CN110179803A - 一种噻吨溴类化合物的应用 - Google Patents
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Abstract
本发明涉及一种噻吨溴类化合物的应用，通过建立的WWP1小分子化合物抑制剂体外筛选体系及蛋白酶谱、圆二色谱、体外自泛素化检测实验发现该噻吨溴类化合物能有效抑制泛素连接酶WWP1与泛素结合酶E2的相互作用。通过MTT实验、LC3斑点实验、流式细胞术以及细胞划痕实验研究发现，该噻吨溴类化合物影响肿瘤细胞的细胞生长、细胞自噬、细胞凋亡及细胞迁移能力。这些实验结果表明该噻吨溴类化合物能有效抑制WWP1的泛素连接酶活性，进而影响细胞的多种生命进程，可作为靶向WWP1的抗肿瘤药物。
Description
技术领域
本发明涉及一种化合物的应用，尤其是一种噻吨溴类化合物的应用。
背景技术
在生物体内，蛋白质参与细胞各项生理功能，蛋白质在发挥功能之后，往往需要被及时清除。泛素-蛋白酶体途径和自噬溶酶体途径是生物体细胞中的蛋白质降解的两种主要方式，泛素-蛋白酶体途径能够选择性、特异性的降解丧失功能的蛋白质。泛素化过程除了介导26S蛋白酶体识别并降解蛋白质之外，还与自噬溶酶体途径降解途径相关。细胞中蛋白质的合成与降解平衡与基因转录、蛋白质翻译、信号传导、生长发育等多种生命活动相关，是维持细胞内稳态的重要机制。
与泛素-蛋白酶体途径相关的疾病病因主要是该途径中的关键酶或底物的突变(功能丧失)导致疾病相关蛋白积累或加速降解。经过深入研究发现，肿瘤发生的发生与发展伴随着细胞周期异常、抑癌蛋白过度降解、癌蛋白聚集的现象，这种现象与泛素-蛋白酶体途径中关键酶或底物的失活或突变息息相关。因此筛选以泛素-蛋白酶体途径中的关键酶为靶点的小分子抑制剂为相应疾病的治疗提供了新策略，成为了新的研究热点。
泛素-蛋白酶体途径中泛素化修饰的泛素链类型以及长短，决定了靶蛋白被蛋白酶体降解或被转移到其他特定部位发挥功能的不同命运。泛素连接酶是确定蛋白质泛素化类型、泛素链长短以及结构的关键酶，在靶蛋白的选择性、特异性识别过程中具有非常重要的作用，因此，泛素连接酶成为近年来研究以及药物开发的重要靶点。
WWP1是众多人体中众多泛素连接酶之一，作为一种致癌因子，在一部分前列腺癌、胃癌、乳腺癌细胞和白血病中的过表达。目前已报道的WWP1的靶蛋白有Smad2、ErbB4、KLF2、p27、KLF5、TβRI、p63等。WWP1可以通过降解p27显著抑制p27诱导的复制衰老，通过降解TβRI负向调控TGF-β信号通路，通过降解KLF2、KLF5促进肿瘤的发生发展。总之WWP1的过表达，导致癌症抑制因子过度降解，促进癌症的发生和发展，这使得WWP1成为治疗相关癌症的靶点。
发明内容
本发明所要解决的技术问题在于提供一种噻吨溴类化合物的应用。
本发明采用的技术方案是：
一种噻吨溴类化合物，其特征在于：为1-Hexadecanaminium,N-[2-[[(4-methoxyphenyl)methyl]-2-pyrimidinylamino]ethyl]-N,N-dimethyl-,bromide(1:1)，具有式(I)所示结构：
上述噻吨溴类化合物的筛选方法，具体步骤如下：
(1)包被泛素结合酶：将待包被蛋白用碳酸盐包被缓冲液稀释至80ng/100μL，混匀后按照每孔100μL加入到96孔板中，37℃孵育2h或4℃过夜孵育，使蛋白附着到孔板底部；
(2)5％脱脂牛奶封闭：将96孔板中牛奶倒掉，加入200μLPBST洗涤缓冲液,水平摇床振荡5min，倒掉并拍净孔内的液体，用PBST重复洗涤4次；80ng/100μL每孔加入150μL用PBS缓冲液配制的5％脱脂牛奶，37℃封闭2h；
(3)WWP1与泛素结合酶共孵育：将96孔板中牛奶倒掉，加入200μLPBST洗涤缓冲液,水平摇床振荡5min，倒掉并拍净孔内的液体，用PBST重复洗涤4次；用PBS缓冲液将WWP1蛋白稀释至364ng/100μL混匀，取300μL蛋白液将小分子化合物稀释至5μmol/L，孵育10min后按照每孔100μL加入到96孔板中，37℃孵育2h；
(4)一抗孵育：将96孔板中液体倒掉，加入120μL PBST洗涤缓冲液,水平摇床振荡5min，倒掉并拍净孔内的液体，用PBST重复洗涤4次；将用PBS稀释的一抗按照每孔100μL加入96孔板中，37℃孵育2h；
(5)二抗孵育：将96孔板中的一抗倒掉，加入120μL PBST洗涤缓冲液，水平摇床振荡5min，倒掉并拍净孔内的液体，用PBST重复洗涤4次；将用PBS稀释好的HRP二抗按照每孔100μL加入96孔板中，37℃避光孵育30min-1h；
(6)显色：将96孔板中的二抗倒掉，加入120μL PBST洗涤缓冲液，水平摇床振荡5min，倒掉并拍净孔内的液体，PBST洗涤3次后换用PBS洗涤一次；将TMB显色液的A液和B液等体积混合后(现用现配)，按每孔100μL加入96孔板中；
(7)终止：待酶标孔内液体蓝色差异最大时，加入100μL2mol/L的硫酸终止反应；
(8)检测：用酶标仪检测各孔的OD450值。
上述噻吨溴类化合物在制备WWP1抑制剂方面的应用。
所述噻吨溴类化合物能够有效抑制WWP1与泛素结合酶的相互作用、抑制WWP1介导的泛素化过程。
上述噻吨溴类化合物在制备WWP1诱导的相关疾病的药物方面的应用。
优选的，上述噻吨溴类化合物的应用，所述疾病为肿瘤疾病。
优选的，上述噻吨溴类化合物的应用，所述疾病为乳腺癌。
所述噻吨溴类化合物能够有效抑制癌细胞的生长和迁移。
所述噻吨溴类化合物能有效抑制自噬降解途径，能有效积累癌细胞中自噬相关蛋白LC3。
所述噻吨溴类化合物能够有效诱导癌细胞凋亡。
本发明的有益效果是：
上述噻吨溴类化合物，能有效抑制WWP1与泛素结合酶的相互作用，抑制WWP1介导的泛素化过程。通过MTT实验、LC3斑点实验、流式细胞术以及细胞划痕实验研究发现，上述噻吨溴类化合物能够影响癌细胞的生长、自噬、凋亡及迁移能力。上述噻吨溴类化合物能有效抑制WWP1介导的蛋白质降解过程，进而影响细胞的多种生命进程，可作为靶向WWP1的全新抗肿瘤药物。
附图说明
图1为本发明WWP1小分子抑制剂筛选体系筛选获得本发明所述噻吨溴类化合物的结果。结果说明，编号为S4355的化合物即为本发明所述的噻吨溴类化合物能有效抑制WWP1与泛素结合酶的相互作用。
图2为本发明自泛素化实验检测本发明所述噻吨溴类化合物对WWP1自泛素化影响的结果。结果说明，编号为S4355的化合物即为本发明所述噻吨溴类化合物能有效抑制WWP1的自泛素化。
图3为本发明MTT实验检测本发明所述噻吨溴类化合物对癌细胞生长影响的结果。结果说明，编号为S4355的化合物即为本发明所述的噻吨溴类化合物能有效抑制癌细胞活力；
图4为本发明细胞划痕实验检测本发明所述噻吨溴类化合物对癌细胞迁移影响的结果。结果说明，编号为S4355的化合物即为本发明所述的噻吨溴类化合物能有效抑制癌细胞迁移。
图5为本发明GFP-LC3斑点实验检测本发明所述噻吨溴类化合物对癌细胞自噬影响的结果。结果说明，编号为S4355的化合物即为本发明所述的噻吨溴类化合物能抑制自噬降解途径；
图6为本发明流式细胞术检测本发明所述噻吨溴类化合物对癌细胞凋亡影响的结果。结果说明，编号为S4355的化合物即为本发明所述的噻吨溴类化合物能诱导癌细胞凋亡。
具体实施方式
下面结合实施例对本发明进一步说明，但实施例不限制本发明的保护范围。
实施例1
(1)噻吨溴类化合物的筛选
①包被泛素结合酶：将待包被蛋白His-HA-UbcH7用碳酸盐包被缓冲液稀释至80ng/100μL，混匀后按照每孔100μL加入到96孔板中，37℃孵育2h或4℃过夜孵育，使蛋白附着到孔板底部；
②5％脱脂牛奶封闭：将96孔板中牛奶倒掉，加入200μL PBST洗涤缓冲液,水平摇床振荡5min，倒掉并拍净孔内的液体，用PBST重复洗涤4次；每孔加入150μL用PBS缓冲液配制的5％脱脂牛奶，37℃封闭2h；
③WWP1与泛素结合酶共孵育：将96孔板中牛奶倒掉，加入200μL PBST洗涤缓冲液,水平摇床振荡5min，倒掉并拍净孔内的液体，用PBST重复洗涤4次；用PBS缓冲液将WWP1截短体蛋白(GST-WW4-end)蛋白稀释至364ng/100μL混匀，取300μL蛋白液将小分子化合物稀释至5μmol/L，孵育10min后按照每孔100μL加入到96孔板中，37℃孵育2h；
④一抗孵育：将96孔板中液体倒掉，加入120μL PBST洗涤缓冲液,水平摇床振荡5min，倒掉并拍净孔内的液体，用PBST重复洗涤4次；用PBS稀将GST一抗按1:10000稀释后，每孔100μL加入96孔板中，37℃孵育2h；
⑤二抗孵育：将96孔板中的一抗倒掉，加入120μL PBST洗涤缓冲液，水平摇床振荡5min，倒掉并拍净孔内的液体，用PBST重复洗涤4次；用PBS将HRP二抗按1：20000后，每孔100μL加入96孔板中，37℃避光孵育30min-1h；
⑥显色：将96孔板中的二抗倒掉，加入120μL PBST洗涤缓冲液，水平摇床振荡5min，倒掉并拍净孔内的液体，PBST洗涤3次后换用PBS洗涤一次；将TMB显色液的A液和B液等体积混合后(现用现配)，按每孔100μL加入96孔板中；
⑦终止：待酶标孔内液体蓝色差异最大时，加入100μL 2mol/L的硫酸终止反应；
⑧检测：用酶标仪检测各孔的OD450值。图1结果显示，编号为S4355的化合物，即为本发明所述噻吨溴类化合物处理后，WWP1与泛素结合酶的相互作用程度减弱，说明本发明所述噻吨溴类化合物能有效抑制WWP1的与泛素结合酶的相互作用。
(2)噻吨溴类化合物对WWP1自泛素化的影响
GST-WW4-end的自泛素化反应体系，如表1所示。
表1体外自泛素化实验体系
按表1将自泛素化体系中的各组分冰浴融化后加到EP管中；混匀离心后将EP管置于30℃水浴1h；加入7.5μL 5×SDS蛋白上样缓冲液，95℃煮5min后混匀离心；进行WesternBlot检测。图2结果显示，编号为S4355的化合物，即为本发明所述噻吨溴类化合物处理后，WWP1的自泛素化条带变弱，说明即为本发明所述噻吨溴类化合物能有效抑制WWP1的自泛素化。
(3)噻吨溴类化合物对癌细胞活力的影响
①96孔板铺板：收集对数期MDA-MB-231细胞，调整细胞悬液的浓度，每孔加入100μL DMEM培养基，铺板使待测细胞密度5000个/孔。
②分别利用不同浓度(0,5,10,20,40μM)的化合物或DMSO处理细胞48和72h。
③每孔加入20μL MTT(现用现配，用PBS配置成5mg/mL的MTT溶液)继续培养4h。
④终止培养，小心吸去孔内的培养基，注意不要吸走沉淀。
⑤每孔加入200μL DMSO溶解沉淀，低速振荡10min，使结晶充分溶解。
⑥利用酶标仪检测490nm波长下各孔的吸光度(A490)。
⑦细胞活力＝实验组吸光值/对照组吸光值。图3的结果显示，编号为S4355的化合物，即为本发明所述噻吨溴类化合物能有效抑制癌细胞活力。
(4)噻吨溴类化合物对癌细胞迁移的影响
①在6孔板中接种适量MDA-MB-231细胞，37℃、5％CO2培养箱中过夜培养；
②待细胞长至60％-70％时，用200μL枪头沿着直尺垂至于培养皿划线，每隔0.5-1cm一道，呈“井”字形；
③用DPBS洗净划下细胞，按照实验设计加入含有小分子化合物的新鲜DMEM培养基。置于37℃、5％CO2培养箱中培养；
④分别在药物处理0、12、24、48h时在显微镜下观察并在同一位置拍照分析实验结果。图4结果显示，本发明所述噻吨溴类化合物能有效抑制癌细胞迁移。
(5)噻吨溴类化合物对癌细胞自噬的影响
①分别消化收集对数期的HeLa(GFP-LC3)细胞和MDA-MB-231细胞；
②分别用计数板计数后调整细胞悬液浓度，HeLa(GFP-LC3)按每孔100μL(包含5000-8000个细胞)加入96孔板中，MDA-MB-231按每孔50万个细胞接种到6孔板中；
③用DPBS填充96孔板边缘孔，将两孔板置于37℃，5％CO2培养箱中过夜培养；
④按照实验设计将旧培养基更换成含上述化合物或DMSO的培养基，于37℃，5％CO2培养箱中继续培养；
⑤HeLa(GFP-LC3)细胞分别在药物处理0、12、24h时利用荧光显微镜观察GFP-LC3斑点的形成情况，并拍照保存；提取MDA-MB-231细胞中的蛋白，然后利用蛋白免疫印迹检测自噬相关蛋白LC3及p62蛋白水平。图5结果显示，编号为S4355的化合物，即为本发明所述的噻吨溴类化合物处理后，
GFP-LC3聚集形成斑点，LC3、P62增加，说明本发明所述的噻吨溴类化合物能抑制自噬降解途径；
(6)Annexin V/PI检测噻吨溴类化合物对癌细胞凋亡的影响
①在6孔板中接种适量MDA-MB-231细胞，孔板置于37℃，5％CO2条件下培养。
②待细胞完全贴壁后，分别用化合物或等量的DMSO处理细胞24h。
③收集细胞。每孔加200μL不含EDTA 0.25％胰酶消化细胞。消化完成后，每孔加1mL培养基终止消化，用移液器反复吹打，使细胞悬浮于培养基中。然后将细胞悬液转移到1.5mL EP管中。
④3000r/min，离心3min，弃去上层培养基，用1mL PBS重悬细胞。
⑤3000r/min，离心3min，收集细胞，移液器尽量除净上清。
⑥用500μL binding buffer重悬细胞，移液器反复吹吸数次，使其成为单细胞悬液。向EP管中分别加入2μL Annexin V和PI染液，避光染色15min。设置不加染液的空白对照和只加2μL Annexin V或2μL PI的单阳对照。
⑦流式细胞仪检测细胞凋亡。图6结果显示，编号为S4355的化合物，即为本发明所述噻吨溴类化合物能诱导癌细胞凋亡。
上述实施例对该一种噻吨溴类化合物的应用进行的详细描述，是说明性的而不是限定性的，可按照所限定范围列举出若干个实施例，因此在不脱离本发明总体构思下的变化和修改，应属本发明的保护范围之内。
Claims (4)
1.一种噻吨溴类化合物在制备WWP1抑制剂方面的应用，所述噻吨溴类化合物具有式(I)所示结构：
2.权利要求1所述噻吨溴类化合物在制备WWP1诱导的相关疾病的药物方面的应用。
3.根据权利要求2所述噻吨溴类化合物的应用，其特征在于：所述疾病为肿瘤疾病。
4.根据权利要求3所述噻吨溴类化合物的应用，其特征在于：所述疾病为乳腺癌。
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