具有新抗菌模式的新型抗耐药菌红霉素的设计合成及其杀菌机制研究

The rising and increasingly prevalent bacterial resistance has led to the loss of the potency of the clinical antibiotics. Discovery of an effective antibacterial mode is crucial to structural modification of the skeleton against the resistance. Erythromycin is a protein synthesis inhibitor by blocking the release of nascent proteins. Recently we found if the bacteriostatic agent erythromycin was appropriately modified with a mode of interaction with the gate-function base A2062 located in the bacterial ribosomal nascent peptide exit tunnel (NPET), the resulting derivatives not only are against resistant pathogens but also become bactericidal agents. Thus we hypothesize the bactericidal activity results from a novel mechanism of modulating the gate-function base followed by selective release of some specific proteins. In this proposal, a new protocol of rational design based on the multi-base of the rRNA will be explored with the aid of the partial covalent molecular docking and the available complex crystal structures of RNA and erythromycins. By this way, we will unravel how to enhance the activity of new erythromycins against resistant bacteria by interacting with the rRNA multi-base including the gate-function base A2062. Furthermore, we will combine the advantage of modulating both the ribosomal bases and the protein networks, and synthesize more potent erythromycin derivatives. Then, X-ray diffraction crystallography plus chemfootprinting and differential proteomics techniques will be utilized to illuminate the effect of the obtained bactericidal erythromycins on the bacterial rRNA bases and the subsequent protein expression profiling, respectively. Finally, optimizing drug-like properties in combination with analysis of the structure-activity relationships will shed light on how to fight effectively against the erythromycin-resistant bacteria. This proposal is not limited by well-known mechanisms targeting at the bacterial ribosome, which account for a half of the clinical antibiotics. The research will pave a new avenue for future design of antibiotics facilitated by the protein networks-based bactericidal mechanism studied herein.

细菌耐药导致抗生素纷纷失效，新抗菌模式成为指导抗耐药菌结构修饰的关键。红霉素的抗菌机制是阻碍新生蛋白从核糖体输出而抑菌，我们前期合成一种新型红霉素，作用细菌核糖体新生肽输出通道门靶点，不但抗耐药菌活性高还呈现杀菌剂特点。故此本申请提出假说：优化的杀菌活性与调控门靶点所致的选择性释放功能蛋白的新途径相关。本项目拟借助部分分子共价对接和核糖体与红霉素复合物的晶体数据虚拟设计，研究亲和核糖体门靶点在内的新型多靶点杀菌红霉素分子的合成策略，揭示门靶点协同其它靶点提高抗耐药菌活性的科学规律；然后利用多种生化手段研究杀菌机制的内在结构基础、差异蛋白质组学揭示调控门靶点对蛋白表达谱的影响，探索调控靶点与调控蛋白相结合的结构修饰新思路；最后优化杀菌红霉素结构的类药性质并提高代谢安全性，系统阐明杀菌红霉素的构效关系。临床抗生素半数以核糖体为靶标，本申请突破传统模式，可为未来提供一个调控蛋白网络的新抗菌模式。

红霉素类药物是临床常用的抗生素，然而耐药菌导致其疗效大大降低。为此，我们开展了新型抗耐药菌大环内酯设计及其作用机制研究。大环内酯为传统的抑菌剂，我们通过结构修饰使得新型红霉素对包括耐药菌在内的多数临床分离致病菌株获得杀菌活性，并首次系统总结了大环内酯结构与杀菌活性关系。目前唯一上市的抗耐药菌红霉素-泰利霉素由于代谢不稳定导致脱靶的肝毒性，这使得其使用大大受限。我们通过对十四元的2-氟酮内酯的构效和构性关系研究，发现比泰利霉素半衰期长、血药浓度高而且体外抗菌活性相当的抗耐药菌化合物，同时其对CYP3A4的抑制浓度较高，药物相互作用风险得以降低。首次设计合成具有作用核糖体和拓扑异构酶双靶点的十五元大环内酯新结构，尽管其对拓扑异构酶的活性比喹喏酮低一个数量级，而且对高水平耐药的组成型耐药菌也无抗菌活性。进一步的，全面研究了新型十四元非酮内酯构效关系后，对最优化合物与核糖体复合物晶体解析，发现了可用作大环内酯抗组成型耐药菌的新位点；作用该位点的化合物与泰利霉素不具有交叉耐药性，对目前临床主要耐药机制流行病菌如含有erm和mef的耐药菌具有较好的抗菌活性。这些成果为今后理性设计具有新机制的新型抗耐药菌分子奠定了坚实基础。