Characterization of an allosteric citalopram-binding site at the serotonin transporter

Department of Psychiatry, Aarhus University, Aarhus, Central Jutland, Denmark
Journal of Neurochemistry (Impact Factor: 4.28). 02/2005; 92(1):21-8. DOI: 10.1111/j.1471-4159.2004.02835.x
Source: PubMed


The serotonin transporter (SERT), which belongs to a family of sodium/chloride-dependent transporters, is the major pharmacological target in the treatment of several clinical disorders, including depression and anxiety. In the present study we show that the dissociation rate, of [3H]S-citalopram from human SERT, is retarded by the presence of serotonin, as well as by several antidepressants, when present in the dissociation buffer. Dissociation of [3H]S-citalopram from SERT is most potently inhibited by S-citalopram followed by R-citalopram, sertraline, serotonin and paroxetine. EC50 values for S- and R-citalopram are 3.6 +/- 0.4 microm and 19.4 +/- 2.3 microm, respectively. Fluoxetine, venlafaxine and duloxetine have no significant effect on the dissociation of [3H]S-citalopram. Allosteric modulation of dissociation is independent of temperature, or the presence of Na+ in the dissociation buffer. Dissociation of [3H]S-citalopram from a complex with the SERT double-mutant, N208Q/N217Q, which has been suggested to be unable to self-assemble into oligomeric complexes, is retarded to an extent similar to that found with the wild-type, raising the possibility that the allosteric mechanism is mediated within a single subunit. A species-scanning mutagenesis study comparing human and bovine SERT revealed that Met180, Tyr495 and Ser513 are important residues in mediating the allosteric effect, as well as contributing to high-affinity binding at the primary site.

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Available from: Connie Sánchez, Sep 28, 2014
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    • "Clomipramine can also inhibit the dissociation of[ 3 H]S-CIT (Plenge et al., 2012). Common for most published allosteric ligands to date is that (i) they bind to the allosteric site with low potency, typically in the micromolar range (Chen et al., 2005a;Chen et al., 2005b;Banala et al., 2013;Plenge et al., 2012), and (ii) they bind to the S1 site with considerably higher affinity, typically within the low nanomolar range. The most potent allosteric ligand reported so far is S-CIT, which impairs[ 3 H]S-CIT dissociation with an IC 50 value of ~5 µM (Plenge et al., 2012). "
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    ABSTRACT: Background and purpose: The serotonin transporter (SERT) is target for antidepressant drugs. SERT possesses two binding sites: the orthosteric (S1) binding site, which is the presumed target for current SERT inhibitors, and an allosteric (S2) site for which potential therapeutic effects are unknown. The antidepressant drug citalopram displays high-affinity S1 binding and low-affinity S2 binding. To elucidate a possible therapeutic role of allosteric inhibition of SERT a drug that specifically targets the allosteric site is required. The purpose of this study was to find a compound bearing higher selectivity towards the S2 site. Experimental approach: We performed a systematic structure-activity relationship study based on the scaffold of citalopram and the structurally closely related congener, talopram, that shows low-affinity S1 binding in SERT. The role of the four chemical substituents, which distinguish citalopram from talopram in conferring selectivity towards the S1 and S2 site, respectively, was assessed by determining the binding of 14 citalopram/talopram analogous to the S1 and S2 binding sites in SERT using membranes of COS7 cells transiently expressing SERT. Key results: The structure-activity relationship study revealed a di-methyl citalopram, which binds to the S1 site with an affinity of 6.4 [4.7;8.8] μM (mean[SEM interval]) and shows an allosteric potency of 3.6 [3.3;3.8] μM, thus bearing ~2-fold selectivity for the allosteric site relative to the S1 site in SERT. Conclusions and implications: The compound could be a useful lead for future synthesis of drugs with high affinity and high selectivity towards the allosteric binding site. This article is protected by copyright. All rights reserved.
    Full-text · Article · Dec 2015 · British Journal of Pharmacology
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    • "*P <0.05 compared with Esc to counteract the action of escitalopram without causing pharmacokinetic interactions (Sanchez 2006; Mnie-Filali et al. 2007). Escitalopram interacts with both the orthosteric and allosteric sites of the SERT, whereas R-citalopram has much weaker binding to the orthosteric site, although its affinity for the allosteric site is comparable to that of escitalopram (Sanchez 2006; Chen et al. 2005). In our study, we found that compared with escitalopram treatment group, the co-culture with R-citalopram and escitalopram could partially increase the level of the phosphorylated tau at different epitopes. "
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    ABSTRACT: To investigate the effect of escitalopram (a widely used and highly efficacious antidepressant from the SSRI class) on tau hyperphosphorylation, HEK293/tau441 cells were pretreated with 4 μM of forskolin for 2 h. Then we treated the cells with different doses of escitalopram (0, 5, 10, 20, 40, 80 μM) for 22 h. We measured the phosphorylation level of tau by Western blotting. It was shown that escitalopram could protect tau from hyperphosphorylation induced by pharmacological activation of protein kinase A (PKA) at a dose of 20, 40, and 80 μM in vitro. Interestingly, the same dose of escitalopram could also increase the level of serine-9-phosphorylated GSK-3β (inactive form) and the phosphorylation level of Akt at Ser473 (active form) with no significant change in the level of total GSK-3β and Akt. Unexpectedly, 5-hydroxytryptamine 1A receptor (5-HT1A) agonist 8-OH-DPAT did not decrease forskolin-induced tau hyperphosphorylation. Our results suggest that escitalopram can ameliorate forskolin-induced tau hyperphosphorylation, which is not through the typical 5-HT1A pathway, and Akt/GSK-3β signaling pathway is involved. These findings may support an effective role of antidepressants in the prevention of dementia associated with depression in patients.
    Full-text · Article · Feb 2015 · Journal of Molecular Neuroscience
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    • "The SERT has two types of binding site, the orthosteric binding site (also referred to as the primary site) to which escitalopram and other SSRIs bind, resulting in inhibition of its uptake function, and one or more allosteric sites (Chen et al., 2005a, 2005b; Sanchez, 2006). Many studies have led to the thorough characterization of the allosteric mechanism of escitalopram (Wennogle and Meyerson, 1982; Plenge and Mellerup, 1997; Chen et al., 2005a, 2005b), although other compounds have also been reported to have allosteric activities at the SERT but are less well characterized (Nandi et al., 2004; Nightingale et al., 2005; Boos et al., 2006). "
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    ABSTRACT: It is known that newer antidepressants, such as the selective serotonin reuptake inhibitors (SSRIs), provide advantages in tolerability over antidepressants such as the tricyclics. However, even within the SSRI class, differences in efficacy or tolerability exist between the individual drugs. Among the three most widely prescribed SSRIs are paroxetine, sertraline, and escitalopram. Escitalopram is commonly referred to as an SSRI, but also has well-documented allosteric properties, and thus can be further classed as an allosteric serotonin reuptake inhibitor. All three antidepressants are efficacious compared with placebo, but there is evidence that escitalopram is more effective than a range of other antidepressants. There are no direct data to regard either paroxetine or sertraline as a superior antidepressant. Escitalopram is superior compared with paroxetine, which has a less favorable tolerability profile. Paroxetine is associated with cholinergic muscarinic antagonism and potent inhibition of CYP2D6, and sertraline has moderate drug interaction issues in comparison with escitalopram. Overall, as an allosteric serotonin reuptake inhibitor that is somewhat different from classical SSRIs, escitalopram is the first choice judged by combined efficacy and tolerability, and nonclinical data have offered possible mechanisms through which escitalopram could be more efficacious, based on its interaction with orthosteric and allosteric binding sites at the serotonin transporter.
    Full-text · Article · Jan 2014 · International clinical psychopharmacology
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