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Development of the reliable methods to detect the interaction of proteins and ligands using diffusion of molecules

开发利用分子扩散检测蛋白质和配体相互作用的可靠方法

基本信息

批准号：
15550076
负责人：
TASHIRO Mitsuru
金额：
$ 1.79万
依托单位：
Department of Chemistry, College of Science and Technology, Meisei University (2005)Tokyo Metropolitan University (2003-2004)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

There is a growing need for efficient methods, capable of reliably identifying compounds in a relatively short time span with desired binding affinity. In designing NMR-based screening methods for lead compounds, it is critical to develop the technique capable of screening a large pool of new drug candidates and identifying lead compounds with high affinity towards the target proteins. an improved version of the WaterLOGSY sequence is proposed, and its efficiency of identifying a ligand bound to a protein is compared with that of STD and NOE-pumping techniques. The effectiveness of this NMR-based screening method is demonstrated using the RNase T_1-inhibitor system.Water-LOGSY experiment was improved by incorporating the double pulsed field gradient spin-echo (DPFGSE) sequence. DPFGSE sequence provides superior selective excitation without phase distortion problems that are usually associated with conventional selective excitation pulses. In the WaterLOGSY spectra, the positive signals from the inhibitors bound to RNase T_1 were clearly observed for the H8 proton of 5'-GMP and the H2 and H8 protons of 3'-AMP. These signals were not observed in the other techniques, which indicate the effectiveness of the DPFGSE-WaterLOGSY.Practical aspects of several ROESY pulse sequences have been investigated to identify bound water molecules of the non-labeled saccharide. To increase the selectivity of water resonance excitation, very weak rf strengths for the selective 180° pulses, corresponding to pulse widths of 500 ms, was used in DPFGSE. Several Cross peaks arising from bound water proton-proton of saccharide spin-spin cross relaxation are observed, and a binding water was identified by NMR.

人们越来越需要有效的方法，能够在相对较短的时间内可靠地鉴定具有所需结合亲和力的化合物。在设计基于 NMR 的先导化合物筛选方法时，开发能够筛选大量新候选药物并鉴定对目标蛋白具有高亲和力的先导化合物的技术至关重要。提出了 WaterLOGSY 序列的改进版本，并将其识别与蛋白质结合的配体的效率与 STD 和 NOE 泵技术进行了比较。使用 RNase T_1 抑制剂系统证明了这种基于 NMR 的筛选方法的有效性。通过合并双脉冲场梯度自旋回波 (DPFGSE) 序列改进了 Water-LOGSY 实验。 DPFGSE 序列提供卓越的选择性激励，而不会出现通常与传统选择性激励脉冲相关的相位失真问题。在 WaterLOGSY 光谱中，对于 5'-GMP 的 H8 质子以及 3'-AMP 的 H2 和 H8 质子，可以清楚地观察到来自与 RNase T_1 结合的抑制剂的阳性信号。在其他技术中没有观察到这些信号，这表明了 DPFGSE-WaterLOGSY 的有效性。已经研究了几种 ROESY 脉冲序列的实际方面，以识别非标记糖的结合水分子。为了提高水共振激发的选择性，在 DPFGSE 中使用了非常弱的选择性 180° 脉冲射频强度（对应于 500 ms 的脉冲宽度）。观察到由糖自旋-自旋交叉弛豫的结合水质子-质子产生的几个交叉峰，并通过NMR鉴定了结合水。