Water molecules in the cannabinoid receptor 2 binding site crucially impact the discovery of novel ligands

The cannabinoid receptor 2 (CB₂R) is of considerable therapeutic and scientific interest. Hence, the discovery of novel molecules that target and modulate this receptor, ideally selectively over its closest relative, the cannabinoid receptor 1, is of great importance. In this study, we aimed to discover novel ligands targeting the CB₂R using large library in silico docking screens. However, since the CB₂R binding site is difficult to target with in silico methods due to its hydrophobic nature, we used a variety of screening approaches, including the placement of water molecules in predicted water sites of the receptor binding site, and screening against multiple docking setups and receptor conformations. We systematically evaluated these different approaches to support future screens at the CB₂R and other receptors. In the present work, each setup contributed different ligands of varying intrinsic activities, leading to an overall improved hit rate compared to that of a single screen. Of the novel ligands of the CB₂R discovered and experimentally confirmed in this study, one series features high-affinity ligands with a previously undescribed scaffold.