Discovery of Novel Adenosine Receptor Agonists That Exhibit Subtype Selectivity

Abstract

Running Title: Investigating adenosine receptor selectivity of N 6 -modified agonists.Keywords: GPCRs; adenosine receptor; selectivity; G proteins; yeast. 2 AbstractA series of N 6 -bicyclic and N 6 -(2-hydroxy)cyclopentyl derivatives of adenosine were synthesized as novel A1R agonists and their A1R/A2R selectivity assessed using a simple yeast screening platform. We observed that the most selective, high potency ligands were achieved through N 6 -adamantyl substitution in combination with 5'-N-ethylcarboxamido or 5'-hydroxymethyl groups. In addition, we determined that 5'-(2-fluoro)thiophenyl derivatives all failed to generate a signaling response despite showing an interaction with the A1R. Some selected compounds were also tested on A1R and A3R in mammalian cells revealing that four of them are entirely A1R-selective agonists. By using in silico homology modeling and ligand docking, we provide insight into their mechanisms of recognition and activation of the A1R. We believe that given the broad tissue distribution, but contrasting signaling profiles, of adenosine receptor subtypes these compounds might have therapeutic potential.3

Computer-aided design of multi-target ligands at A1R, A2AR and PDE10A, key proteins in neurodegenerative diseases

“…It can be observed that the ligand/protein interactions predicted for the active compounds against the A 2A R are also those seen in the co-crystallised ligand/protein crystal structures (PDB ID: 4EIY, [ 33 ] 3EML, [ 49 ] 5IU4, [ 50 ] with a K i value of 0.8 nM for ZM241385, which is the common ligand for the three PDB IDs). Similar was the case for the reported interactions with the A 1 R homology model in the literature (with IC 50 values of 2.9 and 6.2 nM for the reported ligands predicted to bind to the homology model of A 1 R) [ 51 , 52 ].…”

“…3 Docking studies predicted molecular interactions characteristic of the 4,6-substituted 2-amino-pyridin-3-carbonitriles with the A 2A R protein crystal structure (PDB ID: 4EIY), A 1 R homology model, and PDE10A protein crystal structure (PDB ID: 4DDL), which are displayed for representative multi-target ligands with the following combinations: compound 8 (A 1 R–PDE10A), 18 (A 1 R–A 2A R), and 25 (A 2A R–PDE10A): a interactions with A 2A R: the overlaid compounds 18 and 25 exhibit H-bonds via amino and carbonitrile groups with Asn 253 , and the pyridine rings are π-stacked with Phe 168 b interactions with A 1 R: the overlaid compounds 8 and 18 exhibit H-bonds via amino and carbonitrile groups with Asn 254 , and the pyridine rings are π-stacked with Phe 171 c interactions with PDE10A: the overlaid compounds 8 and 25 have the pyridine rings π-stacked with Phe 686 and Phe 719 . The molecular interactions predicted for the active molecules are consistent with observed interactions between co-crystallised ligands and their corresponding protein crystal structures (PDB ID: 4EIY and 4DDL) [ 33 , 34 ] and the interactions with the A 1 R homology model reported in the literature [ 51 , 52 ] …”

“…In addition, compounds 8 and 16 exhibited the desired multi-target profile against A 1 R, A 2A R and PDE10A, which would serve as good starting points for further functional efficacy assessment and analog modification for the improvement of selectivity. In particular, this comprises investigating the signal transduction profiles of these compounds using techniques some of the authors have described before [ 51 ], as well as evaluating functional effects in cAMP assays to determine if these compounds do provide synergistic elevations in intracellular cAMP. One specific functional profile that would be of high interest and which is likely to elevate cAMP levels synergistically via the combination effect on multiple targets simultaneously, is the A 1 R antagonist/A 2A R agonist, and PDE10A inhibitor.…”

Pharmacological Characterisation of Novel Adenosine Receptor A3R Antagonists

“…For testing of potential antagonists, cells received a co-stimulation stimulated with forskolin, agonist and compound/DMSO control. cAMP levels were then determined using a LANCE® cAMP kit as described previously (Knight et al, 2016). In order to reduce evaporation of small volumes, the plate was sealed with a ThermalSeal® film (EXCEL Scientific) at all stages.…”

GLP-1(9-36) mediates the glucagonostatic effect of GLP-1 by promiscuous activation of the glucagon receptor

“…The transfected cells were grown 48 h prior to assays. cAMP accumulation assays: Assays were performed as previously described (19,23,39). In brief, ∆CTR-HEK 293 cells transiently transfected with GCGR or GPR119 were The cells were stimulated with ligands for 15 or 30 min as indicated and cAMP accumulation was measured using a LANCE® cAMP detection assay kit.…”