Development of Artificial Agonists for a Bacterial Riboswitch

细菌核糖开关人工激动剂的开发

• 批准号: 7810909
• 负责人: JULIANE K STRAUSS-SOUKUP
• 金额: $ 12.99万
• 依托单位: CREIGHTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7810909
• 关键词: Acids; Active Sites; Affect; Agonist; Amines; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Infections; Binding; Biological Assay; Catalysis; Catalytic RNA; Cell Wall; Chemicals; Cleaved cell; Coenzymes; Collaborations; Commit; Complex; Deuterium; Development; Elements; Feedback; Financial Support; Functional RNA; Funding; Gene Expression; Gene Expression Alteration; Genes; Genetic; Gram-Positive Bacteria; Grant; Growth; Humulus; Hydrogen; In Vitro; Investigation; Ions; Isotopes; Kinetics; Knowledge; Laboratories; Ligands; Manuscripts; Maps; Measurement; Messenger RNA; Metabolic; Metabolic Pathway; Metabolism; Metals; Play; Process; Property; Protons; RNA; Reaction; Reagent; Recovery; Regulation; Relative (related person); Reporter; Reporter Genes; Resistance; Role; Structure; System; Testing; Time; United States National Institutes of Health; Work; analog; antimicrobial drug; base; combat; design; fighting; functional group; glucosamine 6-phosphate; graduate student; in vivo; inorganic phosphate; insight; meetings; novel; novel strategies; nucleobase; nucleotide analog; pathogen; prospective; public health relevance; reaction rate; research study

DESCRIPTION (provided by applicant): Antibiotic Properties of Artificial Agonists for a Bacterial Riboswitch Notice Number NOT-OD-09-058 Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications 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 currently used, imprecise antibiotics, 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 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 ligand recognition. 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 the original grant were focused 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. Aim 1 has been completed and Aim 2 is nearly finished. In regards to Aim 3, we have begun to test a small number of non-natural ligands, one of which does support glmS self-cleavage. This revision will significantly expand the scope of Aim 3 of the original grant in order to include additional ligand analog syntheses and bacterial growth experiments to test the antibiotic properties of the synthesized artificial agonists. Further expansion of the project will include an investigation of the mechanism of the self-cleavage reaction using kinetic isotope experiments. Both lines of experimentation will further aid in rational design of non-natural metabolite-like compounds that can function as agonists/antibiotics to halt bacterial growth through alteration of gene expression. PUBLIC HEALTH RELEVANCE: 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 and mechanistic studies of riboswitches have enabled detailed studies of ligand recognition by RNA as well as rational design of non-natural agonists that ultimately could function as antibiotics.

描述（由申请人提供）：细菌核糖开关人工激动剂的抗生素特性通知编号no - od -09-058通知标题：NIH宣布竞争性修订申请恢复法案资金的可用性抗生素耐药性的出现要求应用新方法以有效地对抗大量医学相关的细菌感染。目前使用的不精确的抗生素需要被新的、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。被称为核糖开关的RNA结构元件的发现，结合细胞代谢物并控制必需代谢基因的表达，为开发对抗细菌感染的人工激动剂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，而磷酸可能在配体识别中发挥作用。为了开发针对glmS核开关的有效人工激动剂/抗生素，了解核开关-代谢物复合物的结构和功能细节是必不可少的。最初拨款的目的集中在(1)研究金属离子在glmS核开关中的结构和催化作用，(2)破译glmS核开关对配体的识别，以及(3)设计能够刺激glmS核开关自我切割和控制基因表达的非天然激动剂。Aim 1已经完成，Aim 2即将完成。关于Aim 3，我们已经开始测试少量的非天然配体，其中一种确实支持glmS自裂。此次修订将显著扩大原拨款的Aim 3的范围，以便包括额外的配体模拟物合成和细菌生长实验，以测试合成的人工激动剂的抗生素特性。该项目的进一步扩展将包括利用动力学同位素实验研究自解理反应的机制。这两种实验将进一步有助于合理设计非天然代谢物样化合物，这些化合物可以作为激动剂/抗生素，通过改变基因表达来阻止细菌生长。