Negative allosteric modulators of cannabinoid receptor 1: Ternary complexes including CB1, orthosteric CP55940 and allosteric ORG27569

In October 2019, the first X-ray crystal structure of a ternary cannabinoid receptor 1 (CB1) complex (PDB ID: 6KQI) was published, including the well-known orthosteric agonist, CP55940, and the well-studied negative allosteric modulator, ORG27569. Prior to the release of 6KQI, we applied binding pocket analysis and molecular docking to carefully prepared computational models of the ternary CB1 complex, in order to predict the binding site for ORG27569 with the CP55940-bound CB1 receptor. We carefully studied the binding pose of agonist ligands in the CB1 orthosteric pocket, including CP55940. Our computational studies identified the most favorable binding site for ORG27569, in the CP55940-CB1 complex, to be at the intracellular end of the receptor. However, in the 6KQI structure, ORG27569 was found at an extrahelical, intramembrane site on the complex, a site that partially overlaps with the site predicted in our calculations to be second-best. We performed molecular dynamics simulations of the CP55940-bound CB1 complex with ORG27569 at different binding sites. Our analysis of the simulations indicated that ORG27569 bound favorably and stably in each simulation, but, as in the earlier calculations, bound best at the intracellular site, which is different than that found in the crystal structure. These results suggest that the intracellular site might serve as an alternative binding site in CB1. Our studies show that the computational techniques we used are valuable in identifying ligand-binding pockets in proteins, and could be useful for the study of the interaction mode of other allosteric modulators.