CN112480097B - 一种靶向ets结构域蛋白的抑制剂及其应用 - Google Patents

一种靶向ets结构域蛋白的抑制剂及其应用 Download PDF

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CN112480097B
CN112480097B CN202011350630.3A CN202011350630A CN112480097B CN 112480097 B CN112480097 B CN 112480097B CN 202011350630 A CN202011350630 A CN 202011350630A CN 112480097 B CN112480097 B CN 112480097B
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大卫·凯尔文
李成勋
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Shantou University Medical College
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Abstract

本发明涉及靶向抑制剂领域，公开了一种靶向ETS结构域蛋白的抑制剂，可以靶向结合ERG的保守结构域ETS，通过阻断ETS结构域与PARP的结合来激活parthanatos，影响其对下游基因的表达，其中大部分下游基因为细胞因子，同时造成DNA损伤修复基因的上调。通过阻断ETS结构域与其他蛋白的结合，达到治疗相关疾病的目的。

Description

一种靶向ETS结构域蛋白的抑制剂及其应用
技术领域
本发明涉及靶向抑制剂领域，尤其涉及一种靶向ETS结构域蛋白的抑制剂及其应用。
背景技术
2005年，Tomlins等人证实，在前列腺癌中，TMPRSS2：ERG融合非常常见(大约50％)，与TMPRSS2基因的雄激素驱动启动子融合后，ERG在前列腺癌中持续高表达会促进前列腺癌的进展。从高级PIN(10–20％)到癌变(30–80％)，融合的发生频率增加，融合患者中有79％更有可能复发。ERG及其家族成员(ETS1/2，ETV1，ETV6，FLI1和ELK3等)还与其他癌症有关，包括Ewing氏肉瘤和白血病。例如，ERG阳性的急性T淋巴细胞白血病复发的可能性是阴性的四倍。ERG的过度表达是将局部侵袭性癌症转化为转移性癌症的关键因素之一。该家族成员由于均含有高度保守的ETS结构域被称为ETS家族。ETS结构域主要与DNA结合发挥转录调节的功能。
ETS家族除了与癌症密切相关还在病毒的复制和致病过程中发挥重要作用。例如，ETS1和USF1及其他蛋白的相互作用是T细胞中HIV-1LTR完全转录活性所必需的；ETS1与HBV共同作用促进肝细胞癌的转移。但目前，未发现临床上能被广泛应用的靶向含有ETS结构域的蛋白的抑制剂。
发明内容
本发明发现的新型靶向含有ETS结构域的蛋白抑制剂主要是以4-hydroxy-N'-[(1E)-(5-nitrofuran-2-yl)methylidene]benzohydrazide为代表的乙酰肼衍生物及其类似物，以下简称NFZ。
为了解决上述目的，本发明采用的技术方案如下：
一种靶向ETS结构域蛋白的抑制剂，包括如下结构中的一种：
Figure BDA0002801252450000021
其中，R1：硝基呋喃、十氢萘基、萘基、3-6元碳环或者杂环及其低级烷基、低级烷氧基、氰基、烯基、炔基、氨基、硝基、羟基、羧基、巯基、卤素邻间对取代基团中的一种；R2：氢，羟基，氨基，巯基，低级烷基，低级烷氧基，卤素中的一种；R3：氢，低级烷基，低级氧烷基，低级硫烷基，3-6元碳环或者杂环及其低级烷基、低级烷氧基、氰基、烯基、炔基、氨基、硝基、羟基、羧基、巯基、卤素邻间对取代基团中的一种；R4：硝基呋喃，十氢萘基，萘基，3-6元碳环或者杂环及其低级烷基、低级烷氧基、氰基、烯基、炔基、氨基、硝基、羟基、羧基、巯基、卤素邻间对取代基团中的一种；R5：3-6元碳环或者杂环及其低级烷基、低级烷氧基、氰基、烯基、炔基、氨基、硝基、羟基、羧基、巯基、卤素邻间对取代基团中的一种。
本发明发现的新型靶向含有ETS结构域的蛋白抑制剂主要是以4-hydroxy-N'-[(1E)-(5-nitrofuran-2-yl)methylidene]benzohydrazide为代表的乙酰肼衍生物及其类似物，以下简称NFZ。
优选的，所述抑制剂与所述ETS结构域蛋白能产生特异性结合，结合点位于ASN319和PHE375所形成的的孔洞内。
优选的，所述ETS结构域蛋白为ERG蛋白。
实验中发现，NFZ可以结合在ERG的ETS结构域中，特别是ASN319和PHE375所形成的的孔洞内，结合能为-4.73kJ/mol，而这是所有ETS结构域蛋白所特有的结构，即NFZ能与ETS结构域蛋白形成特异性结合。NFZ与含有ETS结构域的蛋白结合后，能有效地阻断ETS结构域与PARP的结合来激活parthanatos，影响其对下游基因的表达，其中大部分下游基因为细胞因子，同时造成DNA损伤修复基因的上调。
一种上述靶向ETS结构域蛋白的抑制剂的应用，应用于制备治疗与ETS结构域蛋白相关疾病的药物。
优选的，所述与ETS结构域蛋白相关疾病包括肿瘤疾病、RNA病毒感染疾病、DNA病毒感染疾病中的一种或多种。
优选的，所述肿瘤疾病包括前列腺癌、尤文氏肉瘤、白血病中的一种或多种；所述RNA病毒感染疾病包括HIV；所述DNA病毒感染疾病包括HBV。
一种包含如上述靶向ETS结构域蛋白的抑制剂的药物组合物。
所述药物组合物包括直接作用于DNA的化疗药物、干扰DNA合成的化疗药物中的一种或多种。
优选的，所述药物组合物包括多柔比星(Mitoxantron)，米托蒽醌(Doxorubicin)，博来霉素(Bleomycin)、氟尿嘧啶(Fluorouracil)中的一种或多种。
NFZ可以增强直接作用于DNA或者干扰DNA合成的化疗药物的作用，特别是增强多柔比星，米托蒽醌，博来霉素和氟尿嘧啶的抑制肿瘤的效果。这些化疗药物单独使用时，能对肿瘤细胞的DNA结构造成破坏或者影响DNA的合成，从而产生大量的DNA损伤。通常，大量DNA损伤会使得PARP过度活化，从而激活parthanatos通路使细胞走向坏死。而ETS结构域蛋白会与PARP结合，减少PARP的活化，从而使得含有DNA损伤的细胞也可以存活。NFZ与ETS结构域蛋白特异性结合后，可以通过阻断PARP与ETS结构域的结合，让PARP成功活化，从而快速激活parthanatos通路导致细胞坏死。可见，NFZ与这类化疗药物有良好的协同杀灭肿瘤细胞的作用。
实施本发明，具有如下有益效果：
本发明通过研究证实，乙酰肼衍生物及其类似物可以靶向结合ERG的保守结构域ETS，通过阻断ETS结构域与PARP的结合来激活parthanatos，影响其对下游基因的表达，其中大部分下游基因为细胞因子，同时造成DNA损伤修复基因的上调。通过阻断ETS结构域与其他蛋白的结合，达到治疗相关疾病的目的。
附图说明
图1是ERG与不同浓度的NFZ利用表面等离子共振技术测得的响应值变化曲线图；
图2是ETS结构域与NFZ的结合模式图；
图3是不同浓度的NFZ抑制TMPRSS2：ERG融合阳性的前列腺癌细胞系VCap的柱形图；
图4是不同浓度的Olaparib和NFZ混合抑制TMPRSS2：ERG融合阳性的前列腺癌细胞系VCap的柱形图；
图5是不同浓度的NFZ、Olaparib+NFZ对TMPRSS2：ERG融合阳性的前列腺癌细胞系VCap的流式分析图；
图6是被NFZ处理后的TMPRSS2：ERG融合阳性的前列腺癌细胞系VCap的转录组测序结果说明图；
图7是NFZ与不同的化疗药物联合抑制TMPRSS2：ERG融合阳性的前列腺癌细胞系VCap的柱形图。
具体实施方式
为了更好地理解本发明，下面结合实施例和附图对本发明做进一步的详细说明，但本领域技术人员了解，下述实施例不是对本发明保护范围的限制，任何在本发明基础上做出的改变和变化，都在本发明的保护范围之内。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
实验材料来源：
Recombinant Human Transcriptional regulator ERG购于CUSABIO公司；SeriesS Sensor Chip CM5购于Cytiva公司；Nifuratel，Nifuroxazide，Nifursol，Andrographolide，Olaparib和CCK8购于MCE；转录组测序由Novogene公司完成。
细胞增殖抑制实验：
待75cm2培养瓶细胞生长汇合度至85％左右时，将细胞消化，以每孔5000个VCap细胞接种于96孔板，完全培养液每孔100μl。待细胞贴壁后，加入完全培养基配制的不同浓度的Nifuratel，Nifuroxazide，Nifursol，Andrographolide溶液，以等量的溶剂DMSO为对照，培养箱培养72h。去除上层培养基，每孔加入10μl CCK8原液和90μl相应的完全培养基，放入培养箱孵育2h，用酶标仪测定在450nm处的吸光度，结果如表1所示。
表1：不同的乙酰肼衍生物及其类似物抑制TMPRSS2：ERG融合阳性的前列腺癌细胞系VCap的IC50值
Table 1.IC50 values of different drugs
Figure BDA0002801252450000051
从表1中可以看出，本发明乙酰肼衍生物及其类似物均能对VCap细胞有较好的抑制杀灭效果，在本次实验中，表中的第二个药物Nifuroxazide的IC50值约为2.56μmol/L，对VCap细胞的抑制杀灭效果最强。
实施例2
实验材料来源同实施例1，下面以Nifuroxazide作为本发明乙酰肼衍生物及其类似物的代表，以下简称NFZ，进行实验测试。其他具有类似结构的乙酰肼衍生物及其类似物药物虽然未做具体实验，本领域人员知晓能获得相识的实验结果。
1.NFZ与ERG亲和力测定
依据美国药典USP40 IMMUNOLOGICAL TEST METHODS—SURFACE PLASMONRESONANCE(1105)的规定，将ERG蛋白固定于CM5芯片上，将NFZ配制成100，50，25，12.5，6.25，3.125，1.5625和0μM/L依次通过实验通道，以等量的溶剂DMSO作为参比，由Biacore8K完成检测。结果如图1所示。
根据图1，通过“稳态法”计算得到NFZ与ERG亲和力KD值为10.8μmol/L。根据响应值的变化可以看出NFZ与ERG的结合属于快速结合并且快速解离的模式。
2.NFZ与ERG的分子对接
从PDB下载的ERG的ETS结构域的3D结构(PDB编码：4IRI)。利用AutoDock4.2进行预处理(加氢，除水，平衡电荷和除去配体等)，生成受体和配体的对接参数文件。采用Lammarckian GA 4.2算法，其余参数设置默认，完成对接。通过Pymol软件进行可视化分析，结果如图2所示。
从图2可以看出，NFZ结合在ERG的ETS结构域，其结合于ASN319和PHE375所形成的的孔洞内，结合能为-4.73kJ/mol。这是所有ETS结构域蛋白所特有的结构，表明NFZ能与ETS结构域蛋白形成特异性结合。研究证实该位点的ASN319和PHE375是ETS与PARP结合的关键氨基酸。
3.细胞增殖抑制
待75cm2培养瓶细胞生长汇合度至85％左右时，将细胞消化，以每孔5000个VCap细胞接种于96孔板，完全培养液每孔100μl。待细胞贴壁后，加入完全培养基配制的不同浓度的NFZ溶液，以等量的溶剂DMSO为对照，培养箱培养72h。去除上层培养基，每孔加入10μlCCK8原液和90μl相应的完全培养基，放入培养箱孵育2h，用酶标仪测定在450nm处的吸光度，结果如图3所示。
从图3可以看出，随着NFZ浓度的增加，VCap细胞的存活数量显著性地减少。在本次实验中，IC50值为2.678±0.268μmol/L。
4.NFZ与PARP抑制剂Olaparib(简称OLP)的相互作用
待75cm2培养瓶细胞生长汇合度至85％左右时，将细胞消化，以每孔5000个VCap细胞接种于96孔板，完全培养液每孔100μl。待细胞贴壁后，加入完全培养基配制的不同浓度的OLP溶液与5μM/L的NFZ，以等量的溶剂DMSO为对照，培养箱培养72h。去除上层培养基，每孔加入10μl CCK8原液和90μl相应的完全培养基，放入培养箱孵育2h，用酶标仪测定在450nm处的吸光度，结果如图4所示。
从图4可以看出，NFZ对VCap细胞的抑制作用随着OLP浓度的增加越来越弱，直至抵消。本实验验证了NFZ对VCap细胞的抑制作用与PARP活性相关，当PARP活性被抑制剂OLP抑制，NFZ对VCap细胞的抑制作用显著减弱。
5.细胞坏死的检测
将VCap细胞接种在六孔板内，待细胞生长汇合度达到85％时加入NFZ。以DMSO为对照，在对照组细胞加入等量DMSO。设置三个不同的NFZ浓度(5μmol/L，10μmol/L和20μmol/L)，作用时间均为72h。其中5μmol/L组设置48h和72h两个时间点。同时设立一个5μmol/LNFZ和OLP 20μmol/L共同作用的组，用酶标仪测定在450nm处的吸光度，结果如图5所示。
从图5可以看出，VCap细胞在不同浓度以及不同时间的NFZ作用下，呈现了相关性的变化。VCap细胞沿着Y轴逐渐分群，代表着碘化丙啶染色的细胞逐渐增多。这表明坏死细胞的增多。OLP抑制了PARP活性，抵消了NFZ诱导的VCap细胞的坏死。
6.转录组测序
以每孔20万VCap细胞接种于6孔板，以等量的溶剂DMSO为对照组，1μmol/L的NFZ为实验组，每组设立三个独立平行的样品，培养箱培养24h后，提取总RNA。将样品以RNA溶液的方式交由诺禾致源(Novogene)公司进行质量检测和测序。用expected_count值在基迪奥生物平台(https://www.omicshare.com/)进行数据的均一化处理，包括CPM值(Counts permillion)的计算和对照组与Nifuroxazide组基因表达的差异分析，结果如图6所示。
从图6可以看出，NFZ通过与ETS结构域的结合，影响了大量相关蛋白的下游基因的表达。其主要作用表现在对含有ETS结构域的蛋白，如ETS1和ERG部分下游基因被下调；干扰了含有ETS结构域的蛋白与其他转录调控因子的共同作用，下调了大量相关的细胞因子；并且导致大量DNA损伤修复基因被上调。
7.化疗药与NFZ的联合作用
待75cm2培养瓶细胞生长汇合度至85％左右时，将细胞消化，以每孔5000个VCap细胞接种于96孔板，完全培养液每孔100μl。待细胞贴壁后，加入完全培养基配制的不同浓度的化疗药溶液与5μM/L的NFZ，以等量的溶剂DMSO为对照，培养箱培养72h。去除上层培养基，每孔加入10μl CCK8原液和90μl相应的完全培养基，放入培养箱孵育2h，用酶标仪测定在450nm处的吸光度，结果如图7所示。
从图7可以看出，NFZ与化疗药的联合用药组比单纯的化疗药组表现出了更低的细胞存活率。证明了NFZ与多柔比星，米托蒽醌，博来霉素，氟尿嘧啶等直接作用于DNA或者干扰DNA合成的化疗药物有良好的协同抑制肿瘤效果。
以上所揭露的仅为本发明的较佳实施例而已，当然不能以此来限定本发明之权利范围，因此依本发明权利要求所作的等同变化，仍属本发明所涵盖的范围。

Claims (5)

1.一种化合物在制备靶向ETS结构域蛋白的抑制剂中的应用，其特征在于，所述化合物的结构为：
Figure FDA0003639636690000011
2.如权利要求1所述的应用，其特征在于，所述化合物与所述ETS结构域蛋白能产生特异性结合，结合点位于ASN319和PHE375所形成的孔洞内。
3.如权利要求1所述的应用，其特征在于，所述ETS结构域蛋白为ERG蛋白。
4.如权利要求1所述的应用，其特征在于，所述抑制剂用于治疗与ETS结构域蛋白相关疾病，所述与ETS结构域蛋白相关疾病为RNA病毒感染疾病、DNA病毒感染疾病中的一种或多种。
5.如权利要求4所述的应用，其特征在于，所述RNA病毒感染疾病包括HIV；所述DNA病毒感染疾病包括HBV。
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