这里,我们描述了对含GluN2B的NMDA受体具有选择性的放射性配体的表征,3-[3H]1-(氮杂环丁烷-1-基)-2-(6-(4-氟-3-甲基-苯基)吡咯并[3,2-b]吡啶-1-基)乙酮([3H]-JNJ-GluN2B-5)。在大鼠皮质膜中,化合物结合到一个位点,并测量了以下动力学参数:缔合速率常数Kon=0.0066±0.0006min-1nM-1,解离速率常数Koff=0.0210±0.0001min-1,表明计算的KD=Koff/Kon=3.3±0.4nM,(平均值±SEM,n=3)。从大鼠皮质的饱和结合实验确定的平衡解离常数为KD为2.6±0.3nM(平均值±SEM,n=3)。与广泛使用的GluN2B放射性配体[3H]-Ro25-6981相反,其对sigma1和sigma2受体的亲和力Ki分别为2和189nM,分别,[3H]-JNJ-GluN2B-5对σ1和σ2受体没有可测量的亲和力(两者的Ki>10μM),提供不同的选择性优势。大鼠[3H]-JNJ-GluN2B-5结合位点的解剖分布,鼠标,狗,猴子,使用体外放射自显影技术研究了人的脑组织,在海马和皮质中显示出高度的特异性结合,在小脑中的结合可以忽略不计。在体外放射性配体结合和体外放射自显影测定中,对含GluN2B受体的选择性增强可转化为良好的信噪比。总之,[3H]-JNJ-GluN2B-5是一种高亲和力GluN2B放射性配体,具有出色的信噪比和前所未有的选择性。
Here, we describe the characterization of a
radioligand selective for GluN2B-containing NMDA receptors, 3-[3H] 1-(azetidin-1-yl)-2-(6-(4-fluoro-3-methyl-phenyl)pyrrolo[3,2-b]pyridin-1-yl)ethanone ([3H]-JNJ- GluN2B-5). In rat cortical membranes, the compound bound to a single site, and the following kinetic parameters were measured; association rate constant Kon = 0.0066 ± 0.0006 min-1 nM-1, dissociation rate constant Koff = 0.0210 ± 0.0001 min-1 indicating calculated KD = Koff/Kon = 3.3 ± 0.4 nM, (mean ± SEM, n = 3). The equilibrium dissociation constant determined from saturation binding experiments in rat cortex was KD of 2.6 ± 0.3 nM (mean ± SEM, n = 3). In contrast to the widely used GluN2B
radioligand [3H]-Ro 25-6981, whose affinity Ki for sigma 1 and sigma 2 receptors are 2 and 189 nM, respectively, [3H]-JNJ-GluN2B-5 exhibits no measurable affinity for sigma 1 and sigma 2 receptors (Ki > 10 μM for both) providing distinct selectivity advantages. Anatomical distribution of [3H]-JNJ-GluN2B-5 binding sites in rat, mouse, dog, monkey, and human brain tissue was studied using in vitro autoradiography, which showed high specific binding in the hippocampus and cortex and negligible binding in the cerebellum. Enhanced selectivity for GluN2B-containing receptors translated to a good signal-to-noise ratio in both in vitro
radioligand binding and in vitro autoradiography assays. In conclusion, [3H]-JNJ-GluN2B-5 is a high-affinity GluN2B
radioligand with excellent signal-to-noise ratio and unprecedented selectivity.