基于靶点活性的双重原位抗分支杆菌素高通量筛选平台的建立以及分支杆菌DPPS抑制剂的筛选和优化研究

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a life-threatening infectious disease with high mortality caused by a single pathogen, which calls for new chemical entities with diverse structure scaffolds targeting novel molecular targets. Current screening methods for antimycobacterials suffer from problems such as difficulty in bacteria engineering, high rates of off-target effect, low screening efficiency and overlook of the in situ molecular environment of the host, which results in an urgent need for new screening method. With Self-Assembled Monolayers for MALDI-TOF Mass Spectrometry as an analytical tool, this proposal aims at developing a target activity-based HTS platform for antimycobacterials against the dual-in-situ model of mycobacterium-infected macrophage. HTS will be performed against mycobacterial decaprenyl diphosphate synthase that is essential in cell wall biosynthesis, followed by lead optimization. With the proposed HTS platform as a technical support, the efficiency and success rate of antimycobacterials discovery will be largely enhanced, and drug candidates with high enzyme activity, high intracellular bacterial growth inhibition and low toxicity will be developed to advance tuberculosis therapy and prognosis.

由结核分枝杆菌引起的结核病是单一致病菌感染导致死亡率最高的感染性疾病，筛选靶向新靶标、化学结构新颖的新化学实体作为新一代抗分支杆菌素对于预防和治疗结核病至关重要。现有的针对抗分枝杆菌素的筛选方法存在细菌模型建立难度高、筛选效率低和易产生脱靶效应等缺陷，且忽略对宿主原位环境的考虑。本项目提出以自组装单层膜结合基质辅助激光解析电离质谱为技术基础，考虑靶点在细菌菌体内和细菌在宿主细胞胞内的双重原位条件，以受分枝杆菌感染的巨噬细胞为实验对象，构建直接检测靶点活性、双重原位的抗分支杆菌素高通量筛选平台。在此基础上，选择分支杆菌细胞壁生物合成中必需的顺式十聚异戊二烯二磷酸合酶为靶点（DPPS），开展抑制剂筛选和优化研究。本项目有望为抗分支杆菌素研发提供技术支撑，提高其研发成功率，同时得到具有高酶活性、高细胞内杀菌活性、低毒性的抗分枝杆菌素，为结核病的有效治疗以及预后提供方案。

由结核分枝杆菌引起的结核病是单一致病菌感染导致死亡率最高的感染性疾病，筛选靶向新靶标、化学结构新颖的新化学实体作为新一代抗分支杆菌素对于预防和治疗结核病至关重要。本项目以分支杆菌细胞壁生物合成中必需的顺式十聚异戊二烯二磷酸合酶为靶点，通过高通量抑制剂筛选，得到微摩尔级抑制剂，并且此抑制剂具有良好的体外抑菌活性以及基于线虫模型的体内活性。在针对抗分支杆菌素的开发上，本项目还以细胞膜和DNA作为双靶向，对具有双重选择性作用机制的抗耐药寡聚物进行了开发。利用细菌DNA位于无膜保护的拟核中，而真核生物的DNA受到核膜的保护这一物种差异性，获得具有更好选择性的抗菌寡聚物，在保证抗菌效果的前提下降低其真核细胞毒性，得到了对巨噬细胞内分支杆菌的良好治疗效果。