Analysis of the Binding Sites of Opioid Receptor by Photoaffinity Labeling

研究目的：最近在嵌合阿片受体的构建中的成功证明了几个细胞外区域的存在，这可能是负责每种类型的阿片受体的不同配体结合谱。然而，通常不可能排除导致配体结合改变的潜在构象变化。本研究的目的是开发和应用基于二氮丙啶的光探针来分析阿片类药物的结合位点。使用这些光反应部分，一对新的光探针，其唯一不同的是在卡宾生成网站的合成。探针的这种结构特征对于映射配体结合口袋内的不同位点是潜在有用的。为了避免使用放射性探针，还开发了一种新的生物素化的光反应性纳洛酮类似物。所有携带二氮丙啶的新探针均以高亲和力可逆地结合于μ-、δ-和κ-受体。利用生物素探针对重组δ受体蛋白进行光亲和标记。通过光化学生物素化蛋白质的荧光检测，成功地实现了目标分子的可视化。

Research Purpose : Recent success in the construction of chimeric opioid receptors demonstrated the presence of several extra cellular regions which could be responsible for the distinct ligand-binding profiles of the each type of opioid receptors. However, it is generally impossible to exclude potential conformational changes which result in alterations of the ligand binding. The purpose of this research is the development and application of diazirine based photoprobes for the analysis of opioid binding sites.Results : A convenient synthesis of m- and p-CF_3-diazirinylbenzoic acid was developed. Using these photoreactive moieties, a novel pair of photoprobes which only differ in the carbene generating sites were synthesized. This structural feature of the probes are potentially useful to map the different sites within the ligand binding pocket. For avoiding the use of radioactive probes, a novel biotinylated photoreactive naltrexone analog was also developed. All new probes bearing diazirine bind reversibly with high affinity at mu-, delta-, and kappa- receptors. The photoaffinity labeling of recombinant delta-receptor protein was investigated with the biotinyl probe. The visualization of target molecule was successfully accomplished by the chemiluminescent detection of photochemically biotinylated proteins.