@article {Emerymol.112.081760, author = {Andrew C Emery and Maribeth V Eiden and Lee E Eiden}, title = {A New Site and Mechanism of Action for the Widely Used Adenylate Cyclase Inhibitor SQ22,536}, elocation-id = {mol.112.081760}, year = {2012}, doi = {10.1124/mol.112.081760}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {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 and 2{\textquoteright},5{\textquoteright}-dideoxyadenosine (ddAd) are effective and potent AC inhibitors in HEK293 cells expressing a reporter gene for CRE, while MDL-12,330A is not. Neuroscreen-1 (NS-1) cells were employed 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-pCPT-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 it acts not only as an AC blocker, but also as an inhibitor of the NCS. The observed {\textquoteright}off-target{\textquoteright} 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 lead us to point out a second target for SQ22,536 that should be considered when interpreting its effects in whole cell and in vivo experiments.}, issn = {0026-895X}, URL = {https://molpharm.aspetjournals.org/content/early/2012/10/10/mol.112.081760}, eprint = {https://molpharm.aspetjournals.org/content/early/2012/10/10/mol.112.081760.full.pdf}, journal = {Molecular Pharmacology} }