Inhibition of prokaryote-specific saccharide biosynthesis in microbial pathogens

描述(申请人提供)：众所周知，抗生素耐药性是与微生物病原体作斗争的一个关键问题。鲜为人知的是抗菌治疗新方法的前进方向。就在最近几年，细菌细胞表面的糖偶联物，包括细胞外壁的脂多糖成分和许多医学上相关的革兰氏阴性细菌的细胞表面N-和O-连接的糖蛋白，已经被详细地表征，并被发现是毒力和致病性所必需的。这项建议旨在进一步定义和利用在这些糖共轭中发现的产生不寻常的微生物特异性碳水化合物积木的途径，从而提供一种新的方法来对抗细菌病原体。本研究主要针对空肠弯曲菌和淋球菌N-和O-连接蛋白糖基化途径中参与UDP-GlcNAc转化为UDP-diNAcBac(UDP-diNAcBac)的酶的抑制剂的开发和体内外验证。由于UDP-diNAcBac是导致细菌糖结合物生物合成的关键中间体，这些抑制剂可以作为选择性化学工具来阐明高度修饰的糖在微生物发病中的基础作用。这项研究的实验方法包括：1.应用基于结构引导的片段筛选(FBS)策略开发有效的UDP-diNAcBac生物合成抑制剂；2.在检测空肠弯曲菌和淋病奈瑟菌体内糖蛋白生物合成、细胞毒性以及抑制糖蛋白生物合成对体内原生生物运动、黏附和侵袭的影响的试验中，评估优化的抑制剂；3.建立空肠弯曲菌和淋病奈瑟菌的线虫模型。如果成功，该动物模型系统将有价值用于评估简单宿主中的抑制活性；4.与阿尔伯塔大学的Szymanski合作，评估空肠弯曲菌UDP-diNAcBac生物合成抑制剂在鸡传染性模型中的效果。这项研究解决了一个核心假设，即病原菌中导致高度修饰的糖组成块(如二-N-乙酰基杆菌胺)的生物合成途径代表着可以在抗击传染病的斗争中利用的“阿喀琉斯之踵”。我们在这些研究中开发的一般原理将适用于将原核生物特有的N-和O-连接糖蛋白作为毒力因子的其他微生物病原体。如果成功，这项研究将确定在面对不断升级的抗生素耐药性的全球危机中抗击传染病的新的酶目标和策略。 与公共卫生相关：半个世纪以来，人类用来击败细菌敌人的常用手段已经动摇，因为常见微生物病原体的抗生素耐药性对公共健康构成了越来越大的威胁。最近的研究澄清了细胞表面糖蛋白的产生途径，这些糖蛋白对致命的革兰氏阴性菌起着关键作用，使它们能够进入并攻击人类细胞。我们的提案将支持通过阻止糖共轭组合体中关键构建块的产生来开发这些与毒力相关的基本途径的工作，从而开发一套全新的抗菌治疗靶点。

DESCRIPTION (provided by applicant): It is well known that antibiotic resistance is a critical issue in the battle against microbial pathogens. Less well known is the way forward to new approaches in antibacterial therapy. Just in the last few years bacterial cell surface glycoconjugates, including the lipopolysaccharide component of the outer cell wall and cell surface N- and O-linked glycoproteins of numerous medically relevant Gram-negative bacterial pathogens, have been characterized in molecular detail and found to be essential for virulence and pathogenicity. This proposal aims to further define and exploit the pathways that produce the unusual microbe-specific carbohydrate building blocks that are found in these glycoconjugates, thus providing a novel approach to combat bacterial pathogens. This research focuses specifically on the development and in vitro and in vivo validation of inhibitors to enzymes involved in the conversion of UDP-GlcNAc into UDP-di-N-acetyl-bacillosamine (UDP- diNAcBac) in the N- and O-linked protein glycosylation pathways of C. jejuni and N. gonorrhoeae. Since UDP-diNAcBac is a critical intermediate in the pathways that result in the biosynthesis of the bacterial glycoconjugates, these inhibitors could be employed as selective chemical tools to elucidate the fundamental roles of highly modified saccharides in microbial pathogenesis. The experimental approach of the proposed research involves: 1. Application of a structure-guided fragment-based screening (FBS) strategy for the development of potent UDP-diNAcBac biosynthesis inhibitors; 2. Evaluation of optimized inhibitors in assays that probe glycoprotein biosynthesis, cell toxicity and the effects of inhibiting glycoprotein biosynthesis on motility, adherence and invasion in the native organism in vivo in C. jejuni and N. gonorrhoeae; 3. Establishment of a C. elegans model for C. jejuni and N. gonorrhoeae infectivity and virulence. If successful, this animal model system will be valuable for to assessing inhibitory activity in a simple host; 4. Assessment of the effect of C. jejuni UDP-diNAcBac biosynthesis inhibitors in the chick infectivity model in collaboration with Szymanski at the University of Alberta. This research addresses the central hypothesis that the biosynthetic pathways in pathogenic bacteria that lead to highly modified sugar building blocks, such as di-N-acetyl-bacillosamine, represent an "Achilles' heel" that can be exploited in the battle against infectious diseases. The general principles that we develop in these studies will be applicable to other microbial pathogens that implement prokaryote-specific N- and O-linked glycoproteins as virulence factors. If successful, the research will identify new enzyme targets and strategies in the global crisis of combating infectious diseases in the face of escalating antibiotic resistance. PUBLIC HEALTH RELEVANCE: The usual means that humans have used for half a century to defeat their bacterial foes have faltered, as antibiotic resistance of common microbial pathogens presents a growing threat to public health. Recent research has clarified pathways in the production of cell surface glycoproteins that play key roles for deadly Gram-negative bacteria, enabling their access and attack on human cells. Our proposal will support work to exploit these essential virulence-associated pathways by blocking production of critical building blocks in the glycoconjugate assemblies, thus developing an entirely new set of targets for antimicrobial therapy.