CN110275010A - 一种用于治疗前列腺癌药物的P38a MAPK信号通路抑制剂的筛选方法 - Google Patents
一种用于治疗前列腺癌药物的P38a MAPK信号通路抑制剂的筛选方法 Download PDFInfo
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Abstract
本发明公开了一种用于治疗前列腺癌药物的P38a MAPK信号通路抑制剂的筛选方法,包括:选择两个小分子数据库Chemdiv和Enamine,分别获取1,425,044和1,969,861个化合物;靶点处理;进行筛选;对所得化合物使用PAINS‑remove进行假阳性筛选:受体蛋白制备;Classifer model分类模型选择;分子对接;人工筛选;对70个中可购买的58个化合物进行P38a MAPK激酶抑制实验得到对P38a有明显抑制效果的化合物。本发明能提高针对P38aMAPK信号通路靶向性、提高对肿瘤杀伤效应,降低副作用和脱靶效应。
Description
技术领域
本发明涉及化学和生命科学技术领域,具体来说涉及一种用于治疗前列腺癌药物的P38a MAPK信号通路抑制剂的筛选方法。
背景技术
目前以小分子化合物数据库为依托平台,通过多种虚拟筛选方式获取能与目标蛋白相互结合并发挥功能的化合物用于临床治疗或科研实验是一种重要的方式。
P38MAPK信号通路属于MAPK信号通路的一员,是一种保守的丝氨酸-苏氨酸蛋白激酶信号通路,参与细胞的增殖分化的调控。在肿瘤的发生和发展中都具有重要的作用。其中,P38a是P38MAPK信号通路中最常见的亚型,它广泛存在于多种组织当中,当有外界压力或一些细胞因子作用时可激活并促进肿瘤的增殖、迁移和分化。因此,找到一种特异性针对P38a MAPK的抑制剂可以有效地抑制肿瘤的生长。
多种P38aMAPK抑制剂被发现用于各种炎症疾病和肿瘤,但是,由于这些抑制剂具有的脱靶效应,会对病人产生比较明显的副作用,因此,现有的P38MAPK信号通路抑制剂在临床上并没有得到广泛的使用,因此,有必要对P38a MAPK信号通路抑制剂进行研究,找到一种具有特异靶向作用的药物为临床治疗和科研研究提供帮助。
Wang L等通过虚拟筛选的方法,通过类药性五原则等筛选手段,从小分子数据库中筛选出了多个mTOR抑制剂,并对其进行细胞功能研究,发现筛选出的抑制剂可以有效的抑制mTOR并抑制肿瘤的生长(Wang L,Chen L,Yu M et al.Discovering new mTORinhibitors for cancer treatment through virtual screening methods and invitro assays.Sci Rep-Uk.2016;6(1):18987),但其所使用的筛选方式较为单一,不能保证这种单一方式筛选出来的化合物能够特异性靶向P38aMAPK,极有可能造成脱靶效应而产生副作用,并且,单一的筛选方式不能有效的筛选出对P38a MAPK激酶有抑制效果的化合物。
发明内容
本发明目的在于克服上述缺点而提供的一种能提高针对P38aMAPK信号通路靶向性、提高对肿瘤杀伤效应,降低副作用和脱靶效应的用于治疗前列腺癌药物的P38a MAPK信号通路抑制剂的筛选方法。
本发明的一种用于治疗前列腺癌药物的P38a MAPK信号通路抑制剂的筛选方法,包括以下步骤:
(1)化合物的获取:选择两个小分子数据库Chemdiv和Enamine,分别获取1,425,044和1,969,861个化合物;
(2)靶点处理:对获得的化合物加氢原子、加电荷、并对带电残基进行质子化;
(3)进行筛选:用分子量小于500、氢键给体数目小于5、氢键受体数目小于10、脂水分配系数小于5和可旋转键的数量不超过10个的类药性五原则对靶点处理过的化合物进行,两个小分子数据库筛选后分别剩余小分子1064103和1889526个;
(4)对以上所得化合物使用PAINS-remove进行假阳性筛选,筛选后分别剩余小分子995581和1834361个:
(5)受体蛋白制备:从PDB数据库(https://www.rcsb.org/)选择3ZS5文件作为MAPK P38α受体蛋白,蛋白质大分子用autodock tools处理,加氢,加电荷,删除水,保留BOG、EDO;
(6)Classifer model分类模型选择:从chembl和bindingDB数据库选择数据,参数设置为:p38a亚型化合物、保留人源、活性数据为激酶测试水平,以IC50表示;对选择的数据去重,化合物包含Na+、K+去掉,去掉活性数据范围不明确化合物;用1uM、5uM、10uM、20uM分别作为截断值来建立基于ECFP_4的贝叶斯模型;选择10uM为活性阈值根据各模型马修斯相关系数(MCC),所得到的值越高,其效果越好,根据测试集结果显示,测试集中效果较好的是LCFP-12模型、EPFP-10模型,将LCFP-12和EPFP-10模型的小分子定义为具有潜在活性,并将其组成P38aMAPK抑制剂数据库;
(7)分子对接:在天河二号超级计算机平台上,使用自建流程(输入参数receptor=/HOME/huiyuan_fnjiang_1/WORKSPACE/ZSF/virtual_screen/conf/protein.pdbqt;center_x=21.792;center_y=36.8;center_z=19.496;size_x=18;size_y=26;size_z=26;num_modes=9)和AutoDock Vina软件进行分子对接,对接位点选择21.723,35.246,15.333°A,活性口袋定义详细参数x_center=21.723,y_center=35.246,z_center=15.333。x_size=40,y_size=40,z_size=40,最终得到得分前2000的化合物;
(8)人工筛选:对前2000的结果进行人工筛选,通过计算分子的类药性质ADME/T性质的估算,排除那些不具有类药性质的分子,主要分析对接化合物:a)有没有和Phe169和Tyr35形成疏水作用,尤其考察有没有形成堆积作用;b)有没有和p38激酶的Met109形成氢键作用,含额外的氢键Gly100位氨基酸;c)有没有和Ala51、Thr106、Lys53形成疏水相互作用;最终遴选107个化合物做后续的数据处理;进行可溶性、已知生物活性及重复性检测:a)溶解性筛选:DS_ADMET_solubility等级在2-4的化合物,筛选剩余82个;b)重复项:重复的小分子三个,筛选剩余79个;c)已有活性筛选:查询CHEMBL库中,没有发现化合物中有关于MAPK P38α的活性实验,筛选剩余79个;结构相似性聚类:把筛选剩余的79个化合物进行结构上相似性的比较,剔除9个在结构上很相似的化合物,最终剩余70个;
(9)对70个中可购买的58个化合物进行P38a MAPK激酶抑制实验:将5μl 10μM浓度的候选化合物与纯化的10μl P38a MAPK混合,在室温反应10分钟,再加入含有羧基荧光素的肽链和三磷酸腺苷,在28℃反应之后加入25μl的终止液,将反应后的液体进行吸光度分析,根据吸光度计算出候选化合物的激酶抑制数据,最终得到对P38a有明显抑制效果的化合物,其中第9号化合物对P38a MAPK的抑制效果最好,其结构通式(I)如下:
上述的用于治疗前列腺癌药物的P38a MAPK信号通路抑制剂的筛选方法,其中:步骤(6)中p38a亚型化合物为MAPK14。
本发明与现有技术相比,具有明显的有益效果,从以上技术方案可知:以超级计算机为平台,对超过三百万的小分子进行筛选,并通过类药性五原则、ECFP_4的贝叶斯模型等多重筛选方式进行筛选,得到有效的抑制P38aMAPK的化合物。对所筛选出的化合物进行P38a MAPK激酶抑制实验和细胞功能实验,结果表明,所筛选出的9号化合物对P38a MAPK具有明显的抑制效应,P38a在10μM 9号化合物的浓度下抑制效率百分比为77%,半数致死浓度为2.78μM,且9号化合物可以明显抑制前列腺癌细胞的增殖和转移,并且增加前列腺癌细胞的凋亡率。
附图说明
图1:9号化合物对前列腺癌细胞增值的影响图。
图2:9号化合物对前列腺癌细胞凋亡和周期的影响图。
图3:9号化合物对前列腺癌细胞迁移的影响图。
图4:测试集结果图。
具体实施方式
一种用于治疗前列腺癌药物的P38a MAPK信号通路抑制剂的筛选方法,包括以下步骤:
(1)化合物的获取:选择两个小分子数据库Chemdiv(上海陶素生化科技有限公司)和Enamine,分别获取1,425,044和1,969,861个化合物;
(2)靶点处理:对获得的化合物加氢原子、加电荷、并对带电残基进行质子化;
(3)进行筛选:用分子量小于500、氢键给体数目小于5、氢键受体数目小于10、脂水分配系数小于5和可旋转键的数量不超过10个的类药性五原则对靶点处理过的化合物进行,筛选结果如下:
(4)对以上所得化合物使用PAINS-remove进行假阳性筛选:
(5)受体蛋白制备:从PDB数据库(https://www.rcsb.org/)选择3ZS5文件作为MAPK P38α受体蛋白,蛋白质大分子用autodock tools处理,加氢,加电荷,删除水,保留BOG、EDO。
(6)Classifer model分类模型选择:从chembl和bindingDB数据库选择数据,参数设置为:p38a亚型(MAPK14)化合物、保留人源、活性数据为激酶测试水平,以IC50表示。对选择的数据去重,化合物包含Na+、K+去掉,去掉活性数据范围不明确化合物。用1uM、5uM、10uM、20uM分别作为截断值来建立基于ECFP_4的贝叶斯模型。选择10uM为活性阈值(如图4)根据各模型马修斯相关系数(MCC),所得到的值越高,其效果越好,根据测试集结果显示,测试集中效果较好的是LCFP-12模型、EPFP-10模型,因此,为了保证结果的准确性,同时将LCFP-12和EPFP-10模型的小分子定义为具有潜在活性,并将其组成P38aMAPK抑制剂数据库;
(7)分子对接:在天河二号超级计算机平台上,使用自建流程(输入参数receptor=/HOME/huiyuan_fnjiang_1/WORKSPACE/ZSF/virtual_screen/conf/protein.pdbqt;center_x=21.792;center_y=36.8;center_z=19.496;size_x=18;size_y=26;size_z=26;num_modes=9)和AutoDock Vina软件进行分子对接,对接位点选择21.723,35.246,15.333°A,活性口袋定义详细参数(x_center=21.723,y_center=35.246,z_center=15.333。x_size=40,y_size=40,z_size=40),最终得到得分前2000的化合物;
(8)人工筛选:对前2000的结果进行人工筛选,通过计算分子的类药性质ADME/T(吸收absorption、器官分布distribution、体内代谢metabolism、排泄excretion和毒性toxicity)性质的估算,排除那些不具有类药性质的分子,主要分析对接化合物:a)有没有和Phe169和Tyr35形成疏水作用,尤其考察有没有形成堆积作用;b)有没有和p38激酶的Met109形成氢键作用,含额外的氢键Gly100位氨基酸;c)有没有和Ala51、Thr106、Lys53形成疏水相互作用,最终遴选107个化合物做后续的数据处理。进行可溶性、已知生物活性及重复性检测:a)溶解性筛选:DS_ADMET_solubility等级在2-4的化合物,筛选剩余82个。b)重复项:重复的小分子三个,筛选剩余79个;c)已有活性筛选:查询CHEMBL库中,没有发现化合物中有关于MAPK P38α的活性实验,筛选剩余:79个;结构相似性聚类:把筛选剩余的79个化合物进行结构上相似性的比较,剔除9个在结构上很相似的化合物,最终剩余70个;
(9)对70个中可购买的58个化合物进行P38a MAPK激酶抑制实验:将10μM浓度的候选化合物与纯化的10μl P38a MAPK混合,在室温反应10分钟,再加入含有羧基荧光素的肽链和三磷酸腺苷,在28℃反应之后加入25μl的终止液,将反应后的液体进行吸光度分析,根据吸光度计算出候选化合物的激酶抑制数据,最终得到对P38a有明显抑制效果的化合物,其中第9号化合物对P38a MAPK的抑制效果最好,其中第9号化合物结构通式(I)如下:
(1)
试验例1:9号化合物对P38a MAPK混合进行激酶抑制实验检测9号化合物对P38aMAPK的抑制效率和抑制活性达50%时的浓度。
准备1x激酶缓冲液(混合50mM HEPES,pH7.5和0.0015%Brij-35)和停止缓冲液(结合100mM HEPES,pH7.5,0.015%Brij-35,0.2%涂层试剂和50mM EDTA)用于检测激酶。然后转移10μl 10μM的9号化合物和90μl 1x激酶缓冲液到96孔板作为中间板。最后,从中间板中吸取5μl到384孔板重复试验。
在激酶反应阶段,将激酶溶液、激酶缓冲液、荧光标记肽、ATP和化合物混合加入到检测板上。添加25μl停止缓冲液,在室温下孵化后从Caliper程序中收集并复制转换数据(如表1)
试验例2:9号化合物对前列腺癌细胞增值的影响。
使用不同浓度9号化合物处理前列腺癌细胞,得出9号化合物对不同前列腺癌细胞的半数致死量(如图1)采用CCK8法测定化合物9的抗增殖活性。A)化合物9处理三株前列腺细胞的抗增殖曲线。B)IC50值是由三个细胞系至少三次独立试验的结果确定的。
DU145、LNCaP和RWPE-1细胞分别加入含10%胎牛血清、2mM左旋谷酰胺,1%的抗生素的DMEM、1640和F12K培养基,置于37℃、5%CO2饱和湿度的培养箱中培养,每3-4天换液或传代。
将三种细胞分别种入96孔板中,分别用不同浓度的9号化合物培养,处理2天后用CCK8检测各孔吸光度计算出9号化合物对前列腺癌细胞的半数致死剂量。
试验例3:9号化合物对前列腺癌细胞凋亡和周期的影响。
为了探明9号化合物对前列腺癌增殖影响的具体机制,我们使用流式细胞术对9号化合物处理过的细胞进行凋亡和周期检测(如图2)细胞与化合物9在浓度为IC50值的DU145和LNCaP下孵育48小时。A)9号化合物与对照处理前列腺癌细胞株的凋亡率。B)前列腺癌细胞凋亡率的定量。C)化合物9处理LNCaP细胞周期与对照进行流式细胞检测,D)对前列腺癌细胞各阶段进行定量。*与对照组比较,P<0.05。与对照组比较,**P<0.01。
将LNCap和DU145细胞与用图1所测得的半数致死剂量在37度恒温孵育48h,重复实验。
采用Annexin v-APC凋亡检测试剂盒。培养细胞收集于1X结合缓冲液中(AnnexinV-APC/7AAD kit,Cat NO.4224750;Multisciences,中国)。在BD FACSCalibur流式细胞仪(BD Biosciences)上进行凋亡分析,每个实验样本至少使用1x106个细胞计数。
4℃70%预冷乙醇固定处理后的前列腺癌细胞过夜。100ug/ml的RNase A和碘化丙钠染色20min,流式细胞术检测。在G0/G1期、S期和G2/M期的细胞数量用Flowjo软件进行量化,不包括计算细胞碎片和固定人工制品。
试验例4:9号化合物对前列腺癌细胞迁移的影响。
为了判断所筛选出来的9号化合物是否还能一直肿瘤的迁移,我们对使用9号化合物的前列腺癌细胞进行transwell迁移实验(如图3)A)9号化合物处理后前列腺癌细胞的迁移能力与对照对比在40倍、200倍显微镜下观察的结果并定量分析了前列腺癌细胞的迁移能力。*与对照组比较,P<0.05。与对照组比较,**P<0.01。
在下室添加10%1640培养基600ul,上室种DU145细胞4*10^4个,用无血清培养基200ul重悬。培养24小时后取出上室,棉签擦去上室表面的细胞,底面的细胞用4%多聚甲醛固定15min,然后后用结晶紫染色,拍照计数9个独立对称视野。数据以三个独立实验的平均值±SD表示。
采用CCK8法测定化合物9的抗增殖活性。A)化合物9处理三株前列腺细胞的抗增殖曲线。B)IC50值是由三个细胞系至少三次独立试验的结果确定的。
细胞与化合物9在浓度为IC50值的DU145和LNCaP下孵育48小时。A)9号化合物与对照处理前列腺癌细胞株的凋亡率。B)前列腺癌细胞凋亡率的定量。C)化合物9处理LNCaP细胞周期与对照进行流式细胞检测,D)对前列腺癌细胞各阶段进行定量。*与对照组比较,P<0.05。与对照组比较,**P<0.01。
A)9号化合物处理后前列腺癌细胞的迁移能力与对照对比在40倍、200倍显微镜下观察的结果并定量分析了前列腺癌细胞的迁移能力。*与对照组比较,P<0.05。与对照组比较,**P<0.01。
以上所述,仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,任何未脱离本发明技术方案内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰,均仍属于本发明技术方案的范围内。
Claims (2)
1.一种用于治疗前列腺癌药物的P38a MAPK信号通路抑制剂的筛选方法,包括以下步骤:
(1)化合物的获取:选择两个小分子数据库Chemdiv和 Enamine,分别获取1,425,044和1,969,861 个化合物;
(2)靶点处理:对获得的化合物加氢原子、加电荷、并对带电残基进行质子化;
(3)进行筛选:用分子量小于500、氢键给体数目小于5、氢键受体数目小于10、脂水分配系数小于5和可旋转键的数量不超过10个的类药性五原则对靶点处理过的化合物进行,两个小分子数据库筛选后分别剩余小分子1064103和1889526个;
(4)对以上所得化合物使用PAINS-remove进行假阳性筛选,筛选后分别剩余小分子995581和1834361个:
(5)受体蛋白制备:从PDB数据库(https://www.rcsb.org/)选择3ZS5文件作为MAPKP38α受体蛋白,蛋白质大分子用autodock tools处理,加氢,加电荷,删除水,保留BOG、EDO;
(6)Classifer model分类模型选择:从chembl和bindingDB数据库选择数据,参数设置为:p38a亚型化合物、保留人源、活性数据为激酶测试水平,以IC50表示;对选择的数据去重,化合物包含Na+、K+去掉,去掉活性数据范围不明确化合物;用1uM、5uM、10uM、20uM分别作为截断值来建立基于ECFP_4的贝叶斯模型;选择10uM为活性阈值根据各模型马修斯相关系数(MCC),所得到的值越高,其效果越好,根据测试集结果显示,测试集中效果较好的是LCFP-12模型、EPFP-10模型,将LCFP-12和EPFP-10模型的小分子定义为具有潜在活性,并将其组成P38aMAPK抑制剂数据库;
(7)分子对接:在天河二号超级计算机平台上,使用自建流程(输入参数receptor = /HOME/ huiyuan_fnjiang_1/ WORKSPACE/ ZSF/ virtual_screen/ conf/ protein.pdbqt;center_x = 21.792;center_y = 36.8;center_z = 19.496;size_x = 18;size_y = 26;size_z = 26;num_modes = 9)和AutoDock Vina软件进行分子对接,对接位点选择21.723,35.246, 15.333˚A,活性口袋定义详细参数x_center = 21.723,y_center = 35.246,z_center = 15.333,x_size = 40,y_size = 40,z_size = 40,最终得到得分前2000的化合物;
(8)人工筛选:对前2000 的结果进行人工筛选,通过计算分子的类药性质ADME/T性质的估算,排除那些不具有类药性质的分子,主要分析对接化合物:a)有没有和Phe169和Tyr35形成疏水作用,尤其考察有没有形成堆积作用;b)有没有和p38激酶的Met109形成氢键作用,含额外的氢键Gly100位氨基酸;c)有没有和Ala51、Thr106、Lys53形成疏水相互作用;最终遴选107个化合物做后续的数据处理;进行可溶性、已知生物活性及重复性检测:a)溶解性筛选: DS_ADMET_solubility 等级在2-4的化合物, 筛选剩余82个;b)重复项:重复的小分子三个,筛选剩余79个;c)已有活性筛选:查询CHEMBL库中,没有发现化合物中有关于MAPK P38α的活性实验,筛选剩余79个;结构相似性聚类:把筛选剩余的79个化合物进行结构上相似性的比较,剔除9个在结构上很相似的化合物,最终剩余70个;
(9)对70个中可购买的58个化合物进行P38a MAPK激酶抑制实验:将5μl 10μM浓度的候选化合物与纯化的10μl P38a MAPK混合,在室温反应10分钟,再加入含有羧基荧光素的肽链和三磷酸腺苷,在28℃反应之后加入25μl的终止液,将反应后的液体进行吸光度分析,根据吸光度计算出候选化合物的激酶抑制数据,最终得到对P38a有明显抑制效果的化合物,其中第9号化合物对P38a MAPK的抑制效果最好,其结构通式(I)如下:
(I)。
2.如权利要求1所述的用于治疗前列腺癌药物的P38a MAPK信号通路抑制剂的筛选方法,其中:步骤(6)中p38a亚型化合物为MAPK14。
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CN111298118A (zh) * | 2020-02-11 | 2020-06-19 | 王雄 | Mapk14抑制剂在制备药物中的应用 |
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