Pharmacophore Identification of the Neuronal Cyclic AMP sensors PKA, Epac2 and NCS‐Rapgef2 Using Novel Adenine‐Based Compounds

In the neuroendocrine NS‐1 cell line, three separate cAMP signaling pathways mediate growth arrest (via Epac2 ‐‐ p38 MAPK), induction of neuronal genes and cell survival (via PKA ‐‐ CREB), and neurite outgrowth (via NCS‐Rapgef2 ‐‐ ERK) (see Emery et al., J. Biol. Chem. 289:10126–39, 2014). There is no identifiable cross‐talk between these three pathways, thus they are parcellated. These three cAMP signaling pathways are activated by a variety of Gs‐coupled receptors expressed in the CNS, which include the PACAP receptor PAC1 (Emery & Eiden, FASEB J, 26, 3199–3211, 2011) and the D1 dopamine receptor (Jiang et al., eNeuro, ENEURO.0248–17.2017). Therefore, a signaling component unique to one pathway could represent a target for intracellular therapeutics to modulate aspects of neuronal behavior for CNS disorders related to addiction (e.g. D1‐dependent signaling to ERK) and anxiety (e.g. PACAP/PAC1 signaling). Using cell‐based assays to monitor the activation of each of the three pathways, we have sought to identify specific inhibitors and activators of NCS‐Rapgef2.Using a battery of biochemical and high‐content assays that distinguish signaling between the three cAMP sensors (PKA, Epac2 and NCS‐Rapgef2), we found that the adenylate cyclase inhibitor 9‐tetrahydrofuranyladenine (SQ,22536; IC50 for forskolin‐induced cAMP accumulation = 10 μM) also inhibits NCS‐Rapgef2 (IC50 = 170 μM, inhibiting 8‐Br‐cAMP‐induced Elk1 activation, reported in Emery et al., Mol Pharmacol 83, 95–105, 2013). To create compounds with selectivity and greater potency at NCS‐Rapgef2, we generated analogs of 9‐tetrahydrofuranyladenine at several positions of the adenine (6‐amino‐purine) moiety. In NS‐1 cells, we found that the compound N6‐phenyl‐9‐tetrahydrofuranyladenine (EL1101) has greatly reduced activity at adenylate cyclase (IC50 > 300 μM) and no detectable activity, either an agonist antagonist, for PKA‐ and Epac2‐dependent signaling. However, EL1101 inhibts 8‐Br‐cAMP‐induced ERK phosphorylation with a greater potently than 9‐tetrahydrofuranyladenine (IC50 = 10 μM for EL1101). At similar potencies (IC50 = 10 μM), EL1101 also blocked ERK phosphorylation by Gs‐coupled GPCR activation (PACAP/PAC1), adenylate cyclase activation by forskolin, and direct activation of NCS‐Rapgef2 with cell‐permeable cAMP analogs (8‐Br‐cAMP). We also employed a phenotypic high‐content assay (cAMP analog‐dependent neurite outgrowth) and found that EL1101 blocked the neuritogenic effect of cAMP analogs at a similar potency observed in biochemical assays (IC50 = 10 μM). We are currently exploring further derivatization of the 9‐tetrahydrofuranyladenine scaffold to create a sufficiently potent and selective NCS‐Rapgef2 inhibitor for use in vivo to block aspects of D1 dopamine receptor‐dependent signaling caused by psychomotor stimulants that require ERK activation.Support or Funding InformationThis work was supported by National Institute of Mental Health Intramural Research Program Grants 1ZIAMH002386 (to L.E.E.) and 1ZIAMH002592 (to M.V.E.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.