高效、高选择性的PDHc E1抑制剂的合理设计、合成与杀菌活性研究

Pyruvate dehydrogenase complex E1-subunit (PDHc E1) from E. coli is chosen as a target of microbicide or fungicide in this project. Analogues of coenzyme TPP (thiamine pyrophosphate ) as a kind of E. coli PDHc E1 inhibitors have been reported, but most of these E. coli PDHcE1 inhibitors showed lower application value as microbicide or fungicide due to no practicability and bad selectivity between mammals and bacteria. Aim at these problems, new analogues of TPP as E. coli PDHc E1 inhibitors are designed according to the difference of interaction of coenzyme TPP and E. coli PDHc E1 comparing with the interaction of TPP and mammals PDHc E1. Based on the three dimensional structure of E. coli PDHc E1 and pig PDHc E1 respectively, the virtual screening or predicting model are established by using molecular simulation technology, QM/MM, MD etc various calculations method. Meanwhile, the method for enzyme assays in vitro including E. coli and pig PDHc E1 will be set up on the basis of the genes clone and express. Triazole-containing 2-methylpyrimidine-4-ylamine derivatives Io as TPP analogues were found to be effective against E. coli PDHc E1 in our previous work. In this project, some compound Io is chosen as hit compound. On the basis of structure Io a number of novel TPP analogues I will be designed and synthesized for examining their enzyme-selective inhibition, microbicidal and fungicidal activity. The mode of action of active compounds I will be studied by enzyme kinetic experiment. Binding modes of the protein-inhibitor interaction will also be studied by molecular docking. Acting site in the active site of E. coli PDHc E1 can be comfirmed by site-specific mutagenesis. Structural optimization will be carried out on the basis of study on structure-activity relationship, and the relationship of enzyme -inhibitory activity and fungicidal activity. The microbicidal or fungicidal property of representative active compounds and their acute toxicity will be further evaluated. It is expected that some effective PDHcE1 inhibitors with practicability, lower acute toxicity and good selectivity between mammals and bacteria can be found by this project, and these active compounds can be used as new lead compounds or potential microbicide or fungicide. This project will provide experiment and theory basis for the biorational design of microbicide or fungicide which acts as novel PDHc E1 inhibitor.

针对现有文献报道的PDHc E1抑制剂作为杀菌剂应用存在选择性和成药性差的关键科学问题，本项目以微生物PDHc E1为杀菌剂靶标，根据辅酶TPP与微生物(大肠杆菌)和人体或猪PDHc E1相互作用的差异，提出设计选择性PDHc E1抑制剂的新思路和TPP类似物的新骨架。采用分子模拟技术和分子生物学实验相结合的方法，基于大肠杆菌和人体或猪PDHc E1的三维结构，模建理论筛选或预测体系指导分子设计，构建大肠杆菌和猪PDHc E1酶抑制活性的体外筛选方法；设计、合成、筛选高选择性、高活性的大肠杆菌PDHc E1抑制剂，研究其选择性机理，探明抑制剂与不同生物源PDHc E1的结合模式；定点突变验证抑制剂与靶酶关键作用位点；研究化合物的酶抑制活性与杀菌活性构效关系及杀菌作用机制；评价高效、高选择性抑制剂作为杀菌剂应用的成药性；探索发现高效、高选择性、低毒的PDHcE1抑制剂与新型杀菌剂先导体。

针对现有文献报道的丙酮酸脱羧酶(PDHcE1)抑制剂作为杀菌剂应用存在选择性差、成药性差的关键科学问题，本项目以微生物PDHc E1为杀菌剂靶标,根据辅酶TPP与微生物(大肠杆菌)和人体(或猪)PDHc-E1相互作用的差异，提出了设计抑制剂的新思路, 设计了14个系列TPP类似物的新骨架,建立了14个系列新型目标化合物的合成方法。采用计算机分子模拟技术模建理论筛选或预测体系指导分子设计, 合成了153个未见文献报道的新型目标化合物,完成了结构表征。采用分子生物学实验技术建立了大肠杆菌PDHc E1酶活体外筛选方法,筛选发现了一批对大肠杆菌PDHc E1的高效抑制剂。同时构建了猪PDHc E1酶抑制活性的体外筛选方法,考察了高活性大肠杆菌PDHc E1抑制剂对猪PDHc E1酶的抑制活性。研究发现了在相同的浓度下,对大肠杆菌PDHc E1具有高效抑制作用(抑制率为 100%)，但对猪PDHc E1无抑制作用或弱抑制作用(抑制率为 0-30%)的高选择性抑制剂。其代表性化合物显示了对班马鱼低毒的特点。通过分子设计合成，构效关系，结构优化，活性验证研究的多次循环，发现了几类优于前期先导骨架具有显著杀细菌和真菌活性的新型先导结构, 以及数个与某些商品化杀菌剂活性相当或更优的高活性化合物。特别是显著优于目前唯一报道的对丙酮酸脱氢酶有抑制作用的广谱杀菌剂克菌丹的活性。进一步采用分子对接、定点突变、酶学分析和荧光光谱分析相结合的方法，探明了本项目合成抑制剂在大肠杆菌和猪PDHc-E1活性空腔中的结合模式。并采用定点突变技术验证了抑制剂与靶酶的关键作用位点。从作用方式上解释了抑制剂的选择性机理，证明了设计思想的合理性和高效性。本项目的研究结果达到了预期的研究目的, 其结果对于创制高效、高选择性、低毒、环境友好的新靶标杀菌剂具有重要的科学意义。此外，本研究意外的发现对大肠杆菌PDHc-E1具有高效抑制活性的部分化合物对蓝藻具有显著的抑制活性。

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