Novel Adenine‐based Inhibitors Define a Pharmacophore with Selectivity for the cAMP Sensor NCS‐Rapgef2 versus PKA, Epac and Adenylate Cyclase in Neuroendocrine Cell Lines
Authors: Eiden Lee E , Alvarez Ryan , Eiden Maribeth V , Xu Wenqin , Emery Andrew C 
Published: November 2023

We have identified in a neuroendocrine cell line, NS‐1, three separate pathways for cAMP signaling mediated through Epac2 via p38 phosphorylation (subserving growth arrest); through PKA via CREB phosphorylation (subserving neuronal survival and neuron‐specific gene expression); and through the novel cAMP sensor NCS‐Rapgef2 (Emery et al., Sci. Signal. 6,281:ra51, 2013) via ERK phosphorylation (subserving neuritogenesis) (Emery et al., J. Biol. Chem. 289:10126–39, 2014). These pathways are parcellated, exhibiting little if any signaling cross‐talk, and are activated by a variety of CNS Gs‐coupled receptors, including the PAC1 PACAP receptor (Emery & Eiden, FASEB J, 26, 3199–3211, 2011) and the D1 dopamine receptor (Eiden et al., FASEB J, 30, supplement 1, 1265.5, 2016). Thus, points within each pathway may eventually represent targets for intracellular therapeutics aimed at modulation of individual components of neuronal behavior, in the context of CNS disorders relating to addiction and anxiety. Accordingly we have embarked on a drug discovery program, using assays developed in cellula for each pathway, for specific inhibitors of NCS‐Rapgef2.The adenylyl cyclase inhibitor SQ22,536 (9‐tetrahydrofuranyladenine) (IC50 = 10 mM to inhibit forskolin‐induced Elk1 activation) has in addition inhibitory activity against NCS‐Rapgef2 (IC50 = 170 mM to inhibit 8‐Br‐cAMP‐induced Elk1 activation): this work was carried out using a battery of high‐content in cellula assays that distinguish signaling through the three intracellular cAMP sensors NCS‐Rapgef2, exchange protein activated by cAMP (Epac), and protein kinase A (PKA) (Emery et al., Mol Pharmacol 83, 95–105, 2013). We have now examined the activities of compounds with modified substituents at several positions of adenine (6‐amino‐purine) to enhance selectivity for NCS‐Rapgef2 by decreasing affinity for adenylyl cyclase (AC) without increasing affinity for PKA or Epac. The compound N6‐phenyl‐9‐tetrahydrofuranyladenine is a potent NCS‐Rapgef2 inhibitor (IC50 = 10 mM to inhibit 8‐Br‐cAMP‐dependent ERK phosphorylation) with greatly diminished AC inhibitory activity (IC50 >300 mM for cyclic AMP measurements following 40 min treatment with IBMX + forskolin), without noticeable inhibitory or stimulatory activity at either PKA or Epac. These measurements were carried out in the neuroendocrine pheochromocytoma (PC12)‐derived neuroendocrine cell line NS‐1, engineered to allow high‐content screening for activation and inhibition of AC, PKA, Epac and NCS‐Rapgef2. Exploration of further modification to create a sufficiently potent and selective NCS‐Rapgef2 inhibitor for use in vivo for blockade of D1 dopamine receptor‐dependent effects of psychomotor stimulants that require signaling through ERK are in progress.Support or Funding InformationThis work was supported by the National Institute of Mental Health Intramural Research Program, Projects MH002386 to L.E.E. and MH002592 to M.V.E.