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


CONCLUSIONS
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.
Tsuruda
Pamela R
PR
Theravance, Inc., South San Francisco, CA 94080, USA. ptsuruda@theravance.com
Yung
Joey
J
Martin
William J
WJ
Chang
Ray
R
Mai
Ngoc
N
Smith
Jacqueline A M
JA
D018759:Adrenergic Uptake Inhibitors / Q000494:pharmacology*; D000465:Algorithms; D001665:Binding Sites; D002460:Cell Line; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008024:Ligands; D009638:Norepinephrine / Q000378:metabolism; D050484:Norepinephrine Plasma Membrane Transport Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D011485:Protein Binding; D011869:Radioligand Assay; D011994:Recombinant Proteins; D012701:Serotonin / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins / Q000187:drug effects* / Q000235:genetics; D017367:Selective Serotonin Reuptake Inhibitors / Q000494:pharmacology*; D013696:Temperature
D018759:Adrenergic Uptake Inhibitors; D005456:Fluorescent Dyes; D008024:Ligands; D009638:Norepinephrine; D050484:Norepinephrine Plasma Membrane Transport Proteins; D011994:Recombinant Proteins; D012701:Serotonin; D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors