Metadynamics simulations and site‐directed mutagenesis determine the binding of cannabidiol to the adenosine <scp> A _2A </scp> receptor

Cannabidiol (CBD), the second most abundant of the active compounds found in the Cannabis sativa plant, is nonaddictive and of increasing interest due to its potential therapeutic utilities. The actions of CBD are mediated by cannabinoid receptors and other targets, both G protein-coupled receptors (GPCRs) and non-GPCR proteins. It has recently been shown that CBD is not an orthosteric ligand of the adenosine A_2A receptor (A_2AR), but rather a negative allosteric modulator. Because of the multitude non-conserved allosteric pockets in GPCRs, the binding mode of CBD to A_2AR remains unknown. To fill this knowledge gap, and due to the therapeutic relevance of CBD and A_2AR, we have used metadynamics simulations and site-directed mutagenesis to identify the binding mode of CBD. These theoretical and experimental results show that the allosteric binding site of CBD is along the ligand binding pathway of adenosine to A_2AR, near the orthosteric binding site.