Structural Characterization of Agonist Binding to an A₃ Adenosine Receptor through Biomolecular Simulations and Mutagenesis Experiments

Abstract

The adenosine A3 receptor (A3R) binds adenosine and is a drug target against cancer cell proliferation. Currently, there is no experimental structure of A3R. Here, we have generated a molecular model of A3R in complex with two agonists, the nonselective 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-β-d-ribofuranuronamide (NECA) and the selective 1-deoxy-1-[6-[[(3-iodophenyl)­methyl]­amino]-9H-purin-9-yl]-N-methyl-β-d-ribofuranuronamide (IB-MECA). Molecular dynamics simulations of the wild-type A3R in complex with both agonists, combined with in vitro mutagenic studies revealed important residues for binding. Further, molecular mechanics-generalized Born surface area calculations were able to distinguish mutations that reduce or negate agonistic activity from those that maintained or increased the activity. Our studies reveal that selectivity of IB-MECA toward A3R requires not only direct interactions with residues within the orthosteric binding area but also with remote residues. Although V1695.30 is considered to be a selectivity filter for A3R binders, when it was mutated to glutamic acid or alanine, the activity of IB-MECA increased by making new van der Waals contacts with TM5. This result may have implications in the design of new A3R agonists.

Pharmacological characterisation of novel adenosine A3 receptor antagonists

“…Combining MD simulations with mutagenesis data, we presented a final binding pose of K18 which appears to be within the orthosteric binding site, involving residues previously described to be involved in binding of A 3 R compounds 35 . 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.…”

“…5. Mutation of residues F168 5.29 , L246 6.51 , N250 6.55 and I268 7.39 abolished agonist induced suppression of forskolin-induced cAMP accumulation and were discontinued in this study 22 . The affinity (pA 2 ) of K18 at the WT and mutant A 3 R were compared in order to determine the potential antagonist binding site ( Fig.…”

“…The Nluc-tagged rat A 3 R pcDNA3.1 + construct, used in the generation of the stable Nluc-tagged rat A 3 R expressing HEK 293 cell line was kindly gifted to us by Stephen Hill and Stephen Briddon (University of Nottingham). All oligonucleotides used for mutagenesis were designed using the online Agilent Genomics 'QuikChange Primer Design' tool (detailed in Stamatis et al 22 ( Table S4)) and purchased from Merck. All constructs were confirmed by in-house Sanger sequencing.…”

“…Relative binding free energies of the complexes between K5, K17, K18, MRS 1220 and A 3 R was estimated by the 1-trajectory MM-PBSA approach 56 . Effective binding energies (ΔG eff ) were computed considering the gas phase energy and solvation free energy contributions to binding-see appendix II 21,22 .…”

In Silico Drug Design for Purinergic GPCRs: Overview on Molecular Dynamics Applied to Adenosine and P2Y Receptors

“…The compounds' adenosine-like scaffold similarly bound the two receptors to the binding mode adenosine or other agonists assumed in the experimental structure of A 1 AR or A 2A AR (Figure 2A,B) [32][33][34][35][36][37]39]: a bidentate H-bond between N250 6.55 (N251 6.55 at mA 3 AR) and the ligands' N7 and exocyclic N 6 H, a π-π stacking between the adenine aromatic scaffold and F168 ECL2 (F169 ECL2 (m)), and the H-bonds of hydroxyls 2 and 3 with H272 7.43 and S271 7.42 (respectively H273 7.43 (m) and S272 7.42 (m)), which were less persistent during MD. Contacts with residues that are involved in agonist binding and/or receptor activation according to site-directed mutagenesis (SDM) studies [70][71][72] were observed during the simulations: L90 3.32 (L91 3.32 (m)), T94 3.36 (T95 3.36 (m)), M177 5.38 (M178 5.38 (m)), W243 6.48 (W244 6.48 (m)), L246 6.51 (L247 6.51 (m)), I268 7.39 (I269 7.39 (m)), in addition to the aforementioned residues. Together with this, MRS7591 transiently interacted with residues of ECL2 and ECL3 in both hA 3 AR and mA 3 AR simulations, possibly explaining the higher affinity of this compound for the receptor in comparison to MRS5776.…”