The development of NMR methods for drug discovery targeting on ribosome recycling.

我们建立了源自5种细菌的RRFs的NMR定位。结果不仅为后续的结构研究提供了基础，而且为药物发现提供了一套原子分辨率的相互作用探针。我们通过核磁共振弛豫分析和纳秒分子动力学模拟研究了RRF的分子间动力学。实验结果揭示了RRF分子在结构域间取向上的柔韧性特征，表明与铰链区结合的化合物会抑制RRF的功能。为了获得CARDD的结构信息，我们对副溶血性弧菌的RRF进行了x线分析。为了阐明RRF的核糖体结合位点，我们对RRF结构域I对应的肽片段（RRF- di）进行了表达和表征。RRF- di与70S核糖体和50S亚基结合，其亲和力与野生型RRF相似。但它不与30S亚基结合。此外，我们还对rrf -核糖体复合体进行了低温电镜分析和化学探针实验。在确定RRF结构和相互作用模式的基础上，通过虚拟筛选，获得了一些候选的RRF抑制剂。为了检测这些化合物，我们开发了一种新的体外筛选系统。

We established NMR assignments of RRFs originated from five bacteria. Resulting assignments bring not only the basis of following structural study, but also the set of interaction probes at an atomic resolution for drug discovery. We investigated inter-molecular dynamics of RRF by NMR relaxation analyses and nanosecond molecular dynamics simulations. The results revealed characteristic flexibility in inter-domain orientation of RRF molecule experimentally, indicating that a compound bound to hinge region would inhibit RRF function. In order to obtain structural information for CARDD, we carried out an X-ray analysis on RRF from Vibrio parahaemolyticus. To elucidate the ribosome binding site of RRF, the peptide fragment corresponding to domain I of RRF (RRF-DI) was expressed and characterized. RRF-DI is bound to 70S ribosome and the 50S subunit with an affinity similar to that of wild-type RRF. But it does not bind to the 30S subunit. Moreover, we carried out a cryo-EM analyses and chemical probing experiments on RRF-ribosome complex. By the virtual screening based on the determined structure and interaction mode of RRF, some candidates for RRF inhibitor were obtained. To assay such compounds, novel screening system in vitro was developed.