Molecular Basis for Selective Serotonin Reuptake Inhibition by the Antidepressant Agent Fluoxetine (Prozac) s

Inhibitors of the serotonin transporter (SERT) are widely used antidepressant agents, but the structural mechanism for inhibitory activity and selectivity over the closely related norepinephrine transporter (NET) is not well understood. Here we use a combination of chemical, biological, and computational methods to decipher the molecular basis for high-affinity recognition in SERT and selectivity over NET for the prototypical antidepressant drug fluoxetine (Prozac; Eli Lilly, Indianapolis, IN). We show that fluoxetine binds within the central substrate site of human SERT, in agreement with recent X-ray crystal structures of LeuBAT, an engineered monoamine-like version of the bacterial amino acid transporter LeuT. However, the binding orientation of fluoxetine is reversed in our experimentally supportedmodel comparedwith the LeuBAT structures, emphasizing the need for careful experimental verification when extrapolating findings from crystal structures of bacterial transporters to human relatives. We find that the selectivity of fluoxetine and nisoxetine, a NET selective structural congener of fluoxetine, is controlled by residues in different regions of the transporters, indicating a complex mechanism for selective recognition of structurally similar compounds in SERT and NET. Our findings add important new information on the molecular basis for SERT/NET selectivity of antidepressants, and provide the first assessment of the potential of LeuBAT as a model system for antidepressant binding in human transporters, which is essential for future structure-based drug development of antidepressant drugs with fine-tuned transporter selectivity. Introduction The early recognition of the serotonin (5-hydroxytryptamine) transporter (SERT) and the norepinephrine transporter (NET) as important targets for antidepressant drugs fostered extensive drug discovery efforts dedicated to the design and synthesis of compounds selectively targeting SERT and/or NET (Kristensen et al., 2011). In 1986, fluoxetine (Prozac; Eli Lilly, Indianapolis, IN) was approved as one of the first selective serotonin reuptake inhibitors (SSRIs) for the treatment of depression, and it has since become widely acknowledged as a breakthrough drug for depression (Wong et al., 1995, 2005). Unlike the tricyclic antidepressants (TCAs), fluoxetine and other SSRI drugs are highly selective for SERT, and today the SSRIs remain among the most widely prescribed antidepressant drugs (Waitekus and Kirkpatrick, 2004; Bauer et al., 2008). Although compounds targeting SERT and NET have had important clinical significance for several decades, the molecular details underlying binding of antidepressants to these transporters are not clearly understood. Structural information of SERT and NET is still lacking, but X-ray crystal structures of the bacterial homolog LeuT (Yamashita et al., 2005; Krishnamurthy and Gouaux, 2012) have proved to be excellent structural templates for its mammalian counterparts and facilitated identification of the location and structure of ligand-binding sites in human transporters (Beuming et al., 2008; Celik et al., 2008; Andersen et al., 2009, 2010; Kaufmann et al., 2009; Koldsø et al., 2010, 2013a; Sinning et al., 2010; Plenge et al., 2012; Severinsen et al., 2012, 2013). Structures of LeuT have also provided direct insight into the binding mechanism of antidepressants. SSRIs and TCAs bind LeuT with low affinity to a site (denoted S2) located in an extracellular facing vestibule (Fig. 1) (Singh et al., 2007; Zhou et al., 2007, 2009), leading to the proposal that antidepressant drugs also bind to the S2 site in human transporters (Zhou et al., 2007, 2009). In contrast, recent structures of LeuBAT, an engineered version of LeuT with residues from SERT inserted into the central substrate site (denoted S1), and the dopamine transporter (DAT) from Drosophila melanogaster, displayed high-affinity binding of antidepressants within the central S1 This work was supported by the Lundbeck Foundation, the Danish National Research Foundation (DNRF59), and the Danish Council for Independent Research, Natural Sciences. Current affiliation: Department of Chemistry, University of Copenhagen, Copenhagen, Denmark. Current affiliation: Department of Biochemistry, University of Oxford, Oxford, United Kingdom. dx.doi.org/10.1124/mol.113.091249. s This article has supplemental material available at molpharm.aspetjournals. org. ABBREVIATIONS: b-CIT, (2)-2b-carbomethoxy-3b-(4-iodophenyl)tropane; DAT, dopamine transporter; IFD, induced-fit docking; NET, norepinephrine transporter; RMSD, root-mean-square deviation; SAR, structure-activity relationship; SERT, serotonin transporter; SSRI, selective serotonin reuptake inhibitor; TCA, tricyclic antidepressant; TM, transmembrane helix; WT, wild-type. 703 http://molpharm.aspetjournals.org/content/suppl/2014/02/10/mol.113.091249.DC1 Supplemental material to this article can be found at: at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from