An Integrative Biophysical and Computational Workflow Uncovers New Allosteric Sites and Modulators of the Human A 2A Adenosine Receptor

Allosteric modulators offer a promising approach for targeting receptor function in pathological contexts. Although numerous orthosteric ligands have been developed, the discovery of allosteric inhibitors remains limited, hindered by challenges with identifying modulators and the perceived lower druggability of allosteric sites. In this study, we employed affinity selection–mass spectrometry (AS–MS) with a chemically diverse library to identify novel allosteric modulators of the human A 2A adenosine receptor (A 2A AR). Subsequent competition binding and orthogonal biophysical assays confirmed the allosteric nature of multiple initial hits, underscoring the sensitivity and utility of the AS–MS approach. Despite exhibiting relatively weak affinity, these compounds modulated cAMP production, supporting the idea that allosteric modulators can exert functional effects without requiring high potency to outcompete endogenous adenosine. Experiments in the presence of an A 2A AR agonist further supported the classification of these compounds as negative allosteric modulators (NAMs), with distinct pharmacological profiles indicative of diverse mechanisms of action. An integrated computational workflow designed to predict allosteric sites and model ligand interactions provided insight into potential binding poses, yielding both intracellular and extracellular allosteric sites that aligned with the experimentally observed pharmacological properties. The identification of these previously uncharacterized NAMs represents an important step toward developing alternative A 2A AR-targeted therapies, enabling pharmacological intervention through receptor sites beyond the orthosteric binding pocket.