含吡啶基喹唑啉酮类化合物C1抑制柑橘溃疡病菌作用靶标及机制研究

Citrus canker (Xanthomonas axonopodis pv. citri, Xac) has caused serious harm to citrus production in China. In our previous work, quinazolinone compound containing pyridine moiety C1 was found to possess good inhibitory activity on Xac. However, the mechanism and molecular target of C1 inhibiting Xac is unclear. This proposal aims to elucidate the action mechanism of Xac against C1, and the main research plans are as following. 1) Preliminarily exploration of the antibacterial action mechanism of Xac against C1 will be carried out through using electronic microscopy as well as other biological experimental methods. 2) Target identification of C1 in Xac will be studied using proteomic, bioinformatic and molecular biology methods, during which 1-2 target protein is expected to be obtained. 3) Pokaryotic expression of target protein will be carried out and the interaction between active compound C1 and target protein will be investigated. 4) A series of new quinazolinone containing pyridine moiety will be designed and synthesized. The biological activities of the new compounds will be assayed against Xac in vitro. The interaction of highly active compounds and target protein will be evaluated, during which 1-2 more potent new structures are expected to be found out.

柑橘溃疡病菌(Xanthomonas axonopodis pv. citri, Xac)对我国柑橘生产造成了严重危害。本课题组前期工作发现吡啶基喹唑啉酮类化合物C1对Xac具有良好的抑制活性。但是，C1抑制柑橘溃疡病菌的作用机制、其作用分子靶标尚不明确。为了探究C1的作用机制，本申请拟开展以下四部分工作：1）通过电镜及生物学实验，初步探讨C1对Xac的抑菌作用机理；2）采用蛋白组学、生物信息学、分子生物学方法，寻找C1抑制Xac的关键靶标蛋白1~2个；3）通过体外原核表达方式获得靶标蛋白，建立化学生物学研究平台，确定靶标蛋白与C1之间的作用模式；4）设计合成一系列新型吡啶基喹唑啉酮衍生物，测试目标化合物对Xac的离体生物活性，采用建立的化学生物学研究平台，探究高活性化合物与靶标蛋白的相互作用，期望获得更高活性的新结构化合物1~2个。

本研究设计合成7个系列共计99个喹唑啉酮衍生物，并开展其抑菌活性及作用机制研究，通过合理设计从中筛选出抑制柑橘溃疡病菌活性优于化合物C1的含二硫键喹唑啉酮先导化合物7个，其中化合物1对柑橘溃疡病菌Xanthomonas axonopodis pv. Citri（Xac）的EC50值为2.62 μg/mL，对水稻白叶枯病菌Xanthomonas oryzae pv. Oryzae（Xoo）的EC50值为0.55 μg/mL。抑菌作用机制研究发现：化合物1通过干扰萜类骨架结构的生物合成MEP途径抑制Xac；通过调控菌毛运动的组胺酸激酶蛋白，干扰菌毛蹭行运动抑制Xoo。同时，还意外发现多个化合物具有较好的肿瘤抑制活性。完成了原计划中的研究内容和量化考核指标。.在该项目资助下，参加国际学术会议1次，做学术报告3人次；独立及协助指导硕士研究生毕业共计16名。以主持人作为通讯作者，标明该项目资助且研究内容与项目一致，发表SCI论文7篇，申报国家发明专利3项；以主持人作为通讯作者，该项目资助下发表其他SCI论文6篇、中文核心文章9篇，申报国家发明专利2项。