CN110433154A - 藤黄酸的新用途 - Google Patents
藤黄酸的新用途 Download PDFInfo
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- CN110433154A CN110433154A CN201910739598.9A CN201910739598A CN110433154A CN 110433154 A CN110433154 A CN 110433154A CN 201910739598 A CN201910739598 A CN 201910739598A CN 110433154 A CN110433154 A CN 110433154A
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- gambogicacid
- mutation
- cell
- sulfydryl
- covalent modification
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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Abstract
本发明公开了藤黄酸的新用途,藤黄酸在制备靶向恢复突变p53功能药物中的应用,即藤黄酸在恢复突变p53野生型功能及巯基共价修饰方面的新用途;藤黄酸可以恢复突变p53的野生型构象及功能,主要表现在藤黄酸可以激活在突变p53背景下p53下游基因PUMA启动子荧光素酶报告基因系统的荧光素酶表达量,下调肿瘤细胞突变p53的表达水平,上调野生型p53(wtp53)及其靶基因PUMA、P21、NOXA、BAX的表达水平;另一方面,藤黄酸具有巯基共价修饰活性并通过发挥此活性而抑制肿瘤,而且藤黄酸也可能通过巯基共价作用而重激活突变p53;这些特征表明藤黄酸具有针对恢复突变p53功能的个性化治疗药物的应用潜力。
Description
技术领域
本发明属于医药领域,涉及藤黄酸的新用途,具体涉及藤黄酸在重激活突变p53及通过发挥巯基共价修饰活性及其在肿瘤个性化靶向医疗技术中应用。
背景技术
癌症(即恶性肿瘤)治疗一直是世界公认的医学难题,即使在医学进步的今天,人们依旧谈癌色变。最新发布的全球癌症统计报告显示,2018年,全球范围内的癌症新发及死亡病例分别高达1810万、960万。目前的肿瘤治疗中,化疗是主要手段之一;而相对于有机合成小分子,天然化合物具有来源广泛、价格低廉、毒副作用低的优势,并且具有调控多个关键信号通路而抑制肿瘤的潜力。因此,筛选具有抗肿瘤活性的天然化合物并深度探索潜在的抗肿瘤机理,可为推进它们的临床应用及其它天然化合物的开发利用提供依据。肿瘤中,抑癌基因TP53高频突变,且突变不仅造成抑癌功能的分离、丧失,更获得驱动肿瘤恶性进展的特性,而由于p53突变所造成的构象改变是微小且可逆的,这使得通过天然化合物重激活mutp53,进而抑制mutp53肿瘤具有可行性。经过近20年的探索,已经发现包括小分子化合物、短肽、提取自植物的天然化合物在内的一大批mutp53重激活剂。其中,包括PRIMA-1、STIMA-1、MIRA-1在内的部分化合物可通过发挥巯基共价修饰活性,与mutp53的关键Cys氨基酸残基(如Cys124、Cys141)发生迈克尔加成反应,进而抑制mutp53的蛋白错误折叠并最终恢复mutp53的野生型功能。
藤黄酸,英文名为Gambogic acid,分子式为C38H44O8,分子量为628.75,提取自藤黄科植物藤黄树的干燥树脂。目前的研究表明,在肝癌、胃癌等多种癌细胞中,藤黄酸可诱导细胞凋亡。在非小细胞肺癌中,藤黄酸可通过抑制TGF-β诱导的上皮间质转化(EMT)而抑制肿瘤侵袭及转移。在乳腺癌细胞中,藤黄酸可促进mutp53通过E3泛素连接酶CHIP介导的蛋白酶体途径降解。在人脐静脉内皮细胞(HUVEC)中,藤黄酸可显著抑制细胞增殖,迁移,侵袭,血管形成,和微血管生长。在动物肿瘤模型和临床试验中,藤黄酸有效抑制肿瘤生长,并且副作用很小,对免疫和造血系统具有低毒性;藤黄酸能够产生组织特异性蛋白酶抑制作用和肿瘤特异性毒性。在类风湿性关节炎大鼠中,藤黄酸通过调控PI3K/Akt/mTOR信号通路而抑制炎症反应。在急性心肌梗死大鼠模型中,藤黄酸通过调控抑制炎症、iNOS和NF-κB/p38通路而发挥心脏保护作用。目前,藤黄酸在恢复mutp53的野生型功能及通过发挥巯基共价修饰活性而抑制肿瘤的作用尚未见报道。
发明内容
为了拓宽化合物藤黄酸的应用领域,本发明目的在于提供了藤黄酸在重激活突变p53及通过发挥巯基共价修饰活性而抑制肿瘤的新用途,进而为开发藤黄酸在突变p53相关肿瘤治疗中的作用提供理论依据,其具体机理为:(1) 藤黄酸重激活突变p53,进而上调p53抑癌信号通路从而抑制肿瘤;(2) 藤黄酸通过巯基共价修饰氧化还原平衡维持者GSH进而使肿瘤细胞的氧化还原失衡,最终抑制肿瘤;并可能通过巯基共价修饰mutp53的关键Cys而重激活突变p53。
本发明另一目的是提供一种药物组合物,含有有效量的权利要求1或3所述的藤黄酸及具药物呈递作用的运载小分子;本发明化合物藤黄酸可与具有药物呈递效果的运载小分子(如PAMAM树形分子)组成复合体,并以组合物形式通过口服或注射等途径施用于肿瘤病人,从而改善肿瘤患者的治疗效果;口服用药时,可将其制成片剂、锭剂、软胶囊、滴丸、微丸、水性或油混悬剂、乳剂、糖浆剂等,为了提升衍生产品的吸收效果或口感,可添加适量药学上可接受的辅料,如填充剂、吸收促进剂、稳定剂、香味剂、色素和甜味剂等;注射用药时,可制成无菌的水性或油性溶液、无菌粉末、脂质体、乳剂、微囊等。
我们通过基因工程技术,将P53 R175H/ P53 R273H 全长编码序列、PUMA启动子序列分别构建至增强型EGFP转染报告载体(pIRES2-EGFP)、双荧光素酶报告基因载体(PEZX-GA01),并通过脂质体转染法将以上两个重组载体转染至H1299(p53 null)细胞,得到稳定表达PUMA启动子荧光素酶报告基因系统的H1299 P53 R175H/ P53 R273H 细胞,并以藤黄酸处理细胞15h,发现藤黄酸处理后,与已报道的mutp53重激活剂PRIMA-1类似,细胞中相对荧光素酶活上升,提示了藤黄酸是潜在的突变p53重激活剂,可以转录激活wtp53靶基因PUMA的表达;随后,我们采用p53构象特异性抗体PAb1620(识别野生型p53)和PAb240(识别突变型p53)进行免疫荧光和免疫沉淀实验,发现藤黄酸明显下调SK-BR-3、HT29细胞突变p53表达水平,上调野生型p53表达水平。进一步的蛋白质印迹实验也表明了藤黄酸处理后,HT29细胞中的p53明显下降,而wtp53靶基因PUMA、p21、BAX、NOXA的蛋白水平明显上调。以上实验结果提示了藤黄酸恢复mutp53的野生型构象及功能。另一方面,MTT实验结果显示,藤黄酸与已知巯基修饰化合物PRIMA-1类似,与外源性巯基供体NAC、GSH合成抑制剂BSO联用后,其IC50分别出现了明显的上升或下降,提示了藤黄酸具有巯基共价修饰活性,并通过共价修饰GSH的游离巯基而使肿瘤细胞中的氧化还原系统失衡,进而抑制肿瘤增殖,并且藤黄酸重激活mutp53可能与其巯基共价修饰活性有关。进一步的体外巯基结合实验显示,藤黄酸可抑制DTNB与NAC游离巯基间的二硫键交换反应(反应产物为在412nm有最大吸收峰的NTB2-二价阴离子),提示了藤黄酸具有巯基结合活性,可与DTNB竞争性结合NAC的游离巯基,并且可能通过巯基共价修饰mutp53的Cys氨基酸残基而重激活mutp53。这为开发藤黄酸通过重激活mutp53及发挥巯基共价修饰活性而抑制肿瘤方面的作用提供理论基础,对于其它mutp53重激活剂、具巯基共价修饰活性抗肿瘤药物的筛选及制备也具有一定的指导意义。
附图说明
图1是化合物藤黄酸处理H1299 p53R175H/p53R273H-PUMA promoter BS2细胞15小时后相对荧光素酶表达结果;其中A图为H1299 p53R175H- PUMA promoter BS2细胞,B图为H1299 p53 R273H-PUMA promoter BS2细胞;
图2是化合物藤黄酸处理SK-BR-3、HT29细胞12h、24h后,免疫荧光实验显示的野生型p53与突变型p53的表达结果;其中A图为SK-BR-3细胞;B图为HT29细胞;
图3是化合物藤黄酸处理HT29细胞24h后,免疫沉淀实验显示的野生型p53与突变型p53的表达结果;
图4是化合物藤黄酸处理HT29细胞12、24、48h后,蛋白质印迹实验显示的p53及PUMA、p21等wtp53靶基因的表达结果;
图5是化合物藤黄酸单独及与NAC或BSO联合处理HT29细胞72h后,MTT实验显示的肿瘤增殖抑制效果;A图为化合物PRIMA1,B图为藤黄酸;
图6是化合物藤黄酸与具巯基修饰活性的小分子DTNB共孵育1h,与DTNB竞争性结合游离巯基的效果。
具体实施方式
下面通过实施例对本发明作进一步详细说明,但本发明的保护范围不局限于所述内容,实施例中方法如无特殊说明均采用常规方法,使用试剂如无特殊说明,均为常规市售试剂或采用常规方法配置的试剂。
实施例1:mutp53重激活剂筛选
1、取1.5×105个处于对数生长期的H1299 p53R175H/ p53R273H-PUMA promoter BS2细胞(稳定表达PUMA启动子荧光素酶报告基因),铺板至6孔板中,以RPMI1640培养基补至2mL,晃匀后,在37℃恒温培养箱培养24h使细胞贴璧。
2、将贴璧完全的H1299 p53R175H/ p53R273H-PUMA promoter BS2细胞从培养箱取出,吸净旧培养基,加入2mL新鲜RPMI 1640培养基及化合物藤黄酸 (终浓度为25、50μM),晃匀后,在37℃恒温培养箱培养15h。
3、检测GLuc的活性
GLuc、SEAP都属于分泌型的蛋白,因此化合物处理完毕后,可轻轻收集细胞培养上清,立即检测GLuc、SEAP的活性;
(1)收集化合物处理完毕的100μL细胞培养上清至1.5mL离心管,然后置于室温;
(2)将10×的GL-S缓冲液取出并在常温下解冻,充分混匀后取适量10×的GL-S缓冲液用超纯水稀释为1×GL-S缓冲液;1×GL-S缓冲液用量为100μL/反应;
(3)避光环境下,往1×GL-S缓冲液加入1/100总体积的Substrate GL(100×),充分混匀,配得GLuc工作液;
(4)将待检测上清及GLuc工作液分别移入25℃杂交炉中温育25min;
(5)取一个干净的不透光96孔酶标板,将温育完毕的上清及工作液取出分别移取10μL、100μL至孔中,用移液枪温和混匀;
(6)混匀完毕后,96孔板放入25℃杂交炉孵育1min,然后将酶标仪的读板模式设置为“化学发光”,检测96孔板中GLuc氧化荧光素所发生的生物萤光。(室温孵育后应争取在5分钟内读板)
4、检测SEAP的活性
(1) 吸取50μL细胞培养上清至新的1.5mL离心管,然后在65℃杂交炉中加热15min,取下置于冰浴备用;
(2)将10×的AP缓冲液取出并在常温下自然解冻,充分混匀后取适量的10×的AP缓冲液用超纯水稀释为1×AP缓冲液,1×GL-S缓冲液的用量为100μL/反应;
(3)避光环境下,往1×AP缓冲液加入1/100总体积的Substrate AP (100×),充分混匀,配得SEAP工作液;
(4)将待检测上清及SEAP工作液分别移入25℃杂交炉中温育10min;
(5)取一个干净的不透光96孔酶标板,将温育完毕的上清及SEAP工作液取出分别移取10μL、100μL至孔中,用移液枪温和混匀;
(6)混匀完毕后,96孔板放入25℃杂交炉孵育10min,然后通过酶标仪检测96孔板中SEAP与底物反应的发光强度。
5、 GLuc活性标准化
计算样品GLuc/SEAP发光强度的比率,使所有样品的GLuc活性标准化。
6、相对荧光强度计算
各样品GLuc活性标准化完毕后,以化合物处理组GLuc活性/Control组GLuc活性,计算相对荧光强度。
结果见图1,从图中1中可以看出藤黄酸处理后,H1299 p53R175H/ p53R273H-PUMApromoter BS2细胞中的相对荧光素酶活明显增强,提示藤黄酸可以重激活突变p53进而转录激活wtp53靶基因PUMA。
实施例2:免疫荧光实验
1、细胞爬片:取灭菌的盖玻片放到6孔板中,然后取2×105个对数生长期的SK-BR-3、HT29细胞种至孔中,晃匀后放入37℃培养箱培养24h使细胞紧密贴于盖玻片;
2、化合物处理:将贴璧完全的SK-BR-3、HT29细胞从培养箱取出,吸净旧培养基,加入2mL新鲜RPMI 1640培养基及化合物藤黄酸(终浓度为0.1、0.25μM),晃匀后,在37℃恒温培养箱培养12h、24h;
3、细胞清洗:化合物处理完毕后,将6孔板取出并吸净旧培养基,然后,用1mL 的1×PBS清洗细胞,重复三次;
4、细胞固定:按照5%多聚甲醛:1×PBS:20%蔗糖=6:3:1(体积比)的比例配制固定液,往每孔中加入1mL固定液并室温静置孵育10min,然后吸净固定液并用1×PBS清洗3次;
5、细胞通透:往孔中加入800μL的去垢剂NP-40(浓度为1%)并室温静置孵育5min,从而在细胞膜上打孔以利于后续抗体进入,然后,吸净NP-40并用1×PBS清洗3次;
6、封闭:往孔中加入1mL的5% BSA室温孵育2h,封闭非特异性位点进而消除非特异性反应。然后,吸净BSA并用1×PBS清洗3次;
7、抗体孵育:参照抗体说明书,将构象特异型p53抗体PAb1620(识别野生型 p53)、PAb240(识别突变型p53)用2%BSA稀释至合适浓度,然后,将100μL抗体稀释液均匀滴至盖玻片上,将6孔板放入4℃层析柜抗体孵育过夜。次日,将6孔板取出,将抗体吸净并用1×PBS清洗3次;
8、标记二抗:避光环境下,将抗鼠荧光二抗IgG488从4℃冰箱取出并用2%BSA溶液按1:300比例稀释。然后,将100μL二抗稀释液均匀滴至盖玻片上,将6孔板用锡箔纸严密包裹后放入27℃杂交炉中孵育1.5h;然后,将抗体吸净并用1×PBS清洗3次;
9、细胞核染色:避光环境下,将核染料DAPI(0.5mg/mL)从-20℃取出解冻,用1×PBS缓冲液按1:500的比例稀释。将稀释后的DAPI按每孔100μL的量匀滴至盖玻片上,放入27℃杂交炉避光孵育15min。经DAPI染核,细胞核呈现亮蓝色,在后续荧光拍摄中利于视野找寻及待测蛋白的亚细胞定位分析,然后,将DAPI吸净并用1×PBS清洗3次;
10、封片:避光环境下,取30uL防淬灭剂至载玻片中央,然后用掰弯的针头将贴于皿底的盖玻片轻轻挑起,控去多余水分后轻置至载玻片上,缓缓将接种有细胞的那面朝下封于防淬灭剂中,避免产生气泡,封片完毕后将成片快速放至暗盒中4℃保存或直接用正置荧光显微镜进行拍照;
11、观察及拍照:避光条件下,将成片从暗盒取出并固定于正置荧光显微镜的载物台上,先通过20倍镜找到细胞所在区域,后转换为油镜以获取更清晰的视野,选择几个具有代表性的视野,调整对比度、曝光强度等参数,进行荧光拍摄并保存;
结果见图2,从图中2中可以看出藤黄酸处理后,SK-BR-3、HT29细胞中突变p53的表达下降,同时野生型p53的表达增加,提示藤黄酸可以恢复突变p53的野生型构象。
实施例3:免疫沉淀实验
1、细胞接种:取生长良好,汇合度80-90%的HT29细胞,按2×106个细胞/皿接种至10cm培养皿,晃匀,放入37℃培养箱孵育24h至细胞贴璧完全;
2、化合物处理:将贴璧完全的HT29 细胞从培养箱取出,吸净旧培养基,加入10mL新鲜1640培养基(含0.25μM的藤黄酸),晃匀,放入37℃培养箱培养24h;
3、收集细胞:用细胞刮收集细胞并将细胞悬液移入15mL的离心管中,4℃、1000g离心5min,弃去上清,以1mL 预冷1×PBS重悬细胞后将悬液转至1.5mL的离心管,4℃,以1000g离心5min,弃去上清并将所留沉淀存于-80℃待用;
4、蛋白提取:以适量RIPA裂解液重悬细胞沉淀后,在冰浴中对样品进行超声破碎,功率设为25%,超声10s,间隔6s,超声10次;然后,样品置于4℃的360°静音混匀器裂解2-3h。4℃、10000g离心30min,小心转移上清至新的1.5mL离心管;
5、预洗:取20μL Protein A+G琼脂糖珠至新1.5mLEP管并以1mL 1×PBS重悬清洗琼脂糖珠后,4℃,8000rpm离心1min,弃上清,重复两次;将4中蛋白样品及1μg的IgG抗体移入EP管中置于360°静音混匀仪4℃预洗2h从而去除与珠子非特异性结合的蛋白,4℃下,5000rpm,离心30min后,将上清转入新的已预冷1.5mL EP管备用;
6、免疫沉淀反应:根据蛋白浓度测定结果,取100-500μg的蛋白样品至新的1.5mL EP管,加入1μg目的抗体(或IgG),移至4℃的360°静音混匀仪中免疫反应过夜;次日,将免疫反应样品完全转移到20μL已用PBS充分清洗的Protein A+G琼脂糖珠中,补加600μL预冷1×PBS,在4℃的360°静音混匀仪继续孵育过夜;
7、清洗:样品从4℃取出,冰浴静置2min后,6000g、4℃,离心1min,弃上清。沿壁加入1mL预冷1×PBS,轻轻清洗珠子后,4℃、6000g,离心1min,弃上清,清洗8-10次;
8、变性及蛋白质印迹: 往样品管中加入16μL 1×PBS及4μL 5×Loading dye,短暂低速离心后,放入沸水浴煮沸7-10min使结合于琼脂糖珠的免疫复合物完全解离。已变性样品冷凝后,离心并进行SDS-PAGE电泳;
结果见图3,从图中3中可以看出藤黄酸处理后,HT29细胞中突变p53的表达下降,同时野生型p53的表达增加,提示藤黄酸可以恢复突变p53的野生型构象。
实施例4:蛋白质印迹实验
1、取2×106个对数生长期的HT29细胞接种至10cm培养皿,于37℃培养箱中培养24h至细胞完全贴璧后,采用Gambogic acid (0.25μM)处理细胞12、24、48h,然后,用细胞刮将细胞刮下,4℃离心,用1×PBS清洗一遍,移至1.5mL离心管中得到细胞沉淀;
2、加入适宜的细胞裂解液,对细胞沉淀进行超声处理,条件为25-35% Amp,超声10s停5s,共10次,如果沉淀未被完全超声彻底可以适当增加超声次数,提取出的蛋白利用考蓝或BCA方法进行定量;
3、配置蛋白上样体系:15μL、20μg,蛋白上样体系中加溴酚蓝(已加β-巯基乙醇)终浓度为1×上样体系;体系配好后,沸水煮7min左右,冷却离心;
4、跑胶:配置12%的分离胶和4.5%的浓缩胶,配置1×的Running Buffer(含1%的SDS),准备上样;
5、转膜:配置转膜液,200mL甲醇+100mL的10×Running Buffer纯水定容到1L,PVDF膜用之前要用甲醇活化;按照板子黑面-海绵-三层滤纸-胶-PVDF膜-三层滤纸-海绵-板子白面顺序制备转膜装置。转膜条件:180mA、300V、3h;
6、封闭,先用1×TBST清洗PVDF膜上的转膜液,然后用5%的牛奶37℃封闭2h或4℃摇床封闭过夜;
7、标一抗:用1×TBST清洗封闭牛奶,然后用配好的p53(DO-1)(1:500)、PUMA(1:100)、p21(1:1000)、NOXA(1:500)、BAX(1:500)、Tubulin(1:1000)抗体4℃摇床过夜孵育;
8、标二抗:PVDF膜用1×TBST清洗后,以HRP标记的二抗常温孵育2h,二抗以1×TBST按1:10000比例稀释;
9、显影:按照1:1比例配制ECL化学发光液,将PVDF膜以发光液孵育30s后,放入进行CCD显影仪显影;
结果见图4,从图中4中可以看出藤黄酸处理后,突变p53表达下降,而wtp53靶基因PUMA、p21、NOXA、BAX的蛋白水平上调,提示藤黄酸可以降解mutp53,也可以转录激活wtp53靶基因。
实施例5:MTT实验
1、细胞铺板:取生长旺盛的HT29细胞制备单细胞悬液,按5×103个/孔将细胞铺至96孔板,轻振板身使细胞在孔中分布均匀后放入37℃培养箱培养至次日使细胞贴璧完全;
2、化合物处理:细胞贴璧之后,根据化合物的母液浓度以及想要检测的工作浓度,吸取相应体积藤黄酸母液至新鲜培养基中,配得一系列浓度梯度的稀释液,充分混匀后,按每孔20μL的量加入96孔板中,联用组加入BSO(终浓度为0.5mM)或NAC(终浓度为10mM),然后将细胞放入37℃培养箱培养72个小时;
3、MTT检测:取出 96孔板并于在避光处加入MTT溶液(终浓度为5μg/mL),细胞放回培养箱继续孵育4h,孵育完毕后,泵吸掉孔中液体并加入二甲基亚砜(加入量为150μL/孔),在振板器充分振荡15min,使蓝紫色甲瓒晶体充分溶解,用酶联免疫检测仪测定波长490nm处的OD值。用Microsoft office/WPS处理实验数据(即Control组、加药组的OD490),计算并比较Gambogic acid单独及BSO或NAC联用后半数抑制浓度IC50的差异;
结果见图5及下表,从图中5中可以看出藤黄酸与外源性巯基供体联用后,与已知巯基修饰化合物PRIMA-1类似,均出现IC50的明显上升;而与还原型谷胱甘肽GSH合成抑制剂BSO联用后,IC50的明显减小,这提示了藤黄酸与PRIMA-1一样具有巯基共价修饰活性,可通过巯基共价修饰氧化还原平衡维持者GSH进而发挥肿瘤抑制功能,也可能通过巯基共价修饰作用恢复mutp53的野生型功能;
。
实施例6:巯基结合实验
1、浓度梯度标准工作液制备:以ddH2O配制1L反应缓冲液(含0.1M磷酸钠,1mM EDTA ,pH=8.0)然后准确称取52.68mg的半胱氨酸一水合盐酸盐(MW=175.6g/moL)充分溶于200mL反应缓冲液中,配得浓度为1.5mM的标准品,并按照下表进行稀释,配得浓度梯度为0-1.5mM的标准工作液;
2、标准曲线绘制:称取8mg 的DTNB粉末溶于2mL反应缓冲液制得Ellman试剂溶液,取若干试管,每个试管中加入50μL的Ellman试剂液和2.5mL的缓冲液。然后,在各试管中加入250μL各浓度的标准工作液,室温下反应1h后用酶标仪检测波长412nm的OD值(A412),根据工作液的浓度及A412,绘制标准曲线,进而表征巯基含量及A412的关系;
3、待测物的巯基结合反应:取若干试管,每个试管中加入50μL的Ellman试剂液和2.5mL的缓冲液;然后,在各试管中分别加入相应的待测液(总体积为250μL);室温下反应1h后用酶标仪检测各反应体系的A412,根据标准曲线计算并比较各反应体系中的游离巯基含量,从而表征PRIMA-1、藤黄酸与DNTB竞争性结合游离巯基的程度;
试管1:1mM的NAC溶液
试管2:1mM的PRIMA-1溶液
试管3:1mM的NAC溶液+1mM的PRIMA-1溶液
试管4:1mM的Gambogic acid溶液
试管5:1mM的NAC溶液+1mM的藤黄酸溶液
其中,设置试管2、4的反应体系是为了排除PRIMA-1、藤黄酸化合物溶液对A412数值的影响。
结果见图6,从图中6中可以看出藤黄酸与已知巯基修饰化合物PRIMA-1类似,A412明显下降可以明显抑制NAC与DTNB间的二硫键交换反应,进而减少反应产物NTB2-二价阴离子(呈黄色,最大吸收峰在412nm处)的生成量,提示了藤黄酸具有巯基结合活性,可能通过巯基共价修饰作用重激活mutp53。
Claims (7)
1.藤黄酸在制备突变p53重激活剂中的应用。
2.根据权利要求1所述的应用,其特征在于:藤黄酸作为突变p53的重激活剂,激活突变p53进而重塑肿瘤细胞中的p53信号通路,从而实现抗肿瘤的目的。
3.藤黄酸作为突变p53重激活剂在制备抗肿瘤药物中的应用。
4.藤黄酸在制备巯基共价修饰药物中的应用。
5.根据权利要求3所述的应用,其特征在于:藤黄酸通过巯基共价修饰氧化还原平衡维持者GSH而使肿瘤细胞的氧化还原失衡,并通过巯基共价修饰突变p53的关键Cys氨基酸残基而重激活突变p53,从而实现抗肿瘤的目的。
6.藤黄酸作为巯基共价修饰剂在制备抗肿瘤药物中的应用。
7.一种药物组合物,包括权利要求1或4所述的藤黄酸以及药物运载体。
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