CN116712433B - 一种抑制去泛素化酶otud3活性的化合物在治疗和/或预防肿瘤中的应用 - Google Patents
一种抑制去泛素化酶otud3活性的化合物在治疗和/或预防肿瘤中的应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域,具体涉及一种抑制去泛素化酶OTUD3活性的化合物在治疗和/或预防肿瘤中的应用。本发明所述的化合物能够抑制OTUD3与泛素结合,促进GRP78蛋白的降解,可以用于治疗癌症和/或病毒感染。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种抑制去泛素化酶OTUD3活性的化合物在治疗和/或预防肿瘤中的应用。
背景技术
泛素-蛋白酶体系统(UPS)对真核生物的生命至关重要,并调节细胞生理学的许多方面。泛素(Ub)通过E1泛素激活酶、E2泛素结合酶和E3泛素连接酶的协同作用与蛋白质底物共价偶联,并被去泛素化酶(DUBs)去除。UPS的功能障碍与多种人类疾病的病因和进展有关,包括癌症、代谢综合征、神经变性、自身免疫、炎症性疾病和感染。UPS在调节蛋白质稳态和功能方面发挥着基本作用,这些功能在癌症进展过程中经常发生改变,从而为抗肿瘤治疗的发展提供了切入点。例如蛋白酶体抑制剂Velcade(硼替佐米)的批准和临床成功验证了UPS是治疗干预的可行靶点。作为UPS途径中蛋白酶体上游的一部分,DUBs可能为开发具有潜在增强特异性和降低毒性的新疗法提供新的机会。DUBs抑制增强了特定底物的泛素化,而不影响全局蛋白或泛素水平,这是DUB抑制剂最令人兴奋和临床应用的用途之一。大约有100个已鉴定的人类DUB,它们通过各自催化结构域的序列相似性聚类为七个家族:USP、OTU、UCH、MJD、JAMM、MINDY、ZUP1。USPs作为DUBs中最大的亚家族,在抑制剂的研究和开发方面最受关注。几种抑制剂已被证实对抗恶性肿瘤有效,特别是靶向USP1、USP7和USP14的抑制剂,在临床应用中显示出巨大的潜力。
OTUD3是OTU的一个成员,近年来吸引了越来越多的兴趣。研究表明,OTUD3在肿瘤发生、神经退行性疾病、代谢和免疫中发挥着关键作用。例如OTUD3在先天抗病毒免疫中起负作用,作为乙酰化依赖性去泛素酶,OTUD3限制先天抗病毒免疫信号传导,小鼠的OTUD3缺陷导致先天免疫增强、病毒载量和发病率降低。另外,在肝细胞癌中OTUD3能够通过去泛素化增强α-辅肌动蛋白4(ACTN4)的稳定性,促进肝细胞癌的生长和转移,再如,SOAT1是胆固醇酯生物合成的关键酶,OTUD3依赖其去泛素化酶活性,通过特异性去除SOAT1的多聚泛素化修饰,维持SOAT1的蛋白稳定性,SOAT1高表达与肝癌、甲状腺癌、头颈癌、胃癌、肾癌、前列腺癌、胰腺癌等不良预后均有关,联合应用SOAT1抑制剂和抗程序性死亡受体1抗体对小鼠黑色素瘤进展的控制效果。综上所述,靶向抑制OTUD3可能是治疗癌症或抗病毒的潜在策略。但是现有技术中还没有关于OTUD3的小分子抑制剂的报道。
发明内容
本申请通过基于结构的虚拟筛选,从10万种OTUD3小分子抑制剂化合物中筛选出18种候选化合物,经验证,最终确定一种化合物OTUDin3能够有效的抑制肺癌细胞增殖。进一步,证明OTUDin3通过增强GRP78在体内外的降解来抑制癌细胞。
本发明的第一方面,提供了一种化合物或其药学上可接受的盐、立体异构体、水合物、前药、溶剂化物或代谢产物在制备治疗和/或预防疾病的产品中的应用,所述的化合物为通式(Ⅰ)结构:
其中,
所述的R1、R2独立的选自:H、烷基或卤原子;优选为H、C1-C6烷基(例如C1、C2、C3、C4、C5、C6)或卤原子。
所述的m、n独立的选自:0-4的整数,例如0、1、2、3或4;
所述的A、B独立的选自:稠合杂环化合物;优选稠合双环化合物;进一步优选双环中一个环为苯环,另一个环为含有C、N、O或S的五元杂环或六元杂环;更进一步优选为
所述的X和Y独立的选自:单键、-O-、-O(C=O)O-、-(C=O)NRa-、-NRa(C=O)-、-NRa-、-O(C=O)-、-(C=O)O-、-C(=O)-、-S(O)q-、-OS(O)qO-、-S-S-、-C(=O)S-、-SC(=O)-、-NRaC(=O)NRa-、-OC(=O)NRa-、-NRaC(=O)O-、-OC(=O)S-、-SC(=O)O-;优选为-(C=O)NH-、-NH(C=O)-或-S(O)2-。
其中,q选自0、1或2;
Ra选自H或烷基。
优选的,所述的A、B独立的选自稠合双环化合物。
进一步优选的,所述的稠合双环化合物,一个环为苯环,另一个环为含有C、N、O或S的五元杂环或六元杂环。
更优选的,所述的A、B独立的选自
在本发明的一个具体实施方式中,所述的化合物中,
所述的A、B独立的选自
所述的X、Y独立的选自:-(C=O)NH-、-NH(C=O)-或-S(O)2-;
所述的m、n独立的选自:0-4的整数,例如0、1、2、3或4;
所述的R1、R2独立的选自:H、C1-C6烷基(例如C1、C2、C3、C4、C5、C6)或卤原子;
优选的,所述的卤原子选自F、Cl、Br、I、At、Ts。
在本发明的一个具体实施方式中,所述的化合物的结构式如式(Ⅱ)所示,
优选的,所述的疾病包括肿瘤或病毒感染。
优选的,所述的肿瘤包括但不限于淋巴瘤、肺癌、宫颈癌、白血病、卵巢癌、鼻咽癌、乳腺癌、子宫内膜癌、结肠癌、肝癌、脑癌、直肠癌、胃癌、膀胱癌、脑胶质瘤、肺癌、支气管癌、骨癌、前列腺癌、胰腺癌、肝和胆管癌、食管癌、肾癌、甲状腺癌、头颈癌、睾丸癌、胶质母细胞瘤、星形细胞瘤、黑色素瘤、骨髓增生异常综合征、以及肉瘤;其中,所述的白血病选自急性淋巴细胞性白血病、急性骨髓性白血病、髓性白血病、慢性淋巴细胞性白血病、多发性骨髓瘤、浆细胞白血病或慢性骨髓性白血病中的一种或两种以上。
进一步优选的,所述淋巴瘤包括霍奇金淋巴瘤和非霍奇金淋巴瘤,包括B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、边缘区B细胞淋巴瘤、T细胞淋巴瘤或瓦尔登斯特伦巨球蛋白血症中的一种或两种以上。
进一步优选的,所述肉瘤包括骨肉瘤、尤文肉瘤、平滑肌肉瘤、滑膜肉瘤、软组织肉瘤、血管肉瘤、脂肪肉瘤、纤维肉瘤、横纹肌肉瘤或软骨肉瘤中的一种或两种以上。
更优选的,所述的肿瘤选自但不限于肺癌、肝癌、甲状腺癌、头颈癌、胃癌、肾癌、前列腺癌、胰腺癌或黑色素瘤中的一种或两种以上。
在本发明的一个具体实施方式中,所述的肿瘤为肺癌。
优选的,所述的肺癌选自小细胞肺癌或非小细胞肺癌。
进一步优选的,所述的非小细胞肺癌包括肺腺癌、肺鳞癌、大细胞癌、腺鳞癌、肉瘤样癌、类癌、涎腺型癌等等。
所述的病毒感染包括急性病毒感染或慢性病毒感染;优选为流感病毒、副流感病毒、疱疹病毒(例如HSV-1、EBV)、麻疹病毒、水泡口炎病毒、乙型肝炎病毒、丙型肝炎病毒、人免疫缺陷病毒、淋巴细胞脉络丛脑膜炎病毒或人乳头瘤病毒。
本发明的第二方面,提供了一种OTUD3抑制剂,所述的OTUD3抑制剂包含通式(Ⅰ)化合物或其药学上可接受的盐、立体异构体、水合物、前药、溶剂化物或代谢产物。
对化合物的限定,同本发明的第一方面。
本发明的第三方面,提供了一种药物或药物组合物,所述的药物或药物组合物包含通式(Ⅰ)化合物或其药学上可接受的盐、立体异构体、水合物、前药、溶剂化物或代谢产物,以及,药物学上可接受的辅料。
对化合物的限定,同本发明的第一方面。
优选的,所述的药学上可接受的辅料选自载体、赋形剂、稀释剂、润滑剂、润湿剂、乳化剂、防腐剂、抗氧化剂、缓冲剂、抑菌剂、使制剂与接受者的血液等渗的溶质、悬浮剂、助悬剂、增溶剂、增稠剂、稳定剂、甜味剂以及香料中的一种或两种以上的组合。
优选的,所述药物或药物组合物的制剂形式可以为糖浆剂、酏剂、悬浮剂、粉剂、颗粒剂、片剂、胶囊、锭剂、水溶液、霜剂、膏剂、洗液剂、凝胶剂或乳剂。所述药物的各种剂型可以按照药学领域的常规生产方法制备。
优选的,药物制剂优选为单位剂量制剂。
单位剂量制剂中药物活性组分的量可从0.001毫克到1000毫克改变或调整,根据活性组分的具体应用和效力而定。如果需要,药物组合物还可包含其它适合的治疗剂。
单位剂量制剂可以配置成活性成分浓度为0.001-50μM,优选1-20μM,例如1、1.25、2.5、4、5、6.1、7.4、7.5、8.1、10、14.8、15、20、30、40、50μM等;
所述的化合物或药物或药物组合物可以按照0.001-100mg/kg(优选5-30mg/kg,进一步优选10-20mg/kg)例如1、2、3、5、10、15、20、25、30、35、40、50、60、70、80、90、100mg/kg等的剂量给药。
所述的药物或药物组合物适于非肠胃给药诸如例如通过静脉内、肌内、皮内和皮下途径给药的制剂包括含水和非水的等渗无菌注射液,其可包含抗氧化剂,缓冲剂,抑菌剂,和使制剂与接受者的血液等渗的溶质,以及含水和非水的无菌悬浮剂,其可包含助悬剂,增溶剂,增稠剂,稳定剂或防腐剂。在本发明的实践中,药物或药物组合物可通过例如静脉输注,经口,局部,腹膜内,膀胱内和鞘内给药。化合物的制剂可存在于单位剂量或者多剂量密封容器诸如安瓿和小瓶中。注射用溶液和悬浮液可从先前所述类型的无菌粉剂、颗粒和片剂制备。
在本发明的环境下,药物或药物组合物的施加剂量通过所用的具体化合物的效力和对象的病况、以及待治疗对象的体重或者体表面积而定。剂量的大小将根据在具体对象中伴随具体化合物给药产生的任何不利副作用的存在、性质和程度而定。在正被治疗的病症的治疗或者预防中确定待给药的化合物的有效量中,医师可以评价诸如化合物的循环血浆水平、化合物毒性和/或疾病进程等因素而定。
所述的药物或药物组合物可以用于治疗肿瘤或病毒感染。
优选的,所述的肿瘤包括但不限于淋巴瘤、肺癌、宫颈癌、白血病、卵巢癌、鼻咽癌、乳腺癌、子宫内膜癌、结肠癌、肝癌、脑癌、直肠癌、胃癌、膀胱癌、脑胶质瘤、肺癌、支气管癌、骨癌、前列腺癌、胰腺癌、肝和胆管癌、食管癌、肾癌、甲状腺癌、头颈癌、睾丸癌、胶质母细胞瘤、星形细胞瘤、黑色素瘤、骨髓增生异常综合征、以及肉瘤;其中,所述的白血病选自急性淋巴细胞性白血病、急性骨髓性白血病、髓性白血病、慢性淋巴细胞性白血病、多发性骨髓瘤、浆细胞白血病或慢性骨髓性白血病中的一种或两种以上。
进一步优选的,所述淋巴瘤包括霍奇金淋巴瘤和非霍奇金淋巴瘤,包括B细胞淋巴瘤、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、边缘区B细胞淋巴瘤、T细胞淋巴瘤或瓦尔登斯特伦巨球蛋白血症中的一种或两种以上。
进一步优选的,所述肉瘤包括骨肉瘤、尤文肉瘤、平滑肌肉瘤、滑膜肉瘤、软组织肉瘤、血管肉瘤、脂肪肉瘤、纤维肉瘤、横纹肌肉瘤或软骨肉瘤中的一种或两种以上。
更优选的,所述的肿瘤选自但不限于肺癌、肝癌、甲状腺癌、头颈癌、胃癌、肾癌、前列腺癌、胰腺癌或黑色素瘤中的一种或两种以上。
优选的,所述的肺癌选自小细胞肺癌或非小细胞肺癌。
进一步优选的,所述的非小细胞肺癌包括肺腺癌、肺鳞癌、大细胞癌、腺鳞癌、肉瘤样癌、类癌、涎腺型癌等等。
所述的病毒感染包括急性病毒感染或慢性病毒感染;优选为流感病毒、副流感病毒、疱疹病毒(例如HSV-1、EBV)、麻疹病毒、水泡口炎病毒、乙型肝炎病毒、丙型肝炎病毒、人免疫缺陷病毒、淋巴细胞脉络丛脑膜炎病毒或人乳头瘤病毒。
本发明的第四方面,提供了一种抑制OTUD3蛋白与泛素结合的方法,所述的方法包括使用上述任一所述的通式(Ⅰ)化合物或其药学上可接受的盐、立体异构体、水合物、前药、溶剂化物或代谢产物,和/或,OTUD3抑制剂,和/或,上述任一所述的药物或药物组合物。
本发明的第五方面,提供了一种通式(Ⅰ)化合物或其药学上可接受的盐、立体异构体、水合物、前药、溶剂化物或代谢产物在制备抑制OTUD3蛋白与泛素结合的产品中的应用。
对化合物的限定,同本发明的第一方面。
优选的,所述的产品包括药物或药物组合物。
进一步优选为上述的药物或药物组合物。
本发明的第六方面,提供了一种促进GRP78蛋白降解的方法,所述的方法包括上述任一所述的通式(Ⅰ)化合物或其药学上可接受的盐、立体异构体、水合物、前药、溶剂化物或代谢产物,和/或,OTUD3抑制剂,和/或,上述任一所述的药物或药物组合物。
本发明的第七方面,提供了一种上述通式(Ⅰ)化合物或其药学上可接受的盐、立体异构体、水合物、前药、溶剂化物或代谢产物在制备促进GRP78蛋白降解的产品中的应用。
对化合物的限定,同本发明的第一方面。
优选的,所述的产品包括药物或药物组合物。
进一步优选为上述的药物或药物组合物。
本发明的第八方面,提供了一种治疗疾病的方法。
优选的,所述的方法包括向患病个体施加通式(Ⅰ)化合物或其药学上可接受的盐、立体异构体、水合物、前药、溶剂化物或代谢产物,和/或,上述任一所述的OTUD3抑制剂,和/或,上述任一所述的药物或药物组合物。
对化合物的限定,同本发明的第一方面。
优选的,所述的疾病为肿瘤或病毒感染,所述的患病个体为患肿瘤或病毒感染的个体。
优选的,所述的患病个体选自人或非人动物。
对肿瘤和病毒的限定,同发明的第一方面。
本发明所述的“药学上可接受的盐”指由药学上可接受的无毒性的酸或碱制备而来的盐,其中的酸或碱包括无机酸或碱或有机酸或碱。所述的无机酸选自盐酸、氢溴酸、磷酸、氢碘酸或硫酸。所述的无机碱选自钙、镁、锂、钠、锌、铝或钾。所述的有机酸选自甲酸、羟基乙酸、丙酸、醋酸、琥珀酸、甲磺酸、乙磺酸、顺丁烯二酸、谷氨酸、苯甲酸、硬脂酸、海藻酸、苯磺酸、葡萄糖醛酸、双羟萘酸或半乳糖醛酸。所述的有机碱选自二乙醇胺、胆碱、普鲁卡因、赖氨酸或1,2-乙二胺。
本发明所述的“立体异构体”包括对映体、非对映体和几何异构体的形式存在。
本发明所述的“水合物”为含有水的化合物,其中水可以以配位键与其他部分相连,也可以是以共价键相结合。
本发明所述的“溶剂化物”是指本发明的化合物与一种或多种溶剂分子的物理结合。该物理结合包括各种程度的离子和共价键合,包括氢键合。在某些情况下,溶剂化物可被分离出来,例如当一个或多个溶剂分子掺入到结晶固体的晶格中。溶剂化物包括溶液相和可分离的溶剂化物。代表性的溶剂化物包括乙醇化物、甲醇化物等。
本发明所述的“代谢产物”是指本发明的化合物在体内生理条件下使其发生化学降解所得产物。
本发明所述的“前药”指药物经过化学结构修饰后得到的在体外或非特定靶位无活性或活性较小、在体内经酶或非酶转化释放出活性药物(例如本发明的化合物)而发挥药效的化合物。
本发明所述的“烷基”代表直链或支链的且不含不饱和键的烃链自由基,且该烃链自由基以单键与分子其它部分连接。典型的烷基基团含有1至20(例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20)个碳原子,例如甲基、乙基、正丙基、异丙基、正丁基、仲丁基、异丁基、叔丁基、正戊基、异戊基、新戊基、叔戊基、正己基、异己基、正庚基、异庚基、正辛基、壬基、癸基、十一烷基、1-甲基十一烷基、十二烷基、十三烷基、十四烷基、十五烷基、十六烷基、十七烷基、十八烷基、十九烷基及二十烷基等。
本发明所述的“个体”可以为人或非人动物,例如非人哺乳动物。
本发明术语“包括”或“包含”是开放式的描述,含有所描述的指定成分或步骤,以及不会实质上影响的其他指定成分或步骤。
本发明术语“和/或”包含该术语所连接的项目的所有组合,应视为各个组合已经单独地在本文列出。例如,“A和/或B”包含了“A”、“A和B”以及“B”。又例如,“A、B和/或C”包含了“A”、“B”、“C”、“A和B”、“A和C”、“B和C”以及“A和B和C”。
本发明所述的“治疗”表示在疾病已开始发展后减缓、中断、阻止、控制、停止、减轻、或逆转一种体征、症状、失调、病症、或疾病的进展或严重性,但不一定涉及所有疾病相关体征、症状、病症、或失调的完全消除。
附图说明
图1:OTUD3示意图,其中,OTU代表卵巢癌相关结构域,UBA代表一个泛素相关结构域;
图2:OTUD3的PDB模型;
图3:OTUD3的OTU结构域与OTUD5的OTU结构域的3D视图及其叠加结构图;
图4:OTUD3的OTU结构域与泛素的复合物和OTUD5的OTU结构域与泛素的复合物的结构图及其叠加结构图,其中,上层为3D视图,下层为填充模型;
图5:OTUD3的OTU结构域与泛素复合物及其对接盒;
图6:筛选过程示意图;
图7:18种化合物与H1299共同孵育24h和48h对细胞增殖的影响;
图8:18种化合物与H460细胞和A549细胞共同孵育48h对细胞增殖的影响;
图9:OTUDin3的结构图;
图10:OTUDin3与OTUD3的OTU结构域复合物;
图11:OTUD3和OTUDin3的OTU结构域相互作用模式图;
图12:OTUDin3对不同细胞的IC50;
图13:加入DMSO和分别加入1μM、10μM、20μM的OTUDin3处理的H1299的细胞形态;
图14:加入DMSO和分别加入0.5μM、1μM、2μM、4μM、10μM的OTUDin3处理后的H1299细胞系和A549细胞系的集落形成实验结果;
图15:加入DMSO和分别加入1μM、5μM、10μM、15μM、20μM的OTUDin3处理后的H1299细胞系的划痕愈合实验结果;
图16:加入DMSO和分别加入1μM、5μM、10μM、15μM、20μM的OTUDin3处理后的H1299细胞系的Transwell检测实验结果;
图17:加入DMSO和分别加入1μM、5μM、10μM、15μM、20μM的OTUDin3处理后的H1299细胞系的流式细胞检测凋亡结果;
图18:加入0、1μM、5μM、10μM、15μM、20μM的OTUDin3处理后的H1299细胞系的凋亡定量结果;
图19:随着OTUDin3浓度增加,Cleaved-PRAP和Cleaved-Caspase-3蛋白表达水平;
图20:加入0、5μM、10μM、15μM、20μM的OTUDin3处理OTUD3敲低的H1299细胞系增殖的实验结果;
图21:加入DMSO和分别加入1μM、2μM的OTUDin3处理OTUD3敲低的H1299细胞系集落形成的实验结果;
图22:加入DMSO和分别加入5μM、10μM的OTUDin3处理OTUD3敲低的H1299细胞系的划痕愈合实验结果;
图23:加入DMSO和分别加入10μM、20μM的OTUDin3处理OTUD3敲低的H1299细胞系的Transwell检测实验结果;
图24:加入DMSO和分别加入10μM、20μM的OTUDin3处理OTUD3敲低的H1299细胞系的流式细胞检测凋亡结果;
图25:随着OTUDin3浓度增加,OTUD3敲低的H1299细胞系Cleaved-PRAP和Cleaved-Caspase-3蛋白表达水平;
图26:OTUDin3处理不同时间,GRP78蛋白的表达水平;
图27:不同浓度OTUDin3处理,GRP78蛋白的表达水平;
图28:OTUDin3逆转了OTUD3的去泛素化作用;
图29:OTUD3与GRP78之间的相互作用的免疫共沉淀实验结果;
图30:OTUD3与GRP78之间的相互作用的GST pull-down实验结果;
图31:OTUD3的二聚化的免疫共沉淀实验结果;
图32:OTUD3的二聚化的GST pull-down实验结果;
图33:OTUDin3处理后对肿瘤生长的影响;
图34:随处理天数增加,不同组的肿瘤体积;
图35:对照组、10mg/kg组和20mg/kg组肿瘤体积的变化;
图36:对照组、10mg/kg组和20mg/kg组肿瘤重量的变化;
图37:随处理天数增加,不同组的小鼠个体重量;
图38:OTUDin3处理后对心、肝、肾、脾、肺器官的影响;
图39:OTUDin3治疗肿瘤的机理示意图;
图40:OTUD3敲低的western blotting结果。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明的部分实施例,而不是全部。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。
本发明中用到的部分材料来源及方法:
1)计算虚拟筛选
OTUD3(PDB ID:4BOU)和OTUD5(PDB ID:3TMP)的三维结构从蛋白质数据库http://www.rcsb.org/中获得。利用AutoDockTools-1.5.7进行分子对接前,制备OTUD3的结构。对接盒被定义为包含S1结合位点。从ChemDiv(http://www.chemdiv.org/)获得了SDF格式的100000个筛选化合物的多样性集。使用Open Babel3.1.1将SDF格式文件转换为PDB格式。然后使用AutoDockTools-1.5.7中的配体准备脚本将转换后的PDB文件转换为PDBQT格式。然后使用Autodock Vina 1.1.2进行后续的分子对接。详细的步骤参见Forli S,Huey R,Pique ME,Sanner MF,Goodsell DS,Olson AJ.Computational protein-ligand dockingand virtual drug screening with the AutoDock suite.Nat Protoc.2016;11(5):905-19。然后根据亲和能对小分子进行排序。
所有结构文件的可视化都使用PyMOL分子图形系统,Version 2.6.0aLLC进行。使用蛋白质-配体相互作用分析器网络工具分析蛋白质-配体相互作用。
2)细胞系和培养条件
细胞系H1299,H1650,H460,A549和HEK293T来自美国模式培养物研究所(ATCC),经STR分析(Promega)验证,并通过支原体检测显示无支原体。H1299、H1650和H460细胞培养于添加10%胎牛血清(FBS)和1%青霉素-链霉素的Roswell Park Memorial Institute(RPMI)1640培养基中。A549和HEK293T细胞在添加10%牛血清和1%青霉素-链霉素的DMEM中培养。所有细胞在37℃ 5% CO2的潮湿环境中生长。除特别注明外,培养基和补充剂均从Gibco购买。
3)试剂和抗体:
OTUDin3购买自ChemDiv(G856-0029)。研究中使用的抗体有抗GAPDH(1:1000,AC033,ab克隆)、抗OTUD3(1:50 00,HPA028543,Sigma)、抗Cleaved-PARP(1:1000,#9544,Cells Signaling Technology)、抗Cleaved-caspase-3(1:50 00,#9661,Cells SignalingTechnology)、抗GRP78(1:1000,11587-1-AP,Proteintech)、抗HA(1:1000,M180-3,MBL)、抗Myc(1:1000,M192-3,MBL)和抗Flag(1:1000,F7425,Sigma)。
4)质粒
将全长OTUD3WT和全长OTUD3C76A突变体克隆到pFlag-CMV-2载体上。将全长OTUD3WT和全长GRP78WT克隆到pCMV-Myc载体中。将GST标记的OTUD3克隆到pGEX-4T-2载体上。
5)慢病毒感染和OTUD3基因敲低
为了生成针对人OTUD3的慢病毒shRNA构建体,将OTUD3shRNA序列克隆到pCDH-puro载体中。这些病毒被用来感染有聚苯乙烯存在的细胞。48h后,H1299细胞在含嘌呤霉素的培养基中培养,选择稳定的克隆。通过western blotting对稳定敲低OTUD3的克隆进行鉴定和验证。
OTUD3shRNA序列:5’-TGGAAATCAGGGCTTAAAT-3’(SEQ ID NO:1)。
6)细胞活力测定
细胞以每孔5000个细胞的密度接种于96孔板中。24小时后,用不同浓度的DMSO或指定化合物处理细胞72小时,然后使用CCK-8Kit(CK001-500T,LABLEAD)评估细胞活力。CCK-8测定时,每孔加入溶解于90μL培养基中的10μLCCK-8。在37℃下培养1h后,用酶标仪(PerkinElmer)测量450nm处的吸光度。IC50值采用GraphPad Prism非线性回归计算。
7)菌落形成测定
将细胞稀释成单细胞悬液,在6孔板中每孔1000个细胞,37℃,5% CO2培养24h,然后用不同浓度的OTUDin3处理细胞10-14天,直至克隆清晰可见。细胞培养板用PBS轻轻洗涤2次,用4%多聚甲醛磷酸盐缓冲液固定15分钟,然后用结晶紫染色15分钟。用ddH2O洗涤多余的染色,室温干燥数小时。对被染色的集落拍照。
8)划痕愈合实验
进行划痕愈合试验以评估细胞的流动性。细胞接种于6孔板。培养24h后,用200μL移液管头手动划孔,PBS洗涤3次。然后,将细胞培养在不含FBS的RPMI-1640培养基中,并用不同浓度的OTUDin3处理。24h后拍摄划痕区域。使用ImageJ软件(NIH)分析两个细胞边缘之间的距离。
9)Transwell检测
在24孔Transwell板中进行细胞侵袭测定,板上有8.0μm聚碳酸酯膜的Transwell小室(Corning),覆盖有60μL基质凝胶基质(Corning)。简言之,将重悬于无血清培养基中的2×105个H1299细胞与不同浓度的OTUDin3一起接种在每个Transwell小室中。将Transwell小室放置在含有具有10%FBS的RPMI 1640培养基的孔中。24小时后,取出未侵入的细胞,将Transwell小室在PBS中洗涤,在4%甲醛中固定20分钟,并用0.1%结晶紫染色20分钟。对孔进行拍照,并对染色的细胞进行计数。
10)基于流式细胞术的细胞凋亡检测
采用Annexin V-FITC/PI细胞凋亡检测试剂盒(Gene-Protein Link)进行基于流式细胞术的细胞凋亡检测。用DMSO配置不同浓度的OTUDin3处理6孔板中的H1299细胞24h,离心收集细胞,用100μL结合缓冲液重悬。然后加入5μL Annexin V FITC和5μL碘化丙啶(PI),室温避光孵育10min,再加入400μL结合缓冲液,1h内流式细胞仪检测样品,数据用FlowJo_V3.8.1软件处理。
11)Western blotting
采用标准技术进行Western blotting分析。用添加磷酸酶和蛋白酶抑制剂的RIPA裂解缓冲液(50mM Tris-Cl pH 8.0,150mM NaCl,1% NP-40,0.5%脱氧胆酸钠和0.1%SDS)收集细胞。蛋白质通过SDS-PAGE分离,并转移到硝化纤维素过滤器(NC)膜(默克Millipore,德国)。用5%脱脂牛奶在室温下封闭膜1小时,然后与指定的抗体孵育过夜。用tris-buffer saline with Tween(TBST)洗涤膜3次,每次10分钟。将NC膜与二抗在室温下孵育1h,然后用TBST洗涤4次,每次10min。最后,使用FluorChem R成像系统(ProteinSimple,USA)检测目标蛋白的表达。
12)体内GRP78泛素化试验
在体内进行GRP78泛素化检测时,将Flag-OTUD3或Flag-OTUD3C76A、Myc-GRP78和HA-Ub转染HEK293T细胞。各组分别用5μM的OTUDin3或等体积的DMSO处理40h。用20μM的蛋白酶体抑制剂MG132(Calbiochem)处理6h后,用RIPA裂解缓冲液裂解细胞,用抗Myc抗体孵育3h,用蛋白A/G琼脂糖微球(Santa Cruz)在4℃下孵育6h。然后用RIPA缓冲液洗涤三次。在SDS-PAGE样品缓冲液中煮沸释放蛋白,用抗HA单克隆抗体(MBL)进行免疫印迹分析。
13)免疫共沉淀实验
HEK293T细胞按照制造商的方案使用聚乙烯亚胺(PEI,Invitrogen)试剂用指定的质粒转染,并用DMSO或不同浓度的OTUDin3处理。在免疫沉淀实验中,细胞用HEPES裂解缓冲液(20mM HEPES,pH 7.2,50mM NaCl,0.5% Triton X100,1mM NaF和1mM二硫代索糖醇)和蛋白酶抑制剂混合物(Roche)进行裂解。使用指定的一抗和蛋白A/G琼脂糖微球(SantaCruz)在4℃下进行免疫沉淀。然后用HEPES裂解缓冲液冲洗免疫复合物四次。裂解物和免疫沉淀物均使用指定的一抗检测,随后使用相关的二抗和SuperSignal West Pico化学发光底物(Thermo)检测。
14)GST pull-down实验
在DMSO或不同浓度的OTUDin3存在下,将与谷胱甘肽-琼脂糖4B微球(来自GE)结合的细菌表达的GST和GST-OTUD3与HEK293T细胞中表达的Myc-GRP78或Flag-OTUD3在4℃下孵育4小时。然后用GST结合缓冲液(100mM NaCl,10mM Tris,50mM NaF,2mM EDTA,0.5mMNa3VO4和1% NP40)洗涤微球4次,洗脱蛋白质,然后进行western blotting或考马斯蓝染色。
15)非小细胞肺癌异种移植研究
动物实验方案经中国北京生命组学研究所动物护理与使用委员会批准。从VitalRiver实验动物科技有限公司购买的6-8周龄雄性BALB/c裸鼠,以RPMI-1640和Matrigel(Corning)1:1的混合物皮下接种6×106个H1299肿瘤细胞。疗效研究是在肿瘤达到约150-250mm3时开始的。采用随机区组设计,将18只荷瘤小鼠按肿瘤大小分为6个区组,每个区随机分为不同组。小鼠给予10或20mg/kg OTUDin3(每隔一天腹腔注射)的给药方案,10%DMSO、10%聚氧乙烯蓖麻油和80%生理盐水作为对照。每隔一天监测小鼠体重和肿瘤体积。用游标卡尺测量肿瘤的长度和宽度。肿瘤体积计算公式为V=0.5×长×宽2,当小鼠肿瘤最大体积达到2000mm3时,采集肿瘤标本。所有动物均严格按照《实验动物护理与使用指南》和《脊椎动物利用与护理原则》处理,所有动物工作均经北京生命组学研究所机构动物护理与使用委员会(IACUC)批准。
16)统计分析
采用GraphPad Prism 7.0软件进行统计分析。所有结果均以多个独立实验的均数±标准差(SD)表示。采用正态性和对数正态性检验。如果数据不符合正态分布标准,则进行非参数检验。非配对Student’s t-tests检验用于两组比较。采用双因素方差分析和Fisher’s LSD检验来确定H1299shNC与H1299shOTUD3之间的统计学差异。小鼠实验中非正态分布的成对样本采用Wilcoxon检验。P值小于0.05认为有统计学意义。
实施例1小分子OTUD3抑制剂的鉴定
针对DUBs的新型靶向抑制剂的发现主要使用小分子文库的高通量筛选(HTS)进行,与常规的药物筛选方法相比,计算机辅助药物设计(CADD)更高效、更经济。CADD将分子对接集成到一个有希望的治疗靶点的配体结合口袋,计算每个对接的小分子化合物的结合能,并有选择地选择最好的候选小分子进行后续实验程序。本申请采用基于分子对接的计算虚拟筛选OTUD3抑制剂。
OTUD3包含一个卵巢癌相关结构域(OTU)和一个泛素相关结构域(UBA),参见图1,其中,OTU结构域是催化结构域,是OTUD3执行去泛素化活性和与底物相互作用所必需的。OTUD3的PDB文件从蛋白质数据库(http://www.rcsb.org/)获得,PDB号为4BOU,见图2,去除所有的非均相原子,选择4BOU的A链进行后续的分子对接。
先前开发的去泛素化酶抑制剂已被发现与进化上保守的去泛素化酶的催化中心结合,导致选择性差。然而,一些抑制剂,如USP14抑制剂IU1-系列和USP7抑制剂FT671,阻断了泛素C端到活性位点的通路,并表现出惊人的优异选择性。因此,如图2所示的结合位点(S1结合位点)被确定为配体结合位点,这是OTUD3去泛素化活性的关键。然而,由于OTUD3与泛素复合物的结构尚未确定,考虑到OTUD3的结构与OTUD5的结构高度相似,且均方根偏差较低将OTUD3的OTU结构域与OTUD5(PDB:3TMP)的OTU结构域的结构叠加,将OTUD3的OTU结构域与泛素复合物的结构与OTUD5的OTU结构域与泛素复合物的结构进行叠加,确定OTUD3的泛素结合位点(图3-4)。然后,设置对接盒,将整个S1结合位点及其周围的所有氨基酸残基包裹起来(图5)。后续分子对接使用Autodock Vina 1.1.2进行。在ChemDiv上筛选了10万种不同的化合物,并确定了18个对接得分最高的候选化合物,用于进一步的生物学实验验证,筛选过程示意图见图6。18个化合物的结构见下表1。
表1
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由于OTUD3的去泛素化作用可以稳定GRP78蛋白,在肺部肿瘤发生中起促进作用,因此OTUD3抑制剂应能抑制NSCLC细胞的增殖。因此,选择细胞增殖试验作为OTUD3抑制剂的进一步筛选策略。将10μM的18种候选化合物与H1299细胞共同孵育24h和48h,只有化合物16可以显著抑制H1299细胞的增殖(图7)。并后续通过H460细胞系和A549细胞系验证了该结果(图8)。将化合物16命名为OTUDin3,结构如图9所示,对OTUDin3进行分子对接。分子对接分析表明,OTUDin3与OTUD3的亲和能为-10.1kcal/mol,OTUDin3被对接到S1结合位点中(图10)。预测的蛋白-配体相互作用模式显示,OTUDin3与OTUD3形成多重相互作用,包括与Asn136和Ile162的疏水作用,与Gln154、Trp160、Tyr177和Tyr183的氢键,以及与Trp160和Tyr177的π堆积键(图11)。
总的来说,我们通过虚拟筛选和细胞增殖试验确定了OTUDin3是OTUD3的抑制剂,适合进一步的研究。
实施例2OTUDin3对NSCLC细胞生长、转移和凋亡的影响
如实施例1所述,OTUDin3对NSCLC细胞有抑制作用。接下来,使用CCK-8细胞计数试剂盒检测OTUDin3对H1299细胞、A549细胞、H460细胞以及H1650细胞的IC50,H1299细胞、A549细胞、H460细胞以及H1650细胞的IC50值分别为8.1、14.8、6.1和7.4μM(见图12)。并以H1299细胞为例,观察其细胞形态(参见图13),确定OTUDin3能够有效的诱导H1299细胞死亡。
对H1299细胞和A549细胞进行集落形成试验,结果如图14所示,OTUDin3以浓度依赖性的方式抑制H1299细胞和A549细胞的生长。
另外,通过划痕愈合实验评估了OTUDin3对H1299细胞运动的影响。结果表示,经过OTUDin3处理显著降低H1299细胞的迁移能力,并呈浓度依赖性(图15)。此外,通过Transwell检测实验评估,OTUDin3以浓度依赖的方式抑制了H1299细胞的侵袭能力(图16)。
接下来,利用不同浓度的OTUDin3处理H1299细胞,使用凋亡试剂盒定量检测早期和晚期凋亡细胞,使用流式细胞术分析凋亡细胞群。结果表示,OTUDin3以浓度依赖性方式诱导H1299细胞凋亡(图17-18)。
对细胞裂解物通过western blotting进行评估,发现OTUDin3以浓度依赖性的方式增加了凋亡相关蛋白Cleaved-PARP和Cleaved-caspase-3的表达水平(图19)。其中,Cleaved-PARP和Cleaved-caspase-3为剪切型蛋白。
为了研究对NSCLC细胞的作用是否主要来源于靶向OTUD3,我们在H1299细胞中敲低OTUD3(图40)。并重复进行上述的实验,CCK-8检测结果显示,与对照组相比,OTUDin3对OTUD3敲低细胞系增殖的抑制作用和集落形成的影响显著降低(图20-21)。划痕愈合和Transwell实验显示,与对照组相比,OTUD3敲低细胞对OTUDin3的敏感性较低(图22-23)。凋亡实验结果显示,OTUD3敲低细胞对OTUDin3的反应较低(图24-25)。
总之,上述结果表明OTUDin3通过靶向OTUD3,有效的抑制NSCLC细胞的生长、迁移和侵袭,还能促进NSCLC细胞的凋亡。
实施例3OTUDin3影响NSCLC细胞生长、转移和凋亡的机制
如上所述,OTUD3通过稳定GRP78蛋白,以促进肺部肿瘤发生。
使用OTUDin3处理处理H1299细胞,然后裂解,使用western blotting检测GRP78的蛋白水平。
具体的,
使用5μM的OTUDin3处理H1299细胞不同时间,然后裂解。Western blotting结果显示,OTUDin3以时间依赖性的方式下调GRP78,24h后下调效果显著(图26)。
使用不同浓度的OTUDin3处理H1299细胞24小时。Western blotting结果显示,OTUDin3以浓度依赖的方式下调GRP78(图27)。这一结果与现有技术中H1299细胞系中OTUD3敲低对GRP78的下调一致。
进一步的,现有技术中记载,OTUD3以依赖于其去泛素化活性的方式上调GRP78,而催化活性不强的突变体C76A失去了上调GRP78的能力。为了验证OTUDin3通过抑制OTUD3的去泛素化活性对GRP78的下调作用,我们进行了GRP78泛素化实验。将Flag-OTUD3野生型(WT,A组)或Flag-OTUD3C76A(B组)、Myc-GRP78和HA-Ub转染HEK293T细胞。A组和B组分别用5μM的OTUDin3或等体积的二甲亚砜(DMSO)处理46h。
GRP78泛素化实验显示,过表达OTUD3WT显著降低GRP78泛素化水平,而催化活性不强的突变体C76A没有降低GRP78泛素化水平。然而,当OTUD3WT过表达细胞用OTUDin3处理时,GRP78泛素化增加,逆转了OTUD3的去泛素化(图28)。
已有研究表明,OTUD3的N端OTU结构域介导了与GRP78的物理相互作用。鉴于OTUDin3与OTU结构域结合,通过co-IP(Co-immunoprecipitation)和GSTpull-down实验进一步研究OTUDin3是否破坏了OTUD3与GRP78之间的相互作用。结果表示,OTUDin3不影响OTUD3与GRP78之间的相互作用(图29-30)。此外,现有技术中公开OTUD3形成同型二聚体来执行去泛素化活性。而Co-IP和GST pull-down实验结果表明OTUDin3也不影响OTUD3的二聚化(图31-32)。
综上所述,OTUDin3不影响OTUD3的二聚化,以及,OTUD3与GRP78的相互作用,但是OTUDin3与OTUD3结合在S1结合位点,干扰了OTUD3和泛素的结合(参见图5、图10),从而起到抑制OTUD3的去泛素化活性和增加GRP78泛素化来增强GRP78的降解(示意图见图39)。这些结果与OTUD3抑制剂的预期作用机制一致。
实施例4OTUDin3抑制体内的NSCLC生长
为了进一步研究OTUDin3对体内非小细胞肺癌的影响,我们在裸鼠体内植入H1299细胞以构建非小细胞肺癌小鼠模型。当肿瘤达到约150~250mm3时,将荷瘤小鼠按肿瘤大小分为6个组,每个组3只。治疗组分别给予10mg/kg和20mg/kg的OTUDin3腹腔注射,每隔一天注射一次。在相同的实验条件下,对照组给予10% DMSO、10%聚氧乙烯蓖麻油和80%生理盐水。结果显示,用OTUDin3治疗可显著抑制肿瘤生长(图33)。与对照组相比,各治疗组肿瘤体积和重量均明显减小(图34-36)。同时,对照组和治疗组小鼠的平均体重无显著差异(图37)。同时,H&E染色结果显示OTUDin3对心、肝、脾、肺、肾等主要脏器无明显影响(图38)。综上所述,OTUDin3在体内抑制非小细胞肺癌生长,且没有明显的健康问题或体重减轻。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
此外,本发明的各种不同的实施方式之间也可以进行任意组合,只要其不违背本发明的思想,其同样应当视为本发明所公开的内容。
Claims (3)
1.一种化合物或其药学上可接受的盐在制备治疗和/或预防非小细胞肺癌产品中的应用,其特征在于,所述的化合物为式(II)结构:
。
2.一种治疗和/或预防非小细胞肺癌的药物或药物组合物,其特征在于,所述的药物或药物组合物包含化合物或其药学上可接受的盐,以及,药物学上可接受的辅料;
所述的化合物为式(II)结构:
所述药物学上可接受的辅料选自载体或赋形剂。
3.如权利要求2的药物或药物组合物,其特征在于,所述药物学上可接受的辅料选自稀释剂、润滑剂、润湿剂、乳化剂、防腐剂、抗氧化剂、缓冲剂、抑菌剂、使制剂与接受者的血液等渗的溶质、悬浮剂、助悬剂、增溶剂、增稠剂、甜味剂以及香料中的一种或两种以上的组合。
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