RIBOSOMAL FUNCTION & ANTIBIOTIC DESIGN

描述（由申请人提供）：最近核糖体的几种x射线晶体结构的可用性在该领域引起了相当大的兴奋。这些结构将有助于设计实验来阐明核糖体合成蛋白质的细节。此外，细菌核糖体是许多类抗生素的靶标，每一类抗生素都会干扰蛋白质合成的生物化学方面。对核糖体结构的研究将在不久的将来引领新型抗生素的开发。这个多学科合作项目的长期目标是利用已知x射线结构的结构信息，更详细地研究核糖体的功能，并开发一种基本和普遍的生物合成过程的新型抑制剂。提出了四个具体目标。1)将采用分子动力学模拟与实验相结合的方法，探索肽键形成过程中解码过程的动态性质，并了解抗生素结合在此过程中的作用。2)合成两个设计用于探索解码过程中rRNA A位点运动的分子，研究rRNA结合。这些化合物是A位点功能的潜在抑制剂，因此可能是抗菌药物。3)将合成氨基糖苷- edta复合物的两个铁配合物，并测试它们结合核糖体和在抗生素结合位点内片段rRNA主干的能力。这些实验将确定这些抗生素结合的位置，并通过检查裂解模式提供有关动力学的信息。4)具体目标4扩展了新抗生素设计的早期成功。通过与核糖体A位点结合，将产生一系列干扰蛋白质合成的分子。为了研究这些分子的抗菌特性，已经设想了一系列具体的分析（对活细菌的抗菌特性评估、翻译试验、细菌膜渗透性试验、细菌中的DNA和RNA功能试验、细胞毒性试验、A位点突变的30S核糖体和携带它们的细菌的试验、以及对30S核糖体和A位点模型与新型抗生素复合的x射线分析）。这一集体努力将阐明核糖体内的各种分子事件以及如何抑制其中一些过程。

DESCRIPTION (provided by applicant): The recent availability of several x-ray crystal structures for the ribosome have created considerable excitement in the field. These structures will be instrumental in devising experiments to elucidate the details of ribosomal synthesis of proteins. Furthermore, the bacterial ribosome is the target of many classes of antibiotics, each of which interferes with an aspect of the biochemistry of protein synthesis. Studies of ribosome structures will lead the way in developing novel classes of antibiotics in the near future. The long-term goal of this multidisciplinary collaborative project is to utilize the structural information from known x-ray structures to study ribosome function in greater detail and to develop novel inhibitors of an essential and universal biosynthetic process. Four specific aims are proposed. 1) A combination of molecular dynamics simulations and experiments will be used to explore the dynamic nature of the decoding process during peptide-bond formation and to understand the role of antibiotic binding in this process. 2) Two molecules that have been designed to explore the motion of the rRNA A site during the decoding process will be synthesized and studied for rRNA binding. These compounds are potential inhibitors of A site function, hence possible antibacterials. 3) Two iron complexes of aminoglycoside-EDTA complexes will be synthesized and tested for their ability to bind the ribosome and fragment the rRNA backbone at sites located within the antibiotic-binding site(s). These experiments will identify the locations in which these antibiotics bind, as well as provide information about dynamics through examination of cleavage patterns. 4) Specific aim 4 expands on earlier success in the design of new antibiotics. A series of molecules that will interfere with protein synthesis by binding to the ribosomal A site will be generated. A host of specific analyses (assessment of antibacterial properties with living bacteria, translation assays, bacterial membrane permeability assay, assays for DNA and RNA function in bacteria, cytotoxicity assays, assays with A-site mutated 30S ribosomes and bacteria that harbor them, and x-ray analyses of the 30S ribosome and the A-site model complexed with the novel antibiotics) have been envisioned for the study of the antibacterial properties of these molecules. This collective effort will shed light both on the various molecular events within the ribosomal and on how to inhibit some of these processes.