶向cGAS。
如图2A所示，在对照组及马来酸茚达特罗-探针组的全蛋白裂解液中，均能够检测到等量的cGAS蛋白，而在对照组和用马来酸茚达特罗-探针下拉的蛋白裂解液中，仅有后者能够检测到cGAS蛋白，说明马来酸茚达特罗-探针能够与cGAS蛋白特异性结合并将其下拉。如图2B所示，通过蛋白印迹法检测敲除内源cGAS蛋白的HT29细胞株及对照组细胞株的cGAS蛋白表达情况，cGAS内源蛋白敲除成功；如图2C所示，敲除cGAS的HT29细胞较对照组细胞对于马来酸茚达特罗的敏感性更弱，在同样浓度同样时间的马来酸茚达特罗处理下，敲除cGAS的HT29细胞生存能力更强，说明马来酸茚达特罗抑制HT29细胞生长是通过cGAS进行的。
体外水平验证马来酸茚达特罗激活cGAS-STING通路。
如图三A所示，随着马来酸茚达特罗处理浓度的升高，细胞内的cGAMP水平也呈现递增趋势，说明马来酸茚达特罗能够靶向激活cGAS产生cGAMP，cGAMP进一步激活STING及下游通路。如图三B所示，随着马来酸茚达特罗处理浓度的升高，cGAS-STING通路STING、TBK1、IRF3蛋白的激活形式pSTING、pTBK1、pIRF3蛋白量均呈现递增趋势，说明马来酸茚达特罗能够激活cGAS-STING通路。如图三C所示，随着马来酸茚达特罗处理浓度的升高，细胞上清中的IFNβ水平也呈现递增趋势，说明马来酸茚达特罗处理能够激活cGAS-STING通路进而激活下游I型干扰素的生成。
参考文献：
1.Donald PT.Indacaterol maleate for the treatment ofchronic obstructive pulmonary disease.Expert Opin.Pharmacother.2010；11(12):2077-2085.2.AlivertiA.et al.
2.The major limitation to exercise performance in COPD is inadequate energy supply to the respiratory and locomotor muscles.Journal ofapplied physiology.2008；105:749-751.3.Barnes PM.et al.
3.Complementary and alternative medicine use among adults:United States,2002.Seminars in Integrative Medicine.2004；2:54-71.4.Sun,L.et al.
4.Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.Science.2013；339:786–791.5.Civril,F.et al.
5.Structural mechanism ofcytosolic DNA sensing by cGAS.Nature.2013；498:332–3376.Woo,S.-R.et al.
6.The STING pathway and the T cellinflamed tumor microenvironment.Trends Immunol. 2015；36:250–256.
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (5)

  1. 马来酸茚达特罗在作为cGAS-STING通路靶向激动剂中的应用。
  2. 马来酸茚达特罗在制备治疗与cGAS-STING通路相关疾病药物中的应用。
  3. 根据权利要求2所述的应用，其特征在于，所述与cGAS-STING通路相关的疾病为直肠癌。
  4. 根据权利要求2所述的应用，其特征在于，所述药物为治疗直肠癌的药物。
  5. 根据权利要求1-4任一所述的应用，其特征在于，所述马来酸茚达特罗的浓度为0-30μM。
PCT/CN2021/135088 2021-12-02 2021-12-02 马来酸茚达特罗在作为cGAS-STING通路靶向激动剂中的应用 WO2023097610A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/135088 WO2023097610A1 (zh) 2021-12-02 2021-12-02 马来酸茚达特罗在作为cGAS-STING通路靶向激动剂中的应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/135088 WO2023097610A1 (zh) 2021-12-02 2021-12-02 马来酸茚达特罗在作为cGAS-STING通路靶向激动剂中的应用

Publications (1)

Publication Number Publication Date
WO2023097610A1 true WO2023097610A1 (zh) 2023-06-08

Family

ID=86611254

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/135088 WO2023097610A1 (zh) 2021-12-02 2021-12-02 马来酸茚达特罗在作为cGAS-STING通路靶向激动剂中的应用

Country Status (1)

Country Link
WO (1) WO2023097610A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107582550A (zh) * 2017-09-12 2018-01-16 浙江大学 茚达特罗在治疗结直肠癌中的应用
CN110013481A (zh) * 2019-04-30 2019-07-16 暨南大学 马来酸茚达特罗在制备抗肿瘤药物中的应用
CN113876772A (zh) * 2021-09-29 2022-01-04 暨南大学 马来酸茚达特罗在作为cGAS-STING通路靶向激动剂中的应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107582550A (zh) * 2017-09-12 2018-01-16 浙江大学 茚达特罗在治疗结直肠癌中的应用
CN110013481A (zh) * 2019-04-30 2019-07-16 暨南大学 马来酸茚达特罗在制备抗肿瘤药物中的应用
CN113876772A (zh) * 2021-09-29 2022-01-04 暨南大学 马来酸茚达特罗在作为cGAS-STING通路靶向激动剂中的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HUAREN SHIJIE, ZAZHI XIAOHUA: "Role of cGAS-STING Signaling Pathway in Colon Cancer", WORLD CHINESE JOURNAL OF DIGESTOLOGY, SHIJE WEI-CHANGBINGXUE ZAZHISHE , TAIYUAN, CN, vol. 28, no. 21, 8 November 2020 (2020-11-08), CN , pages 1084 - 1089, XP093068709, ISSN: 1009-3079 *

Similar Documents

Publication Publication Date Title
Tang et al. The combination of piR-823 and eukaryotic initiation factor 3 B (EIF3B) activates hepatic stellate cells via upregulating TGF-β1 in liver fibrogenesis
CN113876772A (zh) 马来酸茚达特罗在作为cGAS-STING通路靶向激动剂中的应用
Gao et al. BZW2 gene knockdown induces cell growth inhibition, G1 arrest and apoptosis in muscle‐invasive bladder cancers: A microarray pathway analysis
Du et al. MiR-138-1-3p alters the stemness and radiosensitivity of tumor cells by targeting CRIPTO and the JAK2/STAT3 pathway in nasopharyngeal carcinoma
CN109576297A (zh) 一种含wsb1基因启动子和报告基因的重组质粒及其构建方法和应用
Xu et al. TP53-inducible putative long noncoding RNAs encode functional polypeptides that suppress cell proliferation
Li et al. miR-21-5p/Tiam1-mediated glycolysis reprogramming drives breast cancer progression via enhancing PFKL stabilization
WO2023097610A1 (zh) 马来酸茚达特罗在作为cGAS-STING通路靶向激动剂中的应用
CN112402627B (zh) 一种PIWI蛋白相互作用RNA piR-hsa-211106的用途
CN108034655B (zh) 一种长非编码rna及其组合物在诊断/治疗结直肠癌中的应用
Vaklavas et al. Hallmarks and determinants of oncogenic translation revealed by ribosome profiling in models of breast cancer
Ji et al. Single-cell RNA sequencing reveals the lineage of malignant epithelial cells and upregulation of TAGLN2 promotes peritoneal metastasis in gastric cancer
CN114934048A (zh) 一种用于肿瘤治疗靶点和诊断生物标志物的circMTMR14及试剂盒和应用
Wu et al. Integrated proteomic and transcriptomic analysis revealslong noncoding rna hotair promotes hepatocellular carcinoma cell proliferation by regulating opioid growth factor receptor (ogfr)
CN114703287B (zh) CXorf56基因在治疗三阴性乳腺癌中的应用
US20230250425A1 (en) USE OF PIWI-INTERACTING RNA piR-hsa-211106
CN117187246B (zh) 抑制CKAP5基因表达的siRNA及其应用
Zhang et al. Period2-mediated downregulation of ERK/MAPK phosphorylation in nasopharyngeal carcinoma
CN113440617B (zh) 治疗t细胞急性淋巴细胞白血病的药物组合物
Yang et al. The overexpression of cytochrome c oxidase subunit 6C activated by Kras mutation is related to energy metabolism in pancreatic cancer
Lee et al. Mitochondrial SHMT2 is a crucial therapeutic target in dedifferentiated thyroid cancer
WO2021043340A2 (zh) 一种肿瘤标志物aquaporin 2蛋白及其应用
Yu et al. YTHDC2 promotes the apoptosis of colorectal cancer cells through the p38MAPK signaling pathway
CN110967486B (zh) hnRNPC蛋白的S260位点磷酸化作为结直肠癌干性标记物的应用
CN116942796B (zh) 一种长链非编码rna及其编码多肽或其检测试剂在制备诊断和/或治疗肾透明细胞癌产品中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21966042

Country of ref document: EP

Kind code of ref document: A1