title: Functional binding assays for estimation of the intrinsic efficacy of ligands at the 5-HT1A receptor: application for screening drug candidates.
abstract: BACKGROUND
  Determination of the intrinsic efficacy of ligands at the 5-HT1A receptor is important for selecting drug candidates, e.g. in the case of schizophrenia where partial agonism is a favorable property shared by different atypical antipsychotics.
  
  
  METHODS
  Using seven ligands with different intrinsic efficacies and rat hippocampus synaptosomes, we compared critically three "functional" binding assays based on the ternary complex model that considers that the activated conformation of the receptor is the one coupled to G-protein.
  
  
  RESULTS
  The Ki ratio method, based on the difference of affinity of the competing drug when using an antagonist vs. an agonist as radioligand, discriminated the ligands according to their intrinsic efficacies, with values from 77 for the full agonist 5-hydroxytryptamine to 0.09 for the inverse agonist WAY 100,635. The GTP-shift method, based on the decrease of affinity observed with agonists when GTP is added to the competition binding assay, was equally effective in classifying the drugs according to their intrinsic efficacy. The lower sensibility of the GTP-shift assay was investigated and explained by the different ionic conditions used in the two assays and the way competition curves were analyzed. Albeit more direct, the assay based on agonist-stimulated [(35)S]-GTPγS binding to G proteins was more expensive and of greater variability in our hands.
  
  
  CONCLUSIONS
  We conclude that the GTP-shift procedure described herein for 5-HT1A receptors may expedite drug discovery efforts by predicting at the same time the affinity and intrinsic efficacy of ligands through a simple, rapid and economic ligand binding assay.
authors.affiliation: Laboratório de Farmacologia Bioquímica e Molecular, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Brazil. Electronic address: fnoel@pharma.ufrj.br.
authors.forename: François
authors.initials: F
authors.lastname: Noël
authors.affiliation: Laboratório de Farmacologia Bioquímica e Molecular, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Brazil.
authors.forename: Thais E T
authors.initials: TE
authors.lastname: Pompeu
authors.affiliation: Laboratório de Farmacologia Bioquímica e Molecular, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Brazil.
authors.forename: Bruna C
authors.initials: BC
authors.lastname: Moura
mesh_terms: D000818:Animals; D014150:Antipsychotic Agents / Q000494:pharmacology*; D001667:Binding, Competitive / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D008024:Ligands; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D044282:Receptor, Serotonin, 5-HT1A / Q000378:metabolism*; D012559:Schizophrenia / Q000188:drug therapy
chemical_list: D014150:Antipsychotic Agents; D008024:Ligands; D044282:Receptor, Serotonin, 5-HT1A; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins
keywords: (R)-(+)-8-OH-DPAT hydrobromide (PubChem CID: 1220); 5-HT; 5-Hydroxytryptamine creatinine sulfate (PubChem CID: 13781); Antipsychotics; Binding; Buspirone hydrochloride (PubChem CID: 36431); Clozapine (PubChem CID: 2818); Drug; Efficacy; Guanosine 5′-O-(3-thiotriphosphate) (PubChem CID: 252438130); Methods; Schizophrenia; Screening; Serotonin; Spiperone (PubChem CID: 5265); WAY 100,635 maleate (PubChem CID: 11957721); p-MPPF dihydrochloride (PubChem CID: 11957633)