X-Ray Crystallography and Free Energy Calculations Reveal the Binding Mechanism of A2A Adenosine Receptor Antagonists

Abstract

We present a robust protocol based on iterations of free energy perturbation (FEP) calculations, chemical synthesis, biophysical mapping and X-ray crystallography to reveal the binding mode of an antagonist series to the A_2A adenosine receptor (AR). Eight A_2A AR binding site mutations from biophysical mapping experiments were initially analyzed with sidechain FEP simulations, performed on alternate binding modes. The results distinctively supported one binding mode, which was subsequently used to design new chromone derivatives. Their affinities for the A_2A AR were experimentally determined and investigated through a cycle of ligand-FEP calculations, validating the binding orientation of the different chemical substituents proposed. Subsequent X-ray crystallography of the A_2A AR with a low and a high affinity chromone derivative confirmed the predicted binding orientation. The new molecules and structures here reported were driven by free energy calculations, and provide new insights on antagonist binding to the A_2A AR, an emerging target in immuno-oncology.

Keywords: G protein-coupled receptor (GPCR); adenosine receptors; biophysical mapping (BPM); free energy perturbation (FEP).

Figure 1

Binding mode and chemical structures of antagonists ZM241385 (A, crystal structure 4EIY26) and triazine 4 b (B). The experimental pose of the triazine (cyan) was superimposed on the same crystal structure of the receptor shown in panel A (ribbons). Both compounds had been characterized by BPM (residues labelled and depicted in gray sticks). Receptor‐ligand hydrogen bonds are depicted as magenta lines.

Figure 2

Putative binding modes A (green) and B (magenta) of Chromone 14 to the A_2AAR (H‐bonds in magenta). C) Experimental and calculated changes in binding free energies for each mutation in the BPM. The error bars correspond to the s.e.m. of the replica calculations for the calculated values, or are adjusted to the reported value of 0.1 pK _D unit in the case of experimental data.27

Scheme 1

Structure and synthetic pathways employed to assembly chromones 4, 5 and 8.

Figure 3

A) Dual binding mode of caffeine, as extracted from the A_2AAR crystal structure with the A_2AAR (PDB code 5MZP). Colour code is green (binding mode A) and magenta (binding mode B). B) modelled binding modes of Chromone 4 a, following the same colouring Scheme as in panel A.

Figure 4

A) Crystal structures of the A_2AAR and compound 4 d (PDB code: 6ZDR), ligand shown in sticks and sodium ion shown as a sphere. Electron densities of chromones 4 d (B) and 5 d (C; PDB code: 6ZDV). Omit maps are 2 F _o−F _c at 1 sigma (light blue mesh) and Fo‐Fc at 3 sigma (green mesh). Binding mode of compound 4 d (D) and 5 d (E); ligands and the conserved residue N253^6.55 shown as sticks, water molecules in red spheres.

Figure 5

Crystal structure (orange) and modelled coordinates (cyan) of (A) the highest affinity compound 4 d (PDB code: 6ZDR) and (B) the methylated derivative 5 d (PDB code: 6ZDV) with the A_2AAR. H‐bond interactions are indicated in magenta.