Abstract
Nitric oxide (NO) plays an important role in the protection against the onset and progression of various cardiovascular diseases, including hypertension and atherosclerosis, which are associated with an apparently reduced NO bioavailability. Therefore, the NO/cGMP signaling pathway has gained considerable attention and has become a target for new drug development. We have established a rapid, homogeneous, cell-based and highly sensitive nitric oxide reporter assay which is suitable for ultra-high-throughput screening. In our coculture system, endothelial nitric oxide synthase (eNOS) mediated NO generation is monitored in living cells via soluble guanylyl cyclase (sGC) activation and calcium influx through the olfactory cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the intracellular cGMP sensor [1]. Using this NO reporter assay, a fully automated highthroughput screening campaign for stimulators of NO synthesis was performed. The coculture system reflects most aspects of the natural NO/cGMP signaling pathway. Namely, Ca2+-dependent and Ca2+-independent regulation of eNOS activity by G protein-coupled receptor agonists, oxidative stress, phosphorylation, and cofactor availability, as well as NO-mediated stimulation of cGMP synthesis by sGC activation. The reporter assay allows the real-time detection of NO synthesis within living cells and makes it possible to identify and characterize activators and inhibitors of enzymes involved in the NO/cGMP pathway.
Highlights
Address: 1Lead Discovery Wuppertal, Bayer HealthCare AG, D-42096 Wuppertal, Germany and 2Cardiovascular Research, Bayer HealthCare AG, D-42096 Wuppertal, Germany
3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications Meeting abstracts – A single PDF containing all abstracts in this Supplement is available here.
We have established a rapid, homogeneous, cell-based and highly sensitive nitric oxide reporter assay which is suitable for ultra-high-throughput screening
Summary
Address: 1Lead Discovery Wuppertal, Bayer HealthCare AG, D-42096 Wuppertal, Germany and 2Cardiovascular Research, Bayer HealthCare AG, D-42096 Wuppertal, Germany. Email: Frank Wunder* - frank.wunder@bayerhealthcare.com * Corresponding author from 3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications Dresden, Germany.
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