Membrane Lipids Are an Integral Part of Transmembrane Allosteric Sites in GPCRs: A Case Study of Cannabinoid CB1 Receptor Bound to a Negative Allosteric Modulator, ORG27569, and Analogs

[Image: see text] A growing number of G-protein-coupled receptor (GPCR) structures reveal novel transmembrane lipid-exposed allosteric sites. Ligands must first partition into the surrounding membrane and take lipid paths to these sites. Remarkably, a significant part of the bound ligands appears exposed to the membrane lipids. The experimental structures do not usually account for the surrounding lipids, and their apparent contribution to ligand access and binding is often overlooked and poorly understood. Using classical and enhanced molecular dynamics simulations, we show that membrane lipids are critical in the access and binding of ORG27569 and its analogs at the transmembrane site of cannabinoid CB1 receptor. The observed differences in the binding affinity and cooperativity arise from the functional groups that interact primarily with lipids. Our results demonstrate the significance of incorporating membrane lipids as an integral component of transmembrane sites for accurate characterization, binding-affinity calculations, and lead optimization in drug discovery.