Understanding Ligand Binding Selectivity in a Prototypical GPCR Family

Abstract

Adenosine receptors are involved in many pathological conditions and are thus promising drug targets. However, developing drugs that target this GPCR subfamily is a challenging task. A number of drug candidates fail due to lack of selectivity which results in unwanted side effects. The extensive structural similarity of adenosine receptors complicates the design of selective ligands. The problem of selective targeting is a general concern in GPCRs, and in this respect adenosine receptors are a prototypical example. Here we use enhanced sampling simulations to decipher the determinants of selectivity of ligands in A_2a and A₁ adenosine receptors. Our model shows how small differences in the binding pocket and in the water network around the ligand can be leveraged to achieve selectivity.

Figure 1

ZM241385 bound to the binding sites of (a) A_2aR and (b) A₁R in the enhanced-sampling simulations. (c) The salt bridge hindering the pocket entrance is shown as spheres. A zoomed view of the ligands in the binding sites and the key residues is presented in the boxes.

Figure 2

Ligands in this study, ZM241385 and LUF5452.

Figure 3

Analysis of the conformations adopted by ZM241385 and LUF5452 in the binding pocket of A_2aR and A₁R over the unbiased MD simulation. Probability over the space defined by the RMSD of the common heavy atoms of the ligands to the conformation adopted in PDB 5IU4 or 3PWH, after alignment to the backbone of the proteins. TM7 not shown for clarity.

Figure 4

Solvation of the 4-hydroxyphenyl or phenyl groups of the ligands over the unbiased MD simulations, represented by the number of water oxygen atoms within 4 Å from the moieties’ heavy atoms. The plots illustrate the average solvation and its standard deviation as a function of the RMSD of the ligand to an upright, 5IU4-like conformation (see Figure 3). Conformations A and B show the displacement of water molecules in the hydrophobic ECV pocket of A₁R by LUF5452.

Figure 5

Projection of the free energy landscapes onto Z-projection and XY-projection by reweighting. Boxes: Representative conformations of the ligands bound to the main binding site of the receptors, LUF5452 interacting with the hydrophobic ECV pocket in A₁R, and the two molecules breaking the salt bridge at the entrance of the orthosteric site of the proteins.

Figure 6

Representative binding or unbinding paths of the ligands, extracted from the PT-metaD simulations. The ligands need to break the salt bridge at the entrance of the binding site in order to unbind. Extensive interactions with ECL2 are found before complete solvation of the ligands in the fully unbound states.