Molecular interactions of agonist and inverse agonist ligands at serotonin 5-HT_2C G protein-coupled receptors: computational ligand docking and molecular dynamics studies validated by experimental mutagenesis results

To understand molecular determinants for ligand activation of the serotonin 5-HT_2C G protein-coupled receptor (GPCR), a drug target for obesity and neuropsychiatric disorders, a 5-HT_2C homology model was built according to an adrenergic β₂ GPCR (β₂AR) structure and validated using a 5-HT_2B GPCR crystal structure. The models were equilibrated in a simulated phosphatidyl choline membrane for ligand docking and molecular dynamics studies. Ligands included (2<i>S</i>, 4<i>R</i>)-(–)-<i>trans</i>-4-(3’-bromo- and trifluoro-phenyl)-<i>N</i>,<i>N</i>-dimethyl-1,2,3,4-tetrahydronaphthalene-2-amine (3’-Br-PAT and 3’-CF₃-PAT), a 5-HT_2C agonist and inverse agonist, respectively. Distinct interactions of 3’-Br-PAT and 3’-CF₃-PAT at the wild-type (WT) 5-HT_2C receptor model were observed and experimental 5-HT_2C receptor mutagenesis studies were undertaken to validate the modelling results. For example, the inverse agonist 3’-CF₃-PAT docked deeper in the WT 5-HT_2C binding pocket and altered the orientation of transmembrane helices (TM) 6 in comparison to the agonist 3’-Br-PAT, suggesting that changes in TM orientation that result from ligand binding impact function. For both PATs, mutation of 5-HT_2C residues S3.36, T3.37, and F5.47 to alanine resulted in significantly decreased affinity, as predicted from modelling results. It was concluded that upon PAT binding, 5-HT_2C residues T3.37 and F5.47 in TMs 3 and 5, respectively, engage in inter-helical interactions with TMs 4 and 6, respectively. The movement of TMs 5 and 6 upon agonist and inverse agonist ligand binding observed in the 5-HT_2C receptor modelling studies was similar to movements reported for the activation and deactivation of the β₂AR, suggesting common mechanisms among aminergic neurotransmitter GPCRs.