CN117942344A - 作为组胺3型受体拮抗剂的化合物及其应用 - Google Patents
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Abstract
本发明公开了作为组胺3型受体拮抗剂的化合物及其应用，涉及生物化学技术领域。本发明基于分子对接的虚拟筛选方法以及生物学活性测定发现3个化合物对组胺3型受体具有明显的拮抗活性，可将其作为组胺3型受体拮抗剂应用到与组胺3型受体有关的疾病治疗当中，为目前治疗睡眠障碍的药物研究提供新的选择。
Description
技术领域
本发明涉及生物化学技术领域，具体涉及作为组胺3型受体拮抗剂的化合物及其应用。
背景技术
组胺(Histamine)是一种内源性生物胺，通过作用于G蛋白偶联受体(G protein-coupled receptor，GPCR)家族中的四种不同的组胺受体(H1R、H2R、H3R、H4R)介导多种生理和病理过程。组胺H1受体(H1R)和组胺H2受体(H2R)分别与过敏反应和胃酸分泌相关，而组胺H4受体(H4R)主要参与机体的免疫反应。组胺H3受体(H3R)主要存在于脑部的组胺能神经元上，与睡眠、觉醒、学习和记忆、食欲及脑缺血生理过程相关，因此是治疗睡眠性疾病、阿尔兹海默疾病、精神分裂症、心肌缺血以及肥胖的潜在重要药物靶标。
突触后组胺与H3R的结合不仅限制了组胺的通量，也限制了多种其它重要神经递质的通量，例如乙酰胆碱、多巴胺、谷氨酸、去甲肾上腺素、血清素、γ-氨基丁酸(GABA)等(例如，TA Esbenshade等人，Mol.Intervention，v.6，pp.77-882006.)。相比之下，H3R拮抗剂(也称为反向激动剂)可增加这些神经递质通量，用于治疗不同类型的中枢神经系统疾病(例如，认知、精神、神经运动、疼痛等)。目前H3R拮抗剂中仅Pitolisant被批准用于治疗嗜睡，而多数拮抗剂因心脏毒性、磷脂质病等副作用终止于临床实验。所以需要筛选和设计新的靶向H3R药物。
目前已有用于调节拮抗H3R的化合物，比如，公开号为CN112004802A以及CN114042070A的专利申请均公开了调节拮抗H3R的化合物和药物组合物、其制备以及相应的药物组合物。该发明的化合物和/或药物组合物可潜在地用于制备用于预防、治疗、改善患者中与H3R相关的某些障碍或疾病(包括中枢神经系统紊乱或发作性睡眠病)的药物。
目前，还需要筛选更多的化合物可以应用到组胺3型受体拮抗剂、治疗睡眠障碍药物的制备当中，为治疗睡眠障碍的药物研究提供更多的选择。
发明内容
本发明的目的是提供具有组胺3型受体拮抗活性的化合物，将其应用到组胺3型受体拮抗剂、治疗睡眠障碍药物的制备当中。
本发明通过如下技术方案实现上述目的：
采用计算机辅助药物分子设计的手段来发现靶向组胺3型受体的先导化合物。首先利用分子对接的方式对涵盖多种小分子的化合物数据库进行虚拟筛选，得到得分靠前的100个化合物，之后通过Glosenser实验验证化合物对H3R的拮抗效果，得到拮抗效果最好的2个骨架不同的分子，再通过相似性搜索的方式，从更大的化合物数据库中寻找相似分子约600个，进行新一轮的虚拟筛选和Glosenser实验，最终得到一系列相似的活性化合物，本发明对上述筛选到的化合物进行进一步生物学活性测试，发现上述化合物中有3个化合物对组胺3型受体具有明显的拮抗活性，因此本发明提供了上述任一化合物(或其可药用盐)在制备组胺3型受体拮抗剂中的应用。
具有组胺3型受体拮抗活性的化合物在制备组胺3型受体拮抗剂中的应用，所述化合物的化学式为以下任意一种：
(1)
(2)
(3)
本发明还提供了具有组胺3型受体拮抗活性的化合物在制备治疗睡眠障碍、脑缺血性疾病或神经性疾病药物中的应用，所述化合物的化学式为以下任意一种：
(1)
(2)
(3)
在组胺3型受体拮抗剂的应用中，所述化合物的浓度为10-8～10-6M。
优选的，所述睡眠障碍为嗜睡症或发作性睡病。
优选的，所述药物用于拮抗组胺3型受体治疗患者中中枢神经系统紊乱。
本发明还提供了一种组胺3型受体拮抗剂，包括以下任意一种化学式的化合物：
(1)
(2)
(3)
本发明还提供了一种用于治疗睡眠障碍、脑缺血性疾病或神经性疾病的药物组合物，包含：治疗有效量的(1)～(3)中任意一种化学式的化合物或其药学上可接受的盐；和药学上可接受的赋形剂、载体、佐剂和溶剂中的至少一种；
(1)
(2)
(3)
优选的，所述睡眠障碍为嗜睡症或发作性睡病。
本发明基于分子对接的虚拟筛选方法以及生物学活性测定发现3个化合物对组胺3型受体具有明显的拮抗活性，可将其作为组胺3型受体拮抗剂应用到与组胺3型受体有关的疾病治疗当中，为目前治疗睡眠障碍的药物研究提供新的选择。
附图说明
图1为本发明的3个化合物在10-4M浓度下对H3R的拮抗能力/反向激动能力实验结果。
图2为3个化合物在一系列浓度梯度下对H3R下游G蛋白信号通路的拮抗能力/反向激动能力实验结果。
图3为3个化合物在一系列浓度梯度下对H3R下游β-arrestin信号通路的拮抗能力实验结果。
图4为pcDNA3.1-H3R的图谱。
图5为H3R445-V2R的图谱。
具体实施方式
实验例1
(1)基于分子对接的虚拟筛选
实验原理：利用分子对接方法，对化合物数据库中的化合物与H3R之间的相互作用进行预测、分析和评估，从而确定能够和H3R结合的拮抗分子。
实验方法：首先，将H3R(GenBank号：NM_007232)、Gi蛋白以及单链抗体ScFv16(氨基酸序列：QVQLQESGGGLVQPGGSLRLSCAASGFTFSNYKMNWVRQAPGKGLEWVSDISQSGASISYTGSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARCPAPFTRDCFDVTSTTYAYRGQGTQVTVSS)的基因克隆至SF9昆虫细胞(Spodoptera frugiperda 9)中以进行表达。接着，在组胺分子的激活下，利用DDM去垢剂溶解细胞膜，并采用MBP亲和层析、分子排阻层析色谱进行纯化，以获得均一稳定的H3R-Gi蛋白复合物样品。随后，将纯化后的目标蛋白滴加到不同类型的Quantifoil电镜载网上，并利用Vitrobot液体悬浮冷冻制样机制备冷冻样品。此后，利用300kV电镜收集冷冻电镜数据，并利用RELION、cryoSPARC、Coot和Phenix等软件在GPU工作站上进行单颗粒三维重构及模型搭建。最终，成功得到了组胺-H3R-Gi蛋白复合物的电镜结构。
基于组胺-H3R-Gi蛋白复合物的电镜结构，采用Virtualflow工作流中的vina软件进行了基于分子对接的虚拟筛选。虚拟筛选所采用的化合物库是Chemdiv三维结构数据库。选取虚拟筛选打分最佳的4305个化合物，采用聚类方式剔除相似分子。
实验结果：分子对接能够较为准确地确定能和H3R能形成较强相互作用的有机小分子。基于分子对接的预测结果，从商业化合物库中购买了100多个化合物，并进行了后续细胞水平的活性实验Glosenser，从中发现了一批具有明显H3R拮抗活性的小分子化合物，具体见表1。
表1
根据化合物对H3R的拮抗效果，得到拮抗效果最好的2个分子(Chemdiv ID：E146-1043、F668-0335)。
(2)基于相似性搜索的二次分子对接
实验原理：利用Morgen分子指纹来判断分子间相似性，重新搜索化合物库中与已筛选到的高活性分子相似的有机小分子，从而寻找到分子对接遗漏的潜在拮抗分子。
实验方法：根据Morgen分子指纹，对前一步筛选到的2个拮抗分子，在Chemdiv三维结构数据库中进行相似性搜索，选取相似性打分排名靠前的600个化合物，再次与H3R进行分子对接。对此次虚拟筛选打分最佳的100个化合物，采用目视检查的方式剔除结合不佳的分子。
实验结果：Morgen分子指纹基于分子间连接性判断分子相似性，可以较为准确的搜索到相似分子，基于新一轮相似分子的分子对接预测结果，从商业化合物库中购买了50多个化合物，并进行了细胞水平的活性实验Nanobit，从中发现了一批具有明显H3R拮抗活性的小分子化合物，具体见表2。
表2
实施例2H3R拮抗能力评价实验
(1)cAMP-Glosensor实验
实验原理：H3R作为G蛋白耦合膜受体，可以激活Gi蛋白，抑制腺苷酸环化酶活性，进而降低胞内cAMP水平。Glosensor实验使用基因编码的生物传感器，它由cAMP结合域和Photinus荧光素酶两部分组成。该生物传感器与cAMP结合后，构象发生变化，催化荧光底物发出荧光。在与底物预平衡后，瞬时或稳定表达生物传感器的细胞可用于活细胞测定GPCR功能。
实验步骤：利用LIPO转染试剂，分别将含pGloSensorTM-22F cAMP质粒(Promega，E2301)和H3R基因片段(GenBank号：NM_007232)的质粒(pcDNA3.1-H3R，图谱如图4所示)以1∶3的比例转染进HEK293T细胞中，约24h后将其种板至由粘附因子包被过的白底不透明96孔板中。约12h后，将细胞用PBS饥饿20min，而后加入含20μM cAMP底物和10μM Forskolin的平衡溶液(CO2培养基中加入10％FBS)35μL每孔，在37℃培养箱中孵育30min后，加入35μL每孔梯度浓度稀释的化合物(10-4M，10-5M，10-6M，10-7M，10-8M，10-9M，10-10M，0M)，在常温孵育20min，到达平台期，用多功能酶标仪测luminescence。即可分辨不同化合物拮抗H3R的能力强弱。
实验结果：如图1所示，E772-0183、L084-0675、L084-0792是筛选出的具有较强H3R抑制能力的化合物；如图2所示，3种化合物对cAMP的累积产生明显上调作用，表明其对Gi蛋白的功能产生抑制，且呈现剂量依赖关系，这说明所列举的化合物都是活性良好潜在H3R拮抗剂。
(2)TANGO实验
实验原理：除G蛋白外，H3R还可招募β-arrestin产生下游信号。构建H3R质粒，并在其C端插入V2-tail、TEV和四环素控制的激活因子(tetracycline-controlledtransactivator，tTA)；改造HEK293T细胞，敲降其野生型β-arrestin，稳转β-arrestin2和TEV蛋白酶融合蛋白以及tTA调控的荧光酶(称为HTLA细胞)。受体招募β-arrestin2后，TEV蛋白酶识别到TEV并水解对应位点，使得tTA释放入核，促进特定荧光酶释放，催化荧光底物发出荧光。该方法可定量测定H3R剪切体与β-arrestin的相互作用。
实验步骤：利用LIPO转染试剂，将受体质粒(Addgene，66402；图5)转染进HTLA细胞(由中国科学院分子细胞科学卓越创新中心汪胜实验室赠予)中。约24h后将其种板至由粘附因子包被过的白底不透明96孔板中。约4h后，加入梯度配体，buffer为含20mM HEPES(pH＝7.4)的HBSS。12-14h后，用PBS润洗一次，加入稀释10倍的Bright-Glo试剂，常温孵育10min后，用多功能酶标仪测luminescence。
实验结果如图3所示，E772-0183、L084-0675、L084-0792等化合物对组胺介导的β-arrestin2招募产生明显拮抗作用，且呈现剂量依赖关系，这说明所列举的化合物都是活性良好的潜在H3R拮抗剂。
Claims (8)
1.具有组胺3型受体拮抗活性的化合物在制备组胺3型受体拮抗剂中的应用，所述化合物的化学式为以下任意一种：
(1)
(2)
(3)
2.具有组胺3型受体拮抗活性的化合物在制备治疗睡眠障碍、脑缺血性疾病或神经性疾病药物中的应用，所述化合物的化学式为以下任意一种：
(1)
(2)
(3)
3.根据权利要求1所述的应用，其特征在于，所述化合物的浓度为10-8～10-6M。
4.根据权利要求2所述的应用，其特征在于，所述睡眠障碍为嗜睡症或发作性睡病。
5.根据权利要求2所述的应用，其特征在于，所述药物用于拮抗组胺3型受体治疗患者中中枢神经系统紊乱。
6.一种组胺3型受体拮抗剂，其特征在于，包括以下任意一种化学式的化合物：
(1)
(2)
(3)
7.一种用于治疗睡眠障碍、脑缺血性疾病或神经性疾病的药物组合物，包含：治疗有效量的(1)～(3)中任意一种化学式的化合物或其药学上可接受的盐；和药学上可接受的赋形剂、载体、佐剂和溶剂中的至少一种；
(1)
(2)
(3)
8.根据权利要求7所述的药物组合物，其特征在于，所述睡眠障碍为嗜睡症或发作性睡病。
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