CN115819432A - 一种用于宫颈癌治疗的化合物及药物 - Google Patents
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Abstract
本发明公开了一种用于宫颈癌治疗的化合物及包含该化合物的药物。本发明所筛选的小分子化合物可以通过抑制p53被E6/E6AP复合物结合导致的降解来提升p53蛋白的表达水平,而p53蛋白水平上升能够激活p21转录,p21蛋白水平上升又能抑制SiHa细胞的细胞周期,从而抑制HPV16病原体的产生,实现宫颈癌防治的目的。
Description
技术领域
本发明属于技术医药技术领域,具体涉及一种用于宫颈癌治疗的化合物及药物。
背景技术
宫颈癌是妇女中的常见癌症,也是发展中国家妇女死亡的主要原因,几乎所有的子宫颈癌都与高危型HPV持续性感染有关;目前还没有针对HPV感染及相关疾病的特异性治疗方法,主要对皮肤、生殖器疣和晚期宫颈癌前病变的病例采取细胞毒性药物或手术治疗方式。HPV这种病原体通过表达E6、E7癌蛋白破坏细胞周期,为细胞永生化转化铺平道路。HPV E6致癌蛋白在引发被感染细胞往永生化转化方面的效率比E7更加明显,不依靠E7的情况下,单独的E6就能与E6AP的高度保守序列“LXXLL”结合形成异二聚体,募集并诱导抑癌蛋白p53降解,使感染细胞发生永生化转化,因此抑制HPV的致癌功能可通过抑制E6蛋白与E6AP的结合来实现,消除其诱导和转化能力。
然而现有技术中还没有能够有效抑制HPV致癌功能的手段,更缺乏相应的药物。开发一种抑制HPV致癌能力的药物对宫颈癌的防治很有必要。
发明内容
针对上述现有技术,本发明提供一种用于宫颈癌治疗的化合物及药物,以解决现有技术中宫颈癌治疗药物匮乏的问题。
为了达到上述目的,本发明所采用的技术方案是,提供一种如式I所示的化合物,并将该化合物用于制备治疗宫颈癌的药物中。
进一步的,本申请中将如式I所示的化合物用于制备治疗由HPV16感染所引起的宫颈癌的药物中。
本发明还公开了一种用于治疗宫颈癌的药物,该药物包括如式I所示的化合物。
进一步,药物中如式I所示的化合物的有效浓度为10~30μM。
进一步,还包括药学上可接受的盐。
进一步,药物的剂型为注射剂、片剂、胶囊剂、冲剂或栓剂。
本发明的有益效果是:本发明所筛选的小分子化合物可以通过抑制p53被E6/E6AP复合物结合导致的降解来提升p53蛋白的表达水平,而p53蛋白水平上升能够激活p21转录,p21蛋白水平上升又能抑制SiHa细胞的细胞周期,从而抑制HPV16病原体的产生,实现宫颈癌防治的目的。
附图说明
图1为使用SiHa细胞验证虚拟筛选所得化合物的CCK8检测结果;
图2为4号小分子化合物对SiHa细胞的IC50结果;
图3为3、4、6、10、20、21化合物与HPV16 E6蛋白的结合解离情况;
图4为不同浓度的4号化合物与HPV16 E6蛋白结合解离情况;
图5为不同浓度的4号化合物对SiHa细胞周期的影响结果;
图6为不同浓度的4号化合物对SiHa细胞周期影响的量化统计图;
图7为不同浓度的4号化合物对SiHa细胞凋亡的影响;
图8为在不同浓度的4号小分子下SiHa细胞的凋亡比率;
图9和图10为4号化合物对SiHa细胞的P53/P21蛋白和凋亡相关蛋白表达水平的影响。
具体实施方式
下面结合实施例对本发明的具体实施方式做详细的说明。
实施例1:分子筛选
1.靶蛋白的准备
在PDB数据库中搜索HPV E6蛋白的晶体结构,选取用于虚拟筛选的靶蛋白晶体结构,确定小分子化合物作用靶点;α-9属HPV E6蛋白约由150个氨基酸组成,包含三个结构域:N末端结构域、C末端的结构域以及2个锌指结构域。其中,两个锌指结合域围成一个很深的口袋,能够和E6AP蛋白的“LXXLL”序列结合,进而结合人体最重要的抑癌蛋白p53并促使其泛素化降解,因此,在本发明中将E6上的“LXXLL”结合槽作为小分子抑制剂潜在的作用靶点,也是本次虚拟筛选的靶点。
2.分子库的选取和准备
虚拟筛选需要一个分子库,从该分子库中筛选出可能作为药物候选分子的化合物。本发明使用Specs公司提供的小分子化合物数据库进行药物候选分子筛选。考虑到小分子的理化性质差异,在筛选前,需要对分子进行类药性筛选,去掉不适合做后续筛选的分子,保留与真实药物特性相近的分子。类药性筛选的条件:可旋转键数目小于10;相对分子质量小于500;logP大于-2且小于5;氢键供体数目小于5;氢键受体数目小于10。
3.基于DOCK的第一步虚拟筛选
本发明使用DOCK6进行初步筛选,使用Chimera软件对其进行手动参数设置,以实现后续虚拟筛选所需的DOCK对接循环程序。根据对接结果,选取结果最好的前10%分子,进行下一步的虚拟筛选。
4.基于AutoDock Vina的第二步虚拟筛选
用AutoDock Vina进行分子对接时,要使用AutoDockTools软件对受体蛋白和配体进行参数配置,对筛选获得的侯选抑癌小分子化合物进行必要的化学修饰或基团改造,降低其毒性增加其分子活性、稳定性及水溶性等特性;根据动物和临床实验的表现来判断其是否适用于α-9属HPV相关恶性疾病的治疗,向真正的抗癌药物转变。
5.虚拟筛选所得化合物在SIHA细胞水平上的验证以及筛选
通过虚拟筛选,以已知具有E6/E6AP抑制活性的天然产物木犀草素,即自由能排序第21号、specs化合物库中名称为AM-721/20737006的化合物为阳性对照,以DMSO为空白对照,取前20个小分子化合物进行SiHa细胞水平验证。
小分子化合物通过特异性抑制E6/E6AP的结合,从而抑制P53被E6/E6AP复合物特异性导致的泛素化降解,而P53蛋白水平上调会抑制细胞增殖,甚至导致细胞凋亡。SiHa细胞是由HPV16感染所引发宫颈癌的细胞系,将SiHa细胞分别与不同小分子化合物(50μM)共同孵育48小时,然后使用CCK8方法检测细胞活力水平,本发明以细胞抑制率50%为阈值,结果如图1所示。筛选发现有6个小分子化合物对SiHa细胞的增殖有较强的抑制作用,它们分别是编号为3、4、6、10、20、21的化合物,其中以第4号化合物,Spces库编号为AO-022/42598600,对SiHa细胞抑制作用最为明显,4号化合物的结构式如式I所示。
4号小分子化合物对SiHa细胞的IC50结果如图2所示,从图中可以看出,4号小分子化合物浓度达到20μM左右时就能对SiHa细胞的活力达到50%的抑制,并且对SiHa细胞最大抑制效果能达到90%以上。
6.HPV16 E6蛋白与小分子化合物相互作用检测
在上一步细胞活力水平CCK8测试中筛选出了6个可能能够特异性抑制E6/E6AP结合,从而抑制SiHa细胞增殖的小分子化合物,其中以21号化合物木犀草素作为阳性对照。但是SiHa细胞的抑制也可能是由于化合物毒性导致的,所以需要检测E6蛋白是否与小分子化合物有特异性结合,本发明使用生物膜干涉方法来检测E6蛋白与小分子化合物的相互作用。
检测分为E6蛋白与小分子化合物结合阶段和E6蛋白与小分子解离阶段。结合阶段时,将固化了E6蛋白的传感器探头插入不同小分子化合物溶液中,E6蛋白与小分子化合物不断结合,化合物浓度均为20μM,在E6蛋白与化合物结合阶段测得结合常数Kon,此时纵坐标为纳米nm,随着结合的小分子增加而增加;解离阶段时,将固化了E6蛋白的传感器探头插入PBST缓冲液中,E6蛋白与小分子解离,在E6蛋白与小分子解离阶段,测得解离常数Koff,此时纵坐标nm读数随着E6蛋白与小分子的解离而下降。
蛋白与小分子结合解离是一个非常迅速的过程,并且不断地结合解离并快速达到平衡,计算平衡解离常数KD值(KD=Koff/Kon),反映了相互作用结合能力的强弱,结合Kon越大,解离Koof越小,KD值越小,代表结合能力越强;不同小分子化合物与E6蛋白的相互作用检测结果如图3所示,其中锯齿线条为实验初始数据,光滑线条为分析软件拟合。从图3中可以看到E6蛋白与有的小分子在数秒内就可以结合达到饱和,但是小分子化合物3和小分子化合物6与E6蛋白并没有明显的结合与解离现象,所以被认为它们之间并没有相互作用结合,故而也没有KD值;而小分子化合物4、10、20和阳性对照化合物21木犀草素都有明显的结合与解离效果,所以认为它们之间具有相互作用。通过分析软件得到他们的KD值,如表1所示,可以看到在细胞水平抑制效果最好的化合物4与E6蛋白的平衡解离常数最低,达到了微摩尔级别,相互之间结合能力最强。同时也测出了蛋白E6与不同浓度梯度的4号化合物结合情况,结果如图4所示,可以看出,随着4号小分子浓度的增加,从10μM到30μM,E6蛋白与4号小分子的结合也越强,因此确定4号化合物对HPV16具有良好的抑制效果。
7.4号化合物对SiHa细胞周期的影响
分别使用10μM、20μM、30μM的4号小分子处理SiHa细胞48小时,将细胞使用75%乙醇固定,PI染色后上流式细胞仪检测SiHa细胞周期情况,结果如图5所示。DMSO对照组的G0/G1期占78.69%,S期占12.22%,G2/M期占9.09%;10μM的4号小分子化合物处理时,G0/G1期占78.78%,S期占14.47%,G2/M期占6.75%;20μM的4号小分子处理时,G0/G1期占88.16%,S期占6.68%,G2/M期占5.16%;30μM的4号小分子处理时,G0/G1期占91.2%,S期占5.04%,G2/M期占3.76%。不同浓度的4号化合物对SiHa细胞周期影响的量化统计图如图6所示。
可以看到,随着4号小分子化合物的浓度增大,SiHa细胞的S期以及G2/M期不断减少,而G1期不断增多,其中细胞周期S期和G2/M期是细胞DNA合成以及分裂的周期,其比例越多代表细胞增殖分裂越旺盛,这说明随着4号化合物浓度的增加,SiHa细胞的分裂增殖能力不断降低,SiHa细胞周期被停滞在了G1期;这是由于4号化合物抑制了E6与E6AP的相互作用,从而抑制了p53被E6/E6AP相互作用结合导致的泛素化降解,而p53蛋白水平上升激活了下游通路p21,p21蛋白是一种CDK抑制剂,通过抑制CDK4、6/cyclin-D和CDK2/cyclin-E,从而将SiHa细胞周期抑制在G1期。
8.4号化合物对SiHa细胞凋亡的影响
在上一步周期实验证明了4号化合物可以通过抑制p53被E6/E6AP复合物结合导致的降解,p53蛋白水平上升激活p21转录,p21蛋白水平上升抑制SiHa细胞的细胞周期。为了得到更具体的4号化合物诱导SiHa细胞凋亡的情况,使用流式细胞仪对4号化合物诱导SiHa细胞凋亡进行了更深入的研究。分别使用10μM、20μM、30μM的4号小分子处理SiHa细胞48小时,其中第21号小分子木犀草素作为阳性对照,DMSO为空白对照,结果如图7所示。从中可以看出,在不同浓度的4号化合物处理后SiHa细胞都出现了不同程度的凋亡,而且随着4号小分子浓度的增大,凋亡比例也在不断增大。在图8中给出了在不同浓度的4号小分子下SiHa细胞的凋亡比率,从中可以看出,在4号化合物浓度为10μM、20μM、30μM时,SiHa细胞凋亡比例分别为6.15%、14.93%、24.27%。其中4号小分子浓度为30μM时,SiHa细胞凋亡比例达到了24.27%,证明了4号化合物不仅能抑制SiHa细胞的周期来遏制其的恶性增殖,还能诱导SiHa细胞的凋亡。
9.4号化合物对P53和P21蛋白水平含量的影响
上述实验说明了4号化合物能够抑制并凋亡SiHa细胞。为进一步研究4号化合物处理SiHa细胞后p53蛋白与p21蛋白的含量水平是否提高,使用10μM、20μM、30μM的4号化合物处理SiHa细胞48小时,21号化合物木犀草素为阳性对照,DMSO为空白对照。采用westernblot检测p53和p21以及p53通路相关凋亡标记蛋白,结果如图9和10所示。从中可以看出,随着4号化合物浓度的增加,SiHa细胞中的p53蛋白含量也不断增加,p53的下游p21蛋白含量也随着增加,其中p53下游的促凋亡蛋白BAX含量也增加了,cleaved-caspase3出现了裂解激活,说明4号化合物确实提高了p53蛋白水平含量,而p53作为转录因子激活了p21的转录,提高了p21蛋白的水平含量,同时提高了促凋亡蛋白BAX的蛋白水平含量,BCL-2蛋白水平并无明显变化,BAX/BCL-2的比例增大,使得线粒体膜穿孔,其中的细胞色素C被释放进入细胞质基质中,导致了caspase激活级联效应,最终活化cleaved-caspase3,诱导了p53导致的内源性线粒体caspase依耐型凋亡通路的激活。
虽然结合实施例对本发明的具体实施方式进行了详细地描述,但不应理解为对本专利的保护范围的限定。在权利要求书所描述的范围内,本领域技术人员不经创造性劳动即可作出的各种修改和变形仍属本专利的保护范围。
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