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 EC50 or 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.