RNA modification and antibiotic resistance

 描述（由申请人提供）：抗生素在细菌感染治疗中的应用彻底改变了现代医学实践。在此后的几十年里，不适当的控制使用和细菌种群对这些药物产生耐药性的显着能力的结合严重限制了许多抗生素的临床用途。我们现在正处于一个关键时刻，大多数有用的抗生素都具有已知的，有时是广泛的耐药性，并且很少有新的替代品或策略来对抗耐药性问题。氨基糖苷类抗生素是一大类药物的一个重要例子，这些药物对主要的人类病原体具有保留的活性，但耐药性问题继续增加。特别值得关注的是，在过去十年中，越来越多的病原菌携带非常广谱和高水平的氨基糖苷类耐药决定簇。这些16 S核糖体RNA（rRNA）甲基转移酶化学修饰细菌核糖体小亚基（30 S）的16 S rRNA上的氨基糖苷类结合位点。已经鉴定了两个不同的氨基糖苷类耐药性甲基转移酶家族，它们修饰核苷鸟苷1405或腺苷1408上的16 S rRNA，分别产生m7 G1405和m1 A1408修饰。这两种类型的功能类似的自我保护耐药酶早期在氨基糖苷类抗生素生产菌中被发现，人们认为这些基因已经横向转移到病原菌。我们实验室和其他人最近的研究揭示了每种酶的分子结构，包括药物生产者和病原体来源，并提供了与其底物结合的m1 A1408甲基转移酶的第一个视图。然而，更多的证据表明，这种酶家族可能采用不同的机制来实现底物特异性和修饰。此外，关于m7 G1405如何实现等同的分子功能，相对知之甚少。该提案描述了两个相关目标的实验，这些目标将直接解决这些缺陷。在目的1中，我们将使用生物化学，分子生物学和结构生物学方法的组合来确定的m1 A1408甲基转移酶的分子机制，以确定控制的m1 A1408甲基转移酶活性的S-腺苷-L-甲硫氨酸共底物的分子基础，并由30 S亚基基板。目的2将提供第一个详细的分子见解30 S识别和修改的m7 G1405甲基转移酶使用创新的，跨学科的方法组合，包括合成化学，以产生新的SAM类似物，这将使第一次必要的生化和结构研究这些临床重要的耐药酶。总之，这些研究将为氨基糖苷类耐药甲基转移酶的结构、活性和作用机制提供新的和根本性的重要见解，并将直接为未来开发新的策略以对抗它们赋予的耐药性奠定安全的基础。

 DESCRIPTION (provided by applicant): The application of antibiotics to the treatment of bacterial infections revolutionized modern medical practice. In the decades since, a combination of improperly controlled use and the remarkable ability of bacterial populations to develop resistance to these drugs has severely restricted the clinical usefulness of many antibiotics. We are now at a critical juncture where the majority of useful antibiotics have known and sometimes extensive resistance, and few novel replacements or strategies to combat the resistance problem exist. The aminoglycoside antibiotics are one important example of a large group of drugs with retained activity against key human pathogens but where the problem of resistance continues to increase. Of particular concern is the increasing identification over the last decade of pathogenic bacteria harboring exceptionally broad- spectrum and high-level aminoglycoside resistance determinants. These 16S ribosomal RNA (rRNA) methyltransferases chemically modify the aminoglycoside binding site on the 16S rRNA of the small (30S) bacterial ribosomal subunit. Two distinct families of aminoglycoside-resistance methyltransferases have been identified that modify 16S rRNA on nucleoside guanosine 1405 or adenosine 1408, to create m7G1405 and m1A1408 modifications, respectively. Functionally analogous self-protection resistance enzymes of both types were earlier identified in aminoglycoside-producing bacteria, and it is thought that these genes have laterally transferred to the pathogenic bacteria. Recent studies from our lab and others revealed the molecular structures of each enzyme, of both drug producer and pathogenic origin, and provided a first view of an m1A1408 methyltransferase bound to its substrate. However, additional evidence suggests that this enzyme family may employ diverse mechanisms to accomplish substrate specificity and modification. Further, comparatively little is known about how m7G1405 achieve the equivalent molecular functions. This proposal describes experiments in two connected aims that will directly address these deficiencies. In Aim 1 we will define the molecular mechanisms employed by the m1A1408 methyltransferases using a combination of biochemical, molecular biological and structural biology approaches to determine the molecular bases for control of m1A1408 methyltransferase activity by the S-adenosyl-L-methionine cosubstrate, and by the 30S subunit substrate. Aim 2 will provide the first detailed molecular insights into 30S recognition and modification by the m7G1405 methyltransferases using an innovative, interdisciplinary combination of approaches including synthetic chemistry to produce novel SAM analogs that will enable for the first time essential biochemical and structural studies of these clinical important resistance enzymes. Together these studies will provide novel and fundamentally important insights into the structures, activities and mechanisms of action of the aminoglycoside-resistance methyltransferases and will directly lay a secure foundation for future development of new strategies to counter the resistance they confer.