A generally applicable metadynamics scheme for predicting the free-energy profile of ligand binding to G-protein coupled receptors (GPCRs) is described. A common and effective collective variable (CV) has been defined using the ideally placed and highly conserved Trp6.48 as a reference point for ligand-GPCR distance measurement and the common orientation of GPCRs in the cell membrane. Using this single CV together with well-tempered multiple-walker 2 metadynamics with a funnel-like boundary allows an efficient exploration of the entire ligandbinding path from the extracellular medium to the orthosteric binding site, including vestibule and intermediate sites. The protocol can be used with X-ray structures or high-quality homology models for the receptor and is universally applicable to agonists, antagonists, partial and reverse agonists. The root mean square error (RMSE) in predicted binding free energies for 12 diverse ligands in five receptors (a total of 23 data points) is surprisingly small (less than 1 kcal mol − 1 ).The RMSEs for simulations that use receptor X-ray structures and homology models are very similar.

We propose an experimentally testable extended ternary complex model, where direction of the cooperative effect between ligand and intracellular binding partner (positive or negative) and its magnitude are predicted to be a characteristic of the ligand signaling bias.

Molecular-dynamics simulations with metadynamics enhanced sampling reveal three distinct binding sites for arginine vasopressin (AVP) within its V2 -receptor (V2 R). Two of these, the vestibule and intermediate sites, block (antagonize) the receptor, and the third is the orthosteric activation (agonist) site. The contacts found for the orthosteric site satisfy all the requirements deduced from mutagenesis experiments. Metadynamics simulations for V2 R and its V1a R-analog give an excellent correlation with experimental binding free energies by assuming that the most stable binding site in the simulations corresponds to the experimental binding free energy in each case. The resulting three-site mechanism separates agonists from antagonists and explains subtype selectivity.

The cannabinoid CB1 receptor (CB1R) is an abundant metabotropic G-protein-coupled receptor that has been difficult to address therapeutically because of CNS side effects exerted by orthosteric drug candidates. Recent efforts have focused on developing allosteric modulators that target CB1R. Compounds from the recently discovered class of mixed agonistic and positive allosteric modulators (Ago-PAMs) based on 2-phenylindoles have shown promising functional and binding properties as CB1R ligands. Here, we identify binding modes of both the CP 55,940 agonist and GAT228, a 2-phenylindole allosteric modulator, by using our metadynamics simulation protocol, and quantify their affinity and cooperativity by atomistic simulations. We demonstrate the involvement of multiple adjunct binding sites in the Ago-PAM characteristics of the 2-phenylindole modulators and explain their ability to compete with orthosteric agonists at higher concentrations. We validate these results experimentally by showing the contribution of multiple sites on the allosteric binding of ZCZ011, another homologous member of the class, together with the orthosteric agonist.