蝗总科重要害虫章鱼胺受体序列和结构研究及杀虫剂先导化合物的虚拟筛选

英文摘要：. Lack of insecticides targets was one of the bottleneck in grasshopper insecticides research. And the poor specificity, drug resistance and environmental pollution are still serious. So, it is urgent to find a new drug target for designing a safe and special insecticides. Octopamine receptors (OAR) is the species-typical protein in invertebrates as a member of G-coupled protein receptor (GPCR) super-family. OAR is the physiological basis related with swarming and long-distance migratory behaviours. Therefore, OAR is a potential insecticide target gene.. The applicant recently find that the sequence variation between migratory locust (Locusta migratoria) and desert locust (Schistocerca gregaria) OAR proteins is appeared mainly in the second extracellular loop (EL-2) domain, which is also large different between OAR sequences of other insect species. Moreover, EL-2 domain is the very important active site in OAR protein structure. On the basis of the consideration the applicant proposed to conduct the following researches: (1) design the OAR gene specific degenerate primers for the OAR gene amplification and sequencing of Acridoidea main pests; (2) predict the protein structure and the ligand binding mechanism of grasshopper OAR and establish the conserved structure model of EL-2 domain; (3) virtual screen the potential small molecules with high binding affinity and specificity, using molecular docking technology and based on the modeled structure of grasshopper OAR as target. Then find the lead compounds with insecticidal activity by synthesizing and testing of the potential small molecules.. The study of this proposal can provide us with a technical reserves for grasshopper specifically and environmentally friendly insecticides design. The project proposed a new way for the protection of agricultural production and solve the food problem. So it has essential scientific and practical importance.

治蝗药物研究的瓶颈之一是缺乏杀虫剂靶标，现有的杀虫剂特异性差、抗药性和环境问题严重，亟待寻找新型靶标。章鱼胺受体(OAR)是G蛋白偶联受体(GPCR)超家族中无脊椎动物特有蛋白质，与蝗虫聚集和迁飞相关，是潜在的杀虫剂靶标。. OAR的第二胞外环(EL-2)结构域为其重要的活性位点，申请人前期研究发现飞蝗与沙漠蝗的OAR变异集中于该区域，且与其它昆虫差异较大。因此，本项目拟：设计简并引物对蝗总科重要害虫的OAR基因扩增并测序；预测蝗虫OAR的空间结构并建立EL-2的保守结构模型，分析与配体结合的分子机制；以此为靶标，以哺乳类GPCR为阴性对照，利用分子对接技术虚拟筛选出具有高亲和力和特异性的小分子，合成并检测以获得针对蝗虫有杀虫活性的先导化合物。. 本研究将为设计针对蝗虫的安全特效杀虫剂提供技术储备，为保障农业生产和解决粮食问题提供新的途径，具有重大理论意义和实际价值。

章鱼胺（OA）是包括昆虫在内的无脊椎动物特有生物胺，其受体章鱼胺受体（OARs）属于G蛋白偶联受体超家族（GPCRs）中的A类蛋白质。章鱼胺与其受体主要在昆虫体内发挥神经调节作用，参与到多项生理活动中，如捕食、交配、产卵、嗅觉、学习与记忆等，是潜在的杀虫剂药物设计靶标。本项目主要以蝗虫OARs为研究对象，开展了OARs的结构计算及其与配体结合机制分析，以及基于OctβR结构进行先导杀虫剂虚拟筛选的研究。本项目研究完成的主要内容及取得的成果如下：.（1）通过设计简并引物检测东亚飞蝗、花胫绿纹蝗、黄胫小车蝗等害虫的OARs基因信息并进行分析，鉴定得到多种蝗虫的OARs氨基酸序列信息；（2）通过数据挖掘与多序列比对，不同昆虫间OARs差异主要体现在两个高变区，第二个胞外环（EL2）和第三个胞内环（IL3），而在不同蝗虫体内OARs氨基酸序列之间并无明显差异；（3）通过同源建模和分子动力学模拟，构建出OARs家族中的亚类OctβR（与蝗虫聚集、迁飞相关，且其位点突变导致双甲脒药物抗性）的三维结构，并对位于第二胞外环(EL-2)结构域的活性位点进行进一步分析；（4）通过分子对接和受控动力学模拟，分析出配体与受体结合与解离的分子机制，并找到了有助于药物设计的关键氨基酸位点；（5）基于预测OctβR的空间结构基础，利用分子对接技术虚拟筛选得到了若干与蝗虫OctβR有着较高的亲和力的化合物小分子。.本项目研究成果为设计针对蝗虫的安全特效杀虫剂提供了更多的技术储备，为保障农业生产和解决粮食问题提供新的途径，具有重大理论意义和实际价值。项目研究过程中培养硕士生4人，本科生6人；发表SCI期刊论文6篇，参编专著1部，获省科学技术二等奖1项，在审SCI期刊论文2篇。