Structure-based discovery of positive allosteric modulators of the A₁ adenosine receptor

Allosteric modulation of G protein-coupled receptors (GPCRs) is an exciting strategy for developing new therapeutic agents, and it has several advantages over more commonly used orthosteric drugs. Recently determined GPCR structures have revealed allosteric pockets facing the lipid bilayer, enabling rational drug design. Here, we develop a virtual screening strategy to discover ligands of extrahelical binding pockets and apply this approach to the adenosine A₁ receptor (A₁R). The A₁R is a high-value therapeutic target for ischemia-reperfusion injury and chronic neuropathic pain. Developing effective A₁R therapeutics remains challenging due to high structural conservation across orthosteric binding sites and on-target unwanted effects stimulated by prototypical A₁R agonists, such as bradycardia and atrioventricular block. However, A₁R positive allosteric modulators (PAMs) acting through spatially distinct allosteric sites can fine-tune A₁R activity with high subtype selectivity and spatiotemporal specificity, thereby overcoming current limitations. A chemical library of 160 million compounds was computationally docked to the allosteric pocket identified in a cryo-EM structure of the A₁R, and a set of 26 top-ranked compounds were selected for experimental evaluation. Pharmacological evaluation of these, and structure-guided hit optimization, led to the discovery of subtype-selective A₁R PAMs. These compounds demonstrated minimal allosteric agonism and negligible impact on A₁R-mediated beat rate of an orthosteric agonist. The discovered PAMs pave the way for potential treatments for neuropathic pain and ischemia-reperfusion injury without accompanying side effects. Our results demonstrate the utility of a synergistic computational and experimental approach in GPCR drug discovery.