Antibiotic Properties of Artificial Agonists for a Bacterial Riboswitch

细菌核糖开关人工激动剂的抗生素特性

• 批准号: 7980700
• 负责人: JULIANE K STRAUSS-SOUKUP
• 金额: $ 43.58万
• 依托单位: CREIGHTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7980700
• 关键词: Acids; Active Sites; Affect; Agonist; Amines; Ampicillin; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Infections; Binding; Biological Assay; Catalysis; Catalytic RNA; Cell Wall; Chemicals; Cleaved cell; Collaborations; Complex; Development; Elements; Equipment and supply inventories; Feedback; Functional RNA; Gene Expression; Genes; Genetic; Goals; Gram-Positive Bacteria; Grant; Growth; Human; Humulus; In Vitro; Ions; Isotopes; Kinetics; Laboratories; Ligands; Messenger RNA; Metabolic; Metabolic Pathway; Metals; Methods; Organic Synthesis; Property; Protons; RNA; Reaction; Regulation; Reporter Genes; Resistance; Solvents; Structure; Testing; analog; antimicrobial drug; base; combat; design; fighting; glucosamine 6-phosphate; in vivo; inorganic phosphate; insight; novel; novel strategies; pathogen; public health relevance; research study

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 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 their most essential, metabolic pathways. Riboswitches are RNA structural elements that bind cellular metabolites and control expression of essential metabolic genes providing a unique and distinct set of targets for development of artificial agonists to fight bacterial infections. Riboswitches are found in non-coding regions of mRNA molecules, and gene expression is modulated when metabolite binds directly to the RNA. Many riboswitches, once liganded, repress expression of associated or adjacent 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 inactivation of the mRNA. We have shown that the ligand amine and phosphate functionalities are essential for binding of the metabolite to the riboswitch RNA and for catalysis by the catalytic RNA (ribozyme). These requirements for binding and catalysis of the GlcN6P-dependent riboswitch/ribozyme have been shared with our collaborator, Dr. David Berkowitz, to aid in design and organic syntheses of novel ligand analogs. We will test these analogs for their ability to induce glmS self-cleavage and inhibit bacterial growth. Already one ligand analog shows great promise in glmS self-cleavage assays. We also propose to continue our studies of the glmS self-cleavage reaction mechanism as further insight to acid-base catalysis may affect development of glmS ribozyme agonists that satisfy added chemical requirements for binding and activity. The aims of this renewal grant are focused on (1) ligand analog synthesis and characterization of glmS self-cleavage, (2) the structure, function and antibiotic properties of artificial agonists in regards to glmS riboswitch regulation of reporter gene expression and inhibition of bacterial growth, and (3) mechanistic studies of glmS-supported acid-base catalysis through coordinated proton transfer. Information gained from kinetic studies will further inform our continued design of ligand analogs that support glmS riboswitch/ribozyme catalysis and that act as novel antimicrobial agents against some of the hardest to treat human pathogens. 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 analyses 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）：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重要的代谢途径。核糖开关是结合细胞代谢物和控制必需代谢基因表达的RNA结构元件，为开发人工激动剂对抗细菌感染提供了一套独特的靶点。核糖开关存在于mRNA分子的非编码区，当代谢物直接与RNA结合时，基因表达被调节。许多核开关一旦配位，就会抑制参与代谢物合成的相关或邻近基因的表达，从而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失活。我们已经证明，配体胺和磷酸盐的功能对于代谢产物与核糖体开关RNA的结合和催化RNA（核酶）的催化是必不可少的。这些结合和催化glcn6p依赖性核开关/核酶的要求已经与我们的合作者David Berkowitz博士分享，以帮助设计和有机合成新的配体类似物。我们将测试这些类似物诱导glmS自裂和抑制细菌生长的能力。已经有一种配体类似物在glmS自裂试验中显示出巨大的前景。我们还建议继续我们对glmS自裂反应机制的研究，因为对酸碱催化的进一步了解可能会影响glmS核酶激动剂的开发，从而满足结合和活性的附加化学要求。此次更新资助的目的集中在(1)glmS自裂配体模拟物的合成和表征；(2)glmS核开关调节报告基因表达和抑制细菌生长的人工激动剂的结构、功能和抗生素特性；(3)glmS支持的酸碱协同质子转移催化机制研究。从动力学研究中获得的信息将进一步为我们继续设计支持glmS核开关/核酶催化的配体类似物提供信息，并作为对抗一些最难治疗的人类病原体的新型抗菌药物。