Use of De Novo Synthesis Approaches and Structure-guided Design to Optimize Therapeutic Properties of Streptothricin Class Antimicrobials

抗菌素耐药性的迅速出现给细菌感染的治疗带来了巨大的挑战。耐碳青霉烯类肠杆菌科(CRE)、鲍曼不动杆菌和铜绿假单胞菌尤其令人关注。我们显然需要几种新的革兰氏阴性药物，它们在责任和抗菌类别方面是独一无二的，可以使我们的抗菌药物开发组合多样化。这个多PI方案研究的是被称为链霉毒素的天然产物，它含有三个部分：链甲醚、古罗糖胺糖和一条不同长度的β-赖氨酸或多β-赖氨酸链。链霉毒素是在70多年前被发现的，它通过大量的蛋白质错误编码来抑制蛋白质翻译。在初步实验中，我们确定链霉菌素(天然产物混合物也称为营养苏糖素)对多重耐药的革兰氏阴性病原体具有广泛的活性。特别是，对于链霉毒素-F，它只有一个β-赖氨酸部分，我们在体外和体内鉴定了令人信服的活性。然而，在革兰氏阴性病原菌中发现的几种链霉素乙酰转移酶是通过乙酰化β-赖氨酸残基的β-胺来产生链霉素耐药性的。这些观察结果导致了我们的假设，即抗菌活性可以从毒性中分离出来，同时这些抗生素耐药元件可以通过β-赖氨酸部分和其他成分的衍生化/替换来阻断。因此，本提案的目标是使用高效、面向多样性的药物化学合成链霉菌素类似物来进行假说驱动的构效关系研究，以优化这种支架的治疗性能。特别是，我们建议对每个链霉素类似物进行功能分析，以确定对有问题的革兰氏阴性病原体的效力、对原核核糖体的选择性、毒性和代谢稳定性。优先顺序的类似物将在小鼠模型中进行毒性、药物清除和治疗效果的测试。此外，优先考虑的类似物将在基于低温EM的结构和自动对接研究中进行研究，以了解这些分子变体如何差异地结合到鲍曼不动杆菌70S核糖体上。在拟议工作期间，将利用这些研究的结构见解提供迭代反馈，以优化类似物的设计。综上所述，该提案目的的实验将解决链霉菌素文献中缺乏系统探索的问题，并确定能够进行生产性修饰的分子成分，以增强这种支架的性能，作为未来的治疗药物。

The rapid emergence of antimicrobial resistance presents a significant challenge for treatment of bacterial infections. Carbapenem-resistant Enterobacteriaceae (CRE), Acinetobacter baumannii, and Pseudomonas aeruginosa are of particular concern. We are clearly in need of several new Gram-negative agents that are unique in terms of liabilities and antimicrobial class and which can diversify our antimicrobial development portfolio. This multi-PI proposal investigates natural products called streptothricins, which contain three moieties: streptolidine, a gulosamine sugar, and a single β-lysine or poly-β-lysine chain of varying length. Streptothricins were identified over 70 years ago and inhibit protein translation with extensive protein miscoding. In preliminary experiments, we determined that streptothricins (the natural product mixture is also called nourseothricin) are broadly active against multidrug-resistant Gram-negative pathogens. In particular, for streptothricin-F, which has a single β-lysine moiety, we identified compelling activity in vitro and in vivo. However, several streptothricin acetyl transferases, found in low frequency in Gram-negative pathogens, confer streptothricin resistance by acetylation of the β-amine of the β-lysine residue. These observations led to our hypothesis that antibacterial activity can be separated from toxicities and at the same time these antibiotic resistance elements blocked through derivatization/replacement of the β-lysine moiety and other constituents. Therefore, the goals of this proposal are to use an efficient, diversity- oriented, medicinal chemistry synthesis of streptothricin analogues to perform hypothesis-driven structure- activity relationship studies to optimize therapeutic properties of this scaffold. In particular, we propose to functionally profile each streptothricin analogue to determine potency against problematic Gram-negative pathogens, selectivity for prokaryotic ribosomes, toxicity, and metabolic stability. Prioritized analogues will be tested in a mouse model for toxicity, drug clearance, and therapeutic efficacy. Furthermore, prioritized analogues will be investigated in cryo-EM based-structural and auto-docking studies to understand how these molecular variants differentially bind to the A. baumannii 70S ribosome. Structural insights from these studies will be used to provide iterative feedback to optimize design of analogues during the course of the proposed work. Taken together, the experiments in the aims of the proposal will address the lack of systematic exploration in the streptothricin literature and identify molecular constituents that are amenable to productive modification to enhance properties of this scaffold as a future therapeutic.