PGT Inhibitors Mapped From a Tunicamycin Blueprint

描述(申请人提供)：磷酸糖基转移酶(PGTS)是一种膜结合酶，催化磷酸糖基从活化的UDP-糖供体转移到聚戊烯基磷酸受体。PGT催化的反应提供了两亲性的聚戊二磷酸连接的碳水化合物，从而将多糖组装过程进行到膜界面的某个位置。PGT专用于它们作用的UDP-糖的特性，该特性定义了转移的磷酸糖基的性质。这些酶最常见地参与了许多糖共轭组装途径中的关键早期步骤，包括那些导致细菌脂多糖和荚膜多糖、磷壁酸以及N和O连接的糖蛋白的途径。这种糖结合物在微生物病原体与其感染的宿主之间的相互作用中起着至关重要的作用，然而，目前还缺乏选择性的小分子抑制剂，可以用来评估和确定特定的糖结合物在微生物生理学中的意义，并阐明它们在宿主-病原体相互作用中的作用。这项研究将解决作为细菌PGTS抑制剂的新化学试剂的迫切需求，并寻求提供作用于不同UDP-糖供体底物的各种PGTS的选择性抑制剂。在这个探索性的项目中，我们将使用包括抑制剂设计和合成、基于X射线的结构确定和酶分析的协同方法来开发一种有效和选择性的细菌PGT抑制剂的通用方法。研究方法的中心主题依赖于利用天然产物核苷抗生素，特别是衣霉素的分子逻辑，作为结构指导设计和合成作为PGTS选择性抑制剂的一系列新的模块化化学结构的“蓝图”。衣霉素是细菌PGT的双底物类似物抑制剂，WEC A是酵母菌PGT ALG7的同系物，也能抑制PglC-HPU，PglC-HPU是一种来自白喉杆菌的小PGT，IC50为350？30 nm。这些酶催化从UDP-GlcNAc到十一碳烯基磷酸的磷酸糖基转移，从而在各自的生物体中启动内毒素和N-连接糖蛋白的生物合成。首先，我们将重点关注来自不同微生物病原体的三个小PGTS，它们作用于不同的磷酸糖基供体底物(UDP-GlcNAc、UDP-Gal和UDP-Bac)和一个共同的十一烯基磷酸(Development)受体，我们将开发和验证用于组装新抑制剂的易处理和模块化的合成平台。如果这项研究成功，这项研究将带来一种新的抑制剂设计方法，可以广泛应用于一大类PGTS和强大的化学工具，这些工具将为装饰许多微生物病原体细胞表面的复杂糖蛋白结合物的功能意义提供有价值的新见解。

DESCRIPTION (provided by applicant): Phosphoglycosyl transferases (PGTs) are membrane-bound enzymes, which catalyze the transfer of a phosphoglycosyl group from an activated UDP-sugar donor to a polyprenylphosphate acceptor. PGT-catalyzed reactions afford amphiphilic polyprenyldiphosphate-linked carbohydrates, thereby committing glycan assembly processes to a location at the membrane interface. PGTs are specific for the identity of the UDP-sugar that they act on, which defines the nature of the transferred phosphoglycosyl group. These enzymes are most commonly involved in an essential early step that features in many glycoconjugate assembly pathways including those that lead to bacterial lipopolysaccharides and capsular polysaccharides, teichoic acids, and N- and O-linked glycoproteins. Such glycoconjugates play critical roles in the interactions of microbial pathogens with the hosts that they infect, however, there is a dearth of selective small molecule inhibitors, which can be used as tools to assess and define the significance of specific glycoconjugates in microbial physiology and to elucidate their roles in host-pathogen interactions. This research will address the critical need for new chemical agents as inhibitors for bacterial PGTs and seeks to provide selective inhibitors of diverse PGTs that act on different UDP-sugar donor substrates. In this exploratory program we will employ synergistic approaches involving inhibitor design and synthesis, X-ray based structure determination, and enzymatic analysis to develop a general method for the genesis of potent and selective bacterial PGT inhibitors. The central theme of the research approach relies on exploiting the molecular logic of the natural product nucleoside antibiotics, in particular tunicamycin, as a "blue print" for the structure-guided design and synthesis of a new family of modular chemical structures that act as selective inhibitors of PGTs. Tunicamycin is a potent bisubstrate analog inhibitor of the bacterial PGT, Wec A, which is a homolog of the yeast PGT Alg7, and also inhibits PglC-Hpu, a small PGT from H. pullorum with IC50 of 350¿30 nM. These enzymes catalyze phosphoglycosyl transfer from UDP-GlcNAc to undecaprenylphosphate thereby initiating lipopolysaccharide and N-linked glycoprotein biosynthesis in the respective organisms. Focusing first on three small PGTs from different microbial pathogens that act on diverse phosphoglycosyl donor substrates (UDP-GlcNAc, UDP-Gal and UDP-Bac) and a common undecaprenyl phosphate (UndP) acceptor, we will develop and validate tractable and modular synthetic platforms for the assembly of new inhibitors. If successful, this research will lead to a new inhibitor design approach that can be applied broadly to a large family of PGTs and to powerful chemical tools that will afford valuable new insight into the functional significance of complex glycoprotein conjugates that decorate the cell surfaces of many microbial pathogens.