title: A cell-based cGMP assay useful for ultra-high-throughput screening and identification of modulators of the nitric oxide/cGMP pathway.
abstract: 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.
authors.affiliation: Institute of Cardiovascular Research, Bayer HealthCare AG, D-42096 Wuppertal, Germany. frank.wunder@bayerhealthcare.com
authors.forename: Frank
authors.initials: F
authors.lastname: Wunder
authors.forename: Johannes-Peter
authors.initials: JP
authors.lastname: Stasch
authors.forename: Joachim
authors.initials: J
authors.lastname: Hütter
authors.forename: Cristina
authors.initials: C
authors.lastname: Alonso-Alija
authors.forename: Jörg
authors.initials: J
authors.lastname: Hüser
authors.forename: Emanuel
authors.initials: E
authors.lastname: Lohrmann
mesh_terms: D000818:Animals; D001565:Benzoates / Q000494:pharmacology; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D006224:Cricetinae; D006152:Cyclic GMP / Q000378:metabolism*; D054815:Cyclic Nucleotide-Gated Cation Channels; D004353:Drug Evaluation, Preclinical; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000378:metabolism*; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D009830:Olfactory Bulb / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D001565:Benzoates; D054815:Cyclic Nucleotide-Gated Cation Channels; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D007473:Ion Channels; D011994:Recombinant Proteins; D009569:Nitric Oxide; C480588:BAY 58-2667; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D002118:Calcium