Controlling the Dissociation of Ligands from the Adenosine A_2A Receptor through Modulation of Salt Bridge Strength

Abstract

The association and dissociation kinetics of ligands binding to proteins vary considerably, but the mechanisms behind this variability are poorly understood, limiting their utilization for drug discovery. This is particularly so for G protein-coupled receptors (GPCRs) where high resolution structural information is only beginning to emerge. Engineering the human A2A adenosine receptor has allowed structures to be solved in complex with the reference compound ZM241385 and four related ligands at high resolution. Differences between the structures are limited, with the most pronounced being the interaction of each ligand with a salt bridge on the extracellular side of the receptor. Mutagenesis experiments confirm the role of this salt bridge in controlling the dissociation kinetics of the ligands from the receptor, while molecular dynamics simulations demonstrate the ability of ligands to modulate salt bridge stability. These results shed light on a structural determinant of ligand dissociation kinetics and identify a means by which this property may be optimized.

A covalent antagonist for the human adenosine A2A receptor

“…The rational ligand design came from the reported crystal structures of the hA 2A AR bound to ZM241385, providing a clear blueprint of ligand binding interactions [13, 14, 31]. A deep, planar, and narrow cavity holds the aromatic core and furan ring of ZM241385, while the phenylethylamine substituent is directed to the extracellular region (EL2 and EL3).…”

Mapping the allosteric sites of the A2A adenosine receptor

“…The structure of the A 2A AR consists of seven transmembrane helices (TM1‐TM7) connected by three intracellular loops (ICL1–ICL3) and three extracellular loops (ECL1–ECL3) . The receptor has been crystallized in complex with agonists, including adenosine, antagonists, and coupled to a G protein fragment . The A 2A AR has also been crystallized with interacting lipids and sodium ions …”

“…The receptor has been crystallized in complex with agonists, including adenosine, antagonists, and coupled to a G protein fragment . The A 2A AR has also been crystallized with interacting lipids and sodium ions …”

Prediction of consensus binding mode geometries for related chemical series of positive allosteric modulators of adenosine and muscarinic acetylcholine receptors

“…The carboxylic acid of E172 also forms a strong electrostatic interaction with the amino group of the [ 3 H]CCPA ligand. For A 2A R, it has been shown that disruption of the analogous extracellular salt bridge above the orthosteric binding pocket accelerates the dissociation of ligands from A 2A R, while stabilization slows it . In A 2A R, this salt bridge is part of a “triad” of hydrogen bonding interactions that acts as a “cap” over the orthosteric site, preventing influx of water into the receptor that accelerates dissociation .…”

“…Conversely, a common mechanism to both the adenosine and muscarinic receptors is through PAM binding with direct molecular interaction with specific residue side chains that are involved in necessary side‐chain rearrangements required for agonist ligand dissociation. Namely, (1) the adenosine receptors (A 1 R, A 2A R) PAMs were predicted to interact directly with the specific salt bridge residue pairs that have been implicated in ligand dissociation kinetics, and (2) muscarinic receptors PAMs may form direct molecular interaction with analogous salt bridge residues, such as 12 shown in Figure a, where the carboxylic acid group interacts with lysine K392, forming a salt‐bridge electrostatic interaction with residue Y179 (ECL2). Induced fit docking results also show that compounds 13 , 14 , and 19 may also bind and induce new analogous side chain interactions mediated by Q177 (ECL2) that would stabilize the receptor.…”