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

Adenosine A3 receptor (A3R), is a promising drug target against cancer cell proliferation. Currently there is no experimentally determined structure of A3R. Here, we have investigate a computational model, previously applied successfully for agonists binding to A3R, using molecular dynamic (MD) simulations, Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA) and Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) binding free energy calculations. Extensive computations were performed to explore the binding profile of O4-{[3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl]carbonyl}-2-methyl-1,3-thiazole-4-carbohydroximamide (K18) to A3R. K18 is a new specific and competitive antagonist at the orthosteric binding site of A3R, discovered using virtual screening and characterized pharmacologically in our previous studies. The most plausible binding conformation for the dichlorophenyl group of K18 inside the A3R is oriented towards trans-membrane helices (TM) 5 and 6, according to the MM-PBSA and MM-GBSA binding free energy calculations, and by the previous results obtained by mutating residues of TM5, TM6 to alanine which reduce antagonist potency. The results from 14 site-directed mutagenesis experiments were interpreted using MD simulations and MM-GBSA calculations which show that the relative binding free energies of the mutant A3R - K18 complexes compare to the WT A3R are in agreement with the effect of the mutations, i.e. the reduction, maintenance or increase of antagonist potency. We show that when the residues V1695.30, M1775.38, I2496.54 involved in direct interactions with K18 are mutated to alanine, the mutant A3R - K18 complexes reduce potency, increase the RMSD value of K18 inside the binding area and the MM-GBSA binding free energy compared to the WT A3R complex. Our computational model shows that other mutant A3R complexes with K18, including directly interacting residues, i.e. F1685.29A, L2466.51A, N2506.55A complexes with K18 are not stable. In these complexes of A3R mutated in directly interacting residues one or more of the interactions between K18 and these residues are lost. In agreement with the experiments, the computations show that, M1745.35 a residue which does not make direct interactions with K18 is critical for K18 binding. A striking results is that the mutation of residue V1695.30 to glutamic acid maintained antagonistic potency. This effect is in agreement with the binding free energy calculations and it is suggested that is due to K18 re-orientation but also to the plasticity of A3R binding area. The mutation of direct interacting L903.32 in the low region and the non-directly interacting L2647.35 to alanine in the middle region increases the antagonistic potency, suggesting that chemical modifications of K18 can be applied to augment antagonistic potency. The calculated binding energies ΔGeff values of K18 against mutant A3Rs displayed very good correlation with experimental potencies (pA2 values). These results further approve the computational model for the description of K18 binding with critical residues of the orthosteric binding area which can have implications for the design of more effective antagonists based on the structure of K18.