CN112546051A - 一种化合物的应用 - Google Patents
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Abstract
本发明提供了一种化合物的应用,所述化合物用于在体外增强γ分泌酶的活性,所述化合物为N‑[4‑[[(3‑甲氧基吡嗪基)氨基]磺酰基]苯基]‑3‑(5‑硝基‑2‑噻吩基)‑2‑丙烯酰胺,所述化合物的结构式如式(Ⅰ)所示。所述化合物能够在体外细胞模型中显著增强γ分泌酶的活性,从而降低了对γ分泌酶进行检测和分析的难度,能够为后续进一步以γ分泌酶为靶点的大规模药物筛选工作提供良好的条件。
Description
技术领域
本发明属于生物技术领域,涉及一种化合物的应用,具体是化合物N-[4-[[(3-甲氧基吡嗪基)氨基]磺酰基]苯基]-3-(5-硝基-2-噻吩基)-2-丙烯酰胺的应用。
背景技术
阿尔茨海默病(AD)是痴呆的最常见的原因,导致丧失记忆、认识、推论、判断和辨向能力。AD的特征为除在脑中的突触和神经元的损失之外,存在着胞外淀粉状蛋白斑、胞内神经原纤维缠结。淀粉状蛋白斑的主要成分是β-淀粉状蛋白(Aβ),一种4kDa肽。
Aβ的积累被认为是阿尔茨海默病(AD)的发病机理的早期和关键步骤。Aβ引起了一联串的毒性和炎性事件,其最终导致神经元死亡和认识损伤。Aβ肽源于淀粉状蛋白前体蛋白(APP)的蛋白水解。APP蛋白是由大胞外域和短胞质尾区组成的跨膜蛋白,Aβ片断包括APP的胞外和跨膜域的一部分。
APP可以通过两种路线中的任一个加工,非淀粉质源和淀粉质源途径。大部分APP是通过非淀粉质源途径加工的,由此蛋白酶α-分泌酶在Aβ域中劈开APP从而释放了大的可溶性N-末端片段(sAPPα)和非淀粉质源C-末端片段(C83),这种片段进一步由γ-分泌酶加工以产生22-24残基肽(p3)。在淀粉质源途径中,APP是被β-分泌酶(BACE1)劈开的,这形成了较短的N-末端域(sAPPβ)和淀粉质源C-末端(C99),这种C99片段随后被γ-分泌酶劈开。γ-分泌酶是由以下蛋白质的络合物形成的蛋白酶:Presenilin-1(PS-1)、Nicastrin、PEN-2和APH-1。由γ-分泌酶蛋白水解APP中间体产生了不同长度的Aβ肽(Aβ37、Aβ38、Aβ39、Aβ40、Aβ42)。在这些肽中,Aβ42是AD脑中的最小可溶性的、最聚集的物质和毒性低聚物与淀粉状蛋白斑的主要成分。因此,抑制体内γ分泌酶的活性被认为是治疗阿尔茨海默症的重要方式。现有已经公开的报道也都是在探索体内γ分泌酶的活性的抑制剂以及相关的药物。
目前在以γ分泌酶为靶点的研发过程中,最常用的细胞模型包括体外细胞系和原代细胞。体外细胞系中γ分泌酶活性很低,用常规的生化方法很难检测到,需要用超高速离心等方法将γ分泌酶富集起来才可以检测,大大增加了经济和时间成本。原代细胞(例如,原代神经元)中γ分泌酶活性较高,然而分离原代细胞过程复杂,周期长,分离出来的原代细胞数量也很有限,并且取材困难,这些条件限制了原代细胞在大规模药物筛选中的应用。如何在体外细胞模型中增强γ分泌酶的活性,对于在以γ分泌酶为靶点的大规模药物筛选工作中是十分有意义的。
发明内容
鉴于现有技术存在的不足,本发明提供一种化合物的应用,以解决体外细胞模型中γ分泌酶的活性低而不利于对γ分泌酶进行检测和分析的问题。
为实现上述发明目的,本发明提供了一种化合物的应用,所述化合物为N-[4-[[(3-甲氧基吡嗪基)氨基]磺酰基]苯基]-3-(5-硝基-2-噻吩基)-2-丙烯酰胺,所述化合物的结构式如下式(Ⅰ):
其中,所述化合物用于在体外增强γ分泌酶的活性。
具体地,将所述化合物溶解于有机溶剂形成化合物溶液,再将所述化合物溶液加入到体外培养细胞的培养液中,以增强细胞γ分泌酶的活性。
具体地,所述化合物溶液中,所述化合物的浓度为0.5mmol/L~1mmol/L。
具体地,所述有机溶剂为二甲基亚砜。
具体地,将所述化合物溶液加入到体外培养细胞的培养液中,使得所述化合物在所述细胞培养液中的浓度为0.5μmol/L~2μmol/L。
本发明实施例提供的一种化合物的应用,用于在体外增强γ分泌酶的活性。化合物(N-[4-[[(3-甲氧基吡嗪基)氨基]磺酰基]苯基]-3-(5-硝基-2-噻吩基)-2-丙烯酰胺)能够在体外细胞模型中显著增强γ分泌酶的活性,从而降低了对γ分泌酶进行检测和分析的难度,能够为后续进一步以γ分泌酶为靶点的大规模药物筛选工作提供良好的条件。
附图说明
图1是实施例1的三份细胞膜蛋白样品中γ分泌酶的活性检测结果的归一化统计图;
图2是实施例2的三份细胞蛋白样品的Flag的免疫印迹图示;
图3是实施例2的三份细胞蛋白样品的AICD的免疫印迹图示;
图4是图3中的AICD条带灰度值的归一化统计图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面结合附图对本发明的具体实施方式进行详细说明。这些优选实施方式的示例在附图中进行了例示。附图中所示和根据附图描述的本发明的实施方式仅仅是示例性的,并且本发明并不限于这些实施方式。
在此,还需要说明的是,为了避免因不必要的细节而模糊了本发明,在附图中仅仅示出了与根据本发明的方案密切相关的结构和/或处理步骤,而省略了与本发明关系不大的其他细节。
本发明实施例提供一种化合物的应用,所述化合物用于在体外增强γ分泌酶(γ-secretase)的活性。其中,所述化合物的中文名称为N-[4-[[(3-甲氧基吡嗪基)氨基]磺酰基]苯基]-3-(5-硝基-2-噻吩基)-2-丙烯酰胺,英文名称为necrosulfonamide,CAS号为1360614-48-7,分子式为C18H15N5O6S2,所述化合物的结构式如下式(Ⅰ):
在研究细胞程序性坏死(necroptosis)机制的过程中,科学家们发现necroptosis是由RIPK1-RIPK3-MLKL信号通路调控的。细胞在刺激因素,如肿瘤坏死因子TNFα的处理下,即可触发细胞程序性坏死:RIPK1激酶被激活,继而活化的RIPK1与其下游的RIPK3激酶发生相互作用,RIPK1通过磷酸化RIPK3使其激活,同时活化的RIPK3发生自我磷酸化从而使更多的RIPK3蛋白通过磷酸化而激活。活化后的RIPK3与其下游的MLKL蛋白结合并使其磷酸化。磷酸化后的MLKL发生寡聚化形成寡聚物,与此同时MLKL寡聚物在细胞中发生移位,从细胞质中转移至细胞膜上,并且这些寡聚物在细胞膜上形成类似于离子通道的孔,这些孔的大量形成导致细胞膜破裂,从而引起细胞死亡。研究发现,化合物necrosulfonamide可以特异性结合人源MLKL蛋白并抑制由其介导的细胞程序性坏死。
本发明发现了化合物necrosulfonamide的新功能,即化合物necrosulfonamide可以在体外显著增强γ分泌酶的活性。
如式(Ⅰ)的化合物(necrosulfonamide,以下称为化合物(Ⅰ))的应用,具体地,将所述化合物(Ⅰ)溶解于有机溶剂形成化合物溶液,再将所述化合物溶液加入到体外培养细胞的培养液中,以增强细胞γ分泌酶的活性。
在优选地方案中,所述化合物溶液中,所述化合物(Ⅰ)的浓度配制为0.5mmol/L~1mmol/L。其中,所述有机溶剂为二甲基亚砜。
在优选地方案中,将所述化合物溶液加入到体外培养细胞的培养液中,使得化合物(1)在所述细胞培养液中的浓度(即工作浓度)为0.5μmol/L~2μmol/L。
基于以上实施例提供的方案,所述化合物(Ⅰ)能够在体外细胞模型中显著增强γ分泌酶的活性,从而降低了对γ分泌酶进行检测和分析的难度,能够为后续进一步以γ分泌酶为靶点的大规模药物筛选工作提供良好的条件。
实施例1:采用直接法检测γ分泌酶的活性
(1)、将人胚胎肾细胞HEK293细胞铺在10厘米培养皿中,用10毫升培养液培养24小时后使得细胞生长至约60%满底。
(2)、用浓度为1mM的necrosulfonamide(溶剂为二甲基亚砜)溶液加入步骤(1)的HEK293培养液中。其中,控制加入的necrosulfonamide溶液的量使得necrosulfonamide在培养液中的浓度为0.5μM。
(3)、将步骤(2)的样品孵育过夜(约16个小时),然后向样品补充10毫升含有相同浓度的necrosulfonamide的新鲜细胞培养液后再孵育3个小时。
(4)、将步骤(3)培育完成的样品弃上清,用细胞刮子将细胞收集起来,用超高速离心法提取细胞膜蛋白(因γ分泌酶位于膜上),记为膜蛋白样品“NSA0.5μM”,然后用γ分泌酶荧光底物检测试剂盒检测膜蛋白样品中γ分泌酶的活性,获得膜蛋白样品NSA0.5μM的活性测试数据。
(5)、参照以上步骤(1)-(4)的过程,将其中步骤(2)中控制加入的necrosulfonamide溶液的量使得necrosulfonamide在培养液中的浓度为1μM,相应的,步骤(4)中提取的细胞膜蛋白记为膜蛋白样品“NSA1μM”,按照步骤(4)测试后获得膜蛋白样品NSA1μM的活性测试数据。
(6)、作为对比,参照以上步骤(1)-(4)的过程,将其中步骤(2)中,将浓度为0的necrosulfonamide溶液加入到培养液中,即仅加入溶剂二甲基亚砜,相应的,步骤(4)中提取的细胞膜蛋白记为膜蛋白样品“DMSO”,按照步骤(4)测试后获得样品DMSO的活性测试数据。
图1为本实施例的三份细胞膜蛋白样品中γ分泌酶的活性检测结果的归一化统计图,其中的空白对比样品DMSO的活性记为1。从图1可以看出,necrosulfonamide的工作浓度为0.5μM的膜蛋白样品NSA0.5μM的γ分泌酶活性相对于对比样品DMSO有较大的提升,而necrosulfonamide的工作浓度为1μM的膜蛋白样品NSA1μM的γ分泌酶活性相对于对比样品DMSO则有显著的提升。
实施例2:采用间接法检测γ分泌酶的活性
APP和Notch1是γ分泌酶的众多底物中研究最多的两个蛋白底物,因此通过检测γ分泌酶切割底物后产生的产物的量可以间接的反映γ分泌酶的活性。
(1)、分别构建表达APP Swedish突变体和切除胞外段的Notch1蛋白的HEK293稳转细胞株,其中在切除胞外段的Notch1蛋白碳端(C端)连上了一个Flag标签,这两个细胞株分别命名为APPswe-HEK293和N1ΔE-Flag-HEK293。将这两种细胞株分别铺在6孔板中,每孔3毫升培养液培养24小时后使得细胞生长至约60%满底。
(2)、用浓度为1mM的necrosulfonamide(溶剂为二甲基亚砜)溶液分别加入步骤(1)的APPswe-HEK293和N1ΔE-Flag-HEK293培养液中。其中,控制加入的necrosulfonamide溶液的量使得necrosulfonamide在培养液中的浓度为1μM。
(3)、将步骤(2)的样品孵育过夜(约16个小时),然后向样品补充3毫升含有相同浓度的necrosulfonamide的新鲜细胞培养液后再孵育3个小时。
(4)、将步骤(3)培育完成的样品弃上清,将6孔板放在冰上,每孔加入200μL蛋白裂解液(含蛋白酶抑制剂),冰浴20min后,用细胞刮子收集蛋白样品,离心取上清,即为提取的总蛋白,记为蛋白样品“NSA1μM”;测定蛋白浓度后用免疫印迹的方法检测γ分泌酶切割APP(对应细胞株APPswe-HEK293)产生的产物AICD以及γ分泌酶切割Notch1(对应细胞株N1ΔE-Flag-HEK293)产生的产物NICD的量(因NICD和Flag标签融合在一起,Flag的量即代表了NICD的量),获得蛋白样品NSA1μM的测试数据。
(5)、参照以上步骤(1)-(4)的过程,将其中步骤(2)中控制加入的necrosulfonamide溶液的量使得necrosulfonamide在培养液中的浓度为2μM,相应的,步骤(4)提取的总蛋白记为蛋白样品“NSA2μM”,按照步骤(4)测试后获得蛋白样品NSA2μM的测试数据。
(6)、作为对比,参照以上步骤(1)-(4)的过程,将其中步骤(2)中,将浓度为0的necrosulfonamide溶液加入到培养液中,即仅加入溶剂二甲基亚砜,相应的,步骤(4)提取的总蛋白记为蛋白样品“DMSO”,按照步骤(4)测试后获得蛋白样品DMSO的测试数据。
图2为本实施例的三份细胞蛋白样品的Flag(即NICD)的免疫印迹图示,β-actin作为免疫印迹上样的内参蛋白。图3为本实施例的三份细胞蛋白样品的AICD的免疫印迹图示。从图2和图3可以获知,相比于对比样品DMSO,蛋白样品NSA1μM和蛋白样品NSA2μM使用化合物Necrosulfonamide处理APPswe-HEK293和N1ΔE-Flag-HEK293细胞后,γ分泌酶切割产生的产物Flag(即NICD)和AICD的量显著升高,说明γ分泌酶的活性具有显著的提升。
图4为图3中AICD条带灰度值的归一化统计图,其中的空白对比样品DMSO的活性记为1。从图4可以看出,necrosulfonamide的工作浓度为1μM的蛋白样品NSA1μM和工作浓度为2μM的蛋白样品NSA2μM相对于对比样品DMSO来说,AICD的蛋白水平都有显著的提升,说明γ分泌酶的活性具有显著的提升。
综上所述,本发明将化合物(N-[4-[[(3-甲氧基吡嗪基)氨基]磺酰基]苯基]-3-(5-硝基-2-噻吩基)-2-丙烯酰胺)用于在体外细胞模型中增强γ分泌酶的活性,从而降低了对γ分泌酶进行检测和分析的难度,能够为后续进一步以γ分泌酶为靶点的大规模药物筛选工作提供良好的条件。
以上所述仅是本申请的具体实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本申请原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本申请的保护范围。
Claims (5)
2.根据权利要求1所述的应用,其特征在于,将所述化合物溶解于有机溶剂形成化合物溶液,再将所述化合物溶液加入到体外培养细胞的培养液中,以增强细胞γ分泌酶的活性。
3.根据权利要求2所述的应用,其特征在于,所述化合物溶液中,所述化合物的浓度为0.5mmol/L~2mmol/L。
4.根据权利要求2所述的应用,其特征在于,所述有机溶剂为二甲基亚砜。
5.根据权利要求2-4任一所述的应用,其特征在于,将所述化合物溶液加入到体外培养细胞的培养液中,使得所述化合物在所述细胞培养液中的浓度为0.5μmol/L~1μmol/L。
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