CN104744375B - 一种胍类ntr1小分子拮抗剂 - Google Patents
一种胍类ntr1小分子拮抗剂 Download PDFInfo
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Abstract
一种胍类NTR1小分子拮抗剂。本发明涉及药物设计学和药物学领域。本发明通过虚拟筛选和细胞活性测试以及基于受体的结构修饰，提供了具有下列通式I的化合物，其中，R1是疏水性基团；R2是取代芳香环；R3极性取代基团。所述化合物对NTR1具有抑制活性，在制备与NTR1蛋白相关的疾病的药物中具有重要用途。
Description
技术领域
本发明涉及药物设计学和药物学领域，具体而言，涉及经虚拟筛选及细胞活性测试，并进行基于受体的结构修饰得到的一系列胍类NTR1小分子抑制剂的结构。
背景技术
G蛋白偶联受体(G Protein-Coupled Receptor，GPCR)具有七次跨膜结构，在体内广泛分布，参与对各种信号分子的应答活动。GPCR及其信号转导系统通常处于动态平衡，并根据各种生理因素的改变而启动下游信号通路的调节，维持细胞内环境的稳定；在病理条件下，细胞同样通过GPCR接受外界信号对细胞生命活动进行调节。尤其在恶性肿瘤细胞中，GPCR通过介导肿瘤细胞的信号转导过程，对肿瘤细胞的发生、生长、侵袭与转移等过程进行调节，几乎在所有的恶性肿瘤细胞中，都能够检测到某类GPCR分子的非正常表达。GPCR的过度激活或抑制均可能导致细胞内信号转导途径的失调，从而引起紊乱的生理反应，导致异常的生理现象。
GPCR超家族中A类成员神经降压素受体1(NTR1)蛋白主要分布在中央神经系统和肠胃系统中，介导细胞内Gq蛋白信号传导通路，调控细胞内钙离子的浓度水平。该蛋白的天然激动剂神经降压素(NT)是一个13肽，其中六个氨基酸Arg-Arg-Pro-Tyr-Ile-Leu起关键作用。近年来发现NT和NTR1在多种恶性肿瘤(乳腺癌、胰腺癌、结肠癌、肺癌、前列腺癌等)的生长、增殖、侵袭、转移中起到关键的作用。有文献报道在正常乳腺上皮细胞和浸润性乳腺癌的癌组织中均有NT的表达，而NTR1只在乳腺癌的癌组织和肿瘤细胞系中高表达，在乳腺浸润性导管癌中NTR1表达率高达91％。动物实验研究证实NTR1能够促进移植瘤细胞的生长并且能缩短瘤细胞倍增时间，诱导移植瘤的侵袭和转移。因此，NTR1的拮抗剂会对肿瘤的发生发展起到一定的抑制作用，有望成为治疗肿瘤等疾病的有效药物。鼠源NTR1的晶体结构于2012年被成功解析出来，这为基于受体的药物设计和化合物的优化改造提供了有利条件。
本实验室前期已建立一个针对NTR1的含有10000个小分子的数据库，有效地提高了筛选速度。目前，文献中已报道多个针对NTR1靶标的拮抗剂，从已知的拮抗剂出发，有助于建立药效团模型和2D-Fingerprint的筛选方法，从而寻找新型小分子拮抗剂。
发明内容
现有的以NTR1为靶点的新药研发尚未出现上市药物，且由于那时NTR1的晶体结构未被解析，只能根据同源建模得到的三维结构进行药物筛选和设计。本发明目的是基于已经解析的鼠源NTR1蛋白的三维晶体结构，对整合了多个化合物数据库得到的NTR1小分子数据库进行虚拟筛选并对筛选结果进行细胞活性测试，对具有活性的小分子进行基于受体的结构修饰，以寻求对NTR1具有抑制活性的小分子化合物，用于制备与NTR1相关的疾病的药物。
本发明经过虚拟筛选和细胞活性测试及结构修饰，得到一种胍类化合物，结构如通式Ⅰ所示。
其中：
R1是吗啉环、未取代及甲基、卤素、羟基取代的C5-C6环烷烃基、哌啶环、苯环、吡啶环、二甲基氨基、异丙基、叔丁基疏水性基团；R2是由卤素、甲基或乙基取代的苯环或者吡啶环类芳香环；R3是氢，或是氨基、羟基、氨甲基或羟甲基极性基团；n为1-3。
本发明提供的胍类化合物可应用于与NTR1蛋白相关疾病的药物制备中。
本发明筛选并经细胞活性测试得到的具有抑制活性的小分子化合物1为R1是吗啉环，R2是对位氯取代苯环，R3是氢，n为2，其结构如下(化合物1)，IC50值为44.49μM。
本发明的优点和有益效果：
本发明通过虚拟筛选及细胞活性测试，结果证明，本发明所述筛选得到NTR1小分子拮抗剂(化合物1)对细胞内钙离子浓度有较好抑制活性。进而通过对NTR1与配体结合的口袋的亲疏水性、氢键供受体、电荷性质等进行分析，根据化合物1与NTR1对接的结果及打分，在某些部位进行基团的增删及替换，并进行对接验证，从而对化合物1进行结构改造及修饰，得到一系化合物，这些化合物可通过作用于体内的NTR1从而参与其调节机制，用于制备治疗或辅助治疗肿瘤、帕金森疾病、精神分裂症等疾病的药物，具有良好的药物应用前景。
附图说明
图1是化合物1与NTR1的相互作用示意图。
图2是修饰后化合物2与NTR1的相互作用示意图
图3是化合物1的Ca2+荧光值对化合物浓度对数的曲线图。
具体实施方式
下面对本发明的各个方面和特点作进一步的描述。但这些实施例不意味着对本发明有任何限制。
实施例1、NTR1小分子抑制剂的虚拟筛选和细胞活性测试
一、本发明的虚拟筛选的方法包括以下4个步骤：
步骤1：NTR1小分子数据库的构建
整合多个商业小分子数据库(包括ZINC、SPECS、J&K Screening Library、InterBioScreening、Timtec、Msdiscove、Iris-biotech、TCM Database、Ambinter NaturalProducts、AnalytiCon Discovery NP、Princeton NP、Molecular DiversityPreservation International、PUBCHEM)，用Pipeline pilot软件从中剔除重复的分子和结构相似的分子，并根据计算的分子的ADMET性质挑选出吸收好、毒性低的化合物进行Cluster，得到100万个小分子的数据库。用Schrodinger中的Glide模块将这些小分子与NTR1进行SP精度的对接，选取结果中打分前10000的小分子，构成NTR1小分子数据库。
步骤2：药效团模型的构建及筛选
从BindingDB数据库中找出6个活性较好、结构差异较大的化合物(如下所示)作为训练集，利用Discovery Studio3.5中的Create Pharmacophore Automatically模块建立药效团模型，经测试集验证及FitValue值确定最合适的药效团模型，该模型包括2个疏水中心、1个芳香环中心、1个氢键供体。采用Search 3D Database对NTR1小分子数据库进行筛选。
步骤3：Fingerprint筛选
将BindingDB中得到的3个活性化合物(结构式如下)和11个非活性化合物分别采用Discovery Studio3.5中的Fingerprint模块，同NTR1小分子数据库进行2D-Fingerprint，将活性化合物Fingerprint结果中结构与非活性化合物Fingerprint结果中结构相似的化合物除去，再从中挑选结构未经发表和报道的化合物。
步骤4：聚类分析
将步骤2中药效团模型筛选和步骤3中Fingerprint筛选得到的结果用DiscoveryStudio3.5中的Design and Analyze Libraries下的Cluster模块进行聚类分析，共分成99类，从每一类中选出1个分子，得到99个化合物。购买这些化合物并对其进行细胞活性测试。
二、本发明的细胞活性测试方法包括以下2个步骤：
步骤1：NTR1稳转CHO细胞表达体系的构建
用含10％胎牛血清的RPMI1640培养基，37℃，5％CO2，饱和湿度的条件下培养CHO细胞。将含有PUC57-NTR1重组质粒(酶切位点为EcoR Ⅰ和HindⅢ)的DH5α甘油菌，转化，扩增，提取重组质粒PUC57-NTR1，双酶切PUC57-NTR1和表达载体pcDNA3.1(+)，回收目的基因NTR1和表达载体pcDNA3.1(+)，将NTR1和pcDNA3.1(+)连接，连接产物转化DH5α感受态细菌，用氨苄青霉素筛选出阳性克隆，扩增，提取质粒，命名为pcDNA3.1(+)-NTR1。用pcDNA3.1(+)-NTR1重组质粒转染CHO细胞，同时用只转染pcDNA3.1(+)表达载体的CHO细胞作阳性对照。用G418筛选稳定转染的CHO细胞进行克隆，传代扩大培养，得到单克隆化的人源系。
步骤2：化合物对NTR1稳转CHO细胞的活性测试
NT是NTR1的天然配体，NT与NTR1结合后，通过偶联的Gq蛋白激活的信号传递途径可以引起细胞内钙离子浓度的升高，而对NTR1具有抑制活性的小分子化合物则能够抑制细胞内钙离子浓度的升高，因此可以通过检测加入NT和小分子化合物后细胞内的钙离子浓度是否升高，来判断小分子化合物是否对NTR1具有抑制活性。SR48692是已报道的NTR1的选择性拮抗剂，可拮抗NT的活性。
采用钙6试剂盒，首先进行预实验。在96孔板中加入1nM的NT后，分别加入10μM和100μM待测化合物，测定每个化合物在10μM和100μM浓度下的钙流值，选取在10μM有抑制活性的化合物，初步推测其大概的IC50值。然后以此IC50值为中心，按1:2的比例等比拉8个浓度梯度，测定不同浓度下化合物的钙流值，并分别以NT和SR48692作为阴性和阳性对照，得到钙离子荧光值-化合物浓度曲线。根据该曲线采用GraphPad Prism version5.0软件计算小分子化合物的IC50值。
用上述虚拟筛选和细胞测活方法，筛选并得到了对NTR1具有抑制活性的小分子化合物(化合物1)：1-(4-氯苯氧基)-3-{2-亚胺-3-[2-(4-吗啉)乙基]-2,3-二氢-1-苯并咪唑基}-2-丙醇，其IC50值为44.49μM(图3)。
实施例2、基于小分子与受体NTR1的作用方式进行结构修饰。
首先在Discovery Studio3.5中打开NTR1蛋白的三维晶体结构，该结构来自PDB(Protein Data Bank)，PDB号为4GRV。对其进行加氢，去水，删去插入的T4溶菌酶结构，采用Prepare Protein模块对蛋白进行准备，并用Define Site根据配体中的NT找到活性位点。采用Prepare Ligands模块对小分子进行准备。准备完毕，采用Dock Ligands模块的Libdock方法进行对接，对接结果如图1所示。根据该对接结果，化合物1与文献报道的受体的关键氨基酸Arg327、Tyr146形成氢键，且氢键的键长分别为(图1)，该氢键作用对于二者的结合非常重要。将氯取代苯环上的氯替换为异丙基(化合物2)，这两处关键的氢键键长分别缩短为(图2)。键长越短，氢键作用越强，配体与受体的结合越紧密，则配体的活性可能更好。采用实施例1中的细胞活性测试方法对修饰后的化合物2进行测活，并计算其IC50值。测得化合物2的IC50值为39.62μM。
这里以此修饰为例，在于说明具体的实施方法，但并不能作为限制。
Claims (2)
1.一种具有如式Ⅰ所示结构的胍类NTR1小分子拮抗剂在制备与NTR1蛋白相关疾病的药物中的应用，
所述化合物中，R1是吗啉环，R2是对位氯取代苯环，R3是氢，n为2，其结构如下，
所述的应用具体为用于制备治疗或辅助治疗帕金森疾病或精神分裂症疾病的药物。
2.如权利要求1所述的应用，其特征在于所述的胍类NTR1小分子拮抗剂对NTR1靶点具有拮抗作用。
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