卵菌PDHc E1蛋白表达体系的建立及相关抑制剂的设计合成与筛选

Many oomycetes are destructive pathogens that cause damages to a wide range of agriculturally important plants and also to species associated with forestry and natural ecosystems. Currently, chemical control is still one of the most effective methods to control against oomycete diseases. However, only a very few compounds are available for the control of the diseases in field because of resistance problems. Therefore, it is necessary to develop new classes of fungicides with new mode of action to control the diseases and to manage resistance problems. Previous studies have shown that pyruvate dehydrogenase complex E1 component (PDHc E1) which is a pyrophosphate thiamine-dependent (ThDP-dependent) enzyme and plays a crucial role in cellular metabolism, is a promising target for exploring new pesticides. However, the commercial PDHc E1 inhibitors against phytopathogenic oomycetes are rarely reported. Our group has been working on the PDHc E1 inhibitors for many years. According to the difference in the binding of ThDP in PDHc E1 in different organisms, we have successfully discovered the lead compound L1, which shows a potentially high activity against pathogenic microorganisms and a high safety for warm-blooded animals. This project is intended to design and synthesize series of new compounds based on the structure of L1, and to establish the expression and purification system of PDHc E1 of Phytophthora sojae which can be used for high-throughput screen for highly active PDHc E1 inhibitors out of the newly synthesized compounds. Meanwhile, the antifungal activity against the mycelium of P. sojae will be examined and the structure-activity relationship will be analyzed. Then, for compounds with a high activity against P. sojae, the control spectrum and the control efficacy in greenhouse will be determined. The binding mode of the active compounds in PDHc E1 will also be studied to provide information for further design of new PDHc E1 inhibitors. In this study, we are trying to design, synthesis and screen out PDHc E1-inhibitors with high activity against oomycete diseases. The results would be helpful for the control of oomycete diseases and design of new compounds targeting PDHc E1.

植物病原卵菌危害大，且生产中可使用的药剂种类较少。多项研究表明焦磷酸硫胺素ThDP依赖性酶-丙酮酸脱氢酶PDHc E1是一个重要的农药靶标，但迄今尚无针对病原卵菌的PDHc E1抑制剂报道。本课题组在前期研究中根据ThDP在不同生物PDHc E1中结合的差异性，设计合成并发现了对温血动物安全的微生物PDHc E1高效抑制剂L1。本项目拟以大豆疫霉的PDHc E1为靶标，选择L1为先导化合物进行修饰改造，设计合成全新的化合物；首次建立大豆疫霉PDHc E1表达和纯化体系，用于高通量快速筛选所设计合成的新型化合物，并分析构效关系，确定结构优化策略，同时开展对大豆疫霉菌体杀菌活性的筛选以及杀菌谱和温室防效的研究，获得特异性针对卵菌的PDHc E1抑制剂；并明确与抑制活性相关的氨基酸位点，建立蛋白-化合物相互作用模型，为后续高选择性高活性的新型PDHc E1卵菌抑制剂的结构修饰和优化提供科学依据。

丙酮酸脱氢酶PDHcE1是一个重要的农药靶标，但目前国际上并无有关植物病原微生物PDHcE1的研究。本研究选择大豆疫霉为靶标，依次探索了其PDHcE1蛋白（PsPDHcE1）的原核和真核表达体系，首次成功表达了PsPDHcE1的两个亚基蛋白Psα和Psβ，并在体外成功获得了两个亚基融合的二聚体蛋白，但进一步研究表明所获单体蛋白及二聚体蛋白均无丙酮酸脱氢酶的活性，试验未能继续深入下去。水稻白叶枯病菌所引起的水稻白叶枯病是一种严重危害水稻生产的细菌性病害。据统计，当前市场上可用于细菌病害防治的有效药剂较少，且近二十年内几乎无新型的杀细菌剂的报道，故建立以水稻白叶枯病菌PDHcE1（XooPDHcE1）为分子靶标的杀细菌剂筛选体系，用于新型杀细菌剂的快速高通量筛选同样具有十分重要的意义。因此在该项目的支持下，本研究同时选择了水稻白叶枯病菌，首次建立了其XooPDHcE1的表达和纯化体系以及体外酶活测定方法，并首次成功获得了高纯度高活性的XooPDHcE1蛋白，且已用于相应PDHcE1抑制剂的高通量快速筛选。此外，本研究优化设计并合成了多个系列具有新型结构的目标化合物，筛选了它们对XooPDHcE1的抑制活性，并获得了具有一定酶抑制活性的化合物（IC50=3.6μM和14.1μM），可以作为先导化合物进一步优化。同时筛选获得了多个对重要农作物病原菌如水稻白叶枯病菌、烟草青枯病菌、桃褐病菌等具有高抑制活性的化合物，可作为新型杀菌先导或具有潜在应用价值的新型杀菌剂进一步探索，为高活性杀菌剂的研发奠定了基础。