CN108069943A - 一种棉铃虫甾醇载体蛋白2的潜在抑制剂 - Google Patents

一种棉铃虫甾醇载体蛋白2的潜在抑制剂 Download PDF

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CN108069943A
CN108069943A CN201711391154.8A CN201711391154A CN108069943A CN 108069943 A CN108069943 A CN 108069943A CN 201711391154 A CN201711391154 A CN 201711391154A CN 108069943 A CN108069943 A CN 108069943A
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杜馨
蔡俊
王常高
林建国
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Hubei University of Technology
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Abstract

本发明属于绿色新农药筛选领域，具体涉及一类甾醇载体蛋白2（SCP‑2）小分子抑制剂及筛选方法。本发明利用Interbioscreen化合物库，通过计算机高通量筛选，利用分子对接软件筛选打分并计算结合自由能，对所得的化合物结构聚类及视觉分析，得到一类针对SCP‑2蛋白的小分子抑制剂‑7‑((五氟苯基)甲氧基)‑3‑(喹啉‑2‑基)‑2氢‑二氢香豆精，本发明可用于对鳞翅目为主的多种农业害虫的生物防治。

Description

一种棉铃虫甾醇载体蛋白2的潜在抑制剂
技术领域
本发明涉及绿色新农药筛选的领域，具体涉及一种棉铃虫甾醇载体蛋白2抑制剂及筛选。
背景技术
棉铃虫是一种杂食性、抗性极强的重要农业害虫，棉铃虫对于许多农药都发展出一定抗性，包括对于防治棉铃虫的最主要生物农药苏云金芽孢杆菌(Bt)也出现了一定的抗性，因此，市场上急需开发一种新作用靶标的绿色农药。
近年，在开发新靶标农药的创制过程中，昆虫SCP-2蛋白引起人们的研究兴趣。在昆虫体内，胆固醇是细胞膜的结构组分和蜕皮激素合成前体，然而昆虫缺乏胆固醇生物合成过程中两种关键性酶，不能在体内从头合成，为满足生长、发育和繁殖的需要，昆虫必须从食物中获取胆固醇。因此，胆固醇的吸收、转运对于昆虫的生存至关重要。甾醇载体蛋白2(sterol carrier protein-2，SCP-2)属于含有甾醇结合域的蛋白家族，是一种非特异性脂质转运蛋白。SCP-2蛋白在棉铃虫、埃及伊蚊、烟草天蛾、斜纹夜蛾、科罗拉多幼虫等害虫体内均有发现，集中表达于昆虫中肠细胞，参与胆固醇的吸收与转运。研究表明，蚊子SCP-2蛋白抑制剂能阻断蚊子和烟草天蛾幼虫对胆固醇的吸收；某些蚊子SCP-2抑制剂对双翅目、鳞翅目和鞘翅目的敏感昆虫具有致命作用，但对哺乳动物细胞几乎没有毒性；对其他农药具有抗性的昆虫细胞对SCP-2抑制剂表现敏感。因此昆虫SCP-2蛋白可作为开发新农药的靶标位点。
目前，鳞翅目昆虫SCP-2蛋白抑制剂开发研究较少，中国专利“一种棉铃虫甾醇载体蛋白2抑制剂及其虚拟筛选方法”、申请号201610428709.0，该发明涉及一类甾醇载体蛋白2(SCP-2)小分子抑制剂虚拟筛选方法的建立，所筛选到的抑制剂为N2,N7-二(1-萘基)-9氢-2,7-二磺酰胺芴和N2,N7-二(1-萘基)-9-氧-9氢-2,7-二磺酰胺芴，该抑制剂为人工合成药物，未进行体内或体外活性测定，抑制效果尚不明确。本发明涉及的甾醇载体蛋白2小分子抑制剂为天然产物药物，经体内活性测定，具有良好杀虫效果。
发明内容
本发明针对现有技术中存在的不足，利用Interbioscreen化合物库，通过计算机高通量筛选，提供一系列针对棉铃虫甾醇载体蛋白2的小分子抑制剂，达到防治棉铃虫等鳞翅目农业害虫的目的。
为了实现本发明的目的，本发明人通过大量试验研究和不懈的探索，最终获得了如下技术方案：
一种棉铃虫甾醇载体蛋白2的潜在抑制剂，其分子结构通式为：
其中R1的结构为I至VII中的任一种，R1的具体结构如下：
R2的结构为I至III中的任一种，R2的具体结构如下：
如上所述的一种棉铃虫甾醇载体蛋白2的潜在抑制剂，该抑制剂可抑制棉铃虫甾醇载体蛋白2活性。
如上所述的一种棉铃虫甾醇载体蛋白2的潜在抑制剂，该抑制剂可抑制与棉铃虫甾醇载体蛋白2同源蛋白的活性。
如上所述的一种棉铃虫甾醇载体蛋白2的潜在抑制剂，该抑制剂作为杀虫剂可应用于鳞翅目、鞘翅目、双翅目等农业害虫的防治。
一种棉铃虫甾醇载体蛋白2的潜在抑制剂的筛选方法，具体步骤包括：
(1)获取Interbioscreen小分子化合物库；
(2)利用AutoDock Vina软件对Interbioscreen化合物库中小分子和HaSCP-2蛋白进行对接打分，选取结合自由能低于胆固醇结合自由能(-10.7kcal/mol)的小分子；
(3)利用PyMol视图软件对所得化合物进行手工筛选，得到先导HaSCP-2小分子抑制剂7-((五氟苯基)甲氧基)-3-(喹啉-2-基)-2氢-二氢香豆精(7-((perfluorophenyl)methoxy)-3-(quinolin-2-yl)-2H-chromen-2-one)；
(4)以步骤(3)中化合物基本结构进行基于结构的相似性搜索，再次对搜索得到的小分子和HaSCP-2蛋白进行对接打分，并分析比较HaSCP-2蛋白与小分子相互作用；
(5)将筛选得到的化合物进行棉铃虫生物活性测定。
优选地，如上所述的一种棉铃虫甾醇载体蛋白2的潜在抑制剂的筛选方法，其中步骤(3)中的手工筛选方法为：以步骤(2)得到的小分子化合物为基本结构利用NCBI中PubChem以及ZINC二维结构相似性搜索，得到23种化合物，并进行对接及后期分析。
与现有技术相比，本发明的优点为：本发明筛选到的小分子抑制剂为天然产物药物，经体内活性测定本抑制剂对棉铃虫低龄幼虫具有良好杀虫效果。不同于传统化学杀虫剂，本抑制剂抑制昆虫对植物甾醇的吸收，从而影响昆虫幼虫蜕皮率、产卵率以及卵孵化率，不仅能控制原代害虫数量，对二代甚至三代害虫数量也将形成有效控制。
附图说明
图1为棉铃虫SCP-2蛋白配体结合位点图。
图2为抑制剂与HaSCP-2结合模式图，由图可知抑制剂呈“水平”构象，与蛋白活性口袋契合，主要通过疏水作用、范德华力、π-π堆叠、氢键、静电力和扭转力与HaSCP-2蛋白相互作用。
图3为抑制剂处理棉铃虫幼虫第11天幼虫体重图。
图4为不同浓度抑制剂对棉铃虫幼虫和蛹致死效果图。
具体实施方式
为能清楚说明本发明方案的技术特点，下面结合具体实施例，对本发明进行阐述。但是本发明的保护范围并不限于这些实施例。凡是不背离本发明构思的改变或等同替代均包括在本发明的保护范围之内。
1、获取棉铃虫SCP-2蛋白的PDB数据文件和小分子化合物库
从PDB数据库(http://www.rcsb.org/pdb/home/home.do)中获得HaSCP-2的NMR结构(PDBID：4UEI)，选取能量最低的结构并利用AutoDock tools-1.5.6软件对其加氢、计算电荷等预处理。选取Interbioscreen数据库(http://www.Interbioscreen.com/)，利用Schrodinger中LigPrep模块进行OPLS_2005力场优化，加氢和在pH7.0±2.0条件下离子化，去除小分子片段、盐离子等操作，得到的化合物库使用Raccoon v1.0软件进一步转化为pdbqt格式文件。
2、确定活性中心，分析关键氨基酸残基，设定Grid参数
使用Schrodinger中SiteMap程序对配体结合口袋预测，并根据疏水口袋氨基酸残基使用AutoDocktools设定Grid参数，得到的格子；使用AutoDock Vina对天然配体及已知具有抑制活性的小分子对接，结合定点突变分析结果判断关键氨基酸残基(Y51、F53、F89、L99、F110和T128)。
3、对小分子化合物库中小分子对接打分
根据设定的Grid格子参数，使用Vina将胆固醇与HaSCP-2蛋白对接并进行动力学模拟，验证对接方法的可靠性；借助PyRx 0.8程序使用AutoDock Vina进行半柔性对接，得到排名前300化合物(结合自由能≤-11.0kcal/mol)。
4、蛋白配体结合模式分析
使用PyMol 1.7软件对得到的化合物进行可视化分析，根据打分排名、结构相似性和对接构象，减少假阳性率，初步确定化合物7-((五氟苯基)甲氧基)-3-(喹啉-2-基)-2氢-二氢香豆精可作为先导物的小分子抑制剂。
5、结构相似性搜索
以步骤4中得到的化合物利用NCBI中PubChem以及ZINC二维结构相似性搜索，得到23种化合物，并按步骤3、4进行对接及后期分析。
6、生物活性测定
准确称取一定质量的候选抑制剂化合物，以DMSO溶解配制成100mmol/L的母液，实验工作液以无水乙醇稀释成5μmol/L、25μmol/L、125μmol/L、625μmol/L和3125μmol/L系列浓度，空白对照组以无水乙醇处理。抑制剂处理量为20μl/g食物，每个浓度处理24头幼虫，饲料隔天更换，直到幼虫化蛹。待大多数幼虫生长至五龄第二天时称量幼虫体重，化蛹后称量蛹重，化蛾后记录产卵数。在90％幼虫化蛹后停止添加抑制剂。同时每天观察棉铃虫生长死亡情况。
由图3、图4可知，在喂食抑制剂后的第11天，不同浓度抑制剂对棉铃虫幼虫生长均有抑制作用，其中125μM浓度抑制剂处理组棉铃虫幼虫体重为对照组的56％。在喂食抑制剂后的第三天，125μM浓度抑制剂处理组棉铃虫幼虫死亡率达到52％。125μM浓度抑制剂处理组棉铃虫幼虫蛹孵化率为20％。

Claims (4)

1.一种棉铃虫甾醇载体蛋白2的潜在抑制剂，其特征在于，该抑制剂具有结构通式：
其中R1的结构为I至VII中的任一种，R1的具体结构如下：
其中R2的结构为I至III中的任一种，R2的具体结构如下：
2.如权利要求1所述的一种棉铃虫甾醇载体蛋白2的潜在抑制剂，其特征在于，该抑制剂在抑制棉铃虫甾醇载体蛋白2上的应用。
3.如权利要求1所述的一种棉铃虫甾醇载体蛋白2的潜在抑制剂，其特征在于，该抑制剂在抑制与棉铃虫甾醇载体蛋白2高度同源蛋白上的应用。
4.如权利要求1所述的一种棉铃虫甾醇载体蛋白2的潜在抑制剂，其特征在于，该抑制剂作为杀虫剂在鳞翅目类农业害虫防治中的应用。
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