Development of Artificial Agonists for a Bacterial Riboswitch

描述（由申请人提供）：抗生素耐药性的出现要求采用新的方法，以便有效地对抗一系列医学上相关的细菌感染。目前使用的有限抗生素需要被新的、严格的和安全的治疗方法所取代，以对抗今天进化的细菌。消灭细菌的一种方法是瞄准它们最基本的过程之一——新陈代谢。最近发现的RNA结构元件，称为核糖开关，可以结合细胞代谢物并控制必需代谢基因的表达，为开发人工激动剂来对抗细菌感染提供了一个独特的靶点。核糖开关存在于信使RNA的非编码区，当代谢物直接与RNA结合时，基因表达被调节。许多核开关抑制参与代谢物合成的附近基因的表达，提供了一种有效的遗传控制反馈机制。一种特殊的核糖体开关（glmS核糖体开关）与革兰氏阳性细菌细胞壁的组成部分葡萄糖胺-6-磷酸（GlcN6P）结合，并经历自裂导致mRNA的失活。GlcN6P的胺功能似乎直接参与RNA催化，而磷酸可能在RNA对配体的识别中发挥作用。为了开发针对glmS核糖体开关的有效人工激动剂/抗生素，了解核糖体开关-代谢物复合物的结构和功能细节是必不可少的。该基金的目标集中在(1)研究金属离子在glmS核糖体开关中的结构和催化作用，(2)破译glmS核糖体开关对配体的识别，以及(3)设计具有刺激glmS核糖体开关自裂和控制基因表达能力的非天然激动剂。利用核苷酸模拟干扰映射和抑制（NAIM和NAIS分别），glmS核开关，其配体和金属离子之间的一些远程接触将被确定。使用NAIM，将使用修饰感兴趣原子的核苷酸类似物来定义glmS核开关内单个化学基团的生化贡献。使用NAIS，将确定glmS RNA结构内或涉及的特定三级氢键伙伴。核开关的结构-功能研究将有助于合理设计非天然代谢物样化合物，这些化合物可能作为激动剂/抗生素，通过改变基因表达来阻止细菌生长。由于缺乏有效的抗生素，细菌感染的威胁已经成为最重要的问题，因为这些病原体对几乎所有公众可用的抗生素都具有耐药性。我们迫切需要针对不同但特定且必需的代谢途径的新型抗菌药物，例如利用核糖开关控制基因表达的代谢途径。核开关的结构-功能研究将使非天然激动剂的合理设计成为可能，最终可以作为抗生素发挥作用。

DESCRIPTION (provided by applicant): The emergence of antibiotic resistance has required that new approaches be applied in order to effectively fight a host of medically relevant bacterial infections. The limited group of antibiotics, currently in use, need to be replaced with novel, rigorous, and safe treatments in order to combat the evolved bacterium of today. One way to destroy bacteria is to target one of their most essential processes, metabolism. The recent discovery of RNA structural elements, termed riboswitches, that bind cellular metabolites and control expression of essential metabolic genes provides a unique and distinct target for development of artificial agonists to fight bacterial infections. Riboswitches are found in non-coding regions of messenger RNAs, and gene expression is modulated when metabolite binds directly to the RNA. Many riboswitches repress expression of nearby genes involved in the synthesis of the metabolite, providing an efficient feedback mechanism of genetic control. One particular riboswitch (the glmS riboswitch) binds to glucosamine-6-phosphate (GlcN6P), a building block of the cell wall in Gram-positive bacteria, and undergoes self-cleavage resulting in inactivity of the mRNA. The amine functionality of GlcN6P seems to be directly involved in RNA catalysis, whereas the phosphate may play a role in recognition of the ligand by the RNA. In order to develop effective artificial agonists/antibiotics that target the glmS riboswitch, an understanding of the structural and functional details of the riboswitch-metabolite complex is essential. The aims of this grant focus on (1) investigating the structural and catalytic roles of metal ions in the glmS riboswitch, (2) deciphering ligand recognition by the glmS riboswitch, and (3) designing non-natural agonists with the ability to stimulate glmS riboswitch self-cleavage and control gene expression. Using Nucleotide Analog Interference Mapping and Suppression (NAIM and NAIS, respectively) some of the long range contacts between the glmS riboswitch, its ligand, and metal ions will be determined. Using NAIM, the biochemical contribution of a single chemical group within the glmS riboswitch will be defined using nucleotide analogs that modify the atom(s) of interest. Using NAIS, specific tertiary hydrogen bonding partners within or involving the glmS RNA structure will be determined. Structure-function studies of riboswitches will enable rational design of non-natural metabolite-like compounds that might function as agonists/antibiotics to halt bacterial growth through alteration of gene expression. The threat of bacterial infections due to lack of effective antibiotics has come to the forefront as these pathogens become resistant to almost every antibiotic available to the public. The need is great for new classes of anti-microbial agents that target different, but specific and essential, metabolic pathways, such as those which utilize riboswitches to control gene expression. Structure-function studies of riboswitches will enable rational design of non-natural agonists that ultimately could function as antibiotics.