印楝油杀螨物质对细胞色素氧化酶表达及其蛋白特性的影响

Exploration of new structure drugs and new targets drugs has became an important strategy in development of antiparasite drugs. Moreover, the discovery of new targets drugs of enzyme is a hot area in recent years. The new compound, otadecanoic acid-3,4-tetrahydrofuran diester, was demonstrated to possess significant acaricidal activity and the mechanism was associated with the cytochrome oxidase which was composed of several subunits. However, the specific mode of action and the target subunits as well as the characteristic effect of the compound on these subunits are still not clear. The effects of the compound on discrepantly expressed proteins and the level of expression will be analyzed by the techniques of proteomics and real-time reverse-transcription polymerase chain reaction, then the target proteins can be confirmed and which step involved transcription and translation is prevented by the compound can also be determined. Then, co-immunoprecipitation will be employed to obtain the target proteins; the amino acid sequences and structure of the target proteins will be identified by mass spectrum and nuclear magnetic resonance, respectively. This study will elucidate the target proteins and the specific mode of action of otadecanoic acid-3,4-tetrahydrofuran diester and also lay the foundation for developemnt of novel anti-sarcoptid durgs.

全新结构药物和新靶标的发现已成为抗寄生虫感染药物研发的重要策略，其中酶靶标策略为研究热点。本课题组从印楝油中分离得到一种全新结构药物-十八碳酸-3,4-四氢呋喃二酯，确定其杀螨活性与虫体细胞色素氧化酶相关。细胞色素氧化酶由多个亚基组成，而药物究竟作用于哪些亚基、作用的方式及对这些亚基蛋白特性有怎样的影响均不清楚。本研究拟通过蛋白质组学寻找差异表达的蛋白，采用实时荧光定量反转录PCR技术研究其表达量的变化，确定十八碳酸-3,4-四氢呋喃二酯抗疥螨作用的靶蛋白，并探讨其对靶蛋白作用的方式是阻断基因转录过程还是mRNA的翻译过程；在此基础上运用免疫共沉淀技术捕获药物处理前后的靶蛋白，利用质谱鉴定和核磁共振技术研究药物对靶蛋白氨基酸序列或其空间结构等蛋白特性的影响。通过本研究可确证十八碳酸-3,4-四氢呋喃二酯抗疥螨作用的靶蛋白并揭示其作用的具体途径和方式，为筛选新的抗疥螨分子靶标药物奠定基础。

全新结构药物和新靶标的发现已成为抗寄生虫感染药物研发的重要策略，其中酶靶标策略为研究热点。本研究通过对虫体转录组和蛋白质组学研究表明十八碳酸-3,4-四氢呋喃二酯作用虫体后，主要导致三羧酸循环和氧化磷酸化途径中基因差异表达；蛋白质组学研究结果表明其潜在的作用靶点为NADH-Q还原酶、ATP合酶和细胞色素氧化酶等；其对虫体线粒体含量和细胞色素氧化酶含量的影响结果确证了十八碳酸-3,4-四氢呋喃二酯抗疥螨作用的靶标蛋白为细胞色素氧化酶；两组学研究结果进行联合分析，找到了药物可能的作用靶点，为进一步研究十八碳酸-3,4-四氢呋喃二酯的具体作用靶标奠定了基础。项目按照计划任务书完成相关研究内容，共形成5篇SCI论文，分别发表在Scientific Reports, Veterinary Parasitology等杂志上。其中见刊3篇，在投2篇，获得授权专利1项，四川省科技进步三等奖1项，培养博士1名，硕士 3名。该项目的完成为后期构效关系分析、同类物及其作用机理研究奠定了基础。