以ArgR为靶标的新型4-羟基香豆素类抗菌药物构效关系及其机制研究

Antibiotic resistant bacteria infection is the leading cause of death caused by severe infection, novel antibacterial agents are the most important treatment to control the infection induced by antibiotic resistant bacteria, and finding the new drug targets is the breakthrough for development of novel antibacterial agents. Arginine and its metabolic products play important roles in bacterial resistance mechanism. Once arginine bindings to arginine repressor (ArgR), the expression of arginine metabolic pathway key enzyme and the metabolic products are elevated. As the bacterial specific protein, ArgR is the ideal drug target for novel antibacterial agents. However, there is no available antimicrobial agent targeting this protein, and it is still unclear how arginine metabolites and downstream effector molecules involve in the mechanism of drug resistance of bacteria. Our previous study found that 4- hydroxy coumarin compounds had remarkable activity against antibiotic resistant bacteria, and strongly suggested that these compounds could inhibit the expression of arginine metabolic pathway key enzyme through ArgR. This project aims to take ArgR as the breakthrough point, using bioinformatics, morphological and molecular biological techniques, combined with gene mutant bacterial strain in vitro and infection animal model in vivo, to reveal the molecular model of 4-hydroxy coumarin compounds competitive binding to ArgR with arginine, elucidate the signaling pathway of arginine metabolism and downstream effector molecules involved in the drug resistance of bacteria. It will provide an important theoretical basis for ArgR as a new antibacterial drug target and the development of novel antimicrobial agents, as well as provide a novel strategy for the treatment of antibiotic resistant bacteria infection.

耐药细菌感染是造成重症感染死亡的首要原因，新型抗菌药物是有效控制耐药细菌感染的主要手段，而发现新的药物靶标是新型抗菌药物研制的突破口。精氨酸及其代谢产物对细菌耐药至关重要，精氨酸与精氨酸阻遏子(ArgR)结合后，精氨酸代谢通路关键酶表达和代谢产物增加，细菌特有蛋白ArgR是理想的抗菌药物新靶标，但目前尚未发现有效作用该靶点的抗菌药物，精氨酸代谢产物和下游效应分子参与细菌耐药机制也并不清楚。我们前期研究发现4-羟基香豆素化合物能抑制精氨酸代谢通路关键酶的表达，具有显著抗耐药菌活性。本课题拟以ArgR作为切入点，采用生物信息学、形态学和分子生物学等技术，以基因突变菌株和感染动物模型相结合，揭示4-羟基香豆素化合物竞争拮抗精氨酸结合ArgR的分子模式，深入阐明精氨酸代谢产物及下游效应分子参与细菌耐药的信号通路，为发现新药物靶标和研制新型抗菌药物提供重要理论依据，同时为耐药细菌感染提供新的治疗策。

耐药细菌感染是临床抗感染治疗中极为棘手的问题，也是造成重症感染死亡的首要原因，发现抗耐药细菌的新药物靶标和研制新型抗菌药物是其重要突破口。本项目以多种临床感染最常见、危害性大的耐药菌为研究对象，采用药物化学、药理学、生物信息学和分子生物学等实验方法，合成了系列不同化学结构的新型4-羟基香豆素化合物并进行了结构鉴定，香豆素苯环上的取代基对化合物的抗菌活性影响较大，其中强吸电性原子如卤族元素F、Cl、Br、I以及CF3等取代后抗菌活性明显增强；抗菌谱研究发现4-羟基香豆素类衍生物DCH对革兰阴性菌抗菌活性较差，而对多重耐药细菌在内的革兰阳性菌抗菌活性较好；化合物DCH可以在蛋白和基因水平抑制精氨酸代谢通路关键酶及其编码基因arc簇的表达；具有抗菌活性的4-羟基香豆素类衍生物与调控arc基因簇表达的精氨酸抑制子(ArgR) 相互作用亲和力常数在10-8-10-7 之间，其中化合物DCH的Ka为1220 Ms-1，Kd为9.97×10-5 s-1，KD为8.20×10-8 M，显示出较好的亲和性；分子对接结果显示化合物DCH主要与ArgR蛋白分子C端结合口袋的GLN25和 ASP46氨基酸残基结合；化合物DCH能够明显抑制MRSA离体细菌和大鼠尿道插管生物被膜的形成，及生物被膜相关毒力因子icaA，aaP，srtA和atlE的表达，且存在浓度依赖性；在构建的argR基因缺陷MRSA菌株，生物被膜形成能力及相关毒力因子表达明显减少；化合物DCH处理或argR基因敲除后MRSA细菌内精氨酸和鸟氨酸水平明显增高，而亚精胺和精胺水平 明显升高；化合物DCH没有明显的抗凝活性、细胞毒性和急性毒性，短期内不易诱导细菌产生耐药性，有较好的成药潜力。本课题研究结果将为抗耐药菌药物的研发提供新思路和新靶标，具有重要的理论意义和实际应用价值。