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来有效对抗许多医学相关的细菌感染。目前使用的不精确的抗生素需要用新颖、严格且安全的治疗方法取代，以对抗当今进化的细菌。消灭细菌的一种方法是针对它们最重要的代谢途径。核糖开关是RNA结构元件，可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组独特且独特的靶标。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，基因表达就会受到调节。许多核糖开关一旦配位，就会抑制参与代谢物合成的相关或邻近基因的表达，从而提供有效的遗传控制反馈机制。一种特殊的核糖开关（glmS 核糖开关）与 6-磷酸葡萄糖胺 (GlcN6P)（革兰氏阳性细菌细胞壁的组成部分）结合，并经历自我裂解，导致 mRNA 失活。我们已经证明，配体胺和磷酸官能团对于代谢物与核糖开关 RNA 的结合以及催化 RNA（核酶）的催化作用至关重要。这些 GlcN6P 依赖性核糖开关/核酶的结合和催化要求已与我们的合作者 David Berkowitz 博士分享，以帮助新型配体类似物的设计和有机合成。我们将测试这些类似物诱导 glmS 自我裂解和抑制细菌生长的能力。一种配体类似物已经在 glmS 自裂解测定中显示出巨大的前景。我们还建议继续研究 glmS 自裂解反应机制，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，以满足结合和活性的额外化学要求。该续期资助的目的集中在（1）配体类似物合成和glmS自裂解的表征，（2）人工激动剂在glmS核糖开关调节报告基因表达和抑制细菌方面的结构、功能和抗生素特性。 (3) 通过协调质子转移进行 glmS 支持的酸碱催化的机理研究。从动力学研究中获得的信息将进一步为我们继续设计配体类似物提供信息，这些配体类似物支持glmS核糖开关/核酶催化作用，并作为新型抗菌剂对抗一些最难治疗的人类病原体。 公共卫生相关性：由于缺乏有效的抗生素而导致的细菌感染威胁已成为首要问题，因为这些病原体对公众可用的几乎所有抗生素都产生了耐药性。非常需要针对不同但特定且重要的代谢途径的新型抗微生物剂，例如利用核糖开关控制基因表达的途径。核糖开关的结构功能和机制研究使得能够详细分析 RNA 的配体识别以及合理设计最终可用作抗生素的非天然激动剂。