CN116687892A - 天然产物EuonyquinoneA的新用途 - Google Patents
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Abstract
本发明提供天然产物Euonyquinone A的新用途。Euonyquinone A能够浓度依赖性地抑制p‑STAT3‑Y705和IL‑6刺激的p‑STAT3‑Y705，对Ac‑STAT3‑K685和p‑STAT3‑S727的表达都无影响，对STAT3蛋白具有优异的选择性。因此可以制备成STAT3抑制剂以及抗自身免疫性炎症疾病药物。Euonyquinone A对前列腺癌细胞DU145、乳腺癌细胞MDA‑MB‑231和人骨髓白血病细胞MOLM16的增殖抑制活性的IC50分别为2.5μM、0.3μM和2.1μM。因此还可以制备成抗肿瘤药物。Transwell细胞迁移侵袭实验表明Euonyquinone A可以浓度依赖性的抑制乳腺癌细胞MDA‑MB‑231的迁移和侵袭。因此还可以制备成乳腺癌转移抑制剂。
Description
技术领域
本发明属于抗癌药物开发技术领域，具体涉及天然产物Euonyquinone A的新用途。
背景技术
信号传导与转录激活因子3(STAT3)是STAT转录因子家族中的一员，存在于细胞质中，在细胞表面受体向细胞核传递信号中起着重要的作用。在正常细胞中STAT3受到严格的调控，STAT3的持续激活往往与肿瘤的发生、增殖、促进血管生成和抑制细胞凋亡有关，并且还与抑制抗肿瘤免疫反应等致癌功能相关。在大约70％的人类实体瘤和血液瘤中发现持续性的STAT3活化，包括结直肠癌、肺癌、黑色素瘤、乳腺癌、前列腺癌、肾癌、卵巢癌、肝癌、胰腺癌、多发性骨髓瘤和白血病等。此外，组成性STAT3活化与癌症不良预后关系密切。过度活化的STAT3可通过生成和维持肿瘤干细胞(CSC)或上调Bcl-xL、Mcl-1、 Bcl-1等抗凋亡相关蛋白而驱动失控的细胞增殖、存活并促进癌细胞对细胞毒和靶向药物的产生化学耐药。除了参与癌症进程，STAT3也参与类风湿性关节炎、克罗恩病、动脉粥样硬化和炎症性肠病等自身免疫性炎症疾病进展。因此，STAT3 是具有吸引力的癌症及炎症疾病治疗靶点。
STAT3蛋白705位的酪氨酸磷酸化使STAT3激活，两个磷酸化的STAT3单体通过相互的p-Tyr705-SH2作用形成二聚体，进入到细胞核内完成细胞核靶基因的转录。因此，设计靶向STAT3 SH2结构域的抑制剂可以阻止STAT3的二聚和转录活性。在过去的二十多年中，药物研发人员在开发STAT3 SH2结构域 (SH2D)的小分子抑制剂方面做了大量的工作。然而，到达临床研究阶段却显示出非常有限的临床活性。其主要原因可能是之前针对STAT3 SH2结构域的抑制剂在抑制STAT3 Tyr705位点磷酸化的同时，还会影响Ser727的磷酸化水平和Lys685的乙酰化水平。另一个原因是STAT3和其他STAT家族成员具有高度结构同源的SH2结构域，很难获得高选择性的STAT3抑制剂。
Euonyquinone A是从西双版纳特有的卫矛属植物兽毒卫矛(EuonymusglabraRoxb.)中分离得到的一种结构全新的天然产物。研究发现Euonyquinone A 可以在体外和细胞水平抑制多巴脱羧酶的活力，为多巴胺相关疾病的治疗和预防研究提供了新的候选药物和分子探针工具，针对该天然产物更广泛的活性研究还有待进一步开发。
发明内容
经研究，天然产物Euonyquinone A能够浓度依赖性地抑制p-STAT3-Y705 和IL-6刺激的p-STAT3-Y705，对Ac-STAT3-K685和p-STAT3-S727的表达都无影响，对STAT3蛋白具有优异的选择性。以及MST测试结果拟合出 Euonyquinone A与STAT3蛋白亲和力Kd值为0.88uM。因此，本发明提供天然产物Euonyquinone A在制备STAT3抑制剂上的用途。
进一步地，本发明还提供Euonyquinone A在制备抗自身免疫性炎症疾病药物中的用途。
此外，Euonyquinone A对前列腺癌细胞DU145、乳腺癌细胞MDA-MB-231 和人骨髓白血病细胞MOLM16的增殖抑制活性的IC50分别为2.5μM、0.3μM 和2.1μM。因此，本发明还提供了Euonyquinone A在制备抗肿瘤药物中的用途。
进一步地，Transwell细胞迁移侵袭实验表明Euonyquinone A可以浓度依赖性的抑制乳腺癌细胞MDA-MB-231的迁移和侵袭。因此，本发明还提供了 Euonyquinone A在制备乳腺癌转移抑制剂中的用途。
进一步地，上述药物还包括药学上可接受的辅料。
所述药物可以为当前药品领域任何剂型，优选为丸剂、胶囊剂、片剂、粉剂、颗粒剂或口服液。各药物剂型可根据该剂型实际需要选取合适的可接受辅料来制备，这属于本领域常规的剂型制备技术。
通过以上技术方案可知，化合物Euonyquinone A是一个活性佳且具有选择性的STAT3 SH2D抑制剂，有望作为新型选择性STAT3抑制剂开发，并进一步开发成抗肿瘤和抗自身免疫性疾病药物，用于肿瘤和自身免疫性疾病的治疗。
附图说明
图1为本发明实施例2中Western Blot实验结果。
图2为本发明实施例3中Transwell细胞迁移侵袭实验结果。
图3为本发明实施例4中Euonyquinone A对STAT3蛋白采用MST法的亲和力测试拟合图。
具体实施方式
在本发明的描述中，需要说明的是，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
下面结合具体实施方式对本发明作进一步详细的说明，以帮助本领域的技术人员对本发明的发明构思、技术方案有更完整、准确和深入的理解，本发明的保护范围包括但不限于以下实施例，在不偏离本申请的精神和范围的前提下任何对本发明的技术方案的细节和形式所做出的修改均落入本发明的保护范围内。
Euonyquinone A的结构式如下：
Euonyquinone A来源于植物提取，提取方法参照学位论文《兽毒卫矛化学成分及多巴脱羧酶抑制活性研究》
本发明具体实施例通过一系列体外实验可知，化合物Euonyquinone A是一个活性佳且具有选择性的STAT3 SH2D抑制剂。有望作为新型选择性STAT3抑制剂开发，用于肿瘤和自身免疫性疾病的治疗。具体研究实验如下：
实施例1
本实施例考察Euonyquinone A抑制细胞增殖实验(CTG法)，具体步骤如下：
1.将化合物用DMEM培养液稀释成实验设计浓度。
2.将前列腺癌细胞DU145、乳腺癌细胞MDA-MB-231和人骨髓白血病细胞 MOLM16分别经计数后接种于96孔培养板，2000细胞/孔，每孔加入90ul培养液(含血清)培养，再加入不同浓度的10ul药物溶液。
3.培养72h后，每孔加入CTG 100ul。
4.室温静置10min，在酶标仪上测定各孔化学发光值。结果如表1所示。
表1.Euonyquinone A抑制肿瘤细胞增殖的IC50
注：*BP-1-102为文献报道(Proc.Natl.Acad.Sci.USA.2012,109(24):9623-8.)的非选择性STAT3抑制剂。
实施例2
本实施例考察Euonyquinone A的蛋白免疫印迹(Western Blot法)，具体如下：
1.在75cm细胞培养瓶中，正常培养MDA-MB-231细胞，收集样本前24 h，更换新鲜培养基；收集样本时，保证细胞处于对数生长期，密度在80％左右。
2.用PBS清洗3次，加入2ml 0.5％胰酶，消化细胞5min，2000rpm离心 5min，重复操作两次，倒置于纸上尽量吸干液体；接下来的步骤将样本置于冰上操作，每份加入100ul细胞裂解液进行重悬；冰上裂解30min，每隔10min 剧烈震荡至少30s，加速细胞裂解；4℃，12000rpm离心10min，取上清。
3.取一部分上清液，进行BCA法蛋白定量(按照说明进行操作)；
4.以裂解液稀释样品至2.5g/L的蛋白浓度，加入5×上样缓冲液，将混合后的样品95℃，5min充分变性，冷却后准备上样进行电泳(暂不进行电泳即于 -80℃冻存)。
5.电泳凝胶：12％分离胶和4％浓缩胶；
6.SDS-PAGE电泳：电泳时间一般90min，电压为80V。电泳至溴酚兰刚跑出即可终止电泳，拆胶浸于转膜缓冲液中准备转膜；
7.转膜：PVDF膜先以甲醇浸润1min进行激活，然后与滤纸一起浸于转膜缓冲液中备用，装好夹子后400mA转移0.5h；
8.封闭：以5％脱脂奶粉对膜进行封闭40min；
9.一抗孵育：加入一抗与对照抗体，4℃孵育过夜；
10.二抗孵育：PBST洗三次，加HRP标记的二抗，37℃孵育1h；
11.ECL显色：PBST洗三次，按照试剂盒说明书配制显色工作液，取适量加至膜上，室温避光孵育3-30min；
12.成像：以凝胶成像仪进行成像拍照。结果如图2所示。
Western Blot实验结果显示，Euonyquinone A以浓度依赖的方式抑制显著抑制了p-STAT3-Y705(图1A)和IL-6刺激的p-STAT3-Y705(图1D)，并且对 Ac-STAT3-K685、p-STAT3-S727和STAT3总表达量都无显著抑制作用。在 STAT1和STAT5a/b蛋白的WesternBlot实验中(图1B，1C)，Euonyquinone A 对p-STAT1-Y701、p-STAT1-S727、p-STAT5a-Y694、p-STAT5b-Y699、STAT1 和STAT5a/b蛋白总表达量无显著的抑制作用，说明Euonyquinone A对STAT3 较STAT1和STAT5a/b有显著的选择性。
实施例3
本实施例考察Euonyquinone A的Transwell细胞迁移侵袭实验，具体如下：
1.接种前将24孔板和Transwell小室用1×PBS浸泡5min湿润小室；
2.用胰蛋白酶消化细胞后，1％FBS的培养基重悬MDA-MB-231细胞，调整密度1×105细胞/ml，接种到Transwell小室内，每个小室分别加0.2ml各组细胞悬液和10-1000nMEuonyquinone A的DMSO溶液或空白DMSO对照，下层加入0.8ml含10％FBS的完全培养液，置于37℃培养箱中培养；
3.每孔加入1ml 4％多聚甲醛溶液，室温固定15min；
4.吸去固定液，用1×PBS洗涤2次，每孔加入1ml 0.5％结晶紫溶液，染色 60min后用1×PBS洗三次，晾干；
5.用棉签小心擦去Transwell小室上部内没有迁移的细胞，置于显微镜下观察。结果如图2所示。
肿瘤细胞的侵袭和迁移属于肿瘤细胞恶性行的主要特征，是引起恶性肿瘤患者死亡的首要因素。通过Transwell细胞迁移侵袭实验(图2)，考察了 Euonyquinone A抑制肿瘤细胞迁移侵袭的效果，从图2中可知，随着 Euonyquinone A浓度的提高，可以浓度依赖性的抑制乳腺癌细胞MDA-MB-231 的迁移(图2-A)和侵袭(图2-B)。
实施例4
本实施例采用微量热泳动法(MST)测定Euonyquinone A与蛋白结合活性实验，具体如下：
1、配制浓度20μM的蛋白母液，用ddH2O稀释到5μM；
2、column A柱摇匀，1500g，4℃，离心1min后重新加入300μL的Labeling buffersalt溶液洗涤3次；
3、将稀释好的蛋白溶液加入到column A中，1500g，4℃，离心2 min；
4、配置染料：die yielding approx，加25ul 100％的DMSO溶解染料 (600μM)，使用时稀释为25μM；
5、将稀释好的染料与置换好体系的蛋白溶液1：1混合，避光孵育半小时；
6、column B洗脱，将column B下面的冒拧开，加入3ml的MST buffer，液体随时补充；
7、准备10个(左右)1.5ml的EP管，标号，用于接从column B中洗脱好流出的样品，将200μL蛋白染料混合液加入column B中，加入MST buffer加压，让液体顺利流下；
8、预试验检测样品的荧光强度：
每管蛋白稀释10倍，上机后将荧光强度大的两管蛋白混合到一起保证蛋白荧光度在300-500左右，放到-80℃的冰箱中保存；根据预实验结果确定最终稀释浓度；
9、正式实验：
药物稀释：母液10mM，使用0.05％tween20稀释成想要的浓度如：1mM 或100μM；
加样：先在每个管中加入5μL的0.05％的tween 20溶液，之后加入梯度稀释好的药物，每管中加入蛋白5μL；
上机：MST仪器检测荧光强度软件：Mo Control(×86)。结果如图3所示。根据MST测试结果拟合出Euonyquinone A与STAT3蛋白亲和力Kd值为0.88 uM。
综上所述，本发明具体实施例公开的研究数据表明：Euonyquinone A对前列腺癌细胞DU145、乳腺癌细胞MDA-MB-231和人骨髓白血病细胞MOLM16 的增殖抑制活性的IC50分别为2.5μM、0.3μM和2.1μM。在Western Blot实验中，Euonyquinone A浓度依赖性的抑制p-STAT3-Y705和IL-6刺激的 p-STAT3-Y705，对STAT3的表达量无影响，说明其是通过直接作用于SH2结构域抑制p-STAT3-Y705，而不是通过抑制STAT3表达造成的。并且对 Ac-STAT3-K685和p-STAT3-S727的表达都无影响，说明其对STAT3蛋白具有优异的选择性。在STAT1和STAT5a/b的Western Blot实验中，Euonyquinone A 对p-STAT1-Y701、p-STAT1-S727、p-STAT5a-Y694、p-STAT5b-Y699、STAT1 和STAT5a/b的表达均无显著的抑制效果，说明其对STAT3较STAT1和STAT5a/b 有优异的选择性。在Transwell细胞迁移侵袭实验中，Euonyquinone A可以浓度依赖性的抑制乳腺癌细胞MDA-MB-231的迁移和侵袭。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (6)
1.天然产物Euonyquinone A在制备STAT3抑制剂上的用途。
2.Euonyquinone A在制备抗自身免疫性炎症疾病药物中的用途。
3.Euonyquinone A在制备抗肿瘤药物中的用途。
4.Euonyquinone A在制备乳腺癌转移抑制剂中的用途。
5.根据权利要求1～4任意一项所述的用途，其特征在于：还包括药学上可接受的辅料。
6.根据权利要求1～4任意一项所述的用途，其特征在于：所述抑制剂或者所述药物为丸剂、胶囊剂、片剂、粉剂、颗粒剂或口服液。
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