Wunder Frank, Tersteegen Adrian, Rebmann Annegret, Erb Christina, Fahrig Thomas, Hendrix Martin
Cardiovascular Research, Bayer HealthCare AG, Aprather Weg 18a, D-42096 Wuppertal, Germany.
Mol Pharmacol. 2005 Dec;68(6):1775-81. doi: 10.1124/mol.105.017608. Epub 2005 Sep 8.
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.
我们在此报告1-(2-氯苯基)-6-[(2R)-3,3,3-三氟-2-甲基丙基]-1,5-二氢-4H-吡唑并[3,4-d]嘧啶-4-酮（BAY 73-6691）的体外特性，它是磷酸二酯酶9（PDE9）的首个强效且选择性抑制剂，目前正处于治疗阿尔茨海默病的临床前开发阶段。该化合物在体外选择性抑制人（IC50 = 55 nM）和小鼠（IC50 = 100 nM）的PDE9活性，而对其他环核苷酸特异性磷酸二酯酶仅表现出中等活性。我们还报告了稳定转染的PDE9中国仓鼠卵巢细胞系的构建及特性，该细胞系还额外表达可溶性鸟苷酸环化酶（sGC）、嗅觉环核苷酸门控阳离子通道CNGA2和光蛋白水母发光蛋白。在该细胞系中，细胞内cGMP水平可通过CNGA2作为细胞内cGMP传感器，由Ca2+内流诱导的水母发光蛋白发光进行实时监测。这个简单而灵敏的检测系统用于表征新型PDE9抑制剂的细胞活性。在该实验条件下，单独使用BAY 73-6691不会显著提高基础cGMP水平。然而，与sGC激活剂4-[((4-羧基丁基)[2-[(4-苯乙基-苄基)氧基]苯乙基]氨基)甲基]苯甲酸（BAY 58-2667）的次最大刺激浓度联合使用时，该化合物诱导浓度依赖性发光信号和细胞内cGMP积累。PDE9抑制剂显著增强了由sGC激活化合物如BAY 58-2667或5-环丙基-2-[1-(2-氟苄基)-1H-吡唑并[3,4-b]吡啶-3-基]嘧啶-4-胺（BAY 41-2272）产生的cGMP信号，并使相应的浓度-反应曲线向左移动。使用我们新构建的PDE9报告细胞系，我们能够证明BAY 73-6691能够有效穿透细胞并抑制细胞内PDE9活性。
