Structure-activity relationship studies of citalopram derivatives: examining substituents conferring selectivity for the allosteric site in the serotonin transporter (original) (raw)
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Characterization of an allosteric citalopram-binding site at the serotonin transporter
Journal of Neurochemistry, 2005
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 [ 3 H]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 [ 3 H]S-citalopram from SERT is most potently inhibited by S-citalopram followed by R-citalopram, sertraline, serotonin and paroxetine. EC 50 values for S-and R-citalopram are 3.6 ± 0.4 lM and 19.4 ± 2.3 lM, respectively. Fluoxetine, venlafaxine and duloxetine have no significant effect on the dissociation of [ 3 H]Scitalopram. Allosteric modulation of dissociation is independ-ent of temperature, or the presence of Na + in the dissociation buffer. Dissociation of [ 3 H]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.
European Neuropsychopharmacology, 2005
The interaction of the S-and R-enantiomers (escitalopram and R-citalopram) of citalopram, with high-and low-affinity binding sites in COS-1 cell membranes expressing human SERT (hSERT) were investigated. Escitalopram affinity for hSERT and its 5-HT uptake inhibitory potency was in the nanomolar range and approximately 40-fold more potent than R-citalopram. Escitalopram considerably stabilised the [ 3 H]-escitalopram/SERT complex via an allosteric effect at a low-affinity binding site. The stereoselectivity between escitalopram and Rcitalopram was approximately 3:1 for the [ 3 H]-escitalopram/hSERT complex. The combined effect of escitalopram and R-citalopram was additive. Paroxetine and sertraline mainly stabilised the [ 3 H]-paroxetine/hSERT complex. Fluoxetine, duloxetine and venlafaxine have only minor effects. 5-HT stabilised the [ 125 I]-RTI-55, [ 3 H]-MADAM, [ 3 H]-paroxetine, [ 3 H]-fluoxetine and [ 3 H]-venlafaxine/SERT complex to some extent. Thus, escitalopram shows a unique interaction with the hSERT compared with other 5-HT reuptake inhibitors (SSRIs) and, in addition to its 5-HT reuptake inhibitory properties, displays a pronounced effect via an affinity-modulating allosteric site. D
Bioorganic & medicinal chemistry letters, 2017
The recent publication of X-ray structures of SERT includes structures with the potent antidepressant S-Citalopram (S-Cit). Earlier predictions of ligand binding at both a primary (S1) and an allosteric modulator site (S2), were confirmed. We provide herein examples of a series of Citalopram analogs, showing distinct structure-activity relationship (SAR) at both sites that is independent of the SAR at the other site. Analogs with a higher affinity and selectivity than benchmark R-Citalopram (R-Cit) for the S2 versus the S1 site were identified. We deploy structural and computational analyses to explain this SAR and demonstrate the potential utility of the newly emerging X-ray structures within the neurotransmitter:sodium Symporter family for drug design.
Neuroscience Letters, 2005
The serotonin transporter (SERT) is responsible for terminating or modulating the action of serotonin released from the presynaptic neuron and is the major target for most antidepressants including the tricyclic antidepressants and the selective serotonin uptake inhibitors. Two binding sites for uptake inhibitors and serotonin (5-HT) have been found on SERT. At one site, uptake inhibitors bind with high-affinity to SERT, thereby blocking the uptake of 5-HT. The other site is a low-affinity allosteric site, which influences the dissociation of uptake inhibitors, such as imipramine, paroxetine, and citalopram from the first site, when occupied by 5-HT and a few uptake inhibitors like paroxetine and citalopram. In this study, the connection between the high-affinity binding site and the allosteric affinity-modulating site was investigated by introducing 20 single amino acid substitutions into positions of presumed importance. Binding of S-citalopram, both to the high-affinitybinding site and to the allosteric binding site, was measured in these mutants with the purpose of investigating the connection between the two binding sites. The amino acid substitutions did not introduce large changes in the two binding sites, but the results indicate that the two binding sites are independent as mutants were found in which the two binding sites were affected differently. Mutations were found which destabilised the high-affinity binding without changing the allosteric effect (e.g., G128A); mutations which destabilised the high-affinity binding but increased the allosteric effect (e.g., G100A), and mutations which were without effect on the high-affinity binding, but which increased the allosteric effect (e.g., Q562A). It is concluded that the allosteric binding site is independent of the high-affinity-binding site; it may therefore represent a new drug target.
Journal of Biological Chemistry, 2010
The serotonin transporter (SERT) regulates extracellular levels of the neurotransmitter serotonin (5-hydroxytryptamine) in the brain by facilitating uptake of released 5-hydroxytryptamine into neuronal cells. SERT is the target for widely used antidepressant drugs, including imipramine, fluoxetine, and (S)-citalopram, which are competitive inhibitors of the transport function. Knowledge of the molecular details of the antidepressant binding sites in SERT has been limited due to lack of structural data on SERT. Here, we present a characterization of the (S)citalopram binding pocket in human SERT (hSERT) using mutational and computational approaches. Comparative modeling and ligand docking reveal that (S)-citalopram fits into the hSERT substrate binding pocket, where (S)-citalopram can adopt a number of different binding orientations. We find, however, that only one of these binding modes is functionally relevant from studying the effects of 64 point mutations around the putative substrate binding site. The mutational mapping also identify novel hSERT residues that are crucial for (S)-citalopram binding. The model defines the molecular determinants for (S)citalopram binding to hSERT and demonstrates that the antidepressant binding site overlaps with the substrate binding site. . 2 The abbreviations used are: SERT, serotonin transporter; hSERT, human serotonin transporter; 5HT, 5-hydroxytryptamine (serotonin); SSRI, selective serotonin reuptake inhibitor; IFD, induced-fit docking; hNET, human norepinephrine transporter; TM, transmembrane helice; SLC6, solute carrier 6; PBSCM, phosphate-buffered saline containing CaCl 2 and MgCl 2 .
Journal of Medicinal Chemistry, 2013
The serotonin transporter (SERT) is the primary target for antidepressant drugs. The existence of a high affinity primary orthosteric binding site (S1) and a low affinity secondary site (S2) has been described, and their relation to antidepressant pharmacology has been debated. Herein, structural modifications to the N, 4, 5, and 4′ positions of (±)citalopram (1) are reported. All of the analogues were SERT-selective and demonstrated that steric bulk was tolerated at the SERT S1 site, including two dimeric ligands (15 and 51). In addition, eight analogues were identified with similar potencies to S-1 for decreasing the dissociation of [ 3 H]S-1 from the S1 site via allosteric modulation at S2. Both dimeric compounds had similar affinities for the SERT S1 site (K i = 19.7 and 30.2 nM, respectively), whereas only the N-substituted analogue, 51, was as effective as S-1 in allosterically modulating the binding of [ 3 H]S-1 via S2. A dx.doi.org/10.1021/jm4014136 | J. Med. Chem. XXXX, XXX, XXX−XXX
Neuropsychopharmacologia Hungarica, 2020
Discovery and development of the selective serotonin reuptake inhibitors mark a milestone in neuro-pharmacology. Drugs from this class alter the functioning of the serotonin system by the potentiation of serotonin through the negative allosteric modulation of its neuronal uptake by the human serotonin transporter. Selective serotonin reuptake inhibitors show few side effects compared to those caused by traditional antidepressants and they vary in the binding interactions formed during binding. Gener-ally, their binding involves three specific regions of the drug structures, each participating in vital inter-actions, such as salt bridge formation and additional hydrophobic interactions with conserved resi-dues in the central binding site of the target protein. Side effects, however, such as the initial lack of response to treatment, or drowsiness, nausea, and sexual dysfunction occasionally may arise. Addi-tional binding studies, furthermore, highlighted the importance of enantioselectivity in the binding of these compounds, raising concerns about the beneficial application of racemate mixtures of some of these compounds. Therefore, additional characterisation of binding and further structural improve-ment of this class of drugs is necessary. The recently synthesized sertraline salts, and functional deriv-atives of fluoxetine and citalopram show promising results in delivering antidepressant activity as well as in effectively overcoming anorexigenic side-effects in rodent models. Hence, despite certain non-desired effects associated with selective serotonin reuptake inhibitor applications, this class of drugs is considered as first-line medication in the management of major depression, and is carrying an excel-lent potential for the development and refinement of the currently available and novel antidepressant therapies.
European Journal of Pharmacology, 2007
The human 5-HT transporter (hSERT) has two binding sites for 5-HT and 5-HT uptake inhibitors: the orthosteric high-affinity site and a lowaffinity allosteric site. Activation of the allosteric site increases the dissociation half-life for some uptake inhibitors. The objectives of this study were 1) to identify hSERT mutations that inactivate the high-affinity site without affecting the allosteric site and 2) to observe allosteric effects in which hSERT binds R-citalopram with higher affinity than S-citalopram. Wild-type and mutant (Y95F, I172M, and Y95F/I172M) hSERTs were expressed in COS-7 cells, and their 5-HT uptake and uptake inhibitor-binding abilities were studied. The hSERT mutations did not alter affinities for 5-HT or paroxetine, but high-affinity binding of S-citalopram was severely affected, particularly by the I172M, and Y95F/I172M mutations -K i respectively 4 nM (wild-type), 35 nM, 1000 nM, and 17.100 nM (mutants). The allosteric site however, in wild-type hSERT and the three mutants was unaffected by the mutations as attenuation of the dissociation rate of the [ 3 H]-paroxetine:hSERT complex in the presence of Scitalopram or paroxetine was the same for wild-type hSERT and the three mutants. Further, R-citalopram previously thought of as an inactive enantiomer strongly attenuated dissociation of the wild-type [ 3 H]-imipramine:hSERT complex, whereas S-citalopram had almost no effect on this complex. These results suggest that 1: The allosteric site on hSERT is distinct from the site to which S-citalopram binds with high affinity. 2: The allosteric effects of R-citalopram on the dissociation of [ 3 H]-imipramine from hSERT indicate that R-citalopram introduces a conformational change in hSERT.
Basic <html_ent glyph="@amp;" ascii="&"/> Clinical Pharmacology <html_ent glyph="@amp;" ascii="&"/> Toxicology, 2006
Recent results on the in vivo and in vitro pharmacology of escitalopram are summarised. The exact molecular mechanism by which R-citalopram inhibits the effect of S-citalopram on the serotonin transporter remains to be elucidated. Preliminary evidence indicates an effect of R-citalopram on the association of escitalopram with the high affinity primary site, and on its dissociation from the serotonin transporter, via an allosteric mechanism. Escitalopram can be considered as an allosteric serotonin reuptake inhibitor. This serotonin dual action in binding to two sites on the serotonin transporter (both the primary site and the allosteric site) is hypothesised to be responsible for a longer binding to, and therefore greater inhibition of the serotonin transporter by escitalopram.