Yangthara Buranee, Mills Aaron, Chatsudthipong Varanuj, Tradtrantip Lukmanee, Verkman A S
Department of Medicine, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521, USA.
Mol Pharmacol. 2007 Jul;72(1):86-94. doi: 10.1124/mol.107.034496. Epub 2007 Apr 13.
G-protein-coupled receptors (GPCRs) such as the vasopressin-2 receptor (V(2)R) are an important class of drug targets. We developed an efficient screen for GPCR-induced cAMP elevation using as read-out cAMP activation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. Fischer rat thyroid cells expressing CFTR and a halide-sensing yellow fluorescent protein (H148Q/I152L) were transfected with V(2)R. Increased cell Cl(-) conductance after agonist-induced cAMP elevation was assayed using a plate reader from cell fluorescence after solution I(-) addition. The Z' factor for the assay was approximately 0.7 with the V(2)R agonist [deamino-Cys1, Val4, d-Arg8]-vasopressin (1 nM) as positive control. Primary screening of 50,000 small molecules yielded a novel, 5-aryl-4-benzoyl-3-hydroxy-1-(2-arylethyl)-2H-pyrrol-2-one class of V(2)R antagonists that are unrelated structurally to known V(2)R antagonists. The most potent compound, V(2)R(inh)-02, which was identified by screening 35 structural analogs, competitively inhibited V(2)R-induced cAMP elevation with K(i) value of approximately 70 nM and fully displaced radiolabeled vasopressin in binding experiments. V(2)R(inh)-02 did not inhibit forskolin or beta(2)-adrenergic receptor-induced cAMP production and was more than 50 times more potent for V(2)R than for V(1a)R. The favorable in vitro properties of the pyrrol-2-one antagonists suggests their potential usefulness in aquaretic applications. The CFTR-linked cAMP assay developed here is applicable for efficient, high-throughput identification of modulators of cAMP-coupled GPCRs.
G蛋白偶联受体（GPCRs），如血管加压素2型受体（V(2)R），是一类重要的药物靶点。我们开发了一种高效的筛选方法，用于检测GPCR诱导的环磷酸腺苷（cAMP）升高，该方法以囊性纤维化跨膜传导调节因子（CFTR）氯离子通道的cAMP激活作为读数。将表达CFTR和卤化物敏感型黄色荧光蛋白（H148Q/I152L）的Fischer大鼠甲状腺细胞转染V(2)R。在加入溶液I(-)后，使用酶标仪通过细胞荧光检测激动剂诱导的cAMP升高后细胞氯离子传导率的增加。以V(2)R激动剂[脱氨基-Cys1，Val4，d-Arg8]-血管加压素（1 nM）作为阳性对照，该检测方法的Z'因子约为0.7。对50,000种小分子进行初步筛选，得到了一类新型的5-芳基-4-苯甲酰基-3-羟基-1-(2-芳基乙基)-2H-吡咯-2-酮类V(2)R拮抗剂，其结构与已知的V(2)R拮抗剂无关。通过筛选35种结构类似物鉴定出的最有效化合物V(2)R(inh)-02，在结合实验中以约70 nM的K(i)值竞争性抑制V(2)R诱导的cAMP升高，并完全取代放射性标记的血管加压素。V(2)R(inh)-02不抑制福斯高林或β(2)-肾上腺素能受体诱导的cAMP产生，对V(2)R的效力比对V(1a)R高50倍以上。吡咯-2-酮拮抗剂良好的体外特性表明它们在利水应用中具有潜在用途。这里开发的与CFTR相关的cAMP检测方法适用于高效、高通量鉴定cAMP偶联GPCR的调节剂。
