新型结核分枝杆菌选择性抑制剂的构效关系及靶点鉴定研究

The incidence of multidrug-resistant tuberculosis (MDR-TB) and the combined epidemics of HIV and tuberculosis are increasing.This worsening situation make the treatment extremely difficult for patients who are suffering from tuberculosis.Novel antitubercular drug candidates should address such issues as cross-resistance,long-time drug therapy, adverse effects and drug-drug interactions with anti-retrovirus treatment for AIDS. Whole cell-based phenotypic screening has resurged for its high productivity and intrinsic advantages over screening based on biological targets.Forward chemical genetics stratedy like whole genome sequencing offers the possibility of identifying and validating the biological targets of active molecules. In previous study of structural modification of Linezolid, we found a novel bis-oxazolidinone compound which demonstrated excellent and highly selective activities against H37Rv with an MIC value of 0.125mg/L.This property is quite different from the marketed Linezolid and other oxazolidinone drugs in clinical trials. Those oxazolidione drugs possess broad antibacterial spectrum to gram-positive bacteria. However, our compound decreased its potentcy on drug-resistant tuberculosis strains, whose underlying mechanism remained unclear. The present proposal plans to do research work on structure optimization of the hit bis-oxazolidinone compound aiming at improvement of efficacy on the drug-resistant tuberculosis isolates,establishment of the structure -activities relationship, and subsquent design, synthesis and selction of capture compounds for pull-down experiments in the study of chemical proteomics.Furthermore, genome-wide transcriptional analysis of drug treated strains of BCG and H37Rv in different culture conditions, and whole genome sequencing of the mutant strains induced by the active bis-oxazolidinone compounds will be carried out.Canonical correlation analysis will be used to elucidate the protein target(s), their binding mode(s) and drug-resistant machinary. Aslo we would like to analyze the selective mechanism between the species based on our experimental data and the published data, particular those found in NCBI database for gene and protein.

耐药结核菌的出现及与HIV病毒的共感染使得结核病的治疗异常困难。基于细胞水平的表型高通量筛选，进而采用化学蛋白组学等正向化学遗传学手段对活性分子的靶点进行鉴定，已成为抗结核治疗新机制及药物发现的有效策略。课题组在对利奈唑胺结构修饰研究中，偶然发现一个双噁唑烷酮化合物，对结核分枝杆菌H37Rv具有选择性抑制活性（MIC=0.125mg/L），这种特点显著区别于已上市的利奈唑胺和处于临床研究阶段的噁唑烷酮类药物的广谱抗菌性质。但它对临床分离的耐药结核菌株出现不同程度的药敏下降，机制尚不清楚。本项目拟对该双噁唑烷酮化合物做结构优化，在寻找对耐药结核菌效果更优的类似物的同时，阐明构效关系，并据此设计合成活性三功能俘获探针分子，开展基于活性的化学蛋白组学研究，结合转录组和基因组分析结果，筛选确认双噁唑烷酮化合物的作用靶点蛋白,分析其选择性机制，为抗结核药物治疗新机制及候选药物发现奠定实验和理论基础。

耐药结核及与艾滋病的共感染呈现增加态势，使得临床结核病人的治疗异常困难。结核病特别是耐药结核的治疗仍然迫切需要新结构候选分子和抗结核新机制的发现，以应对临床未满足需求。本课题对前期表型筛选得到的噁唑烷酮类抗结核先导分子，以及本课题通过综合利用计算机模拟、活性分子化合物库筛选（包括基于靶点和基于抗菌活性表型的筛选）发现的吡咯、萘醌、嘧啶等新颖骨架类型的苗头分子进行了结构改造，研究了它们的构效关系，并发现了4个先导分子，为该类化合物的成药性研究奠定了基础，若继续深入研究，具有潜在的转化价值。通过新探针分子-MtbClpP1P2的共晶实验和结合模式解析，提供了新探针分子作用于靶点蛋白的直接证据，并且发现新探针分子结合在MtbClpP1P2的P1亚基赤道手柄区。通过序列比对，发现该区域氨基酸残基在不同种系间保守性不高，区别于高保守的活性中心口袋或伴侣蛋白ClpX/ClpC结合域，因此有可能成为一个可靶性位点，实现对正常菌群和宿主细胞的选择性，具有重要的科学意义和潜在的应用价值。综上，本课题研究成果发现了新的抗结核先导分子和结核分枝杆菌酪蛋白水解酶ClpP1P2的新的探针分子结合口袋，可作为潜在的选择性抗结核药物靶标位点进行深入考察，为临床耐药结核的治疗提供了潜在新机遇，为该领域的进一步深入研究奠定了基础。