Insights to the Binding of a Selective Adenosine A₃ Receptor Antagonist Using Molecular Dynamic Simulations, MM-PBSA and MM-GBSA Free Energy Calculations, and Mutagenesis

Abstract

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.

Pharmacological characterisation of novel adenosine A3 receptor antagonists

“…5, Table 5). As described previously 21 , K18 displayed no difference in affinity at I249A 6.54 (compared to WT), whereas M177A 5.40 and V169A 5.30 were significantly smaller. Interestingly, we found an increase in the affinity for L90A 3.32 and L264A 7.34 when compared to WT.…”

“…These experimental findings are reflected in our predicted binding pose of K18 at the WT A 3 R ( Fig. 4) 21 .…”

“…Interestingly we found an increase in K18 affinity at L90A 3.32 and L264A 7.34 when compared to WT. Our detailed MD simulations, results published elsewhere 21 have investigated the selectivity profile of K18 and have demonstrated that K18 failed to bind A 1 R and A 2A R due to a more polar area close to TM5, TM6 when compared to A 3 R.…”

“…We previously reported 22 no detectable G i/o response following co-stimulation with forskolin and NECA or IB-MECA for A 3 R mutants F168A 5.29 , L246A 6.51 , N250A 6.55 and I268A 7.39 and our findings are in line with previous mutagenesis studies investigating residues important for agonist and antagonist binding at the human A 3 R 36,37 . Through performing Schild analysis (results of which were used to inform modelling in Lagarias et al 21 ) we experimentally determined the effect of receptor mutation on antagonist affinity for L90A 3.32 , V169A/E 5.30 , M177A 5.40 , I249A 6.54 and L264A 7.34 A 3 R. The pA 2 value for I249A 6.54 A 3 R is similar to WT, whereas M177A 5.40 and V169A 5.30 are significantly smaller suggesting these residues appear to be involved in K18 binding. Interestingly we found an increase in K18 affinity at L90A 3.32 and L264A 7.34 when compared to WT.…”

“…We have previously investigated the binding mode of K18 at the human A 3 R using a homology model of the human A 3 R and simulations 21 . The computational model showed that the 3-(dichlorophenyl) group is positioned close to V169 5.30 , M177 5.40 , I249 6.54 and L264 7.34 of the A 3 R orthosteric binding site forming attractive vdW interactions.…”