Antidepressant Binding Sites Research Articles

Identification of a novel tricyclic antidepressant binding site within opioid receptor using molecular dynamics and functional assays.

Tricyclic antidepressants (TCAs) had been used as a first-line treatment for mood disorder, but rapidly vanished due to moderate-to-severe adverse effects. Instead, the TCAs showed some promising analgesic effects towards neuropathic diseases, and an outstanding therapeutic effect to patients who suffer from irritable bowel syndrome. However, the mechanism by which those atypical effect are manifested has been unclear. Among the proposed mechanisms were the well-known, pain-related inhibitory G-protein coupled receptor (GiPCR), namely, the opioid receptor (OR). Here, we confirmed that TCA indeed stimulates OR, and commences the Gi signaling. Based on minimal-energy docking, we have identified a putative TCA binding site within OR. So, we predicted the binding site of TCA based on the previously revealed Cryo-EM structure of the activated OR. Within the binding site, an aspartate residue showed the most significant contribution towards the drug binding, and aspartate-to-arginine mutation of the residue severely decreased the OR-Gαi FRET efficiency once robustly induced by AMI.As an alternative way to monitor the Gi-signaling efficacy, we have utilized a well-known functional relationship between Gαi protein and TRPC4. Namely, we measured TRPC4 current in HEK293 cells in which either WT or mutant ORs are co-expressed. All types of TCA elicited μOR-induced TRPC4 current, as did the δ- and κ-ORs. And as expected, TCA-induced current of TRPC4 was not observed in the Asp-Arg mutant of OR. Our findings demonstrate that OR could be proclaimed as a promising target among numerous binding partners of the TCA, and that TRPC4 activation could be expected as a significant downstream pathway.

Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter.

Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode photocrosslinking unnatural amino acids (UAAs) into 75 different positions in hSERT. UAAs are incorporated with high specificity, and functionally active transporters have similar transport properties and pharmacological profiles compared with wild-type transporters. We employ ultraviolet-induced crosslinking with p-azido-L-phenylalanine (azF) at selected positions in hSERT to map the binding site of imipramine, a prototypical tricyclic antidepressant, and vortioxetine, a novel multimodal antidepressant. We find that the two antidepressants crosslink with azF incorporated at different positions within the central substrate-binding site of hSERT, while no crosslinking is observed at the vestibular-binding site. Taken together, our data provide direct evidence for defining the high-affinity antidepressant binding site in hSERT.

Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

LeuT serves as the model protein for understanding the relationships between structure, mechanism and pharmacology in neurotransmitter sodium symporters (NSSs). At the present time, however, there is a vigorous debate over whether there is a single high-affinity substrate site (S1) located at the original, crystallographically determined substrate site or whether there are two high-affinity substrates sites, one at the primary or S1 site and the other at a second site (S2) located at the base of the extracellular vestibule. In an effort to address the controversy over the number of high-affinity substrate sites in LeuT, one group studied the F253A mutant of LeuT and asserted that in this mutant substrate binds exclusively to the S2 site and that 1 mM clomipramine entirely ablates substrate binding to the S2 site. Here we study the binding of substrate to the F253A mutant of LeuT using ligand binding and X-ray crystallographic methods. Both experimental methods unambiguously show that substrate binds to the S1 site of the F253A mutant and that binding is retained in the presence of 1 mM clomipramine. These studies, in combination with previous work, are consistent with a mechanism for LeuT that involves a single high-affinity substrate binding site.

Mutational Mapping and Modeling of the Binding Site for (S)-Citalopram in the Human Serotonin Transporter

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.

Location of the Antidepressant Binding Site in the Serotonin Transporter

The serotonin transporter (SERT) regulates extracellular levels of serotonin (5-hydroxytryptamine, 5HT) in the brain by transporting 5HT into neurons and glial cells. The human SERT (hSERT) is the primary target for drugs used in the treatment of emotional disorders, including depression. hSERT belongs to the solute carrier 6 family that includes a bacterial leucine transporter (LeuT), for which a high resolution crystal structure has become available. LeuT has proved to be an excellent model for human transporters and has advanced the understanding of solute carrier 6 transporter structure-function relationships. However, the precise structural mechanism by which antidepressants inhibit hSERT and the location of their binding pockets are still elusive. We have identified a residue (Ser-438) located within the 5HT-binding pocket in hSERT to be a critical determinant for the potency of several antidepressants, including the selective serotonin reuptake inhibitor citalopram and the tricyclic antidepressants imipramine, clomipramine, and amitriptyline. A conservative mutation of Ser-438 to threonine (S438T) selectively increased the K(i) values for these antidepressants up to 175-fold. The effects of introducing a protein methyl group into the 5HT-binding pocket by S438T were absent or reduced for analogs of these antidepressants lacking a single methyl group. This suggests that these antidepressants interact directly with Ser-438 during binding to hSERT, implying an overlapping localization of substrate- and inhibitor-binding sites in hSERT suggesting that antidepressants function by a mechanism that involves direct occlusion of the 5HT-binding site.

Driving Forces for Ligand Migration in the Leucine Transporter

The potential mechanisms for driving a ligand to and through a transporter are examined for the leucine transporter using the recently published X-ray structure. Through analyses with computational methods, including investigation of electrostatic properties, site and channel identification, and docking studies, a picture emerges whereby dipolar patches which characterize the electrostatic field serve as the primary driving force to attract the ligand to the protein and begin propagation into it. The electrostatic forces are then augmented by hydrophobic forces in the transport stages, with conformational changes in the protein helping to accommodate the migration. We identify 12 sites that might be involved in ligand recognition and migration. One of these sites corresponds to the tricyclic antidepressant binding site observed in the recently published X-ray structures.

Identifying the binding site(s) for antidepressants on the Torpedo nicotinic acetylcholine receptor: [ 3H]2-azidoimipramine photolabeling and molecular dynamics studies

Radioligand binding, photoaffinity labeling, and docking and molecular dynamics were used to characterize the tricyclic antidepressant (TCA) binding sites in the nicotinic acetylcholine receptor (nAChR). Competition experiments indicate that the noncompetitive antagonist phencyclidine (PCP) inhibits [ 3H]imipramine binding to resting (closed) and desensitized nAChRs. [ 3H]2-azidoimipramine photoincorporates into each subunit from the desensitized nAChR with ∼ 25% of the labeling specifically inhibited by TCP (a PCP analog), whereas no TCP-inhibitable labeling was observed in the resting (closed) state. For the desensitized nAChR and within the α subunit, the majority of specific [ 3H]2-azidoimipramine labeling mapped to a ∼ 20 kDa Staphylococcus aureus V8 protease fragment (αV8-20; Ser 173-Glu 338). To further map the labeling site, the αV8-20 fragment was further digested with endoproteinase Lys-C and resolved by Tricine SDS-PAGE. The principal labeled fragment (11 kDa) was further purified by rpHPLC and subjected to N-terminal sequencing. Based on the amino terminus (αMet 243) and apparent molecular weight, the 11 kDa fragment contains the channel lining M2 segment. Finally, docking and molecular dynamics results indicate that imipramine and PCP interact preferably with the M2 transmembrane segments in the middle of the ion channel. Collectively, these results are consistent with a model where PCP and TCA bind to overlapping sites within the lumen of the Torpedo nAChR ion channel.

Kinetic and thermodynamic assessment of binding of serotonin transporter (SERT) inhibitors

Considering their shared mechanism of action, there is surprising diversity in the clinical outcomes of SERT inhibitors. Such behavior may arise from the binding modes of those inhibitors with SERT. We report here on the thermodynamics of ligand‐SERT interactions determined from equilibrium and kinetic binding analyses. All drugs excepting fluvoxamine displayed remarkably similar binding thermodynamics with relatively equal contributions of entropy and enthalpy to free energy of binding. Binding enthalpies correlate with ligand polar surface areas suggesting an allowance for charged/polar interactions without enormous energetic penalties for dehydration. In the case of fluvoxamine, which possesses the greatest conformational flexibility yet binds entropically, flexible interaction is implied such that the entropic penalty expected from conformational restriction is limited. Rapid association was observed for all ligands, reflecting low energy barriers and diffusion‐limited binding reactions. The inhibitors bind in a mutually‐exclusive manner with each other as well as with 5‐HT. Taken together, the antidepressant binding site is probably solvent exposed, possibly along the substrate transport pore as predicted from structural modeling based on the leucine transporter.

What Is an Antidepressant Binding Site Doing in a Bacterial Transporter?

LeuT is a bacterial amino acid transporter belonging to a large family of membrane proteins, including the neurotransmitter transporters that are targets for antidepressant drugs. The high-resolution structure of LeuT has provided an important model for understanding structure and function in this family. Two recent papers found that LeuT can bind tricyclic antidepressants, raising the possibility that it may also serve as a model for the pharmacological properties of neurotransmitter transporters.

Antidepressant binding site in a bacterial homologue of neurotransmitter transporters

Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 A above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the rational design of new inhibitors.

Linking emotion to the social brain

In 2006, research on the neurotransmitter serotonin and its transporter protein (5‐HTT) in the synaptic gap celebrated a series of anniversaries. Forty‐five years earlier, presynaptic neurotransmitter uptake was discovered (Hertting & Axelrod, 1961). Two decades later, 5‐HTT was first linked to depression (Langer et al , 1981), shortly after its identification as a target of antidepressant drugs (Raisman et al , 1979). The sequence of the transporter gene from rats was published 10 years after that (Blakely et al , 1991), which initiated an avalanche of molecular genetic studies on the regulation of emotionality. This research culminated in two reports revealing an association between variations of the 5‐HTT gene ( 5‐HTT and SLC6A4 ) and anxiety‐related traits as well as depression (Collier et al , 1996; Lesch et al , 1996). Since then, clinical studies have further supported the link between variants of 5‐HTT and disorders in the regulation of emotion. Although modest effect sizes—typical of non‐Mendelian traits—polygenic patterns of inheritance, epistatic and epigenetic interactions, and heterogeneity between studies confounded the results, 5‐HTT comprises a model molecule for studying gene–environment interactions in cognitive and psychiatric neuroscience. The demonstration in rhesus macaques that stress early in life uniquely reinforces links between variations of 5‐HTT , behaviour and psychopathology seems to herald in a new era of behavioural genetics. Moreover, the discovery that 5‐HTT is a susceptibility gene for depression is a first step towards explaining the molecular dimensions of personality and behaviour, identifying physiological pathways that lead to other disorders of cognitive function and emotion, and analysing the interactive effects of genes and environment in the development of disease. On the heels of these results from behavioural genetics, novel approaches including neurophysiology, neuropsychology and functional neuroimaging, as well as the inclusion of other phenotypes (such as higher cognitive functions, communication skills, social competence and longevity), have …

Inhibition of the Norepinephrine Transporter by the Venom Peptide χ-MrIA: SITE OF ACTION, Na+ DEPENDENCE, AND STRUCTURE-ACTIVITY RELATIONSHIP

chi-Conopeptide MrIA (chi-MrIA) is a 13-residue peptide contained in the venom of the predatory marine snail Conus marmoreus that has been found to inhibit the norepinephrine transporter (NET). We investigated whether chi-MrIA targeted the other members of the monoamine transporter family and found no effect of the peptide (100 microM) on the activity of the dopamine transporter and the serotonin transporter, indicating a high specificity of action. The binding of the NET inhibitors, [3H]nisoxetine and [3H]mazindol, to the expressed rat and human NET was inhibited by chi-MrIA with the conopeptide displaying a slight preference toward the rat isoform. For both radioligands, saturation binding studies showed that the inhibition by chi-MrIA was competitive in nature. It has previously been demonstrated that chi-MrIA does not compete with norepinephrine, unlike classically described NET inhibitors such as nisoxetine and mazindol that do. This pattern of behavior implies that the binding site for chi-MrIA on the NET overlaps the antidepressant binding site and is wholly distinct from the substrate binding site. The inhibitory effect of chi-MrIA was found to be dependent on Na+ with the conopeptide becoming a less effective blocker of [3H]norepinephrine by the NET under the conditions of reduced extracellular Na+. In this respect, chi-MrIA is similar to the antidepressant inhibitors of the NET. The structure-activity relationship of chi-MrIA was investigated by alanine scanning. Four residues in the first cysteine-bracketed loop of chi-MrIA and a His in loop 2 played a dominant role in the interaction between chi-MrIA and the NET. H alpha chemical shift comparisons indicated that side-chain interactions at these key positions were structurally perturbed by the replacement of Gly-6. From these data, we present a model of the structure of chi-MrIA that shows the relative orientation of the key binding residues. This model provides a new molecular caliper for probing the structure of the NET.

Neuroimaging of serotonin uptake sites and antidepressant binding sites in the thalamus of humans and ‘higher’ animals

This review presents the results of in vitro, ex vivo and in vivo studies carried out primarily for identifying serotonin uptake sites and/or antidepressant binding sites in the brain of humans and ‘higher’ animals, namely nonhuman primates and pigs. Five lines of evidence are considered. First, studies carried out in vitro using synaptosomes or membrane preparations from human, nonhuman primate, and porcine brain have shown that certain thalamic nuclei are major sites of serotonergic neurotransmission in these species. Second, studies carried out in vitro or ex vivo using autoradiography or immunohistochemistry have indicated that the dorsomedial nucleus and some adjacent regions of the thalamus have a particularly high density of binding sites for antidepressant drugs that are selective serotonin reuptake inhibitors (SSRIs). Third, studies carried out in the living brain of nonhuman primates and pigs have found that SSRIs, radiolabeled for use in PET or SPECT, accumulate to a relatively high degree in midline and dorsal nuclei of the thalamus. Fourth, studies carried out using PET or SPECT radioligands in humans have demonstrated that regions in and around the dorsomedial nucleus of the thalamus are principal sites for accumulation of SSRIs. Fifth, studies of behavior of humans suffering from localized tissue damage in the thalamus have reported that symptoms of mania often occur in the patients, in accordance with the notion that an intact thalamus is required for normal regulation of mood. Taken together, the findings are consistent with the hypothesis that serotonergic neurotransmission in the dorsomedial nucleus of the thalamus could be causally involved in the pathophysiology of affective disease as well as in therapeutic actions of SSRIs.

Heterogeneity of Antidepressant Binding Sites on the Recombinant Rat Serotonin Transporter SERT1

Antidepressant drugs block the uptake of serotonin into serotonergic nerve terminals and blood platelets. Here, binding of the tricyclic antidepressant [3H]imipramine to the recombinant rat serotonin transporter SERT1 expressed in human embryonic kidney cells was found to be nonhomogeneous. Scatchard analysis and competition experiments revealed the existence of two distinct antidepressant binding sites. At site 1, [3H]imipramine binding was strictly sodium-dependent with an apparent KD of approximately 10 nM. In contrast, [3H]imipramine binding to site 2 occurred also in the absence of sodium and exhibited a lower affinity. Binding of the nontricyclic antidepressant [3H]citalopram was observed only at site 2. The natural substrate of this carrier, serotonin, competitively inhibited antidepressant binding at both sites; however, its affinity to site 2 was approximately 5-fold lower. These data provide a molecular explanation for the distinct pharmacological actions of different antidepressants.

Interaction of lectins with human platelet serotonin transporter

Lectin affinity chromatography was used to demonstrate the glycoprotein nature of the serotonin (5-HT) transporter. The human platelet transporter protein was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), labelled with [ 3H]cyanoimipramine and chromatographed on lectin columns. Wheat germ, gorse seed and lentil lectin-agarose columns specifically retained 5-HT transporter. Peanut and horse gram lectins were ineffective. Concanavalin A showed high non-specific adsorption. Binding of [ 3H]imipramine to platelet membranes or solubilized 5-HT transporter was not affected by lectins. These data suggest that the lectin interaction with 5-HT transporter is extrinsic to the antidepressant binding site.

Interactions of phospholipids and free fatty acids with antidepressant recognition binding sites in rat brain.

The lipid microenvironment of cell membranes has been shown to regulate both neurotransmitter and hormone receptors. Preincubation of cortical synaptosomal membranes of rat brain with phospholipase A2 (PLA2) increases the number of [3H]imipramine ([3H]IMI) high affinity binding sites without altering Kd (Bmax control: 2.53 +/- 0.28 pmol/mg protein vs Bmax PLA2: 3.66 +/- 0.26 pmol/mg protein). The displacement curves of [3H]IMI binding in synaptosomal membranes with other tricyclic antidepressants are not affected by the presence of PLA2. The effect of PLA2 was prevented by incubation with EGTA (2 x 10(-3)) or bovine serum albumin (BSA; 1:1). In addition, end products of catalytic activity of PLA2 such as unsaturated fatty acids (arachidonic or oleic acids) mimicked the effect of PLA2. These effects were entirely prevented by preincubation with BSA. The in vitro addition of the acidic phospholipid phosphatidylserine isolated from bovine brain (BC-PS) produced a similar increase in Bmax. This action was also blocked by addition of BSA. On the other hand, palmitic acid, a saturated fatty acid, and lysophosphatidylserine (lysoPS) or lysophosphatidylethanolamine (lysoPE) failed to modify [3H]IMI binding sites. The chronic administration of tricyclic antidepressant (AD) resulted in a 25% decrease in [3H]IMI binding sites in synaptosomal membranes. Preincubation of these AD-treated membranes with PLA2 did not alter [3H]IMI binding, whereas the addition of unsaturated free fatty acids (FFA) produced a greater increase in the density of [3H]IMI binding sites in comparison with control membranes. Taken together, these findings suggest that unsaturated free fatty acids could play an important role in the regulation of the number of [3H]IMI high affinity binding sites in the mammalian brain.

Direct effects of ovarian hormones on antidepressant binding sites

Tricyclic antidepressants such as imipramine interact with a membrane binding site associated with the uptake of serotonin. Ovarian hormones estradiol and progesterone were found to affect the concentration and affinity of imipramine binding sites in the hypothalamus but not the cortex of female rats. In vivo and in vitro, estradiol increased the amount of imipramine binding at physiological concentrations; at high concentrations estradiol decreased imipramine binding. Ovariectomy (OVX) had no effect on imipramine binding, consonant with the biphasic dose-response relationship for estradiol. The effects of progesterone were dependent upon the concentration of estradiol. Effects of physiological concentrations of both estradiol and progesterone on imipramine binding in an isolated membrane preparation suggest that the hormones are affecting this aspect of serotonergic neuronal function by nongenomic mechanisms.

Drug inhibition indicates a single-site model of the 5-HT uptake site/antidepressant binding site in rat and human brain

Drug inhibition against [3H]paroxetine binding to rat cortex and human putamen was investigated in saturation experiments. The addition of 5-HT, imipramine, citalopram and clomipramine all produced changes in apparent binding affinity (Kd) without changes in the number of binding sites (Bmax). These data suggest that there is no heterogeneity of specific [3H]paroxetine binding, supporting a single site model of the 5-HT uptake site and antidepressant binding site.

Autoradiography of antidepressant binding sites in the human brain: localization using [ 3h]imipramine and [ 3h]paroxetine

[ 3H]Imipramine and [ 3H]paroxetine were used to label sites associated with serotonin uptake mechanisms in post-mortem brain tissue from control subjects. The anatomical localization of these sites was examined by autoradiography and densities measured by microdensitometry. We found [ 3H]imipramine binding to increase with age in the cortex and amygdala, but to be independent of gender and post-mortem delay. Preliminary results indicate that the binding of both [ 3H]imipramine and [ 3H]paroxetine is diminished in the brain of patients treated with imipramine. The distribution of [ 3H]imipramine and [ 3H]paroxetine high-affinity binding sites was very similar, and correlated well with the distribution of serotonergic presynaptic markers in the brain. The highest densities of binding sites were found in the raphénuclei and the midline thalamic nuclei. Other structures presenting high levels of binding were the substantia nigra, nucleus interpeduncularis, locus coeruleus, nucleus nervi hypoglossi, nucleus nervi facialis, mammillary bodies and other parts of the hypothalamus. In contrast, regions such as the neocortex, hippocampus, amygdala and cerebellum showed low densities of [ 3H]imipramine and [ 3H]paroxetine binding sites. This distribution seems to indicate that the ascending serotonergic pathways are the main site of action of antidepressants.

Effects of antidepressants on monoamine transporters

Langer, Salomon Z. and Schoemaker, Hans: Effects of antidepressants on monoamine transporters. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1988, 12 :193–216 1. 1. Using [ 3H]antidepressants, high affinity binding sites associated with the neuronal transporter for serotonin, noradrenaline, dopamine and adrenaline have been Identified. 2. 2. The association of high affinity [ 3H]imipramine binding with the serotonin transporter in brain and platelets is well established. Although the exact relationship between the [ 3H]imipramine recognition site and the serotonin transporter remains to be elucidated, it appears that the [ 3H]imipramine labelled component of the serotonin transporter represents a novel receptor that functions to modulate serotonin uptake. 3. 3. Most data available to date support the hypothesis that [ 3H]imipramine binding to platelet represents a biological marker in depression. The majority of studies indicate that the Bmax of platelet [ 3H]imipramine binding is lower in depressed, untreated patients than in the control population and that this finding is relatively specific to depression. 4. 4. Among the [ 3H]antidepressant binding sites associated with the other monoaminergic transporters, the recent identification of [ 3H]desipramine binding to the neuronal transporter for adrenaline offers novel perspectives. Thus, given the high affinity for [ 3H]desipramine binding to the adrenaline transporter in the frog heart for not only desipramine but also imipramine and the atypical antidepressants mianserin and iprindol, it is possible that an interaction with the adrenaline transporter is of significance to the clinical effects of antidepressant drugs.