Influence of ligand binding kinetics on functional inhibition of human recombinant serotonin and norepinephrine transporters

Introduction: Monoamine reuptake inhibitors treat a wide range of CNS disorders, including depression,obesity, and pain. The in vitro pharmacological properties of these inhibitors are determined routinely using radioligand binding and/or neurotransmitter uptake assays. Measurements from such studies can be influenced by assay design and ligand-specific characteristics, both of which may contribute to discrepancies in literature reports. Methods: We modified traditional methodologies to identify and account for factors that can confound in vitro potency determinations. Apparent equilibrium binding affinities (pK(i); values) were determined in either HEK293 cells stably-transfected with human recombinant serotonin (SERT) or norepinephrine (NET) transporters, or membranes prepared from these cell lines. Care was taken to ensure that apparent affinities were measured under conditions that minimized ligand depletion and established equilibrium for both the radioligand and the compound of interest. An unlabelled ligand kinetic method was used to approximate inhibitor binding kinetic constants and corresponding dissociation half lives. To measure inhibitory effects on substrate uptake, both radiolabeled neurotransmitter ([(3)H]-5-HT or [(3)H]-NE) and fluorescence-based assays were used. The time-dependent nature of functional inhibition was examined using a fluorescent substrate uptake assay which provided real-time measurements of NET and SERT function. Results: SERT and NET inhibitors displayed a range of affinities, potencies, and inhibition modes by binding and functional uptake assays. Binding kinetic profiles for this panel of inhibitors were diverse, and affected in vitro measures using the former techniques. Discussion: In the present study we describe key features of in vitro assay methodology that can influence the apparent pharmacological profiles of standard SERT and/or NET inhibitors. Such information can serve as a foundation for understanding the in vitro profiles of monoamine reuptake inhibitors in the context of their clinical efficacy and tolerability. (C) 2010 Elsevier Inc. All rights reserved.