CN105440097A - 雷公藤三萜酸C用于制备趋化因子受体2b受体拮抗剂的制药用途 - Google Patents

雷公藤三萜酸C用于制备趋化因子受体2b受体拮抗剂的制药用途 Download PDF

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CN105440097A
CN105440097A CN201510828237.3A CN201510828237A CN105440097A CN 105440097 A CN105440097 A CN 105440097A CN 201510828237 A CN201510828237 A CN 201510828237A CN 105440097 A CN105440097 A CN 105440097A
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ccr2b
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严明
汪豪
高鹏
张陆勇
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China Pharmaceutical University
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Abstract

本发明涉及式(I)的趋化因子受体2b拮抗剂或其药学上可接受的盐。此类化合物可以拮抗趋化因子受体2b，有效地产生抗炎作用。

Description

雷公藤三萜酸C用于制备趋化因子受体2b受体拮抗剂的制药用途
技术领域
本发明涉及雷公藤三萜酸C对趋化因子受体2b拮抗剂的抗炎用途。
背景技术
趋化因子(chemokines)是指具有趋化作用的细胞因子，属于小分子的分泌蛋白超家族(约8-10KDa，含70-90个氨基酸)。它在很多种疾病的病理生理过程中起着重要作用，如多发性硬化、类风湿性关节炎、器官移植排斥、心脑血管疾病、肿瘤以及HIV等。趋化因子通过与受体结合，在某种程度上促进了各种炎症性疾病以及各种自身免疫性疾病的发生和发展。因而通过抑制趋化因子与其受体结合可在一定程度上抑制相关疾病的发生。趋化因子受体拮抗剂的研究已经成为当前新药研究的热点课题之一。
趋化因子受体2b(CCR2b)是单核细胞趋化蛋白-1(MonocyteChemoattractantProtein-1，MCP-1)的受体，MCP-1是重要的趋化类炎症因子，在炎症、风湿性关节炎、多发性硬化、动脉粥样硬化、肺纤维化等多种疾病的发生和发展过程中起着关键作用。因此筛选趋化因子受体2b拮抗剂在一定程度上对研究以上疾病的发病机制及治疗有一定的意义。
发明内容
为了开发趋化因子受体2b拮抗剂从而为炎症的治疗寻找新的候选药物，本发明在采用趋化因子受体2b拮抗剂高通量筛选模型，通过功能性验证寻找先导化合物，发现了一类雷公藤三萜酸C类趋化因子受体2b拮抗剂，为新型的抗炎药开发提供先导化合物。本发明可为此类临床炎症治疗药物的开发提供先导化合物，为新型抗炎药物开发提供线索和实验依据。
本发明的技术方案为：在中国仓鼠卵巢癌细胞(CHO)内稳定转染趋化因子受体2b，建立趋化因子受体2b拮抗剂靶向激动剂高通量筛选模型，初筛，复筛，得到一类具有抗炎作用的候选药物。具体步骤如下：
步骤一：建立及培养稳定转染趋化因子受体2b的CHO细胞株。
步骤二：激动剂模型建立及阳性药验证，确定激动剂的EC80。
步骤三：拮抗剂模型建立及阳性性药验证。
步骤四：采用稳转细胞株和实验确定的最佳检测条件对化合物进行趋化因子受体2b拮抗剂的高通量筛选，得到有明显量效关系的化合物。
附图说明：
图1：激动剂对照量效关系
图2：拮抗剂对照量效曲线
图3：先导化合物量效曲线
具体实施方式
以下结合附图说明本发明的具体实施方式：
1.实验材料：
(1)完全培养：F12；10％FBS。
(2)选择培养：400μg/mLG418。
(3)StimulationBuffer(SB)：试剂盒自带。
(4)主要仪器：CO2培养箱；384孔板；ParadigmTMDetectionPlatform。
2.建立及培养稳定转染趋化因子受体2b的CHOCCR2B细胞株。
3.激动剂模型建立及阳性药验证，确定激动剂的EC80：
(1)配制MCP-1梯度稀释液：确定最佳细胞数时需要用MCP-1对细胞进行刺激，所以应首先将11494nM的MCP-1母液(使用含不低于0.1％BSA的PBS配制)用SB逐级稀释为工作浓度的2倍。
(2)配制不同浓度的细胞稀释液：用SB将细胞分别稀释为40000cells/well、30000cells/well及20000cells/well。
(3)加样：将3.(1)中稀释好的MCP-1溶液按每个浓度2个复孔，5μL每孔加入384孔板中。接着向不同浓度的MCP-1溶液中加入3.(2)配制好的不同浓度的细胞稀释液各5μL。将384孔板在500rpm下离心15s，使10μL试剂混合均匀以充分反应。为防止试剂蒸发造成损失，将TopSeal-A膜贴于板面并将板放于室温下使之孵育60min。
(4)终止试剂：用Lysisbuffer(LB)将IP1-d2及anti-IP1稀释33倍，按1∶1混匀后加入各孔，每孔10μL。将384孔板在500rpm下离心15s，使20μL试剂混合均匀以充分反应。重新贴上TopSeal-A膜并将板放于室温下继续孵育60min。
(5)检测：孵育时间到达后，揭去TopSeal-A膜，将板放于设定好程序的ParadigmTMDetectionPlatform上检测。处理数据，确定最佳细胞数及MCP-1的EC80。
4.拮抗剂验证
(1)CCR2b拮抗剂BMSCCR222使用DMSO配制成0.5mM储备液，用SB逐级稀释为最终浓度的2倍。
(2)配制含最佳细胞数的溶液：用SB将细胞稀释为3.(5)中得到的最佳细胞浓度。
(3)加样：将4.(1)中稀释好的BMSCCR222溶液按每个浓度2个复孔，2.5μL每孔加入384孔板中。将4.(2)配制的细胞溶液按每孔5μL加入384孔板中。将384孔板在500rpm下离心15s，使7.5μL试剂混合均匀以充分反应。为防止试剂蒸发造成损失，将TopSeal-A膜贴于板面并将板放于室温下使之孵育60min。
(4)将MCP-1母液稀释成3.(5)中测定的EC80，每孔2.5μl加入对应的含BMSCCR222的384孔板中。将384孔板在500rpm下离心1min，使10μL试剂混合均匀以充分反应。为防止试剂蒸发造成损失，将TopSeal-A膜贴于板面并将板放于37℃/5％CO2下使之孵育60min。
(5)按3.(4)配制终止试剂。向每孔加入10μLIP1-d2/anti-IP1溶液(1∶1)。将384孔板在500rpm下离心15s，使20μL试剂混合均匀以充分反应。重新贴上TopSeal-A膜并将板放于室温下继续孵育60min。
(6)孵育时间到达后，揭去TopSeal-A膜，将板放于设定好程序的ParadigmTMDetectionPlatform上检测。处理数据，确定拮抗剂的IC50。
5.采用摸索的最佳检测条件在稳转细胞中对趋化因子受体2b拮抗剂进行初筛与复筛。处理数据，计算所筛选化合物IC50。
趋化因子受体2b受体活性筛选实验结果
筛选得到的趋化因子受体2b拮抗剂为雷公藤三萜酸C类化合物，其结构式与IC50如下：
先导化合物对趋化因子受体2b拮抗活性：

Claims (2)

1.式(I)的化合物或其药学上可以接受的盐在趋化因子受体2b拮抗剂的用途；
2.按照权利要求1的用途，作为趋化因子受体2b拮抗剂，用于治疗炎症的用途。
CN201510828237.3A 2015-11-20 2015-11-20 雷公藤三萜酸C用于制备趋化因子受体2b受体拮抗剂的制药用途 Pending CN105440097A (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112516150A (zh) * 2019-09-19 2021-03-19 上海海洋大学 雷公藤三萜酸在制备防治白斑综合征病毒药物的应用

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US20110218143A1 (en) * 2007-11-20 2011-09-08 University Of Florida Research Foundation Compositions and methods for tissue repair

Patent Citations (1)

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US20110218143A1 (en) * 2007-11-20 2011-09-08 University Of Florida Research Foundation Compositions and methods for tissue repair

Non-Patent Citations (3)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112516150A (zh) * 2019-09-19 2021-03-19 上海海洋大学 雷公藤三萜酸在制备防治白斑综合征病毒药物的应用
CN112516150B (zh) * 2019-09-19 2022-06-14 上海海洋大学 雷公藤三萜酸在制备防治白斑综合征病毒药物的应用

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