Conantokin-G类似物的分子设计及抗吗啡成瘾作用研究
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摘要
1.目的
药物依赖不仅是医学问题、生物学问题,也是社会问题。药物依赖是一种慢性复发性脑病,造成依赖的原因包括正性强化因素(欣快感、奖赏效应)、负性强化因素(逃避现实、减轻戒断症状)及条件性强化因素。阿片依赖的本质是代偿性适应,涉及多个脑区、多种神经递质及其受体的改变,包括阿片系统本身、去甲肾上腺素系统、兴奋性氨基酸系统等。N-甲基-D-天冬氨酸受体(NMDAR)是兴奋性谷氨酸受体的药理学亚型之一,由不同比例的NR2和NR3与NR1亚基组成,对ca~(2+)具有高通透性。研究表明NMDAR在突触信号转导、神经可塑性、学习记忆等方面发挥着重要的生理作用,同时许多急慢性神经系统的病理改变与NMDAR功能障碍密切相关。
鉴于NMDAR参与中枢神经系统成瘾药物依赖的奖赏效应,作为中枢神经系统NMDAR特异性拮抗剂的芋螺毒素(CTX)Conantokins家族,有可能应用于治疗药物成瘾。本实验室前期已经证实Conantokin-G(Con-G)能有效干预吗啡诱导的CPP的表达和重建,具有防治吗啡依赖和复吸的潜力。
由于Con-G存在给药不方便、吸收率低和成本高等缺点,本研究旨在利用计算机辅助设计方法设计Con-G类似物,优化多肽结构,并用视频跟踪.计算机自动分析条件性位置偏爱(Conditioned Place Preference,CPP)实验系统对其抗成瘾效果进行评估,期望发现有效的Con-G类似物。
2.材料和方法
2.1实验动物
清洁级成年雄性昆明种小鼠,体重18～22g。
2.2主要药物及试剂
Conantokin G(纯度＞75%) Sigma公司,美国
[Glu~(3,4,7,10,14)]-Conantokin G(纯度＞90%) Sigma公司,美国
[Glu~(3,4,7,10,14)]-Conantokin G[1-13](纯度＞90%)赛百盛公司,中国
盐酸吗啡注射液(批号:041101)沈阳第一制药厂
2.3主要实验装置及软件
计算机模拟软件
InsightⅡ软件ZDCOK模块
Modeller 8v2
NACCESS
视频跟踪-计算机自动分析CPP实验系统
SPSS统计软件
2.4实验方法
2.4.1同源模建
根据序列通过BLAST(PSI-BLAST)搜寻NR2B的同源蛋白,选择同源性较高的参考蛋白2A5S、1Y20、1S50和2F34,然后进行序列比对。根据同源性大小确定参考蛋白2A5S,将比对序列和参数输入计算机,利用Modeller 8v2进行NR2B亚基的同源模建。Modeller 8v2程序可以模建多个目的蛋白结构,根据空间结构和能量最小原则选出较优蛋白进行下一步优化。最后对模建结果进行合理性检测。
2.4.2分子对接
利用计算机模拟技术研究Con-G与NR2B亚基的相互作用情况,研究分子之间相互作用采用ZDOCK程序。蛋白对接采用镶嵌在InsightⅡpackage version 2005(Accelrys~(TM) Inc.)中的ZDOCK模块。选取与活性数据相对符合的构象运用Charmm19力场进行能量优化。通过NACCESS软件计算NR2B受体与Con-G作用前后蛋白质表面溶剂可及性(ASA)的变化。△ASA~i=ASA~i_(NR2B)-ASA~i_(NR2B-Con-G)
2.4.3分子设计
根据分子对接结果得到复合物结合模型,通过观察受体NR2B亚基与配体Con-G相互作用的几何模型与结构特征,计算它们之间重要氨基酸相互作用形成的氢键位点和长短,设计并构建Con-G类似物,得到Glu-Con-G[1-13]。
2.4.4 Glu-Con-G[1-13]、Con-G和Glu-Con-G对CPP表达的影响:
成年雄性昆明小鼠d1、d3、d5、d7腹腔注射(i.p.)吗啡(5mg/kg)后放入白盒内自由活动50 min,d2、d4、d6、d8给予生理盐水后放置黑盒训练50 min。对照组小鼠均给予生理盐水,其它处理同吗啡组小鼠。Glu-Con-G[1-13]、Con-G和Glu-Con-G干预CPP表达组在实验的d9于CPP测试前半小时,侧脑室注射(i.c.v.)不同剂量的Glu-Con-G(0、30、60、120pmol),给药容量为2μl/只,观察其对吗啡诱导CPP表达的干预作用。
2.5实验观察指标及统计分析
摄录的视频文件用Rat/Mice Tracking软件分析位置偏爱指标:白盒停留时间;运动活性指标:总运动距离、穿梭次数。用SPSS统计软件对以上数据进行统计分析,均用均数士标准误表示,P＜0.05视为有统计学差异。
3.结果
3.1同源模建
利用UCLA-DOE服务器上程序对模建结果进行合理性检测,测得Verify,3D分数为136.58,平均每个氨基酸得分0.34,模板蛋白2A5S总分为114.98,平均得分为0.41。模建蛋白Errat plots得分为79.3%,模板得分为94.7%。Ramachandran图中显示79%的氨基酸落在最适合区域,95.7%的氨基酸落在适合的区域中。
3.2分子对接
Con-G17个氨基酸与NR2B亚基对接中发现7组氢键作用。
通过NACCESS软件计算得到复合物与NR2B亚基之间△ASA,Glu420,Ser421,Asp423,Lys458和Asp715的△ASA有了显著的变化,其中Lys458和Asp715的△AsA大于50。
3.3天然偏爱测试
天然偏爱结果显示:各组小鼠在黑盒停留时间(s)长于白盒停留时间(P＜0.05),表明小鼠天然偏爱黑盒;同时各组间比较小鼠的黑盒停留时间无明显差异(P＞0.05)。
3.4 Con-G及其类似物对吗啡诱导小鼠CPP表达的影响
吗啡诱导的CPP表达阶段各组小鼠在伴药盒(白盒)的停留时间同吗啡对照组(M-G0)相比,Con-G(30、60、120 pmol)干预组小鼠的白盒停留时间呈剂量.效应依赖性减少(30 pmol,P＜0.01;60 pmol,P＜0.01;120 pmol;P＜0.01)。Glu-Con-G(30、60、120 pmol)干预组小鼠的白盒停留时间呈剂量-效应依赖性减少(30 pmol,P＜0.01;60 pmol,P＜0.01;120 pmol;P＜0.01)。Glu-Con-G[1-13](30、60、120 pmol)干预组小鼠的白盒停留时间呈剂量-效应依赖性减少(30 pmol,P＜0.01;60 pmol,P＜0.01;120 pmol;P＜0.01)。Con-G及其类似物最小剂量均为30 pmol,药效相当。
4.结论
4.1根据NR2B亚基404-802序列构建了谷氨酸结合位点三维结构。
4.2 Con-G的E2、Gla4、L5、Q9、I12和Q13,NR2B的E420、S421、D423、K458和D715是参与相互作用的重要氨基酸,它们之间形成了七对氢键。
4.3所设计多肽Glu-Con-G[1-13]符合对接模型。
4.4 Glu-Con-G[1-13]与Con-G以及Glu-Con-G剂量相关性抑制吗啡诱导的小鼠条件性位置偏爱表达。
4.5 Glu-Con-G[1-13]在分子大小、合成成本上有改善,可以作为候选药物进行深入研究。
1 Objective
Drug dependence is not only a medical problem,biological problem,but also social issue,and it is a chronic relapsing encephalopathy.Causes of dependency include positive reinforcement factors(euphoria,reward effect),negative reinforcement factors(escape from reality,to reduce withdrawal symptoms) and conditions of the reinforcement factors.Opioid dependence is the essence of compensatory adaptation,involving multiple brain regions,a variety of changes in neurotransmitters and their receptors(including the opioid system itself) norepinephrine system,excitatory amino acid systems(such as N-methyl-D-aspartate (NMDA) receptors),etc.NMDAR is the excitatory glutamate receptor subtype,which consists of a different proportion of NR2,NR3 and NR1 subunits,and it has a high-pass permeability to Ca~(2+).Studies have shown that NMDAR in synaptic signal transduction,neural plasticity,learning and memory,etc.play an important physiological role,while many acute and chronic pathological changes of the nervous system are closely related to NMDA receptor dysfunction.
In view of the central nervous system,NMDA receptors are involved in reward-dependent effects of addictive drugs,as the central nervous system,NMDA receptor-specific antagonist Conantokins family may be applied to treat drug addiction. Our experiments confirmed that Con-G can effectively interfere with the expression of morphine-induced CPP and reconstruction,which may have prevention and treatment of morphine dependence and relapse potential.
Since there are some disadvantages on Con-G,such as the inconvenient administration,low absorption rate and the high cost disadvantages.Therefore,this study is designed using computer-aided to design Con-G analogues,so as to overcome some shortcomings of the original peptide and used using the computer based video tracking CPP system to analyze its anti-assess the effects of drug addiction,which is used to strive for effective screening of the Con-G analogs.
2 Materials and methods
2.1 Animals:
Adult male Kun-Ming mice(18-22g) were supplied by Shanghai Centre of Experimental Animals,Chinese Academy of Sciences.
2.2 Chemical reagents:
Conantokin G(purity＞90%)(Sigma)
[Glu~(3,4,7,10,14)]-Conantokin G(purity＞90%)(Sigma)
[Glu~(3,4,7,10,14)]-Conantokin G[1-13](purity＞90%)(Sbsgene)
Morphine hydrochloride(First Pharmaceutical Company of Shenyang,China);
2.3 Apparatus:
Computer simulation software
ZDOCK in InsightⅡsoftware
Modeller 8v2
NACCESS
Computer based video tracking CPP system
SPSS software
2.4 Methods
2.4.1 Homology modeling
The protein sequence of human NR2B subunit(404-802) was obtained from the National Center for Biotechnology Information(NCBI) server.Homologous proteins with known crystal structures were found by performing a position- specific iterated BLAST(PSI-BLAST) search.Using Modeller 8v2,protein 3D structures were generated by satisfying spatial restraints imposed by the sequence alignment with the template structure.To optimize the local interactions,several models obtained were subjected to a short simulated annealing refinement protocol using Charmml9 force-field.Stereochemical quality of the polypeptide backbone and side chains was evaluated using Ramachandran plots obtained from the RAMPAGE server.Amino acid environment was evaluated using Verify 3D and Errat plots.
2.4.2 Ligand docking
Protein Docking was performed with the ZDOCK software bundled with the InsightⅡpackage version 2005(Accelrys~(TM) Inc.).Con-G was first docked manually by ZDOCK,then computationally by RDOCK for the top 50 ranks.The resulting NR2B/ligand complex was energy-minimized in the absence of solvent using Charmm19 force-field.The accessible surface area(ASA) of NR2B subunit (uncomplexed) and its docked complex with Con-G were calculated using the program NACCESS.Composite coordinates of ligand and NR2B subunit were generated to form the docked complex.The change in accessible surface area for residue was calculated using:
△ASA~i=ASA~i_(NR2B)-ASA~i_(NR2B)-Con-G.
2.4.3 Molecular Design
Based on molecular docking complex combination of model,the geometric model and structural characteristics between NR2B subunit Con-G analogues were designed according to model and structure activity relationship
2.4.4 Effect of Glu-Con-G[1-13],Con-G and Glu-Con-G on the expression of CPP induced by morphine:
Male Kun-Ming mice were administered morphine(5mg/kg,i.p.) in white chamber for 50 mins on day1,3,5 and 7.On day2,4,6,8 mice were given saline in black chamber for 50 mins.The control group was treated with a daily saline injection for eight consecutive days both in the morphine and saline paired chambers.To test the effects of Con-G and its analogues Glu-Con-G,Glu-Con-G[1-13]on the expression of morphine-induced CPP,different dose of Con-G and its analogues Glu-Con-G, Glu-Con-G[1-13](0,30,60,120pmol) were given by intracranial administration 30 mins before the test on day 9.Total volume was 2μl.
2.5 Data analysis and statistics
Place Preference:the time spent in white chamber(TW);Locomotor Activity:the total distance that mice moved in different chambers(TD) and shuttle time(ST);All the data were expressed as the mean±S.E.M,and analyzed with SPSS software.P＜0.05 was considered statistically significant.
3 Results:
3.1 Homology Modeling
In the Ramachandran plot,a total of 79.0%of the residues were in the most favoured region,a total of 95.7%of the residues were in the allowed region.Verify 3D score and Errat plots score were 136.58 and 79.3%,and its reference protein score are 114.98 and 94.7%.
3.2 Ligand Docking
When Con-G structure was docked into the glutamate binding site of NR2B subunit by energy minimization,Con-G structure was nicely fitted into the agonist binding cleft on the NR2B subunit.Several H-bonds were found between NR2B subunit and Con-G.Connolly surfaces of the NR2B subunit and of Con-G were calculated.The decreases of accessible surface area(△ASA) of the docking complex were observed.Considering NR2B subunit,significant change of accessible surface area of Glu420,Ser421,Asp423,Lys458 and Asp715 occurred,Lys458 and Asp715 lost more than 50A ASA when going from the uncomplexed to the complexed state.
3.3 Test of preference in preconditioning phase
Pre-conditioning test showed that all groups mice spent more time in black chamber than in white chamber(P＜0.05),which suggested that mice prefer the black chamber;Meanwhile,mice of the different groups did not show any significant difference(P＞0.05) in the time spent in black chamber.
3.4 Effect of Con-G and its analogues on the expression of morphine induced CPP
After conditioning with morphine for 8 days,on the CPP expression day(d9), compared with the mice in morphine control group(M-G0),A pretreatment with Con-G and its analogue(30,60,120pmol) 30 min before the test,Con-G displayed a dose-effect dependent decrease the expression of morphine induced CPP(30pmol, P＜0.01;60pmol,P＜0.01;120pmol;P＜0.01);Glu-Con-G(30,60,120pmol) displayed a dose-effect dependent decrease the expression of morphine induced CPP(30pmol, P＜0.01;60pmol,P＜0.01;120pmol;P＜0.01);Glu-Con-G[1-13](30,60,120pmol) also displayed a dose-effect dependent decrease the expression of morphine induced CPP (30pmol,P＜0.01;60pmol,P＜0.01;120pmol;P＜0.01).Con-G and its analogues had the same lowest efficient dose.
4 Conclusions
4.1 The glutamate binding site is built according to the sequence of NR2B 404-802.
4.2 Structurally and functionally important residues were identified,including E2, Gla4,L5,Q9,I12 and Q13 of Con-G,they interacted with E420,S421,D423,K458 and D715 of NR2B.Seven H-bonds were also found.
4.3 Glu-Con-G[1-13]is in line with docking model.
4.4 Glu-Con-G[1-13],Con-G and Glu-Con-G,displayed a dose-effect dependent decrease the expression of morphine induced CPP.
4.5 Glu-Con-G[1-13]has an improvement in the molecular size and the difficulty and cost of synthesis which can be used as the next phase of the study key.
药物依赖不仅是医学问题、生物学问题,也是社会问题。药物依赖是一种慢性复发性脑病,造成依赖的原因包括正性强化因素(欣快感、奖赏效应)、负性强化因素(逃避现实、减轻戒断症状)及条件性强化因素。阿片依赖的本质是代偿性适应,涉及多个脑区、多种神经递质及其受体的改变,包括阿片系统本身、去甲肾上腺素系统、兴奋性氨基酸系统等。N-甲基-D-天冬氨酸受体(NMDAR)是兴奋性谷氨酸受体的药理学亚型之一,由不同比例的NR2和NR3与NR1亚基组成,对ca~(2+)具有高通透性。研究表明NMDAR在突触信号转导、神经可塑性、学习记忆等方面发挥着重要的生理作用,同时许多急慢性神经系统的病理改变与NMDAR功能障碍密切相关。
鉴于NMDAR参与中枢神经系统成瘾药物依赖的奖赏效应,作为中枢神经系统NMDAR特异性拮抗剂的芋螺毒素(CTX)Conantokins家族,有可能应用于治疗药物成瘾。本实验室前期已经证实Conantokin-G(Con-G)能有效干预吗啡诱导的CPP的表达和重建,具有防治吗啡依赖和复吸的潜力。
由于Con-G存在给药不方便、吸收率低和成本高等缺点,本研究旨在利用计算机辅助设计方法设计Con-G类似物,优化多肽结构,并用视频跟踪.计算机自动分析条件性位置偏爱(Conditioned Place Preference,CPP)实验系统对其抗成瘾效果进行评估,期望发现有效的Con-G类似物。
2.材料和方法
2.1实验动物
清洁级成年雄性昆明种小鼠,体重18～22g。
2.2主要药物及试剂
Conantokin G(纯度＞75%) Sigma公司,美国
[Glu~(3,4,7,10,14)]-Conantokin G(纯度＞90%) Sigma公司,美国
[Glu~(3,4,7,10,14)]-Conantokin G[1-13](纯度＞90%)赛百盛公司,中国
盐酸吗啡注射液(批号:041101)沈阳第一制药厂
2.3主要实验装置及软件
计算机模拟软件
InsightⅡ软件ZDCOK模块
Modeller 8v2
NACCESS
视频跟踪-计算机自动分析CPP实验系统
SPSS统计软件
2.4实验方法
2.4.1同源模建
根据序列通过BLAST(PSI-BLAST)搜寻NR2B的同源蛋白,选择同源性较高的参考蛋白2A5S、1Y20、1S50和2F34,然后进行序列比对。根据同源性大小确定参考蛋白2A5S,将比对序列和参数输入计算机,利用Modeller 8v2进行NR2B亚基的同源模建。Modeller 8v2程序可以模建多个目的蛋白结构,根据空间结构和能量最小原则选出较优蛋白进行下一步优化。最后对模建结果进行合理性检测。
2.4.2分子对接
利用计算机模拟技术研究Con-G与NR2B亚基的相互作用情况,研究分子之间相互作用采用ZDOCK程序。蛋白对接采用镶嵌在InsightⅡpackage version 2005(Accelrys~(TM) Inc.)中的ZDOCK模块。选取与活性数据相对符合的构象运用Charmm19力场进行能量优化。通过NACCESS软件计算NR2B受体与Con-G作用前后蛋白质表面溶剂可及性(ASA)的变化。△ASA~i=ASA~i_(NR2B)-ASA~i_(NR2B-Con-G)
2.4.3分子设计
根据分子对接结果得到复合物结合模型,通过观察受体NR2B亚基与配体Con-G相互作用的几何模型与结构特征,计算它们之间重要氨基酸相互作用形成的氢键位点和长短,设计并构建Con-G类似物,得到Glu-Con-G[1-13]。
2.4.4 Glu-Con-G[1-13]、Con-G和Glu-Con-G对CPP表达的影响:
成年雄性昆明小鼠d1、d3、d5、d7腹腔注射(i.p.)吗啡(5mg/kg)后放入白盒内自由活动50 min,d2、d4、d6、d8给予生理盐水后放置黑盒训练50 min。对照组小鼠均给予生理盐水,其它处理同吗啡组小鼠。Glu-Con-G[1-13]、Con-G和Glu-Con-G干预CPP表达组在实验的d9于CPP测试前半小时,侧脑室注射(i.c.v.)不同剂量的Glu-Con-G(0、30、60、120pmol),给药容量为2μl/只,观察其对吗啡诱导CPP表达的干预作用。
2.5实验观察指标及统计分析
摄录的视频文件用Rat/Mice Tracking软件分析位置偏爱指标:白盒停留时间;运动活性指标:总运动距离、穿梭次数。用SPSS统计软件对以上数据进行统计分析,均用均数士标准误表示,P＜0.05视为有统计学差异。
3.结果
3.1同源模建
利用UCLA-DOE服务器上程序对模建结果进行合理性检测,测得Verify,3D分数为136.58,平均每个氨基酸得分0.34,模板蛋白2A5S总分为114.98,平均得分为0.41。模建蛋白Errat plots得分为79.3%,模板得分为94.7%。Ramachandran图中显示79%的氨基酸落在最适合区域,95.7%的氨基酸落在适合的区域中。
3.2分子对接
Con-G17个氨基酸与NR2B亚基对接中发现7组氢键作用。
通过NACCESS软件计算得到复合物与NR2B亚基之间△ASA,Glu420,Ser421,Asp423,Lys458和Asp715的△ASA有了显著的变化,其中Lys458和Asp715的△AsA大于50。
3.3天然偏爱测试
天然偏爱结果显示:各组小鼠在黑盒停留时间(s)长于白盒停留时间(P＜0.05),表明小鼠天然偏爱黑盒;同时各组间比较小鼠的黑盒停留时间无明显差异(P＞0.05)。
3.4 Con-G及其类似物对吗啡诱导小鼠CPP表达的影响
吗啡诱导的CPP表达阶段各组小鼠在伴药盒(白盒)的停留时间同吗啡对照组(M-G0)相比,Con-G(30、60、120 pmol)干预组小鼠的白盒停留时间呈剂量.效应依赖性减少(30 pmol,P＜0.01;60 pmol,P＜0.01;120 pmol;P＜0.01)。Glu-Con-G(30、60、120 pmol)干预组小鼠的白盒停留时间呈剂量-效应依赖性减少(30 pmol,P＜0.01;60 pmol,P＜0.01;120 pmol;P＜0.01)。Glu-Con-G[1-13](30、60、120 pmol)干预组小鼠的白盒停留时间呈剂量-效应依赖性减少(30 pmol,P＜0.01;60 pmol,P＜0.01;120 pmol;P＜0.01)。Con-G及其类似物最小剂量均为30 pmol,药效相当。
4.结论
4.1根据NR2B亚基404-802序列构建了谷氨酸结合位点三维结构。
4.2 Con-G的E2、Gla4、L5、Q9、I12和Q13,NR2B的E420、S421、D423、K458和D715是参与相互作用的重要氨基酸,它们之间形成了七对氢键。
4.3所设计多肽Glu-Con-G[1-13]符合对接模型。
4.4 Glu-Con-G[1-13]与Con-G以及Glu-Con-G剂量相关性抑制吗啡诱导的小鼠条件性位置偏爱表达。
4.5 Glu-Con-G[1-13]在分子大小、合成成本上有改善,可以作为候选药物进行深入研究。
1 Objective
Drug dependence is not only a medical problem,biological problem,but also social issue,and it is a chronic relapsing encephalopathy.Causes of dependency include positive reinforcement factors(euphoria,reward effect),negative reinforcement factors(escape from reality,to reduce withdrawal symptoms) and conditions of the reinforcement factors.Opioid dependence is the essence of compensatory adaptation,involving multiple brain regions,a variety of changes in neurotransmitters and their receptors(including the opioid system itself) norepinephrine system,excitatory amino acid systems(such as N-methyl-D-aspartate (NMDA) receptors),etc.NMDAR is the excitatory glutamate receptor subtype,which consists of a different proportion of NR2,NR3 and NR1 subunits,and it has a high-pass permeability to Ca~(2+).Studies have shown that NMDAR in synaptic signal transduction,neural plasticity,learning and memory,etc.play an important physiological role,while many acute and chronic pathological changes of the nervous system are closely related to NMDA receptor dysfunction.
In view of the central nervous system,NMDA receptors are involved in reward-dependent effects of addictive drugs,as the central nervous system,NMDA receptor-specific antagonist Conantokins family may be applied to treat drug addiction. Our experiments confirmed that Con-G can effectively interfere with the expression of morphine-induced CPP and reconstruction,which may have prevention and treatment of morphine dependence and relapse potential.
Since there are some disadvantages on Con-G,such as the inconvenient administration,low absorption rate and the high cost disadvantages.Therefore,this study is designed using computer-aided to design Con-G analogues,so as to overcome some shortcomings of the original peptide and used using the computer based video tracking CPP system to analyze its anti-assess the effects of drug addiction,which is used to strive for effective screening of the Con-G analogs.
2 Materials and methods
2.1 Animals:
Adult male Kun-Ming mice(18-22g) were supplied by Shanghai Centre of Experimental Animals,Chinese Academy of Sciences.
2.2 Chemical reagents:
Conantokin G(purity＞90%)(Sigma)
[Glu~(3,4,7,10,14)]-Conantokin G(purity＞90%)(Sigma)
[Glu~(3,4,7,10,14)]-Conantokin G[1-13](purity＞90%)(Sbsgene)
Morphine hydrochloride(First Pharmaceutical Company of Shenyang,China);
2.3 Apparatus:
Computer simulation software
ZDOCK in InsightⅡsoftware
Modeller 8v2
NACCESS
Computer based video tracking CPP system
SPSS software
2.4 Methods
2.4.1 Homology modeling
The protein sequence of human NR2B subunit(404-802) was obtained from the National Center for Biotechnology Information(NCBI) server.Homologous proteins with known crystal structures were found by performing a position- specific iterated BLAST(PSI-BLAST) search.Using Modeller 8v2,protein 3D structures were generated by satisfying spatial restraints imposed by the sequence alignment with the template structure.To optimize the local interactions,several models obtained were subjected to a short simulated annealing refinement protocol using Charmml9 force-field.Stereochemical quality of the polypeptide backbone and side chains was evaluated using Ramachandran plots obtained from the RAMPAGE server.Amino acid environment was evaluated using Verify 3D and Errat plots.
2.4.2 Ligand docking
Protein Docking was performed with the ZDOCK software bundled with the InsightⅡpackage version 2005(Accelrys~(TM) Inc.).Con-G was first docked manually by ZDOCK,then computationally by RDOCK for the top 50 ranks.The resulting NR2B/ligand complex was energy-minimized in the absence of solvent using Charmm19 force-field.The accessible surface area(ASA) of NR2B subunit (uncomplexed) and its docked complex with Con-G were calculated using the program NACCESS.Composite coordinates of ligand and NR2B subunit were generated to form the docked complex.The change in accessible surface area for residue was calculated using:
△ASA~i=ASA~i_(NR2B)-ASA~i_(NR2B)-Con-G.
2.4.3 Molecular Design
Based on molecular docking complex combination of model,the geometric model and structural characteristics between NR2B subunit Con-G analogues were designed according to model and structure activity relationship
2.4.4 Effect of Glu-Con-G[1-13],Con-G and Glu-Con-G on the expression of CPP induced by morphine:
Male Kun-Ming mice were administered morphine(5mg/kg,i.p.) in white chamber for 50 mins on day1,3,5 and 7.On day2,4,6,8 mice were given saline in black chamber for 50 mins.The control group was treated with a daily saline injection for eight consecutive days both in the morphine and saline paired chambers.To test the effects of Con-G and its analogues Glu-Con-G,Glu-Con-G[1-13]on the expression of morphine-induced CPP,different dose of Con-G and its analogues Glu-Con-G, Glu-Con-G[1-13](0,30,60,120pmol) were given by intracranial administration 30 mins before the test on day 9.Total volume was 2μl.
2.5 Data analysis and statistics
Place Preference:the time spent in white chamber(TW);Locomotor Activity:the total distance that mice moved in different chambers(TD) and shuttle time(ST);All the data were expressed as the mean±S.E.M,and analyzed with SPSS software.P＜0.05 was considered statistically significant.
3 Results:
3.1 Homology Modeling
In the Ramachandran plot,a total of 79.0%of the residues were in the most favoured region,a total of 95.7%of the residues were in the allowed region.Verify 3D score and Errat plots score were 136.58 and 79.3%,and its reference protein score are 114.98 and 94.7%.
3.2 Ligand Docking
When Con-G structure was docked into the glutamate binding site of NR2B subunit by energy minimization,Con-G structure was nicely fitted into the agonist binding cleft on the NR2B subunit.Several H-bonds were found between NR2B subunit and Con-G.Connolly surfaces of the NR2B subunit and of Con-G were calculated.The decreases of accessible surface area(△ASA) of the docking complex were observed.Considering NR2B subunit,significant change of accessible surface area of Glu420,Ser421,Asp423,Lys458 and Asp715 occurred,Lys458 and Asp715 lost more than 50A ASA when going from the uncomplexed to the complexed state.
3.3 Test of preference in preconditioning phase
Pre-conditioning test showed that all groups mice spent more time in black chamber than in white chamber(P＜0.05),which suggested that mice prefer the black chamber;Meanwhile,mice of the different groups did not show any significant difference(P＞0.05) in the time spent in black chamber.
3.4 Effect of Con-G and its analogues on the expression of morphine induced CPP
After conditioning with morphine for 8 days,on the CPP expression day(d9), compared with the mice in morphine control group(M-G0),A pretreatment with Con-G and its analogue(30,60,120pmol) 30 min before the test,Con-G displayed a dose-effect dependent decrease the expression of morphine induced CPP(30pmol, P＜0.01;60pmol,P＜0.01;120pmol;P＜0.01);Glu-Con-G(30,60,120pmol) displayed a dose-effect dependent decrease the expression of morphine induced CPP(30pmol, P＜0.01;60pmol,P＜0.01;120pmol;P＜0.01);Glu-Con-G[1-13](30,60,120pmol) also displayed a dose-effect dependent decrease the expression of morphine induced CPP (30pmol,P＜0.01;60pmol,P＜0.01;120pmol;P＜0.01).Con-G and its analogues had the same lowest efficient dose.
4 Conclusions
4.1 The glutamate binding site is built according to the sequence of NR2B 404-802.
4.2 Structurally and functionally important residues were identified,including E2, Gla4,L5,Q9,I12 and Q13 of Con-G,they interacted with E420,S421,D423,K458 and D715 of NR2B.Seven H-bonds were also found.
4.3 Glu-Con-G[1-13]is in line with docking model.
4.4 Glu-Con-G[1-13],Con-G and Glu-Con-G,displayed a dose-effect dependent decrease the expression of morphine induced CPP.
4.5 Glu-Con-G[1-13]has an improvement in the molecular size and the difficulty and cost of synthesis which can be used as the next phase of the study key.
引文
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