CN109771426A - 千金藤素作为asph的酶活抑制剂的应用 - Google Patents
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Abstract
本发明公开了千金藤素作为天冬氨酰β‑羟化酶(aspartateβ‑hydroxylase,ASPH)的酶活抑制剂的应用。千金藤素可用于制备治疗ASPH过表达相关疾病的药物。所述的疾病为癌症及肝纤维化和肝硬化。所述的癌症包含:肝癌、肝内胆管癌、恶性胶质瘤、小细胞肺癌、结肠癌、胰腺癌。本发明通过蛋白三维结构虚拟筛选,得到能结合进ASPH的酶活结构域的化合物,即千金藤素;该化合物能抑制ASPH酶活性,100μM时的抑制率为80.5%;该化合物能抑制肝癌细胞HepG2、Hep3B和Huh7的增殖,IC50值分别为8.02μM、12.57μM和13.88μM。
Description
技术领域
本发明涉及医药领域,具体涉及千金藤素作为ASPH的酶活抑制剂的应用。
背景技术
千金藤素,别称千金藤碱、头花千金藤碱、千金藤啶碱等,英文名Cepharanthine,分子式为C37H38N2O6,分子量为606.72,CAS登录号为481-49-2,结构式如下:
千金藤素目前作为白细胞增生药,能促进骨髓组织增生,从而升高白细胞;用于白细胞减少症,可使外周血白细胞增多。其作用机制是促进骨髓组织增生,从而产生升高白细胞作用,用于因肿瘤化疗、放疗引起的粒细胞缺乏症和其他原因引起的白细胞减少症。
ASPH全称为天冬氨酰β-羟化酶(aspartateβ-hydroxylase,ASPH),是α-酮戊二酸依赖型双加氧酶家族成员,属Ⅱ型跨膜蛋白,其主要的酶活性功能区域位于C端,ASPH分子的C端片段具有羟化酶活性,能够特异性作用于某些蛋白质中,例如凝血因子和Notch信号通路上的受体与配体等的表皮生长因子(Epidermal Growth Factor,EGF)样结构域,催化其内天冬氨酸或天冬酰胺基团上β碳原子的羟化反应,并发挥各项生物学功能,如作用于Notch信号通路调控细胞生长、黏附与迁移。有研究表明ASPH的过表达与肿瘤细胞形成和肿瘤细胞的增生、侵入及转移有着密切的相关性,被认为是一种潜在的广谱性肿瘤标记物。图1为ASPH对表皮生长因子样结构域的催化反应过程。ASPH在α-酮戊二酸及Fe2+存在的条件下,可将EGF样结构域内的天冬氨酸或天冬酰胺残基上的β碳原子羟基化(A cell-surfaceβ-hydroxylase is a biomarker and therapeutic target for hepatocellularcarcinoma.Hepatology.2014Oct;60(4):1302-13.doi:10.1002/hep.27275)。
ASPH在包括肝癌、肝内胆管癌、恶性胶质瘤、小细胞肺癌、结肠癌、胰腺癌等多种肿瘤组织中较正常组织呈高表达,可影响肿瘤细胞的恶性转化和迁移能力。ASPH还可能参与肝纤维化和肝硬化进程。ASPH高表达病人手术后复发时间更短,生存期更短。已发表的文章结果表明,ASPH基因高表达的肝癌患者(以肿瘤组织比周围正常组织mRNA水平高2倍及以上定义为高表达患者)手术后更短时间复发,生存期更短(Overexpression of aspartyl-(asparaginyl)-beta-hydroxylase in hepatocellul ar carcinoma is associatedwith worse surgical outcome.Hepatology.2010Jul;52(1):164-73.doi:10.1002/hep.23650)。另一篇文献表明,高表达ASPH的肝癌患者(通过ASPH抗体免疫组化染色癌组织中ASPH的表达,如果阳性细胞数大于等于10%,判断该癌组织为高表达),肿瘤复发率更高,患者存活率更小(Hydroxylase Activity of ASPH Promotes Hepatocellul arCarcinoma Metastasis Through Epithelial-to-Mesenchymal TransitionPathway.EBioMedicine.2018May;31:287-298.doi:10.1016/j.ebiom.2018.05.004)。
针对ASPH的小分子抑制剂能抑制肿瘤细胞和小鼠移植肿瘤的生长,ASPH的抑制剂有用于肿瘤治疗的潜力。(A cell-surfaceβ-hydroxylase is a biomarker andtherapeutic target for hepatocellular carcinoma.Hepatology.2014Oct;60(4):1302-13.doi:10.1002/hep.27275)。
发明内容
本发明的目的是提供千金藤素作为ASPH的酶活抑制剂的应用,千金藤素可抑制ASPH对蛋白质中EGF样结构域的羟化酶活性,可用于治疗ASPH过表达的相关疾病。
为了达到上述目的,本发明提供了千金藤素作为ASPH的酶活抑制剂的应用。
千金藤素可应用于制备治疗ASPH过表达相关疾病的药物。
较佳地,所述的疾病为癌症。
较佳地,所述的癌症包含:肝癌。
较佳地,所述的癌症还包含:肝内胆管癌。
较佳地,所述的癌症还包含:恶性胶质瘤、小细胞肺癌、结肠癌、胰腺癌。
较佳地,所述的疾病为:肝纤维化和肝硬化。
较佳地,所述的药物为千金藤素和药学上可接受的辅料形成的药物组合物。
本发明可具有以下有益效果:
(1)本发明通过蛋白三维结构虚拟筛选,得到能结合进ASPH的酶活结构域的化合物,即千金藤素;该化合物能抑制ASPH酶活性,100μM时的抑制率为80.5%;该化合物能抑制肝癌细胞HepG2、Hep3B和Huh7的增殖,IC50值分别为8.02μM、12.57μM和13.88μM。
(2)可通过将千金藤素应用于抑制ASPH过表达的药物中,以治疗ASPH过表达的相关疾病。
附图说明
图1为ASPH对表皮生长因子样结构域的催化反应过程。
图2为ASPH的晶体结构,其在PDB中的ID为5JQY。
图3为对接格点范围。
图4为小分子N-oxalylglycine晶体构象与对接构象。
图5为诱导配合对接结果。
图6a为ASPH酶活结构域和化合物的结合图。
图6b为ASPH酶活结构域和化合物的基团间的相互作用图。
图7为千金藤素对ASPH酶活性的抑制率。
图8为千金藤素对肝癌细胞系增殖抑制的IC50值分析结果。
具体实施方式
以下结合具体实施例,对本发明进一步阐述。应理解,这些实施例仅用于解释本发明而不用于限制本发明的范围。除非另有定义或说明,本发明所述的科学术语与本领域普通技术人员所理解具有相同的含义。
本发明根据ASPH酶活区的蛋白晶体结构,通过计算机模拟筛选能结合ASPH酶活区的小分子来高通量筛选ASPH的酶活抑制剂,筛选得到对ASPH起到抑制作用的化合物,即千金藤素。千金藤素对ASPH的酶活抑制作用显著,通过抑制ASPH的活性,可起到抑制肿瘤细胞增殖的作用。本发明所述的ASPH的过表达是指较正常组织呈高表达。
1.ASPH抑制剂的高通量虚拟筛选过程如下:
1.1靶标分析及受体结构
蛋白质结构数据库(protein data bank,PDB)提供的ASPH已解析的晶体结构共8个(见表1),其中分辨率最高的结构为5JQY,其晶体结构中包含小分子N-oxalylglycine,与ASPH的底物分子α-酮戊二酸(α-ketoglutarate)结构类似,其结合位点即为ASPH底物结合位点。因此,我们选用5JQY,经过软件包中的“Protein Preparation Wizard”模块准备(参数默认)后作为虚拟筛选的受体结构(请参阅图2)。
表1 ASPH已知晶体结构
蛋白准备完毕后,按照软件包中分子对接的Glide模块的流程,先用Receptor Grid Generation生成grid文件:以小分子N-oxalylglycine和凝血因子X(Coagulation factor X)的99-116位残基为格点计算范围(Enclosing box)中心;小分子质心位置范围(Ligand diameter midpoint box)尺寸:格点计算范围尺寸(Enclosing box size):Rotatable Groups:Thr629的-OH,Ser668的-OH;其他参数默认(对接格点范围请参阅图3)。
用Glide的额外精度(XP,extra precision)对接小分子N-oxalylglycine,对接后构象基本能与小分子N-oxalylglycine的晶体构象重合(请参阅图4)。考虑到抑制剂体积较N-oxalylglycine大,而且蛋白质具有柔性,所以选用蛋白部分侧链柔性的诱导配合对接(Induced Fit Docking,IFD)再次进行对接。XP精度下的对接结果基本能够解释ASPH抑制剂的构效关系(请参阅图5);最终,选用Induced Fit Docking后的蛋白构象作为虚拟筛选的受体结构。
1.2.小分子库的虚拟筛选
选择网页http://www.sibcb.ac.cn/cp13-5_3.asp提供的商品化的小分子库,化合物用LigPrep模块处理:加氢、加电荷、生成互变异构体,再进行对接。对接选用Glide,标准精度(standard precision,SP),对接后在打分优于-6的构象中,人工查看,挑选可能的候选化合物。候选化合物包含千金藤素,其与ASPH酶活区相互作用如图6a和图6b所示。图6a为ASPH酶活结构域和化合物的结合图,图6b为两者基团间的相互作用图。
2.针对上面虚拟筛选到的千金藤素进行ASPH酶活抑制实验。具体实验方法参考文献:Zou Q,Hou Y,Wang H,et.al.Hydroxylase Activity of ASPH PromotesHepatocellular Carcinoma Metastasis Through Epithelial to MesenchymalTransition Pathway,EBioMedicine,2018,31:287-298。
实验步骤如下:
2.1EGF样的结构域肽段(DGDQCETSPCQNQGKCKDGLGYETCTCLEGFEGKNC)作为ASPH的底物(由中肽生化公司合成),该肽段作为ASPH的底物参考自文献:A cell-surfaceβ-hydroxylase is a biomarker and therapeutic target for hepatocellularcarcinoma.Hepatology.2014Oct;60(4):1302-13.doi:10.1002/hep.27275。
2.2用转染试剂lipofactamine2000(Thermo公司)和ASPH表达质粒(pcDNA3.1-ASPH)混合,按说明书方法转染HEK293T细胞。ASPH的表达序列为NCBI数据库中https://www.ncbi.nlm.nih.gov/nuccore/NM_004318.4公开的序列。pcDNA3.1(-)载体来自Invitrogen公司。将ASPH的表达序列插入pcDNA3.1(-)载体的多克隆位点构建成ASPH表达载体pcDNA3.1-ASPH。
2.3细胞转染48小时后,收集细胞,用裂解液PierceTMIP Lysis Buffer(Thermo公司,货号:87787)裂解细胞。用1μg ASPH抗体(Thermo公司,货号PA5-40954)和10μl蛋白A磁珠(Thermo公司,货号:88803)按照说明书对ASPH进行免疫共沉淀。
2.4将吸附ASPH的蛋白A磁珠加到以下反应体系:50mM哌嗪-1,4-二乙磺酸(PIPES),pH 7.0,100mM Fe2+,20mMα-酮戊二酸盐(alpha ketoglutarate,α-KG),0.1mg/ml牛血清白蛋白(BSA),100mM EGF样的结构域肽段。因为ASPH是α-酮戊二酸依赖型的酶,因此体系中α-KG的消耗量(起始量-剩余量),对应为ASPH的酶活性,即通过计算α-KG的消耗量,可计算得到ASPH的酶活性。
2.5分别将对照溶剂DMSO和不同浓度化合物(0.1μM、1μM、10μM、100μM的千金藤素)加入上述反应体系中,反应体系50μl,37℃反应30分钟。
2.6α-KG的浓度在反应后用α-KG试剂盒(Biovision公司,货号:K677)根据说明书进行测定。根据浓度算出剩余量。
2.7α-KG的消耗量(起始量-剩余量)对应为酶的活性,以溶剂DMSO组对应的值为1(千金藤素浓度为0),取相对值。实验重复三次,图7为三次实验的代表结果,可见该化合物1μM到100μM的浓度都能明显抑制ASPH的酶活性,千金藤素浓度为100μM时抑制率为80.5%。图7中**表示和DMSO组比较T-test检验P值<0.01。
3.千金藤素对肝癌细胞系增殖抑制和IC50值测定。实验步骤如下:
3.1第一天:分别接种肝癌细胞系HepG2、Hep3B和Huh7,及正常增殖细胞系HEK293T到3707的384孔细胞板中,HepG2、Hep3B和Huh7细胞接种1500个细胞/45μl/孔,HEK293T细胞接种3000个细胞/45μl/孔。不同细胞接种到不同384孔板中;细胞培养过夜,37℃,5%CO2。
3.2第二天:以100%DMSO为稀释溶液,将初始浓度为10mM千金藤素以2倍稀释比例进行梯度稀释,采用安捷伦Bravo自动化液体处理平台进行梯度稀释。浓度梯度依次为:10mM,5mM,2.5mM,1.25mM,0.625mM,0.3125mM,0.156mM,0.078mM,0.039mM,0mM。取0.2μl各浓度加入到50μl细胞培养体系中。化合物最终浓度分别为40μM,20μM,10μM,5μM,2.5μM,1.25μM,0.625μM,0.3125μM,0.156μM,0μM。每个化合物每种浓度以双复孔间的平均值形式采集数据。
3.3千金藤素处理细胞72小时后,计算化合物最终浓度为10μM时千金藤素对细胞的抑制率,以及千金藤素对肝癌细胞系增殖抑制的IC50值。用MultidropTM自动分液器将Celltiter-试剂加入每孔细胞中,通过多功能酶标仪收集每孔细胞活力,即冷发光(luminescence)数值。CellTiter-试剂含有Ultra-GloTM重组萤光素酶及荧光素,可直接加入培养细胞检测。该试剂可自动裂解细胞,以使活细胞中的ATP释放出来,并可保护ATP,防止降解。活细胞中的ATP参与萤光素酶反应,反应产生的光正比于微孔中的活细胞数量。以DMSO组为对照,计算千金藤素对各种肝癌细胞系增殖影响。IC50值由分析软件GraphPad Prism8通过非线性回归(曲线拟合)法(Nonlinear regression(curve fit))计算得出,IC50值计算为软件自带功能。千金藤素对肝癌细胞系增殖抑制和IC50值测定实验至少重复三次,下面为代表结果:
(1)千金藤素对细胞的抑制率结果如表2所示。结果表明,千金藤素最终浓度为10μM时,对HepG2细胞系增殖抑制率达到55.2%,对Hep3B细胞系增殖抑制率达到42.5%,对Huh7细胞系增殖抑制率达到41.5%,而对正常增殖细胞系HEK293T的增殖无影响。
表2千金藤素对肝癌细胞系的增殖抑制率
细胞 | HepG2 | Hep3B | Huh7 | HEK293T |
抑制率(千金藤素浓度:10μM) | 55.2% | 42.5% | 41.5% | 0.02% |
(2)千金藤素对肝癌细胞系增殖抑制的IC50值分析结果如图8和表3所示。图8中,Cmpd5代表千金藤素,横坐标为对数化的化合物浓度,纵坐标为标准化的细胞活力,以DMSO组检测到的细胞活力定为100%。由表3可知,千金藤素对HepG2细胞系增殖抑制的IC50值为8.02μM,对Hep3B细胞系增殖抑制的IC50值为12.57μM,对Huh7细胞系增殖抑制的IC50值为13.88μM。
表3千金藤素对肝癌细胞系增殖抑制的IC50值
综上所述,本发明通过蛋白三维结构虚拟筛选,得到能结合进ASPH的酶活结构域的化合物,即千金藤素;该化合物能抑制ASPH酶活性,100μM时的抑制率为80.5%;该化合物能抑制肝癌细胞HepG2、Hep3B和Huh7的增殖,IC50值分别为8.02μM、12.57μM和13.88μM。
尽管本发明的内容已经通过上述优选实施例作了详细介绍,但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后,对于本发明的多种修改和替代都将是显而易见的。因此,本发明的保护范围应由所附的权利要求来限定。
Claims (8)
1.千金藤素作为ASPH的酶活抑制剂的应用。
2.根据权利要求1所述的千金藤素作为ASPH的酶活抑制剂的应用,其特征在于,千金藤素应用于制备治疗ASPH过表达相关疾病的药物。
3.根据权利要求2所述的千金藤素作为ASPH的酶活抑制剂的应用,其特征在于,所述的疾病为癌症。
4.根据权利要求3所述的千金藤素作为ASPH的酶活抑制剂的应用,其特征在于,所述的癌症包含:肝癌。
5.根据权利要求4所述的千金藤素作为ASPH的酶活抑制剂的应用,其特征在于,所述的癌症还包含:肝内胆管癌。
6.根据权利要求4所述的千金藤素作为ASPH的酶活抑制剂的应用,其特征在于,所述的癌症还包含:恶性胶质瘤、小细胞肺癌、结肠癌、胰腺癌。
7.根据权利要求2所述的千金藤素作为ASPH的酶活抑制剂的应用,其特征在于,所述的疾病为:肝纤维化和肝硬化。
8.根据权利要求2所述的千金藤素作为ASPH的酶活抑制剂的应用,其特征在于,所述的药物为千金藤素和药学上可接受的辅料形成的药物组合物。
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