Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10536305 | Analytical Biochemistry | 2005 | 9 Pages |
Abstract
We have established a rapid, homogeneous, cell-based, and highly sensitive assay for guanosine 3â²-5â²-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.
Related Topics
Physical Sciences and Engineering
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Authors
Frank Wunder, Johannes-Peter Stasch, Joachim Hütter, Cristina Alonso-Alija, Jörg Hüser, Emanuel Lohrmann,