title: Mutagenesis Analysis Reveals Distinct Amino Acids of the Human Serotonin 5-HT2CReceptor Underlying the Pharmacology of Distinct Ligands.
abstract: While exploring the structure-activity relationship of 4-phenyl-2-dimethylaminotetralins (PATs) at serotonin 5-HT2Creceptors, we discovered that relatively minor modification of PAT chemistry impacts function at 5-HT2Creceptors. In HEK293 cells expressing human 5-HT2C-INIreceptors, for example, (-)-trans-3'-Br-PAT and (-)-trans-3'-Cl-PAT are agonists regarding Gαq-inositol phosphate signaling, whereas (-)-trans-3'-CF3-PAT is an inverse agonist. To investigate the ligand-receptor interactions that govern this change in function, we performed site-directed mutagenesis of 14 amino acids of the 5-HT2Creceptor based on molecular modeling and reported G protein-coupled receptor crystal structures, followed by molecular pharmacology studies. We found that S3.36, T3.37, and F5.47 in the orthosteric binding pocket are critical for affinity (Ki) of all PATs tested, we also found that F6.44, M6.47, C7.45, and S7.46 are primarily involved in regulating EC/IC50functional potencies of PATs. We discovered that when residue S5.43, N6.55, or both are mutated to alanine, (-)-trans-3'-CF3-PAT switches from inverse agonist to agonist function, and when N6.55 is mutated to leucine, (-)-trans-3'-Br-PAT switches from agonist to inverse agonist function. Notably, most point-mutations that affected PAT pharmacology did not significantly alter affinity (KD) of the antagonist radioligand [3H]mesulergine, but every mutation tested negatively impacted serotonin binding. Also, amino acid mutations differentially affected the pharmacology of other commercially available 5-HT2Cligands tested. Collectively, the data show that functional outcomes shared by different ligands are mediated by different amino acids and that some 5-HT2Creceptor residues important for pharmacology of one ligand are not necessarily important for another ligand.
authors: Center for Drug Discovery, Department of Pharmaceutical Sciences, and Department of Chemistry and Chemical Biology, Northeastern University , Boston, Massachusetts 02115, United States.
authors: Yue
authors: Y
authors: Liu
authors: Center for Drug Discovery, Department of Pharmaceutical Sciences, and Department of Chemistry and Chemical Biology, Northeastern University , Boston, Massachusetts 02115, United States.
authors: Clinton E
authors: CE
authors: Canal
authors: Center for Drug Discovery, Department of Pharmaceutical Sciences, and Department of Chemistry and Chemical Biology, Northeastern University , Boston, Massachusetts 02115, United States.|Department of Medicinal Chemistry, University of Florida , Gainesville, Florida 32611, United States.
authors: Tania C
authors: TC
authors: Cordova-Sintjago
authors: Center for Drug Discovery, Department of Pharmaceutical Sciences, and Department of Chemistry and Chemical Biology, Northeastern University , Boston, Massachusetts 02115, United States.
authors: Wanying
authors: W
authors: Zhu
authors: Center for Drug Discovery, Department of Pharmaceutical Sciences, and Department of Chemistry and Chemical Biology, Northeastern University , Boston, Massachusetts 02115, United States.|Department of Medicinal Chemistry, University of Florida , Gainesville, Florida 32611, United States.
authors: Raymond G
authors: RG
authors: Booth
mesh_terms: D000596:Amino Acids / Q000235:genetics*; D000704:Analysis of Variance; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D006016:Glycolates / Q000138:chemical synthesis / Q000737:chemistry / Q000493:pharmacokinetics; D057809:HEK293 Cells; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D016296:Mutagenesis / Q000235:genetics*; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D011869:Radioligand Assay; D044405:Receptor, Serotonin, 5-HT2C / Q000235:genetics* / Q000378:metabolism*; D018490:Serotonin Agents / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014162:Transfection; D014316:Tritium / Q000493:pharmacokinetics
chemical_list: D000596:Amino Acids; D006016:Glycolates; D007295:Inositol Phosphates; D008024:Ligands; C528865:N-(2-(3,5-di-tert-butyl-4-hydroxyphenyl)-4-oxothiazolidin-3-yl-(4-chlorophenoxy))acetamide; D044405:Receptor, Serotonin, 5-HT2C; D018490:Serotonin Agents; D014316:Tritium; C010251:inositol-1-pyrophosphate
keywords: 5-HT2C receptor; Serotonin; drug discovery; mutagenesis; pharmacology