Antibiotic Properties 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应用新的方法，以有效地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的，严格的和安全的治疗方法来取代，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代谢途径。核糖开关是RNA结构元件，其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，基因表达受到调节。许多核糖开关，一旦配体，抑制参与代谢产物合成的相关或相邻基因的表达，提供了一个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）与葡萄糖胺-6-磷酸（GlcN 6P）结合，葡萄糖胺-6-磷酸是革兰氏阳性细菌细胞壁的结构单元，并发生自切割，导致mRNA失活。我们已经表明，配体的胺和磷酸功能是必不可少的结合的代谢产物的核糖开关RNA和催化的催化RNA（核酶）。我们的合作者大卫·伯克维茨博士与我们分享了GlcN 6 P依赖性核糖开关/核酶的结合和催化的这些要求，以帮助设计和有机合成新型配体类似物。我们将测试这些类似物诱导glmS自切割和抑制细菌生长的能力。已经有一种配体类似物在glmS自切割测定中显示出巨大的前景。我们还建议继续我们的研究glmS自裂解反应机制，进一步了解酸碱催化可能会影响开发glmS核酶激动剂，满足额外的化学要求的结合和活动。该更新资助的目的集中在（1）glmS自切割的配体类似物合成和表征，（2）关于glmS核糖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，以及（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，并作为针对一些最难治疗的人类病原体的新型抗微生物剂。 公共卫生相关性：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，因为这些病原体对公众可用的几乎所有抗生素都具有耐药性。对于靶向不同但特异性和必需的代谢途径的新型抗微生物剂的需求是巨大的，例如利用核糖开关来控制基因表达的那些。核糖开关的结构-功能和机理研究使得能够详细分析RNA的配体识别以及最终可以作为抗生素发挥作用的非天然激动剂的合理设计。

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.