Wang Yuren, Xu Jun, Uveges Albert, Ramarao Manjunath K, Rogers Kathryn E, Jones Philip G
Neuroscience Discovery Research, Wyeth Research, Princeton, NJ 08543, USA.
Anal Biochem. 2006 Jul 1;354(1):35-42. doi: 10.1016/j.ab.2006.04.005. Epub 2006 May 2.
A binding assay for human fatty acid amide hydrolase (FAAH) using the scintillation proximity assay (SPA) technology is described. This SPA uses the specific interactions of [3H]R(+)-methanandamide (MAEA) and FAAH expressing microsomes to evaluate the displacement activity of FAAH inhibitors. We observed that a competitive nonhydrolyzed FAAH inhibitor, [3H]MAEA, bound specifically to the FAAH microsomes. Coincubation with an FAAH inhibitor, URB-597, competitively displaced the [3H]MAEA on the FAAH microsomes. The released radiolabel was then detected through an interaction with the SPA beads. The assay is specific for FAAH given that microsomes prepared from cells expressing the inactive FAAH-S241A mutant or vector alone had no significant ability to bind [3H]MAEA. Furthermore, the binding of [3H]MAEA to FAAH microsomes was abolished by selective FAAH inhibitors in a dose-dependent manner, with IC50 values comparable to those seen in a functional assay. This novel SPA has been validated and demonstrated to be simple, sensitive, and amenable to high-throughput screening.
本文描述了一种使用闪烁邻近分析（SPA）技术检测人脂肪酸酰胺水解酶（FAAH）的结合分析方法。该SPA利用[3H]R(+)-甲磺酰胺（MAEA）与表达FAAH的微粒体之间的特异性相互作用来评估FAAH抑制剂的置换活性。我们观察到一种竞争性非水解FAAH抑制剂[3H]MAEA特异性结合到FAAH微粒体上。与FAAH抑制剂URB-597共同孵育会竞争性地置换FAAH微粒体上的[3H]MAEA。然后通过与SPA微球的相互作用检测释放的放射性标记物。鉴于从表达无活性FAAH-S241A突变体的细胞或单独的载体中制备的微粒体没有明显的结合[3H]MAEA的能力，该分析对FAAH具有特异性。此外，[3H]MAEA与FAAH微粒体的结合被选择性FAAH抑制剂以剂量依赖性方式消除，IC50值与功能分析中的值相当。这种新型SPA已经得到验证，并证明其简单、灵敏且适用于高通量筛选。
