Background: The glucagon and glucagon-like peptide-1 (GLP-1) receptors are important targets for treating type 2 diabetes.Results: We describe novel glucagon receptor pharmacology, through interaction with the receptor activity-modifying protein-2 (RAMP2).Conclusion: RAMP2 regulates both ligand binding and G protein selectivity of the glucagon receptor.Significance: The effect of RAMP2 should be considered when designing anti-diabetic treatments.

The adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between (adenosine receptors) orthosteric binding sites, obtaining highly selective antagonists is a challenging but critical task. Here we screen 39 potential A3R, antagonists using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Structure–activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazolyl moiety or the aromatic nitrogen heterocycle with nitrogen at α-position to the carbon of carboximidamide group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by molecular dynamic simulations combined with Molecular Mechanics—Poisson Boltzmann Surface Area calculations identified the residues important for binding in the A3R orthosteric site. We demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (< 1 µM) competitive antagonist. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies. These results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Our findings may provide new insights for drug discovery.

Adenosine A3 receptor (A3R) is a promising drug target cancer and for a number of other conditions like inflammatory diseases, including asthma and rheumatoid arthritis, glaucoma, chronic obstructive pulmonary disease, and ischemic injury. Currently, there is no experimentally determined structure of A3R. We explored the binding profile of O4-{[3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl]­carbonyl}-2-methyl-1,3-thiazole-4-carbohydroximamide (K18), which is a new specific and competitive antagonist at the orthosteric binding site of A3R. MD simulations and MM-GBSA calculations of the WT A3R in complex with K18 combined with in vitro mutagenic studies show that the most plausible binding conformation for the dichlorophenyl group of K18 is oriented toward trans-membrane helices (TM) 5, 6 and reveal important residues for binding. Further, MM-GBSA calculations distinguish mutations that reduce or maintain or increase antagonistic activity. Our studies show that selectivity of K18 toward A3R is defined not only by direct interactions with residues within the orthosteric binding area but also by remote residues playing a significant role. Although V1695.30 is considered to be a selectivity filter for A3R binders, when it was mutated to glutamic acid, K18 maintained antagonistic potency, in agreement with our previous results obtained for agonists binding profile investigation. Mutation of the direct interacting residue L903.32 in the low region and the remote L2647.35 in the middle/upper region to alanine increases antagonistic potency, suggesting an empty space in the orthosteric area available for increasing antagonist potency. These results approve the computational model for the description of K18 binding at A3R, which we previously performed for agonists binding to A3R, and the design of more effective antagonists based on K18.

Author Contributions:KB, AK and GL conceived and designed the research; KB performed the mammalian assays; KK conducted radioligand binding experiments, PL, DS, EV and AK performed the molecular dynamic simulations; SH derived the equations for the 'Rapid competitor dissociation kinetics' model; KB, GL and AK analysed data; KB and GL wrote manuscript, AK revised and edited the manuscript. Summary Background and PurposeThe adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between ARs orthosteric binding sites, obtaining highly selective receptor antagonists is a challenging but critical task.Experimental approach 39 potential A3R, antagonists were screened using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Further, a likely binding pose of the most potent antagonist (K18) was determined through molecular dynamics (MD) simulations and consistent calculated binding 2 free energy differences between K18 and congeners, using a homology model of A3R, combined with mutagenesis studies. Key ResultsWe demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (<1 µM) competitive antagonist. Structure-activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazole group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by MD simulations identified the residues important for binding in the A3R orthosteric site. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies. Conclusions and ImplicationsThese results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Word count: 241

The development of therapeutic agonists for G protein-coupled receptors (GPCRs) is hampered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A1 receptors (A1Rs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the A1R-selective agonist, benzyloxy-cyclopentyladenosine (BnOCPA), is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native A1Rs arises from BnOCPA’s unique and exquisitely selective activation of Gob among the six Gαi/o subtypes, and in the absence of β-arrestin recruitment. BnOCPA thus demonstrates a highly-specific Gα-selective activation of the native A1R, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective Gα agonism.