基于PT蛋白黄曲霉毒素生物合成抑制剂设计、合成和生物活性研究

Aflatoxins are extremely toxic, carcinogenic compounds, widely containted and seriously threaten the quality and safety consumption of agri-products. Recent research results indicated that the product template protein of polyketide synthase was the key protein which initiated biosynthesis of aflatoxins and became the aim of the aflatoxins biological control and prevention. Our experiments showed that the natural monoterpene compound of citral could elicit biological effect with the product template protein and showed potent inhibitory activity against aflatoxin biosynthesis. The product template protein of polyketide synthase is an ideal target for selective inhibitive compounds of aflatoxins. Based on the three dimensional structure and biological datas of the product template protein, the purpose of the study is rational design, organic synthesis and biological evaluation of citral derivates on the product template protein activity and aflatoxin biosynthesis. By modeling and docking, the molecular mechanism between potent inhibitive compounds and the product template protein will be investigated and provide the scientific guideline for green and efficient biological control research of aflatoxin.

黄曲霉毒素毒性大，致癌力强，污染广，严重威胁农产品消费安全。最新研究表明，聚酮体合酶PT蛋白是黄曲霉毒素生物合成关键蛋白，是黄曲霉毒素生物合成源头，成为黄曲霉污染防控关键调控点。申请人研究表明，天然单萜类化合物柠檬醛对该PT蛋白活性具有影响，并抑制黄曲霉毒素合成。因此，拟以聚酮体合酶PT蛋白为靶点，天然单萜类化合物柠檬醛为先导物，采用基于PT蛋白三维结构合理分子设计、有机合成方法进行结构修饰、合成系列衍生物，并测定其对PT蛋白活性以及黄曲霉毒素生物合成量的影响；并筛选出高效抑制的柠檬醛衍生物，采用计算机辅助分子对接模拟PT蛋白与衍生物互作稳定空间形式，初步揭示所选化合物抑制PT蛋白活性分子机制，为绿色、高效的黄曲霉毒生物防控研究提供科学指导。

采用生物筛选的方法得到对黄曲霉毒素具有抑制活性的天然单萜类化合物柠檬醛为先导化合物，保留先导物小分子具有活性的分子骨架，优化合成路线和反应条件，合成得到柠檬醛肟酯化合物4个。建立黄曲霉毒素聚酮体合酶PT蛋白生物活性筛选平台，利用SPE C18柱和免疫亲和柱富集净化，HPLC-MS-MS定性定量技术动态监测重要中间体代谢物Ⅰ（C17H14O6）和代谢物Ⅱ（C14H10O6），并分析其化合物质谱裂解规律、确证其结构。采用免疫亲和法富集净化样品中黄曲霉毒素，HPLC-MS-MS方法的AFB1、AFB2、AFG1和AFG2检出限分别为0.002、0.004、0.004 和0.012 µg/kg，加标回收率为87-111%，方法前处理简单，抑制了基质效应，能够满足动态确证性定量监测柠檬醛对黄曲霉毒素生物合成的影响。利用Pks的PT蛋白三维结构，采用计算机辅助设计，研究配体柠檬醛与PT蛋白功能关键氨基酸残基相互作用空间位置、分子间作用力，发现柠檬醛与活性位点氨基酸残基Asn1568之间发生氢键作用，对黄曲霉毒素的生物合成具有抑制作用，为黄曲霉毒素生物防控提供理论指导和技术支撑。