Inhibition of prokaryote-specific saccharide biosynthesis in microbial pathogens

描述（由申请人提供）：众所周知，抗生素耐药性是对抗微生物病原体的关键问题。不太为人所知的是抗菌治疗新方法的发展方向。就在过去几年中，细菌细胞表面糖缀合物，包括许多医学相关的革兰氏阴性细菌病原体的外细胞壁和细胞表面N-和O-连接的糖蛋白的脂多糖组分，已经在分子细节上进行了表征，并发现对于毒力和致病性是必不可少的。该提案旨在进一步定义和利用产生在这些糖缀合物中发现的不寻常的微生物特异性碳水化合物构建块的途径，从而提供一种对抗细菌病原体的新方法。 本研究特别关注在C的N-和O-连接蛋白糖基化途径中参与UDP-GlcNAc转化为UDP-二-N-乙酰基-杆菌胺（UDP- diNAcBac）的酶的抑制剂的开发和体外和体内验证。jejuni和N.淋病由于UDP-diNAcBac是导致细菌糖缀合物生物合成的途径中的关键中间体，因此这些抑制剂可用作选择性化学工具来阐明高度修饰的双磷酸腺苷在微生物发病机制中的基本作用。本研究的实验方法包括：1.应用结构指导的基于片段的筛选（FBS）策略开发有效的UDP-二NAcBac生物合成抑制剂; 2.在检测糖蛋白生物合成、细胞毒性以及抑制糖蛋白生物合成对天然生物体中的运动性、粘附性和侵袭性的影响的试验中，评价优化的抑制剂。jejuni和N.淋病; 3.建立了一个C. elegans模式jejuni和N.淋病的传染性和毒力。如果成功的话，这种动物模型系统将有价值的评估抑制活性在一个简单的主机; 4。对C.与阿尔伯塔大学的Szymanski合作，在鸡传染性模型中使用空肠UDP-diNAcBac生物合成抑制剂。 这项研究解决了一个中心假设，即致病菌中导致高度修饰的糖构建块（如二-N-乙酰基-杆菌胺）的生物合成途径代表了一个可以在对抗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则将适用于其他微生物病原体，实现原核生物特异性N-和O-连接的糖蛋白作为毒力因子。如果成功，该研究将确定新的酶靶点和战略，以应对抗生素耐药性不断升级的全球传染病危机。

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.