Wunder Frank, Stasch Johannes-Peter, Hütter Joachim, Alonso-Alija Cristina, Hüser Jörg, Lohrmann Emanuel
Institute of Cardiovascular Research, Bayer HealthCare AG, D-42096 Wuppertal, Germany.
Anal Biochem. 2005 Apr 1;339(1):104-12. doi: 10.1016/j.ab.2004.12.025.
We have established a rapid, homogeneous, cell-based, and highly sensitive assay for guanosine 3'-5'-cyclic monophosphate (cGMP) that is suitable for fully automated ultra-high-throughput screening. In this assay system, cGMP production is monitored in living cells via Ca2+ influx through the olfactory cyclic nucleotide-gated cation channel CNGA2, acting as the intracellular cGMP sensor. A stably transfected Chinese hamster ovary (CHO) cell line was generated recombinantly expressing soluble guanylate cyclase, CNGA2, and aequorin as a luminescence indicator for the intracellular calcium concentration. This cell line was used to screen more than 900,000 compounds in an automated ultra-high-throughput screening assay using 1536-well microtiter plates. In this way, we have been able to identify BAY 58-2667, a member of a new class of amino dicarboxylic acids that directly activate soluble guanylate cyclase. The assay system allows the real-time cGMP detection within living cells and makes it possible to screen for activators and inhibitors of enzymes involved in the nitric oxide/cGMP pathway.
我们建立了一种快速、均相、基于细胞且高度灵敏的鸟苷 3'-5'-环磷酸（cGMP）检测方法，适用于全自动超高通量筛选。在该检测系统中，通过嗅觉环核苷酸门控阳离子通道 CNGA2 的 Ca2+内流来监测活细胞中的 cGMP 生成，CNGA2 作为细胞内 cGMP 传感器。通过重组产生了一种稳定转染的中国仓鼠卵巢（CHO）细胞系，该细胞系表达可溶性鸟苷酸环化酶、CNGA2 和作为细胞内钙浓度发光指示剂的水母发光蛋白。使用 1536 孔微量滴定板，在全自动超高通量筛选检测中，用该细胞系筛选了超过 900,000 种化合物。通过这种方式，我们能够鉴定出 BAY 58-2667，它是一类直接激活可溶性鸟苷酸环化酶的新型氨基二羧酸成员。该检测系统能够在活细胞内实时检测 cGMP，并使得筛选参与一氧化氮/cGMP 途径的酶的激活剂和抑制剂成为可能。
