A new site and mechanism of action for the widely used adenylate cyclase inhibitor SQ22,536.
We evaluated the efficacy, potency, and selectivity of the three most commonly used adenylate cyclase (AC) inhibitors in a battery of cell lines constructed to study signaling via three discrete cAMP sensors identified in neuroendocrine cells. SQ22,536 [9-(tetrahydrofuryl)-adenine] and 2',5'-dideoxyadenosine (ddAd) are effective and potent AC inhibitors in HEK293 cells expressing a cAMP response element (CRE) reporter gene, and MDL-12,330A [cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine hydrochloride] is not. Neuroscreen-1 (NS-1) cells were used to assess the specificity of the most potent AC inhibitor, SQ22,536, to block downstream cAMP signaling to phosphorylate CREB (via PKA); to activate Rap1 (via Epac); and to activate ERK signaling leading to neuritogenesis (via the newly described neuritogenic cAMP sensor NCS). SQ22,536 failed to inhibit the effects of cAMP analogs 8-Br-cAMP and 8-CPT-2'-O-Me-cAMP on PKA-mediated CREB activation/phosphorylation and Epac-mediated Rap1 activation, indicating that it does not inhibit these cAMP pathways beyond the level of AC. On the other hand, SQ22,536, but not ddAd, inhibited the effects of cAMP analogs 8-Br-cAMP and 8-CPT-cAMP on ERK phosphorylation and neuritogenesis, indicating that it acts not only as an AC blocker, but also as an inhibitor of the NCS. The observed off-target actions of SQ22,536 are specific to cAMP signaling: SQ22,536 does not block the actions of compounds not related to cAMP signaling, including ERK induction by PMA, and ERK activation and neuritogenesis induced by NGF. These data led us to indicate a second target for SQ22,536 that should be considered when interpreting its effects in whole cell and in vivo experiments.
National Institute of Mental Health, 49 Convent Drive, Building 49, Room 5A-38, Bethesda, MD 20892-4090, USA.
Andrew C
AC
Emery
Maribeth V
MV
Eiden
Lee E
LE
Eiden
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000067956:Adenylyl Cyclase Inhibitors; D000242:Cyclic AMP / Q000502:physiology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D016048:Dideoxyadenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000502:physiology; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D007097:Imines / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects / Q000502:physiology; D055099:Neuroendocrine Cells / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D010766:Phosphorylation; D043562:Receptors, G-Protein-Coupled / Q000502:physiology; D015398:Signal Transduction; D050807:ets-Domain Protein Elk-1 / Q000096:biosynthesis
D000067956:Adenylyl Cyclase Inhibitors; D017362:Cyclic AMP Response Element-Binding Protein; D020662:Guanine Nucleotide Exchange Factors; D007097:Imines; D043562:Receptors, G-Protein-Coupled; D050807:ets-Domain Protein Elk-1; C017759:9-(tetrahydro-2-furyl)-adenine; D016048:Dideoxyadenosine; C014925:RMI 12330A; C021008:2',5'-dideoxyadenosine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D000225:Adenine