X-ray structure based evaluation of analogs of citalopram: Compounds with increased affinity and selectivity compared with R-citalopram for the allosteric site (S2) on hSERT.

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.

Exploration of insights, opportunities and caveats provided by the X-ray structures of hSERT.

The recently reported X-ray structures of the human serotonin (5-HT) transporter SERT with bound inhibitors open new opportunities for drug discovery at SERT, selectivity design with respect to other neurotransmitter sodium transporters, and enhanced understanding of the molecular events involved in SERT action. Through computational and structural analyses, we explore the binding and migration of 5-HT at SERT. Consistent with earlier studies of leucine migration at the bacterial homolog of SERT, LeuT, we find multiple potential 'stopover' sites for 5-HT binding at SERT including the two (transmembrane S1 and extracellular vestibule S2) seen in the binding of the SSRI (S)-citalopram (S-Cit) to SERT, as well as other sites. Docking studies reveal the possibility of both hetero- (S-Cit+5-HT) and homo-dimeric (5-HT+5-HT) co-binding at both these sites which may explain earlier published allosteric activity observations and provide novel design strategies. Comparisons with substrate bound X-ray structures of the dopamine transporter reveal a number of potential sources of selectivity, some of which may be 'artificial' including target based, species related, experimental design related, and ligand dependent examples including substrate versus inhibitor related features.

Structure-activity relationship studies of citalopram derivatives: examining substituents conferring selectivity for the allosteric site in the 5-HT transporter.

BACKGROUND AND PURPOSE: The 5-HT transporter (SERT) is a 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 having 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, which 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 that dimethyl citalopram possesses the highest affinity for the allosteric site relative to the S1 site in SERT and has approximately twofold 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.

Molecular determinants for selective recognition of antidepressants in the human serotonin and norepinephrine transporters.

Inhibitors of the serotonin transporter (SERT) and norepinephrine transporter (NET) are widely used in the treatment of major depressive disorder. Although SERT/NET selectivity is a key determinant for the therapeutic properties of these drugs, the molecular determinants defining SERT/NET selectivity are poorly understood. In this study, the structural basis for selectivity of the SERT selective inhibitor citalopram and the structurally closely related NET selective inhibitor talopram is delineated. A systematic structure-activity relationship study allowed identification of the substituents that control activity and selectivity toward SERT and NET and revealed a common pattern showing that SERT and NET have opposite preference for the stereochemical configuration of these inhibitors. Mutational analysis of nonconserved SERT/NET residues within the central substrate binding site was performed to determine the molecular basis for inhibitor selectivity. Changing only five residues in NET to the complementary residues in SERT transferred a SERT-like affinity profile for R- and S-citalopram into NET, showing that the selectivity of these compounds is determined by amino acid differences in the central binding site of the transporters. In contrast, the activity of R- and S-talopram was largely unaffected by any mutations within the central substrate binding site of SERT and NET and in the outer vestibule of NET, suggesting that citalopram and talopram bind to distinct sites on SERT and NET. Together, these findings provide important insight into the molecular basis for SERT/NET selectivity of antidepressants, which can be used to guide rational development of unique transporter inhibitors with fine-tuned transporter selectivity.

Homology modeling of the serotonin transporter: insights into the primary escitalopram-binding site.

The serotonin transporter (SERT) is one of the neurotransmitter transporters that plays a critical role in the regulation of endogenous amine concentrations and therefore is an important target for therapeutic agents affecting the central nervous system. The recently published, high resolution X-ray structure of the closely related amino acid transporter, Aquifex aeolicus leucine transporter (LeuT), provides an opportunity to develop a three-dimensional model of the structure of SERT. We present herein a homology model of SERT using LeuT as the template and containing escitalopram as a bound ligand. Our model explains selectivities known from mutational studies and varying ligand data, which are discussed and illustrated in the paper.

Molecular dynamics simulations of Na+/Cl(-)-dependent neurotransmitter transporters in a membrane-aqueous system.

We have performed molecular dynamics simulations of a homology model of the human serotonin transporter (hSERT) in a membrane environment and in complex with either the natural substrate 5-HT or the selective serotonin reuptake inhibitor escitalopram. We have also included a transporter homologue, the Aquifex aeolicus leucine transporter (LeuT), in our study to evaluate the applicability of a simple and computationally attractive membrane system. Fluctuations in LeuT extracted from simulations are in good agreement with crystallographic B factors. Furthermore, key interactions identified in the X-ray structure of LeuT are maintained throughout the simulations indicating that our simple membrane system is suitable for studying the transmembrane protein hSERT in complex with 5-HT or escitalopram. For these transporter complexes, only relatively small fluctuations are observed in the ligand-binding cleft. Specific interactions responsible for ligand recognition, are identified in the hSERT-5HT and hSERT-escitalopram complexes. Our findings are in good agreement with predictions from mutagenesis studies.

Synthesis and positron emission tomography evaluation of three norepinephrine transporter radioligands: [C-11]desipramine, [C-11]talopram and [C-11]talsupram.

Desipramine (DMI), talopram and talsupram, three of the most potent norepinephrine transporter (NET) inhibitors reported to date, were radiolabeled in high yields and at high specific radioactivity (58-75 GBq/micromol) by the methylation of nor-precursors with [C-11]methyl triflate. The regional brain distribution of each radioligand following intravenous injection into cynomolgus monkey was examined in vivo with positron emission tomography (PET). For all three radioligands, the regional brain distribution of radioactivity was slightly heterogeneous, with higher uptake of radioactivity in the mesencephalon, thalamus and lower brainstem than in striatum. The rank order of maximal brain radioactivity (as percentage of injected dose) was [C-11]DMI (2.7%) > [C-11]talsupram (1.3%) > [C-11]talopram (0.7%). The appearance of radioactive metabolites in plasma was similar for each radioligand (75-85% of radioactivity in plasma at 45 min). These metabolites were all more polar than their parent radioligand. The data show that these radioligands are inferior to existing radioligands for the study of brain NET with PET in vivo.

Escitalopram versus citalopram: the surprising role of the R-enantiomer.

RATIONALE: Citalopram is a racemate consisting of a 1:1 mixture of the R(-)- and S(+)-enantiomers. Non-clinical studies show that the serotonin reuptake inhibitory activity of citalopram is attributable to the S-enantiomer, escitalopram. A series of recent non-clinical and clinical studies comparing escitalopram and citalopram to placebo found that equivalent doses of these two drugs, i.e. containing the same amount of the S-enantiomer, showed better effect for escitalopram. These results suggested that the R-citalopram in citalopram inhibits the effect of the S-enantiomer. OBJECTIVE: To review the pharmacological and non-clinical literature that describes the inhibition of escitalopram by R-citalopram, as well as the implications of this inhibition for the clinical efficacy of escitalopram compared to citalopram. METHODS: The information in this review was gathered from published articles and abstracts. RESULTS: In appropriate neurochemical, functional, and behavioural non-clinical experiments, escitalopram shows greater efficacy and faster onset of action than comparable doses of citalopram. The lower efficacy of citalopram in these studies is apparently due to the inhibition of the effect of the S-enantiomer by the R-enantiomer, possibly via an allosteric interaction with the serotonin transporter. Data from randomised clinical trials consistently show better efficacy with escitalopram than with citalopram, including higher rates of response and remission, and faster time to symptom relief. CONCLUSION: The R-enantiomer present in citalopram counteracts the activity of the S-enantiomer, thereby providing a possible basis for the pharmacological and clinical differences observed between citalopram and escitalopram.