CN113101285A - 一种苯并呋喃酮类化合物的应用 - Google Patents
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Abstract
本发明涉及一种苯并呋喃酮类化合物的应用，属于生物技术领域。利用建立的脯氨酸羟化酶(PHD2)小分子化合物抑制剂体外筛选体系，发现该苯并呋喃酮类化合物能够有效抑制脯氨酸羟化酶(PHD2)的羟化活性。该苯并呋喃酮类化合物通过抑制脯氨酸羟化酶(PHD2)的活性，进而稳定和提高细胞内缺氧诱导因子HIF‑α的蛋白水平。
Description
技术领域
本发明涉及一种苯并呋喃酮类化合物的应用。
背景技术
缺氧是指细胞、组织或生物机体所处环境中氧气含量的严重减少。环境缺氧(高海拔地区或深水潜水)可降低动脉血氧饱和度并诱导全身机体组织缺氧，诱发缺氧性疾病。因此，有效的物理或化学干预方法对于预防缺氧相关疾病至关重要。
缺氧诱导因子(hypoxia inducible factor，HIF)即低氧诱导因子，是一种转录调节因子，由α亚基(HIF-α)和β亚基(HIF-β)构成，普遍存在于人体细胞中，其蛋白和活性水平受氧气含量的调节。在含氧量正常的细胞内HIF蛋白不能稳定存在，半衰期仅为数分钟，在缺氧条件下可以稳定的存在，参与机体的多种生理功能调控。已经证实大约有100多个基因的表达受到转录因子HIF的调控，包括促红细胞生成素(erythropoietin，EPO)、运铁蛋白(transferrin)、血管内皮生长因子(vascular endothelial growth factor，VEGF)等，在红细胞生成、血管生长和细胞分化等方面发挥着重要作用。
脯氨酸羟化酶(prolyl hydroxylase，PHD)属于双加氧酶超家族，目前发现4种亚型，分别为PHD1、PHD2、PHD3和PHD4。研究发现，HIF-α是PHD的底物，是HIF降解反应的限速酶。在正常氧气含量下，PHD可识别并羟基化HIF-α上的脯氨酸残基Pro402和Pro564，然后经VHL蛋白介导的泛素化降解途径导致HIF-α蛋白的降解。但在缺氧条件下，PHD羟基化活性下降，导致HIF-α蛋白降解过程受阻，致使HIF-α蛋白稳定积累，从而改善缺氧诱发的相关生理反应。
发明内容
为解决上述技术问题，本发明的技术方案是：
一种苯并呋喃酮类化合物，为3，5，7-三羟基-2-(3，4，5-三羟基苯基)-4H-1-苯并呋喃-4-酮，3，5，7-Trihydroxy-2-(3，4，5-trihydroxyphenyl)-4H-1-benzopyran-4-one，结构式如下：
上述苯并呋喃酮类化合物作为脯氨酸羟化酶(PHD2)的抑制剂。
优选的，所述苯并呋喃酮类化合物能够有效抑制脯氨酸羟化酶(PHD2)的羟化活性。
上述苯并呋喃酮类化合物靶向抑制脯氨酸羟化酶(PHD2)的羟化活性，进而稳定HIF-α在细胞内的蛋白水平。
本发明的有益效果是：
利用建立的脯氨酸羟化酶(PHD2)小分子抑制剂体外筛选体系对小分子化合物库进行筛选，发现一种小分子化合物，所述小分子化合物为苯并呋喃酮类化合物，能够有效抑制脯氨酸羟化酶(PHD2)羟化活性。通过免疫印迹实验表明该化合物能有效稳定HIF-α在细胞内的蛋白水平。上述结果说明该化合物可以通过抑制脯氨酸羟化酶(PHD2)羟化活性而稳定HIF-α在细胞内的蛋白水平，从而改善缺氧诱发的相关生理反应。
附图说明
图1为本发明利用建立的PHD2小分子抑制剂体外筛选体系筛选抑制剂的结果。结果表明，筛选得到的小分子化合物能够有效抑制PHD2的活性。
图2为本发明所述小分子化合物体外实验半数抑制量的实验结果。IC50为63μM。
图3为利用免疫印迹法检测所述小分子化合物对细胞内HIF-α蛋白水平的影响。结果表明，所述小分子化合物能明显提高细胞内HIF-α蛋白的水平。DMEM组为阴性对照，CoCl2组为阳性对照。
具体实施方式
以下参照具体的实施例来说明本发明。
实施例1
抑制PHD2羟化活性小分子化合物的筛选
①按照建立的PHD2小分子抑制剂体外筛选体系，加入每个组分：Tris-HCl 50mM(pH 8.0)；PHD2蛋白5μg；Fe2+ 100μM；Vc 2mM；α-KAD 20μM，体系总体积为5μl。
②将待筛选的化合物分别加入反应体系中，混匀，室温静置30min。
③将PHD2的底物(FITC-aa)加入反应体系至1μM，避光混匀，室温静置60min。
④将VHL蛋白复合物加入反应体系至700nM，同时用50mM的Tris-HCl(pH 8.0)缓存液补齐最终体系至50μl。
⑤荧光偏振分光光度计，设定激发光波长为530nm；发射光波长为460nm。检测筛选体系的荧光偏振值。
如图1结果显示，化合物I可以有效抑制PHD2羟化酶活性。
实施例2
化合物I对PHD2酶活性半数抑制量确定。
按照建立的PHD2小分子抑制剂体外检测体系，加入每个组分(pH 8.0 Tris-HCl50mM；PHD2蛋白5μg；Fe2+ 100μM；Vc 2mM；α-KAD 20μM，总体积5μl)。化合物I分别设置浓度梯度：(0.3125mM、0.15625mM、0.125mM、0.09375mM、0.0625mM、0.046875mM、0.03125mM、0.015625mM、0.007813mM)。荧光偏振分光光度计，检测偏振值。Graph pad统计、分析数据。
如结果2所示，化合物I体外抑制PHD2酶活性，其半数抑制量(IC50)为63μM。
实施例3
化合物I(3，5，7-三羟基-2-(3，4，5-三羟基苯基)-4H-1-苯并呋喃-4-酮)对人肝细胞系L02内HIF-α水平的影响
①6cm培养皿培养L02细胞：收集对数期L02细胞，按7.5×105个/皿，接种细胞，37℃、5％CO2，过夜培养，待细胞生长至80％。
②化合物I处理L02细胞：在培养基内加入化合物I至浓度250nM，37℃、5％CO2，培养16h。
③提取L02细胞总蛋白：收集L02细胞，提取L02细胞内的总蛋白。
④免疫印记技术分别检测：以β-actin为内参，检测L02细胞内HIF-α蛋白含量。
如图3结果所示，化合物I可以稳定和提高L02细胞内HIF-α蛋白水平。
Claims (3)
1.一种苯并呋喃酮类化合物的应用，所述苯并呋喃酮类化合物为3，5，7-三羟基-2-(3，4，5-三羟基苯基)-4H-1-苯并呋喃-4-酮，具有式(I)所示结构：
2.根据权利要求1所述的苯并呋喃酮类化合物能够抑制脯氨酸羟化酶(PHD2)的活性。
3.根据权利要求1所述的苯并呋喃酮类化合物通过抑制脯氨酸羟化酶(PHD2)的活性来稳定和提高细胞内缺氧诱导因子HIF-α的蛋白水平。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102210670A (zh) * | 2011-04-08 | 2011-10-12 | 中国人民解放军总后勤部卫生部药品仪器检验所 | 黄酮醇类化合物在制备抗缺氧药物或食品中的应用 |
| CN108434139A (zh) * | 2018-02-07 | 2018-08-24 | 南京市儿童医院 | 缺氧诱导因子脯氨酰羟化酶活性抑制剂在制备防治急性肾损伤药物中的应用 |
| CN108743579A (zh) * | 2018-05-31 | 2018-11-06 | 天津科技大学 | 黄酮类产物作为制备治疗癌症药物及抑制剂的应用 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102210670A (zh) * | 2011-04-08 | 2011-10-12 | 中国人民解放军总后勤部卫生部药品仪器检验所 | 黄酮醇类化合物在制备抗缺氧药物或食品中的应用 |
| CN108434139A (zh) * | 2018-02-07 | 2018-08-24 | 南京市儿童医院 | 缺氧诱导因子脯氨酰羟化酶活性抑制剂在制备防治急性肾损伤药物中的应用 |
| CN108743579A (zh) * | 2018-05-31 | 2018-11-06 | 天津科技大学 | 黄酮类产物作为制备治疗癌症药物及抑制剂的应用 |
Non-Patent Citations (1)
| Title |
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Application publication date: 20210713 |
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