title: Systematic errors in detecting biased agonism: Analysis of current methods and development of a new model-free approach.
abstract: Discovering biased agonists requires a method that can reliably distinguish the bias in signalling due to unbalanced activation of diverse transduction proteins from that of differential amplification inherent to the system being studied, which invariably results from the non-linear nature of biological signalling networks and their measurement. We have systematically compared the performance of seven methods of bias diagnostics, all of which are based on the analysis of concentration-response curves of ligands according to classical receptor theory. We computed bias factors for a number of β-adrenergic agonists by comparing BRET assays of receptor-transducer interactions with Gs, Gi and arrestin. Using the same ligands, we also compared responses at signalling steps originated from the same receptor-transducer interaction, among which no biased efficacy is theoretically possible. In either case, we found a high level of false positive results and a general lack of correlation among methods. Altogether this analysis shows that all tested methods, including some of the most widely used in the literature, fail to distinguish true ligand bias from "system bias" with confidence. We also propose two novel semi quantitative methods of bias diagnostics that appear to be more robust and reliable than currently available strategies.
authors.affiliation: Ankara University, Faculty of Medicine, Department of Pharmacology, Molecular biology and Technology development unit, Sıhhiye, Ankara, Turkey.
authors.forename: H Ongun
authors.initials: HO
authors.lastname: Onaran
authors.affiliation: Istituto Superiore di Sanitá, Dip. Farmaco, Rome, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: Ambrosio
authors.affiliation: Ankara University, Faculty of Medicine, Department of Pharmacology, Molecular biology and Technology development unit, Sıhhiye, Ankara, Turkey.
authors.forename: Özlem
authors.initials: Ö
authors.lastname: Uğur
authors.affiliation: Ankara University, Faculty of Medicine, Department of Pharmacology, Molecular biology and Technology development unit, Sıhhiye, Ankara, Turkey.
authors.forename: Erzsebet
authors.initials: E
authors.lastname: Madaras Koncz
authors.affiliation: Istituto Superiore di Sanitá, Dip. Farmaco, Rome, Italy.
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Grò
authors.affiliation: Istituto Superiore di Sanitá, Dip. Farmaco, Rome, Italy.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Vezzi
authors.affiliation: Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Sudarshan
authors.initials: S
authors.lastname: Rajagopal
authors.affiliation: Istituto Superiore di Sanitá, Dip. Farmaco, Rome, Italy.
authors.forename: Tommaso
authors.initials: T
authors.lastname: Costa
mesh_terms: D000322:Adrenergic Agonists / Q000378:metabolism* / Q000494:pharmacology; D015982:Bias; D001681:Biological Assay; D002864:Chromogranins / Q000235:genetics / Q000378:metabolism*; D002976:Clenbuterol / Q000378:metabolism / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism / Q000494:pharmacology; D004837:Epinephrine / Q000378:metabolism / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D007528:Isoetharine / Q000378:metabolism / Q000494:pharmacology; D007545:Isoproterenol / Q000378:metabolism / Q000494:pharmacology; D008024:Ligands; D009010:Monte Carlo Method; D011485:Protein Binding; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012044:Regression Analysis; D000071557:beta-Arrestins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000322:Adrenergic Agonists; D002864:Chromogranins; D008024:Ligands; D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; D000071557:beta-Arrestins; C406732:GNAS protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs; D007545:Isoproterenol; D004298:Dopamine; D002976:Clenbuterol; D004837:Epinephrine; D007528:Isoetharine