Pharmacological Characterisation of Novel Adenosine Receptor A₃R Antagonists

Abstract

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

Insights to the Binding of a Selective Adenosine A3 Receptor Antagonist using Molecular Dynamics Simulations, Binding Free Energy Calculations and Mutagenesis

“…The MD simulations of K18 in complex with WT human A 3 R showed that the most frequent (>20% of the MD trajectory) contacts involved V72 2.23 , L90 3.32 , F168 5.29 , V169 5.30 , M177 5.38 , L246 6.51 , I249 6.54 , N250 6.55 as previously found, 24 and less than 15% was recorded for van der Waals with V65 2.16 , I186 5.47 and L264 7.35 . Hydrophobic contacts were measured in the MD simulations and are showed in the interaction fraction plot ( Figure 2D) when the side chain of a hydrophobic residue fell within 3.6 Å from the ligand.…”

“…The MD simulation with the "up TM5, TM6" conformation (see methods section) as starting structure converged in a stable conformation with an RMSD of less than 2 Å compared to the starting structure. 24 Interestingly, the MD simulations revealed that starting from conformation "up TM1, TM2", K18 rotated phenyl-oxazolyl bond but also N-O bond resulting in a conformation with a dichlorophenyl orientation also towards TM5, TM6 conformation ( Figure 2A) with a relative binding free energy ΔΔG eff = + 3.8 compared to "up TM5, TM6" starting structure conformation ( Figure 2B) according to both MM-PBSA and MM-GBSA calculations.…”

“…L264 7.35 A and L90 3.32 A A 3 R mutant showed an increase in antagonistic potency ( Table 2). 24 When compared to the K18-WT A 3 R complex, the interactions in L90 3.32 A A 3 R with N250 6.55 , F168 5.29 , L246 6.51 , M177 5.38 , I249 6.54 were maintained. Van der Waals interaction with I249 6.54 and the interaction with F168 5.29 showed a particularly increased frequency, the last due to a strong hydrogen bond interaction between the carbonyl group of K18 and the backbone NH groups of F168 5.29 aided by the reorientation of K18 towards TM6 (Figure 8).…”

“…In order to investigate computationally the stability and the interactions for each mutant A 3 R-agonist complex with K18 their complexes in a hydrated POPE bilayer were subjected to MD simulations for 150 ns and MM-GBSA calculations were performed in the resulting trajectories. The previously experimentally determined pA2 values 24 were included in Table 2; it is recalled that an increase in the pA 2 value of K18 for a particular mutant A 3 R (when compared to WT A 3 R) shows that the antagonist was more active and a decrease indicated a reduced potency. The pA2 values and the calculated effective binding free energies (ΔG eff ) 57,56 (Table S1, Figure S3) showed significant correlation for K18 r = -0.82 (95% confidence interval, -0.94 to -0.68 (n=12), p < 0.01) ( Figure 4).…”

“…Mutants L90 3.32 , L264 7.35 A that increase the potency of the studied antagonist agonist have relative binding free energies ΔΔG eff that were -5.6 and -4.8 kcal mol -1 more negative than K18-WT A 3 R. While more accurate computational methods like FEP/MD 61 are available the calculated ΔΔG eff values using the MM-GBSA allowed us to distinguish the reduction, maintenance and increase of K18 potency against the corresponding mutant A 3 R receptors. 24 c Mean±SD (Å); Protein RMSD is calculated for the C α atoms of the α-helices, for the last 50 ns of the trajectories. Frame 0 is used as reference structure.…”