title: Characterization of the first potent and selective PDE9 inhibitor using a cGMP reporter cell line.
abstract: We report here the in vitro characterization of 1-(2-chlorophenyl)-6-[(2R)-3,3,3-trifluoro-2-methylpropyl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidine-4-one (BAY 73-6691), the first potent and selective inhibitor of phosphodiesterase 9 (PDE9), which is currently under preclinical development for the treatment of Alzheimer's disease. This compound selectively inhibits human (IC50 = 55 nM) and murine (IC50 = 100 nM) PDE9 activity in vitro and shows only moderate activity against other cyclic nucleotide-specific phosphodiesterases. We also report the generation and characterization of a stably transfected PDE9 Chinese hamster ovary cell line, additionally expressing soluble guanylate cyclase (sGC), the olfactory cyclic nucleotide-gated cation channel CNGA2 and the photoprotein aequorin. In this cell line, intracellular cGMP levels can be monitored in real-time via aequorin luminescence induced by Ca2+ influx through CNGA2, acting as the intracellular cGMP sensor. This simple and sensitive assay system was used for the characterization of the cellular activity of the new PDE9 inhibitor. BAY 73-6691 alone did not significantly increase basal cGMP levels in this experimental setting. However, in combination with submaximal stimulating concentrations of the sGC activator 4-[((4-carboxybutyl)[2-[(4-phenethyl-benzyl)oxy]phenethyl]amino)methyl] benzoic acid (BAY 58-2667), the compound induced concentration-dependent luminescence signals and intracellular cGMP accumulation. The PDE9 inhibitor significantly potentiated the cGMP signals generated by sGC activating compounds such as BAY 58-2667 or 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-4-ylamine (BAY 41-2272) and induced leftward shifts of the corresponding concentration-response curves. Using our newly generated PDE9 reporter cell line, we could show that BAY 73-6691 is able to efficiently penetrate cells and to inhibit intracellular PDE9 activity.
authors.affiliation: Cardiovascular Research, Bayer HealthCare AG, Aprather Weg 18a, D-42096 Wuppertal, Germany. frank.wunder@bayerhealthcare.com
authors.forename: Frank
authors.initials: F
authors.lastname: Wunder
authors.forename: Adrian
authors.initials: A
authors.lastname: Tersteegen
authors.forename: Annegret
authors.initials: A
authors.lastname: Rebmann
authors.forename: Christina
authors.initials: C
authors.lastname: Erb
authors.forename: Thomas
authors.initials: T
authors.lastname: Fahrig
authors.forename: Martin
authors.initials: M
authors.lastname: Hendrix
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors* / Q000235:genetics; D000818:Animals; D001565:Benzoates / Q000494:pharmacology; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D006152:Cyclic GMP / Q000032:analysis; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D017930:Genes, Reporter; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D014162:Transfection
chemical_list: D001565:Benzoates; D004791:Enzyme Inhibitors; C480588:BAY 58-2667; D015105:3',5'-Cyclic-AMP Phosphodiesterases; C507055:Pde9a protein, mouse; D006152:Cyclic GMP