结核分枝杆菌细胞壁多糖的动态化学修饰及其应用

The corresponding research emphasis of this project is "1. To develop new techniques and methods for accurate marking, detection and identification of new dynamic modifications of biological macromolecules" in the first research direction.. Peptidoglycan (PG) is an essential component of Mycobacterial tuberculosis (TB) cell wall and an important target for anti-TB drug development. However, our knowledge of PG metabolism has been severely hampered by the lack of method for probing its dynamics in host environment. Glucosamine-1-phosphate (GlcN-1-P) is a prokaryote-specific precursor that can be metabolically incorporated into the PG structure of TB. Previously we have shown that several non-natural GlcN-1-P derivatives (e.g. the fluoro-substituted GlcN-1-P, 3-F-GlcN-1-P) could also be employed by the biosynthetic machinery of TB and incorporated into the cell wall (Carbohydr. Res. 2011). In this project we plan to develop a chemical strategy for probing PG in vivo through metabolic incorporation of an azide-containing GlcN-1-P derivative (6-N3-3,4-Ac2-GlcN-1-P) into TB cell wall. Using the existing bioorthogonal chemistry imaging technique, the azide-modified bacteria would allow PG dynamics to be inspected in both living host cells and animals. Furthermore, the proposed approach would enable rapid and high-throughput techniques to be developed for the screening of anti-TB drugs and drug-resistant TB strains. These are also included in the scientific programs of this project.

本项目对应的研究重点是重点资助研究方向（一）中的“1.针对生物大分子新型动态修饰，发展精准标记、检测和鉴定的新技术、新方法”。.结核菌细胞壁肽聚糖是研发抗结核药物的重要靶标，然而由于其结构复杂、难以追踪检测，人们对于其在宿主环境内合成和代谢的动态机制知之甚少，在一定程度上限制了抗结核新药的发现。申请人前期（Carbohydr. Res. 2011）合成了葡萄糖胺-1-磷酸（GlcN-1-P）系列衍生物，明确了其可以作为外源物质进入结核菌特有的代谢途径并被组装到其肽聚糖结构中。本项目拟使用带有叠氮基团的GlcN-1-P衍生物作为外源前体，将叠氮基团修饰在结核菌细胞壁肽聚糖结构上，利用生物正交标记反应发展结核菌肽聚糖的高效标记新方法，实现其在宿主细胞及动物体内的动态示踪，并利用该示踪方法发展抗结核化合物和耐药结核菌的高通量筛选技术，为认识肽聚糖的体内代谢规律、为结核病的防治提供基础性技术储备。

结核菌细胞壁肽聚糖是研发抗结核药物的重要靶标，然而由于其结构复杂、难以追踪检测，人们对于其在宿主环境内合成和代谢的动态机制知之甚少，在一定程度上限制了抗结核新药的发现。本项目围绕重大研究计划中“针对生物大分子新型动态修饰，发展精准标记、检测和鉴定的新技术、新方法”这一指南方向，选择结核分枝杆菌细胞壁肽聚糖为研究靶标，通过化学生物学手段，建立了其高效的实时荧光标记方法，并将该标记体系应用于靶向结核分枝杆菌细胞壁肽聚糖药物的筛选，通过本项目平台，目前已得到5种在细胞水平具有显著抗结核能力的候选药物，目前正在进行深入的药物学研究。本项目的研究成果提供了一种全新的结核分枝杆菌肽聚糖高效标记方法和抗结核药物的高通量筛选技术，不仅为认识肽聚糖的体内代谢规律提供了一种工具，同时还为结核病的防治提供基础性技术储备。