Golla Rajasree, Seethala Ramakrishna
Drug Discovery, Pharmaceutical Research Institute, Bristol-Myers Squibb, Princeton, NJ, USA.
J Biomol Screen. 2002 Dec;7(6):515-25. doi: 10.1177/1087057102238625.
In the new high-throughput screening (HTS) campaign, receptor functional assays, 3',5'-cyclic adenosine monophosphate (cAMP), intracellular Ca(2)+, phosphatidylinositol turnover, and reporter-based assays are being used as primary screens as they are now developed as homogeneous and automation-friendly assays. FlashPlate assay and scintillation proximity assay using radiolabeled cAMP have been used for measuring cAMP. A nonradioactive homogeneous HTS assay using HitHunter trade mark enzyme fragment complementation (EFC) technology was evaluated for measuring cAMP in adherent and suspension cells overexpressing a Galpha(s)-coupled receptor. In the EFC-cAMP assay, the beta-galactosidase (beta-gal) donor fragment-cAMP (ED-cAMP) conjugate complements with the beta-gal enzyme acceptor (EA) fragment to form an active beta-gal enzyme. Binding of ED-cAMP conjugate to the anti-cAMP antibody prevents its complementation with the EA fragment to form an active enzyme. Cyclic AMP in the samples compete with ED-cAMP to bind to the anti-cAMP antibody, thus increasing the free ED-cAMP that can complement with the EA fragment to form an active enzyme that is assayed with a luminescent substrate. Thus, this assay results in a positive signal unlike other technologies, wherein the signal is completed by cAMP in the sample. Glucagon-like peptide (GLP)-1 binds to GLP-1 receptor (with a Kd of 0.2 nM) signals through Galpha(s) to activate adenylate cyclase, which results in an increase of intracellular cAMP (EC(50) of 0.3 nM). GLP-1 stimulation of cAMP levels measured by the EFC method was similar in both adherent and suspension cell formats (EC(50)~0.3 nM) at different cell numbers. The assay was further validated with forskolin, exendin, and several active GLP-1 peptide analogues. The stimulation of cAMP by GLP-1 and forskolin was effectively inhibited by the adenylate cyclase inhibitors MDL-12330A and SQ-22536, confirming that the increased cAMP is through the AC pathway. The assay tolerates dimethyl sulfoxide (DMSO) up to 10%, and tartrazine does not interfere with the assay with the adherent cells up to 1 mM and affects minimally up to 10 microM in suspension cells. The assay is very robust, with a Z' value of 0.7 to 0.8. The assay was validated with several plates of low molecular weight nonpeptide compounds and peptide agonists with different potencies. The suspension cell protocol is a robust homogeneous assay that involves fewer steps than the adherent cell protocol and is suitable for HTS. The cAMP assay using EFC technology is advantageous in that it has a greater dynamic range of detection; is nonradioactive, very sensitive, robust; has minimal interference from DMSO and colored compounds; and is amenable for automation. An added advantage of this assay is that the cAMP is measured as a positive signal, thereby reducing the incidence of false positives.
在新的高通量筛选（HTS）活动中，受体功能测定、3',5'-环磷酸腺苷（cAMP）、细胞内Ca(2)+、磷脂酰肌醇周转和基于报告基因的测定被用作初步筛选，因为它们现在已发展成为均相且便于自动化的测定方法。使用放射性标记的cAMP的FlashPlate测定法和闪烁邻近测定法已用于测量cAMP。评估了一种使用HitHunter商标酶片段互补（EFC）技术的非放射性均相HTS测定法，用于测量过表达Galpha(s)偶联受体的贴壁细胞和悬浮细胞中的cAMP。在EFC-cAMP测定中，β-半乳糖苷酶（β-gal）供体片段-cAMP（ED-cAMP）缀合物与β-gal酶受体（EA）片段互补形成活性β-gal酶。ED-cAMP缀合物与抗cAMP抗体的结合会阻止其与EA片段互补形成活性酶。样品中的环磷酸腺苷与ED-cAMP竞争结合抗cAMP抗体，从而增加可与EA片段互补形成活性酶的游离ED-cAMP，该活性酶用发光底物进行测定。因此，与其他技术不同，该测定产生阳性信号，而其他技术中信号由样品中的cAMP完成。胰高血糖素样肽（GLP）-1与GLP-1受体结合（解离常数Kd为0.2 nM），通过Galpha(s)发出信号以激活腺苷酸环化酶，从而导致细胞内cAMP增加（半数有效浓度EC(50)为0.3 nM）。通过EFC方法测量的GLP-1刺激cAMP水平在不同细胞数量的贴壁细胞和悬浮细胞形式中相似（EC(50)~0.3 nM）。该测定法用福司可林、艾塞那肽和几种活性GLP-1肽类似物进一步验证。腺苷酸环化酶抑制剂MDL-12330A和SQ-22536有效抑制了GLP-1和福司可林对cAMP的刺激，证实cAMP的增加是通过AC途径。该测定法可耐受高达10%的二甲基亚砜（DMSO），柠檬黄在高达1 mM时对贴壁细胞测定法无干扰，在悬浮细胞中高达10 microM时影响最小。该测定法非常稳健，Z'值为0.7至0.8。该测定法用几板具有不同效力的低分子量非肽化合物和肽激动剂进行了验证。悬浮细胞方案是一种稳健的均相测定法，比贴壁细胞方案步骤更少，适用于高通量筛选。使用EFC技术的cAMP测定法的优点在于它具有更大的检测动态范围；是非放射性的，非常灵敏、稳健；受DMSO和有色化合物的干扰最小；并且适合自动化。该测定法的另一个优点是cAMP作为阳性信号进行测量，从而降低了假阳性的发生率。
