Inhibition of Glycoprotein Biosynthesis in Gram-Negative 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。不太为人所知的是在抗菌治疗中前进的新方法，以解决耐药性发展的严重后果。在过去的十年里，细菌细胞表面的糖偶联物，包括几种医学上相关的革兰氏阴性细菌细胞外壁的脂多糖成分和细胞表面N-和O-连接的糖蛋白，已经被详细地表征，并被发现对宿主依赖的毒力和致病性是必不可少的。我们建议采用高通量小分子筛选策略来寻找生物合成二-N-乙酰基杆菌三胺(DiNAcBAc)关键步骤的有效和选择性抑制剂，diNAcBAc是一种高度修饰的糖，是许多革兰氏阴性病原体细胞表面糖偶联物的基本构建块。目前的研究目标是发现PglD酶的小分子抑制剂，PglD是一种依赖乙酰辅酶A的乙酰转移酶，在肠病原菌空肠弯曲杆菌的N-连接蛋白糖基化途径中执行UDP-GlcNAc转化为UDP-diNAcBac的最后一步。表型研究证实，UDP-diNAcBac是最终提供细菌细胞表面N-连接糖蛋白的途径中的一个必需中间体，该蛋白参与宿主细胞的黏附、侵袭和定植。因此，从这些研究中得到的小分子抑制剂将是阐明这些问题的有力的化学工具。 高度修饰的糖类在微生物毒力和致病机制中的基础作用。此外，这些探针将代表开发新治疗剂的新线索，并验证一类新的抗生素靶点。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。

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 that address the serious consequences of resistance development. In the past decade bacterial cell surface glycoconjugates, including the lipopolysaccharide (LPS) component of the outer cell wall and cell surface N- and O-linked glycoproteins of several medically relevant Gram-negative bacteria pathogens, have been characterized in molecular detail and found to be essential for host-dependent virulence and pathogenicity. We propose to employ a high-throughput small-molecule screening strategy to identify potent and selective inhibitors of an essential step in the biosynthesis of di-N-acetylbacillosamine (diNAcBAc), which is a highly modified saccharide that features as an essential building block in the cell-surface glycoconjugates of many Gram-negative pathogens. The current studies target the discovery of small molecule inhibitors of the enzyme PglD, which is an acetyl-CoA-dependent acetyl transferase that carries out the final step in the conversion of UDP-GlcNAc into UDP-diNAcBac in the N-linked protein glycosylation pathway of the enteropathogen Campylobacter jejuni. Phenotypic studies establish that UDP-diNAcBac is an obligatory intermediate in the pathway that ultimately affords bacterial cell-surface N-linked glycoproteins that are involved in host cell adhesion, invasion and colonization. Therefore, smal-molecule inhibitors that result from these studies would be incisive chemical tools for elucidating the fundamental roles of highly modified saccharides in microbial virulence and pathogenesis. Additionally, the probes would represent novel leads in the development of new therapeutic agents and validate a new class of antibiotic target. 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.