植物源杀虫剂苦皮藤素V靶蛋白的功能验证及成药性验证

The pesticide creation based on the molecular target is the forefront of international pesticide research and exploit. In the last program of National Science Foundation of China (30871663), a protein bound with botanical insecticide Celangulin V was isolated from the total protein of the midgut of Mythimna separate larva by affinity chromatography, and identificated as vacuolar-type ATPase (V-ATPase) subunit H mainly by the mass spectrometry analysis. Furthermore, the opinion that the V-ATPase subunit H would be the putative target of Celangulin V was supported by the results of studies on computer molecular docking and RNA interference. But the possibility that the V-ATPase subunit H is the target of Celangulin V is still required further function verification and druggability testing. For this reason, we develop this proposal intending to address this scientific question. In this research, it will be a more detailed understanding of the effects of Celangulin V on the function of V-ATPase subunit H through function verification. And the druggability will be tested molecular docking silico screening-protein affinity screening-bioassay in vivo/in vitro and the resulting insecticidal activity screening. This achievement will provide new ideas for innovating insecticides acting on insect digestive system and a target molecular model, and be a major breakthrough for national pesticide creation.

当代农药科学研究与发展的前沿课题是以分子靶标导向的新农药创制。在已结题的国家基金项目（30871663）研究中，申请人采用亲和层析从东方粘虫中肠蛋白中分离出植物源杀虫剂苦皮藤素V的结合蛋白，经蛋白质谱分析及与家蚕基因组数据库比对，鉴定苦皮藤素V的结合蛋白为V-ATP酶H亚基，并采用分子对接及RNA干扰，进一步佐证V-ATP酶H亚基是苦皮藤素V的疑似作用靶标。但V-ATP酶H亚基是否真是苦皮藤素V的作用靶标尚需进一步进行功能验证和成药性验证。为此，申请人提出本项申请，拟以东方粘虫中肠V-ATP酶H亚基为核心，通过H亚基的功能验证，明确苦皮藤素V对V-ATP酶H亚基功能的影响；通过分子对接虚拟筛选-蛋白亲和筛选-离体/活体生测验证昆虫V-ATP酶H亚基作为杀虫剂靶标的成药性，为创制一类作用于昆虫消化系统的新型杀虫剂提供新思路和靶标分子模型，实现我国农药创制的重大突破。

项目的预期目标是从功能及成药性两个方面验证昆虫中肠V-ATP酶H亚基是植物杀虫剂苦皮藤素V的作用靶标。本项目取得如下主要进展：（1）测定了苦皮藤素V及其类似物对粘虫幼虫中肠细胞跨膜电位及V-ATP酶活性的影响，通过杀虫活性和对V-ATP酶活性抑制率的相关性分析，证明苦皮藤素V抑制昆虫中肠V-ATP酶活性；（2）测定了苦皮藤素V及其类似物和V-ATP酶H亚基蛋白的结合能力并进行了对杀虫活性及酶抑制活性的相关性分析，其结果表明V-ATP酶H亚基是苦皮藤素V的作用靶标；（3）将H亚基的dsRNA导入粘虫幼虫，显著降低了粘虫中肠V-ATP酶H亚基的表达水平，甚至导致目的基因完全沉默，这一遗传学实验结果也验证V-ATP酶H亚基是苦皮藤素V的作用靶标；（4）成功构建出粘虫中肠V-ATP酶H亚基的3D模型并进行了虚拟筛选和活体筛选，发现43#这一高活性化合物。进一步以43#化合物为先导设计合成74个苯磺酰胺类化合物，筛选出HA-4-6，HA-4-15及HA-4-17 三个高活性化合物，其毒力分别是苦皮藤素V的17.7、70.7及36.3倍，而且其对粘虫的作用症状和苦皮藤素V完全一致，从成药性角度证明V-ATP酶H亚基是苦皮藤素V的作用靶标。本项研究这些原创性成果对国内外毒理学的研究，特别是对国内外新型杀虫剂的创制提供了新的靶标，提供了新的思路。本项目已发表标注论文21篇，申请中国发明专利1件，培养博士1名，硕士5名。