CN112233730B - 一种区分PBDEs衍生物对烯酰-ACP还原酶活性效应模型的构建方法 - Google Patents
一种区分PBDEs衍生物对烯酰-ACP还原酶活性效应模型的构建方法 Download PDFInfo
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Abstract
本发明公开了一种区分PBDEs衍生物对烯酰‑ACP还原酶活性效应模型的构建方法,包括受体与配体建模,分子对接,分子动力学模拟;本发明模型计算结果与体外结合活性测定实验结果一致,表明本发明模型的构建方法能够准确计算PBDEs衍生物对烯酰‑ACP还原酶的活性效应。
Description
技术领域
本发明属于有机污染物识别技术领域,具体涉及一种区分PBDEs衍生物对烯酰-ACP还原酶活性效应模型的构建方法。
背景技术
脂肪酸的合成由脂肪酸合酶催化是有机体必须的。脂肪酸合酶是一个复合酶系统,在自然界中能够催化乙酰辅酶A和丙酰辅酶A从头合成脂肪酸,在脂肪酸合酶系统中脂肪酸的延长分别由四种酶催化,其中烯酰-ACP还原酶位于脂肪酸延长的最后一步,是催化烯酰-ACP还原成酰基-ACP(ACP,acyl carrier protein,酰基载体蛋白)必须的酶。
多溴二苯醚(PBDEs,Poly Brominated Diphenyl Ethers)作为一种溴系阻燃剂的一大类阻燃物质,由于其优异的阻燃性能,已经越来越广泛的应用于各种消费产品当中。但是随着在环境样品中不断报道PBDEs的检出,该类化合物所造成的环境问题也越来越受到大家特别是环境科学的关注。PBDEs具有环境持久性,远距离传输,生物可累积性及对生物和人体具有毒害效应等特性,对其环境问题的研究已成为当前环境科学的一大热点。
多溴二苯醚(PBDEs)的衍生物包括羟基化多溴二苯醚(HO-PBDEs)和甲氧基化多溴二苯醚(MeO-PBDEs),在各种环境介质中被大量检出。
目前,PBDEs衍生物与烯酰-ACP还原酶的结合活性需要通过实验来进行测定,如何提供一种能够区分不同PBDEs衍生物对烯酰-ACP还原酶结合活性的预测模型是有待解决的技术问题。
发明内容
本发明提供一种区分PBDEs衍生物对烯酰-ACP还原酶活性效应模型的构建方法,包括,受体与配体建模:删除烯酰-ACP还原酶的B链以及B链中结合的NAD+,删除受体中所有水分子,将结合有NAD+的A链作为下一步的分子对接使用的受体,并将NAD+与TCS加入氢原子,配体分子用sybyl7.3中的Sketch Molecule模块构建,并用sybyl7.3内Minimize模块进行结构优化;
分子对接:使用sybyl7.3中的Surflex-Dock模块将配体分子对接到受体中,对接时,使用Ligand模式,产生受体结合口袋,将配体与受体进行对接;每个配体化合物生成20个结合构象,以得分最高的构象作为最有可能的生物活性构象进行下一步的分子动力学模拟;
分子动力学模拟:在sybyl的Tripos力场下将配体分子分别用分子力学程序minimize进行优化,用powell能量梯度法,原子载电荷用Gasteiger-Huckel电荷,优化后的配体放入database中;在进行分子动力学模拟前,系统先经受5,000步的能量最小化,然后用NVT和NPT来平衡系统;每个系统在NVT系统中被模拟为500ps,在能量最小化的情况下从0到300K逐渐加热,并且在NPT系综中在300K下500ps平衡,然后采用Gromacs5.1.2软件包进行20ns的分子动力学模拟,在分子动力学模拟20ns时分析配体与受体之间的氢键、π-π堆积、卤键相互作用,并每2ps记录一次轨迹。
优选地,还包括在分析配体与受体之间的氢键、π-π堆积、卤键相互作用时,判断配体是否与受体上结合的NAD+或受体的氨基酸残基ALA196形成氢键或π-π堆积;若配体未与受体上结合的NAD+或受体的氨基酸残基ALA196形成氢键或π-π堆积,则分析构象变化,判断PRO154-ASN157helix-loop区、PHE251-ASN257 loop-helix区以及THR206-MET216 helix是否出现相互靠近的现象造成受体口袋入口闭合。
优选地,所述分子对接过程中,阈值(Threshold)设为0.5,膨胀系数(Bloat)为0。
优选地,所述分子对接,其中,所述产生受体结合口袋,为将TCS作为阳性物质,以TCS结合位点为中心产生受体结合口袋,预测构象与真实构象之间的RMSD为
优选地,所述在sybyl的Tripos力场下将配体分子分别用分子力学程序minimize进行优化,其中,最大优化次数为100步,0.05KJ/mol。
优选地,在分子动力学模拟过程中,配体分子以及受体分子中的NAD+分子使用Amber的GAFF力场,具体过程为先使用Gaussian09 D01优化分子的结构,计算静电势,拟合RESP电荷,然后用Ambertools中的模块antechamber生成resp文件,使用acpype.py脚本生成Gromacs可用的分子拓扑文件,受体蛋白质的拓扑文件用pdb2gmx生成,使用Amber99sb.ff力场,以距离受体蛋白质-配体分子表面至少2nm构建盒子,并填充TIPP3P水分子,即受体蛋白质与边缘至少有2nm水分子。
优选地,所述分子动力学模拟,包括以TCS结合位点为中心作为受体结合口袋。
本发明的有益效果:本发明模型计算结果与体外结合活性测定实验结果一致,表明本发明模型的构建方法能够准确计算PBDEs衍生物对烯酰-ACP还原酶的活性效应。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例描述中所需要使用的附图作简单地介绍,其中:
图1为TCS作为配体,分子动力学模拟20ns时的构象图。
图2为TCS作为配体,分子动力学模拟0ns时的构象图。
图3为6MeOBDE17在分子动力学模拟20ns时的构象图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合具体实施例对本发明的具体实施方式做详细的说明。
PBDE衍生物对烯酰-ACP还原酶抑制活性的预测方法:
(1)受体与配体建模:烯酰-ACP还原酶(PDB ID:1C14,FabI)的蛋白质分子结构来源于RCSB蛋白质数据库(http://www.rcsb.org/pdb);在进行对接前删除烯酰-ACP还原酶的B链以及B链中结合的NAD+(烟酰胺腺嘌呤二核苷酸),删除受体中所有水分子,将结合有NAD+的A链作为下一步的分子对接使用的受体,并将NAD+与TCS(Triclosan,三氯生)加入氢原子;所有配体小分子(即PBDEs衍生物及阳性对照物TCS)均用sybyl7.3(Tripos Inc.,St.Louis,MO,USA)中的Sketch Molecule模块构建,并用sybyl7.3内Minimize模块进行结构优化。
(2)分子对接:使用sybyl7.3中的Surflex-Dock模块将配体小分子对接到受体蛋白当中;对接时,使用Ligand模式,使用TCS作为阳性物质,以TCS结合位点为中心产生受体结合口袋,与烯酰-ACP还原酶结构进行对接,阈值(Threshold)为0.5,膨胀系数(Bloat)为0。
对接后得到的结构和结合有TCS的蛋白质晶体结构进行对比,结果两者之间的差异较小,预测构象与真实构象之间的RMSD(均方根偏差)仅为该值小于X射线晶体学分辨率/>这说明了对接过程中参数设置以及对接结果的可靠性。
对接过程中考虑环的柔性,每个配体化合物生成20个结合构象,以得分最高的构象作为最有可能的生物活性构象进行下一步的分子动力学模拟。结构-功能关系受结合后的构象影响,而结合后的构象与配体和受体之间的相互作用有关,在此我们选择分析分子对接后配体与受体之间的氢键、π-π堆积、卤键相互作用,并与实测的体外配体(PBDEs衍生物)与受体(烯酰-ACP还原酶)结合活性测定实验结果进行比较,如表1所示:
表1配体与受体之间的氢键、π-π堆积、卤键相互作用及体外结合活性测定
从表1可以看出,各个配体(PBDEs衍生物)对接的构象虽然与TCS相近,但是它们与蛋白质受体的相互作用却各不相同,配体大多与受体的PHE94残基形成相互作用,但是分子对接未能区分配体对受体抑制能力的有无。
(3)分子动力学(MD)模拟:①在sybyl的Tripos力场下将表1中16个配体分子分别用分子力学程序minimize进行优化,用powell能量梯度法,原子载电荷用Gasteiger-Huckel电荷,最大优化次数100步,0.05KJ/mol,优化后的16个配体放入database中,以TCS结合位点为中心作为受体结合口袋。
②采用Gromacs5.1.2软件包进行MD模拟;在进行动力学模拟前,系统先经受5,000步的能量最小化,然后进行两个阶段平衡:NVT和NPT来平衡系统;每个系统在NVT系统中被模拟为500ps,在能量最小化的情况下从0到300K逐渐加热,并且在NPT系综中在300K下500ps平衡,最后进行20ns的MD模拟,并每2ps记录一次轨迹。
为了评估受体-配体系统在MD模拟中的稳定性,计算系统骨架的均方根偏差(RMSD),从RMSD的变化可以得出所有体系都在20ns之前达到了平衡。且每个系统RMSD值变化都小于0.25nm;分析受体蛋白质在20ns内的各个氨基酸残基的波动情况可以发现,在MD模拟过程中,残基波动主要集中在ASP98-ALA197loop区,PRO154-ASN157loop区以及结合口袋中THR194-MET206helix-loop区之间,这说明这些氨基酸残基在MD模拟过程中变动较大,比较灵活。
在模拟过程中配体小分子以及受体分子中的cofactorNAD+小分子使用Amber的GAFF力场,具体过程为先使用Gaussian09D01先优化小分子的结构,计算静电势,拟合RESP电荷,然后用Ambertools中的模块antechamber生成resp文件,使用acpype.py脚本生成Gromacs可用的分子拓扑文件,蛋白质的拓扑文件用pdb2gmx生成,使用Amber99sb.ff力场,以距离蛋白质-小分子配体表面至少2nm构建盒子,并填充TIPP3P水分子,即蛋白质与边缘至少有2nm水分子。
在模拟20ns时分析配体与受体之间的氢键、π-π堆积、卤键相互作用,并与实测的体外配体(PBDEs衍生物)与受体(烯酰-ACP还原酶)结合活性测定实验结果进行比较,如表2所示:
表2配体与受体之间的氢键、π-π堆积、卤键、疏水相互作用及体外结合活性测定实验结果
从表2可知,对于受体(烯酰-ACP还原酶)具有抑制效应的配体大多会与受体上结合的NAD+或受体的氨基酸残基ALA196形成氢键或π-π堆积,这是预测配体与受体结合的关键因素,但仍有一个例外:配体4OHBDE49对于受体烯酰-ACP还原酶具有抑制作用,但其与NAD+或ALA196没有明显的相互作用。
③分析构象变化:受体与对其有抑制能力的配体结合后,ASP98-ALA197 helix-loop区,PRO154-ASN157helix-loop区以及结合口袋中THR194-MET206 helix-loop区从螺旋结构变为规则卷曲,或者螺旋缩短。而PRO154-ASN157 helix-loop区、PHE251-ASN257loop-helix区以及THR206-MET216helix出现相互靠近的现象,由于该段氨基酸是底物进入活性位点的通道,这几个区域的相互靠近使得受体口袋入口闭合,阻碍了酰基进入活性位点,从而产生抑制作用。而无抑制能力的配体大多无这种构象变化,如图1~3所示。图1为TCS作为配体,分子动力学模拟20ns时,由于上述几个区域相互靠近,口袋入口闭合,因而可预测出TCS对受体产生抑制作用;图2为分子动力学模拟在0ns时(初始状态)的构象,口袋入口是打开状态。图3为6MeOBDE17在分子动力学模拟20ns后的构象,模拟20ns后,口袋入口是打开状态,表明6MeOBDE17对受体无抑制效应。配体4OHBDE49由于出现了PRO154-ASN157helix-loop区、PHE251-ASN257loop-helix区以及THR206-MET216helix相互靠近的现象,使得口袋入口闭合,阻碍了酰基进入活性位点,因此,虽然4OHBDE49未与受体上结合的NAD+或受体的氨基酸残基ALA196形成氢键或π-π堆积,但仍然对受体产生了抑制作用。
体外结合活性测定实验方法为:制备过表达His标记的FabI的大肠杆菌裂解液,当细菌光密度(OD600)达到0.6以后,用1mMIPTG,16℃诱导表达过夜,之后收集细菌并用PBS洗涤一次,在含有500mM NaCl、10mM Tris HCl和0.1%NP-40(pH=8.5)的裂解缓冲液中裂解细菌,14000g离心30min,用Ni-NTA琼脂糖(Qiagen)从上清液中纯化FabI。将含Ni-NTA的树脂转移到Poly-Prep色谱柱(Bio-Red),用洗涤缓冲液(50mM PBS,ph8.2,300mM NaCl和10mm咪唑)平衡两次。在4℃下用FabI裂解物孵育30分钟后,用洗涤缓冲液洗涤树脂两次,用含有50mM PBS、pH 8.2、300mM NaCl和250mM咪唑的缓冲液洗脱FabI蛋白。添加300μM丁基辅酶A,加入100nM重组FabI、400μM NADH和40μM NAD+混合物进行反应。NADH氧化为NAD+的速率用微板阅读器在340nm下每2分钟测量一次。为了测定PBDEs衍生物对FabI酶活性的抑制作用,将不同浓度的PBDEs(0.014-10μM)与100nM重组FabI在冰上孵育60分钟进行反应,之后测定NADH氧化为NAD+的速率。
综上,本发明提供了一种区分PBDEs衍生物对烯酰-ACP还原酶活性效应模型的构建方法,本发明通过受体与配体建模、分子对接、分子动力学模拟,分子动力学模拟包括配体优化、模拟20ns时分析配体与受体之间的氢键、π-π堆积相互作用,判断配体是否与受体上结合的NAD+或受体的氨基酸残基ALA196形成氢键或π-π堆积,若形成上述相互作用则表明配体对受体具有抑制作用,若未形成上述相互作用则进一步分析构象变化,判断PRO154-ASN157helix-loop区、PHE251-ASN257 loop-helix区以及THR206-MET216 helix是否出现相互靠近的现象造成受体口袋入口闭合,若出现则表明配体对受体产生抑制作用,否则说明配体未对受体产生抑制作用,本发明配体PBDEs衍生物包括2OHBDE123、4MeOBDE17、4OHBDE49、5MeOBDE47、6MeOBDE17、6MeOBDE47、6MeOBDE85、6MeOBDE90、6OHBDE17、6OHBDE47、6OHBDE90、6OHBDE137、6OHClBDE17、6OHClBDE68,本发明模型计算结果与体外结合活性测定实验结果一致,表明本发明模型的构建方法能够准确计算PBDEs衍生物对烯酰-ACP还原酶的活性效应。
应说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围,其均应涵盖在本发明的权利要求范围当中。
Claims (8)
1.一种区分PBDEs衍生物对烯酰-ACP还原酶活性效应模型的构建方法,其特征在于:包括,受体与配体建模:删除烯酰-ACP还原酶的B链以及B链中结合的NAD+,删除受体中所有水分子,将结合有NAD+的A链作为下一步的分子对接使用的受体,并将NAD+与TCS加入氢原子,配体分子用sybyl7.3中的Sketch Molecule模块构建,并用sybyl7.3内Minimize模块进行结构优化;
分子对接:使用sybyl7.3中的Surflex-Dock模块将配体分子对接到受体中,对接时,使用Ligand模式,产生受体结合口袋,将配体与受体进行对接;每个配体化合物生成20个结合构象,以得分最高的构象作为最有可能的生物活性构象进行下一步的分子动力学模拟;
分子动力学模拟:在sybyl的Tripos力场下将配体分子分别用分子力学程序minimize进行优化,用powell能量梯度法,原子载电荷用Gasteiger-Huckel电荷,优化后的配体放入database中;在进行分子动力学模拟前,系统先经受5,000步的能量最小化,然后用NVT和NPT来平衡系统;每个系统在NVT系统中被模拟为500ps,在能量最小化的情况下从0到300K逐渐加热,并且在NPT系综中在300K下500ps平衡,然后采用Gromacs5.1.2软件包进行20ns的分子动力学模拟,在分子动力学模拟20ns时分析配体与受体之间的氢键、π-π堆积、卤键相互作用,并每2ps记录一次轨迹;
在分析配体与受体之间的氢键、π-π堆积、卤键相互作用时,判断配体是否与受体上结合的NAD+或受体的氨基酸残基ALA196形成氢键或π-π堆积;若配体未与受体上结合的NAD+或受体的氨基酸残基ALA196形成氢键或π-π堆积,则分析构象变化,判断PRO154-ASN157helix-loop区、PHE251-ASN257 loop-helix区以及THR206-MET216 helix是否出现相互靠近的现象造成受体口袋入口闭合。
2.如权利要求1所述的区分PBDEs衍生物对烯酰-ACP还原酶活性效应模型的构建方法,其特征在于:所述分子对接过程中,阈值(Threshold)设为0.5,膨胀系数(Bloat)为0。
3.如权利要求1所述的区分PBDEs衍生物对烯酰-ACP还原酶活性效应模型的构建方法,其特征在于:所述分子对接,其中,所述产生受体结合口袋,为将TCS作为阳性物质,以TCS结合位点为中心产生受体结合口袋,预测构象与真实构象之间的RMSD为
4.如权利要求1所述的区分PBDEs衍生物对烯酰-ACP还原酶活性效应模型的构建方法,其特征在于:所述在sybyl的Tripos力场下将配体分子分别用分子力学程序minimize进行优化,其中,最大优化次数为100步,0.05KJ/mol。
5.如权利要求1所述的区分PBDEs衍生物对烯酰-ACP还原酶活性效应模型的构建方法,其特征在于:在分子动力学模拟过程中,配体分子以及受体分子中的NAD+分子使用Amber的GAFF力场,具体过程为先使用Gaussian09 D01优化分子的结构,计算静电势,拟合RESP电荷,然后用Ambertools中的模块antechamber生成resp文件,使用acpype.py脚本生成Gromacs可用的分子拓扑文件,受体蛋白质的拓扑文件用pdb2gmx生成,使用Amber99sb.ff力场,以距离受体蛋白质-配体分子表面至少2nm构建盒子,并填充TIPP3P水分子,即受体蛋白质与边缘至少有2nm水分子。
6.如权利要求1所述的区分PBDEs衍生物对烯酰-ACP还原酶活性效应模型的构建方法,其特征在于:所述分子动力学模拟,包括以TCS结合位点为中心作为受体结合口袋。
7.如权利要求1所述的区分PBDEs衍生物对烯酰-ACP还原酶活性效应模型的构建方法,其特征在于:所述配体,为PBDEs衍生物,包括2OHBDE123、4MeOBDE17、4OHBDE49、5MeOBDE47、6MeOBDE17、6MeOBDE47、6MeOBDE85、6MeOBDE90、6OHBDE17、6OHBDE47、6OHBDE90、6OHBDE137、6OHClBDE17、6OHClBDE68。
8.如权利要求1所述的区分PBDEs衍生物对烯酰-ACP还原酶活性效应模型的构建方法,其特征在于:所述受体,为对烯酰-ACP还原酶,包括FabI。
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003065978A2 (en) * | 2001-11-28 | 2003-08-14 | Structural Genomix, Inc. | Crystals and structures of a flavin mononucleotide binding protein (fmnbp) |
CN101381894A (zh) * | 2008-05-30 | 2009-03-11 | 南京大学 | 一种有机物雌激素受体激动和拮抗作用的识别方法 |
KR20090101652A (ko) * | 2008-03-24 | 2009-09-29 | 한국과학기술원 | 시스템 기법을 이용한 비브리오속 미생물의 약물 표적 예측 |
EP2329018A1 (en) * | 2008-04-25 | 2011-06-08 | Novalis Ltd. | Method for the annotation of natural product gene-clusters and for the generation of novel biologically active chemical entities from dna sequences in silico |
CN108351350A (zh) * | 2015-08-25 | 2018-07-31 | 杜克大学 | 使用rna指导型内切核酸酶改善基因组工程特异性的组合物和方法 |
CN109285584A (zh) * | 2018-09-03 | 2019-01-29 | 南京大学 | 区分物质雄激素与抗雄激素效应模型的构建和应用 |
CN110426512A (zh) * | 2019-05-21 | 2019-11-08 | 南京大学 | 区分过氧化酶激活增殖受体γ完全激动剂、部分激动剂、拮抗剂活性的方法 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6684162B2 (en) * | 2000-05-08 | 2004-01-27 | Wyeth | Methods for identifying agents that interact with an active site of acyl carrier protein synthase-acyl carrier protein complex |
US20050004225A1 (en) * | 2003-04-16 | 2005-01-06 | Balendiran Ganesaratnam K. | Oxidoreductase inhibitors and methods of screening and using thereof |
-
2020
- 2020-10-16 CN CN202011111077.8A patent/CN112233730B/zh active Active
- 2020-11-09 US US17/093,580 patent/US11908550B2/en active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003065978A2 (en) * | 2001-11-28 | 2003-08-14 | Structural Genomix, Inc. | Crystals and structures of a flavin mononucleotide binding protein (fmnbp) |
KR20090101652A (ko) * | 2008-03-24 | 2009-09-29 | 한국과학기술원 | 시스템 기법을 이용한 비브리오속 미생물의 약물 표적 예측 |
EP2329018A1 (en) * | 2008-04-25 | 2011-06-08 | Novalis Ltd. | Method for the annotation of natural product gene-clusters and for the generation of novel biologically active chemical entities from dna sequences in silico |
CN101381894A (zh) * | 2008-05-30 | 2009-03-11 | 南京大学 | 一种有机物雌激素受体激动和拮抗作用的识别方法 |
CN108351350A (zh) * | 2015-08-25 | 2018-07-31 | 杜克大学 | 使用rna指导型内切核酸酶改善基因组工程特异性的组合物和方法 |
CN109285584A (zh) * | 2018-09-03 | 2019-01-29 | 南京大学 | 区分物质雄激素与抗雄激素效应模型的构建和应用 |
CN110426512A (zh) * | 2019-05-21 | 2019-11-08 | 南京大学 | 区分过氧化酶激活增殖受体γ完全激动剂、部分激动剂、拮抗剂活性的方法 |
Non-Patent Citations (8)
Title |
---|
5-芳亚甲基-N-(4-羧基苯基)罗丹宁衍生物的微波辅助合成及分子模拟研究;蒋军荣;徐峰;柯中炉;;化学试剂(第05期);481-484、487 * |
Biochemical and molecular biomarkers in integument biopsies of free-ranging coastal bottlenose dolphins from southern Brazil;Righetti, BPH等;《CHEMOSPHERE》;139-149 * |
Combined 3D-QSAR, molecular docking and molecular dynamics study on thyroid hormone activity of hydroxylated polybrominated diphenyl ethers to thyroid receptors β;Li, XL等;《TOXICOLOGY AND APPLIED PHARMACOLOGY》;300-307 * |
Shrinivas D. Joshi等.3D-QSAR and molecular docking studies of 1,3,4-oxadiazoles containing substituted phenoxy fragment as inhibitors of enoyl-acyl carrier protein reductase from Escherichia coli.《Medicinal Chemistry Research 》.2014,4542–4558. * |
基于Metadynamic的分子动力学模拟建立受体活性区分模型;石来昊;《中国优秀硕士学位论文全文数据库》;B027-422 * |
多氯联苯鸡胚肝细胞生物活性非单调剂量-响应关系的QSARs研究;穆云松;张爱茜;高常安;彭素芬;王连生;;中国科学(B辑:化学)(第12期);1105-1112 * |
环境中多溴联苯醚(PBDEs)的代谢转化研究现状;温泉;张俊江;关淼;刘红玲;苏冠勇;于红霞;;环境监控与预警(第04期);34-41 * |
选择性磷酸二酯酶4D(PDE4D)抑制剂 的计算模型建立;刘景陶等;《分子科学学报》;249-256 * |
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