title: Differential Pharmacophore Definition of the cAMP Binding Sites of Neuritogenic cAMP Sensor-Rapgef2, Protein Kinase A, and Exchange Protein Activated by cAMP in Neuroendocrine Cells Using an Adenine-Based Scaffold.
abstract: We recently reported that the adenylate cyclase (AC) inhibitor SQ22,536 (9-tetrahydrofuranyl-adenine) also has inhibitory activity against the neuroendocrine-specific neuritogenic cAMP sensor-Rapgef2 (NCS-Rapgef2), a guanine nucleotide exchanger and activator for the small effector GTPase Rap1. Cell-based assays that distinguish signaling through the three intracellular cAMP sensors NCS-Rapgef2, exchange protein activated by cAMP (Epac), and protein kinase A (PKA), as well as AC, were used. These, collectively, assess the activities of adenine (6-amino-purine) derivatives modified at several positions to enhance selectivity for NCS-Rapgef2 by decreasing affinity for adenylate cyclase (AC), without increasing affinity for PKA or Epac. Testing of each adenine derivative in whole-cell assays incorporates features of cell permeability, target selectivity, and intrinsic potency into a single EC50or IC50, making robust extrapolation to compound activity in vivo more likely. N6-MBC-cAMP is a selective PKA activator (EC50= 265 μM) with low efficacy at NCS-Rapgef2. 8-CPT-2'-O-Me-cAMP and ESI-09 are confirmed as Epac-selective, for stimulation and inhibition, respectively, versus both PKA and NCS-Rapgef2. The compound N6-Phe-cAMP is a full agonist of NCS-Rapgef2 (EC50= 256 μM). It has little or no activity against Epac or PKA. The compound N6-phenyl-9-tetrahydrofuranyladenine is a novel and potent NCS-Rapgef2 inhibitor without activity at PKA, Epac, or ACs, as assayed in the neuroendocrine NS-1 cell line. This line has been engineered to allow high-content screening for activation and inhibition of AC, PKA, Epac, and NCS-Rapgef2 and the cellular activities initiated by these signaling pathway protein components.
authors.affiliation: Section on Molecular Neuroscience, ‡Section on Directed Gene Transfer, and §PET Radiopharmaceutical Sciences Section, National Institute of Mental Health Intramural Research Program , 9000 Rockville Pike, Bethesda, Maryland 20892, United States.
authors.forename: Andrew C
authors.identifier: 0000-0002-6760-2570
authors.initials: AC
authors.lastname: Emery
authors.affiliation: Section on Molecular Neuroscience, ‡Section on Directed Gene Transfer, and §PET Radiopharmaceutical Sciences Section, National Institute of Mental Health Intramural Research Program , 9000 Rockville Pike, Bethesda, Maryland 20892, United States.
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Alvarez
authors.affiliation: Section on Molecular Neuroscience, ‡Section on Directed Gene Transfer, and §PET Radiopharmaceutical Sciences Section, National Institute of Mental Health Intramural Research Program , 9000 Rockville Pike, Bethesda, Maryland 20892, United States.
authors.forename: Maribeth V
authors.initials: MV
authors.lastname: Eiden
authors.affiliation: Section on Molecular Neuroscience, ‡Section on Directed Gene Transfer, and §PET Radiopharmaceutical Sciences Section, National Institute of Mental Health Intramural Research Program , 9000 Rockville Pike, Bethesda, Maryland 20892, United States.
authors.forename: Wenqin
authors.initials: W
authors.lastname: Xu
authors.affiliation: Section on Molecular Neuroscience, ‡Section on Directed Gene Transfer, and §PET Radiopharmaceutical Sciences Section, National Institute of Mental Health Intramural Research Program , 9000 Rockville Pike, Bethesda, Maryland 20892, United States.
authors.forename: Fabrice G
authors.initials: FG
authors.lastname: Siméon
authors.affiliation: Section on Molecular Neuroscience, ‡Section on Directed Gene Transfer, and §PET Radiopharmaceutical Sciences Section, National Institute of Mental Health Intramural Research Program , 9000 Rockville Pike, Bethesda, Maryland 20892, United States.
authors.forename: Lee E
authors.initials: LE
authors.lastname: Eiden
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives*; D000818:Animals; D001665:Binding Sites; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D020662:Guanine Nucleotide Exchange Factors / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D055099:Neuroendocrine Cells / Q000187:drug effects* / Q000378:metabolism; D000071446:Neuronal Outgrowth / Q000187:drug effects; D016716:PC12 Cells; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D014157:Transcription Factors / Q000378:metabolism
chemical_list: C500984:CRTC1 protein, human; D020662:Guanine Nucleotide Exchange Factors; C000599193:Rapgef2 protein, rat; C526973:Rapgef3 protein, rat; D014157:Transcription Factors; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000225:Adenine
keywords: CREB; Cyclic AMP; ERK; Epac; Rapgef2; p38