新型抗MRSA持留菌小分子的构效关系和靶点确证研究

The persister cells are considered as the main reason that results in chronic infections and secondary infections in clinic. Moreover, it has been proved that persister could assist genetic resistance and the continued presence of persisters effectively acts as a reservoir for resistant mutancts. Whole cell-based phenotypic screening has been valued as an important tool of anti-persister drug discovery for its high productivity and intrinsic advantages. Combination of chemical proteomics and transcriptomics offers the possibility of identifying and validating the biological targets of anti-persister agents..In previous study of whole cell-based high-throughput screening of a compound library, a novel active compound DEL22379 was found that it could eradicate ATCC33591 persister cells at a concentration of 32 μg/mL. 1H NMR analysis showed that the commercial product of DEL22379 exists as a mixture of cis-trans isomerism. The further anti-persister activity evaluation of monocomponent indicated that trans-configuration component of DEL22379 displayed higher activity against MRSA persister cells over the cis-configuration component. To seek an more active anti-persister cell agent and figure out the how this kind of molecule work, the present proposal plans to do research work on rational structure optimization of the hit DEL22379 aiming at improvement of efficacy, establishment of the structure -activities relationship, and subsequent design, synthesis and selection of capture compounds for pull-down experiments. Then, based on the chemical proteomics and transcriptomics research, the protein target(s) and their binding mode(s) of this kind of active molecules could be elucidated.

持留菌已成为临床上慢性感染和继发性感染的罪魁，更为细菌由表型变异发展为基因突变的耐药菌株提供了重要条件。基于细菌水平的表型筛选是发现抗持留菌药物的直接有效手段，而结合化学蛋白组学和转录组学方法是研究抗持留菌药物作用机制的有效策略。申请人前期基于抗MRSA持留菌活性评价模型，对化合物实体库进行高通量筛选，发现DEL22379在32μg/mL时可完全清除ATCC33591持留菌。1H NMR结构鉴定表明商购DEL22379为顺反异构共存的混合物。单组分活性研究表明，反式组分抗持留菌活性显著优于顺式组分。为了获得全新的活性更优的抗持留菌化合物及鉴定可能药物靶点，本项目拟对DEL22379进行合理结构优化，在寻找活性更优的抗持留菌化合物的同时，阐明构效关系，并借助于化学蛋白组学和转录组学研究策略，综合分析该类化合物对MRSA持留菌可能的作用靶点，为抗MRSA持留菌药物的研究奠定理论依据和实验基础。

持留菌已成为临床上慢性感染和继发性感染的罪魁，更为细菌由表型变异发展为基因突变的耐药菌株提供了重要条件。申请人前期基于抗MRSA持留菌活性评价模型，发现反式DEL22379的抗持留菌活性高于其顺式组分的抗持留菌活性，申请人据此结论，对DEL22379进行结构优化，合成了100个以上的全新结构的衍生物。并对新合成化合物进行体外抗持留菌活性测试及抗常见革兰氏阳性菌活性测试，探讨了详尽的构效关系。我们对其体外毒性和溶血毒性进行初步考察，首先，对体外活性较好的优选化合物进行的溶血实验发现，该类化合物的溶血毒性较小，如活性最优的化合物353在高浓度时（100μM）时，溶血率仅为1.5%。另外，我们通过MTT法考察了优势化合物的体外细胞毒性，发现该类化合物对部分肿瘤细胞HEK293以及正常细胞L02，都具有一定的细胞抑制活性，提示该类化合物的安全性有待进一步的考察。对该类化合物的成药性进行评价，发现其水溶性极差，因此我们设想将水溶性较差的3位硝基咪唑基团替换为水溶性较好的硝基呋喃基团，为了进一步增强其水溶性，我们在5位引入了一个烷基酰胺基，设计了化合物ZY354，随后对其进行了抗菌效果测试，发现其对三株口腔相关细菌S. mutans，S. gordonii和S. sanguinis的MICs在0.12到0.49 µg/mL；MBCs在0.24到1.95 µg/mL。更重要的是，其对上述三株口腔相关细菌的生物膜也具有杀灭作用，其MBICs在0.24到0.49 µg/mL；MBECs在0.12到31.25 µg/mL。随后对ZY354进行了人细胞毒性测试，发现其与临床常用药物CHX相比具有更低的细胞毒性，其对HOK、HEG、RAW三株细胞的IC50均大于62.5µg/mL。随后对ZY354的抗生物膜活性进行了进一步的评价，对ZY354作用后的生物膜进行了活/死细胞荧光染色标记实验以直观的观察其生物膜杀灭活性。所以，通过优化， ZY354是一个具有较好成药性和潜在的应用前景的优选化合物。